Field of the Invention
The invention relates to use of betulinic acid and its derivatives for inhibiting / preventing cancer growth, novel betulinic acid derivatives and a composition for selectively inhibiting tumors or cancers and, more particularly, to treating leukemias, lymphomas, prostate and ovarian cancer using betulinic acid and derivatives thereof.
Background of the Invention
Significant progress has been achieved in the management of adult acute leukemia. However, long term disease free survival is currently achieved in less than 50% of patients. Optimal therapy has not yet been defined and stratification of therapy according to prognostic factors is only now beginning to be addressed. Acute leukemia (AL) is an uncommon form of malignancy affecting approximately five persons per 100,000 in U.S. annually with 90% mortality rate within one year of diagnosis. Although incidence of leukemia over all has been stable for last 30 years, the increasing age of our population will presumably result in a greater number of cases being reported.
On the other hand, malignant lymphomas are the seventh most common causes of death from cancer in the United States. In 1992, approximately 41,000 new cases were diagnosed, and there were approximately 19,400 deaths from the disease. The incidence of lymphocytic lymphomas is increasing each year; the 50 % increase in incidence between 1973 and 1988 reported by the American Cancer Society was one of the largest increases reported for any cancer. A large portion of the increase has been attributed to the lymphocytic lymphomas developing in association with acquired immunodeficiency syndrome (AIDS).
The early treatment of acute myelogenous leukemia (AML) in adults used corticosteroids, methotrexate, 6-mercaptopurine and vincristine, initially as single agents and subsequently in combinations. The emergence of ara-C as the major single agent in the management of
AML in the late 1960s converted this incurable disease to a potentially curable condition. The subsequent development of anthracyclines, such as daunorubicin, doxorubicin (Adrmmycin), rubidazone and idarubicin allowed new combinations to be developed The vinca alkaloids, alone or in combination with corticosteroids, and epidophyllotoxins, methotrexate, 6-mercaptopurine , cyclophosphamide and L asparaginase have only minor activity in adult AML. Most regimens in adult AL incorporate vincristine, corticosteroids, prednisone or dexamethasone, anthracyclines, and L asparaginase.
The predominant treatments for AL have been in place for 5 to 15 years. No major class of drugs for the management of AL has been discovered. The allogenic and autologous marrow transplant program with monoclonal purging and chemotherapy purging have been applied with no major difference in response or survival rates.
The treatment approach to a particular patient with lymphocytic lymphoma is determined by the tumor histology, the stage of disease, and the physiologic status of the patient. The most active chemotherapy programs for intermediate-grade lymphomas include therapy using different combinations, dosages and schedules of cyclophosphamide, doxorubicin, vincristine, procarbazine, prednisone, bleomycin and methotrexate. Development of new drugs that are selective for particular subsets of leukemia and lymphoma patients is of the highest priority.
Second only to lung cancer as a cause of cancer deaths, prostate cancer has become the most common cancer among American men. It was diagnosed that 316,000 new cases of prostate cancer would be diagnosed in 1996, causing 40,000 deaths. Potential prostate cancer chemopreventive agents under consideration are diverse with respect to source, chemical structure and physiological effects. The candidate drugs under development for treatment of prostate cancer must demonstrate long-term safety and minimum toxicity after chronic administration. Retinoids such as lycopene and 4-hydroxyphenretiminide and diiluoiomethyl ornithine (DFMO) are being evaluated in patients scheduled for radical prostiitectomy Androgen deprivation may also have a role in chemoprevention Other
approaches under study include the vitamin D analogs, a-tocopherol and dietary restriction of fat. Conventional chemotherapeulic agents in hormonally relapsed prostate cancer are Doxorubicin, Kclocona/ole, Cyclophosphamide, Prednisolone, Ksliamustinc, Vinblastine, Paclitaxel, Mitoxantrone, either alone or in combination.
The vast majority of ovarian cancers are epithelial in origin and these account for more than 90% of the estimated 26,700 new cases of ovarian cancers that were diagnosed in 1996 in the United States. Ovarian cancer is the leading cause of death from a gynecologic cancer. From 1970 to 1991, survival rates for patients with ovarian cancer have increased from 36% to 44%. This improvement in survival rate is in large part due to the development of curative platinum-based chemotherapy for patients. The generally accepted treatment for patients with either stage III or IV (advanced-stage) ovarian cancer has been similar : cytoreductive surgery when feasible, followed by chemotherapy. Chemotherapeutic agents from a wide variety of different classes have been shown to produce responses in patients with ovarian cancer. Before the demonstration of marked activity of Paclitaxel in ovarian cancer, platinum compounds were considered to be the most active agents in this disease. Both Paclitaxel and Docetaxel have been demonstrated to have activity in platinum-resistant patients. Gemcitabine, a pyridine antimetabolite, has been shown in phase II trials in Europe to be an active agent.
Under the auspices of a National Cooperative Natural Product Drug Discovery Group supported by the National Cancer Institute, the potential antitumor activity of approximately 2500 extracts derived from globally collected plants was evaluated in a panel of enzyme based assays and in a battery of cultured human tumor cell lines. One such extract, prepared from the stem bark of Ziziphnx mawitiami Lam. (Rhamnaceae), displayed selective cytotoxicity against cultured human melanoma cells (Nature Medicine, Vol. I (10), 1995, WO 96/29068). As a result of bioactivity guided fractionation, betulinic acid, a pentacyclic triterpene, was identified as a melanoma-specific cytotoxic agent. In follow-up studies conducted with athymic mice carrying human melanomas, tumor growth
was completely inhibited without toxicity. As judged by a variety of cellular responses, antitumor activity was mediated by the induction of apoptosis.
A number of triterpenoids, including betulinic acid, have several known medical applications, including use as an anticancer drug. Anderson et al., in WO 95/04526, discuss derivatives of triterpenoids which have been used in cancer therapy, including their activity against polyamines which are required by cells to grow at an optimal rate. Some of these triterpenoids have been found to interfere with enzymatic synthesis of polyamines required for optimal cell growth, and thus inhibit the growth of cancer cells, particularly by inhibiting ornithine decarboxylase (Yasukawa, K. et al. Oncology 48 : 72-76,1991). The anticancer activity of betulinic acid and some derivatives has been demonstrated using mouse sarcoma 180 cells implanted subcutaneously in nude mice ( JP 87,301,580). Choi et al have shown that betulinic acid 3-monoacetate, and betulinic acid methylester exhibit EDsn values of 10.5 and 6.8 (.ig/ml, respectively, against p388 lymphocytic leukemia cells (Choi. Y-Hetal., Planta Medica vol XLVII, pages 511 -513, 1988).
Betulinic acid has been reported also to possess anti-inflammatory activity. The anti-inflammatory activity of betulinic acid is, at least in part, due to its capacity to inhibit enzymes involved in leukotriene biosynthesis, including 5-lipoxygenase (Somatsu, S et al.. Skin and Urology 21 : 138, 1959 and Inoue, H., et al., Chem Pharm Bull. 2 : 897 - 901, 1986).
Betulinic acid and dihydrobetulinic acid acyl derivatives have been found to have potent anti - HIV activity (WO 96/39033). Anti-HIV assays indicated that 3-O-(3\ y -diincthylsuccinyO-betulinic acid and dihydrobetulinic acid both demonstrated extremely potent anti-HIV activity in acutely infected H9 lymphocytes with EC.™ values of less than 1.7 x IO"5 |LiM, respectively.
Thus, betulinic acid and its derivatives have been shown to posses several medicinal properties including anticancer activity. But apart from the conclusive evidence of its anti-
melanoma activity, it has not so far been shown to possess anticancer activity on other cancers. In other words, the actitivity of betulinic acid and its derivatives on all kinds of cancer cells are not uniform but it is very selective. Still, now the applicants, after much research, have identified for the first time the antileukemia and antilymphoma activity of betulinic acid and its derivatives with ED5o values in the range of approximately 0.5 to 4.0 |.ig/ml. Further, the applicants also report for the first time antiprostate cancer and anti-ovarian cancer activity of betulinic acid and its derivatives with EDso values in the range of approximately 0.6 to 6.8 (.ig/ml and 0.4 to 8. l[.ig/ml respectively.
Stun in a 17 of the Invention
The invention provides a method of treating a patient suffering from leukemias, lymphomas, prostate, lung or ovarian cancer employing a pharmaceutically effective dosage of betulinic acid ; novel betulinic acid derivatives and its use for treating cancers, and a composition comprising pharmaceutical acceptable additives and betulinic acid and / or its derivatives.
Detailed Description of the Invention
The present invention provides a pharmaceutical composition useful for killing or inhibiting / preventing multiplication of cancer cells. The active compound, betulinic acid, is isolated by a method comprising the steps of preparing an extract from the bark of Zizi/thnsjujHba Mill. (Rhamnaceae) to mediate a selective cytotoxic profile against human leukemias, lymphomas, prostate, lung ovarian cancer using a panel of human tumor cell lines, preparing novel derivatives of betulinic acid and testing the bioactivity using cultured human leukemia (MOLT-4, Jurkat E6.1, HL60, CEM.CM3), lymphoma cells (BRISTOL-8, U937), prostate cancer cells (DU 145) and ovarian cancer cells (PA-1) as the monitor. In a preferred embodiment, a pharmaceutical acceptable carrier, diligent, or solvent is used The invention provides a method of treatment for humans, mammals, or other animals suffering from cancer or other tumors. The present method comprising
essentially of administering a therapeutically effective dose of the pharmaceutical composition so as to kill or inhibit the multiplication of cancer or tumor cells. The method of trciilmuiH of the present invention may be particularly useful in the treatment of leukemias and lymphomas and in general in the treatment of prostate cancer and ovarian
cancer
Objects of the Invention
The main object of the invention is to provide a method and composition for inhibiting / preventing tumor growth and, particularly, for inhibiting / preventing the growth of leukemias and lymphomas and in general for inhibiting / preventing the growth of prostate and ovarian cancer using a natural product-derived compound and its derivatives
Another object of the invention is to provide a treatment method using betulinic acid and its derivatives to prevent the growth of cancerous cells, wherein betulinic acid or its derivative is administered systcmically.
Yet another object of the invention is to overcome the problem of high toxicity associated with standard chemotherapeutic agents by using a natural product-derived compound, e.g., betulinic acid or its derivatives.
Still another object of the invention is to overcome the problem of insufficient availability associated with synthetic anticancer agents by using the readily available betulinic acid and its semi synthetic derivatives.
One more object of the invention is to overcome the problem of high cost of synthetic anticancer agents by utilizing the readily available natural product derived compound, e.g., betulinic acid and its derivatives which would be of substantially cheaper cost.
J
Another object of the invention relates to describing new activities of betulinic acid (Figure 1 of the accompanying drawings) and its derivatives.
One another object of the invention relates to providing novel betulinic acid derivatives which are used for treating cancer or tumour.
Further object of the invention is to provide a pharmaceutical formulation containing pharmaceutically acceptable additive(s) along with betulinic acid and its derivatives alone or in combination.
These and other objects of the present invention will become apparent from the description of the invention disclosed below, which descriptions are intended to limit neither the spirit or scope of the invention but are only offered as illustrations of the preferred embodiments of the invention.
The present invention also comprises novel betulinic acid derivative used for killing or inhibiting / preventing multiplication of cancer or tumour cells, said derivatives having a basic skeleton of betulinic acid as shown in Figure 2 of the accompanying drawings, wherein R, ri, R2, R?, R4, Rs and R6 is independently or in combination represent the following functional groups in the derivatives : RisH
R, is H, Br, Cl, F or I ;
R2& R., together is O,NNHCr,H5, NNHCr,H2CI3, NNHCr.H^CH^ NNHCfiH4OH
N-OX (X being H,COCFi3,SO2C(,H4CH3,
CO(CH2)nCH,( n = I to 5 ) R2 is H and R, OH, OCOCFh, OCO2(CH2)nCH, (n = 1 to 5)
OCOC(CH.03,OCO(CH2),, Cl,( n = I tolO),OCOCV,Hs
OCOCr,H?Cl3,OCOCH(OCOCH3)CH,, OCO(CHOH)CH,,
R4 is ()H,-OCH3,O(CH2)nCOOCH,,O(CH2)nCOOC2 H5,O (CH2)nCOOH(CH2)nCoCI (n=lto5)OCH2CH2OC2H5,OCH2CH2OH,OCH2CH2OCOCH,,Cl,N,,NHNH2,
Nl INI K',,1 M)Mc, Nl INI IC,,I !>('!,. Nl I,. Nl!(('! I,),,CI h (it ~0 to <))
l<< is 11; Hi
Rr, is Cll.,, -CII2Br, CII2OH. CIK), ClbOCOCII,, COON, C:()U(Cll2),,('()()(.'ll.u
COO(CH2)nCOOC2H5, COO(CH2)n COOC2H (n =1 to 5)
R, and R,, is together >OCH;, or CH-Clk
The preferred novel compounds of the invention are shown in figures 3 to 8 of the drawings in which the meanings of Ri,R2,R?, R4, rs and Re are given below :-Compound of Fig.3 of the drawings :R = H;Ri=H;R2 = H; R, = OCOCH(OCOCH3)CH3 or OCOC(CH3)3 ; R4 = H or CH2COOH
Compound of Fig.4 of the drawings : R = H ; ri = H ; R2 = H ;
R, =()COCH(OCOCH,)CH?,O(CH2),COCI , OCOCH(OCOCH3)CH? or OCOCH2CH, ;
R4 = H or CH2COOCH-,
Compound of Fig.5 of the drawings : R = H ; R, = H ; R2 = O or N-OCOCH,; R3= H
Compound of Fig.6 of the drawings : R = H ; R) - H ; R2 =NOH or NNHC6H5 ; R, = H or CH2COOCH,
Compound of Fig.7 of the drawings : R = H ; R, = Br ; R2 = O ; R3 = H; R, = H ; R5 = H
Compound of Fig.8 of the drawings : R = H ; ri = H ; R2 = H.; R., = OCOCH.,; R., -11; R5 = Br ; Rf, - Br
The invention also relates to methods of preparing the novel compounds and in these methods the term "substrate refers to either betulinic acid or its derivative as starting niiilciiiil General procedures for the preparation of various derivatives starting with betulinic acid or its derivatives as basic substrate unless otherwise specifically mentioned. The procedures mentioned below are either used as single experiment or in combination to produce the derivatives mentioned in the claims
Usual work up following above in the procedures refers to evaporation of the reaction mixture, addition of water, extraction with organic solvent, washing and drying over anhydrous sodium sulphate, evaporation of organic layer.
Example 1
Dried bark of Ziziphm ji(jnha Mill. (Rhamnaceae) was successively extracted with a hydrocarbon solvent at a temperature of approximately 40" to 120"C (preferably 60" to 80"C) for a period of approximately 24 hours. The combined extract was concentrated under vacuum to l/20th to 1/100th (preferably l/30th to l/50th) of its original volume and chilled to 0 - 10"C (preferably 0 - 5"C) for 6 to 24 hours (preferably 12 to 16 hours). The crude betulinic acid thus obtained was separated by filtration. It was purified by either crystallization and /or by derivatization followed by dederivatization. Purified Betulinic acid was characterised by comparing with an authentic sample of betulinic acid with respect to melting point, mixed melting point, IR, UV & NMR spectra.
Preparation of Betulinic acid derivatives
The term substrate in the experimental procedures given below referes to betulinic acid or dihydi obetulinic acid. The general procedure for the preparation of various derivatives starts with betulinic acid and dihydrobetulinic acid derivatives as the basic substrate The following procedures are either used as single experiment or in combination to produce the derivatives of the present invention.
Example 2 Preparation of 3-o-acyl derivatives
Method I : Substrate in organic base is treated with suitable anhydaride at room temperature for approximately 4-16 hours. The reaction was worked up by evaporation of the reaction mixture, addition of water and extracting with the organic solvent. The organic layer was dried over anhydrous sodium sulfate, evaporated and residue crystallized to yield the corresponding pure 3-o-acyl derivatives respectively.
Method II : Substrate in halogenated organic solvent in presence of organic base was treated with suitable acyl chloride as in Method I. The reaction was worked up as described in Method I to yield the corresponding 3-o-acyl derivatives in the pure form.
Example 3 Preparation of 3-oxo derivatives
The substrate was dissolved in the organic solvent and the conventional oxidising agent was added under normal reaction conditions. The reaction was worked up as described in Method I to yield the corresponding 3-oxo derivatives in the pure form.
Example 4 Preparation of 2, 20, 29-tribromo derivative
The 3-oxo derivative was dissolved in halogenated organic solvent. To this was added dropwise liquid bromine dissolved in the same solvent maintaining the temperature between 0-10"C. The reaction mixture was brought to room temperature and stirred for a few hours. The reaction was worked up as described in Method I. The organic layer was washed with 5-10% aqueous alkaline solution and evaporated. The crystallized product yielded respective pure 2-Bromo-3-oxo derivatives.
Example 5 Preparation of 3-oximino derivative
The 3-oxo derivative was taken in an alcoholic solvent. To this was added hydroxylamine hydrochloride and sodium acetate and retluxed for few hours. The reaction mixture was evaporated to dryness. The reaction, worked up as described in Method I yield crude-3-oximino derivative which was crystallized to yield the corresponding pure 3-oximino derivative.
Example 6
Preparation of phenylhydrazone of 3-oxo derivative
Phenylhydrazine was added to 3-oxo derivative dissolved in alcoholic solvent and retluxed
for four hours. The reaction was worked up as described in Method I to yield the corresponding phenylhydrazone derivative in the pure form.
Example 7 Preparation of 17 or 20-carboxyalkyl carboxylate
To the substrate dissolved in dry dimethylibrmamide, added sodium hydride and stirred at room temperature for two hours. A suitable haloalkyl carboxyester was added to the above reaction mixture and further stirred at room temperature for 16-20 hours. The reaction was worked up as described in Method I to yield pure 17 or 20-carboxyalkyl carboxylate derivative.
Example 8 Preparation of 17 or 20-carboxyalkyi carboxylic acid
17 or 20-carboxyalkyl carboxylate was dissolved in an alcoholic solvent to which alcoholic hydroxide solution was added. The mixture was warmed to 40-50°C for 2-4 hours. The reaction was worked up as described in Method I to yield pure 17 or 20-carboxyalkyl carboxylic acid derivative.
Example 9
Preparation of 2- bromo- 3—oxo-derivative:
3-oxo-dihydrobetulinic acid derivatives was dissolved in halogenated organic solvent and added liquid bromine dissolved in the same solvent dropwise maintaining the temperature between 0 -10"C. Reaction mixture brought to room temperature and sustained for few hours. Worked up in usual manner, washed organic layer with 5-10% aqueous alkaline solution followed by water. The evaporation and crystallisation yielded respective pure 2-Bromo-3-oxo derivatives.
Example 10 Preparation OfN-0-Tosyl Or N-O-Acyl Of 3-Oximino Derivative:
3-Oximino derivative is dissolved in dry DMF and added sodium hydride and stirred at room temperature for few hours. Cooled to 0 -5"C and added suitable tosyl chloride or acyl chloride and continued stirring at this temperature for few hours. Usual work up gave the corresponding N-acyl or N-0-tosyl derivative.
Example 11
In vitro cytotoxic activity of betulinic acid and its derivatives was determined by performing the MTT cytotoxicity assay (Mosmann T., J Immunological Methods, 65 : 55 ; IC)H3). Briefly, the cultured tumor cells were separately seeded in a 96-well culture plate and co-incubated with betulinic acid or its derivatives dissolved in methanol, dimethyl tbimamide, dimethyl sulfoxide or isopropyl alcohol with relevant controls at 37"C in a CO: incubator. After 72 hours, the assay was terminated and percent cyotoxicities calculated. As shown in Table I, metabolic activity of leukemia cells (MOLT-4, .lurkat E6.1, HL60, CEM.CM3) was inhibited by betulinic acid, i.e., an ED50 value of about 0.6 -3.0 iig/ml. The ED50 value of betulinic acid for lymphoma cells (BRISTOL-8, U937) was in the range of 0.5 to 1.0 (ig/ml. Further betulinic acid showed an EDso value of I 30 ± 0.55, 1.13 ± 0.35 [ig/ml and 2.20 ± 0.70 ng/ml against L132 (human lung), DUI45 (human prostate) and Malme 3M (human melanoma) respectively. The anticancer activity of betulinic acid on human lung and human prostate cancer has not been reported previously. None of the other cancer cell lines tested was affected by betulinic acid (i.e., values of greater than 10 |.ig/ml).

Example 12
Several derivatives of betulinic acid were prepared and characterised on the basis of spectral data. All the derivatives were screened for antileukemia activity using MOLT-4 as the monitor. Twenty eight of these derivatives were screened for antileukemia, anti-lymphoma, antiprostate and antiovarian cancer activity using Jurkat E6.I, IIL60 & CEM.CM3 (leukemia), U937 & BRISTOL 8 (lymphoma), DU 145 (prostate) and PA-1 (ovary) cell lines respectively. The results are given in Table II.
Example 13
Several derivatives of betulinic acid were prepared by making structural changes at and/or CY C;, Cn, C2o, C29 positions of betulinic acid as described in Example 2. The derivatives were characterised on the basis of spectral data. Table III to Table VIIII comprising of basic skeleton of betulinic acid as indicated in Figures 3 to 8 respectively (as shown in the accompanying drawings) lists the structures of twenty eight derivatives mentioned in Table II.
Fig. 3 of the accompanying drawings wherein R to Ri are shown as follows :
Fig 0 of the accompanying drawings wherein R to R? are shown here below :
Rr, are shown here below
Example 15
Betulinic acid is known to exert its antimelanoma activity by causing apoptosis (programmed cell death). Apoptosis culminates in the fragmentation of the cellular DNA which can be detected by using monoclonal antibodies to the fragmented DNA (mono and poly oligonucleosomes). Apoptosis may be used as an indicator for specific cytotoxicity of the test compound. Apoptosis was investigated as a probable (or one of the probable) mechanism of action of betulinic acid for killing leukemia cells (MOLT-4). Briefly, cells were treated with 5 u,g/ml of betulinic acid or its derivatives in vitro and incubated at .U'V in a CO2 incubator for 24 hours, 48 hours or 72 hours. Untreated cells served as controls. At the end of the incubation, one-half of the cells were lysed with lysis butter after removing the supernatant. The supernatant of the remaining cells was collected without lysing the cells. Both lysate and supernatant were centrifuged at 16,000 rpm to separate intact DNA from fragmented DNA, if any. The supernatants were assayed for DNA fragments while the pellets containing intact DNA were discarded. The DNA fragments were detected by the commercially available kit (Boehringer Mannheim, Cat 1774425) in culture supernatants but not in the lysates indicating the presence of fragmented DNA in culture supernatant followed by the lysis of treated cells. The DNA fragments were not detected in untreated cells or cells treated with 5 fig/ml of betulinic acid / betulinic acid derivatives for 24 hours or 48 hours. This suggests that the process of apoptosis occurs between 48 hours and 72 hours of drug treatment.
Table IX shows the Enrichment factor of apoptosis for betulinic acid and its derivatives against MOLT-4 leukemia cells.
The Enrichment factor is calculated by the formula
Enrichment factor =
mU of the Sample (dying / dead cells)
mU of the corresponding Control cells (cells without treatment)
All derivatives show Enrichment factor less than betulinic acid indicating varying degrees of apoptosis.
Example 16
A suitable formulation of betulinic acid or its derivatives was prepared- as follows. Betulinic acid or its derivatives were solubilised in a minimum volume of methanol. Betulinic acid or its derivatives may also be solubilized in isopropyl alcohol, dimethylformamide, dimethylsulfoxide or any other suitable solvent. Polyvinylpyrolidone was separately solubilised in methanol or a suitable solvent. Both were mixed and evaporated at 30-100°C under vacuum while being stirred continuously. A fluffy mass was obtained which solubilised in water at a concentration of 0.1 to 50 mg per milliliter of water [preferably I - 20 mg betulinic acid (or its derivatives) per milliliter of water]. Betulinic acid (or its derivatives) to PVP ratio was between I : I to I : 20 (preferably between I : 2 to I : 10). Systemic administration refers to oral, rectal, nasal, tiansclennal and parentral (i.e., intramuscular, intraperitoneal, subcutaneous and intravenous). In accordance with good clinical practice, it is preferred to administer the composition at a
dose that will produce anticancer effects without causing undue harmful side effects The composition may be administered either alone or as a mixture with other theiapeutic agents.
The composition may optionally and preferably contain phannaceutically acceptable diluents, fillers, lubricants, excipients, solvents, binders, stabilizers, and the like. The preferred diluents may be selected from lactose, starch, mannitol, sorbitol, microcrystalline cellulose, dibasic calcium phosphate dihydrade, sucrose, calcium sulfate dihydrate, dextrose, inositol, maltose and amylose ; the preferred binders may be selected from maize starch, wheat starch, rice starch, potato starch gelatin, tragacanth, methyl cellulose, hydroxy propyl methyl cellulose, sodium carboxy methyl cellulose and poly vinyl pryolidone ; the preferred filters may be selected from lactose, sucrose, mannitol, sorbitol, calcium phosphates ; the preferred lubricants may be selected from silica, talc, stearic acid or salts thereof such as magnesium staearate, or calcium stearate, and / or polyethylene glycol.
Pharmaceutical compositions which provide from about 10 mg to 1000 mg of the composition per unit dose are preferred and are prepared as tablets, lozenges, capsules, powders, aqueous or oily suspensions, syrups, elixirs, inplants and aqueous solutions by any conventional method.. The nature of the pharmaceutical composition employed will, of course, depend on the desired route of administration. The human.dosage of the composition is in the range of 10 to 200 mg / kg / day and the preferred range is 20 to 50 mg / kg / day.
The acute toxicity study of betulinic acid and its derivatives MJ398-RS, MJ43X-RS and MJ542-RS was carried out on 10 - 12 week old BALB/C mice weighing approximately 25 grams. All animals were acclimatized for 7 days and were fasted 18 hours before and 4 hours after dosing. All experimental animals were observed for 5 days. Observations were made three times on the day of dosing and twice daily thereafter for the remaining 5 days or until reversible toxic signs (if any) subsided. Acute intraperitoneal toxicity study was
conducted at three different dose levels of 200 ing / Kg B.Wt, 400 mg / Kg B.Wt and 600 mg / Kg B.Wt. for betulinic acid, three dose levels of 500 mg / Kg B.Wt., 750 nig / Kg B.Wt and 1000 mg / Kg B.Wt. for MJ398-RS and MJ438-RS and three dose levels of 200 mg / Kg B.Wt., 400 mg / Kg B.Wt and 800 mg / Kg B.Wt. for MJ542-RS to calculate LDjo value. No mortality was recorded in betulinic acid and MJ542-RS. There was no visible toxicity or weight loss. Hence the LD5o of betulinic acid is greater than 600 mg / Kg B.Wt. and the LD50 of MJ542-RS is greater than 800 mg / Kg B.Wt. The LD,,, for MJ398-RS and MJ438-RS was 750 mg / Kg B.Wt.
F.xainplr 17
To test the in vivo ability of betulinic acid to serve as an anticancer agent against leukemia, studies were performed with 6 week old athymic mice each weighing approximately 25 g, injected subcutaneously with approximately 20 million leukemia cells (MOI.T-4) Drug treatment with betulinic acid was initiated on day one, i.e., 24 hours post-injection MOLT-4 cells. Betulinic acid was co-precipitated with polyvinylpyrolidone to increase solubility and bioavailability (Waller, D.P., et al Contraception 22, 183-187, 1980). Six animals were treated with the drug at a dose of 40 mg/kg body weight. A total of six intraperitoneal injections were given every alternate day. Four control animals were treated with the vehicle alone. The mice were weighed and the tumors measured with a vernier calliper every alternate day. The treatment resulted in complete inhibition of tumor growth as compared with the control animals which attained a tumor volume of approximately 900 mm1 by day 30.
Example 18
The effect of betulinic acid was determined on normal cell metabolism in vitro. The panel of normal cells included normal mouse splenocytes, human endothelial cells (ECV and Ea.Hy.926) and lung fibroblasts (MRC-9, passage number 4). A 72-hour MTT cytotoxicity assay was performed as described previously. In each of the cell line, the ED5(,
value of betulinic acid was determined to be greater than 20 jig/ml indicating non toxicity of betulinic acid to normal cells.
The instant invention is shown and described herein in what is considered to be the most practical, and preferred embodiments. It is rccogni/ed, however, that departures may be made therefrom which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure.

We claims
1. A method of treating a patient suffering from leukemias, lymphomas, prostate, lung or ovarian cancer, said method comprising administering a pharmaceutically effective dosage of betulinic acid to the patient.
A^Anethod of treating a patient suffering from cancer such as leukemia, lymphoma,
prostate or ovarian cancer, said method comprising administering a
pharmaceutically effective dosage of betulinic acid derivatives to the patient.
A method as claimed in claim 1 or 2 wherein said patient includes humans,
mammals and other animals.
A method as claimed in claim 1 or 2 wherein the antileukemia and antilymphoma
activity of betulinic acid and its derivatives with ED5() values are in the range of
0.5 to 4.0|ig/ml.
A method as claimed in claim I or 2 wherein the antiprostate and anticancer
activity of betulinic acid and its derivatives with EDso values is in the range of I. I
to 5.5(.ig/ml and 0.5 to 8.1 (ig/ml, respectively.

A method as claimed in claim 1 or 2 wherein betulinic acid, and its derivatives are
administered to the patient along with suitable pharmaceutically acceptable
additives such as carriers diluents, solvents, fillers, lubricants, excipients, binders,
stabilizers, and the like.
A method as claimed in claim 6 wherein the diluents may be selected from lactose,
starch, mannitol, sorbitol, microcrystalline cellulose, dibasic calcium phosphate
dihydrade, sucrose, calcium sulfate dihydrate, dextrose, inositol, maltose and
amylose ; the preferred binders may be selected from maize starch, wheat starch, rice starch, potato starch gelatin, tragacanth, methyl cellulose, hydroxy propyl methyl cellulose, sodium carboxy melhyl cellulose and poly vinyl pryolidnnc ; the preferred filters may be selected from lactose, sucrose, mannitol, sorbitol, calcium phosphates ; the preferred lubricants may be selected from silica, talc, stearic acid or salts thereof such as magnesium staearate, or calcium stearate, and / or polyethylene glycol.
A method as claimed in claim 6 wherein the composition provides 10 mg to 1000
mg per unit of the dose of betulinic acid or its derivatives.
A method as claimed in claim 6 wherein the composition is in the form of tablets,
lozenges, capsules, powders, aqueous or oily suspensions, syrups, elixirs, inplants
and aqueous solutions.
10 A method as claimed in claim I or 2 wherein the dosage for humans is in the range of 10 to 200 mg / kg / day and the preferred range is 20 to 50 mg / kg / day.
A method as claimed in claim 1 or 2 wherein the treatment comprising killing or
inhibiting/preventing the multiplication of cancer or tumor cells.
A method as claimed in claim 1 or 2 wherein the treatment includes administering
betulinic acid or its derivatives to the patient systemically.
13 A method as claimed in claim 1 or 2 wherein the treatment includes causing cell death by Apoptosis.
14. A method as claimed in claim 1 or 2 wherein the treatment is a non-toxic one
15. A method as claimed in claim 2 wherein the betulinic acid derivatives have
formula 2 of the accompanying drawings wherein R, R(, R2, Ri, R4, Rs and R,, is
independently or in combination represent the following functional groups in the
derivatives :
R is H
R, is H, Br, Cl, F or 1 ;
R2 & R, together is O,NNHC6H5, NNHCY,H2Ch, NNHCf,H4OCH3, NNHC6H4OH
N-OX (X being -H-COCH,,SO2Cr,H4CH,,
CO(CH2)nCHi( n = I to 5 ) R2 is H and R? is OH, OCOCH3, OCO2(CH2)nCH, (n = 1 to 5)
OCOC(CH?)3,OCO(CH2)n Cl,( n - 1 tolO),OCOC6H5
OCOCf)H?CI?OCOCH(OCOCH,)CH,, OCO(CHOH)CH3,
R4is()H,-OCH,,O(CH2)nCOOCH,,O(CH2)I,COOC2H5,O(CH2)COOH,O(CH2)nCoCI (n-1 to5)OCH2CH2OC2H5,OCH7CH2OH,OCH2CH2OCOCH,,CI,N3,NHNH2, NHNHCftHzC!.-,. NH2,NH(CH2),,CH, (n =0 to 9) R5 is H or Br
Rr, is CH3, CH2Br, CH2OH. CHO, CH2OCOCH,, COOH, COCOO(CH2)nC()()CH,, COO(CH2)nCOOC2H5, COO(CH2)nCOOH(n=l to 5) R5 and Rfi together : OCH2or >CH-CH,.
16. A method as claimed in claim 14 wherein the betulinic acid derivatives having
formula 3 of the accompanying drawings wherein R = H ; ri = H ; R2 = H ;
R3 = OCOCH(OCOCH3)CH3 or OCOC(CH3)? ; R4 = H or CH2COOH
17 A method as claimed in claim 14 wherein the betulinic acid derivatives are shown In fig.4 of the accompanying drawings wherein R = H;Ri = H;R2 = H; R,=OCOCH(OCOCH;OCH,,O(CH2),COC1, OCOCH(OCOCH3)CH3 or OCOCI \}Cl I, ; R4 11 or C'l b(X)()('l I,
18. A method as claimed in claim 14 wherein the betulinic acid derivatives are
represented in fig.5 of the accompanying drawings wherein R ~ II ; ri ~ II ;
R2 ()or N-OCOril,; R3 = H.
19. A method as claimed in claim 14 wherein the hetulinic acid derivatives are
represented in Tig.0of the accompanying drawings wherein R - \\ ; Rt - II ;
R2 =NOH or NNHCf,H5; R? = H or CH2COOCH,
20. A method as claimed in claim 14 wherein the betulinic acid derivatives are
represented in fig. 7 of the accompanying drawings wherein R = H ; ri = Br;
R2 = O ; R? = H; R4 = H ; R, = H .
21. A method as claimed in claim 14 wherein the betulinic acid derivatives are
represented in fig.8 of the.accompanying drawings in which R = H ; ri = H ;
R2 = H ; R3 = OCOCH3; R4=H; R, = Br ; Rr, = Br .
'42 Novel betulinic acid derivatives used for killing or inhibiting / preventing multiplication of cancer or tumor cells, said derivatives having a basic skeleton of betulinic acid as shown in figure. 2 of the accompanying drawings in which R, RK ~R2, R?, R4, R-> and Rr, is independently or in combination represent the following functional groups in the derivatives : RisH
R, is H, Br, Cl, F or I;
R2& R. is together O,NNHCr,H5, NNHCr,H2CI,, NNHCr,H4OCH?NNHC6H.,()H
N-OX (X being -H-COCH,,SO2Cr,H4CH?,
ro(rN,)nrii,,( n i to 5 >
R2 is H and R., OH, OCOCH,, OCO2(CH2)nCH3 (n - I to 5) OCOC(CH,)?,OCO(CH2)lt Cl,( n = I tol()),OCOCf)H5 OCOC(,H.,CI3.OCOCH(OCOCH.OCH.-,,OCO(CHOH)CH,,
K ., is ( )l !,-( )( 'I I ,,( )(( 'I !..)„< '< M )( 'I I ,,< )(( 'I !..)„( '( H )( ' ,1 1,, ()(('! !.,)„( '< )( )l l,< )(( 'I I .,),,< '<>< I
,NI INI IC,,I IsOCI I,, Nl INI KYI I .CK. Nl 1 ,. Nl I(CI I2)BCI 1, (n =0 to 9) R, is ll;Br
roo(rH2)ncooc2H!i, cocxcu,),, coouoi h to .s>
R, and R6 is together >OCH2 or > CM-CM,.
23. Betulinic acid derivatives as claimed in claim 21 wherein the preferred derivatives
Tiaving formula as shown in figure 3 wherein R = H;Ri = H;R2 = H;
R, = OCOCH(OCOCH,)CH3 or OCOC(CH,), ; R4 = H or CH2COOH
24. Betulinic acid derivatives as claimed in claim 21 wherein the preferred derivatives
are shown in fig. 4 of the drawings wherein R = H;Ri=rH;R2 = H;
R,-OCOCH(OCOCH3)CH,,0(CH2),COCI,OCOCH(OCOCH3)CH?or
OCOCH2CH, ; R4 = H or CH2COOCH3.
25. Betulinic acid derivatives as claimed in claim 21 wherein the preferred derivatives
are fn lig.5 of the drawings wherein R = \\ ; ri = M ; R2 = O or N-OCOCU t ;
R3= H.
Betulinic acid derivatives as claimed in claim 21 wherein the preferred compounds
are represented in fig. 6 of the drawings wherein R = H ; ri = H ; R2 =NOH or
NNHC,,H5 R, - H or CH2COOCH,.
Betulinic acid derivatives as claimed in claim 2 1 wherein the preferred compounds
are represented in fig. 7 of the drawings wherein R = H ; ri = Br ; R2 = O ; R3 = H;
28. Betulinic^acid derivatives as claimed in claim 21 wherein the preferred compounds
-ate represented in fig. 8 of the drawings in which R = H;R, = H; R2 = H;
R? = OCOCH-,; R4 =H; R? = Br ; Rr, = Br
A process for the preparation of 3-O - acyl betulinic acid derivatives, said process
comprising treating betulinic acid or its derivatives with suitable anhydride or acyl
chloride at room temperature for few hours, adding water to the product
obtained above and extracting with organic solvent, the organic layer thus
obtained is dried and evaporated to obtain _said 3:0- acy! betulinic acid
derivaitves. A process as claimed in claim 29 wherein the organic layer is dried over
anhydrate sodium sulphate and evaporated in manner known Der se.
^Ajnethod of producing 3-oxo derivatives/of berjjlinic acid, said method comprising dissolving the betulinic acid orits derivatives in an organic solvent , adding •edtfventional oxidising agent under normal reaction condition and obtaining pure 3-oxo derivatives by any known manner.
32. A method as claimed in claim 3 1 wherein pure 3-oxo-betulinic acid derivatives is obtained by adding water to the reaction mixture, extraction with organic solvent followed by crystillsation from the organic layer.
33 A method for the preparation of 2-bromo-3-oxo- betulinic acid derivatives, said process comprising dissolving 3-oxo-belulinic acid derivatives in halogenated organic solvent, adding dropwise liquid bromine disolved in the same solvent at temperature between 0-10°C , the reaction mixuture thus obtained is brought to room temperature and kept for a few hours and obtaining pure 2-bromo-3-oxo derivatives by a method known per se.
A method of preparing 3-oximino-betulinic acid derivatives, said method
comprising adding alcholic solvent to 3-oxo-betulinic acid derivatives , adding
hydroxylamine hydrochloride and sodium acetate , refluxing the reaction mixture
for few hours and obtaining pure 3- oximino derivatives of betulinic acid by a
method known per se.
A method for the preparation of phenylhydrozone of 3-oxo-betulinic acid
derivatives , said method comprises dissolving 3-oxo-betulinic acid derivatives in
an alcholic solvent and retluxing the reaction mixture for a few hours and
.obtaining pure phenyl hydrozone derivatives by known method.
A method for the preparation 17-carboxyalky carboxylate betulinic acid
'derivatives, said method comprises by dissolving betulinic acid and derivatives in
dry DMF (dimethyl formamide), adding sodium hydride and stirred for two hours at room temperature and then further for 16-20 hours after the addition of haloalkylcarboxyester and obtaining pure 17-carboxyalkyl carboxylate butulinic acid derivatives by any known method.
A method of preparing 17-carboxyalkylcarboxylicacid of betulinic acid derivatives,
said method comprising alcoholic solution of 17-carboxyalkyl carboxylate, adding
alcoholic hydroxide solution to the above solution, heating the reaction mixture
to 40-50T for 2-4 hours and obtaining pure 17-carboxyalkyl carboxylic acid
derivatives by any conventional method. ' *
A method of preparing 2(),2l)-dibromo belulinic acid derivatives, said method
comprising dissolving betulinic acid derivatives in halogenated organic solvent,
adding liquid bromine dissolved in the same solvent dropwise, maintaining the
reaction mixture at a temperature 0-1 ()"(.' and bringing the reaction mixture' to the
room temperature and finally obtaining 20,29-dibromo betulinic acid derivatives
by any conventional method.
A composition as claimed in claim 40 which is in the form of tablets, lozenges, capsules, powders, aqueous or oily suspensions, syrups, elixirs, inplants and aqueous solutions.
A composition as claimed in claim 40 wherein the dosage for humans is in the range of 10 to 200 mg / kg / day and the preferred range is 20 to 50 mg / kg / day.