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WO2015081199A1 - Compositions and methods for treating herpesvirus infection - Google Patents

Compositions and methods for treating herpesvirus infection Download PDF

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Publication number
WO2015081199A1
WO2015081199A1 PCT/US2014/067624 US2014067624W WO2015081199A1 WO 2015081199 A1 WO2015081199 A1 WO 2015081199A1 US 2014067624 W US2014067624 W US 2014067624W WO 2015081199 A1 WO2015081199 A1 WO 2015081199A1
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WIPO (PCT)
Prior art keywords
nhc
group
chromen
alkyl
alkenyl
Prior art date
Application number
PCT/US2014/067624
Other languages
French (fr)
Inventor
Yan Yuan
Jun Xu
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The Trustees Of The University Of Pennsylvania
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Publication of WO2015081199A1 publication Critical patent/WO2015081199A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones

Definitions

  • Kaposi's sarcoma-associated herpesvirus also known as human herpesvirus 8 (HHV-8)
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • KS Kaposi's sarcoma
  • KS Kaposi's sarcoma
  • N. Engl. J. Med. 332 1181-1185, Dupin N, et al. 1995. Lancet. 345:761-762, Schalling M, et. 1995. Nat. Med.
  • the contemporary treatment modalities for KS and other KSHV- associated malignancies include conventional cancer therapies, such as radiation and chemotherapy, or AIDS treatment such as HAART (Antman K, et al. 2000. N. Engl. J. Med. 342: 1027-1038). These cancer therapies in general present serious side effects and tumor response to them is only transient.
  • HPV vaccine in preventing cervical cancer has proven that antiviral orientated therapies have the potential to be used to treat human malignant diseases that are caused by viruses.
  • an antiviral targeting KSHV could prove successful at treating KSHV-associated tumor (Moore PS, et al. 2011. Blood. 117:6973-6974).
  • KSHV-associated disease currently there is no therapy for KSHV-associated disease on the basis of targeting of KSHV.
  • KSHV has two types of replication cycle, latent and lytic replication.
  • latent and lytic replication In KS lesions, most KSHV-transformed spindle-shaped cells are in latent replication phase, but a small percentage of infected cells undergo spontaneous lytic replication (Staskus KA, et al. 1997. J. Virol. 71 :715-719, Sun R, et al. 1999. J. Virol. 73:2232-2242, Zhong W, et al. 1996. Proc. Natl. Acad. Sci. U. S. A. 93:6641- 6646).
  • Increasing evidences suggest that the lytic replication in these cells is necessary for maintaining the stable infection and viral pathogenicity.
  • the majority of KSHV genes including those responsible for malignant cell growth and the alteration of
  • Viral DNA replication is considered an ideal target for antivirals.
  • the mechanisms that control KSHV lytic DNA replication have been reported (Lin CL, et al. 2003. J. Virol. 77:5578-5588, Wang Y, et al. 2004. J. Virol. 78:8615-8629, Wang Y, et al. 2006. J. Virol. 80: 12171-12186, Wang Y, et al. 2008. J. Virol. 82:2867-2882).
  • Topo II topoisomerases I and II
  • MSH2/6, RecQL MSH2/6, RecQL
  • PARP-1 poly[ADP-ribose] polymerase 1
  • Topo II inhibitors namely catalytic Topo II inhibitor, was found to have marked inhibition of KSHV replication with minimal cytotoxicity as indicated by their high selectivity indices (e.g. 31.6 for novobiocin) (Gonzalez-Molleda L, et al. 2012. Antimicrob. Agents Chemother. 56:893-902).
  • Topo II can serve as effective targets for antivirals and catalytic inhibitors of Topo II represent promising antiviral agents for the treatment of malignancies associated with KSHV infection, but clinically useful therapeutics have not been developed. There is thus a need in the art for identifying and generating therapeutics that can be used clinically to treat a KSHV infection. The present invention addresses this unmet need in the art.
  • the invention provides a method of treating a herpesvirus infection. In one embodiment, the invention provides a method of treating a gamma-herpesvirus infection. In one embodiment, the invention provides a method of treating a Kaposi's sarcoma- associated herpes virus (KSHV) infection. In one embodiment, the invention provides a method of treating an Epstein-Barr virus (EBV) infection.
  • KSHV Kaposi's sarcoma- associated herpes virus
  • EBV Epstein-Barr virus
  • the invention provides a method of treating a herpesvirus infection in an individual in need thereof, the method comprising the step of: administering to the individual an effective herpesvirus antiviral amount of a
  • composition comprising at least one compound selected from the group consisting of:
  • each occurrence of R 7 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C 3 -Ci 0 heterocycloalkyl, and C 3 -C 6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted; and X is -CH or O; and
  • each occurrence of R 6 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C 3 -C 10 heterocycloalkyl, and C 3 -C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
  • the compound is selected from the group consisting of:
  • the compound of formula (I) is at least one compound of formula (III):
  • each occurrence of R 7 and R 8 is independently selected from the group consisting of H, -Ci-C 6 alkyl, -Ci-C 6 alkenyl, aryl, heteroaryl, cycloalkyl,
  • each occurrence of R 9 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C 3 -C 10 heterocycloalkyl, and C 3 -C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
  • the compound is selected from the group consisting of (5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan- 2-yl acetate, a salt, solvate, or N-oxide thereof, and combinations thereof.
  • the method further comprises administering to the individual an effective amount of a therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a protease inhibitor, a cytokine, and an immunomodulator.
  • the method further comprises administering to the individual an effective amount of a chemotherapeutic agent.
  • the chemotherapeutic agent is selected from the group consisting of a topoisomerase II inhibitor, an antibiotic, a vinca alkaloid, an
  • the invention also provides a method for inhibiting replication of a herpesvirus infection. In one embodiment, the invention provides a method for inhibiting replication of a gamma-herpesvirus. In one embodiment, the invention provides a method for inhibiting replication of a KSHV. In one embodiment, the invention provides a method for inhibiting replication of an EBV.
  • Figure 1 is a series of images depicting the results for the compound screening experiments.
  • Figure 1 A Thirty-three compounds derived from a virtual screening were assayed for their effects on KSHV lytic DNA replication. BCBL-1 cells were induced with TPA and treated with each of the compounds at the concentration of 20 ⁇ . Forty-eight hours post-induction, the intracellular KSHV genomic copy numbers were determined using quantitative real-time PCR. Seven compounds (marked with *) exhibited considerable inhibitory effects on viral lytic DNA replication.
  • Figure IB Effects of the compounds on KSHV virion production.
  • Figure 1C Effects of the compounds on host cell viability. Uninduced BCBL-1 cells were treated with each of the compounds at 20 ⁇ for 48 hours and cell viability was assessed by trypan blue staining as described in Materials and methods. Compounds are shown with ascending molecular weights.
  • Figure 2 is a series of images demonstrating the effect of (+)-Rutamarin on KSHV replication and its associated cytotoxicity.
  • Figure 2A Chemical structure of (+)-Rutamarin.
  • Figure 2B The effect of (+)-Rutamarin on KSHV lytic replication and its associated cytotoxicity in BCBL-1 cells that were treated with a wide range of concentration of (+)-Rutamarin 3 hour after the lytic replication was induced by TPA. Intracellular KSHV genomic DNA replication (blue), extracellular virion production, and cell viability were determined as described elsewhere herein. Values were compared to those from the control cells (non-drug treatment).
  • Figure 3 is a graph depicting the effects of (+)-Rutamarin and novobiocin on BCBL-1 cell proliferation.
  • BCBL-1 cells starting with 2> ⁇ 10 5 cells/ml
  • (+)-Rutamarin and novobiocin were exposed to (+)-Rutamarin and novobiocin, respectively, at the concentrations of their IC 50 and 5 XIC50.
  • Data were obtained from three independent determinations and presented as means with standard deviations.
  • Figure 4 is a graph depicting the effects of (+)-Rutamarin and novobiocin on BCBL-1 cell cycle progression.
  • BCBL-1 cells starting with 2x l0 5 cells/ml
  • (+)-Rutamarin and novobiocin at the concentrations of their IC 50 and 5xICso for 48 hours.
  • Cell cycle progressions were measured by propidium iodide (PI) staining followed by flow cytometric analysis. The data are presented as means obtained from three independent experiments.
  • Figure 5 is an image showing inhibition of KSHV orz-Zyt-dependent DNA replication with (+)-Rutamarin.
  • BCBL-1 cells were transfected with KSHV ori-Lyt- containing plasmid (pOri-A) and RTA expression vector (pCR3.1-ORF50). The transfected cells were treated with increasing concentrations of (+)-Rutamarin and incubated for 72 hours. Hirt DNAs were extracted and digested with Kpnl/Sacl or Kpnl/Sacl/Dpnl. Dpnl-resistant viral replicated DNA (Rep'd DNA) was detected by Southern blotting with 32 P-labeled pBluescript plasmid.
  • Figure 6 is a series of images demonstrating that identification of (+)-Rutamarin as a catalytic inhibitor of Topo Ila.
  • Figure 6A The Topo II-mediated kinetoplast DNA decatenation assay was performed to evaluate the effect of testing compounds for Topo Ila activity at the concentration of 100 ⁇ . The catenated (cat) and decatenated (decat) DNA positions in gels were indicated. The Topo Ila activities in the reactions in ( Figure 6A) were quantitated and inhibition rates of compounds were calculated and presented in ( Figure 6B). The inhibition of Topo Ila (Figure 6C) and Topo Ila (Figure 6E) activities by (+)-Rutamarin in a wide range of concentration was determined.
  • Figure 7 is a graph depicting the effect of (+)-Rutamarin on ATPase activity of Topo Ila.
  • the inhibitory effect of (+)-Rutamarin on Topo Ila ATPase activity was measured using the malachite green assay as described elsewhere herein.
  • the OD 6 2o represents the ATP hydrolysis level and reflects the inhibition of ATPase activity by (+)- Rutamarin.
  • Figure 8 is a series of images demonstrating the stability properties of simulation system and binding model of (+)- Rutamarin with Topo Ila.
  • Figure 8A RMSD plot for the backbone atoms (light blue) and (+)-Rutamarin (light green) during the MD simulations after the equilibration.
  • FIG. 8B Distance distributions between the Asn95-, Alal67- and conserved water 2 and (+)-Rutamarin in water at 310 K. The cutoff value for the formation of a hydrogen bond is 3.5 A.
  • Figure 8C The time dependence of distance between carboxyl oxygen of Glu87, amide nitrogen of Asn91, the two conserved water molecules and Mg 2+ during the 10 ns MD simulations.
  • Figure 8D The detail binding model between (+)-Rutamarin and the residues in the ATPase domain of Topo ⁇ . Hydrogen bonds are shown in red dot line and magenta sphere represents Mg cation.
  • Figure 9 is an image demonstrating the dinding free energy decomposition results based on MM-GBSA method.
  • the key residues are labeled.
  • the unit of the each residue's contribution to total binding energy is kcal/mol.
  • Figure 10 is an image showing the cytotoxicity of (+)-Rutamarin to peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Figure 11 is an image showing the effect of (+)-Rutamarin on RTA expression.
  • BCBL-1 cells were induced with 20ng/ml TPA. After three hours, the cells were treated with different concentrations of (+) Rutamarin as indicated. Forty-eight hours post-induction, whole cell extracts were prepared and analyzed by Western blot with antibodies against RTA and ⁇ -actin.
  • Figure 12 is a schematic showing the chemical structure of novobiocin, merbarone and (+)-rutamarin.
  • Figure 13 comprising Figures 13A through 13E is a series of images showing the effects of Topo II inhibitors on EBV replication and their associated cytotoxicity in P3HR-1 cells.
  • EBV lytic replication in P3HR-1 was induced with TPA and sodium butyrate.
  • Topo II catalytic inhibitors, novobiocin ( Figure 13 A), merbarone ( Figure 13B) and (+)-rutamarin ( Figure 13C) were added to the cell culture 3 h after the induction.
  • the concentration ranges tested for different inhibitors are: 0.1-1000 ⁇ (novobiocin), 0.1 -760 ⁇ (merbarone), and 0.01-150 ⁇ ((+)-rutamarin).
  • Intracellular EBV DNA, extracellular virion DNA, and cell viability were determined for each concentration point. These values were compared to those from the control cells (non- drug treatment). Mean values of results from at least three independent experiments and standard deviations are presented on the Faxes of dose-response curves. Topo II inhibitor doses are indicated on the x axes as logarithmic scales. The effect of (+)- rutamarin on cell viability of P3HR-1 cells on 2 and 5 days exposure were shown in Figure 13D and Figure 13E.
  • Figure 14 is a series of images showing the effects of Topo II inhibitors on EBV replication and their associated cytotoxicity in Akata-Bxl cells.
  • EBV lytic replication in Akata-Bxl was induced by treatment of cells with anti-IgG.
  • Topo II catalytic inhibitors, novobiocin ( Figure 13 A), merbarone ( Figure 13B) and (+)-rutamarin ( Figure 13C) in wide ranges were added to the cell culture 3 h after the induction.
  • Intracellular EBV DNA, extracellular virion DNA, and cell viability were determined for each concentration point. These values were compared to those from the control cells (non-drug treatment).
  • Figure 15 is an image showing the effects of Topo II inhibitors on P3HR-1 cell proliferation.
  • P3HR-1 cells starting with 2 x 10 5 cells/ml
  • IC 50 and 5 x IC 50 concentrations
  • Figure 16 is a series of images showing inhibition of EBV ori-Lyt-associated DNA replication with Topo II inhibitors.
  • P3HR-1 cells were transfected with an ori-Lyt-containing plasmid (pEBV-oriL) and ZTA expression vector (McZ).
  • Transfected cells were cultured in the absence or presence of increasing concentrations of novobiocin ( Figure 16A), merbarone ( Figure 16B), and (+)- rutamarin ( Figure 16C). After 72 h of incubation, hirt DNAs were extracted from the cells and digested with EcoRI or EcoRI/Dpn I. Dpnl -resistant products of DNA replication (Rep'd DNA) were detected by Southern blotting with digoxigenin - labeled EcoR I - Hind III fragment from the pEBV-oriL plasmid.
  • the present invention relates to the unexpected discovery that a ligand based virtual screening approach identified a set of coumarin derivatives that exhibited inhibitory activity against Kaposi's sarcoma-associated herpesvirus (KSHV) DNA replication.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • compounds of the invention exhibit topoisomerase II (Topo II) inhibitor activity.
  • the compounds of the invention belong to the category of catalytic Topo II inhibitors.
  • the compounds of the invention block KSHV lytic DNA replication.
  • the compounds of the invention can be used to treat viral (such as herpesvirus) infection, as well treating and KSHV infection or related conditions.
  • viral such as herpesvirus
  • the invention should not be liminted to treating herpesvirus infection. Rather, the invention includes treating all types of herpesvirus, for example, gamma-herpesvirus infections including but not limited to two human pathogens Epstein-Barr virus (EBV) and KSHV.
  • EBV Epstein-Barr virus
  • KSHV Pharmaceutically acceptable salts and
  • Herpesvirus is an animal DNA virus and Herpesviridae viruses are classified into three subfamilies, that is, the alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae.
  • Alphaherpesvirinae is classified further into Herpes simplex virus, Varicellovirus, Mardivirus, and Ilto virus and typical ones include HSV-1, HSV-2, varicella/zoster virus, porcine herpes virus 1 (pseudorabies virus), and bovine herpesvirus.
  • Viruses belonging to the betaherpesvirinae include human cytomegalovirus (HCMV), while as viruses belonging to the gammaherpesvirinae, EB virus, Kaposi's sarcoma-associated herpesvirus, and the like are known.
  • Epstein-Barr virus (EB virus) of Lymphocryptovirus genus is typical.
  • the present invention also provides a method for inhibiting gamma- herpesvirus replication in a subject.
  • the present invention also provides a method for inhibiting KSHV replication in a subject.
  • the method comprises administering to the subject a composition of the invention by any suitable route of administration.
  • the method of the present invention is useful for administrating the
  • compositions of the invention to a subject exposed to a gamma-herpesvirus, such as KSHV, and to a subject who is at risk of contact with a gamma-herpesvirus, such as KSHV.
  • a gamma-herpesvirus such as KSHV
  • the compounds of the invention act as a topoisomerase II catalytic inhibitor.
  • Another aspect of the invention pertains to a composition comprising a compound as described herein and a pharmaceutically acceptable carrier or diluent.
  • Another aspect of the present invention pertains to a compound as described herein for use in a method of treatment of the human or animal body by therapy.
  • Another aspect of the present invention pertains to use of a compound, as described herein, in the manufacture of a medicament for use in treatment.
  • the present invention pertains to a method of inhibiting (e.g., catalytically inhibiting) topoisomerase II in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a compound of the invention.
  • the present invention pertains to a method of treating a gamma-herpesvirus infection, such as KSHV infection or related conditions, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of the invention, preferably in the form of a pharmaceutical composition.
  • a gamma-herpesvirus infection such as KSHV infection or related conditions
  • the treatment is treatment of a disease or condition that is ameliorated by the catalytic inhibition of topoisomerase II.
  • the treatment is treatment of a proliferative condition.
  • the treatment is treatment of cancer.
  • an element means one element or more than one element.
  • abnormal when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal” (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.
  • the term "container” includes any receptacle for holding the pharmaceutical composition.
  • the container is the packaging that contains the pharmaceutical composition.
  • the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition.
  • packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical
  • composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product.
  • the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating or preventing a disease in a subject.
  • a "disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a disorder in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • a disease or disorder is "alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
  • the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, including simians and humans.
  • herpesvirus or “herpes virus” or “HV” refers to human herpesviruses and may be used, depending on the context, to refer to one, more, or all of the human herpesviruses, including Human Herpesvirus- 1 (HHV-1, Herpes Simplex Virus- 1, HSV1, HSV-1), HHV-2 (Herpes Simplex Virus-2, HSV2, HSV-2), HHV-3 (Varicella Zoster Virus, VZV), HHV-4 (Epstein-Barr Virus, EBV), HHV-5
  • HHV-1 Human Herpesvirus- 1
  • HHV-2 Herpes Simplex Virus-2, HSV2, HSV-2
  • HHV-3 Varicella Zoster Virus, VZV
  • HHV-4 Epstein-Barr Virus, EBV
  • HHV-5 Human Herpesvirus- 1
  • HHV-2 Herpes Simplex Virus-2, HSV2, HSV-2
  • HHV-3 V
  • a herpesvirus is HHV-1.
  • a herpesvirus is HHV-2.
  • a herpesvirus is HHV-3.
  • a herpesvirus is HHV-4.
  • a herpesvirus is HHV-5.
  • a herpesvirus is HHV-6.
  • a herpesvirus is HHV-7.
  • a herpesvirus is HHV-8.
  • each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8 may refer to all strains of each respective HHV.
  • each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8 may refer to a single strain of that HHV.
  • each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV- 8 may include mutants of that particular HHV.
  • HV is HSV (HHV- 1 and/or HHV-2).
  • KS Kaposi sarcoma
  • Classic (or Mediterranean) KS KS
  • endemic KS KS
  • AIDS-related (epidemic) KS KS
  • iatrogenic (transplant-associated) KS immunosuppression-associated KS.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a human.
  • composition refers to a mixture of at least one compound of the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • “Pharmaceutically acceptable” refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.
  • “Pharmaceutically acceptable carrier” refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.
  • the term "pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
  • Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; ellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic s
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions.
  • the "pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention.
  • Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in
  • salt embraces addition salts of free acids or free bases that are compounds useful within the invention.
  • Suitable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, phosphoric acids, perchloric and tetrafluoroboronic acids.
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, ⁇ -hydroxybutyric, sal
  • Suitable base addition salts of compounds useful within the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, lithium, calcium, magnesium, potassium, sodium and zinc salts.
  • Acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl- glucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding free base compound by reacting, for example, the appropriate acid or base with the corresponding free base.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology, for the purpose of diminishing or eliminating those signs or symptoms.
  • treating a disease or disorder means reducing the frequency or severity with which a sign or symptom of the disease or disorder is experienced by a patient.
  • the terms "effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • an "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.
  • the term “potency” refers to the dose needed to produce half the maximal response (ED 50 ).
  • the term “efficacy” refers to the maximal effect (E max ) achieved within an assay.
  • (+)-rutamarin refers to (5)-2-(6-(2-methylbut- 3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan-2-yl acetate.
  • alkyl by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. Ci- 6 means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups.
  • Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl.
  • Most preferred is (Ci-C 6 )alkyl, particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.
  • substituted alkyls include, but are not limited to, 2,2-difluoropropyl,
  • heteroalkyl by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized.
  • the heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -0-CH 2 -CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -OH, -CH 2 -CH 2 -NH-CH 3 ,
  • Up to two heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 , or -CH 2 -CH 2 -S-S-CH 3
  • alkoxy employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homo logs and isomers.
  • oxygen atom such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homo logs and isomers.
  • halo or halogen alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.
  • cycloalkyl refers to a mono cyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom.
  • the cycloalkyl group is saturated or partially unsaturated.
  • the cycloalkyl group is fused with an aromatic ring.
  • Cycloalkyl groups include groups having from 3 to 10 ring atoms.
  • Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:
  • Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene.
  • Polycyclic cycloalkyls include adamantine and norbornane.
  • cycloalkyl includes "unsaturated nonaromatic carbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon carbon double bond or one carbon carbon triple bond.
  • heterocycloalkyl refers to a heteroalicyclic group containing one to four ring heteroatoms each selected from O, Sand N.
  • each heterocycloalkyl group has from 4 to 10 atoms in its ring system, with the proviso that the ring of said group does not contain two adjacent O or S atoms.
  • the heterocycloalkyl group is fused with an aromatic ring.
  • the heterocycloalky group is fused to a second heterocycle.
  • the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized.
  • the heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure.
  • a heterocycle may be aromatic or non-aromatic in nature.
  • the heterocycle is a heteroaryl.
  • An example of a 3-membered heterocycloalkyl group includes, and is not limited to, aziridine.
  • 4-membered heterocycloalkyl groups include, and are not limited to, azetidine and a beta lactam.
  • 5-membered heterocycloalkyl groups include, and are not limited to, pyrrolidine, oxazolidine and thiazolidinedione.
  • 6-membered heterocycloalkyl groups include, and are not limited to, piperidine, morpholine and piperazine.
  • Other non-limiting examples of heterocycloalkyl groups are:
  • non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1 ,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,
  • aromatic refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized ⁇ (pi) electrons, where n is an integer.
  • aryl employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene.
  • aryl groups include phenyl, anthracyl, and naphthyl. Preferred examples are phenyl and naphthyl, most preferred is phenyl.
  • aryl-(Ci-C3)alkyl means a functional group wherein a one- to three-carbon alkylene chain is attached to an aryl group, e.g.,
  • aryl-CH 2 CH 2 -phenyl Preferred is aryl-CH 2 - and aryl-CH(CH 3 )-.
  • substituted aryl-(Ci-C 3 )alkyl means an aryl-(Ci-C 3 )alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH 2 )-.
  • heteroaryl- (Ci-C 3 )alkyl means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e.g. , -CH 2 CH 2 -pyridyl. Preferred is heteroaryl-(CH 2 )-.
  • substituted heteroaryl-(Ci-C 3 )alkyl means a heteroaryl-(Ci-C 3 )alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl- (CH 2 )-.
  • heteroaryl or “heteroaromatic” refers to a heterocycle having aromatic character.
  • a polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include the following
  • heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
  • polycyclic heterocycles and heteroaryls examples include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5 -quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1 ,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-difiydrobenzofuryl, 1 ,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothien
  • 2-benzimidazolyl benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.
  • substituted means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
  • substituted further refers to any level of substitution, namely mono-, di-, tri-, terra-, or penta-substitution, where such substitution is permitted.
  • the substituents are
  • the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two.
  • the term "optionally substituted” means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.
  • the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NH 2 , -OH, -NH(CH 3 ), -N(CH 3 ) 2 , alkyl (including straight chain, branched and/or unsaturated alkyl), substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted
  • the substituents are independently selected from the group consisting of Ci_ 6 alkyl, -OH, Ci_ 6 alkoxy, halo, amino, acetamido, oxo and nitro. In yet another embodiment, the substituents are independently selected from the group consisting of Ci_ 6 alkyl, Ci_ 6 alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being preferred.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description
  • the invention provides compositions and methods for modulating and treatment of a gamma-herpesvirus infection, such KSHV infection, or KSHV-mediated effects on cellular proliferation and phenotype, comprising inhibiting KSHV lytic replication.
  • the compounds of the invention can be used to treat or prevent KSHV infection, Kaposi's sarcoma (KS) and related cancers and conditions.
  • KS Kaposi's sarcoma
  • the compounds of the invention inhibit both viral DNA synthesis and virion production with relatively low cytotoxicity.
  • the compounds of the invention inhibit the enzymatic activity of Topo ⁇ in a dose-dependent manner. In one embodiment, the compounds of the invention function as a Topo ⁇ catalytic inhibitor.
  • the compounds of the present invention may be synthesized using techniques well-known in the art of organic synthesis.
  • the starting materials and intermediates required for the synthesis may be obtained from commercial sources or synthesized according to methods known to those skilled in the art.
  • the compound of the invention is a compound of formula (I), or a salt, solvate, or N-oxide thereof:
  • each occurrence of R 7 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C3-C10 heterocycloalkyl, and C 3 -C 6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted; and
  • X is -CH or O.
  • the compound of the invention is a compound of formula (II), or a salt, solvate, or N-oxide thereof:
  • each occurrence of R 6 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted.
  • the compound of the invention is a compound of formula (III), or a salt, solvate, or N-oxide thereof:
  • each occurrence of R 7 and R 8 is independently selected from the group consisting of H, -Ci-C 6 alkyl, -Ci-C 6 alkenyl, aryl, heteroaryl, cycloalkyl,
  • each occurrence of R 9 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C 3 -C 10 heterocycloalkyl, and C 3 -C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted.
  • the compound of the invention is selected from the group consisting of:
  • the compound of formula (III) is 2-(6-(2-methylbut-3- en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan-2-yl acetate, a salt, solvate, or N-oxide thereof, and any combination thereof.
  • the compounds of the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration.
  • compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In one embodiment, a mixture of one or more isomer is utilized as the therapeutic compound described herein.
  • compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non- limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
  • N-oxides if appropriate, crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the invention, as well as metabolites and active metabolites of these compounds having the same type of activity.
  • Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like.
  • the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol.
  • the compounds described herein exist in unsolvated form.
  • the compounds of the invention may exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
  • prodrugs In one embodiment, compounds described herein are prepared as prodrugs.
  • a "prodrug” refers to an agent that is converted into the parent drug in vivo.
  • a prodrug upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
  • sites on, for example, the aromatic ring portion of compounds of the invention are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In one embodiment, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
  • Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2 H, 3 H, U C, 13 C, 14 C, 36 C1, 18 F, 123 1, 125 1, 13 N, 15 N,
  • isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies.
  • substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
  • substitution with positron emitting isotopes such as 11 C, 18 F, 15 O and 13 N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
  • the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
  • Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed.
  • each protective group is removable by a different means.
  • Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal.
  • protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions.
  • reducing conditions such as, for example, hydrogenolysis
  • oxidative conditions such as, for example, hydrogenolysis
  • Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile.
  • Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.
  • base labile groups such as, but not limited to, methyl, ethyl, and acetyl
  • carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc.
  • Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as
  • Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts.
  • an allyl-b locked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base- labile acetate amine protecting groups.
  • Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.
  • blocking/protecting groups may be selected from:
  • Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS) and related diseases. Without wishing to be bound by any particular theory, it is believed that blocking lytic KSHV replication hinders KSHV-induced hyperproliferative disorders such as cancer.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • the invention includes methods for treating KSHV infection and related conditions (e.g., KS).
  • the methods for treating KS provided herein inhibit, reduce, diminish, arrest, or stabilize a tumor associated with KS or a symptom thereof.
  • the methods for treating KS provided herein inhibit, reduce, diminish, arrest, or stabilize the blood flow, metabolism, peritumoral inflammation or peritumoral edema in a tumor associated with KS or a symptom thereof.
  • the methods for treating KS provided herein reduce, ameliorate, or alleviate the severity of KS and/or a symptom thereof.
  • the methods for treating KS provided herein cause the regression of a KS tumor, tumor blood flow, tumor metabolism, or peritumoral edema and/or a symptom associated with KS.
  • the methods for treating KS provided herein reduce hospitalization (e.g., the frequency or duration of hospitalization) of a subject diagnosed with KS.
  • the methods for treating KS provided herein reduce hospitalization length of a subject diagnosed with KS.
  • the methods provided herein increase the survival of a subject diagnosed with KS.
  • the methods for treating KS provided herein inhibit or reduce the progression of one or more tumors or a symptom associated therewith.
  • the methods for treating KS provided herein enhance or improve the therapeutic effect of another therapy (e.g., an anti-cancer agent, radiation, chemotherapy, or surgery).
  • the methods for treating KS involve the use of a compound as an adjuvant therapy.
  • the methods for treating KS provided herein improve the ease in removal of tumors (e.g., enhance respectability of the tumors) by reducing vascularization prior to surgery.
  • the methods for treating KS provided herein reduce vascularization after surgery, for example, reduce vascularization of the remaining tumor mass not removed by surgery.
  • the methods for treating KS provided herein prevent recurrence, e.g., recurrence of vascularization and/or tumor growth.
  • the methods for treating KS provided herein reduce or eliminate one, two, or more of the following: skin lesions, nausea, vomiting, abdominal pain, bleeding, difficulty breathing and lymphedema.
  • the methods for treating KS provided herein reduce the growth of a tumor associated with KS.
  • the methods for treating KS provided herein eradicate, remove, or control primary, regional and/or metastatic tumors associated with KS.
  • the methods for treating KS provided herein decrease the number or size of lesions associated with KS.
  • the methods for treating KS provided herein reduce the mortality of subjects diagnosed with KS.
  • the methods for treating KS provided herein increase the tumor-free survival rate of patients diagnosed with KS.
  • the methods for treating KS provided herein increase relapse-free survival. In certain embodiments, the methods for treating KS provided herein increase the number of patients in remission or decrease the hospitalization rate. In other embodiments, the methods for treating KS provided herein maintain the size of the tumor so that it does not increase, or so that it increases by less than the increase of a tumor after administration of a standard therapy as measured by methods available to one of skill in the art, such as measurement of a lesion, photography, X-ray, CT Scan, MRI, PET Scan, bronchoscopy, and endoscopy. In other embodiments, the methods for treating KS provided herein prevent the development or onset of KS, or a symptom associated therewith.
  • the methods for treating KS provided herein increase the length of remission in patients. In particular embodiments, the methods for treating KS provided herein increase symptom-free survival of KS patients. In some embodiments, the methods for treating KS provided herein do not cure KS in patients, but prevent the progression or worsening of the disease.
  • the methods for treating KS achieve one or more of the following: (i) inhibition or reduction in pathological production of VEGF; (ii) stabilization or reduction of peritumoral inflammation or edema in a subject; (iii) reduction of the concentration of VEGF or other angiogenic or inflammatory mediators (e.g., cytokines or interleukins) in biological specimens (e.g., plasma, serum, cerebral spinal fluid, urine, or any other bio fluids); (iv) reduction of the concentration of P IGF, VEGF-C, VEGF-D, VEGF-R, IL-6, IL-8 and/or IL-10 in biological specimens (e.g., plasma, serum, cerebral spinal fluid, urine, or any other biofluids); (v) inhibition or decrease in tumor metabolism or perfusion; (vi) inhibition or decrease in angiogenesis or vascularization; and/or (vii) improvement in quality of life as assessed by methods well known in the art, for e.g, a
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject as determined by methods well known in the art, e.g., MRI. Three dimensional volumetric measurement performed by MRI has been shown to be sensitive and consistent in assessing tumor size (see, e.g., Harris et al, Neurosurgery, June 2008, 62(6): 1314-9), and thus may be employed to assess tumor shrinkage in the methods provided herein.
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least about 20% as assessed by methods well known in the art, e.g., MRI.
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art, e.g., MRI.
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least an amount in a range of from about 10% to about 100%, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art.
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by an amount in a range of from about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 40% to 100%, or any range in between, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art, e.g., MRI.
  • the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject as assessed by methods well known in the art, e.g., dynamic contrast-enhanced MRI (DCE-MRI).
  • DCE-MRI dynamic contrast-enhanced MRI
  • Standard protocols for DCE-MRI have been described (see., e.g., Morgan et al, J. Clin. Oncol, Nov. 1, 2003, 21(21):3955- 64; Leach et al, Br. J. Cancer, May 9, 2005, 92(9): 1599-610; Liu et al, J. Clin. Oncol, August 2005, 23(24): 5464-73; and Thomas et al, J. Clin. Oncol, Jun. 20, 2005,
  • the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, relative to tumor perfusion prior to administration of a compound, as assessed by methods well known in the art, e.g., DCE-MRI.
  • the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject in the range of from about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 40% to 100%, or any range in between, relative to tumor perfusion prior to administration of a compound, as assessed by methods well known in the art.
  • the methods for treating KS provided herein reduce or inhibit KSHV gene transcription or reduce KSHV mRNA levels in tumor biopsies from a subject with KS by about 5%, 10%, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or any range in between, relative to the respective KSHV gene transcription level or KSHV mRNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • the methods for treating KS provided herein reduce or inhibit KSHV gene transcription or reduce KSHV mRNA levels in tumor biopsies from a subject with KS by 5% to 10%>, 10%> to 20%>, 10%> to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between relative to the respective KSHV gene transcription level or KSHV mRNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • the methods for treating KS reduce or inhibit KSHV replication or reduce the number of copies of KSHV in circulating peripheral blood mononuclear cells (PBMC) in a subject with KS by about 5%, 10%>, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or any range in between, relative to the respective KSHV replication level or number of KSHV copies in circulating PBMC observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • PBMC peripheral blood mononuclear cells
  • the methods for treating KS provided herein reduce or inhibit KSHV replication or reduce the number of copies of KSHV in circulating PBMC in a subject with KS by about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between, relative to the respective KSHV replication level or number of KSHV copies in circulating PBMC observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • the methods for treating KS provided herein inhibit or reduce HIV plasma RNA levels in a subject with KS by about 5%, 10%, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in a subject with KS relative to the respective HIV plasma RNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • the methods for treating KS provided herein inhibit or reduce HIV plasma RNA levels in a subject with KS by about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between, relative to the respective HIV plasma RNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • compositions can contain additional compatible pharmaceutically active materials for combination therapy (such as supplementary antimicrobials, antipruritics, astringents, local anesthetics, anticancer, or anti-inflammatory agents), or can contain materials useful in physically formulating various dosage forms of the preferred embodiments, such as excipients, dyes, perfumes, thickening agents, stabilizers, skin penetration enhancers, preservatives or antioxidants.
  • additional compatible pharmaceutically active materials for combination therapy such as supplementary antimicrobials, antipruritics, astringents, local anesthetics, anticancer, or anti-inflammatory agents
  • materials useful in physically formulating various dosage forms of the preferred embodiments such as excipients, dyes, perfumes, thickening agents, stabilizers, skin penetration enhancers, preservatives or antioxidants.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after the onset of a gamma-herpesvirus infection, such as KSHV infection, and related conditions. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions of the present invention may be carried out using known procedures, at dosages and for periods of time effective to treat a gamma-herpesvirus infection, such as KSHV infection, and related conditions in the patient.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a gamma- herpesvirus infection, such as KSHV infection, and related conditions in the patient.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be
  • an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • the compound in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of KSHV infection and related conditions a patient.
  • compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • compositions of the invention comprise a
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
  • Compounds of the invention for administration may be in the range of from about 1 ⁇ g to about 10,000 mg, about 20 ⁇ g to about 9,500 mg, about 40 ⁇ g to about 9,000 mg, about 75 ⁇ g to about 8,500 mg, about 150 ⁇ g to about 7,500 mg, about 200 ⁇ g to about 7,000 mg, about 3050 ⁇ g to about 6,000 mg, about 500 ⁇ g to about 5,000 mg, about 750 ⁇ g to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1 ,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present invention is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a gamma-herpesvirus infection, such as KSHV infection, and related conditions in a patient.
  • a gamma-herpesvirus infection such as KSHV infection
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • routes of administration of any of the compositions of the invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.
  • the compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral
  • formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone,
  • the tablets may be coated using suitable methods and coating materials such as OPADRYTM film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRYTM OY Type, OYC Type, Organic Enteric OY- P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRYTM White,
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorb
  • the powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation.”
  • a binder material For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.
  • Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents.
  • the low melting solids when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium.
  • the liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together.
  • the resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form.
  • Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution.
  • U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties.
  • the granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture.
  • certain flow improving additives such as sodium bicarbonate
  • only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) melt.
  • the present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of KSHV infection and related conditions.
  • a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of KSHV infection and related conditions.
  • a wax/pH-sensitive polymer mix a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
  • the compounds of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used. Additional Administration Forms
  • Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO
  • the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds.
  • the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of KSHV infection and related conditions in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the administration of the inhibitor of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday").
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%- 100%, including, by way of example only, 10%, 15%,20%,25%,30%, 35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%, 95%, or 100%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained.
  • patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
  • the compounds for use in the method of the invention may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD 50 and ED 50 .
  • Active compounds exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such active compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • reaction conditions including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
  • Example 1 Antiviral Activity of (+)-Rutamarin against SHV by Inhibiting Catalytic Activity of Human Topoisomerase II
  • Kaposi's sarcoma-associated herpesvirus is an etiological agent of several AIDS-associated malignancies including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castkeman's disease (MCD). Its lytic replication cycle has been proven to be critical for the pathogenesis of KSHV-associated diseases. In KS lesion, lytic viral replication, production of virion particles and reinfection of endothelial cells are essential to sustain the population of infected cells that otherwise would be quickly lost as spindle cells divide. Thus, antivirals that block KSHV replication could be a strategy in treatment of KSHV-associated diseases. However there is no effective anti-KSHV drug currently available.
  • (+)-Rutamarin exhibits the highest efficiency in blocking KSHV lytic replication.
  • the results presented herein demonstrated that (+)-Rutamarin has human Topo II inhibition activity and belongs to the category of catalytic Topo II inhibitor.
  • (+)-Rutamarin a novel catalytic inhibitor of human Topo ⁇ , namely (+)-Rutamarin.
  • the binding mode of (+)-Rutamarin to the ATPase domain of human Topo ⁇ was established by docking and validated by MD simulations. More importantly, (+)- Rutamarin efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with an IC 50 of 1.12 ⁇ and blocks virion production with an EC 50 of 1.62 ⁇ . It possesses low cytotoxicity as indicated by the selectivity index (SI) of 84.14. This study demonstrated a great potential of (+)-Rutamarin to become an effective drug for treatment of human diseases associated with KSHV infection.
  • SI selectivity index
  • the BCBL-1 and JSC-1 are two primary effusion lymphoma cell lines that are latently infected with KSHV (Renne R, et al. 1996. Nat. Med. 2:342-346, Cannon JS, et al. 2000. J. Virol. 74: 10187-10193).
  • BJAB cell line is a KSHV-negative B cell line isolated from Burkitt's lymphoma.
  • PBMC Peripheral blood mononuclear cells
  • Plasmid pOri-A contains an EcoRI-PstI fragment (nucleotides 22409 to 26491) of KSHV DNA in pBluescript at the EcoRI/PstI site as previously described (Lin CL, et al. 2003. J. Virol. 77:5578-5588).
  • pCR3.1-ORF50 is an RTA expression vector described previously (Lin CL, et al. 2003. J. Virol. 77:5578-5588).
  • JSC-1 cells were induced by 3 mM sodium butyrate (Cannon JS, et al. 2000. J. Virol. 74: 10187- 10193). Three hours post-induction, compounds in dilutions were added to BCBL-1 or JSC-1 cells and subsequently antiviral effects and cytotoxicity were assayed at different time points.
  • KSHV genomic DNA content was quantified using the DNeasy kit according to the manufacturer's protocol (Qiagen).
  • KSHV genomic copy number was quantified by realtime PCR on a Roche LightCycler instrument using the LightCycler FastStart DNA Master plus SYBR green kit with the primers for the detection of L ANA (forward, 5 '- CGCGAATACCGCTATGTACTCA-3'; SEQ ID NO: 1; reverse, 5'- GGAACGCGCCTCATACGA-3'; SEQ ID NO: 2).
  • the intracellular viral genomic DNA in each sample was normalized to GAPDH using the primers directed to GAPDH (forward, 5'-ACATCATCCCTGCCTCTAC -3'; SEQ ID NO: 3; reverse, 5'- TCAAAGGTGGAGGAGTGG -3'; SEQ ID NO: 4).
  • IC 50 half maximal inhibitory concentration
  • BCBL-1 cells after treated or untreated with chemicals were assessed by counting Trypan blue-stained cells 2 or 5 days post-treatment using a light microscope. Cell viabilities were defined relative to control cells (non-drug treated). The half maximal cytotoxic concentration (CC 50 ) was calculated from dose-response curves with Graphpad Prism software.
  • Cell proliferation assay BCBL-1 and BJAB cells (both starting with 2 l0 5 cells/ml) were treated with (+)-Rutamarin for 5 days at two different concentrations: IC 50 and 5xICso. The cells were stained by Trypan blue and counted every day for 5 days. To maintain the exponential growth, fresh medium (supplemented with or without the drug) was added to these cultures every 2 days.
  • BCBL-1 and BJAB cells (both starting with 4 l0 5 cells/ml) were treated with (+)-Rutamarin and novobiocin at two different concentrations: IC 50 and 5 x IC 5 o.
  • IC 50 and 5 x IC 5 o Two days post-treatment, cells were collected and fixed with cold 70% ethanol for 15 min, permeabilized, and stained with PI solution (50 g/ml of propidium iodide, 0.1 mg/ml of RNase A, and 0.05%> of Triton X-100) for 2 hours. Cell cycle progression was measured using a FACStar PLUS cell sorter flow cytometer (Becton Dickinson). ori-Lyt-dependent DNA replication assay
  • BCBL-1 cells were cotransfected with plasmids pOri-A (2.5 ⁇ g) and pCR3.1- ORF50 (2.5 ⁇ g) by nucleoporation (Amaxa) and cultured with different concentrations of (+)-Rutamarin. 72 hours post-transfection, extrachromosomal DNA was prepared from cells using the Hirt DNA extraction method as previously described (Gonzalez -Molleda L, et al.. 2012. Antimicrob. Agents Chemother. 56:893-902). The extracted
  • extrachromosomal DNA was treated with RNase A at 25°C for 30 min, followed by proteinase K at 50°C for 30 min. Five ⁇ g DNA was digested with Kpnl/Sacl or
  • the decatenation assay is designed to measure type II topoisomerase activity and inhibition of the enzyme by testing compounds.
  • the Kinetoplast DNA (kDNA, 200 ng) was incubated in 20 ⁇ reaction containing 50 mM Tris-HCl (pH 7.5), 85 niM KC1, 10 mM MgCl 2 , 0.5 mM Na 2 EDTA, 0.5 mM dithiothreitol, 1 mM ATP, 30 ⁇ g/ml BSA, and 2 unit of Topo ⁇ or Topo ⁇ for 30 min at 37°C in the absence or the presence of (+)-Rutamarin or control compounds (novobiocin and etoposide). The reactions were analyzed by electrophoresis on agarose gels. The decatenated kDNAs were measured with Gel DoxTM XR+ imaging system (BIO-RAD).
  • Malachite green reagent was prepared by mixing malachite green
  • ATP hydrolysis reaction (20 ⁇ ) containing 50 mM Tris-HCl, pH 7.4, 85 mM KC1, 10 mM MgCl 2 , 0.5 mM DTT, 0.5 mM Na 2 EDTA, 30 ⁇ g/mL BSA, 200 ng pBR322 DNA, 2 units Topo Ila, 500 ⁇ ATP and varying concentrations of (+)-Rutamarin was initiated by adding ATP to the reaction and incubated at 37°C for 2 hours. The reaction was stopped by adding 80 ⁇ of the malachite green reagent to the reaction, followed by adding 10 ⁇ of 34% sodium citrate to develop blue-green color change. The absorbance at 620 nm was measured using a plate reader. The OD 62 o was used for representing the ATP hydrolysis level. Molecular docking study
  • the docking program FlexX encoded in SYBYL 7.3 (Tripos Inc.) was applied to identify the potential binding of (+)-Rutamarin to the Topo Ila ATPase domain.
  • the receptor for docking simulation consists of protein chain A, two conserved water and Mg 2+ .
  • ADPNP was redocked into the crystal structure of the ATPase domain of Topo ⁇ .
  • the docked ADPNP has similar binding pose with the cocrystallized ligand, with a root mean square deviation of about 1.6 A.
  • the active sites were defined as all residues within 6.5 A radius of the bound ADPNP.
  • Other FlexX parameters were set to default values. Thirty poses were retained during the docking process. After running FlexX, all the poses were visually inspected, and the most suitable docking pose was selected on the basis of score and interactions with key residues of the active site.
  • the complex of Topo ⁇ and the most suitable docking conformer of (+)-Rutamarin was used as initial coordinates for the subsequent molecular dynamics.
  • the simulation consisted of energy minimization, heat phase, equilibration and production.
  • first phase of the minimization only hydrogen atoms were relaxed for 1000 steps, holding all other atoms restrained.
  • second minimization phase is that the protein backbone atoms were restrained and hydrogen atoms, water molecules and ions were relaxed with 1000 steps.
  • a harmonic restraint of 5kcal mol-1 A-2 was applied.
  • all atoms were freely minimized with 2000 steps.
  • a combined steepest descent and conjugate gradient minimization steps was equal.
  • binding free energy calculations was performed on 1000 snapshot structures extracted at 4ps intervals over the last 4 ns stable MD trajectory. For each snapshot structure, the binding free energy was calculated for both enzyme-inhibitor complexes through the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and generalized Born (MM-GBSA) methods Kollman et al, 2000 Accounts of Chemical Research, 33, 889-897; Swanson et al., 2004 Biophysical Journal, 86, 67-74). In the MM -PBS A and MM-GBSA approach an interaction free energy is defined as
  • Gcomplex, Gprotein, and Gligand are the free energies of the complex, protein and the ligand, respectively.
  • Each free energy term in eq 1 was computed as sum of the absolute free energy in the gas phase (Egas), the solvation free energy (Gsolvation), and the entropy term (TS), using eq 2:
  • Egas was expressed as the sum of changes in the van der Waals energy (Evdw), electrostatic energy (Eele), and the internal energies (Eint) in the gas phase (eq 3).
  • Eint is the energy associated with vibration of covalent bonds and bond angels, rotation of single bond torsional angels (eq 4)
  • the polar contribution (GPB/GB) to the solvation energy was calculated either using the PB and GB model implemented in AMBER 12.
  • the grid size used is 0.5 A.
  • the dielectric constant was set to 1 for interior solute and 80 for exterior water.
  • (+)-Rutamarin inhibits the lytic replication of SHV
  • C31 which is identified as the natural product (+)-Rutamarin (Fig. 2A), exhibited significant inhibitions of both viral DNA synthesis and virion production with relatively low cytotoxicity at the concentration of 20 ⁇ . Therefore it was chosen for further investigation.
  • the half maximal inhibitory concentration (IC 50 ) values of (+)-Rutamarin were determined from dose-response curve of KSHV DNA content in TPA-induced BCBL-1 cells and found to be 1.12 ⁇ (Fig 2B).
  • the effect of (+)-Rutamarin on progeny virion production was also determined by quantification of encapsidated viral DNA in BCBL-1 culture media.
  • the half maximal antiviral effective concentration (EC 50 ) calculated from dose-response curve of extracellular virion is 1.62 ⁇ (Fig 2B).
  • (+)-Rutamarin inhibits KSHV lytic replication regardless of host cells and means of lytic cycle induction
  • experiments were designed to test the effect of (+)-Rutamarin on KSHV replication in JSC-1 cells induced by sodium butyrate for lytic replication.
  • the IC50, EC50 and CC50 of (+)-Rutamarin on butyrate- induced JSC-1 cells were found to be 2.29 ⁇ , 1.40 ⁇ and 94.34 ⁇ , respectively, that are very similar to those obtained with BCBL-1.
  • the cytotoxicity of (+)- Rutamarin to primary lymphocytes was also assessed with peripheral blood mononuclear cells (PBMC).
  • the CC 50 to PBMC was calculated to be 60.91 ⁇ (Fig. 10).
  • (+)-Rutamarin To further investigate the potential of (+)-Rutamarin to become an effective and safe antiviral, the effect of the compound on cell proliferation was assessed. KSHV-carrying BCBL-1 and virus-free BJAB cells were cultured in the presence and the absence of (+)-Rutamarin and live cell numbers were counted over 5 days. (+)-Rutamarin did not exhibit cell growth inhibitory effect in both cell lines at the IC 50 concentration. At an excess concentration (5 x IC 50 ), (+)-Rutamarin showed an adverse effect on cell growth but inhibition extent was less than that of novobioicin (Fig. 3). The effect of (+)- Rutamarin on cell cycle progression was also examined.
  • (+)-Rutamarin inhibits SHV lytic replication by blocking viral ori-L vt-dependent DNA replication
  • (+)-Rutamarin is capable of inhibiting KSHV DNA synthesis and virion production.
  • an ori-Lyt DNA replication assay was conducted. BCBL-1 cells were cotransfected with an on-Zyt-containing plasmid (pOri-A) and an RTA expression vector. Lytic DNA replication was induced by RTA expression (Sun R, et al. 1998. Proc. Natl. Acad. Sci. U. S. A. 95:10866-10871).
  • the transfected cells were cultured in the presence of (+)-Rutamarin at varying concentrations and the effect of (+)-Rutamarin on orz-Zyt-dependent DNA replication was measured by a Dpn I assay (Lin CL, et al. 2003. J. Virol. 77:5578-5588, Wang Y, et al. 2004. J. Virol. 78:8615-8629).
  • DNA was isolated from the treated cells 72 h post-transfection and digested with Kpnl/Sacl and Kpnl/Sacl/Dpnl.
  • Replicated plasmid DNA was distinguished from input plasmid by Dpnl restriction digest, which cleaves input DNA that has been dam + methylated in
  • (+)-Rutamarin blocks KSHV DNA replication through inhibiting RTA expression
  • the effect of (+)-Rutamarin on RTA expression in TPA-induced BCBL-1 cells was examined by Western analysis. The result showed no adverse effect of this compound on RTA expression up to 25 ⁇ (22 times excess over IC 50 ) (Fig. 11).
  • the reduced level of RTA in the treatment with 50 and 100 ⁇ of (+)-Rutamarin may result from the cytotoxicity of the compound in the concentration near its IC 50 (94.24 ⁇ ).
  • (+)-Rutamarin is an inhibitor of human topoisomerase ⁇ .
  • Topo II inhibitor that blocks KSHV on-Zyt-dependent DNA replication through inhibiting catalytic activity of host cell Topo II.
  • Topo II isoforms there are two Topo II isoforms in mammals, Topo ⁇ and Topo ⁇ .
  • a DNA decatenation-based topoisomerase II assay was established.
  • kinetoplast DNA consisting of a large network of interlocked DNA minicircles can be decatenated into separate minicircles in the presence of ATP and Topo II of either isoform.
  • (+)-Rutamarin in a wide range of concentration was added in the reaction and the effect of the compound on Topo II activity was measured by determining decatenated kDNA using agarose gel electrophoresis. Results showed that (+)-Rutamarin is able to inhibit the enzymatic activity of Topo ⁇ in a dose-dependent manner (Fig. 6A and 6B).
  • the IC 50 of (+)-Rutamarin on Topo ⁇ inhibition was calculated as 28.22 ⁇ 6.2 ⁇ (Fig. 6C and 6D).
  • the inhibition rate of (+)- Rutamarin is significantly higher than novobiocin and close to Topo II poison etoposide (Fig. 6A).
  • (+)-Rutamarin did not exhibit any inhibitory effect on Topo ⁇ activity (Fig. 6E).
  • (+)-Rutamarin is a catalytic inhibitor of Topo ⁇ and acts in blocking ATPase activity of the enzyme as novobiocin does
  • the inhibition effect of Rutamarin on ATP hydrolysis of Topo ⁇ was studied using the malachite green assay.
  • the APTase activity of Topo ⁇ was inhibited by (+)-Rutamarin in a dose-dependent manner, suggesting that, like novobiocin, (+)-Rutamarin inhibits human Topo ⁇ by binding to the ATP pocket of the enzyme and blocking its ATP hydrolysis.
  • Fig. 8A illustrates the time dependence of the RMSD values for X-ray reference enzyme structure of backbone atoms (C, CD, N, and O) over the production phase of simulation.
  • the RMSD values of simulation converged after ⁇ 3ns, indicating that the system is stable and equilibrated.
  • the RMSD value of ligand compared docking pose is swinging within 1.8 A, which illustrates that the docking pose is reliable.
  • (+)-Rutamarin forms a hydrogen bond with the side chain of Asn95, Ala 167 and conserved water 2, respectively, and hydrophobically interacts with He 125, Ilel41, Phel42, Alal67, Thr215, Glyl66 and Lysl23 in the catalytic site of Topo ⁇ (Fig. 8D).
  • the distance distributions analysis also showed that the hydrogen bonds between conserved water 2- and Alal67-(+)-Rutamarin are comparatively stable, whereas the hydrogen bond of Asn95-(+)-Rutamarin is unstable (Fig.
  • (+)-Rutamarin as an effective antiviral agent that inhibits SHV ori-L vt-dependent DNA replication with low cytotoxicity
  • (+)-Rutamarin was identified as an effective antiviral agent that inhibits KSHV on-Zyt-dependent DNA replication with low cytotoxicity. It blocks KSHV DNA replication through inhibiting catalytic activity of human Topo ⁇ .
  • KSHV has been proven to be the etiological cause of KS and other KSHV-associated malignancies.
  • the current treatment modalities for KSHV- associated diseases include only traditional cancer therapies.
  • the chemotherapeutics that have been approved by the FDA include liposomal anthracycline products (liposomal doxorubicin or liposomal daunorubicin), paclitaxel and interferon- alpha (Potthoff A, et al. 2007. J. Dtsch. Dermatol. Ges. 5: 1091-1094).
  • liposomal anthracycline products liposomal doxorubicin or liposomal daunorubicin
  • paclitaxel paclitaxel
  • interferon- alpha Pieris factor-alpha
  • IRIS immune reconstitution inflammatory syndrome
  • IRIS-KS is the result of responses by a recovered immune system to KS-causing pathogen, i.e. KSHV
  • KSHV antiretroviral
  • HIV- positive patients with a combination of antiretroviral (HAART) and anti-KSHV therapeutics is expected to yield positive results.
  • antiretroviral (HAART) antiretroviral
  • anti-KSHV therapeutics there are no effective drugs targeting KSHV available.
  • ganciclovir or foscarnet could reduce the incidence of KS in AIDS patients (Glesby MJ, et al. 1996. J Infect Dis. 173: 1477-1480, Mocroft A, et al. 1996. AIDS. 10: 1101-1105).
  • severe side effects renal impairment and bone marrow suppression
  • rapid development of drug resistance have limited the use of these drugs in the treatment of KS. Therefore, there is a need for effective drugs targeting KSHV.
  • Topo II inhibitors are divided into two categories: Topo II poisons, which target the
  • Topo II catalytic inhibitors which disrupt catalytic turnover of the enzyme.
  • Topo II poisions in general exhibit very strong cytotoxicities to host cells.
  • Topo II catalytic inhibitors show less cytotoxicity (such as novobiocin with a CC50 values of 871 ⁇ and merbarone of 212.9 ⁇ to BCBL-1 cells). Both novobiocin and merbarone are effective in halting KSHV DNA synthesis with IC 50 of 27.55 ⁇ and 19.54 ⁇ , respectively. The low
  • Topo II catalytic inhibitors cytotoxicities and high inhibition rates for viral replication of catalytic Topo II inhibitors suggest potentials of Topo II catalytic inhibitors in becoming effective anti-KSHV drugs for treatment of KS and other KSHV-associated diseases (Gonzalez-Molleda L, et al.. 2012. Antimicrob. Agents Chemother. 56:893-902).
  • viruses have tendencies to mutate their genome and therefore develop drug resistance
  • targeting host cellular proteins that viruses rely on for their replication offers the advantage of minimizing drug resistance, and hence constitutes a novel therapeutic strategy.
  • (+)-Rutamarin as a lead which efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with the IC 50 of 1.12 ⁇ and EC 50 of 1.62 ⁇ , 25- and 17- fold lower than those of novobiocin.
  • (+)-Rutamarin exhibits very low cytotoxicity and the selective index (SI) on KSHV lytic replication was calculated as 84.14, three times better that that of novobiocin.
  • SI selective index
  • (+)-Rutamarin is a natural product found in plants such as ruta graveolens L (common rue). It was reported that (+)-Rutamarin possesses anti-proliferation and anti- cancer activities against a variety of tumor cell lines (Yang QY, et al. 2007. J. Asian Nat. Prod. Res. 9:59-65). It is also found to induce the expression and translocation of glucose transporter 4 (GLUT4). As impaired translocation or decreased expression of GLUT4 in response to insulin is one of the major pathological features of type 2 diabetes, the function of (+)-Rutamarin in ameliorating glucose homeostatsis suggests a potential of this compound in anti-type 2 diabetes drug development (Zhang Y, et al. 2012. PLoS
  • (+)-Rutamarin did not show perceptible effect on cell proliferation and cell cycle progression in BCLB-1 and BJAB cells.
  • (+)-Rutamarin exhibited a degree of suppression on cell proliferation with a delay in the S phase.
  • (+)-Rutamarin has an extent of antiproliferative property and may provide an additional benefit if it becomes a drug to treat KSHV-associated malignancies, the moderate anti-proliferation property may not be potent enough to provide anti-tumor activities. It may also be true for all Topo II catalytic inhibitors.
  • (+)-Rutamarin is a novel human Topo Ila catalytic inhibitor
  • Topo II is an ATPase and uses the energy derived from ATP hydrolysis to resolve the winding problem of double-stranded DNA.
  • the detailed binding mode of (+)- Rutamarin and human Topo Ila was identified using molecular docking and MD simulation. The significance of these studies is two-fold. First, the study provided insight into the binding of (+)-Rutamarin to Topo Ila and results confirmed that this compound binds to the ATP pocket of Topo Ila. Second, the result of the study help in designing modification to the compound for more potent antiviral candidates. Energy
  • the major contributing residues can be divided into three clusters, which are consistent with the three binding pockets sites, i.e. (i) Walker A consensus motif (residues 161-167), (ii) Mg 2+ binding area (residues 87, 91 and 94), and (iii) hydrophobic chamber (key residues, He 125, Ilel41, Phel42, Lysl23 and Thr215). All of these residues except Glu87 positively contribute to the binding affinity for (+)-Rutamarin.
  • Topo II topoisomerase II
  • Topo II catalytic inhibitors were evaluated for their potentials in blocking EBV replication and becoming efficacious antiviral agents.
  • Topo II catalytic inhibitors in general exhibited marked inhibition of EBV lytic replication and minimal cytotoxicity.
  • (+)-rutamarin with the best selectivity index (SI > 63) among the inhibitors tested in this study, is effective in inhibiting EBV DNA replication and virion production but shows little adverse effect on cell proliferation, suggesting its potential to become an efficacious and safe drug for the treatment of human diseases associated with EBV infection.
  • the P3HR-1 is a EBV -positive Burkitt lymphoma cell line, a clonally derived subline of Jiyoye (Hinuma et al, 1967, J. Virol, 1 :1045-1051).
  • Akata-Bxl cell line carries a recombinant EBV where the thymidine kinase gene was replaced by a CMV immediate early promoter driven GFP (Guerreiro-Cacais et al., 2007, J. Virol., 81 : 1390- 1400).
  • Cells were cultured in RPMI 1640 medium (Gibico-BRL, Gaithersburg, MD), supplemented with 10% FBS (Gibico-BRL), penicillin (100 U/ml), streptomycin
  • the EBV ori-Lyt plasmid pEBV-oriL was constructed by cloning a 1434 bp fragment carrying EBV ori-Lyt (nucleotides 52385-53819 of EBV genome) into pUC18 vector.
  • the plasmid McZ is a ZTA expression vector in the backbone of pBXGl . Chemicals and cell treatment
  • Novobiocin was purchased from Sigma-Aldrich (St. Louis, MO) and merbarone was purchased from Merck Millipore. (+)-Rutamarin (Figure 12) is extracted from natural plant Ruta graveolens L, supplied by the Guangdong Small Molecule Tangible Library (GSMTL) (Gu et al, 2010, Molecules, 15:5031-5044). Novobiocin was prepared as aqueous stock, while the others were dissolved in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • P3HR-1 cells (4 x 10 5 cells/ml of culture) were treated with 20 ng/ml 12-O-tetradecanoylphorbol 13 -acetate (TP A; Sigma- Aldrich) and 3 mM sodium butyrate (Sigma-Aldrich).
  • TP A 12-O-tetradecanoylphorbol 13 -acetate
  • Sigma-Aldrich 3 mM sodium butyrate
  • Novobiocin, merbarone, and (+)- rutamarin in different concentrations was added to P3HR-1 cells three hours after induction.
  • Akata-Bxl cells were resuspended at 2 x 10 6 /ml in fresh medium and treated with 0.8%(V/V) goat anti-human IgG (Jackson ImmunoResearch).
  • Akata-Bxl cells were adjusted to the density of 4 x 10 5 cells/ml and treated with various concentrations of novobiocin, merbarone, and (+)-rutamarin. The effects of these inhibitors on viral DNA content and virion production were assayed at different time points.
  • EBV genomic DNA was quantified by real-time PCR on a Roche Light Cycler II instrument using the Lightcycler 480 SYBR green I Master kit with primers directed to EBNA1 (forward, 5'- CATTGAGTCGTCTCCCCTTTGGAAT-3 ', SEQ ID NO:6; reverse, 5'- TC AT AAC AAGGTCCTT AATCGC ATC-3 ', SEQ ID NO:7).
  • the intracellular viral genomic DNA in each sample was normalized with the amount of GAPDH determined also by real-time DNA PCR by using primers directed to GAPDH (forward, 5'- AGCCACATCGCTCAGACAC-3', SEQ ID NO:8; reverse, 5'- GCCCAATACGACCAAATCC-3', SEQ ID NO:9).
  • EBV DNA content value from induced cells was subtracted by that from uninduced cells. These corrected values were divided by those from the control, non-drug treatment and then represented on the y axes of dose-response curves: y axis
  • l x phosphate -buffered saline PBS
  • PBS l x phosphate -buffered saline
  • the concentrated viruses were treated with DNase I (TaKaRa) at 37 °C for 1 h followed by proteinase K digestion.
  • the amounts of virion particles from the media were determined by quantifying encapsidated viral DNA by real-time PCR, and values were corrected as described above.
  • the 50% antiviral effective concentration (EC 50 ) for each compound was calculated from the dose- response curve with the aid of GraphPad Prism software.
  • P3HR-1 cells (starting with 2 x 10 5 cells/ml) were treated with topoisomerase inhibitors for 5 days at two different concentrations: IC 50 and an excess concentration (5 x IC 50 ). Cell samples were daily collected, stained with Trypan blue, and counted. To provide a constant cellular growth, fresh medium (supplemented with or without the drug) was added to these cultures every 2 days.
  • P3HR-1 cells (1 x 10 7 ) were transfected with plasmids pEBV-oriL (2.5 ⁇ g) and McZ (2.5 ⁇ g) by nucleoporation (Amaxa) and cultured in the media with each drug in a wide range of concentration. Seventy-two hours post-transfection, extrachromosomal DNAs were prepared from cells using the Hirt DNA extraction method (Hirt, 1967, J. Mol. Biol, 26:365-369). Cells were lysed in 700 ⁇ lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM EDTA, and 0.6% SDS).
  • Chromosomal DNA was precipitated at 4 °C overnight by adding 5 M NaCl to a final concentration of 0.85 M. Cell lysates were centrifuged at 4 °C at 14,000 rpm for 30 min. The supernatant containing
  • extrachromosomal DNA was subjected to phenol-chloroform extraction, followed by ethanol precipitation.
  • the DNA was treated with RNase A at 25 °C for 30 min and then with proteinase K at 50 °C for 30 min. Twelve micrograms of DNA was digested with EcoR I or EcoR I/Dpnl (TaKaRa). The DNAs were separated by electrophoresis on 0.9% agarose gels and transferred onto GeneScreen PLUS membranes (Perkin Elmer, Boston, MA).
  • Southern blot was performed according to an optimized protocol using the DIG high prime DNA labeling and detection starter kit I (Roche). Probes were prepared by excising insert DNA from pEBV-oriL plasmid by restriction digestion and labeled with digoxigenin by random primed DNA synthesis with digoxigenin-dUTP. The membranes were prehybridization in 10 ml of the DIG Easy Hybridization solution, with 50 mg/ml denatured salmon sperm DNA for 4 h at 68 °C. Then, DIG labeled probe was added and hybridization was carried for overnight. The membranes were washed three times for 15 min at 65 °C with washing buffer (2* SSC, 0.1% SDS). Subsequently the membranes were blocked for 1 h and incubated in anti-DIG-AP solution for 1 h and then incubated in 10 ml color substrate solution in the dark.
  • DIG labeled probe was added and hybridization was carried for overnight. The membranes were washed three times for
  • Topo II catalytic inhibitors including novobiocin and merbarone, have potent antiviral activity halting KSHV DNA replication and virion production (Gonzalez-Molleda et al., 2012, Antimicrob. Agents
  • (+)-rutamarin revealed the highest potency in halting EBV DNA replication with a half-maximal inhibitory concentration (IC 50 ) of 3.78 ⁇ and in blocking virion production with a half-maximal antiviral effective concentration (EC50) of 5.40 ⁇ ( Figure 13C and Table 3 A).
  • Table 1 Antiviral activities of Topo II inhibitors and their associated cytotoxicities a in P3HR-1 cells induced with TPA/butyrate and Akata-Bxl cells induced with Anti-IgG.
  • aIC 5 o represents the half maximal inhibitory concentration for EBV DNA replication.
  • EC50 denotes the half maximal effective concentration for blocking EBV virion production.
  • CC50 refers to the concentration of the compound that causes 50% cell death after specific exposure time. They were determined by nonlinear regression analysis of dose response curves and are expressed as mean values of results from at least three independent experiments. All these parameters are presented in ⁇ units.
  • R 2 correlation coefficient.
  • c SI selectiveivity index
  • (+)-rutamarin again exhibited the highest antiviral potency in Akata-Bxl cells with the IC50 and EC50 of (+)-Rutamarin of 2.38 ⁇ and 2.94 ⁇ , respectively ( Figure 14 and Table 3B).
  • Cytotoxicities of these three inhibitors were examined in parallel with their inhibition of EBV DNA replication and virion production in P3HR-1 and Akata- Bxl cells. Cells treated with these inhibitors at different concentrations were subjected to the trypan blue exclusion method to assess the numbers of viable cells and nonviable cells in culture. The half-maximal cytotoxic concentrations (CC 50 ) were determined based on the results. Novobiocin and merbarone exhibited relatively low cytotoxicity to P3HR-1 cells with CC 50 values of 518.5 ⁇ and 137.6 ⁇ , respectively ( Figure 13A and B).
  • (+)-rutamarin the highest concentration used in this study (150 ⁇ ) showed little cytotoxicity in 2 and 5 days culture, suggesting that the CC 50 value of (+)-rutamarin to P3HR-1 cell is greater than 150 ⁇ ( Figure 13D and E). Similar results were obtained with Akata-Bxl cells ( Figure 14D and 14E).
  • topoisomerase II inhibitors tested in this study have been demonstrated to inhibit EBV DNA synthesis and virion production.
  • Experiments where designed to assess whether these inhibitors indeed block viral ori-Lyt-dependent DNA replication To address this question, P3HR-1 cells were cotransfected with an EBV ori- Lyt-containing plasmid (pEBV-oriL) and a ZTA expression vector (McZ). Expression of ZTA sufficiently drives latent EBV into lytic replication cycle (Countryman and Miller, 1985, Proc. Natl. Acad. Sci. U.S.A., 82:4085-4089 and Rooney et al, 1988, Proc. Natl. Acad. Sci. U.S.A., 85:9801-9805).
  • the transfected cells were cultured in the absence or the presence of each inhibitor at various concentrations, usually with two concentration lower than IC 50 , and two higher concentrations.
  • the ori-Lyt-dependent DNA replication and the drug effects were measured by a Dpn I assay (Gonzalez-Molleda et al., 2012, Antimicrob. Agents Chemother, 56:893-902).
  • DNA was isolated from the treated cells 72 h post-transfection and digested with EcoR I and EcoR I/Dpn I.
  • Replicated plasmid DNA can be distinguished from input plasmid by Dpnl restriction digest, which cleaves input DNA that has been dam + methylated in Escherichia coli but leaves intact the DNA that has been replicated at least one round in eukaryotic cells
  • Human topoisomerase II catalytic inhibitors are potent in inhibiting EBV replication
  • EBV is associated with a number of human diseases.
  • the lytic replication of EBV is directly linked to infectious mononucleosis, chronic active EBV infection (CAEBV), oral hairy leukoplakia and to an increased risk of EBV-associated
  • CAEBV nasopharyngeal carcinoma
  • Chronic active EBV infection is characterized by chronic or recurrent infectious mononucleosis-like symptoms that persist for a long time and by an unusual pattern of anti-EBV antibodies, with life-threatening complications, such as virus-associated hemophagocytic syndrome and lymphoma (Kimura et al., 2001, Blood, 98:280-286).
  • the patients usually have a markedly elevated EBV DNA level in the blood (10 3 - 10 7 copies/ml), indicating active lytic viral replication. So far no satisfactory therapy is available for CAEBV.
  • Antiviral or immunomodulatory agents such as acyclovir, ganciclovir, vidarabine, interferon-a, and interleukin-2
  • Acyclovir ACV
  • Acyclovir which inhibits herpesviral DNA polymerase, is commonly used for treatment of CAEBV (Gershburg and Pagano, 2005, J. Antimicrob. Chemother, 56:277-281) but found generally inefficient for this disease.
  • drug resistance frequently occurs as a major problem among immunosuppressed hosts, mainly those who have received prolonged ACV therapy (Andrei et al, 2012, J.
  • topoisomerases could be therapeutic targets for blocking replication of herpesviruses including EBV and treatment of the infection-associated human diseases. It has been reported previously that Topo I inhibitor camptothecin is able to inhibit replication of herpes simplex virus type 2 (Yamada et al, 1990, Arch. Virol., 110: 121-127).
  • Topo II catalytic inhibitor ICRF193 and Topo II poison teniposide were found to be able to block herpes simplex virus type 1 from replication (Ebert et al, 1990, J. Virol, 64:4059-4066 and Hammarsten et al, 1996, J. Virol, 70:4523-4529).
  • the results presented herein demonstrate that KSHV replication can be effectively blocked by Topo I inhibitor camptothecin, Topo II poisons etoposide and ellipticine as well as Topo II catalytic inhibitors novobiocin, merbarone and (+)- rutamarin (Gonzalez-Molleda et al, 2012, Antimicrob.
  • Topo I inhibitors and Topo II poisons in general possess considerable toxicities to host cells (Gonzalez-Molleda et al, 2012, Antimicrob. Agents Chemother. 56:893-902).
  • viruses have tendencies to mutate their genome and therefore develop drug resistance.
  • An antiviral that targets a cellular protein such as Topo II offers the advantage of minimizing drug resistance and hence constitutes an important, novel therapeutic strategy.
  • Novobiocin is an old antibiotic drug against staphylococcal infection (Kirby et al, 1956, AMA Arch. Intern. Med., 98: 1-7). Novobiocin has also been used in combination with chemotherapeutical agents for the treatment of several cancers (Eder et al, 1991, Cancer Res., 51 :510-513 and Kennedy et al, 1995, J. Clin. Oncol, 13: 1136- 1143). The results presented herein reveal its antiviral potency against EBV, suggesting a new usage for an old drug.
  • (+)-Rutamarin is a natural product and newly identified Topo II catalytic inhibitor (Xu et al., 2014, Antimicrob. Agents Chemother, 58:563-573). Among three inhibitors tested in this study, (+)-Rutamarin exhibited the highest potency in anti-EBV activity. (+)-Rutamarin effectively inhibits EBV replication with an IC 50 of 2.38 ⁇ and an EC 50 of 2.94 ⁇ (in Akata-Bxl cells). It possesses relative low cytotoxicity to P3HR- 1 cells with a CC50 greater than 150 ⁇ . Without wishing to be bound by any particular theory, experiments can be desgined to optimize the (+)-Rutamarin structure in order to further elevate its antiviral activity and improve its solubility, making it an anti-EBV drug lead.

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Abstract

The invention provides compositions and methods for treating a herpesvirus infection. The invention also provides compositions and methods for treating Kaposi's sarcoma or Kaposi's sarcoma-associated herpes virus infection by administering an effective amount of a compound that inhibits topoisomerases Ilα to an individual in need thereof. For example, the compound that inhibits topoisomerases Ilα is (+)- Rutamarin, an anolog, or a derivative thereof.

Description

TITLE OF THE INVENTION
Compositions and Methods for Treating Herpesvirus Infection
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT
This invention was made with government support under grant no.
R01AI052789 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application Serial No. 61/909,712, filed November 27, 2013, the content which is incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a member of human γ-herpesvirus family. It has been proven to be the etiologic agent of Kaposi's sarcoma (KS), a multicentric malignant neoplasm of endothelial origin (Chang Y, et al. 1994. Science. 266: 1865-1869, Moore PS, et al. 1995. N. Engl. J. Med. 332: 1181-1185, Dupin N, et al. 1995. Lancet. 345:761-762, Schalling M, et. 1995. Nat. Med. 1 :707-708, Chuck S, et al. 1996. J. Infect. Dis. 173:248-25). Although classic KS is a rare disease and in general non-fatal, it often occurs in immunocompromised patients who are under immunosuppression treatment after organ transplantation. In AIDS epidemic, KS has become the most common AIDS-associated malignancy and led to significant mortality (Antman K, et al. 2000. N. Engl. J. Med.
342: 1027-1038). Approximately 20% of AIDS patients develop KS during the course of their disease and AIDS-associated KS is estimated to contribute to 10% of death of AIDS patients (Krentz HB, et al 2005. HIV Med. 6:99-106). Besides KS, two B-cell-associated lymphoproliferative disorders, namely primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD) are also related with KSHV infection and mainly occur in AIDS patients (Antman K, et al. 2000. N. Engl. J. Med. 342:1027-1038, Cesarman E, et al. 1999. Seminar in Cancer Biology . 9: 165-174). The incidence of KSHV-associated MCD has risen since the advent of HAART (Powles T, et al. 2009. Ann. Oncol. 20:775-779).
The contemporary treatment modalities for KS and other KSHV- associated malignancies include conventional cancer therapies, such as radiation and chemotherapy, or AIDS treatment such as HAART (Antman K, et al. 2000. N. Engl. J. Med. 342: 1027-1038). These cancer therapies in general present serious side effects and tumor response to them is only transient. The success of HPV vaccine in preventing cervical cancer has proven that antiviral orientated therapies have the potential to be used to treat human malignant diseases that are caused by viruses. Thus, an antiviral targeting KSHV could prove successful at treating KSHV-associated tumor (Moore PS, et al. 2011. Blood. 117:6973-6974). However, currently there is no therapy for KSHV-associated disease on the basis of targeting of KSHV.
Like all herpesviruses, KSHV has two types of replication cycle, latent and lytic replication. In KS lesions, most KSHV-transformed spindle-shaped cells are in latent replication phase, but a small percentage of infected cells undergo spontaneous lytic replication (Staskus KA, et al. 1997. J. Virol. 71 :715-719, Sun R, et al. 1999. J. Virol. 73:2232-2242, Zhong W, et al. 1996. Proc. Natl. Acad. Sci. U. S. A. 93:6641- 6646). Increasing evidences suggest that the lytic replication in these cells is necessary for maintaining the stable infection and viral pathogenicity. The majority of KSHV genes including those responsible for malignant cell growth and the alteration of
microenvironment express only in lytic phase (Cesarman E, et al. 2000. J. Exp. Med. 191 :417-422). Furthermore, the lytic replication is necessary in sustaining the population of latently infected cells that otherwise would be quickly lost by segregation of latent viral episomes as spindle cells divide (Grundhoff A, et al. 2004. J. Clin. Invest. 113: 124- 136). Thus, KSHV lytic replication and constant primary infection to fresh cells are crucial not only for viral propagation but also for viral pathogenesis.
Viral DNA replication is considered an ideal target for antivirals. The mechanisms that control KSHV lytic DNA replication have been reported (Lin CL, et al. 2003. J. Virol. 77:5578-5588, Wang Y, et al. 2004. J. Virol. 78:8615-8629, Wang Y, et al. 2006. J. Virol. 80: 12171-12186, Wang Y, et al. 2008. J. Virol. 82:2867-2882). It has been found that several host cellular proteins, including topoisomerases I and II (Topo I and II), MSH2/6, RecQL, and poly[ADP-ribose] polymerase 1 (PARP-1), are involved in KSHV lytic DNA replication (Wang Y, et al. 2008. J. Virol. 82:2867-2882). It has been demonstrated that both Topo I and Topo II are indispensible for KSHV lytic replication and specific inhibitors to Topo I and II can effectively inhibit KSHV lytic replication. One category of Topo II inhibitors, namely catalytic Topo II inhibitor, was found to have marked inhibition of KSHV replication with minimal cytotoxicity as indicated by their high selectivity indices (e.g. 31.6 for novobiocin) (Gonzalez-Molleda L, et al. 2012. Antimicrob. Agents Chemother. 56:893-902).
Topo II can serve as effective targets for antivirals and catalytic inhibitors of Topo II represent promising antiviral agents for the treatment of malignancies associated with KSHV infection, but clinically useful therapeutics have not been developed. There is thus a need in the art for identifying and generating therapeutics that can be used clinically to treat a KSHV infection. The present invention addresses this unmet need in the art.
SUMMARY OF THE INVENTION
The invention provides a method of treating a herpesvirus infection. In one embodiment, the invention provides a method of treating a gamma-herpesvirus infection. In one embodiment, the invention provides a method of treating a Kaposi's sarcoma- associated herpes virus (KSHV) infection. In one embodiment, the invention provides a method of treating an Epstein-Barr virus (EBV) infection.
In one embodiment, the invention provides a method of treating a herpesvirus infection in an individual in need thereof, the method comprising the step of: administering to the individual an effective herpesvirus antiviral amount of a
pharmaceutical composition comprising at least one compound selected from the group consisting of:
(i) a compound of formula (I):
Figure imgf000005_0001
wherein in formula (I):
each occurrence of R1 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, -S(=0)2R7, -NHS(=0)2R7, - C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, - OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, -C(OH)(R7)2, - C(CH3)2(CH=CH2), -C(CH3)2OC(=0)CH3, and -C(NH2)(R7)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, -S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, - C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, -
C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups, or two or more adjacent R1, R2, R3, R4, R5,and R6 groups combine to form a first ring optionally substituted with 0-4 R1 groups, wherein the first ring is optionally fused to a second ring optionally substituted with 0-4 R1 groups;
each occurrence of R7 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-Ci0 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted; and X is -CH or O; and
a compound of formula (II):
Figure imgf000006_0001
wherein in formula (II):
each occurrence of R1, R2, R3, R4, and R5 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR6, -SR6, -S(=0)R6, - S(=0)2R6, -NHS(=0)2R6, -C(=0)R6, -OC(=0)R6, -C02R6, -OC02R6, -CH(R6)2, -N(R6)2, -C(=0)N(R6)2, -OC(=0)N(R6)2, -NHC(=0)NH(R6), -NHC(=0)R6, -NHC(=0)OR6, - C(OH)(R6)2, and -C(NH2)(R6)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR6, -SR6, -S(=0)R6, -S(=0)2R6, -NHS(=0)2R6, -C(=0)R6, -OC(=0)R6, -C02R6, -OC02R6, -CH(R6)2, -N(R6)2, -
C(=0)N(R6)2, -OC(=0)N(R6)2, -NHC(=0)NH(R6), -NHC(=0)R6, -NHC(=0)OR6, - C(OH)(R6)2, and -C(NH2)(R6)2; and
each occurrence of R6 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
a salt, solvate, or N-oxide thereof, and any combinations thereof.
In one embodiment, the compound is selected from the group consisting of:
2H-chromen-2-one;
7-hydroxy-2H-chromen-2-one;
7-amino-4-methyl-2H-chromen-2-one; 7-hydroxy-4-methyl-2H-chromen-2-one;
6.7- dihy droxy-2H-chromen-2 -one ;
7 , 8 -dihy droxy-2H-chromen-2 -one ;
2H-furo [2,3 -h] chromen-2-one;
7H-furo [3 ,2-g] chromen-7-one;
6- hydroxy-7-methoxy-2H-chromen-2-one;
7.8- dihydroxy-6-methoxy-2H-chromen-2-one;
9-methoxy-7H-furo [3 ,2-g] chromen-7-one;
9-methoxynaphtho[2,3-£]furan-7(8H)-one;
7- hydroxy-6,8-dimethoxy-2H-chromen-2-one;
7-(diethylamino)-4-methyl-2H-chromen-2-one;
7- methoxy-8-(3-methylbut-2-en-l-yl)-2H-chromen-2-one;
8- (2,3-dihydroxy-3-methylbutyl)-7-hydroxy-2H-chromen-2-one;
4-((3 -methylbut-2-en- 1 -yl)oxy)-7H-furo [3 ,2-g] chromen-7-one;
9- ((3 -methylbut-2-en- 1 -yl)oxy)-7H-furo [3 ,2-g] chromen-7-one;
6b-ethyl-10a-hydroxy-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2-¾]pyridine-
6,10(10aH)-dione;
10a-hydroxy-3,6b-dimethyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
10a-hydroxy-2,6b-dimethyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
6-(2,3-dihydroxy-3-methylbutyl)-5,7-dimethoxy-2H-chromen-2-one;
4-hydroxy-3-(3-oxo- 1 -phenylbutyl)-2H-chromen-2-one;
6b-ethyl-10a-hydroxy-3-methyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
6b-ethyl-10a-hydroxy-2-methyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
(E)-2-(8-oxo-3,8-dihydro-2H-furo[3,2-A]chromen-2-yl)propan-2-yl 2-methylbut- 2-enoate;
12a-hydroxy-8b-methyl-8b,9-dihydro-8H- benzo[7*,8*]chromeno[3*,4*:4,5]furo[3,2-¾]pyridine-8,12(12aH)-dione; 3,9-dihydroxy-2-(3-methylbut-2-en-l-yl)-6H-benzofuro[3,2-c]chromen-6-one; 7-hydroxy-6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-2H-chromen-2-one;
6-methoxy-7-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-2H-chromen-2-one;
(5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2- yl)propan-2-yl acetate;
9-hydroxy-5-(3,4,5-trimethoxyphenyl)-5,5a,8a,9- tetrahydromro[3\4^6,7]naphtho[2,3-(i][l,3]dioxol-6(8H)-one;
9-((7,8-dihydroxy-2-methylhexahydropyrano[3,2-(i][l,3]dioxin-6-yl)oxy)-5-(3,5- dimethoxy-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)phenyl)-5,5a,8a,9-tetrahydromro[3^4^6,7]naphtho[2,3- ][l,3]dioxol-6(8H)-one; a salt, solvate, or N-oxide thereof, and combinations thereof.
In one embodiment, the compound of formula (I) is at least one compound of formula (III):
Figure imgf000008_0001
wherein in formula (III):
each occurrence of R1 is independently selected from the group consisting of Η, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR9, -SR9, -S(=0)R9, -S(=0)2R9, -NHS(=0)2R9, - C(=0)R9, -OC(=0)R9, -C02R9, -OC02R9, -CH(R9)2, -N(R9)2, -C(=0)N(R9)2, - OC(=0)N(R9)2, -NHC(=0)NH(R9), -NHC(=0)R9, -NHC(=0)OR9, -C(OH)(R9)2, - C(CH3)2(CH=CH2), -C(CH3)2OC(=0)CH3, and -C(NH2)(R9)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR9, -SR9, -S(=0)R9, -S(=0)2R9, -NHS(=0)2R9, -C(=0)R9, -OC(=0)R9, -C02R9, -OC02R9, -CH(R9)2, -N(R9)2, - C(=0)N(R9)2, -OC(=0)N(R9)2, -NHC(=0)NH(R9), -NHC(=0)R9, -NHC(=0)OR6, - C(OH)(R9)2, and -C(NH2)(R9)2;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups;
each occurrence of R7 and R8 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl,
heterocycloalkyl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups, or R7 and R8 combine to form a ring optionally substituted with 0-4 R1 groups; and
each occurrence of R9 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
a salt, solvate, or N-oxide thereof, and any combinations thereof.
In one embodiment, the compound is selected from the group consisting of (5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan- 2-yl acetate, a salt, solvate, or N-oxide thereof, and combinations thereof. In one embodiment, the method further comprises administering to the individual an effective amount of a therapeutic agent.
In one embodiment, the therapeutic agent is selected from the group consisting of a protease inhibitor, a cytokine, and an immunomodulator.
In one embodiment, the method further comprises administering to the individual an effective amount of a chemotherapeutic agent.
In one embodiment, the chemotherapeutic agent is selected from the group consisting of a topoisomerase II inhibitor, an antibiotic, a vinca alkaloid, an
anthracycline, and a taxane.
The invention also provides a method for inhibiting replication of a herpesvirus infection. In one embodiment, the invention provides a method for inhibiting replication of a gamma-herpesvirus. In one embodiment, the invention provides a method for inhibiting replication of a KSHV. In one embodiment, the invention provides a method for inhibiting replication of an EBV.
BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
Figure 1, comprising Figures 1A through 1C, is a series of images depicting the results for the compound screening experiments. (Figure 1 A) Thirty-three compounds derived from a virtual screening were assayed for their effects on KSHV lytic DNA replication. BCBL-1 cells were induced with TPA and treated with each of the compounds at the concentration of 20 μΜ. Forty-eight hours post-induction, the intracellular KSHV genomic copy numbers were determined using quantitative real-time PCR. Seven compounds (marked with *) exhibited considerable inhibitory effects on viral lytic DNA replication. (Figure IB) Effects of the compounds on KSHV virion production. Five days post-induction, the KSHV virions in supernatants were collected and measured by quantifying encapsidated viral DNA in preparations. (Figure 1C) Effects of the compounds on host cell viability. Uninduced BCBL-1 cells were treated with each of the compounds at 20 μΜ for 48 hours and cell viability was assessed by trypan blue staining as described in Materials and methods. Compounds are shown with ascending molecular weights.
Figure 2, comprising Figures 2A through 2C, is a series of images demonstrating the effect of (+)-Rutamarin on KSHV replication and its associated cytotoxicity. (Figure 2A) Chemical structure of (+)-Rutamarin. (Figure 2B) The effect of (+)-Rutamarin on KSHV lytic replication and its associated cytotoxicity in BCBL-1 cells that were treated with a wide range of concentration of (+)-Rutamarin 3 hour after the lytic replication was induced by TPA. Intracellular KSHV genomic DNA replication (blue), extracellular virion production, and cell viability were determined as described elsewhere herein. Values were compared to those from the control cells (non-drug treatment). Mean values of results from three independent experiments and standard deviations are presented on the Y axe of dose-response curves. (Figure 2C) The effect of (+)-Rutamarin on KSHV lytic replication and its associated cytotoxicity in JSC-1 cells that were induced with 3mM sodium butyrate. Intracellular KSHV genomic DNA replication, extracellular virion production, and cell viability were determined as described elsewhere herein.
Figure 3 is a graph depicting the effects of (+)-Rutamarin and novobiocin on BCBL-1 cell proliferation. BCBL-1 cells (starting with 2>< 105 cells/ml) were exposed to (+)-Rutamarin and novobiocin, respectively, at the concentrations of their IC50 and 5 XIC50. Data were obtained from three independent determinations and presented as means with standard deviations.
Figure 4 is a graph depicting the effects of (+)-Rutamarin and novobiocin on BCBL-1 cell cycle progression. BCBL-1 cells (starting with 2x l05 cells/ml) were exposed to (+)-Rutamarin and novobiocin at the concentrations of their IC50 and 5xICso for 48 hours. Cell cycle progressions were measured by propidium iodide (PI) staining followed by flow cytometric analysis. The data are presented as means obtained from three independent experiments. Figure 5 is an image showing inhibition of KSHV orz-Zyt-dependent DNA replication with (+)-Rutamarin. BCBL-1 cells were transfected with KSHV ori-Lyt- containing plasmid (pOri-A) and RTA expression vector (pCR3.1-ORF50). The transfected cells were treated with increasing concentrations of (+)-Rutamarin and incubated for 72 hours. Hirt DNAs were extracted and digested with Kpnl/Sacl or Kpnl/Sacl/Dpnl. Dpnl-resistant viral replicated DNA (Rep'd DNA) was detected by Southern blotting with 32P-labeled pBluescript plasmid.
Figure 6, comprising Figures 6A through 6E, is a series of images demonstrating that identification of (+)-Rutamarin as a catalytic inhibitor of Topo Ila. (Figure 6A) The Topo II-mediated kinetoplast DNA decatenation assay was performed to evaluate the effect of testing compounds for Topo Ila activity at the concentration of 100 μΜ. The catenated (cat) and decatenated (decat) DNA positions in gels were indicated. The Topo Ila activities in the reactions in (Figure 6A) were quantitated and inhibition rates of compounds were calculated and presented in (Figure 6B). The inhibition of Topo Ila (Figure 6C) and Topo Ila (Figure 6E) activities by (+)-Rutamarin in a wide range of concentration was determined. (Figure 6D) The IC50 of (+)-Rutamarin on Topo Ila inhibition was calculated as 28.22±6.2 μΜ. Data are means and standard deviations for three experimental replicates. (Figure 6E) (+)-Rutamarin exhibits no obvious inhibitory effect on human Topo Πβ. Rut: (+)-Rutamarin; Eto: Etoposide; Nov: Novobiocin.
Figure 7 is a graph depicting the effect of (+)-Rutamarin on ATPase activity of Topo Ila. The inhibitory effect of (+)-Rutamarin on Topo Ila ATPase activity was measured using the malachite green assay as described elsewhere herein. The OD62o represents the ATP hydrolysis level and reflects the inhibition of ATPase activity by (+)- Rutamarin.
Figure 8, comprising Figures 8A through 8F, is a series of images demonstrating the stability properties of simulation system and binding model of (+)- Rutamarin with Topo Ila. (Figure 8A) RMSD plot for the backbone atoms (light blue) and (+)-Rutamarin (light green) during the MD simulations after the equilibration.
(Figure 8B) Distance distributions between the Asn95-, Alal67- and conserved water 2 and (+)-Rutamarin in water at 310 K. The cutoff value for the formation of a hydrogen bond is 3.5 A. (Figure 8C) The time dependence of distance between carboxyl oxygen of Glu87, amide nitrogen of Asn91, the two conserved water molecules and Mg2+ during the 10 ns MD simulations. (Figure 8D) The detail binding model between (+)-Rutamarin and the residues in the ATPase domain of Topo Πα. Hydrogen bonds are shown in red dot line and magenta sphere represents Mg cation. (Figure 8E) The polar and hydrophobic surface profile of the ATPase domain of Topo Πα with (+)-Rutamarin. Polar and hydrophobic area are depicted. (Figure 8F) Superposition of (+)-Rutamarin with ADPNP in ligand-binding pocket.
Figure 9 is an image demonstrating the dinding free energy decomposition results based on MM-GBSA method. The key residues are labeled. The unit of the each residue's contribution to total binding energy is kcal/mol.
Figure 10 is an image showing the cytotoxicity of (+)-Rutamarin to peripheral blood mononuclear cells (PBMC). PBMC were isolated from whole blood of the health volunteer and cultured in in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cells were treated with different concentrations of (+)-Rutamarin as indicated for 48 hour. The cell viabilities were measured by cell counting with trypan blue staining. The CC50 was calculated as 60.91 μΜ.
Figure 11 is an image showing the effect of (+)-Rutamarin on RTA expression. BCBL-1 cells were induced with 20ng/ml TPA. After three hours, the cells were treated with different concentrations of (+) Rutamarin as indicated. Forty-eight hours post-induction, whole cell extracts were prepared and analyzed by Western blot with antibodies against RTA and β-actin.
Figure 12, comprising Figures 12A through 12C, is a schematic showing the chemical structure of novobiocin, merbarone and (+)-rutamarin.
Figure 13, comprising Figures 13A through 13E is a series of images showing the effects of Topo II inhibitors on EBV replication and their associated cytotoxicity in P3HR-1 cells. EBV lytic replication in P3HR-1 was induced with TPA and sodium butyrate. Topo II catalytic inhibitors, novobiocin (Figure 13 A), merbarone (Figure 13B) and (+)-rutamarin (Figure 13C) were added to the cell culture 3 h after the induction. The concentration ranges tested for different inhibitors are: 0.1-1000 μΜ (novobiocin), 0.1 -760 μΜ (merbarone), and 0.01-150 μΜ ((+)-rutamarin). Intracellular EBV DNA, extracellular virion DNA, and cell viability were determined for each concentration point. These values were compared to those from the control cells (non- drug treatment). Mean values of results from at least three independent experiments and standard deviations are presented on the Faxes of dose-response curves. Topo II inhibitor doses are indicated on the x axes as logarithmic scales. The effect of (+)- rutamarin on cell viability of P3HR-1 cells on 2 and 5 days exposure were shown in Figure 13D and Figure 13E.
Figure 14, comprising Figures 14A through 14E, is a series of images showing the effects of Topo II inhibitors on EBV replication and their associated cytotoxicity in Akata-Bxl cells. EBV lytic replication in Akata-Bxl was induced by treatment of cells with anti-IgG. Topo II catalytic inhibitors, novobiocin (Figure 13 A), merbarone (Figure 13B) and (+)-rutamarin (Figure 13C) in wide ranges were added to the cell culture 3 h after the induction. Intracellular EBV DNA, extracellular virion DNA, and cell viability were determined for each concentration point. These values were compared to those from the control cells (non-drug treatment). Mean values of results from at least three independent experiments and standard deviations are presented on the Faxes of dose-response curves. Topo II inhibitor doses are indicated on the x axes as logarithmic scales. The effect of (+)-rutamarin on cell viability of Akata-Bxl cells on 2 and 5 days exposure were shown in Figure 14D and Figure 14E.
Figure 15 is an image showing the effects of Topo II inhibitors on P3HR-1 cell proliferation. P3HR-1 cells (starting with 2 x 105 cells/ml) were exposed to different inhibitors as indicated at two concentrations (IC50 and 5 x IC50) and counted every day for five days. Data were obtained from three independent determinations and presented as means with standard deviations.
Figure 16, comprising Figures 16A through 16C, is a series of images showing inhibition of EBV ori-Lyt-associated DNA replication with Topo II inhibitors. P3HR-1 cells were transfected with an ori-Lyt-containing plasmid (pEBV-oriL) and ZTA expression vector (McZ). Transfected cells were cultured in the absence or presence of increasing concentrations of novobiocin (Figure 16A), merbarone (Figure 16B), and (+)- rutamarin (Figure 16C). After 72 h of incubation, hirt DNAs were extracted from the cells and digested with EcoRI or EcoRI/Dpn I. Dpnl -resistant products of DNA replication (Rep'd DNA) were detected by Southern blotting with digoxigenin - labeled EcoR I - Hind III fragment from the pEBV-oriL plasmid.
DETAILED DESCRIPTION
The present invention relates to the unexpected discovery that a ligand based virtual screening approach identified a set of coumarin derivatives that exhibited inhibitory activity against Kaposi's sarcoma-associated herpesvirus (KSHV) DNA replication. In one embodiment, compounds of the invention exhibit topoisomerase II (Topo II) inhibitor activity. In one embodiment, the compounds of the invention belong to the category of catalytic Topo II inhibitors.
In one embodiment, the compounds of the invention block KSHV lytic DNA replication. Among other things, the compounds of the invention can be used to treat viral (such as herpesvirus) infection, as well treating and KSHV infection or related conditions. However, the invention should not be liminted to treating herpesvirus infection. Rather, the invention includes treating all types of herpesvirus, for example, gamma-herpesvirus infections including but not limited to two human pathogens Epstein-Barr virus (EBV) and KSHV. Pharmaceutically acceptable salts and
stereoisomers of the compounds also are included in the invention.
Herpesvirus is an animal DNA virus and Herpesviridae viruses are classified into three subfamilies, that is, the alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae. Alphaherpesvirinae is classified further into Herpes simplex virus, Varicellovirus, Mardivirus, and Ilto virus and typical ones include HSV-1, HSV-2, varicella/zoster virus, porcine herpes virus 1 (pseudorabies virus), and bovine herpesvirus.Viruses belonging to the betaherpesvirinae include human cytomegalovirus (HCMV), while as viruses belonging to the gammaherpesvirinae, EB virus, Kaposi's sarcoma-associated herpesvirus, and the like are known. As viruses belonging to the gammaherpesvirinae, Epstein-Barr virus (EB virus) of Lymphocryptovirus genus is typical.
The present invention also provides a method for inhibiting gamma- herpesvirus replication in a subject. The present invention also provides a method for inhibiting KSHV replication in a subject. The method comprises administering to the subject a composition of the invention by any suitable route of administration. For example, the method of the present invention is useful for administrating the
compositions of the invention to a subject exposed to a gamma-herpesvirus, such as KSHV, and to a subject who is at risk of contact with a gamma-herpesvirus, such as KSHV.
In one embodiment, the compounds of the invention act as a topoisomerase II catalytic inhibitor. Another aspect of the invention pertains to a composition comprising a compound as described herein and a pharmaceutically acceptable carrier or diluent. Another aspect of the present invention pertains to a compound as described herein for use in a method of treatment of the human or animal body by therapy. Another aspect of the present invention pertains to use of a compound, as described herein, in the manufacture of a medicament for use in treatment.
In one embodiment, the present invention pertains to a method of inhibiting (e.g., catalytically inhibiting) topoisomerase II in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a compound of the invention.
In one embodiment, the present invention pertains to a method of treating a gamma-herpesvirus infection, such as KSHV infection or related conditions, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of the invention, preferably in the form of a pharmaceutical composition.
In one embodiment (e.g., of use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of a disease or condition that is ameliorated by the catalytic inhibition of topoisomerase II.
In one embodiment (e.g., of use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of a proliferative condition. In one embodiment, the treatment is treatment of cancer.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
"About," as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%), ±1%), or ±0.1%) from the specified value, as such variations are appropriate to perform the disclosed methods.
The term "abnormal," when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.
As used herein, the term "container" includes any receptacle for holding the pharmaceutical composition. For example, in one embodiment, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical
composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating or preventing a disease in a subject. A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, including simians and humans.
The term "herpesvirus" or "herpes virus" or "HV" refers to human herpesviruses and may be used, depending on the context, to refer to one, more, or all of the human herpesviruses, including Human Herpesvirus- 1 (HHV-1, Herpes Simplex Virus- 1, HSV1, HSV-1), HHV-2 (Herpes Simplex Virus-2, HSV2, HSV-2), HHV-3 (Varicella Zoster Virus, VZV), HHV-4 (Epstein-Barr Virus, EBV), HHV-5
(Cytomegalovirus, CMV, HCMV), HHV-6, HHV-7, HHV-8 (Kaposi's Sarcoma- associated Herpesvirus, KSHV). In some embodiments, a herpesvirus is HHV-1. In some embodiments, a herpesvirus is HHV-2. In some embodiments, a herpesvirus is HHV-3. In some embodiments, a herpesvirus is HHV-4. In some embodiments, a herpesvirus is HHV-5. In some embodiments, a herpesvirus is HHV-6. In some embodiments, a herpesvirus is HHV-7. In some embodiments, a herpesvirus is HHV-8. In some embodiments, each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8 may refer to all strains of each respective HHV. In some
embodiments, each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8 may refer to a single strain of that HHV. In some embodiments, each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV- 8 may include mutants of that particular HHV. In some embodiments HV is HSV (HHV- 1 and/or HHV-2). As used herein the term "Kaposi sarcoma" or "KS" refers to any form of KS, including classic (or Mediterranean) KS, endemic KS, AIDS-related (epidemic) KS, or iatrogenic (transplant-associated) KS or immunosuppression-associated KS.
The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound of the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
"Pharmaceutically acceptable" refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability. "Pharmaceutically acceptable carrier" refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.
As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; ellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein,
"pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in
Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
As used herein, the term "salt" embraces addition salts of free acids or free bases that are compounds useful within the invention. Suitable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, phosphoric acids, perchloric and tetrafluoroboronic acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable base addition salts of compounds useful within the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, lithium, calcium, magnesium, potassium, sodium and zinc salts. Acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl- glucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding free base compound by reacting, for example, the appropriate acid or base with the corresponding free base.
A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology, for the purpose of diminishing or eliminating those signs or symptoms.
As used herein, "treating a disease or disorder" means reducing the frequency or severity with which a sign or symptom of the disease or disorder is experienced by a patient.
As used herein, the terms "effective amount," "pharmaceutically effective amount" and "therapeutically effective amount" refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
An "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.
As used herein, the term "potency" refers to the dose needed to produce half the maximal response (ED50).
As used herein, the term "efficacy" refers to the maximal effect (Emax) achieved within an assay.
As used herein, the term "(+)-rutamarin" refers to (5)-2-(6-(2-methylbut- 3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan-2-yl acetate. As used herein, the term "alkyl," by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. Ci-6 means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. Most preferred is (Ci-C6)alkyl, particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.
As used herein, the term "substituted alkyl" means alkyl as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH2, -N(CH3)2, -C(=0)OH, trifluoromethyl, -C≡N, -C(=0)0(C C4)alkyl, -C(=0)NH2, -S02NH2, -C(=NH)NH2, and -N02, preferably containing one or two substituents selected from halogen, -OH, alkoxy, -NH2, trifluoromethyl, -N(CH3)2, and -C(=0)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl,
2-carboxycyclopentyl and 3-chloropropyl.
As used herein, the term "heteroalkyl" by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -0-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-NH-CH3,
-CH2-S-CH2-CH3, and -CH2CH2-S(=0)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or -CH2-CH2-S-S-CH3
As used herein, the term "alkoxy" employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homo logs and isomers. Preferred are (C1-C3) alkoxy, particularly ethoxy and methoxy.
As used herein, the term "halo" or "halogen" alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.
As used herein, the term "cycloalkyl" refers to a mono cyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In one embodiment, the cycloalkyl group is saturated or partially unsaturated. In another embodiment, the cycloalkyl group is fused with an aromatic ring. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:
Figure imgf000023_0001
Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbornane. The term cycloalkyl includes "unsaturated nonaromatic carbocyclyl" or "nonaromatic unsaturated carbocyclyl" groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon carbon double bond or one carbon carbon triple bond.
As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a heteroalicyclic group containing one to four ring heteroatoms each selected from O, Sand N. In one embodiment, each heterocycloalkyl group has from 4 to 10 atoms in its ring system, with the proviso that the ring of said group does not contain two adjacent O or S atoms. In another embodiment, the heterocycloalkyl group is fused with an aromatic ring. In another embodiment, the heterocycloalky group is fused to a second heterocycle. In one embodiment, the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or non-aromatic in nature. In one embodiment, the heterocycle is a heteroaryl.
An example of a 3-membered heterocycloalkyl group includes, and is not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, and are not limited to, azetidine and a beta lactam. Examples of 5-membered heterocycloalkyl groups include, and are not limited to, pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-membered heterocycloalkyl groups include, and are not limited to, piperidine, morpholine and piperazine. Other non-limiting examples of heterocycloalkyl groups are:
Figure imgf000024_0001
Figure imgf000024_0002
Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1 ,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,
4,7-dihydro-l,3-dioxepin, and hexamethyleneoxide. As used herein, the term "aromatic" refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized π (pi) electrons, where n is an integer.
As used herein, the term "aryl," employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. Preferred examples are phenyl and naphthyl, most preferred is phenyl.
As used herein, the term "aryl-(Ci-C3)alkyl" means a functional group wherein a one- to three-carbon alkylene chain is attached to an aryl group, e.g.,
-CH2CH2-phenyl. Preferred is aryl-CH2- and aryl-CH(CH3)-. The term "substituted aryl-(Ci-C3)alkyl" means an aryl-(Ci-C3)alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH2)-. Similarly, the term "heteroaryl- (Ci-C3)alkyl" means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e.g. , -CH2CH2-pyridyl. Preferred is heteroaryl-(CH2)-. The term "substituted heteroaryl-(Ci-C3)alkyl" means a heteroaryl-(Ci-C3)alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl- (CH2)-.
As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include the following
moieties:
Figure imgf000025_0001
Examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
Examples of polycyclic heterocycles and heteroaryls include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5 -quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1 ,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-difiydrobenzofuryl, 1 ,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly
2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (particularly
2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.
As used herein, the term "substituted" means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. The term "substituted" further refers to any level of substitution, namely mono-, di-, tri-, terra-, or penta-substitution, where such substitution is permitted. The substituents are
independently selected, and substitution may be at any chemically accessible position. In one embodiment, the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two.
As used herein, the term "optionally substituted" means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.
In one embodiment, the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, alkyl (including straight chain, branched and/or unsaturated alkyl), substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=0)2alkyl, -C(=0)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -C(=0)N[H or alkyl]2, -
OC(=0)N[substituted or unsubstituted alkyl]2, -NHC(=0)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -NHC(=0)alkyl, -N[substituted or unsubstituted alkyl]C(=0)[substituted or unsubstituted alkyl], -NHC(=0)[substituted or unsubstituted alkyl], -C(OH) [substituted or unsubstituted alkyl]2, -OC(=0)[substituted or unsubstituted alkyl, or substituted or unsubstituted alkenyl], -C(=0) [substituted or unsubstituted alkyl], and -C(NH2) [substituted or unsubstituted alkyl]2. In another embodiment, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CH(CH3)2, -CF3, -CH2CF3, -OCH3, -OCH2CH3, -OCH(CH3)2, -OCF3, - OCH2CF3, -S(=0)2-CH3, -C(=0)NH2, -C(=0)-NHCH3, -NHC(=0)NHCH3, -C(=0)CH3, -
OC(=0)CH3, and -C(=0)OH. In yet one embodiment, the substituents are independently selected from the group consisting of Ci_6 alkyl, -OH, Ci_6 alkoxy, halo, amino, acetamido, oxo and nitro. In yet another embodiment, the substituents are independently selected from the group consisting of Ci_6 alkyl, Ci_6 alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being preferred.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description
The invention provides compositions and methods for modulating and treatment of a gamma-herpesvirus infection, such KSHV infection, or KSHV-mediated effects on cellular proliferation and phenotype, comprising inhibiting KSHV lytic replication. In one embodiment, the compounds of the invention can be used to treat or prevent KSHV infection, Kaposi's sarcoma (KS) and related cancers and conditions.
In one embodiment, the compounds of the invention inhibit both viral DNA synthesis and virion production with relatively low cytotoxicity.
In one embodiment, the compounds of the invention inhibit the enzymatic activity of Topo Πα in a dose-dependent manner. In one embodiment, the compounds of the invention function as a Topo Πα catalytic inhibitor.
Compounds of the Invention
The compounds of the present invention may be synthesized using techniques well-known in the art of organic synthesis. The starting materials and intermediates required for the synthesis may be obtained from commercial sources or synthesized according to methods known to those skilled in the art.
In one aspect, the compound of the invention is a compound of formula (I), or a salt, solvate, or N-oxide thereof:
Figure imgf000028_0001
(I)
wherein in formula (I):
each occurrence of R1 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, -S(=0)2R7, -NHS(=0)2R7, - C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, - OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, -C(OH)(R7)2, - C(CH3)2(CH=CH2), -C(CH3)2OC(=0)CH3, and -C(NH2)(R7)2; wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, -S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, - C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups, or two or more adjacent R1, R2, R3, R4, R5,and R6 groups combine to form a first ring optionally substituted with 0-4 R1 groups, wherein the first ring is optionally fused to a second ring optionally substituted with 0-4 R1 groups;
each occurrence of R7 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted; and
X is -CH or O.
In another aspect, the compound of the invention is a compound of formula (II), or a salt, solvate, or N-oxide thereof:
Figure imgf000029_0001
wherein in formula (II) each occurrence of R1, R2, R3, R4, and R5 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR6, -SR6, -S(=0)R6, - S(=0)2R6, -NHS(=0)2R6, -C(=0)R6, -OC(=0)R6, -C02R6, -OC02R6, -CH(R6)2, -N(R6)2, -C(=0)N(R6)2, -OC(=0)N(R6)2, -NHC(=0)NH(R6), -NHC(=0)R6, -NHC(=0)OR6, - C(OH)(R6)2, and -C(NH2)(R6)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR6, -SR6, -S(=0)R6, -S(=0)2R6, -NHS(=0)2R6, -C(=0)R6, -OC(=0)R6, -C02R6, -OC02R6, -CH(R6)2, -N(R6)2, - C(=0)N(R6)2, -OC(=0)N(R6)2, -NHC(=0)NH(R6), -NHC(=0)R6, -NHC(=0)OR6, - C(OH)(R6)2, and -C(NH2)(R6)2; and
each occurrence of R6 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted.
In another aspect, the compound of the invention is a compound of formula (III), or a salt, solvate, or N-oxide thereof:
Figure imgf000030_0001
wherein in formula (III):
each occurrence of R1 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR9, -SR9, -S(=0)R9, -S(=0)2R9, -NHS(=0)2R9, - C(=0)R9, -OC(=0)R9, -C02R9, -OC02R9, -CH(R9)2, -N(R9)2, -C(=0)N(R9)2, - OC(=0)N(R9)2, -NHC(=0)NH(R9), -NHC(=0)R9, -NHC(=0)OR9, -C(OH)(R9)2, - C(CH3)2(CH=CH2), -C(CH3)2OC(=0)CH3, and -C(NH2)(R9)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR9, -SR9, -S(=0)R9, -S(=0)2R9, -NHS(=0)2R9, -C(=0)R9, -OC(=0)R9, -C02R9, -OC02R9, -CH(R9)2, -N(R9)2, - C(=0)N(R9)2, -OC(=0)N(R9)2, -NHC(=0)NH(R9), -NHC(=0)R9, -NHC(=0)OR6, - C(OH)(R9)2, and -C(NH2)(R9)2;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups;
each occurrence of R7 and R8 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl,
heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups, or R7 and R8 combine to form a ring optionally substituted with 0-4 R1 groups; and
each occurrence of R9 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted. In one embodiment, the compound of the invention is selected from the group consisting of:
2H-chromen-2-one;
7-hydroxy-2H-chromen-2-one;
7-amino-4-methyl-2H-chromen-2-one;
7-hydroxy-4-methyl-2H-chromen-2-one;
6.7- dihy droxy-2H-chromen-2 -one ;
7 , 8 -dihy droxy-2H-chromen-2 -one ;
2H-furo[2,3-A]chromen-2-one;
7H-furo [3 ,2-g] chromen-7-one;
6- hydroxy-7-methoxy-2H-chromen-2-one;
7.8- dihydroxy-6-methoxy-2H-chromen-2-one;
9-methoxy-7H-furo [3 ,2-g] chromen-7-one;
9-methoxynaphtho[2,3-£]furan-7(8H)-one;
7- hydroxy-6,8-dimethoxy-2H-chromen-2-one;
7-(diethylamino)-4-methyl-2H-chromen-2-one;
7- methoxy-8-(3-methylbut-2-en-l-yl)-2H-chromen-2-one;
8- (2,3-dihydroxy-3-methylbutyl)-7-hydroxy-2H-chromen-2-one;
4-((3-methylbut-2-en-l-yl)oxy)-7H-furo[3,2-g]chromen-7-one;
9- ((3-methylbut-2-en-l-yl)oxy)-7H-furo[3,2-g]chromen-7-one;
6b-ethyl-10a-hydroxy-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2-¾]pyridine-
6,10(10aH)-dione;
10a-hydroxy-3,6b-dimethyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
10a-hydroxy-2,6b-dimethyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
6-(2,3-dihydroxy-3-methylbutyl)-5,7-dimethoxy-2H-chromen-2-one;
4-hydroxy-3-(3-oxo- 1 -phenylbutyl)-2H-chromen-2-one;
6b-ethyl-10a-hydroxy-3-methyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione; 6b-ethyl-10a-hydroxy-2-methyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
(E)-2-(8-oxo-3,8-dihydro-2H-furo[3,2-A]chromen-2-yl)propan-2-yl 2-methylbut- 2-enoate;
12a-hydroxy-8b-methyl-8b,9-dihydro-8H- benzo[7^8*]chromeno[3^4^4,5]furo[3,2-¾]pyridine-8,12(12aH)-dione;
3,9-dihydroxy-2-(3-methylbut-2-en-l-yl)-6H-benzofuro[3,2-c]chromen-6-one; 7-hydroxy-6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-2H-chromen-2-one;
6-methoxy-7-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-2H-chromen-2-one;
(5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2- yl)propan-2-yl acetate;
9-hydroxy-5-(3,4,5-trimethoxyphenyl)-5,5a,8a,9- tetrahydroi iro[3\4^6,7]naphtho[2,3-(i][l,3]dioxol-6(8H)-one;
9-((7,8-dihydroxy-2-methylhexahydropyrano[3,2-(i][l,3]dioxin-6-yl)oxy)-5-(3,5- dimethoxy-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)phenyl)-5,5a,8a,9-tetrahydromro[3^4^6,7]naphm^
a salt, solvate, or N-oxide thereof, and any combination thereof.
In one embodiment, the compound of formula (III) is 2-(6-(2-methylbut-3- en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan-2-yl acetate, a salt, solvate, or N-oxide thereof, and any combination thereof.
The structures and corresponding names of the compounds are illustrated in Table 1.
Table 1 : Compounds of the present invention
Figure imgf000033_0001
Figure imgf000033_0002
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Preparation of the Compounds of the Invention
Compounds of formula (I), (II), or (III) may be prepared by the general schemes described herein, using the synthetic method known by those skilled in the art. The following examples illustrate non-limiting embodiments of the invention.
The compounds of the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration. In one embodiment, compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In one embodiment, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In another embodiment, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non- limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
The methods and formulations described herein include the use of
N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the invention, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In one embodiment, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In another embodiment, the compounds described herein exist in unsolvated form.
In one embodiment, the compounds of the invention may exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
In one embodiment, compounds described herein are prepared as prodrugs. A "prodrug" refers to an agent that is converted into the parent drug in vivo. In one embodiment, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In another embodiment, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
In one embodiment, sites on, for example, the aromatic ring portion of compounds of the invention are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In one embodiment, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, UC, 13C, 14C, 36C1, 18F, 1231, 1251, 13N, 15N,
15 17 18 32 35
1J0, "0, 100, JT, and JJS. In one embodiment, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet another embodiment, substitution with positron emitting isotopes, such as 11 C, 18 F, 15 O and 13 N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
In one embodiment, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.
Compounds described herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources, or are prepared using procedures described herein. In one embodiment, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected in order to avoid their unwanted
participation in reactions. Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In another embodiment, each protective group is removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal.
In one embodiment, protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions. Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.
In one embodiment, carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as
2,4-dimethoxybenzyl, while co-existing amino groups are blocked with fluoride labile silyl carbamates.
Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts. For example, an allyl-b locked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base- labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.
Typically blocking/protecting groups may be selected from:
Figure imgf000042_0001
ally* aitoe Me
Figure imgf000042_0002
Boc PM8 tnty} ^-W ¥moc
Other protecting groups, plus a detailed description of techniques applicable to the creation of protecting groups and their removal are described in Greene & Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference for such disclosure.
Methods
Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS) and related diseases. Without wishing to be bound by any particular theory, it is believed that blocking lytic KSHV replication hinders KSHV-induced hyperproliferative disorders such as cancer.
In one embodiment, the invention includes methods for treating KSHV infection and related conditions (e.g., KS). In particular embodiments, the methods for treating KS provided herein inhibit, reduce, diminish, arrest, or stabilize a tumor associated with KS or a symptom thereof. In other embodiments, the methods for treating KS provided herein inhibit, reduce, diminish, arrest, or stabilize the blood flow, metabolism, peritumoral inflammation or peritumoral edema in a tumor associated with KS or a symptom thereof. In some embodiments, the methods for treating KS provided herein reduce, ameliorate, or alleviate the severity of KS and/or a symptom thereof. In particular embodiments, the methods for treating KS provided herein cause the regression of a KS tumor, tumor blood flow, tumor metabolism, or peritumoral edema and/or a symptom associated with KS. In other embodiments, the methods for treating KS provided herein reduce hospitalization (e.g., the frequency or duration of hospitalization) of a subject diagnosed with KS. In some embodiments, the methods for treating KS provided herein reduce hospitalization length of a subject diagnosed with KS. In certain embodiments, the methods provided herein increase the survival of a subject diagnosed with KS. In particular embodiments, the methods for treating KS provided herein inhibit or reduce the progression of one or more tumors or a symptom associated therewith. In specific embodiments, the methods for treating KS provided herein enhance or improve the therapeutic effect of another therapy (e.g., an anti-cancer agent, radiation, chemotherapy, or surgery). In certain embodiments, the methods for treating KS involve the use of a compound as an adjuvant therapy. In certain embodiments, the methods for treating KS provided herein improve the ease in removal of tumors (e.g., enhance respectability of the tumors) by reducing vascularization prior to surgery. In particular embodiments, the methods for treating KS provided herein reduce vascularization after surgery, for example, reduce vascularization of the remaining tumor mass not removed by surgery. In some embodiments, the methods for treating KS provided herein prevent recurrence, e.g., recurrence of vascularization and/or tumor growth.
In specific embodiments, the methods for treating KS provided herein reduce or eliminate one, two, or more of the following: skin lesions, nausea, vomiting, abdominal pain, bleeding, difficulty breathing and lymphedema. In some embodiments, the methods for treating KS provided herein reduce the growth of a tumor associated with KS. In certain embodiments, the methods for treating KS provided herein eradicate, remove, or control primary, regional and/or metastatic tumors associated with KS. In other embodiments, the methods for treating KS provided herein decrease the number or size of lesions associated with KS. In particular embodiments, the methods for treating KS provided herein reduce the mortality of subjects diagnosed with KS. In other embodiments, the methods for treating KS provided herein increase the tumor-free survival rate of patients diagnosed with KS. In some embodiments, the methods for treating KS provided herein increase relapse-free survival. In certain embodiments, the methods for treating KS provided herein increase the number of patients in remission or decrease the hospitalization rate. In other embodiments, the methods for treating KS provided herein maintain the size of the tumor so that it does not increase, or so that it increases by less than the increase of a tumor after administration of a standard therapy as measured by methods available to one of skill in the art, such as measurement of a lesion, photography, X-ray, CT Scan, MRI, PET Scan, bronchoscopy, and endoscopy. In other embodiments, the methods for treating KS provided herein prevent the development or onset of KS, or a symptom associated therewith. In other embodiments, the methods for treating KS provided herein increase the length of remission in patients. In particular embodiments, the methods for treating KS provided herein increase symptom-free survival of KS patients. In some embodiments, the methods for treating KS provided herein do not cure KS in patients, but prevent the progression or worsening of the disease.
In particular embodiments, the methods for treating KS achieve one or more of the following: (i) inhibition or reduction in pathological production of VEGF; (ii) stabilization or reduction of peritumoral inflammation or edema in a subject; (iii) reduction of the concentration of VEGF or other angiogenic or inflammatory mediators (e.g., cytokines or interleukins) in biological specimens (e.g., plasma, serum, cerebral spinal fluid, urine, or any other bio fluids); (iv) reduction of the concentration of P IGF, VEGF-C, VEGF-D, VEGF-R, IL-6, IL-8 and/or IL-10 in biological specimens (e.g., plasma, serum, cerebral spinal fluid, urine, or any other biofluids); (v) inhibition or decrease in tumor metabolism or perfusion; (vi) inhibition or decrease in angiogenesis or vascularization; and/or (vii) improvement in quality of life as assessed by methods well known in the art, for e.g, a questionnaire.
In certain aspects, the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject as determined by methods well known in the art, e.g., MRI. Three dimensional volumetric measurement performed by MRI has been shown to be sensitive and consistent in assessing tumor size (see, e.g., Harris et al, Neurosurgery, June 2008, 62(6): 1314-9), and thus may be employed to assess tumor shrinkage in the methods provided herein. In specific embodiments, the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least about 20% as assessed by methods well known in the art, e.g., MRI. In certain embodiments, the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art, e.g., MRI. In particular embodiments, the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least an amount in a range of from about 10% to about 100%, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art. In particular embodiments, the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by an amount in a range of from about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 40% to 100%, or any range in between, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art, e.g., MRI.
In particular aspects, the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject as assessed by methods well known in the art, e.g., dynamic contrast-enhanced MRI (DCE-MRI). Standard protocols for DCE-MRI have been described (see., e.g., Morgan et al, J. Clin. Oncol, Nov. 1, 2003, 21(21):3955- 64; Leach et al, Br. J. Cancer, May 9, 2005, 92(9): 1599-610; Liu et al, J. Clin. Oncol, August 2005, 23(24): 5464-73; and Thomas et al, J. Clin. Oncol, Jun. 20, 2005,
23(18):4162-71) and can be applied in the methods provided herein. In specific embodiments, the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, relative to tumor perfusion prior to administration of a compound, as assessed by methods well known in the art, e.g., DCE-MRI. In particular embodiments, the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject in the range of from about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 40% to 100%, or any range in between, relative to tumor perfusion prior to administration of a compound, as assessed by methods well known in the art.
In certain embodiments, the methods for treating KS provided herein reduce or inhibit KSHV gene transcription or reduce KSHV mRNA levels in tumor biopsies from a subject with KS by about 5%, 10%, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or any range in between, relative to the respective KSHV gene transcription level or KSHV mRNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art. In particular embodiments, the methods for treating KS provided herein reduce or inhibit KSHV gene transcription or reduce KSHV mRNA levels in tumor biopsies from a subject with KS by 5% to 10%>, 10%> to 20%>, 10%> to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between relative to the respective KSHV gene transcription level or KSHV mRNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
In other embodiments, the methods for treating KS provided herein reduce or inhibit KSHV replication or reduce the number of copies of KSHV in circulating peripheral blood mononuclear cells (PBMC) in a subject with KS by about 5%, 10%>, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or any range in between, relative to the respective KSHV replication level or number of KSHV copies in circulating PBMC observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art. In particular embodiments, the methods for treating KS provided herein reduce or inhibit KSHV replication or reduce the number of copies of KSHV in circulating PBMC in a subject with KS by about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between, relative to the respective KSHV replication level or number of KSHV copies in circulating PBMC observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
In certain embodiments, the methods for treating KS provided herein inhibit or reduce HIV plasma RNA levels in a subject with KS by about 5%, 10%, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in a subject with KS relative to the respective HIV plasma RNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art. In particular embodiments, the methods for treating KS provided herein inhibit or reduce HIV plasma RNA levels in a subject with KS by about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between, relative to the respective HIV plasma RNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
The compounds of the invention can additionally employ adjunct components conventionally found in pharmaceutical compositions in their art-established fashion and at their art-established levels. Thus, for example, the compositions can contain additional compatible pharmaceutically active materials for combination therapy (such as supplementary antimicrobials, antipruritics, astringents, local anesthetics, anticancer, or anti-inflammatory agents), or can contain materials useful in physically formulating various dosage forms of the preferred embodiments, such as excipients, dyes, perfumes, thickening agents, stabilizers, skin penetration enhancers, preservatives or antioxidants. Administration/Dosage/Formulations
The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of a gamma-herpesvirus infection, such as KSHV infection, and related conditions. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions of the present invention to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a gamma-herpesvirus infection, such as KSHV infection, and related conditions in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a gamma- herpesvirus infection, such as KSHV infection, and related conditions in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the therapeutic situation. A non- limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of KSHV infection and related conditions a patient.
In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one
embodiment, the pharmaceutical compositions of the invention comprise a
therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier.
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DMSO alone. In one embodiment, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
Compounds of the invention for administration may be in the range of from about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1 ,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
In one embodiment, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a gamma-herpesvirus infection, such as KSHV infection, and related conditions in a patient.
Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
Routes of administration of any of the compositions of the invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral
administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
Oral Administration
For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
For oral administration, the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone,
hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY- P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,
32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid). Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation." For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.
Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution.
U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) melt.
The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of KSHV infection and related conditions. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release. Parenteral Administration
For parenteral administration, the compounds of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used. Additional Administration Forms
Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO
03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404;
WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO
98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.
Controlled Release Formulations and Drug Delivery Systems
In one embodiment, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form. For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
In one embodiment of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug
administration.
As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
Dosing
The therapeutically effective amount or dose of a compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of KSHV infection and related conditions in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
A suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the inhibitor of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%- 100%, including, by way of example only, 10%, 15%,20%,25%,30%, 35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%, 95%, or 100%.
Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained. In one embodiment, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
The compounds for use in the method of the invention may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Active compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such active compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application. It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.
The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.
EXPERIMENTAL EXAMPLES
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
Example 1 : Antiviral Activity of (+)-Rutamarin against SHV by Inhibiting Catalytic Activity of Human Topoisomerase II
Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of several AIDS-associated malignancies including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castkeman's disease (MCD). Its lytic replication cycle has been proven to be critical for the pathogenesis of KSHV-associated diseases. In KS lesion, lytic viral replication, production of virion particles and reinfection of endothelial cells are essential to sustain the population of infected cells that otherwise would be quickly lost as spindle cells divide. Thus, antivirals that block KSHV replication could be a strategy in treatment of KSHV-associated diseases. However there is no effective anti-KSHV drug currently available.
Using a ligand based virtual screening approach, more than 7200 compound structures were screened. A set of coumarin derivatives that showed activities in inhibition of KSHV DNA replication equal to or greater than that of novobiocin was identified. In particular, (+)-Rutamarin exhibits the highest efficiency in blocking KSHV lytic replication. The results presented herein demonstrated that (+)-Rutamarin has human Topo II inhibition activity and belongs to the category of catalytic Topo II inhibitor.
The results presented herein demonstrate the discovery and characterization of a novel catalytic inhibitor of human Topo ΙΙα, namely (+)-Rutamarin. The binding mode of (+)-Rutamarin to the ATPase domain of human Topo Πα was established by docking and validated by MD simulations. More importantly, (+)- Rutamarin efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with an IC50 of 1.12 μΜ and blocks virion production with an EC50 of 1.62 μΜ. It possesses low cytotoxicity as indicated by the selectivity index (SI) of 84.14. This study demonstrated a great potential of (+)-Rutamarin to become an effective drug for treatment of human diseases associated with KSHV infection.
The materials and methods employed in these experiments are now described.
Materials and Methods
Cells and Plasmids
The BCBL-1 and JSC-1 are two primary effusion lymphoma cell lines that are latently infected with KSHV (Renne R, et al. 1996. Nat. Med. 2:342-346, Cannon JS, et al. 2000. J. Virol. 74: 10187-10193). BJAB cell line is a KSHV-negative B cell line isolated from Burkitt's lymphoma. These B cells were grown in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gibco- BRL), penicillin-streptomycin (50 units/ml), and fungizone (1.25 g/ml amphotericin B and 1.25g/ml sodium deoxycholate). Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of a health donor and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin-streptomycin, and fungizone.
Plasmid pOri-A contains an EcoRI-PstI fragment (nucleotides 22409 to 26491) of KSHV DNA in pBluescript at the EcoRI/PstI site as previously described (Lin CL, et al. 2003. J. Virol. 77:5578-5588). pCR3.1-ORF50 is an RTA expression vector described previously (Lin CL, et al. 2003. J. Virol. 77:5578-5588).
Chemicals and treatment of cells
The chemicals used in this study are from the Guangdong Small Molecule
Tangible Library (GSMTL) (Gu Q, et al. 2010. Molecules. 15:5031-5044). Compounds were dissolved in dimethyl sulfoxide (DMSO) and serially diluted before adding into BCBL-1, JSC-1 or BJAB cell cultures (both were 4 l05 cells/ml). The final DMSO concentration in culture medium was maintained at 1%. For lytic replication, BCBL-1 cells were induced by tetradecanoylphorbol acetate (TP A) as previously reported
(Gonzalez-Molleda L, et al. 2012. Antimicrob. Agents Chemother. 56:893-902). JSC-1 cells were induced by 3 mM sodium butyrate (Cannon JS, et al. 2000. J. Virol. 74: 10187- 10193). Three hours post-induction, compounds in dilutions were added to BCBL-1 or JSC-1 cells and subsequently antiviral effects and cytotoxicity were assayed at different time points.
Analysis of intracellular KSHV genomic DNA content and chemical effects TPA-induced and uninduced BCBL-1 cells were harvested at 48 hours post-induction and total DNA was purified using the DNeasy kit according to the manufacturer's protocol (Qiagen). KSHV genomic copy number was quantified by realtime PCR on a Roche LightCycler instrument using the LightCycler FastStart DNA Masterplus SYBR green kit with the primers for the detection of L ANA (forward, 5 '- CGCGAATACCGCTATGTACTCA-3'; SEQ ID NO: 1; reverse, 5'- GGAACGCGCCTCATACGA-3'; SEQ ID NO: 2). The intracellular viral genomic DNA in each sample was normalized to GAPDH using the primers directed to GAPDH (forward, 5'-ACATCATCCCTGCCTCTAC -3'; SEQ ID NO: 3; reverse, 5'- TCAAAGGTGGAGGAGTGG -3'; SEQ ID NO: 4).
The half maximal inhibitory concentration (IC50) values of compounds were determined from dose-response curve of KSHV DNA content values from TPA- induced and chemical-treated cells. The viral DNA contents were subtracted by those from uninduced cells, divided by that from the control cells of no drug treatment and then represented on the Y axes of dose-response curves: Y axis value = (TP AX - no
ΤΡΑχ)/(ΤΡΑο - no TPA0), where X is any concentration of the drug and 0 represents non-drug treatment. The IC50 on viral DNA synthesis for each compound was calculated with the aid of GraphPad Prism software.
Analysis of extracellular KSHV virion production and chemical effects Five days post-induction with TPA, BCBL-1 culture media were collected and extracellular virions were pelleted from the medium supernatant. To remove contaminating DNA outside viral particles, the concentrated viruses were treated with Turbo DNase I (Ambion) at 37°C for 1 h followed by proteinase K digestion. Virion DNA was extracted with phenol-chloroform, precipitated with ice-cold ethanol, and then dissolved in Tris-EDTA (TE) buffer. The KSHV genomic copy numbers were determined by real-time PCR, and values were corrected as described above. The half maximal antiviral effective concentration (EC50) values were calculated from dose- response curves with GraphPad Prism software.
Cytotoxicity assay.
The viabilities of BCBL-1 cells after treated or untreated with chemicals were assessed by counting Trypan blue-stained cells 2 or 5 days post-treatment using a light microscope. Cell viabilities were defined relative to control cells (non-drug treated). The half maximal cytotoxic concentration (CC50) was calculated from dose-response curves with Graphpad Prism software. Cell proliferation assay BCBL-1 and BJAB cells (both starting with 2 l05 cells/ml) were treated with (+)-Rutamarin for 5 days at two different concentrations: IC50 and 5xICso. The cells were stained by Trypan blue and counted every day for 5 days. To maintain the exponential growth, fresh medium (supplemented with or without the drug) was added to these cultures every 2 days.
Cell cycle assay
BCBL-1 and BJAB cells (both starting with 4 l05 cells/ml) were treated with (+)-Rutamarin and novobiocin at two different concentrations: IC50 and 5xIC5o. Two days post-treatment, cells were collected and fixed with cold 70% ethanol for 15 min, permeabilized, and stained with PI solution (50 g/ml of propidium iodide, 0.1 mg/ml of RNase A, and 0.05%> of Triton X-100) for 2 hours. Cell cycle progression was measured using a FACStar PLUS cell sorter flow cytometer (Becton Dickinson). ori-Lyt-dependent DNA replication assay
To assess the effect of (+)-Rutamarin on orz-Zyt-dependent DNA replication, BCBL-1 cells were cotransfected with plasmids pOri-A (2.5 μg) and pCR3.1- ORF50 (2.5 μg) by nucleoporation (Amaxa) and cultured with different concentrations of (+)-Rutamarin. 72 hours post-transfection, extrachromosomal DNA was prepared from cells using the Hirt DNA extraction method as previously described (Gonzalez -Molleda L, et al.. 2012. Antimicrob. Agents Chemother. 56:893-902). The extracted
extrachromosomal DNA was treated with RNase A at 25°C for 30 min, followed by proteinase K at 50°C for 30 min. Five μg DNA was digested with Kpnl/Sacl or
Kpnl/Sacl/Dpnl (New England Bio- Labs), separated on 1% agarose gel, and transferred onto GeneScreen membranes (Perkin Elmer, Boston, MA). The Southern blots were hybridized with a 32P-labeled pBluescript plasmid in 5><SSC, 2xDenhardt's solution, 1% SDS, and 50 μg/ml denatured salmon sperm DNA at 68°C.
Kinetoplast DNA decatenation assay
The decatenation assay is designed to measure type II topoisomerase activity and inhibition of the enzyme by testing compounds. The Kinetoplast DNA (kDNA, 200 ng) was incubated in 20 μΐ reaction containing 50 mM Tris-HCl (pH 7.5), 85 niM KC1, 10 mM MgCl2, 0.5 mM Na2EDTA, 0.5 mM dithiothreitol, 1 mM ATP, 30 μg/ml BSA, and 2 unit of Topo Πα or Topo Πβ for 30 min at 37°C in the absence or the presence of (+)-Rutamarin or control compounds (novobiocin and etoposide). The reactions were analyzed by electrophoresis on agarose gels. The decatenated kDNAs were measured with Gel Dox™ XR+ imaging system (BIO-RAD).
Malachite green assay for ATPase activity of Topo Ila
Malachite green reagent was prepared by mixing malachite green
(0.0812%, wt/vol), polyvinyl alcohol (2.32%, wt/vol), ammonium molybdate (5.72%, wt/vol, in 6M HC1), and ultrapure water in the ratio of 2: 1 : 1 :2. ATP hydrolysis reaction (20 μΐ) containing 50 mM Tris-HCl, pH 7.4, 85 mM KC1, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM Na2EDTA, 30 μg/mL BSA, 200 ng pBR322 DNA, 2 units Topo Ila, 500 μΜ ATP and varying concentrations of (+)-Rutamarin was initiated by adding ATP to the reaction and incubated at 37°C for 2 hours. The reaction was stopped by adding 80 μΐ of the malachite green reagent to the reaction, followed by adding 10 μΐ of 34% sodium citrate to develop blue-green color change. The absorbance at 620 nm was measured using a plate reader. The OD62o was used for representing the ATP hydrolysis level. Molecular docking study
The X-ray crystal structure of the ATPase domain of human Topo Ila in complex with ADPNP (PDB ID: 1ZXM, resolution = 1.87A) was retrieved from the Protein Data Bank (Wei H, et al. 2005. J. Biol. Chem. 280:37041-37047). Only chain A was kept for docking study. The missing loop (residues from 345 to 350) was added and optimized using Prepare Protein Module Encoded in Discovery Studio 3.5 (Accelrys Inc.) (Spassov VZ, et al. 2008. Protein Eng. Des. Sel. 21 :91-100). The initial structures of (+)- Rutamarin and ADPNP were optimized using MMFF force field (Cheng A, et al. 2000. J. Mol. Graph. Model. 18:273-282), and the Powell method was used for energy
minimization by default parameters in Discovery Studio 3.5.
The docking program FlexX encoded in SYBYL 7.3 (Tripos Inc.) was applied to identify the potential binding of (+)-Rutamarin to the Topo Ila ATPase domain. Previous study suggested that two conserved waters were not neglected to distinguish the active/moderate/inactive ligands (Baviskar AT, et al. 2011. J. Med. Chem. 54:5013-5030). In the present study, the receptor for docking simulation consists of protein chain A, two conserved water and Mg2+. To validate the molecular docking protocol, ADPNP was redocked into the crystal structure of the ATPase domain of Topo Πα. The docked ADPNP has similar binding pose with the cocrystallized ligand, with a root mean square deviation of about 1.6 A. The active sites were defined as all residues within 6.5 A radius of the bound ADPNP. Other FlexX parameters were set to default values. Thirty poses were retained during the docking process. After running FlexX, all the poses were visually inspected, and the most suitable docking pose was selected on the basis of score and interactions with key residues of the active site. The complex of Topo Πα and the most suitable docking conformer of (+)-Rutamarin was used as initial coordinates for the subsequent molecular dynamics. Molecular dynamics (MP) simulations
MD simulations were performed in AMBER 12 (Amaro RE, et al. 2011. Nat. Commun. 2:388. doi: 10.1038/ncommsl390) with the AMBER ff99SB force field (Hornak V, et al. 2006. Proteins. 65:712-725, Newhouse EI, et al. 2009. J. Am. Chem. Soc. 131 : 17430-17442). The docked structure of Topo Πα ATPase domain with (+)- Rutamarin was used as the initial coordinates for MD simulations. Parameters for (+)- Rutamarin was obtained from ANTECHAMBER module using the Generalized Amber Force Field (GAFF) (Zhang W, et al. 2003. J. Phys. Chem. B. 107:9071-9078) with RESP charge-fitting procedure with input from Hartree-Fock calculations at the 6-31G* level by using the Gaussian03 program (Frisch M J, et al. 2004. Gaussian 03, (Revision E.01). Gaussian Inc. Pittsburgh. PA). All hydrogen atoms of the protein were added using tleap module, considering ionizable residues set at their default protonation states at a neutral pH. The system was solvated in truncated octahedron box of TIP3P water molecules with margin distance of 10 A. Neutralizing counter ions were added to the simulation system. The detail MD simulations can be found in Supplementary Materials. The MM-PBSA and MM-GBSA methods (Kollman PA, et al. 2000. Acc. Chem. Res. 33:889-897) in the AMBER 12 Suite were used to calculate the binding free energies. MD simulations and Binding free energy analysis
The simulation consisted of energy minimization, heat phase, equilibration and production. During the first phase of the minimization, only hydrogen atoms were relaxed for 1000 steps, holding all other atoms restrained. The second minimization phase is that the protein backbone atoms were restrained and hydrogen atoms, water molecules and ions were relaxed with 1000 steps. In first two minimizations, a harmonic restraint of 5kcal mol-1 A-2 was applied. In the last minimization phase, all atoms were freely minimized with 2000 steps. In each minimization, a combined steepest descent and conjugate gradient minimization steps was equal. Subsequently, the system was linearly heated to 31 OK with lOOps NVT ensemble using a langevin thermostat, with a collision frequency of 5.0 ps-1, and harmonic restraints of 5kcal mol-lA-2 on the backbone atoms. Then, a further lOOps run at 310K was conducted in NPT ensemble with lkcal mol-lA-2, and pressure controlled using a Berendsen barostat (Berendsen et al, 1984, Journal of Chemical Physics, 81, 3684-3690) with a coupling constant of 1 ps and a target pressure of 1 atm. The system was again equilibrated to free simulation for 100 ps. Finally, a production simulation run for 10 ns was performed at 310K and fully unrestrained. All hydrogen atoms were constrained using the SHAKE algorithm
(Miyamoto et al, 1992, J Comput Chem, 13, 8952-8962) and the time step was set at 2fs. Long-range electrostatic interactions were included on every step using the Particle Mesh Ewald algorithm (Norberto de Souza et al, 1997 J Biomol Struct Dyn, 14, 607-611) with a 4th order B-spline interpolation, a grid spacing of < 1.OA, and a direct space cutoff of 10 A. Coordinate trajectories were recorded every 1 ps for the whole MD run.
For (+)-Rutamarin-bound system, binding free energy calculations was performed on 1000 snapshot structures extracted at 4ps intervals over the last 4 ns stable MD trajectory. For each snapshot structure, the binding free energy was calculated for both enzyme-inhibitor complexes through the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and generalized Born (MM-GBSA) methods Kollman et al, 2000 Accounts of Chemical Research, 33, 889-897; Swanson et al., 2004 Biophysical Journal, 86, 67-74). In the MM -PBS A and MM-GBSA approach an interaction free energy is defined as
AGbinding =Gcomplex-[Gprotein + Gligand] (1)
Where Gcomplex, Gprotein, and Gligand are the free energies of the complex, protein and the ligand, respectively. Each free energy term in eq 1 was computed as sum of the absolute free energy in the gas phase (Egas), the solvation free energy (Gsolvation), and the entropy term (TS), using eq 2:
G =Egas+ Gsolvation- TS (2)
Egas was expressed as the sum of changes in the van der Waals energy (Evdw), electrostatic energy (Eele), and the internal energies (Eint) in the gas phase (eq 3). Eint is the energy associated with vibration of covalent bonds and bond angels, rotation of single bond torsional angels (eq 4)
Egas = Eint+Evdw+ Eele (3)
Eint =Ebond+Gangel +Etorsion (4)
The solvation free energy, Gsolvation, is approximated as the sum of the polar contribution (GPB/GB) and nonpolar contribution (Gnonpolar) using continuum solvent methods:
Gsolvation= GPB/GB + Gnonpolar (5)
Gnonpolar= y SASA + b (6)
The polar contribution (GPB/GB) to the solvation energy was calculated either using the PB and GB model implemented in AMBER 12. The grid size used is 0.5 A. The dielectric constant was set to 1 for interior solute and 80 for exterior water. The nonpolar contributions (Gnonpolar) were estimated using eq6, where SASA is the solvent- accessible surface area that was estimated using the linear combination of pairwise overlaps (LCPO) (Weiser et al, 1999 J Comput Chem, 20, 217-230); the probe radius of 1.4 A, γ= 0.0072 kcal · mol-1 · A-2, and b=0 kcal/mol (eq 6).
The calculation of the entropic contribution is computationally expensive and omitted in this study because it requires extremely well minimized structures for a normal-mode analysis or large numbers of snapshots for a quasi-harmonic analysis (Perez et al, 2011 Journal of Physical Chemistry, 115, 15339-15354). Furthermore, the binding free energy decomposition was performed on a per-residue basis using the molecular mechanics generalized Born/surface area (MM-GBSA) method (Tsui et al., 2000
Biopolymers, 56, 275-291; Rastelli et al, 2010 J Comput Chem, 31, 797-810; Zoete et al, 2010 Journal of Molecular Recognition, 23, 142-152). This decomposition was carried out only for molecular mechanics and solvation energies but not for entropies.
The results of the experiments are now described.
(+)-Rutamarin inhibits the lytic replication of SHV
To search for novel Topo II inhibitor(s) that effectively block KSHV lytic DNA replication, a ligand based virtual screening using an in-house 3D molecular superimposing algorithm WEGA (Yan X, et al. 2013. Journal of Chemical Information and Modeling. 53: 1967-1978) was conducted against the molecular structures in the compound repository, Guangdong Small Molecule Tangible Library (GSMTL) (Gu Q, et al. 2010. Molecules. 15:5031-5044). Using novobiocin structure as template, approximate 7200 compounds from the GSMTL were screened. Top thirty-three compounds, which are most three-dimensionally similar to novobiocin, were selected and subsequently tested for antiviral activities. Exponentially growing BCBL-1 cells were induced into lytic replication with TPA. Three hours after induction, the cells were exposed to each of the compounds as well as novobiocin at a concentration of 20 μΜ. Forty-eight hours post-induction, KSHV genomic DNA content in the cells treated with these compounds were analyzed by quantitative real time PCR. Treatment with novobiocin resulted in 42% inhibition of viral replication compared to the control cells treated with no drug. Seven compounds showed similar or better inhibitory effects in comparison to novobiocin on viral DNA replication (Fig 1A). Four of these, C28, C31, C32 and C33, also displayed strong activities in blocking virion production (Fig. IB). With the exception of C31 , the other three compounds possess significant cytotoxicity as demonstrated by low cell viability rates with the treatment (Fig. 1C). C31, which is identified as the natural product (+)-Rutamarin (Fig. 2A), exhibited significant inhibitions of both viral DNA synthesis and virion production with relatively low cytotoxicity at the concentration of 20 μΜ. Therefore it was chosen for further investigation. The half maximal inhibitory concentration (IC50) values of (+)-Rutamarin were determined from dose-response curve of KSHV DNA content in TPA-induced BCBL-1 cells and found to be 1.12 μΜ (Fig 2B). The effect of (+)-Rutamarin on progeny virion production was also determined by quantification of encapsidated viral DNA in BCBL-1 culture media. The half maximal antiviral effective concentration (EC50) calculated from dose-response curve of extracellular virion is 1.62 μΜ (Fig 2B).
Cytotoxicity of (+)-Rutamarin on BCBL-1 cells was examined in parallel to inhibition of KSHV DNA replication and virion production. Cells treated with (+)-Rutamarin at differing concentrations were subjected to the trypan blue exclusion method to determine cell viability in response to (+)-Rutamarin treatment. The half maximal cytotoxic concentration (CC50) was determined to be 94.24 μΜ (Fig 2B). Low cytotoxicity of (+)- Rutamarin results in an appreciable selectivity index (SI = CC50/IC50) of 84.14.
To make sure that (+)-Rutamarin inhibits KSHV lytic replication regardless of host cells and means of lytic cycle induction, experiments were designed to test the effect of (+)-Rutamarin on KSHV replication in JSC-1 cells induced by sodium butyrate for lytic replication. The IC50, EC50 and CC50 of (+)-Rutamarin on butyrate- induced JSC-1 cells were found to be 2.29 μΜ, 1.40 μΜ and 94.34 μΜ, respectively, that are very similar to those obtained with BCBL-1. In addition, the cytotoxicity of (+)- Rutamarin to primary lymphocytes was also assessed with peripheral blood mononuclear cells (PBMC). The CC50 to PBMC was calculated to be 60.91 μΜ (Fig. 10).
Evaluation of (+)-Rutamarin for its effect on host cell proliferation and cell cycle progression
To further investigate the potential of (+)-Rutamarin to become an effective and safe antiviral, the effect of the compound on cell proliferation was assessed. KSHV-carrying BCBL-1 and virus-free BJAB cells were cultured in the presence and the absence of (+)-Rutamarin and live cell numbers were counted over 5 days. (+)-Rutamarin did not exhibit cell growth inhibitory effect in both cell lines at the IC50 concentration. At an excess concentration (5 x IC50), (+)-Rutamarin showed an adverse effect on cell growth but inhibition extent was less than that of novobioicin (Fig. 3). The effect of (+)- Rutamarin on cell cycle progression was also examined. BCBL-1 cells treated with (+)- Rutamarin were stained with propidium iodide and subject to flow cytometric analysis. At the IC50 concentration, no effect was observed on cell cycle pattern with either (+)- Rutamarin or novobiocin in comparison with vehicle control. At an excess concentration (5 x IC50) of (+)-Rutamarin, a slight increase in the S phase was observed in comparison to untreated cells. However, the influence of (+)-Rutamarin on cell cycle progression was observed to be less than that of novobiocin (Fig. 4). Overall, these results demonstrated that (+)-Rutamarin has minimal effects on host cell proliferation and cell cycle progression in its pharmaceutical dose range. (+)-Rutamarin inhibits SHV lytic replication by blocking viral ori-L vt-dependent DNA replication
It was demonstrated that (+)-Rutamarin is capable of inhibiting KSHV DNA synthesis and virion production. To confirm if the inhibitory effect of (+)- Rutamarin on KSHV lytic replication is directly due to the blocking of on-Zyt-dependent DNA replication, an ori-Lyt DNA replication assay was conducted. BCBL-1 cells were cotransfected with an on-Zyt-containing plasmid (pOri-A) and an RTA expression vector. Lytic DNA replication was induced by RTA expression (Sun R, et al. 1998. Proc. Natl. Acad. Sci. U. S. A. 95:10866-10871). The transfected cells were cultured in the presence of (+)-Rutamarin at varying concentrations and the effect of (+)-Rutamarin on orz-Zyt-dependent DNA replication was measured by a Dpn I assay (Lin CL, et al. 2003. J. Virol. 77:5578-5588, Wang Y, et al. 2004. J. Virol. 78:8615-8629). In brief, DNA was isolated from the treated cells 72 h post-transfection and digested with Kpnl/Sacl and Kpnl/Sacl/Dpnl. Replicated plasmid DNA was distinguished from input plasmid by Dpnl restriction digest, which cleaves input DNA that has been dam+ methylated in
Escherichia coli but leaves intact the DNA that has been replicated at least one round in eukaryotic cells. Thus, only newly replicated plasmid DNA in BCBL-1 cells, which is resistant to Dpn I digestion, can be detected in Southern blot analysis. Replicated pOri-A DNA was detected as a Dpn I resistant DNA band in the cells that were cotransfected with pOri-A and RTA expression vector, However, the replicated DNA decreased and diminished with elevated concentration of (+)-Rutamarin in the cell culture (Fig. 5). This result supports that (+)-Rutamarin inhibits KSHV ori-Lyt-dependent DNA replication. To rule out the possibility that (+)-Rutamarin blocks KSHV DNA replication through inhibiting RTA expression, the effect of (+)-Rutamarin on RTA expression in TPA-induced BCBL-1 cells was examined by Western analysis. The result showed no adverse effect of this compound on RTA expression up to 25 μΜ (22 times excess over IC50) (Fig. 11). The reduced level of RTA in the treatment with 50 and 100 μΜ of (+)-Rutamarin may result from the cytotoxicity of the compound in the concentration near its IC50 (94.24 μΜ).
(+)-Rutamarin is an inhibitor of human topoisomerase ΙΙα.
Experiments were designed to determine whether (+)-Rutamarin is a Topo
II inhibitor that blocks KSHV on-Zyt-dependent DNA replication through inhibiting catalytic activity of host cell Topo II. Furthermore, there are two Topo II isoforms in mammals, Topo Πα and Topo Πβ. In order to determine which Topo II isoform (+)- Rutamarin affected, a DNA decatenation-based topoisomerase II assay was established. In this system, kinetoplast DNA consisting of a large network of interlocked DNA minicircles can be decatenated into separate minicircles in the presence of ATP and Topo II of either isoform. (+)-Rutamarin in a wide range of concentration was added in the reaction and the effect of the compound on Topo II activity was measured by determining decatenated kDNA using agarose gel electrophoresis. Results showed that (+)-Rutamarin is able to inhibit the enzymatic activity of Topo Πα in a dose-dependent manner (Fig. 6A and 6B). The IC50 of (+)-Rutamarin on Topo Πα inhibition was calculated as 28.22±6.2 μΜ (Fig. 6C and 6D). At the concentration of 100 μΜ, the inhibition rate of (+)- Rutamarin is significantly higher than novobiocin and close to Topo II poison etoposide (Fig. 6A). In contrast, (+)-Rutamarin did not exhibit any inhibitory effect on Topo Πβ activity (Fig. 6E).
To confirm if (+)-Rutamarin is a catalytic inhibitor of Topo Πα and acts in blocking ATPase activity of the enzyme as novobiocin does, the inhibition effect of Rutamarin on ATP hydrolysis of Topo Πα was studied using the malachite green assay. As shown in Fig. 7, the APTase activity of Topo Πα was inhibited by (+)-Rutamarin in a dose-dependent manner, suggesting that, like novobiocin, (+)-Rutamarin inhibits human Topo Πα by binding to the ATP pocket of the enzyme and blocking its ATP hydrolysis. Binding model of (+)-Rutamarin in ATP binding pocket of Topo Πα
The mode of (+)-Rutamarin binding in the ATPase domain of Topo Πα was investigated by docking and validated by MD simulations. Fig. 8A illustrates the time dependence of the RMSD values for X-ray reference enzyme structure of backbone atoms (C, CD, N, and O) over the production phase of simulation. The RMSD values of simulation converged after ~3ns, indicating that the system is stable and equilibrated. The RMSD value of ligand compared docking pose is swinging within 1.8 A, which illustrates that the docking pose is reliable. Previous studies indicated that Mg2+ is necessary for ATPase domain to hydro lyze ATP molecule, and is stabilized by two conserved water molecules and residues Asn 91 and Glu87 (Wei H, et al. 2005. J. Biol. Chem. 280:37041- 37047, Baviskar AT, et al. 2011. J. Med. Chem. 54:5013-5030). The time evolution of oxygen- and nitrogen-Mg2+ distances for these residues and the water molecules are shown in Fig. 8C. The oxygen- and nitrogen-Mg2+ distances for these residues and the water molecules stabilized at 1.9-2.0 A during the simulation, suggesting that Mg2+ is stabilized by these factors. The simulation results presented herein are consistent with previous experiment result, suggesting that the model is reliable and reasonable.
The details of binding of (+)-Rutamarin to the ATPase domain of Topo Πα at an atomic level were revealed in a conformation clustering analysis. The results suggest that (+)-Rutamarin forms a hydrogen bond with the side chain of Asn95, Ala 167 and conserved water 2, respectively, and hydrophobically interacts with He 125, Ilel41, Phel42, Alal67, Thr215, Glyl66 and Lysl23 in the catalytic site of Topo Πα (Fig. 8D). The distance distributions analysis also showed that the hydrogen bonds between conserved water 2- and Alal67-(+)-Rutamarin are comparatively stable, whereas the hydrogen bond of Asn95-(+)-Rutamarin is unstable (Fig. 8B). Solvent accessible surface area (SASA) analysis suggests that the polar surface of (+)-Rutamarin is complementary with the polar surface of the binding pocket of Topo Πα (Fig. 8E). Superimposition of (+)-Rutamarin and cocrystallized ADPNP suggests that (+)-Rutamarin occupies the ATP binding pocket in a similar fashion as an ATP molecule (Fig. 8F). The acetyl groups of (+)-Rutamarin, like the three phosphate groups of ATP, involves in interaction with the highly conserved Walker A consensus motif GXXGXG (SEQ ID NO. 5) at the residues 161-166 of Topo Πα (Fig. 8D and 8F) (Walker JE, et al. 1982. EMBO J. 1 :945-951). This conserved motif is important for the binding of ATP as well as for the catalytic inhibitors acting on ATPase domain (Baviskar AT, et al. 201 1. J. Med. Chem. 54:5013- 5030). This notion is supported by a site-directed mutagenesis result that the mutations in Argl62Gln and Tyrl65Ser in the ATP binding site lead to drug resistance (Wessel I, et al. 1999. Cancer Res. 59:3442-3450, Wessel I, et al. 2002. FEBS Lett. 520: 161-166). These observations, in consistent to the results of the DNA decatenation assay and the malachite green assay, support that (+)-Rutamarin functions as a Topo Πα catalytic inhibitor.
Molecular mechanics Poisson-Boltzmann surface area (MM -PBS A) and mechanics generalized Born Surface area (MM-GBSA) methods were employed to calculate the binding free energies and to gain information on the different components of interaction energy that contribute to (+)-Rutamarin binding. The results are listed in Table 2. AGMM PBSA and AGMM GBSA for Topo IIa-(+)-Rutamarin complex are -29.90 and -24.66 kcal/mol, respectively. AGMM-PBSA, accounting for total relative binding free energy equal to AGgas + AGsoiv, comes mainly from van der Waals component, whereas electrostatic contribution is very less. This notion is also reflected by the results of MM- GBSA method (Table 1), that shows that (+)-Rutamarin is a comparatively hydrophobic molecule and can form favorable hydrophobic interacts with residues of Topo Πα catalytic pocket.
Table 2. Binding free energy for Topo Πα- (+)-Rutamarin complex along with its different energy components based on MM-PBSA and MM-GBSA method.
Energy ter s MM-PBSA* MM-GBSA"
-40.12 (2.14) -40.22 (2.26)
-0.35 (0.22) -0.42 (0.15)
39.99 (1.93) 22.09 (1.66)
,\ F ■ . -29.43 0.37) -6.11 (0.34)
-40.47 (2.41) -40.64 (2.2S)
10.56 (1.28) 15,98 (1.14)
-29.91 (138) -24.66 (1.69) All energies are in kcal/mol. a Molecular mechanics Poisson-Boltzmann surface area (MMPBSA); b Molecular mechanics generalized Born Surface area (MM-GBSA); c Non-bonded van der Waals; d Non-bonded electrostatics; e Polar component to solvation; f Non-polar component to solvation; g total gas phase energy; h sum of nonpolar and polar contributions to solvation; i final estimated binding free energy calculated from the terms above. Standard errors of the mean are given in parentheses.
(+)-Rutamarin as an effective antiviral agent that inhibits SHV ori-L vt-dependent DNA replication with low cytotoxicity
In the current study, (+)-Rutamarin was identified as an effective antiviral agent that inhibits KSHV on-Zyt-dependent DNA replication with low cytotoxicity. It blocks KSHV DNA replication through inhibiting catalytic activity of human Topo Πα. The salient features and implications of this finding are as follows. There is a need for effective drugs targeting KSHV
KSHV has been proven to be the etiological cause of KS and other KSHV-associated malignancies. However, the current treatment modalities for KSHV- associated diseases include only traditional cancer therapies. For classic KS, the chemotherapeutics that have been approved by the FDA include liposomal anthracycline products (liposomal doxorubicin or liposomal daunorubicin), paclitaxel and interferon- alpha (Potthoff A, et al. 2007. J. Dtsch. Dermatol. Ges. 5: 1091-1094). However, the majority of these agents have serious side effects and the tumor responses to these chemotherapeutic regimens are only transient. In the current AIDS epidemic, even though KS has become a major AIDS-associated malignancy and remains an important cause of morbidity and mortality in AIDS patients, little effort has been made to develop more effective drugs for KS, as there was a general belief that AIDS-associated-KS will be disappeared if AIDS were under control with the advent of highly active antiretroviral therapy (HAART). However, despite its dramatic decrease in frequency since the advent of HAART, KS remains the most common AIDS-associated cancer (Boshoff C, et al. 2002. Nat. Rev. Cancer. 2:373-382). In addition, as experience with HAART has grown, a new HAART-associated syndrome emerged. In a subset of HIV-seropositive individuals, starting HAART in the setting of advanced HIV infection results in a paradoxical clinical worsening of existing infection or the appearance of a new condition including KS in a process known as immune reconstitution inflammatory syndrome (IRIS) (Shelburne SA, 3rd, et al. 2003. AIDS Rev. 5:67-79). IRIS is thought to be the direct result of a reconstituted immune system recognizing pathogens or antigens that were previously present, but clinically asymptomatic. The current regimen for IRIS- associated KS is a combination therapy with HAART plus chemotherapeutics such as liposomal doxorubicin. Since IRIS-KS is the result of responses by a recovered immune system to KS-causing pathogen, i.e. KSHV, treatment of KSHV-seropositive, HIV- positive patients with a combination of antiretroviral (HAART) and anti-KSHV therapeutics is expected to yield positive results. However, at this time there are no effective drugs targeting KSHV available. Earlier studies on cohorts of HIV-seropositive subjects suggested that treatment with anti-herpesviral drugs ganciclovir or foscarnet could reduce the incidence of KS in AIDS patients (Glesby MJ, et al. 1996. J Infect Dis. 173: 1477-1480, Mocroft A, et al. 1996. AIDS. 10: 1101-1105). However, severe side effects (renal impairment and bone marrow suppression) as well as rapid development of drug resistance have limited the use of these drugs in the treatment of KS. Therefore, there is a need for effective drugs targeting KSHV.
( +)-Rutamarin is an antiviral drug candidate for KSHV-associated diseases
It has been reported that host cellular Topo II is required for KSHV DNA replication and thus Topo II inhibitors effectively block KSHV DNA synthesis
(Gonzalez-Molleda L, et al. 2012. Antimicrob. Agents Chemother. 56:893-902). Topo II inhibitors are divided into two categories: Topo II poisons, which target the
topoisomerase-DNA intermediate (cleavable complex), and Topo II catalytic inhibitors, which disrupt catalytic turnover of the enzyme. Although both categories possess inhibitory activities on KSHV DNA replication, Topo II poisions in general exhibit very strong cytotoxicities to host cells. In contrast, Topo II catalytic inhibitors show less cytotoxicity (such as novobiocin with a CC50 values of 871 μΜ and merbarone of 212.9 μΜ to BCBL-1 cells). Both novobiocin and merbarone are effective in halting KSHV DNA synthesis with IC50 of 27.55 μΜ and 19.54 μΜ, respectively. The low
cytotoxicities and high inhibition rates for viral replication of catalytic Topo II inhibitors suggest potentials of Topo II catalytic inhibitors in becoming effective anti-KSHV drugs for treatment of KS and other KSHV-associated diseases (Gonzalez-Molleda L, et al.. 2012. Antimicrob. Agents Chemother. 56:893-902). In addition, given that viruses have tendencies to mutate their genome and therefore develop drug resistance, targeting host cellular proteins that viruses rely on for their replication offers the advantage of minimizing drug resistance, and hence constitutes a novel therapeutic strategy.
In the present study, new Topo II catalytic inhibitors that are potent in inhibiting viral replication and low cytotoxic were screened. This effort led to
identification of (+)-Rutamarin as a lead which efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with the IC50 of 1.12 μΜ and EC50 of 1.62 μΜ, 25- and 17- fold lower than those of novobiocin. In addition, (+)-Rutamarin exhibits very low cytotoxicity and the selective index (SI) on KSHV lytic replication was calculated as 84.14, three times better that that of novobiocin. In addition, the molecular weight of (+)- Rutamarin (MW=356.42) is smaller than that of novobiocin (MW=634.61), the mass concentration differences of their IC50 and EC50 would be greater. These data
demonstrate a great potential of (+)-Rutamarin to become an effective antiviral agent with low cytotoxicity.
(+)-Rutamarin is a natural product found in plants such as ruta graveolens L (common rue). It was reported that (+)-Rutamarin possesses anti-proliferation and anti- cancer activities against a variety of tumor cell lines (Yang QY, et al. 2007. J. Asian Nat. Prod. Res. 9:59-65). It is also found to induce the expression and translocation of glucose transporter 4 (GLUT4). As impaired translocation or decreased expression of GLUT4 in response to insulin is one of the major pathological features of type 2 diabetes, the function of (+)-Rutamarin in ameliorating glucose homeostatsis suggests a potential of this compound in anti-type 2 diabetes drug development (Zhang Y, et al. 2012. PLoS
One. 7:e31811). This study is the first one to demonstrate the capability of this compound in inhibition of viral replication with a potential to be an antiviral.
Anti-proliferation potential o f f+)-Rutamarin
At the dose of IC50 for inhibiting KSHV replication, (+)-Rutamarin did not show perceptible effect on cell proliferation and cell cycle progression in BCLB-1 and BJAB cells. At an excess dose (5xIC50,), (+)-Rutamarin exhibited a degree of suppression on cell proliferation with a delay in the S phase.
The effect of (+)-Rutamarin on cell proliferation in a variety of tumor cell lines was estimated and inhibitory activities against A549, Bel7402, HepG2, HCT8 cell proliferation were reported (Yang QY, et al. 2007. J. Asian Nat. Prod. Res. 9:59-65). Without wishing to be bound by any particular theory, although (+)-Rutamarin has an extent of antiproliferative property and may provide an additional benefit if it becomes a drug to treat KSHV-associated malignancies, the moderate anti-proliferation property may not be potent enough to provide anti-tumor activities. It may also be true for all Topo II catalytic inhibitors. This notion is supported by studies and observations on some Topo II catalytic inhibitor including novobiocin and merbarone, which exhibit anti- proliferation activities in culture cells but show no significant effects on tumor development in clinic trials (Eder JP, et al. 1991. Cancer Res. 51 :510-513, Chang AY, et al. 1993. J. Natl. Cancer Inst. 85:388-394).
The moderate effect of (+)-Rutamarin on cell proliferation, as well as its low cytotoxicity, may result from redundancy of Topo II activities in mammalian cells. Mammals express two isoforms of Topo II, alpha and beta, which are highly homologous but display differences in expression and subcellular localization in the time of mitosis. The similar function and domain structure of the two isoforms make it possible that Topo Πα may be able to partially compensate the function loss of Topo Πα for cell survival in the presence of an inhibitor such as (+)-Rutamarin. However, KSHV may be dependent on only Topo Πα for its lytic replication, therefore explaining the sensitivity of KSHV replication to Topo Πα catalytic inhibitors. ( +)-Rutamarin is a novel human Topo Ila catalytic inhibitor
Topo II is an ATPase and uses the energy derived from ATP hydrolysis to resolve the winding problem of double-stranded DNA. The detailed binding mode of (+)- Rutamarin and human Topo Ila was identified using molecular docking and MD simulation. The significance of these studies is two-fold. First, the study provided insight into the binding of (+)-Rutamarin to Topo Ila and results confirmed that this compound binds to the ATP pocket of Topo Ila. Second, the result of the study help in designing modification to the compound for more potent antiviral candidates. Energy
decomposition analysis led to identification of key residues contributing to binding affinity at the active site. As shown in Fig. 9, the major contributing residues can be divided into three clusters, which are consistent with the three binding pockets sites, i.e. (i) Walker A consensus motif (residues 161-167), (ii) Mg2+ binding area (residues 87, 91 and 94), and (iii) hydrophobic chamber (key residues, He 125, Ilel41, Phel42, Lysl23 and Thr215). All of these residues except Glu87 positively contribute to the binding affinity for (+)-Rutamarin. The incompatibility between the carboxyl group of Glu87 and the acetyl groups of (+)-Rutamarin suggests that Glu87 is unfavorable for the binding affinity to (+)-Rutamarin (Fig. 8D). These results provide a theory basis for (+)- Rutamarin lead optimization.
Example 2: Antiviral activity of topoisomerase II catalytic inhibitors against Epstein-Barr virus
Herpesviruses require several cellular proteins for their lytic DNA replication including topoisomerase II (Topo II). Thus, Topo II could be an effective drug target against herpesviral infection. In this study, several Topo II catalytic inhibitors were evaluated for their potentials in blocking EBV replication and becoming efficacious antiviral agents. Topo II catalytic inhibitors in general exhibited marked inhibition of EBV lytic replication and minimal cytotoxicity. In particular, (+)-rutamarin, with the best selectivity index (SI > 63) among the inhibitors tested in this study, is effective in inhibiting EBV DNA replication and virion production but shows little adverse effect on cell proliferation, suggesting its potential to become an efficacious and safe drug for the treatment of human diseases associated with EBV infection.
The materials and methods employed in these experiments are now described.
Materials and Methods
Cells and Plasmids
The P3HR-1 is a EBV -positive Burkitt lymphoma cell line, a clonally derived subline of Jiyoye (Hinuma et al, 1967, J. Virol, 1 :1045-1051). Akata-Bxl cell line carries a recombinant EBV where the thymidine kinase gene was replaced by a CMV immediate early promoter driven GFP (Guerreiro-Cacais et al., 2007, J. Virol., 81 : 1390- 1400). Cells were cultured in RPMI 1640 medium (Gibico-BRL, Gaithersburg, MD), supplemented with 10% FBS (Gibico-BRL), penicillin (100 U/ml), streptomycin
(100 μ^ιηΐ).
The EBV ori-Lyt plasmid pEBV-oriL was constructed by cloning a 1434 bp fragment carrying EBV ori-Lyt (nucleotides 52385-53819 of EBV genome) into pUC18 vector. The plasmid McZ is a ZTA expression vector in the backbone of pBXGl . Chemicals and cell treatment
Novobiocin was purchased from Sigma-Aldrich (St. Louis, MO) and merbarone was purchased from Merck Millipore. (+)-Rutamarin (Figure 12) is extracted from natural plant Ruta graveolens L, supplied by the Guangdong Small Molecule Tangible Library (GSMTL) (Gu et al, 2010, Molecules, 15:5031-5044). Novobiocin was prepared as aqueous stock, while the others were dissolved in dimethyl sulfoxide (DMSO).
For induction of EBV lytic replication, P3HR-1 cells (4 x 105 cells/ml of culture) were treated with 20 ng/ml 12-O-tetradecanoylphorbol 13 -acetate (TP A; Sigma- Aldrich) and 3 mM sodium butyrate (Sigma-Aldrich). Novobiocin, merbarone, and (+)- rutamarin in different concentrations was added to P3HR-1 cells three hours after induction. Akata-Bxl cells were resuspended at 2 x 106/ml in fresh medium and treated with 0.8%(V/V) goat anti-human IgG (Jackson ImmunoResearch). Three hours post- induction, Akata-Bxl cells were adjusted to the density of 4 x 105 cells/ml and treated with various concentrations of novobiocin, merbarone, and (+)-rutamarin. The effects of these inhibitors on viral DNA content and virion production were assayed at different time points.
Analysis of intracellular EBV genomic DNA content and chemical effects Two days post-treatment, cells were collected from induced and uninduced cultures and total DNA was purified from the cells using the TaKaRa
MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa). EBV genomic DNA was quantified by real-time PCR on a Roche Light Cycler II instrument using the Lightcycler 480 SYBR green I Master kit with primers directed to EBNA1 (forward, 5'- CATTGAGTCGTCTCCCCTTTGGAAT-3 ', SEQ ID NO:6; reverse, 5'- TC AT AAC AAGGTCCTT AATCGC ATC-3 ', SEQ ID NO:7). The intracellular viral genomic DNA in each sample was normalized with the amount of GAPDH determined also by real-time DNA PCR by using primers directed to GAPDH (forward, 5'- AGCCACATCGCTCAGACAC-3', SEQ ID NO:8; reverse, 5'- GCCCAATACGACCAAATCC-3', SEQ ID NO:9). EBV DNA content value from induced cells was subtracted by that from uninduced cells. These corrected values were divided by those from the control, non-drug treatment and then represented on the y axes of dose-response curves: y axis
value = (INDUCE^ - NONINDUCE )/(INDUCEo - NONINDUCE0), where INDUCE means TPA + Butyrate treatment or anti-IgG induction, is any concentration of the drug and 0 represents non-treatment. The 50% DNA replication inhibitory concentration (IC50) for each compound was calculated from the dose-response curve with the aid of Graphpad Prism software.
Analysis of extracellular EBV virion production and chemical effects Five days post-induction, cell culture media were collected and virion particles were cleared by passing through 0.45 um filters and extracellular virions were then pelleted from the medium supernatant of the cultures as detailed earlier (Gonzalez- Molleda et al., 2012, Antimicrob. Agents Chemother., 56:893-902) and were
resuspended in l x phosphate -buffered saline (PBS) in 1/100 of the original volume. To remove any contaminating DNA outside viral particles, the concentrated viruses were treated with DNase I (TaKaRa) at 37 °C for 1 h followed by proteinase K digestion. The amounts of virion particles from the media were determined by quantifying encapsidated viral DNA by real-time PCR, and values were corrected as described above. The 50% antiviral effective concentration (EC50) for each compound was calculated from the dose- response curve with the aid of GraphPad Prism software.
Cytotoxicity assay The cell viabilities of P3HR-1 and Akata-Bxl cells after treated or untreated with chemicals were assessed by counting Trypan blue stained cells 2 or 5 days post treatment using a light microscope. Cell viabilities were defined relative to control cells (non-drug treated) and represented on the y axes of dose-response curves: y axis value = NONIDUCE NONINDUCE0, where is any drug concentration and 0 represents non-treatment. The 50% cytotoxic concentration (CC50) for each compound was calculated from these dose-response curves with the aid of Graphpad Prism software. Cell proliferation assay
P3HR-1 cells (starting with 2 x 105 cells/ml) were treated with topoisomerase inhibitors for 5 days at two different concentrations: IC50 and an excess concentration (5 x IC50). Cell samples were daily collected, stained with Trypan blue, and counted. To provide a constant cellular growth, fresh medium (supplemented with or without the drug) was added to these cultures every 2 days.
Transient-transfection DNA replication assay
To assay the effect of each compound on ori-Lyt-dependent DNA replication, P3HR-1 cells (1 x 107) were transfected with plasmids pEBV-oriL (2.5 μg) and McZ (2.5 μg) by nucleoporation (Amaxa) and cultured in the media with each drug in a wide range of concentration. Seventy-two hours post-transfection, extrachromosomal DNAs were prepared from cells using the Hirt DNA extraction method (Hirt, 1967, J. Mol. Biol, 26:365-369). Cells were lysed in 700 μΐ lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM EDTA, and 0.6% SDS). Chromosomal DNA was precipitated at 4 °C overnight by adding 5 M NaCl to a final concentration of 0.85 M. Cell lysates were centrifuged at 4 °C at 14,000 rpm for 30 min. The supernatant containing
extrachromosomal DNA was subjected to phenol-chloroform extraction, followed by ethanol precipitation. The DNA was treated with RNase A at 25 °C for 30 min and then with proteinase K at 50 °C for 30 min. Twelve micrograms of DNA was digested with EcoR I or EcoR I/Dpnl (TaKaRa). The DNAs were separated by electrophoresis on 0.9% agarose gels and transferred onto GeneScreen PLUS membranes (Perkin Elmer, Boston, MA).
Southern blot was performed according to an optimized protocol using the DIG high prime DNA labeling and detection starter kit I (Roche). Probes were prepared by excising insert DNA from pEBV-oriL plasmid by restriction digestion and labeled with digoxigenin by random primed DNA synthesis with digoxigenin-dUTP. The membranes were prehybridization in 10 ml of the DIG Easy Hybridization solution, with 50 mg/ml denatured salmon sperm DNA for 4 h at 68 °C. Then, DIG labeled probe was added and hybridization was carried for overnight. The membranes were washed three times for 15 min at 65 °C with washing buffer (2* SSC, 0.1% SDS). Subsequently the membranes were blocked for 1 h and incubated in anti-DIG-AP solution for 1 h and then incubated in 10 ml color substrate solution in the dark.
The results of the experiments are now described.
Evaluation of Topo II catalytic inhibitors for their effects on EBV DNA replication and virion production
It has been demonstrated elsewhere herein that Topo II catalytic inhibitors, including novobiocin and merbarone, have potent antiviral activity halting KSHV DNA replication and virion production (Gonzalez-Molleda et al., 2012, Antimicrob. Agents
Chemother, 56:893-902). In addition, a ligand-based viral screen using novobiocin as the template and an in-house 3D molecular superimposing algorithm led to identify a novel Topo II catalytic inhibitor, namely (+)-rutamarin. The study demonstrated that (+)- rutamarin is more potent than novobiocin in blocking KSHV DNA replication and virion production (Xu et al., 2014, Antimicrob. Agents Chemother, 58:563-573). Given that EBV and KSHV share a similar lytic DNA replication mechanism and both require host Topo II for their DNA replication (Kawanishi, 1993, J. Gen. Virol, 74:2263-2268, Wang et al, 2008, J. Virol, 82:2867-2882, and Wang et al, 2009, J. Virol, 83:8090-8098), experiments were designed to explore if these Topo II catalytic inhibitors can effectively block EBV lytic replication and have any potential to become antiviral drugs against EBV. P3HR-1 cells that are latently infected by EBV were induced with TPA and butyrate for lytic viral replication. Three hours after induction, the cells were treated with various concentrations of novobiocin, merbarone, and (+)-rutamarin. The effects of these inhibitors on the viral DNA synthesis and on virion production were examined 2 days and 5 days post-induction, respectively. All three inhibitors were found to effective block EBV DNA synthesis as well as virion production (Figure 13A-13C and Table 3 A). Among them, (+)-rutamarin revealed the highest potency in halting EBV DNA replication with a half-maximal inhibitory concentration (IC50) of 3.78 μΜ and in blocking virion production with a half-maximal antiviral effective concentration (EC50) of 5.40 μΜ (Figure 13C and Table 3 A).
Table 1 : Antiviral activities of Topo II inhibitors and their associated cytotoxicitiesa in P3HR-1 cells induced with TPA/butyrate and Akata-Bxl cells induced with Anti-IgG.
Figure imgf000082_0001
aIC5o represents the half maximal inhibitory concentration for EBV DNA replication. EC50 denotes the half maximal effective concentration for blocking EBV virion production. CC50 refers to the concentration of the compound that causes 50% cell death after specific exposure time. They were determined by nonlinear regression analysis of dose response curves and are expressed as mean values of results from at least three independent experiments. All these parameters are presented in μΜ units. R2: correlation coefficient.
b Cytotoxicity was measured after 2 days of drug treatment.
c SI (selectivity index) was calculated as the ratio of CC50/IC50.
d Cytotoxicity was measured after 5 days of drug treatment.
e NA means not available. To make sure that novobiocin, merbarone and (+)-rutamarin inhibit EBV lytic replication regardless of host cells and means of lytic cycle induction, experiments were designed to test the effect of these inhibitors on EBV replication in Akata-Bxl cells induced by anti-human IgG for lytic replication (Daibata et al., 1990, J. Immunol., 144:4788-4793). The inhibition of EBV lytic DNA replication and virion production by these three Topo II inhibitors in Akata-Bxl cells are very similar to the results obtained with P3HR-1 cells. In particular, (+)-rutamarin again exhibited the highest antiviral potency in Akata-Bxl cells with the IC50 and EC50 of (+)-Rutamarin of 2.38 μΜ and 2.94 μΜ, respectively (Figure 14 and Table 3B).
Evaluation of Topo II inhibitors for their cytotoxicities and effects on host cell proliferation
Cytotoxicities of these three inhibitors were examined in parallel with their inhibition of EBV DNA replication and virion production in P3HR-1 and Akata- Bxl cells. Cells treated with these inhibitors at different concentrations were subjected to the trypan blue exclusion method to assess the numbers of viable cells and nonviable cells in culture. The half-maximal cytotoxic concentrations (CC50) were determined based on the results. Novobiocin and merbarone exhibited relatively low cytotoxicity to P3HR-1 cells with CC50values of 518.5 μΜ and 137.6 μΜ, respectively (Figure 13A and B). For (+)-rutamarin, the highest concentration used in this study (150 μΜ) showed little cytotoxicity in 2 and 5 days culture, suggesting that the CC50 value of (+)-rutamarin to P3HR-1 cell is greater than 150 μΜ (Figure 13D and E). Similar results were obtained with Akata-Bxl cells (Figure 14D and 14E). The low cytotoxicities and high inhibition rates for both viral DNA replication and virion production, represented by the selectivity indices (CC50/IC50) higher than 39.6 in P3HR-1 and 63 in Akata-Bxl cells for (+)- rutamarin (Table 3 A and 3B), suggest the potential of (+)-rutamarin to be an effective anti-EBV drug candidate for treatment of EBV-associated diseases.
To further investigate the potentials of Topo II catalytic inhibitors to become safe anti-EBV drug candidates, experiments were designed to assess the effects of these inhibitors on cell proliferation. P3HR-1 cells were cultured in the absence and the presence of novobiocin, merbarone and (+)-rutamarin at two different concentrations, the IC50 and an excess concentration (5 x IC50) for 5 days. Cell samples were daily collected and counted. Merbarone was found to be able to inhibit cell proliferation in both concentrations; novobiocin did not exhibit much inhibitory effect on cell growth at the concentration of IC50 but the cell proliferation rate was dramatically decreased at the excess concentration (5 x IC50). In contrast, little effect was observed with (+)-rutamarin at both IC50 and 5 x IC50 concentrations on cell proliferation (Figure 15).
Validation of Topo II inhibitors for their effects on EBV ori-Lyt-dependent DNA replication
The topoisomerase II inhibitors tested in this study have been demonstrated to inhibit EBV DNA synthesis and virion production. Experiments where designed to assess whether these inhibitors indeed block viral ori-Lyt-dependent DNA replication. To address this question, P3HR-1 cells were cotransfected with an EBV ori- Lyt-containing plasmid (pEBV-oriL) and a ZTA expression vector (McZ). Expression of ZTA sufficiently drives latent EBV into lytic replication cycle (Countryman and Miller, 1985, Proc. Natl. Acad. Sci. U.S.A., 82:4085-4089 and Rooney et al, 1988, Proc. Natl. Acad. Sci. U.S.A., 85:9801-9805). The transfected cells were cultured in the absence or the presence of each inhibitor at various concentrations, usually with two concentration lower than IC50, and two higher concentrations. The ori-Lyt-dependent DNA replication and the drug effects were measured by a Dpn I assay (Gonzalez-Molleda et al., 2012, Antimicrob. Agents Chemother, 56:893-902). In brief, DNA was isolated from the treated cells 72 h post-transfection and digested with EcoR I and EcoR I/Dpn I.
Replicated plasmid DNA can be distinguished from input plasmid by Dpnl restriction digest, which cleaves input DNA that has been dam + methylated in Escherichia coli but leaves intact the DNA that has been replicated at least one round in eukaryotic cells
(Gonzalez-Molleda et al., 2012, Antimicrob. Agents Chemother, 56:893-902). Thus, only newly replicated plasmid DNA in P3HR-1 cells is resistant to Dpn I digestion and can be detected in Southern blot analysis. Replicated DNA was detected in the cells that were cotransfected with pEBV-oriL and ZTA expression vector (Figure 16A-16C). EcoR I digested Hirt DNAs served as controls for equal transfection of input plasmids in each chemical concentration. Novobiocin, merbarone and (+)-rutamarin potently blocked the newly replicated DNA in a dose-dependent manner (Figure 5A-5C). In conclusion, Topo II catalytic inhibitors that were tested were found to be able to inhibit the ori-Lyt- dependent DNA replication, suggesting that these inhibitors act in blocking EBV ori-Lyt- dependent DNA replication.
Human topoisomerase II catalytic inhibitors are potent in inhibiting EBV replication
EBV is associated with a number of human diseases. The lytic replication of EBV is directly linked to infectious mononucleosis, chronic active EBV infection (CAEBV), oral hairy leukoplakia and to an increased risk of EBV-associated
nasopharyngeal carcinoma (Al Tabaa et al., 2011, J. Clin. Virol., 52:33-37, Dardari et al., 2000, Int. J. Cancer, 86:71-75 and Lau et al, 1993, Virology, 195:463-474). For example, CAEBV is an often fatal disorder that is rare in USA but occurs more frequently in Asia and South America (Katano et al., 2004, Blood, 103: 1244-1252). Chronic active EBV infection is characterized by chronic or recurrent infectious mononucleosis-like symptoms that persist for a long time and by an unusual pattern of anti-EBV antibodies, with life-threatening complications, such as virus-associated hemophagocytic syndrome and lymphoma (Kimura et al., 2001, Blood, 98:280-286). The patients usually have a markedly elevated EBV DNA level in the blood (103- 107 copies/ml), indicating active lytic viral replication. So far no satisfactory therapy is available for CAEBV. Antiviral or immunomodulatory agents, such as acyclovir, ganciclovir, vidarabine, interferon-a, and interleukin-2, have been trialed for CAEBV with limited success (Kimura et al, 2003, J. Infect. Dis., 187:527-533). Acyclovir (ACV), which inhibits herpesviral DNA polymerase, is commonly used for treatment of CAEBV (Gershburg and Pagano, 2005, J. Antimicrob. Chemother, 56:277-281) but found generally inefficient for this disease. Moreover, drug resistance frequently occurs as a major problem among immunosuppressed hosts, mainly those who have received prolonged ACV therapy (Andrei et al, 2012, J. Virol, 86:2641-2652 and Field and Vere Hodge, 2013, Br. Med. Bull, 106:213-249). Some studies suggested that ganciclovir, which is a guanosine analog, might be a more effective drug than ACV for treating lytic EBV infection in patients (Andrei et al., 2012, J. Virol., 86:2641-2652 and Keever- Taylor et al, 2003, Cytotherapy, 5:323-335). Unfortunately, the use of ganciclovir is limited by nephrotoxicity as well as hematological toxicity (Gilbert et al., 2002, Drug Resist. Updat, 5:88-114 and Williams et al., 2003, Antimicrob. Agents Chemother, 47:2186-2192). Thus, there is an urgent need for efficient antivirals against EBV for treatment of EBV-associated human diseases.
EBV as well as other herpesviruses do not encode topoisomerases and rely on host cell topoisomerases activity for their DNA replication (Kawanishi, 1993, J. Gen. Virol, 74:2263-2268). Thus topoisomerases could be therapeutic targets for blocking replication of herpesviruses including EBV and treatment of the infection-associated human diseases. It has been reported previously that Topo I inhibitor camptothecin is able to inhibit replication of herpes simplex virus type 2 (Yamada et al, 1990, Arch. Virol., 110: 121-127). Both Topo II catalytic inhibitor ICRF193 and Topo II poison teniposide (VM26) were found to be able to block herpes simplex virus type 1 from replication (Ebert et al, 1990, J. Virol, 64:4059-4066 and Hammarsten et al, 1996, J. Virol, 70:4523-4529). The results presented herein demonstrate that KSHV replication can be effectively blocked by Topo I inhibitor camptothecin, Topo II poisons etoposide and ellipticine as well as Topo II catalytic inhibitors novobiocin, merbarone and (+)- rutamarin (Gonzalez-Molleda et al, 2012, Antimicrob. Agents Chemother, 56:893- 902 and Xu et al, 2014, Antimicrob. Agents Chemother, 58:563-573). However, Topo I inhibitors and Topo II poisons in general possess considerable toxicities to host cells (Gonzalez-Molleda et al, 2012, Antimicrob. Agents Chemother. 56:893-902). The results presented herein demonstrate that human topoisomerase II catalytic inhibitors are potent in inhibiting EBV replication and some of them exhibit little cytotoxicities, suggesting some Topo II catalytic inhibitors are promising antiviral candidates for EBV- associated human diseases. Furthermore, viruses have tendencies to mutate their genome and therefore develop drug resistance. An antiviral that targets a cellular protein such as Topo II offers the advantage of minimizing drug resistance and hence constitutes an important, novel therapeutic strategy.
Novobiocin is an old antibiotic drug against staphylococcal infection (Kirby et al, 1956, AMA Arch. Intern. Med., 98: 1-7). Novobiocin has also been used in combination with chemotherapeutical agents for the treatment of several cancers (Eder et al, 1991, Cancer Res., 51 :510-513 and Kennedy et al, 1995, J. Clin. Oncol, 13: 1136- 1143). The results presented herein reveal its antiviral potency against EBV, suggesting a new usage for an old drug.
(+)-Rutamarin is a natural product and newly identified Topo II catalytic inhibitor (Xu et al., 2014, Antimicrob. Agents Chemother, 58:563-573). Among three inhibitors tested in this study, (+)-Rutamarin exhibited the highest potency in anti-EBV activity. (+)-Rutamarin effectively inhibits EBV replication with an IC50 of 2.38 μΜ and an EC50 of 2.94 μΜ (in Akata-Bxl cells). It possesses relative low cytotoxicity to P3HR- 1 cells with a CC50 greater than 150 μΜ. Without wishing to be bound by any particular theory, experiments can be desgined to optimize the (+)-Rutamarin structure in order to further elevate its antiviral activity and improve its solubility, making it an anti-EBV drug lead.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:
1. A method of treating a herpesvirus infection in an individual in need thereof, the method comprising the step of: administering to the individual an effective KSHV antiviral amount of a pharmaceutical composition comprising at least one compound selected from the group consisting of:
(iii)a compound of formula (I):
Figure imgf000088_0001
wherein in formula (I):
each occurrence of R1 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, -S(=0)2R7, -NHS(=0)2R7, - C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, - OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, -C(OH)(R7)2, - C(CH3)2(CH=CH2), -C(CH3)2OC(=0)CH3, and -C(NH2)(R7)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, -S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, - C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R/)2, -OC(=0)N(R/)2, -NHC(=0)NH(R/), -NHC(=0)R\ -NHC(=0)OR/, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups, or two or more adjacent R1, R2, R3, R4, R5,and R6 groups combine to form a first ring optionally substituted with 0-4 R1 groups, wherein the first ring is optionally fused to a second ring optionally substituted with 0-4 R1 groups;
each occurrence of R7 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted; and
X is -CH or O; and
(iv)a compound of formula (II):
Figure imgf000089_0001
wherein in formula (II):
each occurrence of R1, R2, R3, R4, and R5 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR6, -SR6, -S(=0)R6, - S(=0)2R6, -NHS(=0)2R6, -C(=0)R6, -OC(=0)R6, -C02R6, -OC02R6, -CH(R6)2, -N(R6)2, -C(=0)N(R6)2, -OC(=0)N(R6)2, -NHC(=0)NH(R6), -NHC(=0)R6, -NHC(=0)OR6, - C(OH)(R6)2, and -C(NH2)(R6)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR6, -SR6, -S(=0)R6, -S(=0)2R6, -NHS(=0)2R6, -C(=0)R6, -OC(=0)R6, -C02R6, -OC02R6, -CH(R6)2, -N(R6)2, - C(=0)N(R6)2, -OC(=0)N(R6)2, -NHC(=0)NH(R6), -NHC(=0)R6, -NHC(=0)OR6, - C(OH)(R6)2, and -C(NH2)(R6)2; and each occurrence of R6 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
a salt, solvate, or N-oxide thereof, and any combinations thereof.
2. The method of claim 1, wherein the compound is selected from the group consisting of:
2H-chromen-2-one;
7-hydroxy-2H-chromen-2-one;
7-amino-4-methyl-2H-chromen-2-one;
7-hydroxy-4-methyl-2H-chromen-2-one;
6.7- dihy droxy-2H-chromen-2 -one ;
7 , 8 -dihy droxy-2H-chromen-2 -one ;
2H-furo[2,3-A]chromen-2-one;
7H-furo [3 ,2-g] chromen-7-one;
6- hydroxy-7-methoxy-2H-chromen-2-one;
7.8- dihydroxy-6-methoxy-2H-chromen-2-one;
9-methoxy-7H-furo [3 ,2-g] chromen-7-one;
9-methoxynaphtho[2,3-£]furan-7(8H)-one;
7- hydroxy-6,8-dimethoxy-2H-chromen-2-one;
7-(diethylamino)-4-methyl-2H-chromen-2-one;
7- methoxy-8-(3-methylbut-2-en-l-yl)-2H-chromen-2-one;
8- (2,3-dihydroxy-3-methylbutyl)-7-hydroxy-2H-chromen-2-one;
4-((3-methylbut-2-en-l-yl)oxy)-7H-furo[3,2-g]chromen-7-one;
9- ((3-methylbut-2-en-l-yl)oxy)-7H-furo[3,2-g]chromen-7-one;
6b-ethyl-10a-hydroxy-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2-¾]pyridine-
6,10(10aH)-dione;
10a-hydroxy-3,6b-dimethyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
10a-hydroxy-2,6b-dimethyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione; 6- (2,3-dihydroxy-3-methylbutyl)-5,7-dimethoxy-2H-chromen-2-one;
4-hydroxy-3-(3-oxo- 1 -phenylbutyl)-2H-chromen-2-one;
6b-ethyl-10a-hydroxy-3-methyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2-
£]pyridine-6, 10(10aH)-dione;
6b-ethyl-10a-hydroxy-2-methyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
(E)-2-(8-oxo-3,8-dihydro-2H-furo[3,2-A]chromen-2-yl)propan-2-yl 2-methylbut- 2-enoate;
12a-hydroxy-8b-methyl-8b,9-dihydro-8H- benzo[7^8*]chromeno[3^4^4,5]furo[3,2-¾]pyridine-8,12(12aH)-dione;
3,9-dihydroxy-2-(3-methylbut-2-en-l-yl)-6H-benzofuro[3,2-c]chromen-6-one;
7- hydroxy-6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-2H-chromen-2-one;
6-methoxy-7-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-2H-chromen-2-one;
(5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2- yl)propan-2-yl acetate;
9-hydroxy-5-(3,4,5-trimethoxyphenyl)-5,5a,8a,9- tetrahydrofuro [3 ',4': 6 ,7]naphtho [2,3 -d] [ 1 ,3 ]dioxol-6(8H)-one;
9-((7,8-dihydroxy-2-methylhexahydropyrano[3,2-(i][l,3]dioxin-6-yl)oxy)-5-(3,5- dimethoxy-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)phenyl)-5,5a,8a,9-tetrahydroi iro[3^4^6,7]naphtho[2,3- ][l,3]dioxol-6(8H)-on^ a salt, solvate, or N-oxide thereof, and combinations thereof.
3. The method of claim 1 , wherein the compound of formula (I) is at least one compound of formula (III):
Figure imgf000091_0001
(III)
wherein in formula (III): each occurrence of R1 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR9, -SR9, -S(=0)R9, -S(=0)2R9, -NHS(=0)2R9, - C(=0)R9, -OC(=0)R9, -C02R9, -OC02R9, -CH(R9)2, -N(R9)2, -C(=0)N(R9)2, - OC(=0)N(R9)2, -NHC(=0)NH(R9), -NHC(=0)R9, -NHC(=0)OR9, -C(OH)(R9)2, - C(CH3)2(CH=CH2), -C(CH3)2OC(=0)CH3, and -C(NH2)(R9)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR9, -SR9, -S(=0)R9, -S(=0)2R9, -NHS(=0)2R9, -C(=0)R9, -OC(=0)R9, -C02R9, -OC02R9, -CH(R9)2, -N(R9)2, - C(=0)N(R9)2, -OC(=0)N(R9)2, -NHC(=0)NH(R9), -NHC(=0)R9, -NHC(=0)OR6, - C(OH)(R9)2, and -C(NH2)(R9)2;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups;
each occurrence of R7 and R8 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl,
heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R/)2, -OC(=0)N(R/)2, -NHC(=0)NH(R/), -NHC(=0)R\ -NHC(=0)OR/, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups, or R7 and R8 combine to form a ring optionally substituted with 0-4 R1 groups; and
each occurrence of R9 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
a salt, solvate, or N-oxide thereof, and any combinations thereof.
4. The method of claim 3, wherein the compound is selected from the group consisting of (5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2- g]chromen-2-yl)propan-2-yl acetate, a salt, solvate, or N-oxide thereof, and combinations thereof.
5. The method of claim 1, further comprising administering to the individual an effective amount of a therapeutic agent.
6. The method of claim 5, wherein the therapeutic agent is selected from the group consisting of a protease inhibitor, a cytokine, and an immunomodulator.
7. The method of claim 1, further comprising administering to the individual an effective amount of a chemotherapeutic agent.
8. The method of claim 7, wherein the chemotherapeutic agent is selected from the group consisting of a topoisomerase II inhibitor, an antibiotic, a vinca alkaloid, an anthracycline, and a taxane.
9. The method of claim 1, wherein the herpesvirus is selected from the group consisting of Kaposi's sarcoma-associated herpes virus (KSHV) and Epstein- Barr virus (EBV).
10. A method for inhibiting replication of a herpesvirus in an individual, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising at least one compound selected from the group consisting of:
(i) a compound of formula (I):
Figure imgf000094_0001
wherein in formula (I):
each occurrence of R1 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, -S(=0)2R7, -NHS(=0)2R7, - C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, - OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, -C(OH)(R7)2, - C(CH3)2(CH=CH2), -C(CH3)2OC(=0)CH3, and -C(NH2)(R7)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, -S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, - C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups, or two or more adjacent R1, R2, R3, R4, R5,and R6 groups combine to form a first ring optionally substituted with 0-4 R1 groups, wherein the first ring is optionally fused to a second ring optionally substituted with 0-4 R1 groups;
each occurrence of R7 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted; and
X is -CH or O; and
(ii) a compound of formula (II):
Figure imgf000095_0001
wherein in formula (II):
each occurrence of R1, R2, R3, R4, and R5 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR6, -SR6, -S(=0)R6, - S(=0)2R6, -NHS(=0)2R6, -C(=0)R6, -OC(=0)R6, -C02R6, -OC02R6, -CH(R6)2, -N(R6)2, -C(=0)N(R6)2, -OC(=0)N(R6)2, -NHC(=0)NH(R6), -NHC(=0)R6, -NHC(=0)OR6, - C(OH)(R6)2, and -C(NH2)(R6)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR6, -SR6, -S(=0)R6, -S(=0)2R6, -NHS(=0)2R6, -C(=0)R6, -OC(=0)R6, -C02R6, -OC02R6, -CH(R6)2, -N(R6)2, - C(=0)N(R6)2, -OC(=0)N(R6)2, -NHC(=0)NH(R6), -NHC(=0)R6, -NHC(=0)OR6, - C(OH)(R6)2, and -C(NH2)(R6)2; and
each occurrence of R6 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
a salt, solvate, or N-oxide thereof, and any combinations thereof.
11. The method of claim 10, wherein the compound is selected from the group consisting of:
2H-chromen-2-one;
7-hydroxy-2H-chromen-2-one;
7-amino-4-methyl-2H-chromen-2-one;
7-hydroxy-4-methyl-2H-chromen-2-one;
6.7- dihy droxy-2H-chromen-2 -one ;
7 , 8 -dihy droxy-2H-chromen-2 -one ;
2H-furo[2,3-A]chromen-2-one;
7H-furo [3 ,2-g] chromen-7-one;
6- hydroxy-7-methoxy-2H-chromen-2-one;
7.8- dihydroxy-6-methoxy-2H-chromen-2-one;
9-methoxy-7H-furo [3 ,2-g] chromen-7-one;
9-methoxynaphtho[2,3-£]furan-7(8H)-one;
7- hydroxy-6,8-dimethoxy-2H-chromen-2-one;
7-(diethylamino)-4-methyl-2H-chromen-2-one;
7- methoxy-8-(3-methylbut-2-en-l-yl)-2H-chromen-2-one;
8- (2,3-dihydroxy-3-methylbutyl)-7-hydroxy-2H-chromen-2-one;
4-((3-methylbut-2-en-l-yl)oxy)-7H-furo[3,2-g]chromen-7-one;
9- ((3-methylbut-2-en-l-yl)oxy)-7H-furo[3,2-g]chromen-7-one;
6b-ethyl-10a-hydroxy-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2-¾]pyridine-
6,10(10aH)-dione;
10a-hydroxy-3,6b-dimethyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
10a-hydroxy-2,6b-dimethyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
6-(2,3-dihydroxy-3-methylbutyl)-5,7-dimethoxy-2H-chromen-2-one;
4-hydroxy-3-(3-oxo- 1 -phenylbutyl)-2H-chromen-2-one;
6b-ethyl-10a-hydroxy-3-methyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione; 6b-ethyl-10a-hydroxy-2-methyl-6b,7-dihydro-6H-chromeno[3',4':4,5]furo[3,2- £]pyridine-6, 10(10aH)-dione;
(E)-2-(8-oxo-3,8-dihydro-2H-furo[3,2-A]chromen-2-yl)propan-2-yl 2-methylbut- 2-enoate;
12a-hydroxy-8b-methyl-8b,9-dihydro-8H- benzo[7^8*]chromeno[3^4^4,5]furo[3,2-¾]pyridine-8,12(12aH)-dione;
3,9-dihydroxy-2-(3-methylbut-2-en-l-yl)-6H-benzofuro[3,2-c]chromen-6-one;
7-hydroxy-6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-2H-chromen-2-one;
6-methoxy-7-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-2H-chromen-2-one;
(5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2- yl)propan-2-yl acetate;
9-hydroxy-5-(3,4,5-trimethoxyphenyl)-5,5a,8a,9- tetrahydroi iro[3\4^6,7]naphtho[2,3-(i][l,3]dioxol-6(8H)-one;
9-((7,8-dihydroxy-2-methylhexahydropyrano[3,2-(i][l,3]dioxin-6-yl)oxy)-5-(3,5- dimethoxy-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)phenyl)-5,5a,8a,9-tetrahydrof½o[3^4^
a salt, solvate, or N-oxide thereof, and combinations thereof.
12. The method of claim 10, wherein the compound of formulat (I) is at least one compound of formula (III):
Figure imgf000097_0001
wherein in formula (III):
each occurrence of R1 is independently selected from the group consisting of Η, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR9, -SR9, -S(=0)R9, -S(=0)2R9, -NHS(=0)2R9, - C(=0)R9, -OC(=0)R9, -C02R9, -OC02R9, -CH(R9)2, -N(R9)2, -C(=0)N(R9)2, - OC(=0)N(R9)2, -NHC(=0)NH(R9), -NHC(=0)R9, -NHC(=0)OR9, -C(OH)(R9)2, - C(CH3)2(CH=CH2), -C(CH3)2OC(=0)CH3, and -C(NH2)(R9)2;
wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl or heterocycloalkyl group is optionally substituted with 0-5 substituents, each of which is independently selected from the group consisting of -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR9, -SR9, -S(=0)R9, -S(=0)2R9, -NHS(=0)2R9, -C(=0)R9, -OC(=0)R9, -C02R9, -OC02R9, -CH(R9)2, -N(R9)2, - C(=0)N(R9)2, -OC(=0)N(R9)2, -NHC(=0)NH(R9), -NHC(=0)R9, -NHC(=0)OR6, - C(OH)(R9)2, and -C(NH2)(R9)2;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups;
each occurrence of R2, R3, R4, R5,and R6 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups;
each occurrence of R7 and R8 is independently selected from the group consisting of H, -Ci-C6 alkyl, -Ci-C6 alkenyl, aryl, heteroaryl, cycloalkyl,
heterocycloalkyl, -Ci-C6 heteroalkyl, F, CI, Br, I, -CN, -N02, -OR7, -SR7, -S(=0)R7, - S(=0)2R7, -NHS(=0)2R7, -C(=0)R7, -OC(=0)R7, -C02R7, -OC02R7, -CH(R7)2, -N(R7)2, -C(=0)N(R7)2, -OC(=0)N(R7)2, -NHC(=0)NH(R7), -NHC(=0)R7, -NHC(=0)OR7, - C(OH)(R7)2, and -C(NH2)(R7)2, wherein the alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, cycloalkyl, or heterocycloalkyl group is optionally substituted with 0-5 R1 groups, or R7 and R8 combine to form a ring optionally substituted with 0-4 R1 groups; and each occurrence of R9 is independently selected from the group consisting of H, Ci-C6 alkyl, Ci-C6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
a salt, solvate, or N-oxide thereof, and any combinations thereof.
13. The method of claim 12, wherein the compound is selected from the group consisting of (5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2- g]chromen-2-yl)propan-2-yl acetate, a salt, solvate, or N-oxide thereof, and combinations thereof.
14. The method of claim 10, wherein the herpesvirus is selected from the group consisting of Kaposi's sarcoma-associated herpes virus (KSHV) and Epstein- Barr virus (EBV).
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