WO2009023201A1 - Rigid derivatives of triptolide as anticancer, immune suppressant, anti-fibrosis, and cns protectant agents - Google Patents

Abstract

Compounds having the Formula I: are useful for inducing cell death (apoptosis), in immune suppression, in treating fibrosis and protecting CNS cell death. In Formulate I, R1 is OX and R2 - R7 is H or OX. X is COY, COOY, or CONHY.

Description

RIGID DERIVATIVES OF TRIPTOLIDE AS ANTICANCER, IMMUNE SUPPRESSANT, ANTI-FIBROSIS, AND CNS PROTECTANT AGENTS

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit under 35 U.S.C. § 119(e)(l) of provisional application 60/965,025 filed August 16, 2007, which application is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION This invention relates to rigid derivatives of triptolide useful as anticancer, immune suppressant, anti-fibrosis, and CNS protective agents.

REFERENCES

Chin, J. et al, Transplant Proc. 21:3351 (1989). Cooper, J. R. et al , The Biochemical Basis of Neuropharmacology, 6th Edition,

Oxford University Press, New York, (1991).

Dai, D. et al, U.S. Patent No. 6,943,259 (2005).

Fidler, J. M. et al, PCT Pub. No. WO 2005/020887 (2005).

Fidler, J. M. et al, U.S. Patent No. 6,620,843 (2003). Gleichmann, E. et al, Immunol Today 5:324 (1984).

He, Q. et al, Beijing Da Xue Xue Bao 35:252 (2003).

Jung et al, U.S. Patent No. 5,972,998 (1999).

Jung et al, U.S. Patent No. 6,004,999 (1999).

Korngold, R., J. Exp. Med. 148:1687 (1978). Krishna, G. et al, Am. J. of Pathology 158(3): 997 (2001).

Kupchan, S. M. et al, J. Am. Chem. Soc. 94:7194 (1972).

Kupchan, S. M. et al, U.S. Patent No. 4,005,108 (1977).

Lipsky, P. E., et al, U.S. Patent No. 5,294,443 (1994).

Ma, B. et al, J. Chin. Pharm. Sci. 1(2): 12 (1992). Murase, N. et al, Transplantation 55:701 (1993).

Musser, J. H., U.S. Patent No. 6,150,539 (2000).

Ono, K. et al, J. Thor. Cardiovasc. Surg. 57(2): 225-29 (1969).

Pu, L. etal, Zhongguo YaoliXuebao 11:76 (1990). Qi, Y. M. et al, U.S. Patent No. 5,962,516 (1999). Qian, et al, U.S. Patent No. 5,430,054 (1995). Veber, DF, et al, J Med. Chem., 45, 2615 (2002). Wang, J. et al, Transplantation Proc. 23:699 (1991). Wang, X. et al, PCT Pub. No. WO 2002/17931 (2002).

Wiedmann, T.W.T., U.S. Patent No. 5,843,452, (1998). Yang, D. et al., Int. J. Immunopharmacology 14:963 (1992).

BACKGROUND OF THE INVENTION The plant Tripterygium wilfordii (Tw) contains many natural products that are biologically active. In particular, the compound triptolide first isolated from the Tw plant (Kupchan et al, 1972) has potent anticancer and immunosuppressive activities (Yang et al., 1992). Also noted is that various extracts of the plant have been used to treat autoimmune diseases such as rheumatoid arthritis, and modulate male fertility. See, for example, Wiedmann et al, 1998, Lipsky et al, 1994, and Qian, et al, 1995.

In general, the biological activity of triptolide derivatives are less than that of native triptolide. See, for example, Jung et al, 1999, Kupchan et al, 1977. However, these compounds often provide other benefits relative to native triptolide, in areas such as pharmacokinetics or biodistribution, by virtue of their activity as prodrugs and/or differences in lipid or aqueous solubility. See, for example, Musser, 2000, and Qi et al., 1999, and references cited therein.

As described above, various derivatives and analogs of triptolide are known in the art, and many are therapeutically useful. However, there has been little attention paid to rigid derivatives. For example, Qi, Y. M. et al, U.S. Patent No. 5,962,516 (1999), esterifies the 14-hydroxyl group on triptolide. The resulting compounds from Qi, Y. M. et al, consist of flexible chains with rotatable carbon-carbon bonds. Indeed, the preferred example is thel4-succinate derivative of triptolide with 4 additional rotatable bonds all part of the added appendage.

Veber, in an analysis of over 1100 drug candidates, found that reduced molecular flexibility, as measured by the number of rotatable bonds, is a good predictor of oral bioavailability [Veber, DF, et. al, J Med. Chem., 45, 2615 (2002)]. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the anti acute leukemia cell (MV-4-11) activity of MRx 105. Figure 2 shows the anti acute leukemia cell (MV-4-11 activity of MRx 106.

SUMMARY OF THE INVENTION

The compounds of this invention are different from those described above in that the added appendage(s) are rigid, that is, the derivatives have a minimum number of rotatable bonds in the added appendage. These rigid derivatives provide benefits relative to native triptolide and previous derivatives of triptolide in areas of pharmacokinetics (PK) and absorption, distribution, metabolism and excretion (ADME), by virtue of their activity as prodrugs that are resistant to metabolic conversion and greater predicted oral bioavailability. Rigid triptolide prodrugs, in which the new rigid feature is located on the appendage of the prodrug, have different rates of conversion to the active drug triptolide, different PK and ADME characteristics because of different octanol/water partition coefficients and different metabolic stabilities.

In one aspect, the invention provides compounds that are useful as anticancer, immune modulator, fibrosis, and neurodegenerative therapy. The compounds are derivatives of triptolide represented by Formula I:

where:

R¹ is OX and R² - R⁷ is H or OX with the proviso that at least five of R² - R⁷ are H. X is COY, COOY, or CONHY.

Y is defined as C3-C12 moiety containing a mono-, bi-, or tricyclic ring system that is unsubstituted or substituted with Z. The mono-, bi- or tricyclic ring system can be saturated or unsaturated.

Z is defined as CO₂R⁸ or N(R⁸)₂, lower alkyl or halogen. Halogen is defined as F, Cl, Br or I. R⁸ = H or lower alkyl.

Lower alkyl is defined as C1-C6, unbranched or branched. C1-C6, unbranched or branched means methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert- butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 2,2- dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4methylpentyl, 1,1- diemthylbutyl, 2,2-dimethylbutyl, 3 ,3 -dimethylbutyl, 1 , 1 ,2-trimethylpropyl, 1 ,2,2- trimethylpropyl.

Preferably, the stereochemistry at R¹ is the β-configuration. The stereochemistry at R² can be of either the α or β-configuration. Preferably, the stereochemistry at R⁴ is the α-configuration. Preferably, the stereochemistry at R⁵ is the β-configuration. The stereochemistry at R⁷ can be of either the α or β-configuration.

Preferred embodiments include compounds in which R¹ = OX and each of R²-R⁷ is hydrogen.

Preferred embodiments for Y include but are not limited to: C3 - cyclopropane, C4- cyclobutane, methylcyclopropane, C5 - cyclopentane, methylcyclobutane, C6 - cyclohexane, methylcyclopentane, C7- cycloheptane, methylcyclohexane, bicyclo[2.2.1]heptane, bicyclo[2.2.1]hept-2-ene, C8 - cyclooctane, methylcycloheptane, 1,1,2-trimethyl-cyclopentane, octahydro-pentalene, bicycle[2.2.2]octane, bicycle[3.2.1]octane, C9 - cyclononane, methylbicycle[2.2.2]octane, octahydro-indene, ClO - cyclodecane, methyloctahydro-indene, bicycle[4.2.2]decane, adamantane, CI l - cycloundecane, bicycle[3.3.3]undecane, decahydro-cyclopentalene, methyladamantane, and C 12 - cyclododecane, dodecahydro-benzocyclooctane, dodecahydro-acenaphthylene and dodecahydro-s-indacene.

In other aspects, the invention provides a method of effecting immune suppression, and a method of inducing apoptosis in a cell, which is especially useful in anticancer therapy. In additional other aspects, the invention provides a method for suppressing fibrosis and a method for protecting neurological cells. The methods comprise administering to a subject in need of such treatment, or contacting said cell, respectively, with an effective amount of a compound having the Formulate I as described herein. Alternatively, the invention encompasses the use of a compound of Formula I 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 Formulate I described herein.

DETAILED DESCRIPTION OF THE INVENTION Triptolide Derivatives Numbering Scheme

For the purposes of the current disclosure, the following numbering scheme is used for triptolide and triptolide derivatives (see Figure II):

The compounds of the invention result from reaction of triptolide (14-OH) or hydroxyl triptolides that contain one additional hydroxyl at C2, C5, Cl 6, Cl, Cl 5 or C- 19 with one or two COY, COOY, CONHY or CON(Y)₂ groups, where Y is defined as C3-C12 moiety containing a mono-, bi-, or tricyclic ring system that is unsubstituted or substituted with Z. The mono-, bi- or tricyclic ring system can be saturated or unsaturated. More specifically, the compounds of the invention are represented by Formulate I below: Formula I

where: R¹ is OX and R² - R⁷ is H or OX with the proviso that at least five of R² - R⁷ are H.

X is COY, COOY, or CONHY.

Y is defined as C3-C12 moiety containing a mono-, bi-, or tricyclic ring system that is unsubstituted or substituted with Z. The mono-, bi- or tricyclic ring system can be saturated or unsaturated. Z is defined as CO₂R⁸ or N(R⁸)₂, lower alkyl or halogen.

Halogen is defined as F, Cl, Br or I. R⁸ = H or lower alkyl.

Lower alkyl is defined as C1-C6, unbranched or branched.. C1-C6, unbranched or branched means methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert- butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 2,2- dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4methylpentyl, 1,1- diemthylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2- trimethylpropyl.

Preferably, the stereochemistry at R¹ is the β-configuration. The stereochemistry at R² can be of either the α or β-configuration. Preferably, the stereochemistry at R⁴ is the α-configuration. Preferably, the stereochemistry at R⁵ is the β-configuration. The stereochemistry at R⁷ can be of either the α or β-configuration.

Preferred embodiments for Y include but are not limited to: C3 - cyclopropane, C4- cyclobutane, methylcyclopropane, C5 - cyclopentane, methylcyclobutane, C6 - cyclohexane, methylcyclopentane, C7- cycloheptane, methylcyclohexane, bicyclo[2.2.1]heptane, bicyclo[2.2.1]hept-2-ene, C8 - cyclooctane, methylcycloheptane, 1 ,1 ,2-trimethyl-cyclopentane, octahydro-pentalene, bicycle[2.2.2]octane, bicycle[3.2.1]octane, C9 - cyclononane, methylbicycle[2.2.2]octane, octahydro-indene, ClO - cyclodecane, methyloctahydro-indene, bicycle[4.2.2]decane, adamantane, CI l - cycloundecane, bicycle[3.3.3]undecane, decahydro-cyclopentalene, methyladamantane and C 12 - cyclododecane, dodecahydro-benzocyclooctane, dodecahydro-acenaphthylene and dodecahydro-s-indacene.

Preparation The compounds of the invention may be prepared from triptolide or hydroxyl triptolides. The latter compounds include tripdiolide (2-hydroxy triptolide, α- or β- configured), and 16-hydroxytriptolide, which, along with 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 isolating 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).

The 5-hydroxy derivative of triptolide can be prepared by metabolic conversion. Incubation of triptolide with Cunninghamella blakesleana, as described by L. Ning et al. {Tetrahedron 59(23):4209-4213, 2003), produces the 5α-hydroxytriptolide as well as lβ- hydroxytriptolide, triptolidenol (15-hydroxytriptolide), 19α- and 19β-hydroxytriptolide. The products are isolated by standard procedures, i.e., extraction of the filtered culture broth with ethyl acetate, concentration, and silica gel chromatography of the residue. Compounds of Formulate I can be prepared from triptolide C 14-OH or hydroxyl triptolides with additional hydroxyl group at C2 (R²), C16 (R³), C5 (R⁴), Cl (R⁵), C15 (R⁶), or C19 (R⁷). Compounds of Formula I are rigid mono-, bi-, or tricyclic esters, or rigid mono-, bi-, or tricyclic carbonates or carbamates of triptolide or hydroxyl triptolides. Descriptions for the preparation of esters, carbonates and carbamates are provided in books such as Advanced Organic Chemistry 4^th Edition by Jerry March, Wiley, (1992) and references cited therein. For example, alcoholysis of anhydrides are shown on pages 392 and 393 and esterifϊcation of carboxylic acid is located on pages 393-396. Also March on page 392, states that carbonates can be obtained by the use of phosgene as the acyl halide. On page 891 of March, it is noted that carbamates can be prepared when isocyanates are treated with alcohols.

These reactions generally proceed as illustrated below.

Mono-, bi-, or tricyclic Derivatives

Mono-, bi-, or tricyclic derivatives are where the added carbon skeleton consists of a rigid mono-, bi-, or tricyclic ring system. The chemistry of how to prepare mono-, bi-, or tricyclic ester, carbamate and carbonate derivatives follows.

For example, triptolide, having a C 14 β-hydroxyl can be reacted with an appropriate mono-, bi-, or tricyclic activated acid to give C14 β- mono-, bi-, or tricyclic esters. The generalized reaction is shown in Scheme I.

Scheme I Mono-, Bi-, or Tricyclic Esters from Acids

For specific examples involving the reaction of mono-, bi-, or tricyclic activated acids to give mono-, bi-, or tricyclic esters, see Example 1 Rigid Esters.

In addition, ester derivatives can be prepared from mono-, bi-, or tricyclic anhydrides, see Scheme H

Scheme π Mono-, Bi-, or Tricyclic Esters from Anhydrides

For specific examples involving the reaction of mono-, bi-, or tricyclic anhydrides to give mono-, bi-, or tricyclic esters, see Example 2 Carboxy-substituted Rigid Esters.

Mono-, bi-, or tricyclic carbamate derivatives require a different type of reagent. For example, treatment of triptolide with a mono-, bi-, or tricyclic isocyanate generates the mono-, bi-, or tricyclic carbamate indicated in Scheme UI.

Scheme DI Mono-, Bi-, or Tricyclic Carbamates from Isocyanates

For specific examples involving the reaction of mono-, bi-, or tricyclic isocyanates to give mono-, bi-, or tricyclic carbamates, see Example 3 Monocyclic Carbamates.

Mono-, bi-, or tricyclic carbonates can be made in general from the reaction with phosgene followed by reaction with the appropriate alcohol, see Scheme IV.

Scheme IV Mono-, Bi-, or Tricyclic Carbonates Reaction Sequence

The synthesis of specific examples of mono-, bi-, or tricyclic carbonates is shown in Example 4 Mono-, Bi-, or Tricyclic Carbonates. Biological Evaluations Cytotoxic Evaluation

The cytotoxic activity of a compound of Formula I can be evaluated using the Alamar Blue fluorescence cytotoxicity assay as described in Example 5. The cytotoxic activity of these compounds that is mediated by programmed cell death can be evaluated using the Terminal deoxynucleotidyl transferase apoptosis assay, as described in Example 6, or the Annexin V apoptosis assay as described in Example 7.

Immunosuppressive activity The immunosuppressive activity of these compounds can be evaluated with the IL-2 inhibition assay using ELISA analysis, as described in Example 8, or the IL-2 inhibition assay using reporter gene analysis, as described in Example 9.

Anti-fibrogenic activity The anti-fibrogenic activity of these compounds can be evaluated with the TGF-β inhibition assay using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, as described in Example 10, or the TGF-β inhibition assay using ELISA analysis, as described in Example 11. The anti-fibrogenic activity of these compounds can be evaluated with the rat chronic kidney transplant rejection model, as described in Example 12.

CNS protective and/or neuroprotective activity

The CNS protective and/or neuroprotective activity of these compounds can be evaluated with the in vitro glutamate excitotoxicity model, as described in Example 13. 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 in Example 14. 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 in Example 15. 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 in Example 16. 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 in Example 17.

Therapeutic Formulations

For the end-use application of compounds of Formulae I and II, in addition to the active pharmaceutical ingredients, the pharmaceutical preparations can contain one or more pharmaceutically acceptable carriers (additives) and/or diluents, which, as used herein, includes any and all solvents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide, as well as phosphate buffer solutions; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and other non-toxic compatible substances employed in pharmaceutical formulations known to those skilled in the art. Such formulations containing these compounds of the invention may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as tablets, capsules, powders, sustained- release formulations, solutions, suspensions, emulsions, ointments, lotions, or aerosols.

Routes of administration 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. Liquid compositions may 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.

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.

Dosage 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 (19th Ed., Williams & Wilkins, 1995). The composition to be administered will contain a quantity of the selected compound in an effective amount for affecting immune suppression in a subject or apoptosis in a targeted cell.

Therapeutic Purposes

Compounds of the present invention possess potent anticancer and immune suppressive activities and may be employed for many therapeutic purposes. Thus, the invented compounds described may be used to treat the following disorders:

Cancer

The properties of the compounds within the scope of the invention make them 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 solid and liquid cancer can be treated with a compound of this invention, including but not limited to, carcinoma 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, xeroderma pigmentosum, keratoacanthoma, thyroid follicular cancer, Kaposi's sarcoma and non-small cell lung cancer.

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.

In another aspect, more benefit may be obtained by administering the invention compositions in combination or sequentially with one or more other conventional anticancer 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. 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).

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.

Immune Disorders

As immune suppressants, the compounds of the present invention are useful when administered for the prevention of immune-mediated tissue or organ graft rejection. Immune regulatory 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.

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, photoallergy 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.

In treating an autoimmune disorder, optimum dosages of the composition containing the active ingredient 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.

As mentioned above, 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).

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.

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 immune suppression level, e.g., by a mixed lymphocyte reaction involving allogeneic lymphocytes, or by taking a biopsy of the transplanted tissue. 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 Fi mice with parental spleen cells; the mice develop a GVHD syndrome characterized by splenomegaly and immune suppression (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.

Other Disorders:

The compounds of the present invention are also useful in the treatment of certain CNS diseases and fibrogenic disorders.

Central Nervous System

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.

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. 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.

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-a and IL-Ib). 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.

Lung diseases

In another aspect, the compounds of the present invention may also be used in the treatment of certain lung diseases. Krishna et al reported that a water-soluble triptolide derivative inhibits bleomycin-mediated fibrosis, which suggested a potential role in the treatment of patients with pulmonary fibrosis (Krishna, 2001). Other examples of respiratory diseases that can be treated include sarcoidosis, fibroid lung, and idiopathic interstitial pneumonia. Further examples of lung diseases that may use the compounds of this invention for treatment include Severe Acute Respiratory Syndrome (SARS) and acute respiratory distress syndrome (ARDS). Fidler et al. described the use of certain triptolide derivatives in the treatment of SARS, as described in PCT Publication WO 2005/020887. EXAMPLES

Example 1 Rigid Esters

To a solution of 1-adamantylacetic acid (Fluka, 01826) in methylene chloride containing DCC, slowly add triptolide and follow the reaction by TLC. Upon completion, workup the reaction using standard methods to yield the indicated substituted halo ester.

Rigid esters can also be prepared from activated acids. To a solution of triptolide in methylene chloride with a slight molar excess of triethylamine, add cyclobutane carbonylchloride (Aldrich, 95706). Concentrate the reaction and filter off the resulting triethylamine hydrochloride. Wash the remaining solution is with dilute sodium bicarbonate. Concentrate and purify to obtain the 14-cycolbutane carbonylester indicated.

Example 2 Carboxy-substituted Rigid Esters

Triptolide anhydride derivative MRx 105 was prepared with triptolide and cis- cyclohexan-1, 2-dicarboxylic anhydride as shown below:

Reaction

One by one 160mg triptolide, 190mg cis-cyclohexan-1, 2-dicarboxylic anhydride, 25mg 4-dimethyaminopridine (DMAP), 10ml distilled dichloromethane and 0.5mg tri- ethylamine, were added to a clean, dry 50ml flask. Upon refluxing with stirring for 15 hours under nitrogen gas, the reaction was complete as indicated by TLC.

The solvent was concentrated to dryness by evaporation. The remaining residue was dissolved by adding 20ml of ethyl acetate and 10ml of saturated solution of sodium bicarbonate after stirring for ten minutes. The resulting organic phase was extracted again with 10ml saturated solution of sodium bicarbonate, and the aqueous layers were combined.

Dilute hydrochloric acid to the aqueous layer was added until the pH is adjusted to 3.0, then was extracted with dichloromethane (20ml twice). The dichloromethane extracts were combined, washed with water (10 ml), dried with sodium sulfate (anhydrous) and after filtration, concentrated. A yellowish solid (250mg) was obtained. The yellowish solid was purified by silica gel column chromatography yielding 214mg of a yellow solid (94.5 %). Conditions for column chromatography were mobile phase acetyl acetate/hexane (1/1), column length 25cm, column inner diameter 2cm, and silica gel 200-300 mesh. Chemical analysis of MRx 105 product is summarized in the following.

High Pressure Liquid Chromatography

HPLC column condition were ODS chromatography column, 5um, 250* 4.6mm, mobile phase 40mmol ammonia acetate solution (pH 5.7-5.8 adjusted with acetic acid), acetonitrile (62:38), wavelength 220nm, sample lOul and flow rate 1.0 ml/min The results were two main peaks (78:22) with the two peaks total at 98 %. Mass Spectrum

With TOF-MS+, one MW of 515.2255 was detected. With TOF, one MW of 513.1290 was detected. With LC-MS, the two peaks of HPLC show the same fragmentation patterns of this MS method, including 515, 537(+Na). NMR

With IH-NMR the spectra were consistent with diasteriomeric mixture of cis- cyclohexan-1, 2-dicarboxylic acids. With 13C-NMR: Three carbonyl signals (172, 175, 179) , two double-bonds signals

(125, 160). Conclusion

Based on the above analysis, the final product, MRx 105, is a diastereomeric mixture (78/22). The ration of the two peaks appear to result from a steric preferred facial and/or regio reaction of cis-cyclohexan-1, 2-dicarboxylic anhydride with the 14-hydroxyl group of triptolide.

Preparation of MRxI 06

Triptolide anhydride derivative MRx 106 was prepared with triptolide and anhydride 3-oxabicyc 1 o[3.1.0]hexane-2, 4-dione as shown below: Scheme

Reaction

One by one, 360mg triptolide, 948mg anhydride 3-oxabicyc lo[3.1.0]hexane-2, 4- dione, 40mg DMAP, 10ml distilled dichloromethane and 0.5mg tri-ethylamine were added to a clean, dry 50ml flask. The reaction was refluxed under nitrogen gas (N2) for

8 hrs, and then stir at room temperature for 3 days. The reaction mixture was then concentrated to dryness by evaporation. The remaining residue was dissolved by adding 20ml of acetyl acetate, and then the resulting solution was extracted with 10ml saturated solution of sodium bicarbonate while stirring for ten minutes. The extraction was repeated with 10ml saturated solution of sodium bicarbonate. The aqueous extracts were combined.

Dilute hydrochloric acid was added to the aqueous extract until the pH was adjusted 5 to 3.0. The resulting aqueous mixture was extracted with dichloromethane, 20ml twice, and the extracts were combined, and washed with water (10ml). The organic phase was filtered, dried with sodium sulfate (anhydrous) and concentrated to give a yellowish solid (420 mg).

The yellowish solid was purified by silica gel column chromatography to yield 10 370mg, of off-white solid (88%). The mobile phase was ethyl acetate/hexane 1/1, column length 25cm, column inner diameter 2cm, silica gel 200-300 mesh. High Pressure Liquid Chromatography

Column condition were ODS chromatography column, 5um , 2504.6mm, mobile phase 40mmol/l ammonia acetate solution (pH 2 adjusted with phosphic acid), 15 acetonitrile (70:30), wavelength 220nm, sample lOul, and flow rate: l.Oml/min. Two main peaks accounting for a total of 99.2% (81.8 + 17.5) of peak area were observed. Mass Spectrum

EI-MS shows masses of 472.4 (P+) and 473.4 (P+l). 20 NMR

With IH-NMR the spectra were consistent with diasteriomeric mixture of 3- oxabicyclo[3.1.0]hexane-2, 4-dione acids.

13CNMR: Three carbonyl signals ( 169,173,174), two double-bonds signals (125, 159), and 5 CH2 signals (DEPT) were indicated. 25 Conclusion:

Based on the above analysis, the final product, MRxI 06, is a diastereomeric mixture (82/18) and the two peaks total %99. Like MRxlO5 above, the ratio of the two peaks appear to result from a steric preferred facial and/or regio reaction of cis-cyclohexan-1, 2- dicarboxylic anhydride with the 14-hydroxyl group of triptolide. 30 Surprising Results of Little Reaction or Complex Product Formation with Exo- and Endo Stereoisomers of Cis-5-Norbornene 2, 3-dicarboxylic Anhydrides

Using the same reaction conditions and methods as with MRx 105 and MRx 106 but employing cis-5-norbornene-2, 3-dicarboxylic anhydride as the reactive reagent gave little reaction with the Exo stereoisomer, and a complex mixture of products with the Endo stereoisomer of the reagent. Exo Anhydride Derivative

With c/s-S-norbornene-exo^S-dicarboxylic anhydride and employing reaction conditions as indicated for MRx 105 and MRx 106, little reaction was observed as monitored by TLC. Endo Anhydride Derivative

Complex Mixture With c/s-5-norbornene-ew*/o-2, 3-dicarboxylic anhydride and employing reaction conditions as indicated for MRx 105 and MRx 106, there was reaction and spots were detected by TLC.

HPLC of the products indicated 4 peaks. One set of peaks is possibly cis adducts and other set of peaks is possibly trans adducts. The doubling of the HPLC peaks could be due to epimerization of the ester methine in both the cis and tans

In a similar manner, reaction of triptolide with (±)-camphoric acid anhydride (available from Fluka Company, 17996) would give a mixture of 14- triptolide 14-β-(±)- camphoric acid regio-esters with the predominate derivative indicated first below. Note both products are expected to give diastereo isomers.

Example 3 Rigid Carbamates

To a solution of triptolide and triethylamine in methylene chloride, slowly add a solution of 1- adamantyl isocyante (Aldrich, 375063) in methylene chloride. Follow by TLC until reaction is complete. Standard methods are employed to isolate the indicated 1 -adamantyl carbamate .

Example 4 Rigid Carbonates

Carefully treat triptolide dissolved in methylene chloride at lowered temperature with phosgene. The resulting acid chloride need not be isolated. Slowly add (-)-endo-2- norborneol (Aldrich, 543519) to the reaction mixture. Follow the reaction by TLC. Isolate the resulting rigid carbonate derivative of triptolide shown below using standard methods.

Likewise, use (trans-4-(dimethyl(amino)cyclohexanol (Combi-Blocks, AM-1615) subsequent to the acid chloride generation step above to prepare the following carbonate.

Example 5. Alamar Blue Fluorescence Cytotoxicity Assay

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 μl of the test compounds at 2x, and the plates are incubated at 37⁰C 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.

Example 6. Terminal Deoxynucleotidyl Transferase Apoptosis Assay

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 μl of the test compounds, and the plates are incubated at 37⁰C in a 5% CO₂ 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.

Example 7: Annexin V Apoptosis Assay

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 μl of the test compounds. The plates are incubated for 24 hours at 37⁰C in a 5% CO₂ incubator after which the cells are washed twice by centrifugation in 2% FCS in PBS and 500μl of binding buffer is used to resuspend the cells from each well according to the Annexin V assay procedure (Bio Vision, Inc., Mountain View, CA). Next, 5ul of the fluorescein isothiocyanate (FITC) conjugate of Annexin V (Bio Vision, 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.

Example 8: IL-2 Inhibition Assay Using ELISA Analysis 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 (#TEB-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 μg/ml Phytohemagglutinin (the optimally stimulatory concentration of purified PHA from Sigma) in the presence of the test compound at 37⁰C in a 5% CO₂ incubator in duplicate 1 ml culture wells. The culture supernatants are collected and stored frozen at -20 ⁰C 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 hEL-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. Example 9: IL-2 Inhibition Assay Using Reporter Gene Expression Analysis

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 37⁰C 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 MgSO₄) 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.

Example 10: TGF-β Inhibition Assay Using RT-PCR Analysis

Normal human lung fibroblasts (NHLFs) treated in vitro with bleomycin are induced to increased TGF-β 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 37⁰C 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-β 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-β/GAPDH mRNA is derived from a scan of the agarose gel and plotted for purposes of comparison of the level of TGF-β gene expression.

Example 11 : TGF-β Inhibition Assay Using ELISA Analysis

NHLFs treated with bleomycin in vitro are induced to increased TGF-β 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 37⁰C 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 culture supernatants for ELISA analysis of human TGF-β. The culture supernatants are collected and stored frozen at -20 ⁰C or lower until assayed. The concentration of human TGF-β in the culture supernatants are measured in a conventional ELISA assay using an anti-TGF-β monoclonal antibody and hTGF-β standard in an ELISA kit (R&D Systems, BD Pharmingen, BioSource International or Promega Corp., Madison, WT). The data are expressed as ng/ml of TGF-β.

Example 12: Rat chronic kidney transplant rejection model 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-β by RT-PCR as described in Example 10, 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.

Example 13: In vitro glutamate excitotoxicity model Test compounds are dissolved in DMSO at a concentration of 20 mM. Further dilutions are done in RPMIl 640 medium (GIBCO, Rockville, MD) supplemented with 10% FCS. PC 12 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 μl/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 ODs_7O 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 (see Example 7). Additionally, reactive oxygen species (ROS) formation and the decrease of mitochondrial membrane potential may be assessed (as described in Example 17: In vitro beta-amyloid (Abeta) toxicity assay) as parameters of glutamate-induced excitotoxicity.

Example 14: In vivo MPTP treatment model in mice

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 1-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 OX42 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.

Example 15: In vitro LPS treatment model of neuron-glial cultures

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 μM. Production of TNF-α and EL-lβ induced by LPS are assayed in 2-4 hour samples using TNFα-specific IL-lβ-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.

Example 16: BDNF in situ ELISA assay system with neuron-glial cultures

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 E_max 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 E_max BDNF in situ immunoassay system after washing away the cultivated cells. The data are expressed as pg/ml of BDNF.

Example 17: In vitro beta-amyloid (Abeta) toxicity assay

Test compounds are dissolved in DMSO at a concentration of 20 mM. Further dilutions are done in medium supplemented with 10% FCS. PC 12 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 (see Example 13) and apoptosis is determined using Annexin V and FACS analysis as described in Example 7. 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 μg/ml). To measure DCF fluorescence, cells are read on a fluorescent plate reader (such as the CytoFluor 2350 Millipore, Bedford, MA) at 485 ran excitation and 530 nm emission wavelength. Mitochondrial membrane potential are measured using the voltage-sensitive dye JC-I (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.

Example 18 MTT assay result for MRx compounds with MV4-11 human AML cells The purpose of this experiment was to observe the anti acute leukemia cell (MV-4-

11) activity of 2 samples in vitro. Two samples were used, MRxlO5 (m.w. 514.56) and MRx 106 (m.w. 472.47) (See Example 2). They were weighted, dissolved in DMSO as stock solution in a sterile test tube, and stored at -2O⁰C. Further dilutions were done

before use with the culture medium. The drug 5-FU was used as a positive control. Cells of MV4-11 human AML cell line were grown in sterile cell culture bottles in

IMDM medium with 10% fetal bovine serum, including 100kU/L penicillin and streptomycin, in a 5% CO₂ humidified incubator at 37°C. The cell line was used as a suspension culture and transferred as a generation every 4-5 days.

The cells at the logarithm growth period and suspended with IMDM medium, were added (5000-8000 cells) in lOOμl portions to each well in 96-well plates and incubated for several hours in a 5% CO₂ humidified incubator at 37°C. To each well was added lOOμl of various concentrations of the test compounds, each concentration had 4 parallel tracks. The plates were incubated for 72-96 hours in a 5% CO₂ humidified incubator at 37°C. After the incubation period, 20μl of 5mg/ml MTT reagent was added to all wells and incubated at 37⁰C for 4 hours. The 96-well plates were centrifuged at 3000 rpm forl 0 min and decanted the supernatant carefully, and then, 200μl of DMSO reagent was added to all wells and the plates were returned to the incubator for an additional 6 hours. The absorbance was measured at OD570 ran using a microplate reader.

The data were showed with X±s. The following formula was used to calculate % viability: % viability = (Control OD-Sample OD)/ Control ODx 100%

A calibration curve was prepared using % viability as ordinate and the data of concentration of the test compound as abscissa. The half-effect concentration was calculated (IC₅₀: μg/ml) with logistic regression. The anticancer effect was expressed with I_max and ICso- The results are shown in Table 1-1 and 1-2 and Figures 1 and 2.

Table 1-1 Anti acute leukemia cell (MV-4-11) activity of MRx 105

Concentration A570nm-OD

Suppression rate Main parameter (μg/ml) (Xts)

Blank 0.916 0.108

MRx 105 0.003 1.191 ± 0.055 -30.06% _ 0.1492μg/

MRx 105 0.009 1.177 ± 0.141 -28.56% ^EC5°- ml

MRxlO5 0.027 1.087 ± 0.059 -18.65%

MRxI 05 0.082 0.917 ± 0.073 -0.08% T _ OOO/

Amax ^~ OO /o

MRx 105 0.247 0.408 ± 0.060 55.50%

MRx 105 0.741 0.122 ± 0.022 86.73%

MRx 105 2.222 0.110 ± 0.003 87.96%

MRx 105 6.667 0.116 ± 0.006 87.31%

MRx 105 20 0.112 ± 0.006 87.77%

5-FU 0.625 0.152 ± 0.01 83.46%

5-FU 2.5 0.119 ± 0.01 87.06%

5-FU 10 0.118 ± 0.007 87.09% Table 1-2 Anti acute leukemia cell (MV-4-11) activity of MRx 106

Concentration A570nm-OD Suppression rate Main parameter (μg/ml) (X±s)

Blank 0.916 ± 0.108

MRxlOό 0.003 1.270 ± 0.038 -38.71% EC₅₀= ⁰ MRx 106 0.009 1.179 ± 0.062 -28.69% mJl ⁸²W

MRx 106 0.027 1.135 ± 0.105 -23.94%

MRx 106 0.082 1.021 ± 0.052 -11.52%

MRx 106 0.247 0.506 ± 0.018 44.80%

MRx 106 0.741 0.137 ± 0.003 85.01%

MRx 106 2.222 0.114 0.006 87.58%

MRxlOό 6.667 0.116 ± 0.004 87.33%

MRx 106 20 0.117 ± 0.007 87.22%

5-FU 0.625 0.152 ± 0.01 83.46%

5-FU 2.5 0.119 ± 0.01 87.06%

5-FU 10 0.118 ± 0.007 87.09%

Accordingly, novel derivatives of triptolide and methods of using the same are disclosed. From the foregoing, it will be appreciated that, although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.

Claims

IT IS CLAIMED:

1. A compound having the Formula I:

where:

R¹ is OX and R² - R⁷ is H or OX with the proviso that at least five of R² - R⁷ are H. X is COY, COOY, or CONHY.

Y is defined as C3-C12 moiety containing a mono-, bi-, or tricyclic ring system that is unsubstituted or substituted with Z. The mono-, bi- or tricyclic ring system can be saturated or unsaturated.

Z is defined as CO₂R⁸ or N(R⁸)₂, lower alkyl or halogen.

Halogen is defined as F, Cl, Br or I.

R⁸ = H or lower alkyl. Lower alkyl is defined as C 1 -C6, unbranched or branched. C 1 -C6, unbranched or branched means methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert- butyl, n-pentyl, 1 -methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 2,2- dimethylpropyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 4methylpentyl, 1,1- diemthylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2- trimethylpropyl.

2. The compound of claim 1, wherein R = OX and each of R >2 -R τ,7 is hydrogen.

3. The compound of claim 2, where: X is COY or COOY.

4. The compound of claim 3 where:

Y is defined as C3-C5 moiety containing a monocyclic ring system that is, unsubstituted or substituted with Z. The monocyclic ring system can be saturated or unsaturated.

5. The compound of claim 3, where:

Y is defined as C6-C12 moiety containing a bi-cyclic ring system that is unsubstituted or substituted with Z. The bi-cyclic ring system can be saturated or unsaturated.

6. The compound of claim 3, where:

Y is defined as C9-C12 moiety containing a tricyclic ring system that is unsubstituted or substituted with Z. The tricyclic ring system can be saturated or unsaturated.

7. The compound of claim 3, where:

Y is defined as C3-C12 moiety containing a mono-, bi-, or tricyclic ring system that is substituted with Z. The mono-, bi- or tricyclic ring system can be saturated or unsaturated.

8. The compound of claim 7, where:

Z is CO₂R⁸ or N(R⁸)₂.

9. The compound of claim 8, where: Z is CO₂H

10. The compound of claim 8, where: Z is N(CH₃)₂

11. A method of treating a disorder in a mammal, comprising administering to the mammal in need thereof an effective amount of a compound of any one of claims 1-10, wherein the disorder is a cancer, an immune disease, a fibrogenic disease, or a CNS disease.

12. The method of claim 11 , wherein the said mammal is a human.

13. The method of claim 11 , wherein the disorder is a type of cancer, whereby apoptosis is induced in a cell when contacting said cell with an effective amount of a compound having a structure I.

14. The method of claim 13, wherein the disorder is a type of cancer involving solid tumors, whereby apoptosis is induced in a tissue when contacting said cell with an effective amount of a compound having a structure I.

15. The method of claim 13, wherein the disorder is a type of cancer involving liquid tumors, whereby apoptosis is induced in a cell when contacting said cell with an effective amount of a compound having a structure I.

16. The method of claim 11 wherein the disorder is an immune disease, whereby immune suppression is achieved using an effective amount of a compound having structure I.

17. The method of claim 11 wherein the disorder is a fibrogenic disease, whereby inhibition of fibrosis is achieved using an effective amount of a compound having structure I.

18. The method of claim 11 wherein the disorder is a CNS disease, whereby inhibition of neurotoxicity or neurodegeneration is achieved using an effective amount of a compound having structure I.

19. A pharmaceutical composition comprising a compound of any one of claims 1-10.