WO1997008162A1

WO1997008162A1 - Beta-lapachone derivatives as antitumor agents

Info

Publication number: WO1997008162A1
Application number: PCT/US1996/013656
Authority: WO
Inventors: Arthur Pardee; Chiang J. Li
Original assignee: Dana-Farber Cancer Institute
Priority date: 1995-08-24
Filing date: 1996-08-23
Publication date: 1997-03-06
Also published as: AU6901296A

Abstract

We have now discovered that compounds of the following formulae (I) and (II) can be used to selectively stimulate the death of mammalian prostate cancer cells, and thus are useful in treating prostate cancer wherein R and R1 are each independently selected from the group consisting of hydrogen, hydroxy, thio, halogen, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted aryl, and substituted and unsubstituted alkoxy, and salts thereof, wherein the dotted double bond between the ring carbons to which R and R1 are bonded represent an optional ring double bond. Preferred compounds of formula (I) include those in which at least one of the substituents R and R1 is hydrogen and/or at least one of said substituents is allyl. Specifically preferred compounds include β-lapachone (i.e., R and R1 both being hydrogen), allyl-β-lapachone, particularly 3-allyl-β-lapachone (i.e., R being allyl and R1 being hydrogen) and 3-bromo-β-lapachone (i.e., R being bromo and R1 being hydrogen).

Description

BETA-LAPACHONE DERrVATTVES AS ANTTTUMOR AGENTS

BACKGROUND OF THE INVENTION

The present invention is directed to a method of stimulating the death of prostate tumor cells. Preferably, the method is used to treat individuals suffering from prostate cancer.

Prostate cancer is a serious cause of death worldwide.

Androgen independent prostate cancer is presently the most common cancer in men in the United States with 38,000 deaths anticipated for the USA in 1994, and is a significant condition worldwide. Approximately 50% of patients are presented with metastatic disease. However, the only existing treatment for metastatic disease is hormonal therapy, which is not curative. Thus, the metastatic disease is typically fatal.

Hormonal therapy consisting of different approaches to blocking the action of androgen on the prostate tumor is effective in controlling only the growth of tumor cells that depend on androgen for growth (hormone-dependent tumor). Unfortunately, hormone-dependent tumor inevitably progresses to more advanced hormone-independent tumor, which cannot be controlled by current treatment. Difficulties in treating prostate cancer arise from a variety of reasons. Although such androgen ablation is a standard therapy for metastatic prostate cancer it is rarely entirely successful because in most individuals the cancer is heterogeneous comprising both androgen dependent and androgen independent cancer cells. Thus, the therapy does not eliminate the androgen independent cells.

Chemotherapy, which has been used to treat a number of other cancers, has not proven successful. This is because the vast majority of these androgen independent cells are not actively proliferating and standard chemotherapeutic agents work by selectively killing actively proliferating cells.

Radiation therapy, which also is selective for rapidly proliferating cells, has also not proven effective. Surgery has also not proven an effective means for treating advanced disease states. Accordingly, it would be desirable to have new methods for stimulating the death of these slow proliferating cancer cells. It would be particularly desirable to have a new means of treating individuals suffering from prostate cancer, particularly androgen independent cancer.

Mapachone (3,4-dihydro-s,3-dimethyl-2H-naphthol[1 ,3-b] pyran- 5,6-clone) is a simple plant product with a chemical structure different from currently used anti-cancer drugs. It is obtained by sulfuric acid treatment of the naturally occurring lapachol, which is readily isolated from Tabebuia avellanedae growing mainly in Brazil, or is easily synthesized from lomatiol, isolated from seeds of lomatia growing in Australia (Hooker, S., et. al., J. Am. Chem. Soc, 58: 1 181 -1 190 (1936); Goncalves de Lima, O., et al., Rev. Inst. Antibiot. Univ. Recife.

4:3-17 (1962)).

Mapachone has been shown to have a variety of pharmacological effects. /Mapachone is a topoisomerase I inhibitor but acts by a different mechanism than camptothecin. Numerous β- lapachone derivatives have been synthesized and tested as anti-viral and anti-parasitic agent (Goncalves, A.M., et al., Mol. Biochem. Parasitology, 1 : 167-176 (1980); Schaffner-Sabba, K., et al., J. Med. Chem., 27:990-994 (1984); Li, C, et al., Proc. Natl. Acad. Sci. USA, 90: 187-1842 (1993)). Mapachone and its derivatives, e.g. 3-a.lyl-0- lapachone, show anti-trypanosomal effects (Goncalves, A.M., et al., supra), the mechanism of which is unclear. It significantly prolongs the survival of mice infected with Rauscher leukemia virus, probably through inhibition of reverse transcriptase (Schaffner-Sabba, K., et al., supra; Schuerch, A.R., et al., J. Biochem., 84: 197-205 (1978)). We taught that /Mapachone also inhibits gene expression directed by the long terminal repeat (LTR) of the human immunodeficiency virus type 1 , and viral replication (Li, C, et al., supra). /Mapachone has also been shown to be a DNA repair inhibitor which sensitizes cells to DNA damaging agents (Boorstein, R.J., et al., Biochem. Biophys. Res.

Commun., 1 18:828-834, (1984); Boothman, D.A., et al., J. Cancer Res., 49:605-612 (1989)). yff-lapachone is well tolerated in dogs, rats, mice, and chickens. The maximum tolerated dose, when given p.o daily for one month, is 200 mg/kg in rats, and 100 mg/kg in dogs. Higher doses cause gastric ulceration and loss of erythrocytes, but not signs of bone marrow suppression (Ciba-Geigy, personal communication). The previous experience with this compound in humans has been limited.

SUMMARY OF THE INVENTION

We have now discovered that compounds of the following formulae I and II can be used to selectively stimulate the death of mammalian prostate cancer cells, including both androgen dependent prostate cancer cells and androgen independent prostate cancer (CaP) cells, and thus are useful in treating prostate cancer:

wherein R and R, are each independently selected from the group consisting of hydrogen, hydroxy, thio (SH), halogen, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted aryl, and substituted and unsubstituted alkoxy, and salts thereof, wherein the dotted double bond between the ring carbons to which R and R, are bonded represent an optional ring double bond. Preferred compounds of formula I include those in which at least one of the substituents R and R, is hydrogen and/or at least one of said substituents is allyl. Specifically preferred compounds include yff-lapachone (i.e., R and R, both being hydrogen), allyl-/?- lapachone, particularly 3-allyl-β-lapachone (i.e. R being allyl and R, being hydrogen) and 3-bromo-/Mapachone (i.e. R being bromo and R, being hydrogen).

The present invention further provides a method for inducing cell apoptosis in vivo which comprises contacting the cell with an effective amount of the compound. The cell is preferably a human prostate cell. More preferably, the cell is a human prostate cancer cell.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of /Mapachone on survival of human prostate cancer cells O, PC-3 cells; O, DU145 cells. Cell survival was determined by the colony formation assay described below.

Figures 2A, 2B, 2C and 2D show induction of apoptosis by β- lapachone in human prostate cancer cells. DNA laddering, typical feature of apoptosis, was induced in PC-3, D 145 (2A) and LNCaP cells (2B). In 2A, cells were treated with 4 μWΛ /Mapachone for 4 hours, followed by incubation in drug-free medium for 4 hours (lane 2,7), 12 hours (lane 3,8), 20 hours (lane 4,9), 44 hours (lane 5, 10). As controls, cells were treated with equal volume of DMSO (lane 1 ,6).

DNA was extracted and subjected to electrophoresis. In 2B, LNCaP cells were treated with /Mapachone for 4 hours at concentrations of O μM (lane 1 ), 0.5 μM (lane 2), 2 μM (lane 3), 4 /M (lane 4), followed by drug free incubation for 20 hours. To quantify apoptotic fraction, LNCaP cells were treated with or DMSO (2C) or 8 μM /Mapachone (2D) for 1 hour, followed by incubation in drug-free media for 23 hours before they were subjected to flow cytometric analysis.

Figure 3 shows lack of correlation between /Mapachone induced apoptosis and the expression of p53 and bcl-2. A. Expression pattern of p53 and bcl-2 in DU-145 cells (lane 1 ) and PC-3 cells (lane 2). B, β- lapachone did not induce expression of p53 and p21 . Lane 1 to 4: PC- 3 cells; lane 5 to 8: DU-145 cells. Cells were treated with DMSO (lane 1 ,5), or /Mapachone at 2 //M (lane 2,6), 4 μM (lane 3, 7) and 8 //M (lane 4, 8) for 1 hour, followed by incubation in drug free media for 23 hours. Expressions of p53, p21 , and bcl-2 were determined by Western blot assay as described below.

Figures 4A, 4B, and 4C shows induction of apoptosis (A) and differentiation in HL-60 cells (B,C) by 0-lapachone. In 4A, HL-60 cells were treated with 8 μM Mapachone for 24 hours (lane 1 ). 16 hours (lane 2), 8 hours (lane 3), 4 hours (lane 4), 2 hours (lane 5), 0 hours (lane 6). Cellular DNA was extracted and subjected to gel electrophoresis. To analyze morphological changes induced by β- lapachone, HL-60 cells were treated with ethanol (1/1000, v/v) (4B) or

0,8 μM /Mapachone dissolved in ethanol (4C) for 6 days before harvest. A thin film of cells were spread on a slide, and stained with modified Wright-Giemsa Stain (Sigma).

Figure 5 shows the effect of /Mapachone on tumor volume. The bar represents episodes of administration of Mapachone.

Figure 6 shows the effect in vivo of /Mapachone on prostate tumors AT-3.1. Figure 7 shows the effect in vivo of /Mapachone on prostate tumors AT-3.1.

DETAILED DESCRIPTION OF THE INVENTION We have now discovered that compounds of the following formulae I and II can be used to stimulate the death of mammalian prostate cancer cells. The mammal is preferably a human. Thus, the compounds having the following formulae are useful in treating prostate cancer:

wherein R and R, are each independently selected from the group consisting of hydrogen, hydroxy, thio (SH), halogen (e.g. fluoro, chloro and bromo), substituted and unsubstituted aryl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkyl and substituted and unsubstituted alkoxy, and salts thereof, wherein the dotted double bond between the ring carbons to which R and R, are bonded represent an optional ring double bond. The alkyl groups preferably have from 1 to about 15 carbon atoms, more preferably from 1 to about 10 carbon atoms, still more preferably from 1 to about

6 carbon atoms. As used herein, the term alkyl unless otherwise modified refers to both cyclic and noncyclic groups, although of course cyclic groups will comprise at least three carbon ring members. Straight or branched chain noncyclic alkyl groups are generally more preferred than cyclic groups. Straight chain alkyl groups are generally more preferred than branched. The alkenyl groups preferably have from 2 to 15 carbon atoms, more preferably from 2 to about 10 carbon atoms, still more preferably from 2 to about 6 carbon atoms. Especially preferred alkenyl groups have 3 carbon atoms (i.e., 1- propenyl or 2-propenyl), with the allyl moiety being particularly preferred. Phenyl and naphthyl are generally preferred aryl groups. Alkoxy groups include those alkoxy groups having one or more oxygen linkage and preferably have from 1 to 15 carbon atoms, more preferably from 1 to about 6 carbon atoms. Said substituted R and R. groups may be substituted at one or more available positions by one or more suitable groups such as, for example, alkyl groups such as alkyl groups having from 1 to 10 carbon atoms or from 1 to 6 carbon atoms, alkenyl groups such as alkenyl groups having from 2 to 10 carbon atoms or 2 to 6 carbon atoms, aryl groups having from 6 to 10 carbon atoms, halogen such as fluoro, chloro and bromo, and N, O and S, including heteroalkyl, e.g., heteroalkyl having one or more of said hetero atom linkages (and thus including alkoxy, aminoalkyl and thioalkyl) and from 1 to 10 carbon atoms or from 1 to 6 carbon atoms.

Compounds of formulae I and II can readily be made or obtained. (See Pardee, A., et al., Cancer Research, 49, 1-8 (1989); Schaffner- Sabba, K., et al., Journal of Medicinal Chemistry, 27, no. 8 990-994

(1984); S. Hooker, 58, 1 181-1 197 (1936).

Preferred compounds of formula I include /Mapachone, 3-allyl-/?- lapachone, 3-bromo-)-Mapachone, 3-OH-/Mapachone and 3-allyl-/?- lapachone and 3-bromo- Mapachone are more preferred. Preferred compounds of formula II include 3-bromo-alpha- lapacheone (compound 4 of Table 1 ).

We have tested a variety of human cancer cells for their sensitivity to compounds of formula I, e.g. /Mapachone, a novel inhibitor of DNA topoisomerase I (Li, C.J., et al., supra). Human prostate cancer cells were the cancer cells that was most sensitive to /Mapachone and its derivatives, these compounds exhibiting enhanced death rates. We believe these cells were induced to undergo a process of programmed cell death (apoptosis) specifically activated in prostate cells after they are deprived of testosterone (Martikainen, P., et al., Cancer Res. 51 :4693-4700 (1991)). Results in the nude mouse model demonstrate that compounds of formulae I and II such as /Mapachone can inhibit human prostate tumor growth. This compound also causes suppression of survival, albeit at slightly higher concentrations, in human ovary and breast cells. However, typical apoptosis was not detected in any other human cancer cells of epithelial origins including colon, kidney, lung, breast, or ovary exposed to the drug other than the prostate cells. Human hematopoietic leukemia cells (HL-60) were induced to undergo either apoptosis or differentiation depending on the concentration used.

The prevalence of p53 inactivation and/or bcl-2 expression in human tumors is generally believed to be at least partially responsible for the general ineffectiveness of current chemo- and radiation therapy for cancer (Berchem, G.J., et al., Cancer Res. 55:735-738 (1995); Lowe, S., et al., Science 266:807-810 (1994)). It is thus important to develop novel anti-cancer drugs that induce cell death in a p53 independent manner. One way such compounds could work is through activation of p53 targeting genes, e.g. p21 (SDI1 /WAF1 /Cip1 ), by a p53 independent pathway (Johnson, M., et al. Mol. Carcinogenesis 1 1 :59-64 (1994)). We have shown that compounds of formulae I and II such as /Mapachone and its derivatives induced apoptosis in the absence of p53 expression (Fig. 3A) indicating that it may be p53 independent. There was no significant induction of p53 and p21 (Fig.

3B) in human prostate cancer cells during apoptosis, suggesting that the induced apoptosis occurs independent of the p53 pathway, β- lapachone and its derivatives induced cell death that aiso did not correlate with bcl-2 expression and was not protected by ectopically overexpressed bcl-2. These results thus indicate the existence of a cell death program independent of both p53 and bcl-2 can be activated by compounds of formulae I and II such as /Mapachone and its derivatives.

In general, for the treatment of androgen independent prostate cancer, a suitable effective dose of one or more compounds of formulae I or II will be preferably in the range of 10 to 500,000 μg per kilogram body weight of recipient per day, more preferably in the range of 1000 to 50,000 μg per kilogram body weight per day, most preferably in the range of 5000 to 25,000 μg per kilogram body weight per day. The desired dose is suitably administered once or several more sub-doses administered at appropriate intervals throughout the day, or other appropriate schedule. These sub-doses may be administered as unit dosage forms, for example, containing 1 to 20,000 μg, preferably 10 to 10,000 μg per unit dosage form.

Accordingly, one would use an effective amount of these compounds in a method to stimulate the death of prostate tumor cells, particularly androgen independent prostatic tumor cells. One would select a subject having prostatic tumor cells and administer an effective amount of one of the compounds of formula I such as 3-allyl-/?- lapachone, 3-halo-/Mapachone or /Mapachone to treat the prostatic tumor cell. Preferably, the subject is a human. Still more preferably, the human has CaP.

In treating metastatic disease, because selective nature of the compounds of formulae I or II, one can administer the compounds intravenously.

Administration of the compounds of the invention may be by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual) and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) with oral or parenteral being preferred. It will be appreciated that the preferred route may vary with, for example, the condition and age of the recipient.

The administrative ingredients may be used in therapy in conjunction with other medicaments.

While one or more compounds of formulae I or II may be administered alone, they also may be present as part of a pharmaceutical composition. The compositions of the invention comprise at least one compound of formulae I or II together with one or more acceptable carriers thereof and optionally other therapeutic ingredients, including those therapeutic agents discussed supra. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual) or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes and may be prepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the to be administered ingredients with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion or packed in liposomes and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Compositions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.

Compositions suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising one or more compounds of formulae I or II and a pharmaceutically acceptable carrier. A suitable topical delivery system is a transdermal patch containing the ingredient to be administered.

Compositions suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Compositions suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

To ensure solubility, the compounds are preferably dissolved in a non-ionic solubilizer such as an ethylene oxide ester-ether and fatty acid glycerides commercially available as Cremphor EL (BASF).

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.

All documents mentioned herein are incorporated herein by reference.

The present invention is further illustrated by the following examples. These examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof. EXAMPLES General Comments

The following reagents and procedures were employed as specified in the examples.

Chemicals

/Mapachone was kindly provided by Dr. A. Matter (CIBA-GEIGY Ltd., Switzerland). It was dissolved in dimethyl sulfoxide (DMSO) at 20 mM concentration, aliquoted, and kept at -20°C.

Cell Cultures

All cell lines used in this study are obtained from the American Type Culture Collection (Rockville, MD.) unless specified otherwise. Cells were maintained at 37°C in 5% CO2 in complete humidity. Human prostrate tumor cells PC-3, DU145, and LNCaP were grown in

Dulbecco's modified Eagle's Medium (Life Technologies, Inc.) supplemented with 10% FCS and 2 mM L-glutamine. HL-60 (human promyelocytic leukemia cell line) was cultured in RPMI medium with 10% heat inactivated FCS. MCF-7 and 21 MT (human breast epithelial cell line), kindly provided by Dr. R. Sager (Dana-Farber Cancer Institute,

Boston, MA), were cultured in MEM alpha medium (Life Technologies, Inc.) supplemented with 10% FCS, 2 mM L-glutamine, and 1 mg/ml insulin. AD 2780s (human ovary carcinoma), a generous gift from Dr. K.J. Scanlon (City of Hope Medical Center, Duarte), 293 (human kidney epithelial cell line), SW11 16 (human colon adenocarcinoma), and human lung carcinoma cell lines (H596, H520) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% FCS and 2 mM L-glutamine. Hela and Hela- bcl-2 cells (Meikrantz, W., et al., Proc. Natl. Acad. Sci. USA, 91 :3754- 3758 (1994), kindly provided by Drs. W. Meikrantz and R. Schlegel (Harvard School of Public Health, Boston, MA), were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% FCS, 2 mM L-glutamine, and 800 //g/ml of G418.

Colony Formation Assay

Exponentially growing cells were seeded (2000 cells/dish) in 60 mm culture dishes and allowed to attach for 48 hours. B-lapachone was added in less than 5 μ\ volume (corresponding to a final DMSO concentration of less than 0.1 %) directly to dishes from concentrated working solutions in DMSO. Control dishes received DMSO alone at equal volume. After 1 to 4 h, cells were rinsed and drug free medium was added to the cells. Cultures were observed daily for 10 to 20 days, cells were fixed and stained with modified Wright-Giemsa Stain (Sigma). Colonies of greater than 30 cells were scored as survivors.

Aαarose Gel Electrophoresis of Apoptotic DNA The method of Wesselborg, S., et al., J. Immunol., 150:4338 (1993) was used. Cells were treated with /Mapachone, and then incubated in drug-free media. They were harvested and lysed in 50 mM Tris-HCl, 10 mM EDTA, 0.5% SDS, 0.5 mg/ml proteinase K, and 0.15 ng/ml RNase. The supernatant of the cell lysate was loaded onto a 2% agarose gel. The electrophoresis was carried out at 24 V for 16 hours. The gel was stained with ethidium bromide. A Polaroid picture was taken after detaining the gel for 1 hour.

Flow Cytometry Analvsis

Cytofluorometric analysis of apoptosis and cell cycle analysis was performed by propodium iodide staining of nuclei as reported previously (Li, CJ., et al., Science 268:429-431 (1995)). Western Blot Analvsis

Nuclear extract was prepared from exponentially growing cells (Dignam, J.D., et al., Nucleic Acids Res., 1 1 : 1475 (1983)). The ECL assay system was used to detect p53 and bcl-2 levels. Briefly, nuclear protein samples (10 μg per sample) were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel and then electrophoretically transferred to a nitrocellulose membrane. The blot was blocked, washed, and incubated with p53, bcl-2, or p21 (Cip1 /Waf1 ) antibody (Oncogene Science, Cambridge, MA) at 1 : 1000 dilution. The filter was then incubated with a secondary antibody that was conjugated with horseradish peroxidase. Finally, the filter was developed with detection reagents (RPN 2109:Amersham) and exposed to a hyperfilm- ECL (RPN 2103).

Animal Administration In the in vivo animal experiments, /Mapachone was formulated into solution with Cremophor EL (BASF).

Example 1 Effects of jg-Lapachone on Survival of Human Cancer Cells Human carcinoma cell lines of different histotypes were used to test the anti-survival effect of /Mapachone. Androgen independent human prostate tumor cells PC-3 and DU145 were treated with β- lapachone in vitro. Cell survival was determined by colony formation assay, /Mapachone inhibits proliferation of both cell lines with an IC₁₀₀ of 4 to 8 μM (Figure 1 ). LNCaP cells were equally sensitive to β- lapachone. 21 MT (a human breast carcinoma cell line) and AD2780s (a human ovary carcinoma cell line) were also relatively sensitive to the antiproliferative effect of /Mapachone (IC₁₀₀, 16 μM). Proliferation of SW1 16, a human colon adenocarcinoma cell line, was not significantly inhibited by /Mapachone up to 128 μM, the maximum concentration used. Other cell lines tested, which included H596, H520 (human lung carcinoma cell lines) and 293 (a human kidney epithelial cell line) were also relatively resistant to /Mapachone (IC₁₀₀ > 32/vM).

Seventeen derivatives of /Mapachone were tested in PC-3 and DU145 cells in the manner described above. 3-allyl-/Mapachone and 3- bromo-/Mapachone were found to be more active than /Mapachone against prostate cells (Table 1 ).

TABLE 1

Antisurvival effect of /Mapachone and its derivatives against human prostate cancer cells

TABLE 1 (Cont.)

Antisurvival effect of Mapachone and its derivatives against human prostate cancer cells

TABLE 1 (Cont.)

TABLE 1 (Cont.)

TABLE 1 (Cont.)

TABLE 1 (Cont.)

TABLE 1 (Cont.)

TABLE 1 (Cont.)

Compound of the present invention.

Example 2 induction of Apoptosis by ^-Lapachone in Human Prostate Cancer Cells

Extensive cell death was observed in proliferating human prostate cancer cells after treatment with /Mapachone. To determine whether this cell death occurs through necrosis or apoptosis, cells were harvested by trypsinization and their genomic DNA was subjected to gel electrophoresis. As shown in Fig. 2A and 2B, /Mapachone induced a typical DNA laddering in human prostate cancer cells, consistent with cell death by apoptosis, a process that is specifically activated in prostate cells after they are deprived of testosterone (Kyprianou, N., et al., Cancer Surv., 1 1 :265-77, (1991 )). This β- lapachone induced apoptosis was observed in every prostate cell line tested, including PC-3, DU145, and LNCaP. To test whether quiescent cells are similarly sensitive to /Mapachone, both PC-3 and DU145 cells were serum starved for 48 hours before drug treatment. Apoptosis was similarly induced in non-proliferating cells (data not shown). To determine the percentage of apoptotic cells, the fraction of sub-G1 cells was quantitated with flow cytometry analysis. As shown in Fig. 2C, Mapachone induced apoptosis in 68% of LNCaP cells by 24 hours after initial treatment with /Mapachone. In PC-3 cells, apoptosis occurs in 62% by 24 hours (data not shown). Apoptosis, as determined by DNA laddering and chromosome condensation, was not detected in β- lapachone treated 21 -MT (human breast epithelial cell line), H520 (human lung carcinoma cell lines), SW1 1 16 (human colon adenocarcinoma), A2780s (human ovary carcinoma) cells. Example 3

Apoptosis Induced by yff-Lapachone is Independent of Expression of p53 and bcl-2.

Expression of bcl-2 has been implicated in the resistance of cancer cells to chemotherapeutic drugs including prostate cells (Berchem, G.J., et al., Cancer Res. 55:735-738 (1995)). To determine whether apoptosis in prostate cancer cells is due to lack of bcl-2 expression, we measured bcl-2 expression by Western blot assay. As shown in Fig. 3, bcl-2 expression is high in PC-3 and low in DU145 cells, which does not correlate with their sensitivity to /Mapachone induced apoptosis. Hela cells with ectopic overexpression of bcl-2 (hela-bcl-2) (Meikrantz, W., et al., supra) was not significantly resistant to /Mapachone in comparison with its parental cell line (IC100 was 16 μM in parental cells, and 32 μM in Hela-bcl-2 cells) (data not shown).

p53 status has been shown to be important for apoptosis in cancer cells provoked by ionizing radiation and chemotherapeutic drugs (Lowe, S., et al., Science 266:807-810 (1994)). p 53 was expressed in DU145 cells, but was not detectable in PC-3 cells (Fig. 3A), although apoptosis was induced in both cell lines (Fig. 2A). β- lapachone treatment did not significantly induce expression of p53 (Fig. 3B). These results suggest that apoptosis induced by ^-lapachone is not dependent on P53 expression. Expression of p21 is not significantly upregulated in prostate cancer cells undergoing apoptosis

(Fig. 3B).

The effect of /Mapachone was also tested in hematopoietic cancer cells. Treatment of HL-60 (p53 -), a human leukemia cell line, with /Mapachone also induced apoptosis. Chromosomal laddering was detectable with 4 hours after /Mapachone treatment (Fig. 4A). At sub- apoptotic doses, /Mapachone induced an increase in the G1 fraction (data not shown) and morphological in HL-60 cells (Fig. 4B).

Example 4 Inhibition of Human Prostate Tumor Growth In Vivo

/Mapachone was tested against human prostate tumor in nude mice. A hormone refractory human prostate adenocarcinoma cell line (PC-3) was inoculated into nude mice. Treatment was initiated when tumor reached 100 to 250 mm³. Without drug treatment (mouse 5), the tumor grew rapidly and the mouse died when the tumor reached about 400 mm³ (Fig. 5). /Mapachone, given at 500 mg/kg, stopped tumor growth (mouse 2, Fig. 5). At intermediate dosage (250 mg.kg, mouse 1 and 3), /Mapachone also showed intermediate inhibition on tumor growth (Fig. 5). Mouse 4 did not develop an appreciable tumor mass under the treatment with /Mapachone (500 mg/kg). No sign of drug toxicity was observed.

In addition, the in vivo efficacy of Mapachone were determined in a widely used prostate tumor model, Dunning R-3327 rat prostate adenocarcinoma. A highly metastatic and malignant clone (RT-3.1 ) of

Dunning R-3327 prostate adenocarcinoma cells were inoculated into Copenhagen rats. Solid tumor masses were formed one week later. Rats in the treatment group received one dose of β-lapachone at 50 mg/kg, I.p. Tumor necrosis was observed 24 hours after drug treatment in every treated rats, but not in untreated controls. Tumor volume was measured. The results are set forth in Figures 6 and 7.

The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention.

Claims

What is claimed is:

1. A method of treating prostate cancer in a human comprising administering to said human an effective treatment amount of a compound of the following formulae I or II:

wherein R and R, are each independently selected from the group consisting of hydrogen, hydroxy, thio, halogen, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted aryl, and substituted and unsubstituted alkoxy, and salts thereof, wherein the dotted double bond between the ring carbons to which R and R^ are bonded represent an optional ring double bond.

2. The method of claim 1 , wherein the prostate cancer is androgen independent prostate cancer.

3. The compound described in claim 1 , wherein at least one of R and R, is hydrogen.

4. The compound described in claim 1 , wherein at least one of R and R, is alkenyl.

5. The compound described in claim 1 , where at least one of R and R, is allyl.

6. The compound described in claim 1 , where the compound of formula I is selected from the group consisting of /Mapachone, 3-allyl- /Mapachone, 3-OH- Mapachone and 3-bromo-/Mapachone.

7. The compound described in claim 6, where the compound of formula I is 3-allyl-/Mapachone.

8. A method of inducing apoptosis of a cell in vivo comprising contacting said cell with an effective amount of a compound of the following formulae I or II:

wherein R and R, are each independently selected from the group consisting of hydrogen, hydroxy, thio, halogen, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted aryl, and substituted and unsubstituted alkoxy, and salts thereof, wherein the dotted double bond between the ring carbons to which R and R, are bonded represent an optional ring double bond.

9. The method of claim 8, wherein the cell is a human prostate cell.

10. The compound described in claim 8, wherein at least one of R and _T is hydrogen.

1 1 . The compound described in claim 8, wherein at least one of R and R_T is alkenyl.

12. The compound described in claim 8, where at least one of R and R, is allyl.

13. The compound described in claim 8, where the compound of formula I is selected from the group consisting of /Mapachone, 3-allyl- /Mapachone, 3-OH-/Mapachone and 3-bromo-/Mapachone.