WO2015051268A2

WO2015051268A2 - Hiv protease inhibitors against picornavirus infection

Info

Publication number: WO2015051268A2
Application number: PCT/US2014/059089
Authority: WO
Inventors: Xiao-Fang Yu
Original assignee: The Johns Hopkins University
Priority date: 2013-10-04
Filing date: 2014-10-03
Publication date: 2015-04-09
Also published as: WO2015051268A3

Abstract

The present invention is directed to methods for treating viral infections. In particular, the present invention is directed to methods for treating picornavirus infection using HIV protease inhibitors.

Description

HIV PROTEASE INHIBITORS AGAINST PICORNAVIRUS INFECTION

CROSS REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Serial No. 61/886,765, filed October 4, 2013; and of U.S. Serial No. 61/940,755, filed February 17, 2014, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0002] The present invention relates generally to methods and compositions for treating and preventing viral infection, and more particularly to methods and compositions using HIV protease inhibitors.

BACKGROUND INFORMATION

[0003] Enteroviruses, which are members of the Picornaviridae family, cause a wide spectrum of clinical syndromes ranging from mild fever to respiratory infections, meningitis, encephalitis, paralytic poliomyelitis and myocarditis. An estimated 10 to 30 million enteroviral infections occur annually in America, causing significant short-term mobility and economic impact. Life -threatening enteroviral infections may occur, especially in high-risk individuals such as immunocompromised patients.

[0004] Enterovirus 71 (EV71) is a member of the Picornaviridae family and is one of the main causes of Hand, Foot and Mouth disease, an epidemic widely seen in the Asia-Pacific regions. The lack of treatment or prevention options make this a major public health issue in the affected countries and underscore the need for effective drugs for this infection.

[0005] Enterovirus 68 (EV68, EV-D68, HEV68) is another member of the Picornaviridae family that causes respiratory illness, and varies from mild to severe. Initial symptoms are similar to those for the common cold, including a runny nose, sore throat, cough, and fever. As the disease progresses, more serious symptoms may occur, including difficulty breathing as in pneumonia, reduced alertness, a reduction in urine production, and dehydration, and may lead to respiratory failure. As with EV71 there is a lack of treatment or prevention options that makes this a major public health issue.

[0006] Other members of the Picornaviridae family are coxsackieviruses.

Coxsackieviruses are among the leading causes of aseptic meningitis. In general, group A coxsackieviruses tend to infect the skin and mucous membranes, causing herpangina, acute hemorrhagic conjunctivitis, and hand, foot, and mouth (HFM) disease. In particular, coxsackievirus A16 (CA16, CV-A16) is a main cause of Hand, Foot and Mouth disease. Group B coxsackieviruses tend to infect the heart, pleura, pancreas, and liver, causing pleurodynia, myocarditis, pericarditis, and hepatitis (inflammation of the liver not related to the hepatotropic viruses). Coxsackie B infection of the heart can lead to pericardial effusion. Both group A and group B coxsackieviruses can cause nonspecific febrile illnesses, rashes, upper respiratory tract disease, and aseptic meningitis. As with other enteroviruses, there is a lack of treatment or prevention options that makes this a major public health issue.

[0007] HIV-1 protease inhibitors (Pis) have widely been in use for treatment of HIV- 1 infected patients. Currently there are nine FDA approved human immunodeficiency virus (HIV)-l Pis available namely saquinavir, ritonavir, lopinavir, indinavir, nelfmavir, atazanavir, amprenavir, darunavir and tipranavir which are used in various HAART regimens. These drugs are potent inhibitors of the HIV-1 protease enzyme that is essential for processing the viral polyprotein and maturation of viral particles. Specifically targeting this enzyme hinders the cleavage of the polyprotein which ultimately leads to the formation of non- infectious virions. Through various clinical trials, population and cohort studies, these drugs have been shown to be effective in reducing the viral load and increasing the CD4 T cell counts of HIV-1 infected patients. Hence they are effectively able to slow down the progression of the infection to the AIDS phase. Also numerous biosafety and toxicity studies were conducted to validate drug safety and tolerance. These data thus provide ample evidence of their antiviral capacity and establish them as safe, validated and well documented drug candidates for repositioning. SUMMARY OF THE INVENTION

[0008] The present disclosure is based on the seminal discovery that HIV protease inhibitors are not only useful for treatment of viral infection by certain lentiviruses, such as HIV, but also for treatment of infection by picornaviruses, such as EV71, EV68,

coxsackievirus A16, hepatitis A virus, rhinovirus and poliovirus.

[0009] Accordingly, in one aspect, the present invention provides a method of treating picomavirus infection. The method includes administering to a subject a therapeutically effective amount of an HIV protease inhibitor, thereby treating the picomavirus infection.

[0010] In another aspect, the invention provides a method of preventing picomavirus infection. The method includes administering to the subject a therapeutically effective amount of an HIV protease inhibitor, thereby preventing the picomavirus infection.

[0011] In another aspect, the invention provides a method of inhibiting picomavirus replication. The method includes contacting a picomavirus with a HIV protease inhibitor, thereby inhibiting replication of the picomavirus.

[0012] In another aspect, the invention provides a method of treating a picomavirus mediated disease in a subject. The method includes administering to the subject a therapeutically effective amount of an HIV protease inhibitor, thereby treating the picomavirus mediated disease.

[0013] In yet another aspect, the invention provides a kit for treating or preventing a picomavirus infection. The kit includes a) an HIV protease inhibitor; and instructions for administering the inhibitor to a subject.

[0014] In various embodiments of the invention, the picomavirus is an aphthovirus, cardiovirus, hepatovirus or enterovirus. The enterovirus may be a rhinovirus, poliovirus, echovirus, or coxsackievirus. In some embodiments, the enterovirus is enterovirus 71 (EV71), enterovirus 68 (EV68, EV-D68, HEV68), coxsackievirus A16 (CVA16 or CA16) or hepatitis A vims (HAV) 1. [0015] In various embodiments of the invention, the HIV protease is a HIV-1 protease inhibitor, such as nelfmavir, ritonavir, indinavir, amprenavir, atazanvir, and lopinavir.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A is a pictorial diagram illustrating potential mechanisms of inhibition of EV71 infection in one embodiment of the invention. Figure IB is a pictorial representation depicting a phylogentic tree showing the genetic relationship of viruses that may be inhibited in various embodiments of the invention.

[0017] Figure 2 is a pictorial diagram illustrating experimental methodology in one embodiment of the invention.

[0018] Figure 3 is a series of pictorial and graphical representations presenting

experimental results of EV71 viral inhibition in one embodiment of the invention.

[0019] Figure 4 is a series of pictorial and graphical representations presenting

[0020] Figure 5 is a pictorial representation illustrating the chemical structure of various HIV-1 protease inhibitors in embodiments of the invention.

[0021] Figure 6 is a pictorial representation illustrating the chemical structure of various HIV-1 protease inhibitors in embodiments of the invention.

[0022] Figure 7 is a graphical representation presenting experimental results of EV71 viral inhibition in one embodiment of the invention.

[0023] Figure 8 is a graphical representation presenting experimental results of EV71 viral inhibition in one embodiment of the invention.

[0024] Figure 9 is a pictorial representation presenting experimental results of EV71 viral inhibition in one embodiment of the invention. Vera cells infected with EC71 BrCr in the absence (lane 3) or presence of various concentrations of nelfmavir (lanes 5-10) are shown. Vera cells with no virus infection are shown in lane 2. [0025] Figures 1 OA- IOC are pictorial representations presenting experimental results of viral inhibition in one embodiment of the invention. Figure 10A shows cytopathic effect (CPE) in vero cells not infected with virus (coxsackievirus A16, CA16) or treated with drug. Figure 10B shows CPE in vero cells infected with CA16 without treatment with drug. Figure IOC shows CPE in vero cells infected with CA16 with treatment with drug.

[0026] Figures 1 lA-11C are pictorial representations presenting experimental results of viral inhibition in one embodiment of the invention. Figure 11 A shows CPE in vero cells not infected with virus (hepatitis A virus, HAV) or treated with drug. Figure 1 IB shows CPE in vero cells infected with HAV without treatment with drug. Figure 11C shows CPE in vero cells infected with HAV with treatment with drug.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides methods and compositions for treating or preventing viral infections. The methods and compositions of the invention make use of HIV protease inhibitors and the discovery that such inhibitors are effective in the treatment of picomaviruses. In particular, the data presented herein illustrate that nelfmavir is an effective treatment for enterovirus infection.

[0028] Before the present compositions and methods are further described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0029] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. [0030] 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 invention, the preferred methods and materials are now described.

[0031] In one aspect, the present invention provides a method of preventing or treating picornavirus infection. The method includes administering to a subject a therapeutically effective amount of an HIV protease inhibitor, thereby treating the picornavirus infection.

[0032] In another aspect, the invention provides a method of inhibiting picornavirus replication. The method includes contacting a picornavirus with a HIV protease inhibitor, thereby inhibiting replication of the picornavirus.

[0033] In another aspect, the invention provides a method of treating a picornavirus mediated disease in a subject. The method includes administering to the subject a therapeutically effective amount of an HIV protease inhibitor, thereby treating the picornavirus mediated disease.

[0034] Compounds of the formulae that are provided herein, for example, formula (1), may be prodrugs. For example, compounds wherein at least one of Q₄-(¾ is -O-J, as defined hereafter, may be used as prodrugs, which can serve to improve the pharmaceutical properties of the compounds, such as pharmacokinetic properties, for example, improved bioavailability or solubility. The preparation of the prodrugs may be carried out by reacting a compound of the formula (1), wherein at least one of Q₄-Qs is -O-H, with, for example, an activated amino acyl, phosphoryl or hemisuccinyl derivative.

[0035] All temperatures stated herein are in degrees Celsius (° C). All units of measurement employed herein are in weight units, except for liquids which are in volume units.

[0036] The term "alkyl" as used herein refers to straight or branched chain groups, preferably, having one to eight, more preferably having one to six, and most preferably having from one to four carbon atoms. The term "C₁-C6 alkyl" represents a straight or branched alkyl chain having from one to six carbon atoms. Exemplary Ci-C₆ alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, neo- pentyl, hexyl, isohexyl, and the like. The term "Ci-C₆ alkyl" includes within its definition the term "Ci-C₄ alkyl."

[0037] The term "cycloalkyl" represents a saturated or partially saturated, mono- or poly- carbocylic ring, preferably having 5-14 ring carbon atoms. Exemplary cycloalkyls include monocyclic rings having from 3-7, preferably 3-6, carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. An exemplary cycloalkyl is a C5-C7 cycloalkyl, which is a saturated hydrocarbon ring structure containing from five to seven carbon atoms.

[0038] The term "alkoxyl" represents -O-alkyl. An example of an alkoxyl is a Ci-C₆ alkoxyl, which represents a straight or branched alkyl chain having from one to six carbon atoms attached to an oxygen atom. Exemplary Ci-C₆ alkoxyl groups include methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, sec-butoxyl, t-butoxyl, pentoxyl, hexoxyl, and the like. Ci-C₆ alkoxyl includes within its definition a Ci-C₄ alkoxyl.

[0039] The term "aryl" as used herein refers to a carbocyclic or heterocyclic, aromatic, 5- 14 membered monocyclic or polycyclic ring. Exemplary aryls include phenyl, naphthyl, anthryl, phenanthryl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl, thiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, benzo[b]thienyl, naphtho[2,3-b]thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxyalinyl, quinzolinyl, benzothiazolyl, benzimidazolyl,

tetrahydroquinolinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, and phenoxazinyl.

[0040] The term "aryloxyl" represents [0041] The term "hydrolyzable group" is a group, which when bonded to an oxygen, forms an ester, which can be hydro lyzed in vivo to a hydroxyl group. Exemplary

hydrolyzable groups, which are optionally substituted, include acyl function, sulfonate function and phosphate function. For example, such hydrolyzable groups may include blocked or unblocked amino acid residue, a hemisuccinate residue, and a nicotinate residue.

[0042] The term "halogen" represents chlorine, fluorine, bromine or iodine. The term "halo" represents chloro, fluoro, bromo or iodo.

[0043] The term "carbocycle" represents an aromatic or a saturated or a partially saturated 5-14 membered monocyclic or polycyclic ring, such as a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic ring, wherein all the ring members are carbon atoms.

[0044] The term "heterocycle" represents an aromatic or a saturated or a partially saturated, 5-14 membered, monocylic or polycyclic ring, such as a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic ring, having from one to three heteroatoms selected from nitrogen, oxygen and sulfur, and wherein any nitrogen and sulfur heteroatoms may optionally be oxidized, and any nitrogen heteroatom may optionally be quatemized. The heterocyclic ring may be attached at any suitable heteroatom or carbon atom. Examples of such heterocycles include decahydroisoquinolinyl, octahydro-thieno[3,2-c]pyridinyl, piperidinyl, piperazinyl, azepinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, isobenzofuranyl, furazanyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, thianthrenyl, triazinyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, chromenyl, xanthenyl, isoquinolinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, benzoazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinylsulfone, oxadiazolyl, triazolyl, tetrahydroquinolinyl, tetrahydrisoquinolinyl, phenoxathienyl, indolizinyl, isoindolyl, indazolyl, purinyl, isoquinolyl, quinolyl,

phthalazinyl, naphthyridinyl, quinoxyalinyl, quinzolinyl, tetrahydroquinolinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,

phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, and phenoxazinyl. [0045] The term "thioether" includes S-aryl, such as phenylthio and naphthylthio; S- heterocycle where the heterocycle is saturated or partially saturated; S-(Cs-C7)-cycloalkyl; and S-alkyl, such as Ci-C₆ alkylthio. In the thioether, the -aryl, the -heterocycle, the - cycloalkyl, and the -alkyl can optionally be substituted. An example of a thioether is "Ci-C₆ alkylthio", which represents a straight or branched alkyl chain having from one to six carbon atoms attached to a sulfur atom. Exemplary Ci-C₆ alkylthio groups include methylthio, ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, t-butylthio, pentylthio, hexylthio, and the like.

[0046] The term "mercapto" represents -SH.

[0047] The term "amino" represents -NLiL₂, wherein Li and L₂ are preferably independently selected from oxygen, carbocycle, heterocycle, alkyl, sulfonyl and hydrogen; or NC(0)L₃, wherein L₃ is preferably alkyl, alkoxyl, hydrogen or NLiL₂. The aryl, alkyl and alkoxyl groups can optionally be substituted. An example of an amino is C₁-C₄ alkylamino, which represents a straight or branched alkyl chain having from one to four carbon atoms attached to an amino group. Exemplary C₁-C₄ alkylamino groups include methylamino, ethylamino, propylamino, isopropylamino, butylamino, sec-butylamino, and the like.

Another example of an amino is di(Ci-C₄)alkylamino, which represents two straight or branched alkyl chains, each having from one to four carbon atoms attached to a common amino group. Exemplary di(Ci-C₄)alkyl amino groups include dimethylamino,

ethylmethylamino, methylpropylamino, ethylisopropylamino, butylmethylamino, sec- butylethylamino, and the like. An example of an amino is C₁-C₄ alkylsulfonylamino, which has a straight or branched alkyl chain having from one to four carbon atoms attached to a sulfonylamino moiety. Exemplary C₁-C₄ alkylsulfonylamino groups include

methylsulfonylamino, ethylsulfonylamino, propylsulfonylamino, isopropylsulfonylamino, butylsulfonylamino, sec-butylsulfonylamino, t-butylsulfonylamino, and the like.

[0048] The term "acyl" represents L₆C(0)L4, wherein L₆ is a single bond, -O or -N, and further wherein L4 is preferably alkyl, amino, hydroxyl, alkoxyl or hydrogen. The alkyl and alkoxyl groups can optionally be substituted. An exemplary acyl is a C₁-C₄ alkoxycarbonyl, which is a straight or branched alkoxyl chain having from one to four carbon atoms attached to a carbonyl moiety. Exemplary C₁-C4 alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, and the like.

Another exemplary acyl is a carboxy wherein L₆ is a single bond and L₄ is alkoxyl, hydrogen, or hydroxyl. A further exemplary acyl is N-(Ci-C₄)alkylcarbamoyl (L₆ is a single bond and L₄ is an amino), which is a straight or branched alkyl chain having from one to four carbon atoms attached to the nitrogen atom of a carbamoyl moiety. Exemplary N-(Ci- C₄)alkylcarbamoyl groups include N-methylcarbamoyl, N-ethylcarbamoyl, N- propylcarbamoyl, N-isopropylcarbamoyl, N-butylcarbamoyl, and N-t-butylcarbamoyl, and the like. Yet another exemplary acyl is N,N-di(Ci-C₄)alkylcarbamoyl, which has two straight or branched alkyl chains, each having from one to four carbon atoms attached to the nitrogen atom of a carbamoyl moiety. Exemplary N,N-di(Ci-C₄)alkylcarbamoyl groups include N,N-dimethylcarbamoyl, Ν,Ν-ethylmethylcarbamoyl, N,N-methylpropylcarbamoyl, N,N-ethylisopropylcarbamoyl, Ν,Ν-butylmethylcarbamoyl, N,N-sec-butylethylcarbamoyl, and the like.

[0049] The term "polycyclic" refers to a polycyclic ring system, wherein at least two rings are connected together. As used herein, "polycyclic' refers to any ring system, which may be any degree of saturation, aromatic, aliphatic and optionally contain a heteroatom. The systems can be further classified according to the number of rings present (e.g., 2 = bicyclic, 3 = tricyclic, 4 = tetracyclic, etc.) and the way the rings are connected together. (1)

Substituted rings systems: These ring systems have no common atoms. In these polycyclic systems, the smaller ring can be regarded as a substituent of the larger ring. By way of example only, biphenyl is a substituted polycyclic ring system. (2) Spiro ring systems: Spiro ring systems share a single common atom. Hence the rings join at a single "point." The connecting atom is also called the spiroatom, most often a quaternary carbon ("spiro carbon"). By way of example, the spiro compound consisting of a cyclohexane ring and a cyclopentane ring is called spiro[4.5]decane. (3) Fused ring systems: Fused ring systems share two common atoms in one common bond, hence the rings share one side. Examplea of fused polycyclic ring systems include, but are not limited to, Naphthalene, Benzofuran, Indole, Benzothiophene, Quinoline and Anthracene. (4) Bridged ring systems: Bridged ring systems share more than two common atoms, i.e., bridged ring systems contain interlocking rings. By way of example, Adamantane, Amantadine, Biperiden, Memantine, Methenamine, Rimantadine and Norbornane are all bridged ring systems.

[0050] The term "sulfmyl" represents -SO-L5, wherein L₅ is preferably alkyl, amino, aryl, cycloalkyl or heterocycle. The alkyl, aryl, cycloalkyl and heterocycle can all optionally be substituted.

[0051] The term "sulfonyl" represents -SO₂-L5, wherein L₅ is preferably alkyl, aryl, cycloalkyl, heterocycle or amino. The alkyl, aryl, cycloalkyl and heterocycle can all optionally be substituted. An example of a sulfonyl is a C₁-C₄ alkylsulfonyl, which is a straight or branched alkyl chain having from one to four carbon atoms attached to a sulfonyl moiety. Exemplary C₁-C₄ alkylsulfonyl groups include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl, sec-butylsulfonyl, t-butylsulfonyl, and the like.

[0052] As indicated above, many of the groups are optionally substituted. Examples of substituents for alkyl and aryl include mercapto, thioether, nitro (N0₂), amino, aryloxyl, halogen, hydroxyl, alkoxyl, and acyl, as well as aryl, cycloalkyl, and saturated and partially saturated heterocycles. Examples of substituents for heterocycle and cycloalkyl include those listed above for alkyl and aryl, as well as aryl and alkyl.

[0053] Exemplary substituted aryls include a phenyl or naphthyl ring substituted with one or more substituents, preferably one to three substituents, independently selected from halo, hydroxy, morpholino(Ci-C₄)alkoxy carbonyl, pyridyl (Ci-C₄)alkoxycarbonyl, halo (Ci- C₄)alkyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, carboxy, C₁-C₄ alkoxycarbonyl, carbamoyl, N-(Ci- C₄)alkylcarbamoyl, amino, C₁-C₄ alkylamino, di(Ci-C₄)alkylamino or a group of the formula -(CH₂)_a-R⁷ where a is 1, 2, 3, or 4, and R⁷ is hydroxy, C₁-C₄ alkoxy, carboxy, C₁-C₄ alkoxycarbonyl, amino, carbamoyl, C₁-C₄ alkylamino or di(Ci-C₄)alkylamino.

[0054] Another substituted alkyl is halo(Ci-C₄)alkyl, which represents a straight or branched alkyl chain having from one to four carbon atoms with 1-3 halogen atoms attached to it. Exemplary halo(Ci-C₄)alkyl groups include chloromethyl, 2-bromoethyl, 1- chloroisopropyl, 3-fluoropropyl, 2,3-dibromobutyl, 3-chloroisobutyl, iodo-t-butyl, trifluoromethyl, and the like.

[0055] Another substituted alkyl is hydroxy(Ci-C₄)alkyl, which represents a straight or branched alkyl chain having from one to four carbon atoms with a hydroxy group attached to it. Exemplary hydroxy(Ci-C₄)alkyl groups include hydroxymethyl, 2-hydroxyethyl, 3- hydroxypropyl, 2-hydroxyisopropyl, 4-hydroxybutyl, and the like.

[0056] Yet another substituted alkyl is C₁-C₄ alkylthio(Ci-C₄)alkyl, which is a straight or branched C₁-C₄ alkyl group with a C₁-C₄ alkylthio group attached to it. Exemplary C₁-C₄ alkylthio(Ci-C₄)alkyl groups include methylthiomethyl, ethylthiomethyl, propylthiopropyl, sec-butylthiomethyl, and the like.

[0057] Yet another exemplary substituted alkyl is heterocycle(Ci-C₄)alkyl, which is a straight or branched alkyl chain having from one to four carbon atoms with a hetero cycle attached to it. Exemplary heterocycle(Ci-C₄)alkyls include pyrrolylmethyl,

quinolinylmethyl, 1-indolylethyl, 2-furylethyl, 3-thien-2-ylpropyl, 1-imidazolylisopropyl, 4- thiazolylbutyl, and the like.

[0058] Yet another substituted alkyl is aryl(Ci-C₄)alkyl, which is a straight or branched alkyl chain having from one to four carbon atoms with an aryl group attached to it.

Exemplary aryl(Ci-C₄)alkyl groups include phenylmethyl, 2-phenylethyl, 3 -naphthyl -propyl, 1-naphthylisopropyl, 4-phenylbutyl, and the like.

[0059] The heterocycle can, for example, be substituted with 1, 2 or 3 substituents independently selected from halo, halo(Ci-C₄)alkyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, carboxy, C₁-C₄ alkoxycarbonyl, carbamoyl, N-(Ci-C₄)alkylcarbamoyl, amino, C₁-C₄ alkylamino, di(Ci-C₄)alkylamino or a group having the structure -(CH₂)_a-R⁷ where a is 1, 2, 3, or 4, and R⁷ is hydroxy, Cj-C₄ alkoxy, carboxy, Cj-C₄ alkoxycarbonyl, amino, carbamoyl, C₁-C₄ alkylamino or di(Ci-C₄)alkylamino.

[0060] Examples of substituted heterocycles include 3-N-t-butyl carboxamide

decahydroisoquinolinyl, 6-N-t-butyl carboxamide octahydro-thieno[3,2-c]pyridinyl, 3- methylimidazolyl, 3-methoxypyridyl, 4-chloroquinolinyl, 4-aminothiazolyl, 8- methylquinolinyl, 6-chloroquinoxalinyl, 3-ethylpyridyl, 6-methoxybenzimidazolyl, 4- hydroxyfuryl, 4-methylisoquinolinyl, 6,8-dibromoquinolinyl, 4,8-dimethylnaphthyl, 2- methyl- 1 ,2,3 ,4-tetrahydroisoquinolinyl, N-methyl-quinolin-2-yl, 2-t-butoxycarbonyl- 1,2,3,4- isoquinolin-7-yl, and the like.

[0061] Exemplary heterocyclic ring systems represented by A or B include (1) 5- membered monocyclic ring groups, such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl, thiazolyl, and the like; (2) 6-membered monocyclic groups such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinly, triazinyl, and the like; and (3) polycyclic heterocyclic rings groups, such as decahydroisoquinolinyl, octahydro-thieno [3,2-c] pyridinyl, benzo[b]thienyl, naphtho[2,3-b]thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, and fully or partially saturated analogs thereof.

[0062] A cycloalkyl may be optionally substituted with 1 , 2 or 3 substituents

independently selected from halo, halo(Ci-C₄)alkyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, carboxy, C₁-C₄ alkoxycarbonyl, carbamoyl, N-(Ci-C₄)alkylcarbamoyl, amino, C₁-C₄ alkylamino, di(Ci-C₄)alkylamino or a group having the structure -(CH₂)_a-R⁷ where a is 1, 2, 3, or 4, and R⁷ is hydroxy, C₁-C₄ alkoxy, carboxy, C₁-C₄ alkoxycarbonyl, amino, carbamoyl, C₁-C₄ alkylamino or di(Ci-C₄)alkylamino. Exemplary substituted cycloalkyl groups include 3- methylcyclopentyl, 4-ethoxycyclohexyl, 5-carboxycyclo-heptyl, 6-chlorocyclohexyl, and the like.

[0063] Exemplary substituted hydrolyzable groups include N-benzyl glycyl, N-Cbz-L- valyl, and N-methyl nicotinate.

[0064] Exemplary compounds of formula (1) include those compounds of formula I in each of the incorporated applications Serial Nos. 08/137,254, 08/133,696, and 08/133,543 that fall within the scope of formula (1) as defined herein. Other exemplary compounds include those set forth in the claims of U.S. Pat. Nos. 5,484,926; 5,827,891; 5,834,467;

5,846,993; 5,952,343; and 6,271,235. The disclosure of each of these patents and patent applications is hereby incorporated by reference in its entirety, although in the case of any contradictions, the disclosure of the present application controls.

[0065] In one preferred embodiment, the compounds useful in the methods of the present invention have at least two asymmetric centers, each of which is denoted by an asterisk in the formula below:

[0066] As a consequence of these asymmetric centers, the compounds useful in the methods of the present invention can occur in any of the possible stereoisomeric forms, and can be used in mixtures of stereoisomers, which can be optically active or racemic, or can be used alone as essentially pure stereisomers, i.e., at least 95% pure. All asymmetric forms, individual stereoisomers and combinations thereof, are within the scope of the present invention.

[0067] The individual stereoisomers may be prepared from their respective precursors by the procedures described above, by resolving the racemic mixtures, or by separating the diastereomers. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known in the art. Further details regarding resolutions can be found in Jacques et al, Enantiomers, Racemates, and Resolutions, John Wiley & Sons 1981.

[0068] Preferably, the compounds useful in the methods of the present invention are substantially pure, i.e, over 50%> pure. More preferably, the compounds are at least 75% pure. Even more preferably, the compounds are more than 90% pure. Even more preferably, the compounds are at least 95% pure, more preferably, at least 97% pure, and most preferably, at least 99% pure. [0069] As used herein, a "pharmaceutical formulation," "pharmaceutical carrier,"

"pharmaceutical diluent," and "pharmaceutical excipient" is a formulation containing an HIV protease inhibitor or a formulation containing a combination of an HIV protease inhibitor and a carrier, diluent, excipient, or salt which is compatible with other ingredients of the formulation, and not deleterious to the recipient thereof.

[0070] As used herein, a "pharmaceutical composition" or "pharmaceutical formulation" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a "pharmaceutical composition" or "pharmaceutical formulation" is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.

[0071] As mentioned above, the methods of the instant invention include, for example, the pharmaceutically acceptable salts of the compounds defined by formula (1). A compound of this invention may possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.

[0072] The term "pharmaceutically acceptable salt(s)", as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the HIV protease inhibitors described herein. The HIV protease inhibitors described herein that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate,

gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1 , 1 '-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

[0073] A "pharmaceutically acceptable salt" of compounds of the above formula are substantially non-toxic to living organisms. Exemplary pharmaceutically acceptable salts include those salts prepared by reaction of the compounds useful in the methods of the present invention with a mineral or organic acid or an inorganic base. The reactants are generally combined in a mutual solvent, such as diethylether or benzene, for acid addition salts, or water or alcohols for base addition salts. The salts normally precipitate out of solution within about one hour to about ten days and can be isolated by filtration or other conventional methods. Such salts are known as acid addition and base addition salts.

[0074] Examples of pharmaceutically acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate,

methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, g-hydroxybutyrate, glycollate, tartrate, methane-sulfonate, propanesulfonate, naphthalene- 1 -sulfonate, napththalene-2-sulfonate, mandelate, and the like.

[0075] Acids that may be employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.

[0076] Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids, such as hydrochloric acid and hydrobromic acid, and those formed with organic acids, such as maleic acid and methanesulfonic acid. Methanesulfonic acid or mesylate is particularly preferred. [0077] Base addition salts include those derived from inorganic and organic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium

hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate and the like. The potassium and sodium salt forms are particularly preferred.

[0078] It should be recognized that the particular counterion forming a part of any salt of this invention is not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.

[0079] The terms "inhibit" or "inhibiting" mean prohibiting, treating, alleviating, ameliorating, halting, restraining, slowing or reversing the progression, or reducing the severity of a pathological symptom related to or resultant from a viral infection. As such, these methods include both medical therapeutic (acute) and/or prophylactic (prevention) administration as appropriate.

[0080] The term "a therapeutically effective amount" for treating a particular disease or infection means an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration may be required to achieve the desired amelioration of symptoms.

[0081] The term "treatment" means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use as antiviral agents.

[0082] The term "amelioration" of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

[0083] The term "subject" as used herein refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0084] In various embodiments, any HIV protease inhibitor, or combination thereof, may be used to practice the present invention. As used herein, HIV protease inhibitors prevent viral replication by selectively binding to viral proteases and blocking proteolytic cleavage of protein precursors that are necessary for the production of infectious viral particles.

Examples of HIV protease inhibitors include HIV-1 protease inhibitors, such as, but not limited to one more of the following: nelfinavir, ritonavir, saquinavir, indinavir, amprenavir, atazanvir, fosamprenavir, lopinavir, tipranavir, darunavir, ritonavir, lopinavir, simeprevir, telaprevir, and boceprevir. Additional examples of HIV protease inhibitors which may be utilized in practicing the present invention are those disclosed in U.S. Patent Nos. 5,952,343, 5,196,438, 5,541,206, 5,413,999, 5,484,926, 5,585,397, 5,914,332, 5,849,911, 6,248,775 and 7,671,032 and are incorporated herein by reference. In one embodiment the inhibitor is nelfinavir.

[0085] In general, HIV protease inhibitors have been determined to inhibit infectivity and reduce replication of picornaviruses. Picornaviruses are non-enveloped, positive-stranded R A viruses with an icosahedral capsid. The genome RNA is unusual because it has a protein on the 5' end that is used as a primer for transcription by RNA polymerase.

Picornaviruses are grouped into a number of genera. Most notably, HIV protease inhibitors are used to inhibit infection of viruses of the generas. In various embodiments, viruses targeted for treatment include those of the genera aphthovirus, cardiovirus, hepatovirus and enterovirus, including for example, rhinovirus, poliovirus, echovirus and coxsackievirus (coxsackie A viruses and coxsackie B viruses). In various embodiments, viruses targeted for treatment include enterovirus 71 (EV71), enterovirus 68 (EV68, EV-D68, HEV68), coxsackievirus A16 (CVA16 or CA16) and hepatitis A virus (HAV) 1.

[0086] Any number of general and standard viral assays can be used to identify protease inhibitors of particular interest. The following method is preferred but is not the only method available to those of ordinary skill.

[0087] Methods

[0088] Anti-viral assay. Vero cells (ATCC, cat. no. CCL-81) are grown in modified Eagle's medium (MEM) supplemented with 10% FBS and 3% L-glutamine at 37°C with 5% C0₂. The EV71BrCr (ATCC, VR-1775), EV71H (ATCC, VR-1432), CA16 (JGV, 95: 1083- 1093), or Hepatitis A virus (ATCC, VR-1402) viruses (100 TCID₅₀/ml, 0.1 ml) are used to infect Vero cells (10⁵) seeded in 24-well

in the absence or presence of various concentrations of drug for 7 days. The CPE of Vero cells are observed by light microscopy and cell counting. Cell supematants are detected by western blot using anti-EV71 VP1 antibodies.

[0089] Cell counting. Vero cells are first trypsinized for 5 minutes at 37°C and 5% C0₂ saturation, and then washed with DMEM supplemented with 10% FBS. The cell mixture is centrifuged at 1000 rpm for 5 minutes to obtain the cell pellet. The supernatant is discarded and the pellet resuspended in 1ml of the medium. Cells and 0.1% trypan blue are mixed in a 1 : 1 ratio and loaded onto a hemocytometer for enumeration. Cells that appear bright, devoid of the blue stain and with retention of their round morphology are considered live. Cells that appear blue are considered dead. Cell concentration (cells/ml) are determined using a standard formula.

[0090] Western blot analysis. Supernatant is harvested from the infected cell monolayers for determining the antiviral effect of the candidate drug. 60uL of Laemeli sample buffer is added to 180uL of each supernatant sample and boiled for 10 minutes to denature the proteins. All samples are vortexed thoroughly and subjected to electrophoresis using 0.75mm 12%) sodium dodecyl sulfate polyacrylamide gel. Samples along with the appropriate protein ladder are run at 130V for 70 minutes and subsequently the resolved proteins are transferred to a polyvinylidene difluoride (PVDF) membrane through a semi-dry transfer process. Blots are blocked with 5% BSA solution and then probed with a 1 : 1000 diluted mouse monoclonal antibody against EV71 VP1 protein overnight at 4°C. This is then followed by washing and labeling with a rabbit derived anti-mouse HRP conjugated secondary antibody. Blots are developed using NBT and BCIP solutions.

[0091] HIV protease inhibitors and pharmaceutically acceptable salts, solvates, esters and prodrugs thereof as discussed herein may be formed into appropriate dosage forms depending on the intended administration routes, and specifically prepared primarily into any one of the preparation forms including injections such as intravenous injection and intramuscular injection, preparations for oral administration such as hard or soft capsules, tablets, granules, powders, lozenges, pills, particulates, wafers, aqueous or oily suspensions, syrups, elixirs and troches, preparations for pulmonary inhalation, preparations for rectal administration, fatty suppositories, and aqueous suppositories.

[0092] The compounds of the present invention are of the formula (1):

wherein:

Qi and Q₂ are independently selected from hydrogen and substituted and

unsubstituted alkyl and aryl, and Qi and Q₂ may form a ring with G, Q₃ is selected from mercapto and substituted and unsubstituted alkoxyl, aryloxyl, thioether, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, and aryl,

Q₄-Qs are independently selected from hydrogen, hydroxyl, mercapto, nitro, halogen, -O-J, wherein J is a substituted or unsubstituted hydrolyzable group, and substituted and unsubstituted alkoxyl, aryloxyl, thioether, acyl, sulfmyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle and aryl, and further wherein any one of Q₄-(¾ may be a member of a spiro ring and any two of Q₄-(¾ may together be members of a ring,

Y and G are independently selected from oxygen, -NH, -N-alkyl, sulfur, selenium, and two hydrogen atoms,

D is carbon or nitrogen,

E is carbon or nitrogen,

Q is selected from hydrogen, halogen, hydroxyl, mercapto, and substituted and unsubstituted alkoxyl, aryloxyl, thioether, amino, alkyl, and aryl, wherein Q may form part of a ring,

A is a carbocycle or heterocycle, which is optionally further substituted, and B is a carbocycle or heterocycle, which is optionally further substituted, or a pharmaceutically acceptable salt thereof.

[0093] The invention also relates to compounds of formula (1), wherein all variables are the same as those defined above for formula (1) with the exception of D, which is carbon or nitrogen, and is singly bonded to each of the adjacent ring atoms.

[0094] The invention more particularly relates to preferred compounds of formula (1) wherein: at least one of Qi and Q₂ is substituted or unsubstituted alkyl and the other is as defined above,

Q₃ is selected from thioether and aryl, Q₄-Q₈ are independently selected from hydrogen, hydroxyl, halogen, -O-J, wherein J is a substituted or unsubstituted hydrolyzable group, and substituted and unsubstituted acyl, alkoxyl, amino and alkyl, and further wherein any one or more of Q₄-Qs may form part of a ring,

Y and G are each oxygen, D is nitrogen,

E is carbon or nitrogen, Q is hydrogen,

A is a carbocycle or heterocycle that is an aromatic or partially saturated, 5-7 membered mono-ring, which is optionally further substituted, and B is a heterocycle that is a saturated or partially saturated, 8-12 membered poly- ring, which is optionally further substituted, or a pharmaceutically acceptable salt thereof.

[0095] The invention even more particularly relates to compounds of the formula (1) wherein: one of Qi and Q₂ is substituted or unsubstituted alkyl, preferably t-butyl, and the other is hydrogen,

Q₃ is selected from thioaryl and aryl, preferably thiophenyl and phenyl, Q₄ is alkyl, preferably methyl,

Q₅ is hydroxyl or -O-J, wherein J is a hydrolyzable group, or substituted or unsubstituted alkoxyl or amino,

Q₆-Qs are independently selected from hydrogen, hydroxyl, halogen, -O-J, wherein J is a substituted or unsubstituted hydrolyzable group, and substituted and unsubstituted alkoxyl, acyl, amino and alkyl, and further wherein any one or more of Q₆-Qs may form part of a ring,

Y and G are each oxygen, D is nitrogen, E is carbon, Q is hydrogen,

A is a carbocycle that is an aromatic, 5-6 membered monocyclic ring, preferably phenyl, which is optionally further substituted, and B is a heterocycle that is a saturated, 6-14 membered monocyclic or polycyclic ring, which is optionally further substituted, preferably of the formula

wherein Mi and M₂ are independently selected from hydrogen, mercapto, hydroxyl, and substituted and unsubstituted thioether, alkyl, alkoxyl, aryloxyl, amino, five membered heterocycle and carbocycle, sulfmyl, sulfonyl, and acyl, and wherein Mi and M₂ optionally form a ring having up to 10 members, wherein preferably Mi and M₂ independently have from zero to eight non-hydrogen atoms; or a pharmaceutically acceptable salt thereof.

[0096] Preferred compounds of the formula (1) include those wherein: one of Qi and Q₂ is tertiary alkyl, preferably t-butyl, and the other is hydrogen, Q₃ is thiophenyl, phenyl, naphthyl, or thionaphthyl, Q₄ is methyl,

Q₅ is hydroxyl, amino, or -O-J, wherein J is a substituted or unsubstituted hydrolyzable group,

Y and G are each oxygen,

D is nitrogen,

E is carbon,

Q is hydrogen,

A is phenyl, which is optionally further substituted, and B is a heterocycle that is a saturated, 9-10 membered bi-ring, preferably decahydroisoquinolinyl or octahydrothieno[3,2,-c]pyridinyl, or a pharmaceutically acceptable salt thereof.

[0097] According to certain embodiments, the portion of formula (1):

is designated as Z or Z¹, and/or the portion of formula (1):

is designated as X or X¹.

[0098] According to certain of those embodiments, the compounds have the formula 1(A):

Z is a group having the structure

where: a is 1, 2, 3, 4, or 5; b is 1, or 2; c is 1, or 2; d is 1, 2, 3, or 4; each R is independently hydrogen, hydroxy, thiol, halo, amino, Cj-C₄ alkylamino, di(Ci-C4)alkylamino, nitro, carboxy, Ci-C₆ alkyl, Ci-C₆ alkoxy, Ci-C₆ alkylthio, halo(Ci- C₄)alkyl, hydroxy(Ci-C₄)alkyl, Ci-C₆ alkylthio(Ci-C6)alkyl, Ci-C₄ alkoxycarbonyl, carbamoyl, N-(Ci-C₄)alkylcarbamoyl, Ci-C₄ alkylsulfonyl, N,N-di-(Ci-C₄)alkylcarbamoyl, or Ci-C₄ alkylsulfonylamino;

A¹ and A² are independently -CH₂- or -N(R⁸)-;

A³ and A⁴ are independently -CH- or -N-; A⁵ and A⁶ are independently - CH₂- or -N(R⁹)-;

7 8

A and A are independently -CH- or -N-;

Q

R is hydrogen or C₁-C₄ alkyl;

R⁹ is hydrogen or C₁-C₄ alkyl;

R¹ is aryl, or -S-aryl;

X is a group having the structure:

R is hydrogen, C₁-C4 alkyl, or -CH₂-pyridyl; R is a group having the structure:

p is 4 or 5;

R⁴ at each occurrence is independently hydrogen, Ci-C₆ alkyl or hydroxy(Ci- C₄)alkyl; and

R⁵ and R⁶ are independently selected from hydrogen, hydroxy, Ci-C₆ alkyl, Ci-C₆ alkoxy, or hydroxy(Ci-C₄)alkyl; with the provisos that:

(1) one of A¹ and A² must be -N(R⁸)-;

(2) A¹ and A² cannot both be -N(R⁸)-;

(3) A³ and A⁴ cannot both be -N-;

(4) one of A⁵ and A⁶ must be -N(R⁹)-;

(5) A⁵ and A⁶ cannot both be -N(R⁹)-;

(6) A⁷ and A⁸ cannot both be -N-; or a pharmaceutically acceptable salt thereof.

[0099] According to certain of those embodiments, the compounds have the formula 1(B):

wherein:

R¹ is aryl, or -S-aryl;

X¹ is a group having the formula:

₂

R is hydrogen, halo, or C₁-C4 alkyl; R is a group having the structure:

p is 4 or 5;

R⁴ at each occurrence is independently hydrogen, Ci-C₆ alkyl or hydroxy (Ci- alkyl; and

R⁵ and R⁶ are independently selected from hydrogen, hydroxy, Ci-C₆ alkyl, Ci alkoxy, or hydroxy (C₁-C4) alkyl;

Z¹ is a group having the structure:

where: a is 1, 2, 3, 4, or 5; b is 1, or 2; c is 1, or 2; d is 1, 2, 3, or 4; each R⁷ is independently hydrogen, hydroxy, thiol, halo, amino, C₁-C₄ alkylamino, di(Ci-C₄)alkylamino, nitro, carboxy, Ci-C₆ alkyl, Ci-C₆ alkoxy, C₁-C₄ alkylthio, halo(Ci- C₄)alkyl, hydroxy(Ci-C₄)alkyl, C₁-C₄ alkylthio(Ci-C₄)alkyl, C₁-C₄ alkoxycarbonyl, carbamoyl, N-(Ci-C₄)alkylcarbamoyl, C₁-C₄ alkylsulfonyl, N,N-di(Ci-C₄)alkylcarbamoyl, or C₁-C₄ alkylsulfonylamino;

A¹ and A² are independently -CH₂- or -N(R⁸)-;

A³ and A⁴ are independently -CH- or -N-;

A⁵ and A⁶ are independently -CH₂ - or -N(R⁹)-;

7 8

A and A are independently -CH- or -N-;

Q

R is hydrogen or Cj-C₄ alkyl; R⁹ is hydrogen or Cj-C₄ alkyl; T² is hydrogen, or C₁-C4 alkyl; with the provisos that:

(1) one of A¹ and A² must be -N(R⁸)-;

(2) A¹ and A² cannot both be -N(R⁸)-;

(3) A³ and A⁴ cannot both be -N-;

(4) one of A⁵ and A⁶ must be -N(R⁹)-;

(5) A⁵ and A⁶ cannot both be -N(R⁹)-;

[00100] Preferred species of the formula (1) include: [3S-(3R*,4aR*,8aR*,2'S*,3*S*)]-2- [2'-hydroxy-3'-phenylthiomethyl-4'-aza-5' -oxo-5'-(2"-methyl-3"-hydroxyphenyl)pentyl] decahydroisoquinoline-3-N-t-butylcarboxamide and its pharmaceutically acceptable salts, especially its methanesulfonic acid salt, and its prodrug analogs, wherein the 3" hydroxy is converted to -O-J, as defined above, especially the dihydrogen phosphate hydrochloride salt; and [6S-(6R^!i:,3aS^!i:,7aR^!i:,2^,S^!i:,3^,S^!i:)]-2-[2^,-hydroxy-3^,-phenylthiomethyl-4^,aza-5^,- oxo-5'-(2"- methyl-3"-hydroxyphenyl)pentyl]-octahydro-thieno[3,2-c]pyridine-6-N-t-butylcarboxamide and its pharmaceutically acceptable salts, especially methanesulfonic acid salt, and its prodrug analogs, wherein the 3" hydroxy is converted to -O-J, as defined above. [00101] The present invention further provides methods comprising the use of pharmaceutical formulations comprising an effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, such as a diluent or excipient.

[00102] Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

[00103] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[00104] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[00105] Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[00106] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[00107] Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavoring agents. [00108] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a nontoxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are

conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[00109] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.01 mg/g of body weight of an HIV protease inhibitor. In other embodiments, the drug may comprise between about 0.1% to about 75% of the weight of the unit, or between about 2% to about 20%, for example, and any range derivable therein. In other non- limiting examples, a dose may also comprise from about 1 μg/kg/body weight, about 100 μg/kg/body weight, about 500 μg/kg/body weight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 300 mg/kg/body weight, about 350 mg/kg/body weight, about 400 mg/kg/body weight, about 450 mg/kg/body weight, about 500 mg/kg/body weight, about 600 mg/kg/body weight, about 700 mg/kg/body weight, about 800 mg/kg/body weight, about 900 mg/kg/body weight, about 1000 mg/kg/body weight, about 2000 mg/kg/body weight to about 5000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 350 mg/kg/body weight to about 1000 mg/kg/body weight, about 50 μg/kg/body weight to about 500 mg/kg/body weight, and the like, can be administered. [00110] HIV protease inhibitors are useful as antiviral agents and, thus, may be used in methods to prevent or treat viral infections in animals. Treatment typically includes administering a pharmaceutically effective amount of an HIV protease inhibitor to a subject in need of such treatment, thereby inhibiting viral growth or replication in the patient. Such a composition typically contains from about 0.1 to 90% by weight (such as 1 to 20% or 1 to 10%) of an HIV protease inhibitor.

[00111] The methods of the present invention prevent or treat a viral infection in a subject by administering a therapeutically effective amount of a HIV protease inhibitor. A

"therapeutically effective amount" or "efficacious amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, and the like, of the subject to be treated. Typically a therapeutically effective amount should produce a serum concentration of drug of from about 0.1 ng/ml to about 50-100 μg/ml. In various embodiments, the dosage administered is sufficient to result in a serum concentration level of the drug in the subject of greater than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 μΜ. The pharmaceutical compositions typically should provide a dosage of from about 0.01 mg to about 2000 mg of compound per kilogram of body weight per day. The drug may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. [00112] In various aspects, individuals who have been clinically diagnosed as infected with a virus, particularly a picornavirus, are suitable for treatment with the methods of the present invention. Individuals who are infected with a picornavirus are generally identified

(diagnosed) as having a picornavirus R A in their blood, and/or having an anti-picornavirus antibody in their serum. In some embodiments, individuals of interest for treatment according to the invention have detectable picornavirus titer indicating active viral replication, they may also have a picornavirus titer of at least about 10⁵, at least about 5 X 10⁵, or at least about 10⁶, or greater than 2 million genome copies of picornavirus per milliliter of serum.

[00113] Whether a subject method is effective in treating a virus infection, can be determined by measuring viral load, or by measuring a parameter associated with virus infection. Viral load can be measured by measuring the titer or level of virus in serum. These methods include, but are not limited to, a quantitative polymerase chain reaction (PCR) and a branched DNA (bDNA) test. For example, quantitative assays for measuring the viral load (titer) of picornavirus RNA have been developed.

[00114] In another aspect, the present invention provides a kit for treating or preventing a picornavirus infection. The kit includes a HIV protease inhibitor as described herein and instructions for administering the inhibitor to a subject.

[00115] The kit may also be diagnostic in nature and include reagents for detection of picornavirus infection. Such reagents include for example, molecules, e.g., antibodies or fragments thereof, that specifically bind to a member of the picornavirus family, or product thereof. The molecule may be immobilized on a suitable solid support. The kit, may also include reagents for staining picornaviruses as well.

[00116] As such, in various aspects, the methods of the present invention may include determining whether an individual suffers from a picornavirus related disease, comprising obtaining a sample from said individual and detecting a picornavirus or functional part, derivative or analogue thereof in the sample by hybridizing and/or amplifying a nucleic acid of the virus or functional part, derivative or analogue with a primer and/or probe and detecting hybridized and/or amplified product.

[00117] In various aspects, the methods of the present invention may include determining whether an individual suffers from an picornavirus related disease, comprising obtaining a sample from said individual and detecting a picornavirus virus or functional part, derivative or analogue thereof in said sample by specifically binding of said virus or functional part, derivative or analogue with an antibody and detecting bound product.

[00118] The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE 1

HIV PROTEASE INHIBITORS AGAINST EV71 INFECTION IN-VITRO

[00119] Current HIV protease inhibitors are FDA approved, are safe, well tolerated at different doses for both adults and children and have established pharmacokinetic data. Additionally, present 3C protease inhibitors have shown marginal pharmacokinetics. Thus, we evaluated the protective efficacy of HIV- 1 protease inhibitors (HIV Pis) against EV71 infection in vitro.

[00120] EV71, belongs to the enterovirus genus of the Picornaviridae family and has a single stranded positive sense RNA genome. EV71 is the etiological agent for Hand, Foot & Mouth Disease, a recurring epidemic that affects millions in the Asia-Pacific region. It's primarily seen in children <5yrs of age and is transmitted through the oro-fecal route.

Currently no specific treatment and vaccine options are available.

[00121] EV71 attaches to B2 scavenger receptor on the host cell for invasion. Once the genome is released in the cytoplasm, it can be translated into a single polyprotein with 3 regions (pl,p2,p3). These are further processed by different viral proteases to yield structural and nonstructural proteins. [00122] Possible mechanisms of inhibition are diagrammed in Figure 1 A. One plausible mechanism is virus associated in that that drug acts on 3 C or 2 A protease of EV71. Another plausible mechanism is host cell mediated in that the drug may act on a cellular signaling pathway, induce inhibition and apoptosis of infected cells or affect cellular factors.

[00123] Experimental methodology is illustrated in Figure 2. Briefly, vero cells are infected with EV71 virions. HIV-1 protease inhibitor is introduced to infected vero cells and free virion formation is measured.

[00124] 5 HIV protease inhibitors were screened. Nelfinavir exhibited inhibition similar to rupintrivir, a human rhinovirus (HRV) 3C protease inhibitor (positive control). In case of H strain, inhibition was seen in both rupintrivir and nelfinavir (Figure 3) as indicated by the lack of Vpl band and marginal CPE. Similar inhibition was seen against EV71 BrCR strain (Figure 4). Nelfinavir displayed protective activity against both strains of EV71 infection in vitro. It's activity was found to be comparable to rupintrivir.

[00125] Nelfinavir (10 μΜ) was also shown to completely block EV71 inducted cytopathic effect (CPE) in vero cells (Figure 7). Additionally, nelfinavir (10 μΜ) was shown to completely block EV71 replication in vero cells as detected by monitoring major viral protein VP1 in culture supernatants (Figure 8). Finally, nelfinavir (4 μΜ) was also shown to completely block EV71 replication in vero cells as detected by monitoring major viral protein VP1 in culture supernatants. Even at concentrations of 1 μΜ nelfinavir blocked EV71 replication by more than 50% (Figure 9). Notably patients treated with nelfinavir typically have a serum concentration of the drug of greater than about 15 μΜ.

EXAMPLE 2

HIV PROTEASE INHIBITORS AGAINST COXSACKIEVIRUS A16 (CA16)

INFECTION IN-VITRO

[00126] Using the methodology of Example 1, nelfinavir was tested for its ability to inhibit CA16 infection in vero cells. Nelfinavir (10 μΜ) was shown to completely block CA16 induced CPE in vero cells (Figures 1 OA- IOC). EXAMPLE 3

HIV PROTEASE INHIBITORS AGAINST HEPATITIS A VIRUS (HAV) AND EV-

D68 INFECTION IN-VITRO

[00127] Using the methodology of Example 1, nelfmavir was tested for its ability to inhibit HAV infection in vera cells. Nelfmavir (10 μΜ) was shown to completely block HAV induced CPE in vera cells (Figures 1 lA-11C).

[00128] Using the methodology of Example 1, nelfmavir was also tested for its ability to inhibit EV-D68 (Fermon strain, ATCC VR-561) infection in HeLa cells. Nelfmavir (2 μΜ) was observed to block EV-D68 induced CPE in HeLa cells.

[00129] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A method of treating picomavirus infection in a subject comprising administering to the subject a therapeutically effective amount of an HIV protease inhibitor, thereby treating the picomavirus infection.

2. The method of claim 1, wherein the picomavirus is selected from the group consisting of aphthovirus, cardiovirus, hepatovirus and enterovirus.

3. The method of claim 1, wherein the picomavirus is an enterovirus selected from rhino vims, polio vims, echo vims and coxsackievims.

4. The method of claim 3, wherein the picomavims is enterovims 71 (EV71), enterovims 68 (EV68, EV-D68, HEV68), coxsackievims A16 (CVA16 or CA16) or hepatitis A vims (HAV) 1.

5. The method of claim 1, wherein the subject is a mammal.

6. The method of claim 5, wherein the mammal is a human.

7. The method of claim 1, wherein the HIV protease inhibitor is selected from the group consisting of nelfinavir, ritonavir, indinavir, amprenavir, atazanvir, and lopinavir.

8. The method of claim 7, wherein the HIV protease inhibitor is nelfinavir.

9. The method of claim 1, wherein the HIV protease inhibitor is administered in a dosage of about 0.0001-1000 mg/kg, about 0.01-1000 mg/kg, or about 0.1-100 mg/kg.

10. The method of claim 1, wherein the HIV protease inhibitor is administered orally.

11. The method of claim 1 , wherein the HIV protease inhibitor is administered daily, weekly, biweekly or monthly.

12. The method of claim 11, wherein the HIV protease inhibitor is administered at least once or twice daily.

13. A method of preventing picomavirus infection in a subject comprising administering to the subject a therapeutically effective amount of an HIV protease inhibitor, thereby preventing the picomavirus infection.

14. The method of claim 13, wherein the picomavirus is selected from the group consisting of aphthovirus, cardiovirus, hepatovirus and enterovirus.

15. The method of claim 13, wherein the picomavirus is an enterovirus selected from rhino vims, polio vims, echo vims and coxsackievims.

16. The method of claim 15, wherein the picomavims is enterovims 71 (EV71), enterovims 68 (EV68, EV-D68, HEV68), coxsackievims A16 (CVA16 or CA16) or hepatitis A vims (HAV) 1.

17. The method of claim 13, wherein the subject is a mammal.

18. The method of claim 17, wherein the mammal is a human.

19. The method of claim 13, wherein the HIV protease inhibitor is selected from the group consisting of nelfinavir, ritonavir, indinavir, amprenavir, atazanvir, and lopinavir.

20. The method of claim 19, wherein the HIV protease inhibitor is nelfinavir.

21. The method of claim 13, wherein the HIV protease inhibitor is administered in a dosage of about 0.0001-1000 mg/kg, about 0.01-1000 mg/kg, or about 0.1-100 mg/kg.

22. The method of claim 13, wherein the HIV protease inhibitor is administered orally.

23. The method of claim 13, wherein the HIV protease inhibitor is administered daily, weekly, biweekly or monthly.

24. The method of claim 23, wherein the HIV protease inhibitor is administered at least once or twice daily.

25. A method of inhibiting picomavirus replication comprising contacting a picomavirus with a HIV protease inhibitor, thereby inhibiting replication of the picomavirus.

26. The method of claim 25, wherein the picomavirus is selected from the group consisting of aphthovirus, cardiovirus, hepatovirus and enterovirus.

27. The method of claim 25, wherein the picomavirus is an enterovirus selected from rhino vims, polio vims, echo vims and coxsackievims.

28. The method of claim 27, wherein the picomavims is enterovims 71 (EV71), enterovims 68 (EV68, EV-D68, HEV68), coxsackievims A16 (CVA16 or CA16) or hepatitis A vims (HAV) 1.

29. The method of claim 25, wherein the HIV protease inhibitor is selected from the group consisting of nelfinavir, ritonavir, indinavir, amprenavir, atazanvir, and lopinavir.

30. The method of claim 29, wherein the HIV protease inhibitor is nelfinavir.

31. The method of claim 25, wherein the HIV protease inhibitor is present at a concentration of about 0.001-1000 μΜ, about 0.01-1000 μΜ, or about 0.1-100 μΜ.

32. A method of treating a picomavims mediated disease in a subject comprising administering to the subject a therapeutically effective amount of an HIV protease inhibitor, thereby treating the picomavims mediated disease.

33. The method of claim 32, wherein the picomavirus is selected from the group consisting of aphthovirus, cardiovirus, hepatovirus and enterovirus.

34. The method of claim 32, wherein the picomavirus is an enterovirus selected from rhino vims, polio vims, echo vims and coxsackievims.

35. The method of claim 34, wherein the picomavims mediated disease is heart disease, diabetes, hepatitis, viral cold, food and mouth disease or poliomyelitis.

36. The method of claim 32, wherein the HIV protease inhibitor is selected from the group consisting of nelfinavir, ritonavir, indinavir, amprenavir, atazanvir, and lopinavir.

37. The method of claim 36, wherein the HIV protease inhibitor is nelfinavir.

38. The method of claim 32, wherein the HIV protease inhibitor is administered at a dosage resulting in a semm concentration of about 0.001-1000 μΜ, about 0.01-1000 μΜ, or about 0.1-100 μΜ.

39. A kit for treating or preventing a picomavims infection comprising:

a) an HIV protease inhibitor; and b) instmctions for administering the inhibitor to a subject.

40. The kit of claim 39, wherein the picomavims is selected from the group consisting of aphthovims, cardiovims, hepatovims and enterovims.

41. The kit of claim 39, wherein the picomavims is an enterovims selected from rhinovims, poliovims, echovims and coxsackievims.

42. The kit of claim 41, wherein the picomavirus is enterovirus 71 (EV71), enterovirus 68 (EV68, EV-D68, HEV68), coxsackievirus A16 (CVA16 or CA16) or hepatitis A virus (HAV) 1.

43. The kit of claim 39, wherein the HIV protease inhibitor is selected from the group consisting of nelfinavir, ritonavir, indinavir, amprenavir, atazanvir, and lopinavir.

44. The kit of claim 43, wherein the HIV protease inhibitor is nelfinavir.

45. The kit of claim 39, wherein the HIV protease inhibitor formulated for oral administration.

46. The kit of claim 39, wherein the instructions specify that the HIV protease inhibitor is administered in a dosage resulting in a serum concentration of the subject of about 0.001- 1000 μΜ, about 0.01-1000 μΜ, or about 0.1-100 μΜ.

47. The kit of claim 32, wherein the wherein the instructions specify that the HIV protease inhibitor is administered daily, weekly, biweekly or monthly.

48. The kit of claim 39, further comprising reagents for detecting the picomavirus.

49. The method according to claim 1, 13 or 32, further comprising detecting the picomavirus.

50. The method of claim 49, wherein detecting is performed before administering the HIV protease inhibitor.

51. The method of claim 49, wherein detecting is performed after administering the HIV protease inhibitor.

52. The method of claim 1, wherein the HIV protease inhibitor is a compound of the formula:

wherein:

Qi and Q₂ are each independently selected from hydrogen and substituted and unsubstituted alkyl and aryl;

Q₃ is selected from mercapto and substituted and unsubstituted alkoxyl, aryloxyl, thioether, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, and aryl;

Q₄, Qs, Q₆, Q₇, and Qs are each independently selected from hydrogen, hydroxyl, mercapto, nitro, halogen, -O-J, where J is a substituted or unsubstituted hydrolyzable group, and substituted and unsubstituted alkoxyl, aryloxyl, thioether, sulfmyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, aryl, and L₆C(0)L4, where L.sub.6 is a single bond, -O or -N, and further where L₄ is alkyl, hydroxyl, alkoxyl or hydrogen; and further wherein any one or more of Q₄, Q₅, Q₆, Q₇, and Qs may be a member of a spiro ring, and any two of Q₄, Q₅, Q₆, Q₇, and Qs may together be members of a ring;

E is carbon or nitrogen;

Q is selected from hydrogen, halogen, hydroxyl, mercapto, and substituted and unsubstituted alkoxyl, aryloxyl, thioether, amino, alkyl, and aryl, where Q may form part of a ring;

is a monocyclic or polycyclic carbocycle or heterocycle, which is optionally further substituted, and when

is heterocycle, each heterocyclic ring has one to three heteroatoms independently selected from nitrogen, oxygen, and sulfur; and

is a 5-membered monocyclic heterocycle optionally having one or two additional heteroatoms independently selected from nitrogen, oxygen and sulfur, which is optionally further substituted;

or a prodrug or pharmaceutically acceptable salt of said compound.

53. The method of claim 1, wherein the HIV protease inhibitor is a compound of the formula:

wherein: Qi and Q₂ are independently selected from hydrogen and substituted and unsubstituted alkyl and aryl, and Qi and Q₂ may form a ring with G;

Q4-Q8 are independently selected from hydrogen, hydroxyl, mercapto, nitro, halogen, -O-J, wherein J is a substituted or unsubstituted hydrolyzable group, and substituted and unsubstituted alkoxyl, aryloxyl, thioether, sulfmyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, aryl, and L₆C(0)L₄, wherein L₆ is a single bond, -O or -N, and further wherein L₄ is alkyl, hydroxyl, alkoxyl or hydrogen; and further wherein any one or more of Q₄-Qs may be a member of a spiro ring and any two of Q₄-Qs may together be members of a ring;

Y and G are independently selected from oxygen, -N-H, -N-alkyl, sulfur, selenium, and two hydrogen atoms;

D is nitrogen;

E is carbon or nitrogen;

Q is selected from hydrogen, halogen, hydroxyl, mercapto, and substituted and unsubstituted alkyoxyl, aryloxyl, thioether, amino, alkyl, and aryl, wherein Q may form part of a ring;

A is a carbocycle or heterocycle, which is optionally further substituted, and when A is heterocycle, each ring has from one to three hetero atoms independently selected from nitrogen, oxygen, and sulfur; and

B is

wherein Bi and B₂ form part of a ring having 6 to 10 members, which ring is optionally further substituted and optionally has from one to three hetero atoms indendently selected from nitrogen, oxygen, and sulfur;

or a pharmaceutically acceptable salt thereof.

54. The method of claim 1, wherein the HIV protease inhibitor is a compound of the formula:

wherein:

Q₃ is substituted or unsubstituted aryl or thioether; Q4, Qs, Q₆, Q7, and Qs are each independently selected from hydrogen, hydroxyl, mercapto, nitro, halogen, -O-J, where J is a substituted or unsubstituted hydrolyzable group, and substituted and unsubstituted alkoxyl, aryloxyl, thioether, sulfmyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, aryl, and L₆C(0)L₄, wherein L₆ is a single bond, -O or -N, and further where L₄ is alkyl, hydroxyl, alkoxyl or hydrogen; and further wherein any two of Q₄, Q₅, Q₆, Q7, and Qs may together be members of a ring; and

Bi and B₂ form part of a ring having 6 to 10 members, which ring is optionally substituted and optionally has from one to three hetero atoms independently selected from nitrogen, oxygen, and sulfur;

or a prodrug or pharmaceutically acceptable salt of said compound.

55. The method of claim 1, wherein the HIV protease inhibitor is a compound of the formula:

wherein:

Q3 is selected from mercapto and substituted and unsubstituted alkoxyl, aryloxyl, thioether, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, and aryl;

Q4, Qs, Q6, Q7, and Qs are each independently selected from hydrogen, hydroxyl, mercapto, nitro, halogen, -O-J, where J is a substituted or unsubstituted hydrolyzable group, and substituted and unsubstituted alkoxyl, aryloxyl, thioether, sulfmyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, aryl, and L₆C(0)L4, where L₆ is a single bond, -O or -N, and further where L₄ is alkyl, hydroxyl, alkoxyl or hydrogen; and further wherein any one or more of Q₄, Q₅, Q₆, Q₇, and Qs may be a member of a spiro ring, and any two of Q₄, Q₅, Q₆, Q7, and Qs ub.8 may together be members of a ring;

E is carbon or nitrogen;

Q⁹ is selected from hydrogen, halogen, hydroxyl, mercapto, and substituted and unsubstituted alkoxyl, aryloxyl, thioether, amino, alkyl, and aryl, where Q⁹ may form part of a ring;

is a 6-membered monocyclic carbocycle or heterocycle, which is optionally further substituted, and

when

is heterocycle, the ring has one to three heteroatoms independently selected from nitrogen, oxygen and sulfur, which is optionally further substituted; wherein at least one of

is a heterocycle;

or a prodrug or pharmaceutically acceptable salt of said compound.

The method of claim 3, 15, 27 or 34, wherein the picomavirus is rhinovirus.

The method of claim 3, 15, 27 or 34, wherein the picomavirus is poliovirus.

The kit of claim 41, wherein the picomavirus is rhinovirus.

The kit of claim 41, wherein the picomavirus is poliovirus.