WO2018222351A1

WO2018222351A1 - Triptolide lactone derivatives as immunomodulators and anticancer agents

Info

Publication number: WO2018222351A1
Application number: PCT/US2018/031598
Authority: WO
Inventors: John H. Musser
Original assignee: Pharmagenesis, Inc.
Priority date: 2017-05-27
Filing date: 2018-05-08
Publication date: 2018-12-06

Abstract

Disclosed herein are triptolide derivative compounds, and compositions comprising said compounds, as well as methods of making and methods of using said compounds and compositions, which are medicinally and pharmaceutically useful as antiproliferative, anticancer and immunosuppressive therapeutics.

Description

TRIPTOLIDE LACTONE DERIVATIVES

AS IMMUNOMODULATORS AND ANTICANCER AGENTS

TECHNICAL FIELD

[0001] The present disclosure relates generally to medicinally useful and pharmaceutically acceptable compositions as immunosuppressive, anti-inflammatory and anticancer agents, as well as to methods of making and methods of using said compositions.

INTRODUCTION

[0002] Immunosuppressive agents are widely used in the treatment of autoimmune disease and in treating or preventing transplantation rejection, including the treatment of graft-versus-host disease (GVHD). Common immunosuppressive agents include azathioprine, corticosteroids, cyclophosphamide, methotrexate, 6-mercaptopurine, vincristine, and cyclosporin A. In general, none of these drugs are completely effective, and most are limited by severe toxicity. For example, cyclosporin A, a widely used agent, is significantly toxic to the kidney. In addition, doses needed for effective treatment may increase the patient's susceptibility to infection by a variety of opportunistic invaders.

[0003] The compound triptolide, obtained from the Chinese medicinal plant Tripterygium wilfordii (TW), and certain derivatives and prodrugs thereof, have been identified as having immunosuppressive activity, e.g. in the treatment of autoimmune disease, and in treating or preventing transplantation rejection, including the treatment of graft-versus-host disease (GVHD). See, for example, U.S. Patent Nos. 5,962,516 (Immunosuppressive compounds and methods), 5,843,452 (Immunotherapy composition and method), 5,759,550 (Method for suppressing xenograft rejection), 5,663,335 (Immunosuppressive compounds and methods), 5,648,376 (Immunosuppressant diterpene compound), and 6,150,539 (Triptolide prodrugs having high aqueous solubility), each of which is herein incorporated by reference in its entirety. Triptolide and certain derivatives and prodrugs thereof have also been reported to show anticancer activity; see, for example, Kupchan et al., 1972, 1977, as well as U.S. Patent No. 6,620,843, each of which is hereby incorporated by reference in its entirety.

[0004] Thus, despite a history of preparations of derivatives and prodrugs of triptolide having beneficial pharmacokinetics and/or biodistribution, e.g. by virtue of differences in lipid or aqueous solubility, or via their activity as prodrugs, the bioactivity and usefulness of triptolide derivatives is often significantly reduced as compared to native triptolide. A need remains for the claimed medicinally useful compositions described herein. [0005] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.

BRIEF SUMMARY

[0006] In some aspects, the present disclosure provides compounds useful for immunosuppressive, anti-inflammatory and/or anticancer therapies, wherein the compounds are derivatives of triptolide represented by formula I:

where

Ri = lower arylacyl, or monsaccharidylacyl;

R2 = OH, F or taken together with R₃ to = O; and

X = H or OH, with the proviso that not more than one X = OH within the same structure.

[0007] In some aspects, the present disclosure provides compounds useful for immunosuppressive, anti-inflammatory and/or anticancer therapies, wherein the compounds are derivatives of triptolide represented by structure II:

Ri = lower arylacyl, or monsaccharidylacyl;

R2 = OH, F or taken together with R₃ to = O; and

X = H or OH, with the proviso that not more than one X = OH within the same structure. [0008] In some aspects, the present disclosure provides compounds useful for immunosuppressive, anti-inflammatory and/or anticancer therapies, wherein the compounds are derivatives of triptolide represented by structure III:

where

Ri = lower arylacyl, or monsaccharidylacyl;

R2 = OH, F or taken together with R3 to = O; and

X = H or OH with the proviso that not more than one X = OH within the same structure.

[0009] In some aspects, a composition is provided, said composition comprising the derivative of triptolide having the structure I, structure II or structure III.

[0010] In some aspects, a method of producing the triptolide derivative having the structure I, structure II or structure III is provided, said method described in detail in the detailed description set forth below.

[0011] In some aspects, the present disclosure provides a method of effecting immunosuppression in a subject, by administering to a subject in need of such treatment, an effective amount of a compound having the structure I, structure II or structure III as described above. In some aspects, a method to modulate the immune system is provided. In some aspects, a method of treating immune system dysfunction is provided, the method comprising orally administering to a mammal an amount of the triptolide derivative composition effective to treat immune system dysfunction.

[0012] In some aspects, the present disclosure provides a method of inducing apoptosis in a cell, which is useful in antiproliferative therapy, especially anticancer therapy. In accordance with this method, the cell is contacted with an effective amount of a compound having structure I, structure II or structure III as described above. Alternatively, the present disclosure encompasses the use of a compound of structure I, structure II or structure III for effecting immunosuppression or for inducing apoptosis in a cell, or for preparation of a medicament for effecting immunosuppression or for inducing apoptosis in a cell. The compound is typically provided in a pharmaceutically acceptable carrier. Specific embodiments of the methods and uses may employ any of the specific embodiments of structure I, structure II or structure III described above.

[0013] In some embodiments, the method further comprises administering at least one additional therapeutic agent in combination with the triptolide derivative composition.

[0014] In some embodiments, the mammal is a human.

[0015] Additional embodiments of the presently disclosed methods and compositions, and the like, will be apparent from the following detailed description, drawings, examples, and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present disclosure. Additional aspects and advantages of the present disclosure are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.

DETAILED DESCRIPTION

[0016] Various aspects and embodiments of the present disclosure are set forth and described more fully hereinbelow. Such aspects are meant to be exemplary and illustrative, not limiting in scope, and may be embodied in many different forms; these aspects and embodiments are not to be construed as limited to those explicitly set forth herein. Rather, these aspects and embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0017] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g. ; A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Morrison and Boyd, Organic Chemistry (Allyn and Bacon, Inc., current edition); J. March, Advanced Organic Chemistry (McGraw Hill, current edition); Remington: The Science and Practice of Pharmacy, A. Gennaro, Ed., 20^th Ed.; Goodman & Oilman The Pharmacological Basis of Therapeutics, J. Griffith Hardman, L. L. Limbird, A. Gilman, 10^th Ed. "Synthesis of peptides and peptidomimetics" Methods of organic chemistry (Houben-Weyl): additional and supplementary volumes to the 4th edition, 2004, Goodman, Murray; Toniolo, Claudio; Moroder, Luis; Felix, Aurthur; Thieme Medical Publishers Inc.

I. Definitions

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the methodologies and materials reported in the publications which might be used in connection with the present disclosure.

[0019] As used in this specification and in the appended claims, the following terms are intended to have the following meanings:

[0020] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" includes a single molecule as well as two or more of the same kind of molecule, reference to an "excipient" includes a single excipient as well as two or more excipients, and the like.

[0021] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure; each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included. For example, if a range of 1% to 8% is stated, it is intended that 2%, 3%, 4%, 5%, 6%, and 7% are also explicitly disclosed, as well as the range of values greater than or equal to 1% and the range of values less than or equal to 8%. Similarly, if a range of 1 μιη to 8 μιη is stated, it is intended that 2 μιη, 3 μιη, 4 μιη, 5 μιη, 6 μιη, and 7 μιη are also explicitly disclosed, as well as the range of values greater than or equal to 1 μιη and the range of values less than or equal to 8 μιη.

[0022] The compounds provided in the present disclosure can be prepared from triptolide or 14- fluorotriptolide, using various schemes presented hereinbelow.

[0023] The phrase "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or RNA molecule in single or double stranded form, particularly a DNA encoding a protein or protein fragment according to the present disclosure. An "isolated nucleic acid sequence" refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.

[0024] The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, three- dimensional structure or origin. Thus, a "fragment" or "portion" of a protein may still be referred to as a "protein" or may be referred to as a "polypeptide" or a "peptide." An "isolated protein" is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

[0025] The terms "purified," "substantially purified," and "isolated," as used herein, refer to the state of being substantially free of other side-products or byproducts of triptolide, or similar or dissimilar compounds from which the triptolide derivative was synthesized. Preferably, "purified," "substantially purified," and "isolated" mean that the composition comprises at least 0.5%, 1%, 5%, 10%, 20%, 30% or 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the mass, by weight, of a given triptolide derivative. In some embodiments, these terms refer to compositions in which the triptolide derivative comprises at least 95% of the mass, by weight, of a given sample. Any degree of purification or concentration greater than that which occurs after the synthetic reaction, including the purification away from other associated structures or compounds are within the meaning of "isolated." The triptolide derivative compositions described herein may be isolated from or otherwise mixed or associated with structures, enantiomers, salt forms or other compounds with which they are not normally associated, according to a variety of methods and processes known to those of skill in the art.

[0026] Further, the term "substantially purified," as used herein, refers to isolated or separated triptolide derivative compositions that are removed from their chemical synthesis environment, and are at least 60% free, at times 75% free, at times 90% free, at times 95% free and at times 99% free from other components with which they were otherwise or previously associated or have become associated prior to the purification process.

[0027] The term "pharmaceutical grade" means that certain specified biologically active and/or inactive components in a drug composition comprising the triptolide derivative compounds disclosed herein must be within a certain specified absolute and/or relative concentration range and/or that the components must exhibit certain activity levels as measured by a disease-, disorder- or condition-specific bioactivity assay. The disease, disorder or condition may afflict a human or an animal. [0028] As used herein, "components" means discrete compounds (i.e. chemicals) which either are present naturally in a composition or have been added to the composition so as to prepare a pharmaceutical grade composition having components within a defined bioactivity range(s) and/or efficacy range.

[0029] "Active component(s)" refers to one or more component(s) to which the observed activity in a disease-specific bioassay is attributed, and accounts for a substantial portion of the observed activity of the composition. In some embodiments, the summation of the active components' activities accounts for the majority (greater than 50%) of the observed biological activity.

[0030] As used herein, "fractions" typically refers to a group of components or a class of structurally similar compounds having similar parameters and/or similar measureable characteristics, such as solubility, molecular weight range, polarity range, adsorption coefficients, binding characteristics, chemical reactivity or selective solubility. Fractions may comprise a class of related components. Most frequently, fractions will be the product of chromatographic separation techniques, i.e., flash chromatography, preparative high performance liquid chromatography (HPLC), preparative gas chromatography, preparative thin layer chromatography, affinity chromatography, size exclusion chromatography, liquid-liquid chromatography e.g., counter-current chromatography or centripetal chromatography. In the methods disclosed herein, an aliquot may be separated into both biologically active and inactive components.

[0031] The clinical indication for (or bioactivity of) the composition may have relevance to any disease, disorder or condition of humans or other animals. The methods disclosed herein are particularly useful in producing pharmaceutical grade triptolide-derived compounds, as agents or drugs for treatment, amelioration and/or prevention of human or veterinary diseases, disorders or conditions. Exemplary indications include, but are not limited to, allergic/ inflammatory disorders, cardiovascular disorders, cancer, central nervous system (CNS) disorders, gastrointestinal disorders, metabolic disorders, nausea, or a disorder, disease or infection induced by a microbial organism or a virus.

[0032] "Mammal" includes humans and non-human mammals, such as companion animals (cats, dogs, and the like) and farm animals (cattle, horses, sheep, goats, swine, and the like).

[0033] "Morbidity" refers to conditions, such as diseases or disorders that compromise the health and well-being of an organism, such as an animal. Morbidity-susceptibility or morbidity- associated genes are genes that, when altered, for example, by a variation in nucleotide sequence, facilitate the expression of a specific disease clinical phenotype. Thus, morbidity susceptibility genes have the potential, upon alteration, of increasing the likelihood or general risk that an organism will develop a specific disease.

[0034] "Mortality" refers to the statistical likelihood that an organism, particularly an animal, will not survive a full predicted lifespan. Hence, a trait or a marker, such as a polymorphism, associated with increased mortality is observed at a lower frequency in older than younger segments of a population.

[0035] "Disease" includes any unhealthy condition of an animal, including a condition detrimental to health resulting from medical therapy (a "side-effect"), and can include autoimmune diseases and conditions of the internal organs, such as, for example the kidney.

[0036] "Predisposition to develop a disease or disorder" means that a subject having a particular genotype and/or haplotype has a higher likelihood than one not having such a genotype and/or haplotype for developing a particular disease or disorder.

[0037] "Ameliorating" or "ameliorate" refers to any indicia of success in the treatment of a pathology or condition, including any objective or subjective parameter such as abatement, remission or diminishing of symptoms or an improvement in a patient's physical or mental well- being. Amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination and/or a psychiatric evaluation.

[0038] "Treating" or "treatment" of a disease includes preventing the disease from occurring in an animal that may be predisposed to the disease but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), inhibiting the disease (slowing or arresting its development), providing relief from the symptoms or side-effects of the disease (including palliative treatment), and relieving the disease (causing regression of the disease).

[0039] A "therapeutically effective amount" means the amount that, when administered to an animal for treating a disease, is sufficient to effect treatment for that disease.

[0040] The active compound(s) and composition(s) of the present disclosure will generally be used in an amount effective to treat or prevent the particular disease being treated. The composition may be administered therapeutically to achieve therapeutic benefit or prophylactically to achieve prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated, e.g., eradication or amelioration of the underlying disease, disorder or allergy, and/or eradication or amelioration of one or more of the symptoms associated with the underlying disease, disorder or allergy, such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. For example, administration of an active compound to a patient suffering from an allergy provides therapeutic benefit not only when the underlying allergic response is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the allergy following exposure to the allergen. Therapeutic benefit also includes halting or slowing the progression of the condition, disorder, disease or allergy, regardless of whether improvement is realized.

[0041] The amount of active compound(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular active compound, etc. Determination of an effective dosage is well within the capabilities of those skilled in the art. Initial dosages may be estimated initially from in vitro assays. Initial dosages can also be estimated from in vivo data, such as animal models.

[0042] The amount of extract in the compositions can vary according to factors such as type of disease, age, sex, and weight of the subject. Dosage regimens may be adjusted to optimize a therapeutic response. In some embodiments, a single bolus may be administered; several divided doses may be administered over time; the dose may be proportionally reduced or increased; or any combination thereof, as indicated by the exigencies of the therapeutic situation and factors known one of skill in the art. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. Dosage regimens may 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 the dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.

[0043] The terms "administration" or "administering" refer to a method of incorporating a composition into the cells or tissues of a subject, either in vivo or ex vivo to diagnose, prevent, treat, or ameliorate a symptom of a disease. In one example, a compound can be administered to a subject in vivo parenterally. In another example, a compound can be administered to a subject by combining the compound with cell tissue from the subject ex vivo for purposes that include, but are not limited to, assays for determining utility and efficacy of a composition. When the compound is incorporated in the subject in combination with one or active agents, the terms "administration" or "administering" can include sequential or concurrent incorporation of the compound with the other agents such as, for example, any agent described above. A pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral such as, for example, intravenous, intradermal, intramuscular, and subcutaneous injection; oral; inhalation; intranasal; transdermal; transmucosal; and rectal administration.

[0044] An "effective amount" of a compound of the present disclosure can be used to describe a therapeutically effective amount or a prophylactically effective amount. An effective amount can also be an amount that ameliorates the symptoms of a disease. A "therapeutically effective amount" refers to an amount that is effective at the dosages and periods of time necessary to achieve a desired therapeutic result and may also refer to an amount of active compound, prodrug or pharmaceutical agent that elicits any biological or medicinal response in a tissue, system, or subject that is sought by a researcher, veterinarian, medical doctor or other clinician that may be part of a treatment plan leading to a desired effect. In some embodiments, the therapeutically effective amount may need to be administered in an amount sufficient to result in amelioration of one or more symptoms of a disorder, prevention of the advancement of a disorder, or regression of a disorder. In one example, treatment of an inflammatory disorder or an autoimmune disorder characterized by inflammation, a therapeutically effective amount preferably refers to the amount of a therapeutic agent that provides a measurable response of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of a desired action of the composition. The term "treating" refers to the administering one or more therapeutic or prophylactic agents taught herein.

[0045] A "prophylactically effective amount" refers to an amount that is effective at the dosages and periods of time necessary to achieve a desired prophylactic result such as, preventing or inhibiting the severity of a platelet or white blood cell count drop, or reducing the nadir of the drop. Typically, a prophylactic dose is used in a subject prior to the onset of a disease, or at an early stage of the onset of a disease, to prevent or inhibit onset of the disease or symptoms of the disease. A prophylactically effective amount may be less than, greater than, or equal to a therapeutically effective amount.

[0046] In some embodiments, the administration can be oral. In other embodiments, the administration can be subcutaneous injection. In some embodiments, the administration can be intravenous injection using a sterile isotonic aqueous buffer. In some embodiments, the administration can include a solubilizing agent and a local anesthetic such as lignocaine to ease discomfort at the site of injection. In other embodiments, the administrations may be parenteral to obtain, for example, ease and uniformity of administration. [0047] In some embodiments, a therapeutically or prophylactically effective amount of a composition may range in concentration from about 0.001 nM to about 0.10 M; from about 0.001 nM to about 0.5 M; from about 0.01 nM to about 150 DM; from about 0.01 nM to about 500 DM; from about 0.01 nM to about 1000 DM, or any range therein. In some embodiments, the compositions may be administered in an amount ranging from about 0.001 mg/kg to about 500 mg/kg; from about 0.005 mg/kg to about 400 mg/kg; from about 0.01 mg/kg to about 300 mg/kg; from about 0.01 mg/kg to about 250 mg/kg; from about 0.1 mg/kg to about 200 mg/kg; from about 0.2 mg/kg to about 150 mg/kg; from about 0.4 mg/kg to about 120 mg/kg; from about 0.15 mg/kg to about 100 mg/kg, from about 0.15 mg/kg to about 50 mg/kg, from about 0.5 mg/kg to about 10 mg/kg, or any range therein, wherein a human subject is assumed to average about 70 kg.

[0048] Dosage amounts will typically be in the range of from about 1 mg/kg/day to about 100 mg/kg/day, 200 mg/kg/day, 300 mg/kg/day, 400 mg/kg/day or 500 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active compound, its bioavailability, the mode of administration and various factors discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound(s) which are sufficient to maintain therapeutic or prophylactic effect. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of active compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective local dosages without undue experimentation.

[0049] The compounds can be administered in dosage units. The term "dosage unit" refers to discrete, predetermined quantities of a compound that can be administered as unitary dosages to a subject. A predetermined quantity of active compound can be selected to produce a desired therapeutic effect and can be administered with a pharmaceutically acceptable carrier. The predetermined quantity in each unit dosage can depend on factors that include, but are not limited to, (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of creating and administering such dosage units.

[0050] The compound(s) may be administered once per day, a few or several times per day, or even multiple times per day, depending upon, among other things, the indication being treated and the judgment of the prescribing physician.

[0051] Preferably, the active compound(s) will provide therapeutic or prophylactic benefit without causing substantial toxicity. Toxicity of the active compound(s) may be determined using standard pharmaceutical procedures. The dose ratio between toxic and therapeutic (or prophylactic) effect is the therapeutic index. Active compound(s) that exhibit high therapeutic indices are preferred.

[0052] "Transmucosal" or similar terms means passage of a permeant into and through the mucosa to achieve effective therapeutic blood levels or deep tissue levels of a drug.

[0053] "Chemical enhancer," "penetration enhancer," "permeation enhancer," and the like shall be inclusive of all enhancers which increase the flux of a permeant, drug, or other molecule across the mucosa and is limited only by functionality. In other words, all cell envelope disordering compounds, solvents, steroidal detergents, bile salts, chelators, surfactants, non- surfactants, fatty acids, and any other chemical enhancement agents are intended to be included. The flux of a drug or analyte across the mucosa can be increased by changing either the resistance (the diffusion coefficient) or the driving force (the gradient for diffusion). Flux may be enhanced by the use of so-called penetration or permeation or chemical enhancers.

[0054] Permeation enhancers are comprised of two primary categories of components, i.e., cell- envelope disordering compounds and solvents or binary systems containing both cell-envelope disordering compounds and solvents. As discussed above, other categories of permeation enhancer are known, however, such as steroidal detergents, bile salts, chelators, surfactants, non- surfactants, and fatty acids.

[0055] Combinations of enhancers consisting of diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate and methyl laurate are disclosed in U.S. Pat. No. 4,973,468 as enhancing the transdermal delivery of steroids such as progestogens and estrogens. A dual enhancer consisting of glycerol monolaurate and ethanol for the transdermal delivery of drugs is shown in U.S. Pat. No. 4,820,720. U.S. Pat. No. 5,006,342 lists numerous enhancers for transdermal drug administration consisting of fatty acid esters or fatty alcohol ethers of C2 to C4 alkanediols, where each fatty acid/alcohol portion of the ester/ether is of about 8 to 22 carbon atoms. U.S. Pat. No. 4,863,970 shows penetration-enhancing compositions for topical application comprising an active permeant contained in a penetration-enhancing vehicle containing specified amounts of one or more cell-envelope disordering compounds such as oleic acid, oleyl alcohol, and glycerol esters of oleic acid; a C2 or C3 alkanol and an inert diluent such as water.

[0056] Other permeation enhancers, not necessarily associated with binary systems include DMSO or aqueous solutions of DMSO such as taught in Herschler, U.S. Pat. No. 3,551,554; Herschler, U.S. Pat. No. 3,711,602; and Herschler, U.S. Pat. No. 3,711,606, and the azones (n- substituted-alkyl-azacycloalkyl-2-ones) such as noted in Cooper, U.S. Pat. No. 4,557,943. [0057] "Permeant," "drug," or "pharmacologically active agent" or any other similar term means any chemical or biological material or compound, inclusive of peptides, suitable for transmucosal administration by the methods previously known in the art and/or by the methods taught in the present present disclosure, that induces a desired biological or pharmacological effect, which may include but is not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as preventing an infection, (2) alleviating a condition caused by a disease, for example, alleviating pain or inflammation caused as a result of disease, and/or (3) either alleviating, reducing, or completely eliminating the disease from the organism. The effect may be local, such as providing for a local anaesthetic effect, or it may be systemic. This disclosure is not drawn to novel permeants or to new classes of active agents. Rather it is limited to the mode of delivery of agents or permeants which exist in the state of the art or which may later be established as active agents and which are suitable for delivery by the present disclosure. Such substances include broad classes of compounds normally delivered into the body, including through body surfaces and membranes, including skin. In general, this includes but is not limited to: antiinfectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; anorexics; antihelminthics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; Antidiabetic agents; antidiarrheals; antihistamines; antiinflammatory agents; antimigraine preparations; antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics; antispasmodics; anticholinergics; sympathomimetics; xanthine derivatives; cardiovascular preparations including potassium and calcium channel blockers, beta-blockers, alpha-blockers, and antiarrhythmics; antihypertensives; diuretics and antidiuretics; vasodilators including general coronary, peripheral and cerebral; central nervous system stimulants; vasoconstrictors; cough and cold preparations, including decongestants; hormones such as estradiol and other steroids, including corticosteroids; hypnotics; immunosuppressives; muscle relaxants; parasympatholytics; psychostimulants; sedatives; and tranquilizers. By the method of the present disclosure, both ionized and nonionized drugs may be delivered, as can drugs of either high or low molecular weight.

[0058] "Buccal" drug delivery is meant delivery of a drug by passage of a drug through the buccal mucosa into the bloodstream. Preferably, buccal drug delivery is effected herein by placing the buccal dosage unit on the upper gum or opposing inner lip area of the individual undergoing drug therapy.

[0059] "Excipients" or "vehicles" as used herein refer to any excipients or vehicles suitable for oral or buccal drug administration, and include any such materials known in the art, e.g. , any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is nontoxic and which does not interact with other components of the composition in a deleterious manner.

[0060] "Pharmaceutically acceptable excipient" means a diluent, adjuvant, excipient or vehicle that is useful in preparing and/or administering a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for human pharmaceutical use as well as for veterinary use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. These pharmaceutical carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0061] Examples of pharmaceutical carriers include, but are not limited to, sterile liquids, such as water, oils and lipids such as, for example, phospholipids and glycolipids. These sterile liquids include, but are not limited to, those derived from petroleum, animal, vegetable or synthetic origin such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water can be a preferred carrier for intravenous administration. Saline solutions, aqueous dextrose and glycerol solutions can also be liquid carriers, particularly for injectable solutions. A carrier is pharmaceutically acceptable after approval by a state or federal regulatory agency or listing in the U.S. Pharmacopeial Convention or other generally recognized sources for use in subjects.

[0062] Suitable pharmaceutical excipients include, but are not limited to, starch, sugars, inert polymers, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting agents, emulsifying agents, pH buffering agents, or a combination thereof.

[0063] The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as, for example, pharmaceutical grades mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. See Martin, E. W. Remington's Pharmaceutical Sciences. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the carrier is suitable for parenteral administration. In other embodiments, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. In other embodiments, the pharmaceutically acceptable carrier may comprise pharmaceutically acceptable salts.

[0064] Pharmaceutical formulations for parenteral administration may include liposomes. Liposomes and emulsions are delivery vehicles or carriers that are especially useful for hydrophobic drugs. Depending on biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. Furthermore, one may administer the drug in a targeted drug delivery system such as, for example, in a liposome coated with target-specific antibody. The liposomes will bind to the target protein and be taken up selectively by the cell expressing the target protein.

[0065] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for a high drug concentration. In some embodiments, the carrier can be a solvent or dispersion medium including, but not limited to, water; ethanol; a polyol such as for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like; and, combinations thereof. The proper fluidity can be maintained in a variety of ways such as, for example, using a coating such as lecithin, maintaining a required particle size in dispersions, and using surfactants.

[0066] In some embodiments, isotonic agents can be used such as, for example, sugars; polyalcohols including, but not limited to, mannitol, sorbitol, glycerol, and combinations thereof; and sodium chloride. Sustained absorption characteristics can be introduced into the compositions by including agents that delay absorption such as, for example, monostearate salts, gelatin, and slow release polymers. Carriers can be used to protect active compounds against rapid release, and such carriers include, but are not limited to, controlled release formulations in implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used such as, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, polycaprolactone, polyglycolic copolymer (PLG), and the like. Such formulations can generally be prepared using methods known to one of skill in the art.

[0067] The compounds may be administered as suspensions such as, for example, oily suspensions for injection. Lipophilic solvents or vehicles include, but are not limited to, fatty oils such as, for example, sesame oil; synthetic fatty acid esters, such as ethyl oleate or triglycerides; and liposomes. Suspensions that can be used for injection may also contain substances that increase the viscosity of the suspension such as, for example, sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, a suspension may contain stabilizers or agents that increase the solubility of the compounds and allow for preparation of highly concentrated solutions.

[0068] In some embodiments, a sterile and injectable solution can be prepared by incorporating an effective amount of an active compound in a solvent with any one or any combination of desired additional ingredients described above, filtering, and then sterilizing the solution. In another embodiment, dispersions can be prepared by incorporating an active compound into a sterile vehicle containing a dispersion medium and any one or any combination of desired additional ingredients described above. Sterile powders can be prepared for use in sterile and injectable solutions by vacuum drying, freeze-drying, or a combination thereof, to yield a powder that can be comprised of the active ingredient and any desired additional ingredients. Moreover, the additional ingredients can be from a separately prepared sterile and filtered solution. In another embodiment, the extract may be prepared in combination with one or more additional compounds that enhance the solubility of the extract.

[0069] In some embodiments, the compounds can be administered by inhalation through an aerosol spray or a nebulizer that may include a suitable propellant such as, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or a combination thereof. In one example, a dosage unit for a pressurized aerosol may be delivered through a metering valve. In another embodiment, capsules and cartridges of gelatin, for example, may be used in an inhaler and can be formulated to contain a powderized mix of the compound with a suitable powder base such as, for example, starch or lactose.

[0070] A "dietary supplement" means a product intended to supplement the diet that contains one or more of the following dietary ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a dietary substance for use by man to supplement the diet by increasing the total dietary intake, or a concentrate, metabolite, constituent, extract, or combination of any of the aforementioned ingredients.

[0071] "Alkyl" refers to a saturated acyclic monovalent radical containing carbon and hydrogen, which may be linear or branched. Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl, n-heptyl, and isopropyl. "Cycloalkyl" refers to a fully saturated cyclic monovalent radical containing carbon and hydrogen, which may be further substituted with alkyl. Examples of cycloalkyl groups are cyclopropyl, methyl cyclopropyl, cyclobutyl, cyclopentyl, ethylcyclopentyl, and cyclohexyl. "Lower alkyl" refers to such a group having one to six carbon atoms, preferably one to four carbon atoms.

[0072] "Alkenyl" refers to an acyclic monovalent radical containing carbon and hydrogen, which may be linear or branched, and which contains at least one carbon-carbon double bond (C=C). "Alkynyl" refers to an acyclic monovalent radical containing carbon and hydrogen, which may be linear or branched, and which contains at least one carbon-carbon triple bond (C≡C). "Lower alkenyl" or "lower alkynyl" such a group having two to six carbon atoms, preferably two to four carbon atoms. [0073] "Acyl" refers to a radical having the form -(C=0)R, where R is alkyl (alkylacyl) or aryl (arylacyl). "Acyloxy" refers to a group having the form -0(C=0)R.

[0074] "Aryl" refers to a monovalent aromatic radical having a single ring (e.g., benzene) or two condensed rings (e.g., naphthyl). As used herein, aryl is preferably monocyclic and carbocyclic (non-heterocyclic), e.g. a benzene (phenyl) ring or substituted benzene ring. By "substituted" is meant that one or more ring hydrogens is replaced with a group such as a halogen (e.g. fluorine, chlorine, or bromine), lower alkyl, nitro, amino, lower alkylamino, hydroxy, lower alkoxy, or halo(lower alkyl).

[0075] "Lower arylacyl" refers to an aromatic or heteroaromatic system consisting of one to three rings coupled via a carbonyl moiety with or without one to three interspersing methylenes. In some embodiments, a "lower arylacyl" may be an aromatic or heteroaromatic system consisting of one or two rings coupled via a carbonyl moiety.

[0076] "Monsaccharidylacyl" refers to a monosaccharide coupled via a carbonyl moiety.

[0077] "Arylalkyl" refers to an alkyl, preferably lower (Ci-C₄ , more preferably Ci-C₂ ) alkyl, substituent which is further substituted with an aryl group; examples are benzyl and phenethyl.

[0078] "Lower alkanol" means refers to an alcohol having one to six carbon atoms. Examples of lower alkanols are methanol, ethanol, butanol, and isopropanol.

[0079] In some embodiments, the lower alkanol can be added to the solvent to a concentration of about 70% lower alkanol at about room temperature to create the precipitate. In some embodiments, the precipitation is done by first using a lower concentration of about 35% lower alkanol in a first precipitation step, and then using a higher concentration of about 70% lower alkanol in a second precipitation step. The concentrations of lower alkanol used in the precipitations can range from, for example, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%- 70%, 70%-80%, 80%-90%, or any concentration therein.

[0080] A "heterocycle" refers to a non-aromatic ring, preferably a 5- to 7-membered ring, whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen and sulfur. Preferably, the ring atoms include 3 to 6 carbon atoms. Such heterocycles include, for example, pyrrolidine, piperidine, piperazine, and morpholine.

[0081] For the purposes of the current disclosure, the following numbering scheme is used for triptolide and triptolide derivatives:

[0082] The compounds of the present disclosure can be prepared from triptolide or 14- fluorotriptolide, as described hereinbelow. Triptolide can be obtained from the root xylem of the Chinese medicinal plant Tripterygium wilfordii (TW) or from other known sources. The TW plant is found in the Fujian Province and other southern provinces of China; TW plant material can generally be obtained in China or through commercial sources in the United States. Methods for preparing triptolide, tripdiolide and 16-hydroxytriptolide are known in the art and are described, for example, in Kupchan et al. (1972, 1977); Lipsky et al. (1994); Pu et al. (1990); and Ma et al. (1992).

[0083] By way of example, see Scheme 1 below, where acylation be controlled by regioselective reactions and diacylation can be controlled via the use of two or more equivalents of base and acylating agents.

Scheme 1 - Regioselectivity in Acylation and Diacylation

Formula I

Examples

Formula II Examples

Formula III Examples

[0084] In general, relative amounts of specific structures shown in Scheme 1 can be controlled by the stoichiometry of RiX and base, and by the choice of base, solvent and X of RiX_. For example, one could use more than 2 or more equivalents of RiX and base to generate the diacylation structures of Structure III. One could also use selective de-acylation reactions to remove Ri to yield Structure I or Structure II.

[0085] Further, according to Barry M. Trost, "the regioselectivity in the acylation of metal enolates in general is dependent on the acylating reagent (acetic anhydride favors O-acylation better than acetyl chloride), the metal (K favors O- acylation while Mg favors C-acylation), the solvent (chelating solvent such as 1,2-dimethoxy ethane favors O-acylation while non-polar solvent such as toluene favors C-acylation), and the reaction temperature (higher temperature favors O-acylation while lower temperature favors C-acylation)." (Barry M. Trost and Jiayi Xu (2007) /. Org. Chem. 72(24):9372-9375; House HO, et al. (1973) /. Org. Chem. 38:514; Olofson RA, et al. (1978) /. Org. Chem. 43:2073; Taylor RJK. (1985) Synthesis. 4:364; Bairgrie LM, et al. (1988) Tetrahedron Lett. 29: 1673; Zhong M, and Brauman JI. (1996) /. Am. Chem. Soc. 118:636). Thus, compounds of Structures I and II can be prepared, selectively.

[0086] Scheme 2 shows a specific example using PG691 as starting material to yield the di- acylation product, PG800mw, and subsequently the mono-O-acylation product PG580mwl. This approach avoids the need for selectivity in mono-0 versus mono-C-acylation. heme 2

[0087] Scheme 3 shows a selective synthetic method of enol esters (O-acylated products) from silyl enol ether and acid chloride in the presence of CuCl. This reaction proceeds smoothly in DMI (1, 3-dimethyl-2-imidazolidinone). (Hajime Ito, et al. (1998) Tetrahedron Lett. 39(35):6295-6298).

Scheme 3

[0088] Scheme 4 shows that starting with fluorine analog of PG490, known as PG763, obviates the need for a substrate protection approach. This 14-fluoro derivative in the beta conformation is used throughout as a specific example. Other starting materials that are considered include the 14-alpha fluoro, and the 14-difluoro derivatives. The benefit of these fluoro derivatives used as starting materials is that protection/deprotection synthetic steps are not needed.

Scheme 4

[0089] Combining the starting material of Scheme 4, PG763, and the first two reaction steps of Scheme 3 gives the O-benzoyl derivative, PG466mw2 shown in Scheme 5. Scheme 5

[0090] Glucose derivatives are used throughout as a specific monosaccharide example. Other monosaccharides that are considered include allose, altrose, mannose, gulose, idose, galactose and talose in alpha or beta form, and pyranose or fucanose form. Disaccharides and oligo saccharides may also be considered.

[0091] Benzoyl chloride derivatives are also used throughout as a specific acyl halide. Other acyl halides that are considered include aromatic (naphthoyl, biphenoyl), heteroaromatic (pyridinoyl, quinolinoyl), aliphatic (acetoyl, hexanoyl), cyclic and bicyclic alkane (cyclohexanoyl, bicyclo[2.2.2]octanoyl), heterocyclics (pyridinoyl, pyrrolidinoyl) halides (CI. Br). The anhydrides of the previous are also considered.

[0092] The octanol-water partition coefficient (clogP) may also be considered in choosing preferred oral or preferred intravenous infusion route of administration. For example, the disclosed compounds may be chosen for oral route of administration if the clog is near 5.0 (Lipinski Rule), or for intravenous infusion route of administration if the clog is less than -2.0 (water soluble). See the Table in the Experimental for specific examples.

[0093] Monosaccharides may be based on Galactose (Gal) and Arabinose (Ara). The Gal and Ara can be in the pyranose and furanose forms, respectively.

[0094] A "modification" of a monsaccharidylacyl group or monosaccharide substituent of a triptolide derivative refers to a methylesterified monosaccharide, an acetylated monosaccharide or a methylesterified peracylated monosaccharide, for example. The monosaccharide (i.e., a single sugar residue) may be naturally occurring, or may be a synthetic analog.

[0095] It will be clear to the skilled artisan that previously described, art-known triptolides differ from those presently disclosed, in that art-known triptolides are O-monoderivatives, primarily 14-O-derivatives, whereas the presently disclosed triptolides are lactone derivatives, preferably C-derivatives and O, C di-derivatives.

[0096] Illustrative publications (incorporated by reference herein, each in its entirety) include co-owned U.S. Patents 7,863,464 and 8,426,616, and U.S. Patent Application serial number 14/776,606. Also of interest is a research publication by Q.-L. He, I. Minn, Q. Wang, P. Xu, S. A. Head, E. Datan, B. Yu, M. G. Pomper, and J. O. Liu, "Targeted Delivery and Sustained Antitumor Activity of Triptolide through Glucose Conjugation." (2016) Angew. Chem., 128(39): 12214-12218.

III. Biological Activities

[0097] Cytotoxic Activity

[0098] The following procedure shows how to measure the cytotoxic activity of the presently disclosed compounds. The testing may involve incubation in mouse serum, human serum, or no such incubation in serum. For comparison, the corresponding curves are used.

[0099] Immunomodulating and Antiinflammatory Treatment / IL-2 suppressive activity

[0100] The following shows how to measure the IL-2 suppression of the presently disclosed compounds incubated in mouse serum, humane serum, or without incubation.

[0101] As immunosuppressants, the compounds of the present invention are useful when administered for the treatment of autoimmune disease and prevention of immune-mediated tissue or organ graft rejection. Immunoregulatory abnormalities have also been shown to exist in a wide variety of autoimmune and chronic inflammatory diseases. Thus, the regulation of the immune response by the compounds of the invention would also find utility in the treatment of these diseases.

[0102] 1. The presently disclosed compounds are useful for the treatment of human diseases such as dysregulation of the immune system and other immune system abnormalities. Immunoregulatory abnormalities have been shown to exist in a wide variety of autoimmune and chronic inflammatory diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type I and II diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, Graves ophthalmopathy and asthma.

[0103] Although the underlying pathogenesis of each of these conditions may be quite different, they have in common the appearance of a variety of autoantibodies and self-reactive lymphocytes. Such self-reactivity may be due, in part, to a loss of the homeostatic controls under which the normal immune system operates.

[0104] Similarly, following a bone-marrow transplant or other transplant of hematopoietic stem cells from a donor tissue source containing mature lymphocytes, the transferred lymphocytes recognize the host tissue antigens as foreign. These cells become activated and mount an attack upon the host (a graft-versus-host response) that can be life-threatening. Moreover, following an organ transplant, the host lymphocytes recognize the foreign tissue antigens of the organ graft and mount cellular and antibody-mediated immune responses (a host- versus-graft response) that lead to graft damage and rejection. [0105] Examples for the treatment and prevention of the resistance by transplantation of organs or tissues such as heart, kidney, liver, skin, cornea, lung, pancreas, intestine, limb, muscle, nerve, duodenum, small -bowel, pancreatic-islet cell, including xenotransplants, etc. Other examples include graft-versus-host disease; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes uveitis, juvenile-onset or recent-onset diabetes mellitus, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, and the like.

[0106] Further uses may include the treatment and prophylaxis of inflammatory and hyperproliferative skin diseases and cutaneous manifestations of immunologically mediated illnesses, such as psoriasis, atopic dermatitis, pemphigus, urticaria, cutaneous eosinophilias, Lupus erythematosus, acne and Alopecia areata; various eye diseases such as conjunctivitis, uveitis, keratitis, sarcoidosis, etc.; inflammation of mucous and blood vessels such as gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory bowel diseases, and necrotizing enterocolitis; intestinal inflammations/allergies such as Coeliac diseases, Crohn's disease and ulcerative colitis; renal diseases such as interstitial nephritis, Good-pasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy; hematopoietic diseases such as idiopathic thrombocytopenia purpura and autoimmune hemolytic anemia; skin disease such as dermatomyositis and cutaneous T cell lymphoma; circulatory diseases such as arteriosclerosis and atherosclerosis; nephrotic syndrome such as glomerulonephritis; renal diseases such as ischemic acute renal insufficiency and chronic renal insufficiency; and Behcet's disease. Also within the scope of the present disclosure is a combination therapy comprising a compound presently disclosed and one or more immunosuppressant agents. These immunosuppressant agents within the scope of the present disclosure include, but are not limited to, IMUREK™, azathioprine sodium, brequinar sodium, SPANIDIN™, gusperimus trihydrochloride (also known as deoxyspergualin), mizoribine (also known as bredinin), CELLCEPT™, mycophenolate mofetil, NEORAL™, Cyclosporin A (also marketed as a different formulation of Cyclosporin A under the trademark SANDIMMUNE™), PROGRAF™, tacrolimus (also known as FK-506) and RAPEVIMUNE™, sirolimus (also known as rapamycin), leflunomide (also known as HWA-486), glucocortcoids, such as prednisolone and its derivatives, antibody therapies such as orthoclone (OKT3) and Zenapax and antithymyocyte globulins, such as thymoglobulins.

[0107] 2. The compounds of the present disclosure may also be used in the treatment of cancer. As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in mammals especially humans, including leukemias, sarcomas, carcinomas and melanoma. Examples of cancers are cancer of the brain, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and medulloblastoma.

[0108] The term "leukemia" refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.

[0109] Additional cancers which can be treated with compounds according to the present disclosure include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.

[0110] 3. The compounds of the present disclosure may also be used in the treatment of certain CNS diseases. Glutamate fulfills numerous physiological functions, but also plays a role in the pathophysiology of different neurological and psychiatric diseases. Glutamate excitotoxicity and neurotoxicity have been implicated in hypoxia, ischemia and trauma, as well as in chronic neurodegenerative or neurometabolic diseases, Alzheimer's dementia, Huntington's disease and Parkinson' s disease. Compounds of this disclosure are envisioned to antagonize the neurotoxic action of glutamates and thus may be a novel therapy for these diseases.

[0111] Recent evidence from MS patients in relapse suggests an altered glutamate homeostasis in the brain of patients with MS. Neurotoxic events occur in MS, and they can be responsible for oligodendrocyte and neuronal cell death in patients with this demyelinating disease. Antagonizing glutamate receptor-mediated excitotoxicity by treatment with compounds of this disclosure may have therapeutic implications in MS patients. Other CNS diseases such as Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy may be treated with the compounds of the present disclosure. [0112] 4. The compounds of the present disclosure may also be used in the treatment of certain lung diseases. Idiopathic pulmonary fibrosis (PF) is a progressive interstitial lung disease with no known etiology. PF is characterized by excessive deposition of intracellular matrix and collagen in the lung interstitium and gradual replacement of the air sacs or "alveoli" of the lungs by scar tissue as a result of inflammation and fibrosis. As the disease progresses the increase in scar tissue interferes with the ability to transfer oxygen from the lungs to the bloodstream. Accordingly, the compounds of the present disclosure may be useful for treatment of PF. Treatment of other respiratory diseases, such as sarcoidosis, fibroid lung, and idiopathic interstitial pneumonia is also considered.

[0113] Other diseases involving the lung envisioned to be treatable by the presently disclosed compounds include but are not limited to Severe Acute Respiratory Syndrome (SARS) and acute respiratory distress syndrome (ARDS). In particular, with respect to SARS, the reduction of virus content (SARS-CoV) before the peak of the disease process and the usefulness of corticosteroid treatment, as noted below, suggest that the development of the most severe, life-threatening effects of SARS may result from the exaggerated response of the body to the infection (immune hyperactivity) rather than effects of the virus itself. (See also co-owned US provisional application 60/483,335, which is incorporated herein by reference). Corticosteroid treatment has been used in SARS patients to suppress the massive release of cytokines that may characterize the immune hyperactive phase, in the hope that it will stop the progression of pulmonary disease in the next phase. Corticosteroid treatment has produced good clinical results in reduction of some of the major symptoms of SARS. However, there are several treatment-related side effects, and there is a clear need for more selective immunosuppressive and/or antiinflammatory agents. Other lung diseases include allergies and reversible obstructive airway disease, which includes condition such as asthma (for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or inveterate asthma (for example, late asthma and airway hyper- responsiveness), bronchitis and the like.

[0115] Leukopenia, Anemia and Thrombocytopenia

[0116] Blood is a multifaceted body fluid and the medium through which essential nutrients are delivered to tissues throughout the body. On average, the adult human body contains more than 5 liters of blood. Blood flows freely through the veins and arteries because it is over half liquid plasma; the remainder of blood volume consists mostly of solid cells and cell fragments, which are suspended in the plasma (http://en.wikipedia.org/wiki/Blood). Major blood disorders include anemia, leukopenia, and thrombocytopenia. [0117] Leukopenia

[0118] Leukopenia is a decrease in the number of white blood cells (WBCs) found in the blood, which places individuals at increased risk of infection. Neutropenia, a subtype of leukopenia, refers to a decrease in the number of circulating neutrophil granulocytes, the most abundant white blood cells. The terms leukopenia and neutropenia may occasionally be used interchangeably, as the neutrophil count is an indicator of infection risk. Low white cell count may be due to acute viral infections, such as with a cold or influenza. It can be associated with chemotherapy, radiation therapy, myelofibrosis and aplastic anemia. HIV and AIDS are also a threat to white cells. Other causes of low white blood cell count include systemic lupus erythematosus, Hodgkin's lymphoma, some types of cancer, typhoid, malaria, tuberculosis, dengue, rickettsial infections, enlargement of the spleen, folate deficiencies, psittacosis and sepsis. Many other causes exist, such as deficiency in certain minerals, such as copper and zinc. Drugs that can impact the number and function of white blood cells include clozapine, bupropion, Minocycline, valproic acid, lamotrigine, and metronidazole. Other drugs that depress WBC count are immunosuppressants, such as sirolimus, mycophenolate mofetil, tacrolimus, cyclosporine and TNF inhibitors. Interferons used to treat multiple sclerosis, such as Rebif, Avonex, and Betaseron, can also cause leukopenia. Chemotherapy targets cells that grow rapidly, such as tumors, but can also impact white blood cells, because they are characterized by bone marrow as rapid growing. A common side effect of cancer treatment is neutropenia, the lowering of neutrophils (a specific type of white blood cell)( http://en.wikipedia.org/wiki/Leukopenia) .

[0119] Anemia

[0120] Anemia is a decrease in number of red blood cells (RBCs). Anemia in its broadest sense is also less than the normal quantity or quality of hemoglobin in the blood. It includes a decreased oxygen-binding ability of each hemoglobin molecule due to deformity or a lack in numerical development. Since all human cells depend on oxygen for survival, varying degrees of anemia can have a wide range of clinical consequences. Anemia is the most common blood disorder, goes undetermined in many people, and symptoms can be minor. Most commonly, people report feelings fatigue or shortness of breath on exertion. In very severe anemia, the body may increase cardiac output. Anemia is typically diagnosed on a complete blood count that reports the number of RBCs and the hemoglobin level. There are more than 400 types of anemia, which are divided into three causes: blood loss, decreased RBC production, or destruction of RBCs. RBCs can be lost through bleeding, which can occur slowly over a long period of time, and can often go undetected. This kind of chronic bleeding commonly results from gastrointestinal conditions such as ulcers, hemorrhoids, gastritis (inflammation of the stomach), and cancer, use of nonsteroidal anti-inflammatory drugs (NSAIDS) such as aspirin or ibuprofen, or menstruation and childbirth in women, especially if menstrual bleeding is excessive and if there are multiple pregnancies. With anemia caused by decreased or faulty RBC production, the body may produce too few blood cells or the blood cells may not function correctly. In either case, anemia can result. RBCs may be faulty or decreased due to abnormal RBCs or a lack of minerals and vitamins needed for RBCs to work properly. Conditions associated with these causes of anemia include sickle cell anemia, iron-deficiency anemia, vitamin deficiency, bone marrow and stem cell problems (http://en.wikipedia.org/wiki/ Anemia).

[0121] Thrombocytopenia

[0122] Thrombocytopenia is the medical term for a low blood platelet count. Platelets (thrombocytes) are colorless blood cells that play a role in blood clotting. Platelets stop blood loss by clumping and forming plugs in blood vessel holes. Thrombocytopenia may be mild and cause few signs or symptoms. In rare cases, the number of platelets may be so low that dangerous internal bleeding can occur. Thrombocytopenia usually improves when the underlying cause is treated. Sometimes medications, surgery or a blood transfusion can help treat chronic thrombocytopenia. Low platelet counts, thrombocytopenia, can be caused by a variety of reasons and can be divided into decreased platelet production, increased platelet destruction or consumption, or increased splenic sequestration. Decreased platelet production is usually related to a bone marrow problem. In some of these conditions, red blood cell and white blood cell productions may also be affected. Viral infections affecting the marrow for example: parvovirus, rubella, mumps, varicella (chickenpox), hepatitis C, Epstein-Barr virus, and HIV. Aplastic anemia is a general term used when the bone marrow fails to produce any blood cells. This can be caused by some viral infections (parvovirus or HIV), medications (gold, chloramphenicol, Dilantin, valproate (Depakote), or radiation. Chemotherapy drugs frequently cause thrombocytopenia. Some other drugs can suppress platelet production, such as thiazide diuretics. Cancers of the bone marrow and blood or cancers of the lymph nodes can cause various degrees of thrombocytopenia. Long term alcohol can cause direct toxicity of the bone marrow. Deficiency of vitamin B12 and folic acid can result in low platelet production by the bone marrow. Increased platelet destruction or consumption can be seen a number of medical conditions. Many medications can cause low platelet count by causing immunologic reaction against platelets, and include sulfonamide antibiotics, carbamazepine, digoxin, quinine, quinidine, acetaminophen, rifampin,and heparin. Idiopathic thrombocytopenic purpura (ITP) is a condition where the immune system attacks platelets. Some rheumatologic condition, such as systemic lupus erythematosus (SLE) or other autoimmune conditions, can cause platelet destruction including transfusion of blood products and organ transplantation, thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), HELLP syndrome, and disseminated intravascular coagulopathy (DIC). Splenic sequestration can also lead to low platelet counts as a result of enlargement or change in function of the spleen for a variety of reasons. Common causes of thrombocytopenia due to splenic enlargement may include advanced liver disease with portal hypertension (cirrhosis, for example, from chronic hepatitis B or C) and blood cancers (leukemias or lymphomas)

(http://en.wikipedia.org/wiki/Tlirombocytopenia).

[0123] Immune Disorders

[0124] An immune disorder is a dysfunction of the immune system. These disorders can be characterized by the component(s) of the immune system affected, by whether the immune system is overactive or underactive, or by whether the condition is congenital or acquired. According to the International Union of Immunological Societies, more than 150 primary immunodeficiency diseases (PIDs) have been characterized. However, the number of acquired immunodeficiencies exceeds the number of PIDs. It has been suggested that most people have at least one primary immunodeficiency (Casanova and Abel. 2007, "Primary immunodeficiencies: a field in its infancy." Science 317(5838):617-9). However, due to redundancies in the immune system, many of these go undetected.

[0125] Autoimmune diseases arise from an abnormal immune response of the body against substances and tissues normally present in the body. This may be restricted to certain organs (e.g. in autoimmune thyroiditis) or involve a particular tissue in different places (e.g. Goodpasture's disease which may affect the basement membrane in both the lung and the kidney). The treatment of autoimmune diseases is typically with immunosuppression— medication that decreases the immune response. A large number of autoimmune diseases are recognized. A major understanding of the underlying pathophysiology of autoimmune diseases has been the application of genome wide association scans that have identified a striking degree of genetic sharing among the autoimmune diseases. A partial list of some autoimmune disorders include lupus, scleroderma, certain types of hemolytic anemia, vasculitis, type one diabetes, Graves disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, Pernicious anemia, some types of myopathy and late Lyme disease.

[0126] There are a large number of immunodeficiency syndromes that present clinical and laboratory characteristics of autoimmunity. The decreased ability of the immune system to clear infections may be responsible for causing autoimmunity through perpetual immune system activation. Examples include common variable immunodeficiency (CVID) where multiple autoimmune diseases are seen, e.g. inflammatory bowel disease, autoimmune thrombocytopenia and autoimmune thyroid disease, and familial hemophagocytic lymphohistiocytosis (FHL), an autosomal recessive primary immunodeficiency, where pancytopenia, rashes, lymphadenopathy and hepatosplenomegaly are commonly seen (http://en.wikipedia.org/wiki/Immune_disorder).

[0127] Autoimmune Diseases and Utility

[0128] The utility of this disclosure is to modulate the immune system that is overactive or underactive. The goals of treatment are to reduce symptoms, control the autoimmune process and to maintain the body's ability to fight disease. Which treatments are used depends on the specific disease and symptoms. Some patients may need supplements to replace a hormone or vitamin that the body is lacking. Examples include thyroid supplements, vitamins such as B12, or insulin injections. If the autoimmune disorder affects the blood, you may need blood transfusions. Medicines prescribed to control or reduce the immune system's response include corticosteroids (such as prednisone) and nonsteroid drugs such as azathioprine, cyclophosphamide, mycophenolate, sirolimus, or tacrolimus.

[0129] Examples of autoimmune diseases include, but are not limited to, arthritis such as rheumatoid arthritis, osteoarthritis, hyperuricemia and arthritis associated with acute gout, chronic gout and systemic lupus erythematosus; human endothelial disorders such as psoriasis, eczematous dermatitis, Kaposi's sarcoma as well as proliferative disorders of smooth muscle cells; various eye diseases (autoimmune and otherwise) such as keratoconjunctivitis, vernal conjunctivitis, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis corneae, corneal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' opthalmopathy, Vogt- Koyanagi-Harada syndrome, sarcoidosis, multiple myeloma, etc.; obstructive airway diseases, which includes conditions such as chronic obstructive pulmonary disease (COPD), asthma (for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or inveterate asthma (for example, late asthma and airway hyper- responsiveness), bronchitis, allergic rhinitis; Other treatable conditions would include but are not limited to ischemic bowel diseases, inflammatory bowel diseases, necrotizing enterocolitis, intestinal lesions associated with thermal burns and leukotriene B₄-mediated diseases; intestinal inflammations/allergies such as Coeliac diseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis; food-related allergic diseases which have symptomatic manifestation remote from the gastro-intestinal tract (e.g., migraine, rhinitis and eczema); renal diseases such as interstitial nephritis, Goodpasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy; nervous diseases such as multiple myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy; endocrine diseases such as hyperthyroidism and Basedow's disease; hematic diseases such as pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and anerythroplasia; bone diseases such as osteoporosis; respiratory diseases such as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skin disease such as dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T cell lymphoma; circulatory diseases such as arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa and myocardosis; collagen diseases such as scleroderma, Wegener's granuloma and Sjogren's syndrome; adiposis; eosinophilic fasciitis; periodontal disease such as lesions of gingiva, periodontium, alveolar bone and substantia ossea dentis; nephrotic syndrome such as glomerulonephritis; male pattern aleopecia or alopecia senilis by preventing epilation or providing hair germination and/or promoting hair generation and hair growth; muscular dystrophy; Pyoderma and Sezary's syndrome. Additional examples of autoimmune diseases are cystic fibrosis, type I diabetes, ischemia-reperfusion injury, post-angioplasty restenosis, and the like.

[0130] In treating an autoimmune disorder, optimum dosages of the composition containing the active ingredients can be determined by routine experimentation according to methods known in the art. For example, the subject is given a dosage level sufficient to reduce symptoms and improve patient comfort periodically such as once every week.

[0131] The compositions and method of the invention are also useful for the treatment of inflammatory conditions such as asthma, both intrinsic and extrinsic manifestations, for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, particularly chronic or inveterate asthma (for example, late asthma and airway hyperresponsiveness). The composition and method may also be used for treatment of other inflammatory conditions, including traumatic inflammation, inflammation in Lyme disease, chronic bronchitis (chronic infective lung disease), chronic sinusitis, sepsis associated acute respiratory distress syndrome, and pulmonary sarcoidosis. For treatment of respiratory conditions such as asthma, the composition is preferably administered via inhalation, but any conventional route of administration may be useful.

[0132] Other immune disorders treatable with the compositions of the invention include the prevention of immune-mediated tissue or organ graft rejection. This application would include, but not be limited to, solid organ transplants (such as heart, kidney and liver), tissue grafts (such as skin, intestine, pancreas, gonad, bone, and cartilage), and cellular transplants (such as cells from pancreas, brain and nervous tissue, muscle, skin, bone, cartilage and liver).

[0133] The compositions are useful for inhibiting rejection of a solid organ transplant, tissue graft, or cellular transplant from an incompatible human donor, thus prolonging survival and function of the transplant, and survival of the recipient. This use would include, but not be limited to, solid organ transplants (such as heart, kidney and liver), tissue grafts (such as skin, intestine, pancreas, gonad, bone, and cartilage), and cellular transplants (such as cells from pancreas, brain and nervous tissue, muscle, skin, bone, cartilage and liver).

[0134] The compositions are useful for inhibiting rejection of a solid organ transplant, tissue graft, or cellular transplant from an incompatible human donor, thus prolonging survival and function of the transplant, and survival of the recipient. This use would include, but not be limited to, solid organ transplants (such as heart, kidney and liver), tissue grafts (such as skin, intestine, pancreas, gonad, bone, and cartilage), and cellular transplants (such as cells from pancreas, brain and nervous tissue, muscle, skin, bone, cartilage and liver).

[0135] In particular, the pharmaceutical compositions of this invention are useful in preventing chronic allograft nephropathy, or chronic rejection. Chronic allograft nephropathy after kidney transplantation is caused by progressive interstitial fibrosis. Transforming growth factor TGF- D plays a key role in chronic rejection, which is the leading cause of long-term renal graft loss. Immunosuppressive/anti-rejection drugs used to treat organ transplant recipients cause kidney damage leading to chronic rejection. The most commonly used drug for prevention of transplant rejection, CsA, causes the kidneys to become fibrous and unable to function. Most kidney transplant patients show at least some reduction in kidney function. CsA induces the production of TGF- D , which leads to kidney fibrosis, a major factor in the kidney damage of chronic rejection. Other clinically used immunosuppressive drugs FK-506 and rapamycin act through the same mechanism to produce chronic rejection. The compounds of this invention, as an active pharmaceutical ingredient of the compositions, can be used to prevent chronic rejection in solid organ transplantation such as kidney transplantation by inhibition of the production of TGF- D , and suppression of organ fibrosis, and preservation of organ function, as well as suppression of immune-mediated rejection.

[0136] The compositions are also useful for inhibiting xenograft (interspecies) rejection; i.e. in preventing the rejection of a solid organ transplant, tissue graft, or cellular transplant from a non-human animal, whether natural in constitution or bioengineered (genetically manipulated) to express human genes, RNA, proteins, peptides or other non-native, xenogeneic molecules, or bioengineered to lack expression of the animal's natural genes, RNA, proteins, peptides or other normally expressed molecules. The invention also includes the use of a composition as described above to prolong the survival of such a solid organ transplant, tissue graft, or cellular transplant from a non-human animal.

[0137] For treating transplantation rejection, such as rejection of heart, kidney, liver, cellular, and bone marrow transplants, the treatment is typically initiated perioperatively, either soon before or soon after the surgical transplantation procedure, and is continued on a daily dosing regimen, for a period of at least several weeks, for treatment of acute transplantation rejection. During the treatment period, the patient may be tested periodically for immunosuppression level, e.g. , by a mixed lymphocyte reaction involving allogenic lymphocytes, or by taking a biopsy of the transplanted tissue.

[0138] Similarly, following a bone-marrow transplant or other transplant of hematopoietic stem cells from a donor tissue source containing mature lymphocytes, the transferred lymphocytes recognize the host tissue antigens as foreign. These cells become activated and mount an attack upon the host (a graft-versus-host response) that can be life-threatening. Moreover, following an organ transplant, the host lymphocytes recognize the foreign tissue antigens of the organ graft and mount cellular and antibody- mediated immune responses (a host-versus-graft response) that lead to graft damage and rejection.

[0139] The compositions of the present invention are useful in applications for which triptolide and its prodrugs and other derivatives have proven effective, e. g. in immunosuppression therapy, as in, treating or inhibiting allograft rejection, xenograft rejection, and graft-versus-host disease, and treating an autoimmune disease such as rheumatoid arthritis, or treating or preventing graft-versus-host disease (GVHD).

[0140] Also within the scope of the invention is a combination therapy comprising a compound or compositions of this invention with one or more conventional immunosuppressive agents. These immunosuppressant agents within the scope of this invention include, but are not limited to Imurek (azathioprine sodium), brequinar sodium, Spanidin (gusperimus trihydrochloride, also known as deoxyspergualin), mizoribine (also known as bredinin), Cellcept (mycophenolate mofetil), Neoral (Cyclosporin A; also marketed as a different formulation under the trademarks andimmune), Prograf (tacrolimus, also known as FK-506), Rapimmune (sirolimus, also known as rapamycin), leflunomide (also known as HWA-486), Zenapax, glucocortcoids, such as prednisolone and its derivatives, antibodies such as orthoclone (OKT3), and antithymyocyte globulins, such as thymoglobulins. All of these are more conventional immunosuppressant compounds, with the exception of OKT3 and the antithymocyte globulins. The compounds are useful as potentiators when administered concurrently with another immunosuppressive drug for immunosuppressive treatments as discussed above. In addition, there are more recently developed immunosuppressive MAbs that have activity in a variety of diseases involving the immune system, which can be used in combination with the compounds and compositions of this invention. These MAbs include Adalimumab (Humira; Trudexa), Afelimomab (Simponi), Afutuzumab, Atlizumab, Basiliximab (Simulect), Bectumomab, Bertilimumab, Brentuximab (brentuximab vedotin), (Adcetris), Briakinumab, Canakinumab (Ilaris), Certolizumab (Cimzia), Clenoliximab, Dacetuzumab],Daclizumab (Zenapax), Epratuzumab (Lymphocyde^a), Efalizumab (Raptiva, formerly Xanelim), Fontolizumab Fresolimumab], Gemtuzumab ozogamicin conjugate (Mylotarg), Ibritumomab (Zevalin), Infliximab (Remicade)], Inolimomab, Inotuzumab ozogamicin conjugate, Keliximab, Lerdelimumab, Lintuzumab (Zamyl), Lumiliximab, Mepolizumab], Metelimumab, Moxetumomab pasudotox, Nerelimomab Omalizumab (Xolair) , Ocrelizumab, Odulimomab, Ofatumumab (Arzerra), Otelixizumab, Pascolizumab, Reslizumab (Cinquil), Rituximab (Rituxan, MabThera), Secukinumab, Siplizumab, Teplizumab, Tocilizumab (Actemra), Tositumomab (Bexxar), Ustekinumab (Stelara), Veltuzumab, Visilizumab (Nuvion), Zanolimumab, Zolimomab, Natalizumab (Tysabri, Antegren), and Vedolizumab.

[0141] The compositions of the invention may also be administered in combination with a conventional anti-inflammatory drug (or drugs), where the drug or amount of drug administered is, by itself, ineffective to induce the appropriate suppression or inhibition of inflammation.

[0142] Immunosuppressive activity of compounds in vivo can be evaluated by the use of established animal models known to those skilled in the art. Such assays may be used to evaluate the relative effectiveness of immunosuppressive compounds and to estimate appropriate dosages for immunosuppressive treatment. These assays include, for example, a well-characterized rat model system for allografts, described by Ono and Lindsey (1969), in which a transplanted heart is attached to the abdominal great vessels of an allogeneic recipient animal, and the viability of the transplanted heart is gauged by the heart's ability to beat in the recipient animal. A xenograft model, in which the recipient animals are of a different species, is described by Wang (1991) and Murase (1993). A model for evaluating effectiveness against GVHD involves injection of normal F¹ mice with parental spleen cells; the mice develop a GVHD syndrome characterized by splenomegaly and immunosuppression (Korngold, 1978; Gleichmann, 1984). Single cell suspensions are prepared from individual spleens, and microwell cultures are established in the presence and absence of concanavalin A to assess the extent of mitogenic responsiveness. Anticancer Treatment

[0143] As described above, the properties of the compounds within the scope of the invention make them extremely useful as anti-cancer agents. Accordingly, in one embodiment, the present invention provides a method of inhibiting the proliferation of a cancer cell comprising administering an effective amount of a compound of the invention to a cell or animal in need thereof. Various types of cancer can be treated with a compound of this invention, including but not limited to, all types of cancer or neoplasm or malignant tumors found in mammals especially humans, including, carcinomas (such as bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma.

[0144] The compositions may be administered to a patient afflicted with cancer and/or leukemia by any conventional route of administration, as discussed above. The method is useful to slow the growth of tumors, prevent tumor growth, induce partial regression of tumors, and induce complete regression of tumors, to the point of complete disappearance. The method is also useful in preventing the outgrowth of metastases derived from solid tumors. Treatment of breast, colon, lung, pancreatic and prostate tumors is particularly contemplated.

[0145] The term "leukemia" refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. [0146] Included, for example, are cancers involving cells derived from reproductive tissue (such as Sertoli cells, germ cells, developing or more mature spermatogonia, spermatids or spermatocytes and nurse cells, germ cells and other cells of the ovary), the lymphoid or immune systems (such as Hodgkin's disease and non-Hodgkin's lymphomas), the hematopoietic system, and epithelium (such as skin, including malignant melanoma, and gastrointestinal tract), solid organs, the nervous system, e. g. glioma, and musculo-skeletal tissue. The compounds may be used for treatment of various cancer cell types, including, but not limited to, brain, including medulloblastoma, head and neck, breast, colon, small cell lung, large cell lung, thyroid, testicle, bladder, prostate, liver, kidney, pancreatic, esophogeal, stomach, ovarian, cervical or lymphoma tumors.

[0147] The compositions may be administered to a patient afflicted with cancer and/or leukemia by any conventional route of administration, as discussed above. The method is useful to slow the growth of tumors, prevent tumor growth, induce partial regression of tumors, and induce complete regression of tumors, to the point of complete disappearance. The method is also useful in preventing the outgrowth of metastases derived from solid tumors.

[0148] The compositions of the invention may be administered as sole therapy or with other supportive or therapeutic treatments not designed to have anti-cancer effects in the subject. The method also includes administering the invention compositions in combination with one or more conventional anti-cancer drugs or biologic protein agents, where the amount of drug (s) or agent (s) is, by itself, ineffective to induce the appropriate suppression of cancer growth, in an amount effective to have the desired anti-cancer effects in the subject. Such anti-cancer drugs include actinomycin D, camptothecin, carboplatin, cisplatin, cyclophosphamide, cytosinearabinoside, daunorubicin, doxorubicin, etoposide, fludarabine, 5-fluorouracil, hydroxyurea, gemcitabine, irinotecan, methotrexate, mitomycin C, mitoxantrone, paclitaxel, taxotere, teniposide,topotecan, vinblastine, vincristine, vindesine, and vinorelbine as the more conventional anti-cancer compounds. Anti-cancer biologic protein agents include tumor necrosis factor (TNF), TNF- related apoptosis inducing ligand (TRAIL), other TNF-related or TRAIL-related ligands and factors, interferon, interleukin-2, other interleukins, other cytokines, chemokines, and factors, antibodies (usually monoclonal antibodies - MAbs) to tumor-related molecules or receptors (such as anti-HER2 antibody), and agents that react with or bind to these agents (such as members of the TNF super family of receptors, other receptors, receptor antagonists, and antibodies with specificity for these agents and antibodies with other reactivities). MAbs are used in cancer treatment to flag cancer cells for immune cell targeting, trigger cancer cell- membrane destruction, block cell growth, prevent blood vessel growth (anti-angiogenesis), block immune system inhibitors (checkpoint inhibitors), directly attack cancer cells, deliver radiation treatment (targeted radioimmunotherapy), deliver chemotherapy, and bind cancer and immune cells (induction of ADCC, etc.). Some of the anti-cancer MAbs with a diversity of targets relevant to cancer proliferation, survival and spread are as follows: Avastin (Bevacizumab), Rituxan (Rituximab), Herceptin (Trastuzumab), Erbitux (Cetuximab), Vectibix (Panitumumab), Zevalin (Ibritumomab), Bexxar (Tositumomab), Arzerra (Ofatumumab), Yervoy (Ipilimumab), Adcetris (Brentuximab Vedotin), Perjeta (Pertuzumab), and Kadcyla (Ado-trastuzumab Emtansine).

[0149] Antitumor activity in vivo of a particular composition can be evaluated by the use of established animal models, as described, for example, in Fidler et al., U. S. Patent No. 6,620,843. Clinical doses and regimens are determined in accordance with methods known to clinicians, based on factors such as severity of disease and overall condition of the patient.

[0150] In another aspect, more benefit may be obtained by administering the invention compositions in combination or sequentially with one or more other conventional anti-cancer drugs or biologic protein agents. Such anti-cancer drugs include, but are not limited to, actinomycin D, carboplatin, cisplatin, cyclophosphamide, hydroxyurea, gemcitabine, mitomycin C, mitoxantrone, paclitaxel, taxotere, vinblastine, vincristine, vindesine, and vinorelbine. Anticancer biologic protein agents include tumor necrosis factor (TNF), TNF-related apoptosis inducing ligand (TRAIL), other TNF-related or TRAIL-related ligands and factors, interferon, interleukin-2, other interleukins, other cytokines, chemokines, and factors, antibodies to tumor- related molecules or receptors (such as anti-HER2 antibody), and agents that react with or bind to these agents (such as members of the TNF super family of receptors, other receptors, receptor antagonists, and antibodies with specificity for these agents).

[0151] Antitumor activity in vivo of a particular composition can be evaluated by the use of established animal models, as described, for example, by Fidler et al. in U.S. Patent No. 6,620,843. Clinical doses and regimens are determined in accordance with methods known to clinicians, based on factors such as severity of disease and overall condition of the patient.

[0152] In addition to these anti-cancer MAbs, checkpoint inhibitors have recently become more important in treating certain cancers by relieving inhibition of the immune system. Checkpoint inhibitors (also known as immune checkpoint modulators) are intended to reduce the effectiveness of checkpoint proteins. They could have a variety of mechanisms of action, but they allow the immune system to recognize and be activated by the other molecules on the surface of cancer cells. Checkpoint inhibition now involves CTLA-4 blockade, PD-1 inhibitors and PD-L1 inhibition. The first checkpoint antibody approved by the FDA was Ipilimumab, which blocks the immune checkpoint molecule CTLA-4. Nivolumab and Pembrolizumab are two leading PD-1 inhibitors. Atezolizumab, Avelumab and Durvalumab are the leading PD-L1 inhibitors.

[0153] Checkpoint inhibitors have shown greater activity with cancers that are characterized by larger numbers of genetic mutations, including melanoma and lung cancer. Although it might be thought that agents with immunosuppressive activity might inhibit the immune responses engendered by treatment with checkpoint inhibitors, it is possible that the cytotoxic activity of the compound described in this invention will enhance the activity of checkpoint inhibitors by inducing cytotoxicity, necrosis and apoptosis that will enhance anti-cancer immune responses. Additionally, autoimmune phenomena are a significant life-threatening side effect of the unleashed immune system with the use of checkpoint inhibitors, and the compounds described in this invention may be useful in controlling these life threatening autoimmune responses that sometimes accompany the anti-cancer activity. These compounds could be very useful in combination with checkpoint inhibitor treatment for certain cancers.

IV. Therapeutic Compositions

[0154] Pharmaceutical formulations and administration

[0155] Formulations containing the triptolide derivatives of the present disclosure may take the form of solid, semi-solid, lyophilized powder, tablets, capsules, powders, sustained-release formulations, liquid dosage forms, such as solutions or suspensions, emulsions, ointments, lotions, or aerosols, preferably in unit dosage forms suitable for simple administration of precise dosages. The compositions typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, or adjuvants.

[0156] In general, the purified compounds and compositions of the present disclosure will be administered in therapeutically effective amounts by oral administration, either singly or in conjunction with of at least one other therapeutic agent capable of treating an immunoregulatory abnormality, an autoimmune or chronic inflammatory disease or disorder. A therapeutically effective amount may vary widely depending on the disease, its severity, the age and relative health of the animal being treated, and other factors. A person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine a therapeutically effective amount of the compositions of the present disclosure for a given disease or disorder.

[0157] In general, the purified compounds and compositions of the present disclosure will be administered alone or as pharmaceutical formulations, which in some embodiments may be administered orally. In pharmaceutical formulations, the composition comprising the purified compounds and compositions of the present disclosure may occur in combination with another active agent and/or an oral excipient. Suitable oral excipients are well known to persons of ordinary skill in the art.

[0158] If formulated, the amount of a compound in the composition may vary widely depending on the type of composition, size of a unit dosage, kind of excipients, and other factors well known to those of ordinary skill in the art. In general, if administered alone the amount of compound may vary from 400mg to 4grams.

[0159] Preferably, the composition includes about 0.5% to 75% by weight of a compound or compounds of the present disclosure, with the remainder consisting of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.

[0160] The composition may be administered to a subject orally, transdermally or parenterally, e.g., by intravenous, subcutaneous, intraperitoneal, or intramuscular injection. For use in oral liquid preparation, the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline. For parenteral administration, an injectable composition for parenteral administration will typically contain the triptolide derivative in a suitable intravenous solution, such as sterile physiological salt solution.

[0161] Liquid compositions can be prepared by dissolving or dispersing the triptolide derivative (about 0.5% to about 20%) and optional pharmaceutical adjuvants in a pharmaceutically acceptable carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension.

[0162] The compound may also be administered by inhalation, in the form of aerosol particles, either solid or liquid, preferably of respirable size. Such particles are sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 10 microns in size, and preferably less than about 5 microns in size, are respirable. Liquid compositions for inhalation comprise the active agent dispersed in an aqueous carrier, such as sterile pyrogen free saline solution or sterile pyrogen free water. If desired, the composition may be mixed with a propellant to assist in spraying the composition and forming an aerosol. CLogP Description

The Table shown shows the clog P values for the indicated compounds.

[0163] As described, for example, in Panchagnula et al. (2000), the partition coefficient or logP of a pharmaceutical agent can affect its suitability for various routes of administration, including oral bioavailability. Some compounds, for example the acylaryl derivatives described herein, are expected to have higher logP values than the parent compound, triptolide, making them better candidates for oral availability. Other compounds, for example the acylmonosaccharides described herein are expected to have lower logP values than the parent compound triptolide, making better candidates for intravenous administration due to enhanced water solubility.

[0164] Methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (20th Ed., Lippincott Williams & Wilkins, 2000). The composition to be administered will contain a quantity of the selected compound in an effective amount for effecting immunosuppression in a subject or apoptosis in a targeted cell.

V. Immunomodulating and Antiinflammatory Treatment

[0165] The present disclosure includes the use of the disclosed compounds as immunosuppressive agents, e.g. as an adjunct to transplant procedures or in treatment of autoimmune disease. The compounds of the present disclosure are effective to inhibit immune responses, such as production of cytokines, in cells or organisms. Immunoregulatory abnormalities have been shown to exist in a wide variety of autoimmune and chronic inflammatory diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type I and II diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, Graves ophthalmopathy and asthma. Although the underlying pathogenesis of each of these conditions may be quite different, they have in common the appearance of a variety of autoantibodies and self-reactive lymphocytes. Such self-reactivity may be due, in part, to a loss of the homeostatic controls under which the normal immune system operates.

[0166] Similarly, following a bone-marrow transplant or other transplant of hematopoietic stem cells from a donor tissue source containing mature lymphocytes, the transferred lymphocytes recognize the host tissue antigens as foreign. These cells become activated and mount an attack upon the host (a graft-versus-host response) that can be life-threatening. Moreover, following an organ transplant, the host lymphocytes recognize the foreign tissue antigens of the organ graft and mount cellular and antibody-mediated immune responses (a host- versus-graft response) that lead to graft damage and rejection.

[0167] One result of an autoimmune or a rejection reaction is tissue destruction caused by inflammatory cells and the mediators they release. Anti-inflammatory agents such as NSAIDs act principally by blocking the effect or secretion of these mediators but do nothing to modify the immunologic basis of the disease. On the other hand, cytotoxic agents, such as cyclophosphamide, act in such a nonspecific fashion that both the normal and autoimmune responses are shut off. Indeed, patients treated with such nonspecific immunosuppressive agents are as likely to succumb from infection as they are from their autoimmune disease.

[0168] The compositions of the present disclosure are useful in applications for which triptolide and its prodrugs and other derivatives have proven effective, e.g. in immunosuppression therapy, as in treating an autoimmune disease, preventing transplantation rejection, or treating or preventing graft-versus-host disease (GVHD). See, for example, co- owned U.S. Patent No. 6,150,539, which is incorporated herein by reference. Triptolide and the present derivatives are also useful for treatment of other inflammatory conditions, such as traumatic inflammation, and in reducing male fertility.

[0169] The compositions are useful for inhibiting rejection of a solid organ transplant, tissue graft, or cellular transplant from an incompatible human donor, thus prolonging survival and function of the transplant, and survival of the recipient. This use would include, but not be limited to, solid organ transplants (such as heart, kidney and liver), tissue grafts (such as skin, intestine, pancreas, gonad, bone, and cartilage), and cellular transplants (such as cells from pancreas, brain and nervous tissue, muscle, skin, bone, cartilage and liver).

[0170] The compositions are also useful for inhibiting xenograft (interspecies) rejection; i.e. in preventing the rejection of a solid organ transplant, tissue graft, or cellular transplant from a non-human animal, whether natural in constitution or bioengineered (genetically manipulated) to express human genes, RNA, proteins, peptides or other non-native, xenogeneic molecules, or bioengineered to lack expression of the animal's natural genes, RNA, proteins, peptides or other normally expressed molecules. The present disclosure also includes the use of a composition as described above to prolong the survival of such a solid organ transplant, tissue graft, or cellular transplant from a non-human animal.

[0171] Also included are methods of treatment of autoimmune diseases or diseases having autoimmune manifestations, such as Addison's disease, autoimmune hemolytic anemia, autoimmune thyroiditis, Crohn's disease, diabetes (Type I), Graves' disease, Guillain-Barre syndrome, systemic lupus erythematosis (SLE), lupus nephritis, multiple sclerosis, myasthenia gravis, psoriasis, primary biliary cirrhosis, rheumatoid arthritis and uveitis, asthma, atherosclerosis, Hashimoto's thyroiditis, allergic encephalomyelitis, glomerulonephritis, and various allergies.

[0172] Further uses may include the treatment and prophylaxis of inflammatory and hyperproliferative skin diseases and cutaneous manifestations of immunologically mediated illnesses, such as psoriasis, atopic dermatitis, pemphigus, urticaria, cutaneous eosinophilias, acne, and alopecia areata; various eye diseases such as conjunctivitis, uveitis, keratitis, and sarcoidosis; inflammation of mucous and blood vessels such as gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory bowel diseases, and necrotizing enterocolitis; intestinal inflammations/allergies such as Coeliac diseases and ulcerative colitis; renal diseases such as interstitial nephritis, Good-pasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy; hematopoietic diseases such as idiopathic thrombocytopenia purpura and autoimmune hemolytic anemia; skin diseases such as dermatomyositis and cutaneous T cell lymphoma; circulatory diseases such as arteriosclerosis and atherosclerosis; renal diseases such as ischemic acute renal insufficiency and chronic renal insufficiency; and Behcet's disease.

[0173] The compositions and method of the present disclosure are also useful for the treatment of inflammatory conditions such as asthma, both intrinsic and extrinsic manifestations, for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, particularly chronic or inveterate asthma (for example, late asthma and airway hyperresponsiveness). The composition and method may also be used for treatment of other inflammatory conditions, including traumatic inflammation, inflammation in Lyme disease, chronic bronchitis (chronic infective lung disease), chronic sinusitis, sepsis associated acute respiratory distress syndrome, and pulmonary sarcoidosis. For treatment of respiratory conditions such as asthma, the composition is preferably administered via inhalation, but any conventional route of administration may be useful.

[0174] In treating an autoimmune condition, the patient is given the composition on a periodic basis, e.g., 1-2 times per week, at a dosage level sufficient to reduce symptoms and improve patient comfort. For treating rheumatoid arthritis, in particular, the composition may be administered by intravenous injection or by direct injection into the affected joint. The patient may be treated at repeated intervals of at least 24 hours, over a several week period following the onset of symptoms of the disease in the patient. The dose that is administered is preferably in the range of 1-25 mg/kg patient body weight per day, with lower amounts being preferred for parenteral administration, and higher amounts being preferred for oral administration. Optimum dosages can be determined by routine experimentation according to methods known in the art.

[0175] For therapy in transplantation rejection, the method is intended particularly for the treatment of rejection of heart, kidney, liver, cellular, and bone marrow transplants, and may also be used in the treatment of GVHD. The treatment is typically initiated perioperatively, either soon before or soon after the surgical transplantation procedure, and is continued on a daily dosing regimen, for a period of at least several weeks, for treatment of acute transplantation rejection. During the treatment period, the patient may be tested periodically for immunosuppression level, e.g., by a mixed lymphocyte reaction involving allogeneic lymphocytes, or by taking a biopsy of the transplanted tissue.

[0176] In addition, the composition may be administered chronically to prevent graft rejection, or in treating acute episodes of late graft rejection. As above, the dose administered is preferably 1-25 mg/kg patient body weight per day, with lower amounts being preferred for parenteral administration, and higher amounts for oral administration. The dose may be increased or decreased appropriately, depending on the response of the patient, and over the period of treatment, the ability of the patient to resist infection.

[0177] In treatment or prevention of graft-versus-host disease, resulting from transplantation into a recipient of matched or mismatched bone marrow, spleen cells, fetal tissue, cord blood, or mobilized or otherwise harvested stem cells, the dose is preferably in the range 0.25-2 mg/kg body weight/day, preferably 0.5-1 mg/kg/day, given orally or parenterally. [0178] Also within the scope of the present disclosure is a combination therapy comprising a compound of formula I, II or III and one or more conventional immunosuppressive agents. These immunosuppressant agents within the scope of this disclosure include, but are not limited to, Imurek® (azathioprine sodium), brequinar sodium, Spanidin™ (gusperimus trihydrochloride, also known as deoxyspergualin), mizoribine (also known as bredinin), Cellcept® (mycophenolate mofetil), Neoral® (Cyclosporin A; also marketed as a different formulation under the trademark Sandimmune®), Prograf™ (tacrolimus, also known as FK-506), Rapimmune® (sirolimus, also known as rapamycin), leflunomide (also known as HWA-486), Zenapax®, glucocortcoids, such as prednisolone and its derivatives, antibodies such as orthoclone (OKT3), and antithymyocyte globulins, such as thymoglobulins. The compounds are useful as potentiators when administered concurrently with another immunosuppressive drug for immunosuppressive treatments as discussed above. A conventional immunosuppressant drug, such as those above, may thus be administered in an amount substantially less (e.g. 20% to 50% of the standard dose) than when the compound is administered alone. Alternatively, the presently described compound and immunosuppressive drug are administered in amounts such that the resultant immunosuppression is greater than what would be expected or obtained from the sum of the effects obtained with the presently disclosed compound used alone. Typically, the immunosuppressive drug and potentiator are administered at regular intervals over a time period of at least 2 weeks.

[0179] The compositions of the present disclosure may also be administered in combination with a conventional anti-inflammatory drug (or drugs), where the drug or amount of drug administered is, by itself, ineffective to induce the appropriate suppression or inhibition of inflammation.

[0180] Immunosuppressive activity of compounds in vivo can be evaluated by the use of established animal models known in the art. Such assays may be used to evaluate the relative effectiveness of immunosuppressive compounds and to estimate appropriate dosages for immunosuppressive treatment. These assays include, for example, a well-characterized rat model system for allografts, described by Ono and Lindsey (1969), in which a transplanted heart is attached to the abdominal great vessels of an allogeneic recipient animal, and the viability of the transplanted heart is gauged by the heart's ability to beat in the recipient animal. A xenograft model, in which the recipient animals are of a different species, is described by Wang (1991) and Murase (1993). A model for evaluating effectiveness against GVHD involves injection of normal Fl mice with parental spleen cells; the mice develop a GVHD syndrome characterized by splenomegaly and immunosuppression (Korngold, 1978; Gleichmann, 1984). Single cell suspensions are prepared from individual spleens, and microwell cultures are established in the presence and absence of concanavalin A to assess the extent of mitogenic responsiveness.

VI. Anticancer Treatment

[0181] The present disclosure includes the use of the compounds as cytotoxic agents, particularly to treat cancers. As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in mammals especially humans, including leukemias, sarcomas, carcinomas and melanoma.

[0182] The term "leukemia" refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.

[0183] Included, for example, are cancers involving cells derived from reproductive tissue (such as Sertoli cells, germ cells, developing or more mature spermatogonia, spermatids or spermatocytes and nurse cells, germ cells and other cells of the ovary), the lymphoid or immune systems (such as Hodgkin's disease and non-Hodgkin's lymphomas), the hematopoietic system, and epithelium (such as skin, including malignant melanoma, and gastrointestinal tract), solid organs, the nervous system, e.g. glioma (see Y.X. Zhou et al., 2002), and musculoskeletal tissue. The compounds may be used for treatment of various cancer cell types, including, but not limited to, brain, including medulloblastoma, head and neck, breast, colon, small cell lung, large cell lung, thyroid, testicle, bladder, prostate, liver, kidney, pancreatic, esophogeal, stomach, ovarian, cervical or lymphoma tumors. Treatment of breast, colon, lung, and prostate tumors is particularly contemplated.

[0184] The compositions may be administered to a patient afflicted with cancer and/or leukemia by any conventional route of administration, as discussed above. The method is useful to slow the growth of tumors, prevent tumor growth, induce partial regression of tumors, and induce complete regression of tumors, to the point of complete disappearance. The method is also useful in preventing the outgrowth of metastases derived from solid tumors.

[0185] The compositions of the present disclosure may be administered as sole therapy or with other supportive or therapeutic treatments not designed to have anti-cancer effects in the subject. The method also includes administering the compositions in combination with one or more conventional anti-cancer drugs or biologic protein agents, where the amount of drug(s) or agent(s) is, by itself, ineffective to induce the appropriate suppression of cancer growth, in an amount effective to have the desired anti-cancer effects in the subject. Such anti-cancer drugs include actinomycin D, camptothecin, carboplatin, cisplatin, cyclophosphamide, cytosine arabinoside, daunorubicin, doxorubicin, etoposide, fludarabine, 5-fluorouracil, hydroxyurea, gemcitabine, irinotecan, methotrexate, mitomycin C, mitoxantrone, paclitaxel, taxotere, teniposide, topotecan, vinblastine, vincristine, vindesine, and vinorelbine. Anti-cancer biologic protein agents include tumor necrosis factor (TNF), TNF-related apoptosis inducing ligand (TRAIL), other TNF-related or TRAIL-related ligands and factors, interferon, interleukin-2, other interleukins, other cytokines, chemokines, and factors, antibodies to tumor-related molecules or receptors (such as anti-HER2 antibody), and agents that react with or bind to these agents (such as members of the TNF super family of receptors, other receptors, receptor antagonists, and antibodies with specificity for these agents).

[0186] Antitumor activity in vivo of a particular composition can be evaluated by the use of established animal models, as described, for example, in Fidler et al., U.S. Patent No. 6,620,843. Clinical doses and regimens are determined in accordance with methods known to clinicians, based on factors such as severity of disease and overall condition of the patient.

VII. Other Indications

[0187] The compounds of the present disclosure may also be used in the treatment of certain CNS diseases. Glutamate fulfills numerous physiological functions, including an important role in the pathophysiology of various neurological and psychiatric diseases. Glutamate excitotoxicity and neurotoxicity have been implicated in hypoxia, ischemia and trauma, as well as in chronic neurodegenerative or neurometabolic diseases, Alzheimer's dementia, Huntington's disease and Parkinson's disease. In view of the reported neuroprotective effects of triptolide, particularly protection from glutamate-induced cell death (Q. He et al., 2003; X. Wang et al., 2003), compounds of the present disclosure are envisioned to antagonize the neurotoxic action of glutamates and thus may be a novel therapy for such diseases.

[0188] Regulation and function of the mammalian central nervous system is governed by a series of interdependent receptors, neurons, neurotransmitters, and proteins. The neurons play a vital role in this system for, when externally or internally stimulated, they react by releasing neurotransmitters that bind to specific proteins. Common examples of endogenous small molecule neurotransmitters such as glutamate, acetylcholine, adrenaline, dopamine, serotonin, and gamma- aminobutyric acid are well known, as are the specific receptors that recognize these compounds as ligands (Cooper, 1991). According to the reported neuroprotective effects of triptolide, particularly protection from glutamate-induced cell death (He, 2003 and Wang, 2002), compounds of the invention are envisioned to antagonize the neurotoxic action of glutamates and thus may be a novel therapy for many CNS diseases. Examples of these diseases include, but are not limited to, hypoxia, ischemia and trauma, as well as in chronic neurodegenerative or neurometabolic diseases, Alzheimer's dementia, Huntington's disease and Parkinson's disease.

[0189] AD is a progressive neurodegenerative disease causing a gradual loss of cognitive function in affected individuals. AD is characterized by senile plaques, neurofibrillary tangles, reactive microglial cells that are closely associated with senile plaques, dystrophic neurites and severely disrupted glutamatergic neurotransmission. Cerebral amyloid angiopathy due to beta- amyloid is one of the specific pathological features of AD. The deposition of beta-amyloid and the toxic cellular response to beta-amyloid aggregates are major pathogenic factors in the development of AD. Neurodegeneration in AD caused by inflammation involves activation of the brain's resident immune cells, microglia, by the aberrant beta- amyloid proteins to produce a variety of proinflammatory factors. These compounds may moderate neurodegeneration by inhibiting the induction of apoptosis in cerebral neurons resulting from the pathogenic actions of aggregates of beta-amyloid in AD. These compounds may inhibit the activation of microglia by the aberrant beta-amyloid proteins and suppress the production of proinflammatory neurotoxic factors, thereby reducing the pathogenic effects of beta-amyloid in AD.

[0190] Like AD, PD is a progressive neurodegenerative disease, characterized by resting tremor, slowness of movement, rigidity and postural instability as a result of progressive loss of dopaminergic neurons in the substantia nigra pars compacta. The cause for this loss of neurons is largely unknown, but considerable evidence supports the suggestion that brain inflammation participates in the pathogenesis of PD. Glial mediated inflammation has been implicated in this process. Activated microglia play a major role in neurodegeneration by releasing cytotoxic compounds that include reactive oxygen species, nitrite, proteases, and proinflammatory cytokines (including TNF-D and IL-1□). The higher density of microglial cells in the substantia nigra compared to other mid-brain areas renders the dopaminergic system most susceptible to damage caused by inflammation, and microglial cells are most likely involved in this process. These compounds may suppress the activation of microglia in response to proinflammatory stimuli, and inhibit the production of proinflammatory neurotoxic factors.

[0191] Glutamate excitotoxicity and neurotoxicity have been implicated in hypoxia, ischemia and trauma, as well as in chronic neurodegenerative or neurometabolic diseases, Alzheimer's dementia, Huntington's disease and Parkinson's disease. In view of the reported neuroprotective effects of triptolide, particularly protection from glutamate-induced cell death (Q. He et al. , 2003; X. Wang et al., 2003), compounds of the invention are envisioned to antagonize the neurotoxic action of glutamates and thus may be a novel therapy for such diseases.

[0192] There is also evidence from MS patients in relapse that suggests an altered glutamate homeostasis in the brain. Neurotoxic events occurring in MS patients can be responsible for oligodendrocyte and neuronal cell death. Antagonizing glutamate receptor-mediated excitotoxicity by treatment with compounds of this invention may have therapeutic implications in MS patients. Other CNS diseases such as Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy may also be treated with the compounds of the present invention.

[0193] The compounds of the present invention may also be used in the treatment of certain lung diseases. Idiopathic pulmonary fibrosis (PF) is a progressive interstitial lung disease with no known etiology. PF is characterized by excessive deposition of intracellular matrix and collagen in the lung interstitium and gradual replacement of the alveoli by scar tissue as a result of inflammation and fibrosis. As the disease progresses, the increase in scar tissue interferes with the ability to transfer oxygen from the lungs to the bloodstream. A 14-succinimide ester of triptolide has been reported to block bleomycin-induced PF (G. Krishna et al., 2001). Accordingly, the compounds of the present invention may be useful for treatment of PF. Treatment of other respiratory diseases, such as sarcoidosis, fibroid lung, and idiopathic interstitial pneumonia is also considered.

[0194] The compositions and method of the invention are also useful for the treatment of asthma, both intrinsic and extrinsic manifestations. For treatment of asthma, the composition is preferably administered via inhalation. The composition and method may also be used for treatment of other inflammatory conditions, such as traumatic inflammation, including traumatic inflammation accompanying head or neck injury.

[0195] Malignant cells are known to have accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is mediated by facilitative glucose transporter (GLUT) proteins. Cancer cells were found to have significantly higher expression of GLUT proteins than the corresponding normal epithelial cells at both the mRNA and protein level (Barron C, Tsiani C and Tsakiridis T. (2012) BMC Proceedings 6 (Suppl 3):P4) Enhanced tumor uptake of glucose is facilitated by the overexpression of glucose transporter proteins, is observed widely in tumor tissue. Elevated GLUT1 expression has been described in many cancers. (Macheda ML, Rogers S, Best JD. (2005) /. Cell Physiol. 202:654-662). [0196] The glucose conjugates described here are intended to selectively target tumor cells overexpressing glucose transporters as well as to exhibit increased water solubility compared to triptolide and derivatives like MRxl02.

VIII. Assays

Cytotoxic Evaluation

[0197] The cytotoxic activity of a compound of Formula I can be evaluated using the Alamar Blue fluorescence cytotoxicity assay as described hereinbelow. The cytotoxic activity of these compounds that is mediated by programmed cell death can be evaluated using the Terminal deoxynucleotidyl transferase apoptosis assay, or the Annexin V apoptosis assay as described hereinbelow.

Immunosuppressive activity

[0198] The immunosuppressive activity of these compounds can be evaluated with the IL-2 inhibition assay using ELISA analysis, as described hereinbelow, or the IL-2 inhibition assay using reporter gene analysis, as described hereinbelow.

Anti-fibrogenic activity

[0199] The anti-fibrogenic activity of these compounds can be evaluated with the TGF- D inhibition assay using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, as described hereinbelow, or the TGF- D inhibition assay using ELISA analysis, as described hereinbelow. The anti-fibrogenic activity of these compounds can be evaluated with the rat chronic kidney transplant rejection model, as described hereinbelow.

CNS protective and/or neuroprotective activity

[0200] The CNS protective and/or neuroprotective activity of these compounds can be evaluated with the in vitro glutamate excitotoxicity model, as described hereinbelow. The CNS protective and/or neuroprotective activity of these compounds relating to protection from neurotoxic agents, and with possible relevance to Parkinson's Disease, can be evaluated with the in vivo MPTP treatment model in mice, as described hereinbelow. The CNS protective and/or neuroprotective activity of these compounds relating to protection from inflammatory stimuli and/or neurotoxic agents, and with possible relevance to Parkinson's Disease, can be evaluated with the in vitro LPS treatment model of neuron-glial cultures, as described hereinbelow. The CNS protective and/or neuroprotective activity of these compounds can be evaluated with the BDNF in situ ELISA assay system with neuron-glial cultures, as described hereinbelow. The CNS protective and/or neuroprotective activity of these compounds can be evaluated with the NGF ELISA assay system with astrocyte cultures, as described hereinbelow. The CNS protective and/or neuroprotective activity of these compounds relating to Alzheimer's Disease and beta-amyloid-induced neurodegeneration can be evaluated with the in vitro beta-amyloid (Abeta) toxicity assay, as described hereinbelow.

Alamar Blue Fluorescence Cytotoxicity Assay

[0201] Test compounds are dissolved in DMSO at a concentration of 20 mM, and a range of serial dilutions of the test compounds in medium supplemented with 10% fetal calf serum (FCS). Additionally, the test compounds may be incubated in human plasma for a period of time (e.g., 24 hours) prior to dilution. Vehicle alone, and the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle), may be used as controls. Cells from an exponentially expanding culture of the Jurkat human T lymphocyte cell line (#TIB-152 obtained from American Type Culture Collection, Manassas, VA) at a concentration of 1 x 10⁶ cells/ml (1 x 10⁵ cells/well) are added to microwells of 96-well culture plates containing 100 Dl of the test compounds at 2x, and the plates are incubated at 37°C in a 5% CO₂ incubator. After 24 hours, an appropriate volume of diluted Alamar Blue reagent (CellQuanti-Blue reagent from BioAssay Systems, Hayward, CA, Alamar Blue reagent from BioSource International (Invitrogen Corporation), Camarillo, CA, CellTiter Blue reagent from Promega Corporation, Madison, WI, or equivalent) is added to all wells and the plates are returned to the incubator for an additional 4 hours. The supematants are harvested from the culture wells and read in a fluorescence plate reader using 560 nm for excitation and 590 nm for emission. The fluorescence is a measure of the conversion by viable cells of the Alamar Blue reagent to a fluorescent compound that can diffuse out of the cells into the supernatant. The data are presented as fluorescence vs. concentration of test compound. The concentration of test compound inducing 50% reduction in viable cells (ID50) is calculated from these dose response curves.

Terminal Deoxynucleotidyl Transferase Apoptosis Assay

[0202] Test compounds are dissolved in DMSO at a concentration of 20 mM. Further dilutions are done in medium supplemented with 10% FCS. Additionally, the test compounds may be incubated in human plasma for a period of time (e.g., 24 hours) prior to dilution. Vehicle alone, and the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle), may be used as controls. Cells from an exponentially expanding culture of the Jurkat human T lymphocyte cell line (#TIB-152 from American Type Culture Collection) at a concentration of 1 x 106 cells/ml (1 x 105 cells/well) are added to wells containing 100 Dl of the test compounds, and the plates are incubated at 37oC in a 5% C02 incubator. After 24 hours, the plates are fixed in paraformaldehyde, treated with ethanol, incubated with the enzyme terminal deoxynucleotidyl transferase and fluorescein labeled- deoxyuridined (Fl-dUTP), and treated with ribonuclease with appropriate washing steps as required. The cells are suspended in medium containing propidium iodide to distinguish intact apoptotic cells. The process allows 3' end labeling of DNA molecules that were nicked during the DNA fragmentation phase of apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL labeling). Apoptosis is analyzed using a flow cytometer. Cells positive for Fl-dUTP are considered to be apoptotic, and the data are calculated as percent apoptotic cells.

Annexin V Apoptosis Assay

[0203] Test compounds were dissolved in DMSO at a concentration of 20 mM. Further dilutions were done in medium supplemented with 10% FCS. Additionally, the test compounds may be incubated in human plasma for a period of time (e.g., 24 hours) prior to dilution. Vehicle alone, and the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle), may be used as controls. Cells from an exponentially expanding culture of the Jurkat human T lymphocyte cell line (#TIB-152 from American Type Culture Collection) at a concentration of 1 x 10⁶ cells/ml (1 x 10⁵ cells/well) are washed by centrifugation and dilution in complete medium and added to wells containing 100 Dl of the test compounds. The plates are incubated for 24 hours at 37°C in a 5% CO₂ incubator after which the cells are washed twice by centrifugation in 2% FCS in PBS and 500 Dl of binding buffer is used to resuspend the cells from each well according to the Annexin V assay procedure (BioVision, Inc., Mountain View, CA). Next, 5ul of the fluorescein isothiocyanate (FITC) conjugate of Annexin V (BioVision, Inc.) and 5ul of propidium iodide (BioVision, Inc.) are added to each sample before 5 minutes of incubation in the dark. Apoptosis is analyzed using a flow cytometer. Cells positive for Annexin V binding are considered to be apoptotic, the cells positive for propidium iodide are considered to be necrotic, and the data are calculated as percent apoptotic cells.

IL-2 Inhibition Assay Using ELISA Analysis

[0204] Test samples are diluted to 1 mM in complete tissue culture medium. Aliquots are placed in microculture plates and serial dilutions are prepared so that the final concentration encompasses the range of 0.001 to 10,000 nM in log increments. Additionally, the test compounds may be incubated in human plasma for a period of time (e.g., 24 hours) prior to dilution. Vehicle alone, and the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle), may be used as controls. Cells from an exponentially expanding culture of the Jurkat human T lymphocyte cell line (#TIB-152 from American Type Culture Collection) at a concentration of 1 x 10⁷ cells/ml (1 x 10⁶ cells/well) are stimulated with 50 ng/ml Phorbol 12-Myristate 13-Acetate (PMA, Sigma, St. Louis, MO) and 10 □ g/ml Phytohemagglutinin (the optimally stimulatory concentration of purified PHA from Sigma) in the presence of the test compound at 37°C in a 5% CO2 incubator in duplicate 1 ml culture wells. The culture supernatants are collected and stored frozen at -20 °C or lower until assayed. The concentration of human IL-2 (hIL-2) in the culture supernatants are measured in a conventional ELISA assay using an anti-hIL-2 monoclonal antibody and hIL-2 standard in an ELISA kit (R&D Systems (Minneapolis, MN), BD Pharmingen (San Diego, CA), or BioSource International (Camarillo, CA)). The data are expressed as ng/ml of IL-2.

IL-2 Inhibition Assay Using Reporter Gene Expression Analysis

[0205] Test samples are diluted to 1 mM in complete tissue culture medium. Aliquots are placed in microculture plates and serial dilutions are prepared so that the final concentration encompasses the range of 0.001 to 10,000 nM in log increments. Additionally, the test compounds may be incubated in human plasma for a period of time (e.g., 24 hours) prior to dilution. Vehicle alone, and the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle), may be used as controls. Jurkat T-cells transfected by electroporation with reporter and expression plasmids are incubated in the presence of the test compound and are stimulated for 6-12 h with 2 mM ionomycin (Iono, Calbiochem, San Diego, CA) plus either 20 ng/ml phorbol 12-myristate 13-acetate (PMA; Calbiochem) or antibody to CD3 (clone HIT3a, Pharmingen) at 37°C in a 5% CO2 incubator. The cells are then resuspended in 50 ml of lysis buffer (1 % Triton X- 100, 0.1 mM HEPES, pH 7.6, 1 mM dithiothreitol, and 2 mM EDTA, pH 8.0) for 10 min at 4 °C. The cell lysates are centrifuged at 13,000 rpm for 10 min, the supernatants are collected by centrifugation as whole cell extracts, and the Bradford reagent (Bio-Rad) is used to measure protein concentration. The cell extracts are mixed with luciferase reaction mixtures (1 mg/ml bovine serum albumin, 5 mM ATP, pH 7.6, 25 mM glycylglycine, and 15 mM MgS0₄) and 100 ml of 1 mM D-luciferin (Analytical Luminescence Laboratory, San Diego, CA) based on the amount of protein and triplicate determinations of luminescence are each read for 20 s using a luminometer.

TGF- D Inhibition Assay Using RT-PCR Analysis

[0206] Normal human lung fibroblasts (NHLFs) treated in vitro with bleomycin are induced to increased TGF- D gene expression, as measured by RT-PCR. Anti-fibrogenic activity can be assayed by incubating NHLFs in vitro in fibroblast growth medium with bleomycin (Blenoxane,

Mead Johnson Oncology Products, Bristol-Myers Squib, Princeton, NJ, 15 U/Vial) at 37°C in a

5% CO2 incubator for 16-24 hrs in the presence of the test compound or the test compound previously incubated in human plasma for a period of time (e.g., 24 hours) and harvesting for RNA extraction. Total RNA is extracted from the cells using a commercial purification kit (Promega, Madison, WI), and reverse- transcription into cDNA is conducted and then amplified in a PCR thermal cycler (such as MiniCycle PCR system (Biorad Laboratories, Hercules, CA) or Applied Biosystems PCR System 9700 (Applied Biosystems, Foster City, CA)) with appropriate denaturation, primer annealing, and primer extension. TGF- D and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense and antisense oligonucleotides are used and PCR products are separated by electrophoresis on 2% agarose gel with ethidium bromide and are visualized with an electronic UV Transilluminator (Ultra-Lum, Inc., Claremont, CA). The ratio of TGF- □ /GAPDH mRNA is derived from a scan of the agarose gel and plotted for purposes of comparison of the level of TGF-□ gene expression.

TGF- D Inhibition Assay Using ELISA Analysis

[0207] NHLFs treated with bleomycin in vitro are induced to increased TGF- D gene expression, as measured by RT-PCR. Anti-fibrogenic activity can be assayed by incubating NHLFs in vitro in fibroblast growth medium with bleomycin (Blenoxane, Mead Johnson Oncology Products, Bristol-Myers Squib, 15 U/Vial) at 37°C in a 5% C0₂ incubator for 16-24 hrs in the presence of the test compound or the test compound previously incubated in human plasma for a period of time (e.g., 24 hours) and harvesting culture supernatants for ELISA analysis of human TGF- D . The culture supernatants are collected and stored frozen at -20 °C or lower until assayed. The concentration of human TGF- D in the culture supernatants are measured in a conventional ELISA assay using an anti-TGF- D monoclonal antibody and hTGF- β standard in an ELISA kit (R&D Systems, BD Pharmingen, BioSource International or Promega Corp., Madison, WI). The data are expressed as ng/ml of TGF-D .

Rat chronic kidney transplant rejection model

[0208] Adult Fisher 344 rats are used as donors and adult Lewis rats are used as recipients. Lewis rat recipients are bilaterally nephrectomized and grafted with a kidney freshly removed from a Fisher 344 donor rat using end-to-side anastomoses between the donor renal artery and recipient abdominal aorta and the donor renal vein and the recipient inferior vena cava, as described (Chin, 1989). This renal transplant model exhibits reproducible pathological changes characteristic of chronic graft rejection by day 90-140 after transplantation when calcineurin inhibitors cyclosporine or FK506 are used at the appropriate dose level and duration. All animals receive cyclosporine treatment (0.75 mg/kg/day s.c.) or FK506 (1 mg/kg/day) from days 1-10 after transplantation to prevent acute rejection. Transplanted kidneys are removed at necropsy, fixed in formaldehyde, embedded in paraffin and sectioned, and individual slides are stained with hematoxylin-eosin or trichrome. The slides are analyzed for histopathology and evaluated for the severity of chronic rejection on a 1-4 scale. The kidneys are also evaluated by gene expression analysis for TGF- D by RT-PCR as described herein, except that extraction procedures appropriate to animal organs rather than a suspension of single cells are utilized. The anti-fibrogenic effect of the test compound and the activity in preventing, or reducing the severity of, chronic graft rejection are evaluated by using this model and treating with the test compound p.o. daily starting on the day of transplantation. Interstitial fibrosis is usually observed in the transplanted kidneys in this model, and the anti-fibrogenic effect of the test compound is demonstrated by a decrease in the incidence and severity of interstitial fibrosis and a reduction in the severity of chronic rejection. Tubular atrophy, glomerular atrophy, cortical scarring and neointimal thickening are also characteristic hallmark changes of chronic rejection observed in this model, and these can also be evaluated for evidence of an effect of the compound upon chronic rejection.

In vitro glutamate excitotoxicity model

[0209] Test compounds are dissolved in DMSO at a concentration of 20 mM. Further dilutions are done in RPMI1640 medium (GIBCO, Rockville, MD) supplemented with 10% FCS. PC12 pheochromocytoma cells (American Type Culture Collection) are treated with various concentrations of glutamate (usually in the range of 1-100 mM), which induces cytotoxicity and apoptosis. The cultures are also incubated with vehicle, one of a series of concentrations of the test compounds after incubation in human plasma, or the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle). Usually the concentration range for the compounds is 0.001 - 1000 nM. After 24 hours, cell viability is assayed using the MTT assay and apoptosis is determined using Annexin V and FACS analysis. Cytotoxicity of the compounds was determined in a standard MTT assay using Cell Proliferation Kit I (#1 465 007, Roche Diagnostics, Mannheim, Germany). After incubation and washing, the cultures are supplemented with 10 Dl/well MTT reagent for 4h and then with 0.1 ml/well solubilizing reagent for an additional 16h. Optical density at 570 nm (OD570) is measured on a ThermoScan microplate reader (Molecular Devices, Menlo Park, CA). The data are presented as OD570 values versus concentration of the compounds, and can be used to calculate percent cytotoxicity or percent viability. Annexin V and FACS analysis is used to assess apoptosis. Additionally, reactive oxygen species (ROS) formation and the decrease of mitochondrial membrane potential may be assessed (as described in the in vitro beta-amyloid (Abeta) toxicity assay) as parameters of glutamate-induced excitotoxicity. In vivo MPTP treatment model in mice

[0210] Mice are treated with the test compound or vehicle the day before MPTP (1 -methyl - 4-phenyl-l,2,3,6-tetrahydropyridine) injection and daily for 7 days. MPTP crosses the blood brain barrier and is metabolized in the astrocytes to its toxic metabolite l-methyl-4-phenyl-2,3- dihydropyridinium (MPP+), by monoamine oxidase-B (MAO-B). Treatment with high dose MPTP rapidly produces oxidative stress in the dopaminergic neurons and causes a cytotoxic effect. The dopaminergic neurons can be visualized as tyrosine hydroxylase immune reactive cells using labeled anti-tyrosine hydroxylase antibody and histopathology, and the microglia can be recognized using an 0X42 antibody recognizing the CR3 receptor and histopathology. This assay can be conducted at a contract research organization such as SkeleTech, Inc. (MDS Pharma Services, Bothell, WA) or in an individual laboratory animal facility.

In vitro LPS treatment model of neuron-glial cultures

[0211] Test compounds are dissolved in DMSO at a concentration of 20 mM. Further dilutions are done in Dulbecco's MEM supplemented with 10% FCS. Embryonic mesencephalic neuron-glial cultures are obtained from timed pregnant Sprague-Dawley rats on embryonic day 14 by culturing cells dissociated from ventral mesencephalic tissues in 24-well culture plates precoated with poly- lysine in Dulbecco's MEM medium. Seven-day cultures are treated with vehicle, one of a series of concentrations of the test compounds, one of a series of concentrations of the test compounds after incubation in human plasma, or the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle) and either medium (untreated) or 5 mg/ml of LPS. Usually the concentration range for the compounds is 0.001 - 100 nM. Accumulation of nitrite, an indicator of NO production induced by LPS, is assayed at 24 and 48 hours using the Griess reaction (the Promega Greiss Reagent System, Promega Corporation, Madison WI.). The nitrite concentration is expressed in □ M. Production of TNF- D and IL-1 D induced by LPS are assayed in 2-4 hour samples using TNFD -specific IL- 1□ -specific ELISA assays. The data are expressed as ng/ml of these cytokines. Dopaminergic neurons can be visualized by histopathology as tyrosine hydroxylase immune reactive cells using labeled anti-tyrosine hydroxylase antibody and histopathology to assess the prevention of the loss of these neurons, and the microglia can be recognized by histopathology using an 0X42 antibody recognizing the CR3 receptor.

BDNF in situ ELISA assay system with neuron-glial cultures

[0212] Test compounds are dissolved in DMSO at a concentration of 20 mM. Further dilutions are done in Dulbecco's MEM medium supplemented with 10% FCS. Embryonic mesencephalic neuron-glial cultures are obtained from timed pregnant Sprague-Dawley rats on embryonic day 14 by culturing cells dissociated from ventral mesencephalic tissues in Dulbecco' s MEM medium in 96-well ELISA plates that had been UV-sterilized and coated with anti-BDNF monoclonal antibody (Promega Corporation) for the Promega Emax BDNF in situ immunoassay system. Cultures are treated with vehicle, one of a series of concentrations of the test compounds, one of a series of concentrations of the test compounds after incubation in human plasma, or the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle) soon after initiation or up to 7 days later. Usually the concentration range for the compounds is 0.0001 - 100 nM. The cultures are incubated for 1-3 days and BDNF is measured using the Promega Emax BDNF in situ immunoassay system after washing away the cultivated cells. The data are expressed as pg/ml of BDNF.

NGF ELISA assay system with astrocyte cultures

[0213] Test compounds are dissolved in DMSO at a concentration of 20 mM. Further dilutions are done in Dulbecco' s MEM medium supplemented with 10% FCS. Astrocyte cultures are initiated, using neocortices from term fetal or early neonatal Sprague-Dawley rats (Luo et al., 1998) by dissociating, purifying and culturing the cells in Dulbecco' s MEM medium. Cultures are treated with vehicle, one of a series of concentrations of the test compounds, one of a series of concentrations of the test compounds after incubation in human plasma, or the highest concentration of diluted human plasma used in the compound assay (after incubation with vehicle) soon after initiation or up to 7 days later. Usually the concentration range for the compounds is 0.0001 - 100 nM. The cultures are incubated for 1-3 days and NGF is measured using NGF-specific ELISA assays (NGF Emax NGF ImmunoAssay System, Promega; ChemiKine NGF Sandwich Elisa Kit, Chemicon). The data are expressed as pg/ml of NGF.

In vitro beta-amyloid (Abeta) toxicity assay

[0214] Test compounds are dissolved in DMSO at a concentration of 20 mM. Further dilutions are done in medium supplemented with 10% FCS. PC12 pheochromocytoma cells are treated with various concentrations of Abeta (ranging from 10(-5) to 100 micromol/L) for 48 hours. Additionally, the cell cultures are treated over the same period with vehicle, one of a series of concentrations of the test compounds, one of a series of concentrations of the test compounds after incubation in human plasma, or the highest concentration of diluted human plasma used in the compound assay. Cell viability is assayed using the MTT assay and apoptosis is determined using Annexin V and FACS analysis as described herein. The neuroprotective effect of the test compounds against cytotoxicity and apoptosis by Abeta cells is determined by the reduction in these parameters in PC 12 cells. The toxicity of Abeta is accompanied by the production of reactive oxygen intermediates (ROI) and changes in mitochondrial potential. To determine the level of ROS, the cultures are pre-incubated for 15 min with DCH2F (5 Dg/ml). To measure DCF fluorescence, cells are read on a fluorescent plate reader (such as the CytoFluor 2350 Millipore, Bedford, MA) at 485 nm excitation and 530 nm emission wavelength. Mitochondrial membrane potential are measured using the voltage- sensitive dye JC-1 (Molecular Probes, Invitrogen Corporation, Carlsbad, CA). With decreasing membrane potential, less JC-aggregates are formed and the emission spectrum changes from 590 to 530 nm.

EXAMPLES

[0215] The following non-limiting examples highlight the features of the present disclosure and are used to illustrate the presently disclosed compounds, compositions and methods. However, this disclosure shall in no way be considered to be limited to the particular embodiments described below.

[0216] All commercially available materials were used as received. All isolated materials were characterized by protein content, sugar content, sugar residue analysis, molecular weight distribution, and sugar linkage analysis.

[0217] The methods to make PG691 and PG763 are shown first. PG691 and PG763 can be used as starting materials to prepare the remaining examples.

[0218] Synthesis of PG691 - Protection of 14-OH of PG490

[0219] To a solution of PG490 (0.56 g, 1.6 mmol) in DMSO (8.5 mL, 0.12 mol) were added acetic acid (28 mL, .49 mol) and acetic anhydride (5.6 mL, 59 mol). The clear colorless solution was stirred at room temperature for five days. The reaction mixture was poured into 200 mL of water. The acidic mixture was neutralized with portion wise addition of solid sodium bicarbonate. The mixture was extracted with ethyl acetate (150 mL x 3) and the combined organic solution was dried over anhydrous sodium sulfate. Concentration under reduced pressure gave the crude product as oil. Silica gel column chromatography purification (3:2 hexanes/ethyl acetate) gave PG691 (0.45 g, 69%) as white foam. ^!H NMR (CDC1₃)□ 0.83 (d, /

= 6.8 Hz, 3H), 1.01 (d, / = 6.8 Hz, 3H), 1.10 (s, 3H), 1.20 (m, 1H), 1.61 (m, 1H), 1.92 (dd, / = 14.7, 13.4 Hz, 1H), 2.19 (s, 3H), 2.10-2.42 (m, 4H), 2.70 (m, 1H), 3.24 (d, / = 5.5 Hz, 1H), 3.51 (d, / = 3.1 Hz, 1H), 3.68 (s, 1H), 3.79 (d, / = 3.1 Hz, 1H), 4.68 (m, 2H), 4.95 (d, 7 = 11.8 Hz, 1H), 5.09 (d, J = 11.8 Hz, 1H)

[0220] Synthesis of PG763 - Preparation of 14-deoxy-14 -fluoro triptolide

PG490 PG763

[0221] To a solution of PG490 (triptolide, 17.3 mg, 0.048 mmol) in dichloromethane (1.0 ml) at 0°C was added (diethylamino)sulfur trifluoride (DAST, 100 μΐ, 0.763 mmol) under N₂. The reaction mixture was stirred at 0°C for 2 hrs, and saturated NaHC0₃ solution (0.8 ml) was then added. The reaction mixture was extracted with 3 x 2 ml of dichloromethane. The combined organic layer was dried over anhydrous NaS0₄ and concentrated under vacuum. The desired product (PG763) was obtained in quantitative yield.

[0222] Analytical TLC Rf = 0.78 (ethyl acetate/hexanes/methanol 1: 1 :0.1). IR (KBr):

3031.0, 2961.2, 2942.4, 2873.8, 1764.6, 1680.9, 1449.3, 1438.3, 1172.2, 1098.1, 1074.5, 1057.0,

1047.1, 1034.2, 1018.2, 1005.6, 987.3, 972.3, 923.9, 909.0, 743.6, 586.0, 566.0, 539.8, 527.6 cm^"1. *H NMR (300 MHz, CDC1₃): δ = 5.16 (d, 1H, 14-CH), 4.70 (q, 2H, 19-CH₂), 3.80 (d, 1H, 11-CH), 3.73 (d, 1H, 7-CH), 3.50 (t, 1H, 12-CH), 2.70 (m, 1H, 5-CH), 2.34 (d, 1H, 2-CHb), 2.27 - 2.02 (m, 3H, 6-CHb, 2-CHa and 15-CH), 1.95 (m, 1H, 6-CHa), 1.56 (dd, 1H, 1-CHb), 1.24 (m, 1H, 1-CHa), 1.11 (s, 3H, 20-CH₃), 1.10 (d, 3H, 17-CH₃), 0.91 (d, 3H, 16-CH₃) ppm.

EXAMPLE 1 :

Acylation PG691 to give Di-O, C-acyl, PG800mw

[0223] One way to run this reaction is to use 1 equivalent of both LDA and acyl chloride to give a mixture of PG691, both mono-C-acyl and mono-O-acyl, and di-O, C-acyl derivatives. Shown below is the use of 2 equivalent of both LDA and acyl chloride to give the di-O, C-acyl derivative followed by acid hydrolysis to give the mono C-acyl derivative after purification.

[0224] PG1580mw

[0225] To a solution of PG691 (73.1 mg, 0.174 mmol) in anhydrous THF (5 mL) is added a solution of LDA in heptane/THF/ethyl benzene (0.17 mL of 2.0 M solution, 0.34 mmol) dropwise at -78° C. The resulting solution is stirred at this temperature for 15 min followed by the dropwise addition of the indicated acylchloride chloride, PG616, (209 mg, 0.34 mmol). The reaction is stirred at -78 °C for 2 h. The reaction is quenched with water and the mixture is extracted with ethyl acetate (25 mL x 3). The combined organic solution is dried over anhydrous over anhydrous sodium sulfate. Concentration under reduce pressure followed by purification by column chromatography (silica gel, 1: 1 hexanes/ethyl acetate) gives an oil, PG1580mw.

[0226] PG1520

[0227] To a solution of the O, C-diacylated-methylthiomethyl protected material, PG1580mw, (167.6 mg, 0.106 mmol) in 1.5 mL acetonitrile/water (4: 1) is added mercury (II) chloride (0.285 g, 1.05 mmol) in one portion. The resulting solution is stirred at room temperature overnight. White solid precipitated from the solution is removed by filtration through Celite and rinsed with ethyl acetate. The EtOAc solution is washed with 5% aqueous NH₄OAc twice. The organic phase is dried (Na₂S0₄) and concentrated under reduce pressure to give the crude product. Purification by column chromatography (silica gel, 1 : 1 hexanes/ethyl acetate) gives the O-acyl product, PG1520. [0228] PG800mw

[0229] The oil, PF1520mw, is dissolved in THF and is treated with 10% palladium on carbon and hydrogen gas under pressure at room temperature until disappearance of starting material is indicated by TLC. Column purification on silica gel, using hexanes/ethyl acetate/methanol as an eluent gives, after concentration, the product, PG800mw.

[0230] Calc. H NMR for PG800mw is indicated below:

Mono-C-acyl PG580mwl

[0231] Dissolve PG800mw in wet acetone and add dilute ethereal HC1. After TLC has indicated reaction is complete by disappearance of starting material, neutralize slowly with dilute sodium bicarbonate, extract with ethyl acetate (3x), dry with anhydrous sodium sulfate and concentrate under reduce pressure gives an oil. Column purification gives the product, PG580mwl. [0232] Calc. H NMR of PG580mwl is indicated below:

[0233] Starting with PG763 instead of PG691, the method to prepare PG802 can be done as is described in Example 1. The major difference is the omission of the unnecessary de- protection step that uses HgC¾, CH₃CN and ¾0.

EXAMPLE 4:

Mono-C-acyl PG580mwl

[0234] Starting with PG802 instead of PG800, the method to prepare PG582 can be done as is shown in Example 2.

EXAMPLE 5:

Synthesis of PG464mw

[0235] PG492

[0236] To a solution of PG691 (197mg, 0.469 mmol) in anhydrous THF (5 mL) is added a solution of LDA in heptane/THF/ethyl benzene (0.25 mL of 2.0 M solution, 0.50 mmol) dropwise at -78° C. The resulting solution is stirred at this temperature for 10 min followed by the dropwise addition of neat chlorotrimethylsilane (65 DL, 0.52 mmol). The reaction was stirred at -78 °C for 1 h and warmed to room temperature. The resulting solution is quenched with water and is extracted with EtOAc (10 mL x 2). The combined organic solution was dried over anhydrous over anhydrous sodium sulfate. Concentration under reduce pressure gave an oil. Column purification (silica gel, 1 : 1 hexanes/ethyl acetate) gives the indicated trimethylsilyl derivative, PG492. [0237] PG524

[0238] To a solution of Cul in l,3-dimethyl-2-imidazolidinone (TMI) and THF with benzoylchoride (80 Dl), is added the indicated trimethlysilyl derivative, PG492 (196.8 mg, 0.4mmol). The reaction is stirred overnight at room temperature. Methylene chloride is added and the mixture is extracted with water (4x) and the organic phase is concentrated to an oil. The oil is purified by column chromatography (silica gel, 1:1 hexanes/ethyl acetate) to give the indicated O-acylated-methylthiomethyl protected material, PG524.

[0239] PG464

[0240] To a solution of the O-acylated-methylthiomethyl protected material, PG524mw, (55.6 mg, 0.106 mmol) in 1.5 mL acetonitrile/water (4:1) is added mercury (II) chloride (0.285 g, 1.05 mmol) in one portion. The resulting solution is stirred at room temperature overnight. White solid precipitated from the solution is removed by filtration through Celite and rinsed with ethyl acetate. The EtOAc solution is washed with 5% aqueous NH₄OAc twice. The organic phase is dried (Na₂S0₄) and concentrated under reduce pressure to give the crude product. Purification by column chromatography (silica gel, 1 : 1 hexanes/ethyl acetate) gives the O-acyl product, PG464mw.

[0241] Calc. Η NMR for PG464mw is indicated below:

EXAMPLE 6:

[0242] PG580mw2

PG580mw2

[0243] Starting with PG492mw and employing the acylation conditions of Example 5, PGlOOOmw is obtained. The major exception is the use PG616mw instead of benzoyl chloride. Then using the de-protection (HgCl₂, CH₃CN, ¾0) followed by de-benzylation (Pd/C, ¾) of Example 1, PG580mw2 is obtained.

[0244] Refer to Example 5 for the following. Starting with PG763 instead of PG691, and using the silylating conditions (LDA leq, THF, -78°C, then Me₃SiCl, leq) PG434mw is obtained. Treating PG434mw to the conditions (CuCl, DMI, rt) and using PG616mw instead of benzoyl chloride gives PG942. Employing hydrogenolysis conditions (H2, Pd/C) of Example 1 gives PG580mw2.

[0245] Using the diacylation conditions of Example 1 and starting with PG763, PG570 mw is obtained.

[0246] Starting with PG570 of Example 9 and using conditions of Example 2. PG466mwl is obtained.

Starting with PG763 and using the silylation conditions of Example 5, PG466mw2

[0248] While various specific embodiments have been illustrated and described, it will be appreciated that equivalents of the specifically disclosed materials and techniques will also be applicable and various changes can be made without departing from the spirit and scope of the present disclosure; such equivalents are intended to be included within the following claims. Therefore, it is to be understood that the disclosure is not to be limited to the specific embodiments disclosed herein, as such are presented by way of example. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0249] All literature and similar materials cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, internet web pages and other publications cited in the present disclosure, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose to the same extent as if each were individually indicated to be incorporated by reference. In the event that one or more of the incorporated literature and similar materials differs from or contradicts the present disclosure, including, but not limited to defined terms, term usage, described techniques, or the like, the present disclosure controls.

Claims

WHAT IS CLAIMED IS:

1. A compound having the structure I:

where

Ri = lower arylacyl, or monsaccharidylacyl;

R2 = OH, F or taken together with R3 to = O; and

X = H or OH, with the proviso that not more than one X OH within the same structure.

2. A compound having the structure II:

where

Ri = lower arylacyl, or monsaccharidylacyl;

R2 = OH, F or taken together with R₃ to = O; and

3. A compound having the structure III:

where Ri = lower arylacyl, or monsaccharidylacyl;

R2 = OH, F or taken together with R3 to = O; and

4. A triptolide derivative composition, comprising a compound having structure I, II or

III.

5. A method of producing a triptolide derivative, comprising:

(a) scheme 1;

(b) scheme 2;

(c) scheme 3;

(d) scheme 4; or

(e) scheme 5.

6. A method of treating an inflammatory disease or disorder in a mammal in need thereof, comprising orally administering to the mammal an amount of the triptolide derivative composition of any of Claims 1 to 5, in an amount effective to treat the inflammatory disease or disorder.