WO2004052875A1 - Tyrostatin and related anticancer drugs - Google Patents

Abstract

The present invention relates to novel compounds having antineoplastic activity, and methods for their synthesis. The compounds are of the formula (I) wherein R is selected from the group consisting of NH2, NH3Cl, NH-Cys, NH-Gly, NH-Phe, NH-Ser, NH-Trp, NH-Tyr, and NH-Val, and pharmaceutically acceptable salts thereof.

Description

TITLE OF THE INVENTION Tyrostatin and Related Anticancer Drugs RELATED APPLICATION DATA This application is based on and claims the benefit of U.S. Provisional Patent Application No. 60/431,563 filed on December 5, 2002.

INTRODUCTION Financial assistance for this invention was provided by the United States Government via Outstanding Investigator Grant CA44344-05-12 and CA90441-01 awarded by the National Institutes of Health; the Arizona Disease Control Research Commission Contract No. 9815; and in part by the National Cancer Institute, National Institutes of Health Contract NOl-CO-56000; and private contributions. Thus, the United States Government has certain rights in this invention.

FIELD OF THE INVENTION This invention relates to novel compounds having usefulness in the treatment of cancer, believed to be resulting from the compounds' effects on tubulin polymerization. The compounds are the antineoplastic agent S^-methylenedioxy-S^-dimethoxy-S^amino-Z- stilbene and derived amino acid amides

BACKGROUND OF THE INVENTION Combretastatin A-2 (la) represents one of the key antineoplastic and cancer vascular targeting stilbenes, designated combretastatins 1-6, that was isolated from the South African bush willow tree Combretum caffrum. (Lin, CM., et al., Interactions of Tubulin with Potent Natural and Synthetic Analogs of the Antimitotic Agent Combretastatin: A Structure-activity Study, Mol. Pharmacol, 1988, 34, 200-208; Lin, CM., et al, Antimitotic Natural Products Combretastatin A-4 and Combretastatin A-2; Studies on the Mechanism of Their Inhibition of the Binding of Colchicine to Tubulin, Biochem., 1989, 28, 6984-6991; Pettit, G.R., et al, Isolation, Structure, and Synthesis of Combretastatin A-2, A-3, and B-2, Can. J. Chem., 1987, 65, 2390-2396.) Initially, combretastatin A-4 (2a) was selected for preclinical development, based on a spectrum of promising biological properties that were greatly enhanced by a structural modification to sodium combretastatin A-4 phosphate prodrug (2b CA4P). (Pettit, G.R., et al, Antineoplastic Agents 322. Synthesis of Combretastatin A-4 Prodrugs, Anti-Cancer Drug Design, 1995, 10, 299-309; Pettit, G.R., et al, Antineoplastic Agents 389, New Synthesis of Combretastatin A-4 Prodrug, Anti-Cancer Drug Design, 1998, 13, 183-191.) For example, 2b caused a 100-fold decrease in tumor blood flow to the P22 carcinosarcoma in the rat while causing no significant blood flow reduction in normal heart, kidney and small intestine. (Galbraith, S.M., et al, Effects of Combretastatin A4 Phosphate on Endothelial Cell Morphology In Vitro and Relationship to Tumour Vascular Targeting Activity In Vivo, Anticancer Res., 2001, 21, 93-102.) Other recent preclinical findings include the antivascular and antitumor effects produced by 2b against non-small cell lung cancer in vivo. (Boehle, A.S., et al, Combretastatin A-4 Prodrug Inhibits Growth of Human Non-small Cell Lung Cancer in a Murine Xenotransplant Model, Annals of Thoracic Surgery, 2001, 71, 1657-1665.) Most importantly, the initial Phase I human cancer clinical trials of 2b have been quite successful. (Remick, S.C., et al, Phase I Pharmacokinetics Study of Single Dose Intravenous (IV) Combretastatin A-4 Prodrug (CA4P) in Patients With Advanced Cancer, Molecular Targets and Cancer Therapeutics Discovery, Development, and Clinical Validation, Proceedings of the AACR-NCI-EORTC International Congress, Washington, DC, 1999, #16, ρ.4; Rustin, G.J.S., et al, Combretastatin A-4 phosphate (CA4P) Selectively Targets Vasculature in Animal and Human Tumors, Molecular Targets and Cancer Therapeutic Discovery, Development and Clinical Validation Proceedings of the AACR-NCI-EORTC International Congress, Washington, D.C, 1999, #14, ρ.4; Griggs J., et al, Targeting Tumour Vasculature: The Development of Combretastatin A4, The Lancet Oncology, 2001, 2, 82-87.)

The discovery of combretastatin A-4 has prompted synthesis of many structural variations. (Pettit, G.R. et al, Isolation and Structure of the Strong Cell Growth and Tubulin Inhibitor Combretastatin A-4, Experimentia, 1989, 45, 209-211; Gwaltney S.L.II, et al, Novel Sulfonate Analogues of Combretastatin A-4: Potent Antimitotic Agents, Bioorg. & Med. Chem. Lett., 2001, 11, 871-874; Maya A., et al, Design, synthesis and cytotoxic activities of naphthyl analogs of combretastatin A-4, Bioorg. & Med. Chem. Lett., 2000, 10, 2549-2551; Pinney, K.G., et al, Synthesis and Biological Evaluation of Aryl Azide Derivatives of Combretastatin A-4 as Molecular Probes for Tubulin, Bioorg. & Med. Chem., 2000, 8, 2417-2425.) One important variant replaces the 3'-phenol of the B ring with an amine (2c) which subsequently resulted in a water soluble hydrochloride salt (2d) given the code name AC-7739. (Ohsumi, K., et al, Novel Combretastatin Analogues Effective Against Murine Solid Tumors: Design and Structure- Activity Relationships, J. Med. Chem., 1998, 41, 3022-3032.) In 1999 interesting amino acid amide derivatives of 2c that showed anticancer activity against murine Colon 26 adenocarcinoma cells were published. (Ohsumi, K., et al, Synthesis and Antitumor Activities of Amino Acid Prodrugs of Amino-Combretastatins, Anti-Cancer Drug Design, 1999, 14, 539-548.) Both 2d and a serine derivative 2e, code name AC7700, have undergone further evaluation. (Grimaudo, S., et al, Effects of AC7739, a Water-Soluble Amino Derivative of Combretastatin A-4, in Multidrug-Resistant and Apoptosis-Resistant Leukemia Cell Lines, Blood 2001, 98, 445; Ohno, T., et al, Antitumor Effect of Combretastatin A-4 Derivative, AC7700, Against Rat Liver Cancer, Gastroenterology 2001, 120, 2831.)

In the present invention the early study of the combretastatin A-4 3 '-amino derivative has been extended to combretastatin A-2, and have synthesized 2,3-methylenedioxy-5,4¹- dimethoxy^-amino-Z-stilbene (lc) and a selection of amino acid amide derivatives (3a-g) for purposes of an SAR investigation directed at locating promising candidate(s) for preclinical development.

Thus, disclosed herein are methods for the synthesis of certain novel compounds, including 2,3-methylenedioxy-5,4¹-dimethoxy-3 ^amino-Z-stilbene (lc) and a variety of amino acid amide derivatives (3a-g), including compound 3f, denominated by the inventors as Tyrostatin. The compounds disclosed herein are believed to be promising candidates for preclinical development in the treatment of cancer.

SUMMARY OF THE INVENTION

Described herein are several novel compounds and methods for their synthesis. The compounds include S^-Methylenedioxy-S^-dimethoxy-S^amino-Z-stilbene (lc) a d its hydrochloride (Id), as well as the glycine amide 3b, and tyrosine amide 3f, the later compound 3f denominated Tyrostatin. The compounds appear promising for pharmaceutical use, particularly in the treatment of cancer in mammals.

Amine lc, hydrochloride Id, glycine amide 3b, and tyrosine amide 3f had the highest level (GI₅₀ 10^"2 to 10^"3 μg/ml) of activity against a panel of six human and one animal cancer (P388) cell lines. Amine lc and its hydrochloride Id potently inhibited tubulin polymerization by binding at the colchicine site while the amides had little activity against purified tubulin. Nevertheless, most of the amides caused a marked increase in the mitotic index of treated cells, indicating that tubulin was their intracellular target.

The synthesis methods involve the following. The nitro-stilbene intermediate 6a was obtained via a Wittig reaction using phosphonium salt 4 and 3~nitro-4-methoxy-benzaldehyde 5. A one step reduction using zinc in acetic acid produced the synthetic objective, amine lc. The coupling of this amine with various Fmoc amino acids, followed by cleavage of the examine protecting group, resulted in a series of new cancer cell growth inhibitory amides. DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the x-ray molecular structure of amine (lc) with the numbering scheme and thermal ellipsoids drawn at 40% probability level.

Figure 2 illustrates the chemical structures of various related compounds, including compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Novel compounds having potential utility in the pharmaceutical field are described herein. In particular, the compounds appear to have utility in the treatment of cancer and other diseases, such as in the treatment of microbial infections.

The compounds were synthesized using the process herein described, and comprise the following basic structure, wherein R is selected from the group consisting of NH₂, NH₃C1, NH-Cys, NH-Gly, NH-Phe, NH-Ser, NH-Trp, NH-Tyr, and NH-Val,

As used herein, the terms Cys, Gly, Ph, Ser, Trp, Tyr and Val refer respectively to the amino acids Cysteine, Glycine, Phenylalanine, Serine, Tryptophan, Tyrosine, and Valine. It is contemplated that other amino acids could be substituted for the foregoing amino acids in the compound of the invention.

More specifically, compounds of the following structures appear to be most promising:

lc

3b

3f

The following reaction scheme should assist in the understanding of the following written description of the method for synthesizing compounds according to the invention.

Scheme 1

1d

^aReagents and Conditions: (a) n-BuLi, THF, Ar, 0°C; (b) 3-nitro-4-methoxybenzaldehyde (5) in THF, Ar; (c) benzil, benzene, 254 nm lamp, Ar; (d) zinc, acetic acid; (e) 1M HCI in diethyl ether, ethyl acetate.

Scheme 2^a

7a, N-ct -Fmoc-Cys(STrt)

7b, N-oc -Fmoc-Gly

7c, N-α -Fmoc-Phe

7d, N-α-Fmoc-SerCOBu') 7e, N-α-Fmoc-Trp(NBoc) 7f, N-α-Fmoc-TyrfOBu') 7g, N-α-Fmoc-Val

7b b 3b 7c 3c

7g 3g

7d c, b 3d 7e ----- 3e 7f 3f , b

7a 3a

^ε 'Reagents and Conditions: (a) Fmoc-amino acid, PyBrOP, DIPEA, Ar; (b) TAEA, DCM; (c) TFA, DCM; (d) TFA, triethylsilane, DCM.

Synthesis Methods

Chemistry. The key precursor S^-methylenedioxy-S^-dimethoxy-S'-nitro-Z- stilbene (6a) was obtained (4- 6) by employing a Wittig reaction sequence. The required cis-stilbene was obtained in a Z to E ratio of 1.4:1. Photochemical isomerization, converted the trans-stilbene to the cis isomer in 62% yield. (Pettit, G.R, et αl, Isolation, Structure, and Synthesis of Combretastatin A-2, A-3, and B-2. Can. J. Chem, 1987, 65, 2390-2396; Waldeck, D.H., Photoisomerization Dynamics of Stilbenes, Chem. Rev., 1991, 91, 415-436.) Reduction of nitrostilbene 6a with zinc in acetic acid afforded amine lc. (Ohsumi, K., et al, Novel Combretastatin Analogues Effective Against Murine Solid Tumors: Design and Structure-Activity Relationships, J. Med. Chem., 1998, 41, '3022-3032.) Following its purification by column chromatography, the S^-methylenedioxy-S^-dimethoxy-S^amino- Z-stilbene crystallized (1:9, ethyl acetate:hexane), and was subjected to X-ray crystal structure determination.

Treatment of amine lc, in ethyl acetate, with 1 N HCI in diethyl ether yielded the amine hydrochloride (Id). Interestingly, this hydrochloride derivative was found to be essentially insoluble in water. Conversion of amine lc to a selection of Fmoc-amino acid amides was readily accomplished using PyBrOP as the coupling reagent. (Coste, J., et al, Oxybenzotiϊazole Free Peptide Coupling Reagents for N-methylated Amino Acids, Jet. Jett., 1991, 32, 1967-1970.) All Fmoc deprotection was performed using tris(2- aminoethyl)amine. (Carpino, L.A., et al, Tris(2-aminoethyl)amine as a Substitute for 4- (aminomethyl)piperidine in the FMOC/polyamine Approach to Rapid Peptide Synthesis, J. Org. Chem., 1990, 55, 1673-1675.) The acid sensitive side chain protecting groups on Cys, Ser, Trp, and Tyr were removed with trifluoroactic acid in dichloromethane. The trityl group on Cys was deprotected in the presence of the triethylsilane.

Biological Evaluation. The cancer cell growth inhibitory activities of the protected amides and related compounds are summarized in Table 1. Amine (lc) was more active than the 3'-phenol combretastatin A-2. (Pettit, G.R., et al, Isolation, Structure, and Synthesis of Combretastatin A-2, A-3, and B-2, Can. J. Chem., 1987, 65, 2390-2396.) This result mirrors combretastatin A-4 published data reporting increased activity of amine 2c (IC₅o 5.1 nM) over phenol 2a (IC₅o 18.0 nM) in a murine colon 26 Adenocarcinoma Cell Study. (Ohsumi, K., et al, Novel Combretastatin Analogues Effective Against Murine Solid Tumors: Design and Structure-Activity Relationships, J. Med. Chem., 1998, 41, 3022-3032.) Although

10 Oshumi et al. reported the serine derivative (2e) of amine 2c to be the most active among their amino acid derivatives of combretastatin A-4, we found that our serine derivative lc displayed only marginal activity. (Ohsumi, K., et al, Synthesis and Antitumor Activities of Amino Acid Prodrugs of Amino-Combretastatins, Anti-Cancer Drug Design, 1999, 14, 539- 548.) Against the minipanel of cancer cell lines listed in Table 1, amine lc, hydrochloride salt Id, glycine amide 3b, and tyrosine amide 3f provided the strongest cancer cell line growth inhibition, while the remaining amides were 1.5 - 10-fold less active.

Because of the well-documented interactions of combretastatins A-2 (la) and A-4 (2a) with tubulin, the newly synthesized analogs (lc and d, 3a-g, 6a and b, 7a-g) were examined for their effects on both tubulin assembly and the binding of [³H]colchicine to tubulin in comparison with the two natural products. Only amine lc and its hydrochloride salt Id had significant, and essentially identical, effects in either reaction (See Table 2; only data for the more cytotoxic amides are shown here; colchicine binding studies with 6a and b and 7a,c-g were performed with the compounds at 100 μM). Consistent with their overall cytotoxicity, the effects of lc and d on the tubulin-based reactions were intermediate between those of the more potent combretastatin A-4 and the less potent combretastatin A-2.

Because the cytotoxic activities of the amides 3a-g in the human tumor cell lines and of 7b in the murine P388 line were overall not that different from the activities of lc and d in these lines, the inventors investigated to determine whether another cellular target might be involved in their activity. As a preliminary approach to this question, the inventors evaluated these amides, the parent compounds lc and d, and the combretastatins for their effects on the mitotic index of MCF-7 cells. An increase in the mitotic index generally represents an effect at the tubulin level. As shown in Table 2, like the combretastatins (la, 2a) and the amine (lc) and its salt (Id), the amides 3a-g, and 7b caused a marked rise in the mitotic index of the MCF-7 cells (data shown for drug treatment for 12 hours). The values obtained ranged from

11 35-57% mitotic cells, defined as those displaying condensed chromosomes, as compared with 4% in untreated cultures. This suggests that either there is a markedly enhanced uptake of these amides by the cell or that the amides are reconverted to lc by an extracellular or intracellular amidase.

All of the new compounds were screened against the bacteria Stenotrophomonas maltophilia, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Neisseria gonorrhoeae, and the fungi Candida albicans and Cryptococcus neoformans, according to established broth microdilution susceptibility assays. (National Committee for Clinical Laboratory Standards, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved Standard M7-A4, Wayne, PA: NCCLS, 1997; National Committee for Clinical Laboratory Standards, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard M27-A, Wayne, PA: NCCLS, 1997.) In broth micrO^'dilution assays, glycine amide 3b exhibited the broadest antimicrobial spectrum, targeting pathogenic yeasts and bacteria. Conclusions

Present evidence indicates that the 3 ^l -amino counterpart (lc) of combretastatin A-2 and derived glycine amide (3b) and tyrosine amide (3f) correspond to new stilbenes with a very high level of inhibition against a mini-panel of cancer cell lines. The potential here was further supported by the potent inhibition of tubulin polymerization exhibited by amine lc. Experimental Section

Materials and Methods. Ether refers to diethyl ether and Ar to argon gas. All solvents were redistilled, and 3-nitro-4-methoxybenzaldehyde was obtained from Alfa AESAR (Ward Hill, MA). Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrOP), O-Boc-N-α-Fmoc-L-tryptophan, O-tert-butyl-N-α-Fmoc-L-tyrosine, N-α-Fmoc-

12 glycine, and S-trityl-N-α-Fmoc-L-cysteine were obtained from Calbiochem-Novabiochem Corporation (San Diego, CA). Acetic acid, N-butyllithium (2.5 M solution in hexanes), diisopropylethylamine (DIPEA), triethylsilane (TES), and trifluoroacetic acid (TFA) were obtained from Acros Organics (Fisher Scientific, Pittsburgh, PA). All other reagents were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI).

Reactions were monitored by thin layer chromatography using Analtech silica gel GHLF Uniplates visualized under long-wave and short-wave UV irradiation. Solvent extracts of aqueous solutions were dried over anhydrous magnesium sulfate. Where appropriate, the crude products were separated by column chromatography, flash (230-400 mesh ASTM) or gravity (70-230 mesh ASTM) silica from E. Merck.

Melting points are uncorrected and were determined on an Electrothermal 9100 apparatus. Optical rotations were recorded using a Perkin-Elmer 241 polarimeter. The [α]o-

1 9 1 values are given in 10^" deg cm g^" . The IR spectra were obtained using a Mattson Instruments 2020 Galaxy Series FT-IR. The 1H- and ¹³C-NMR spectra were recorded employing Varian Gemini 300, Varian Unity 400, or Varian Unity 500 instruments using a deuterated solvent and were referenced either to TMS or the solvent. HRMS data were recorded with a Jeol LCmate mass spectrometer. Elemental analyses were determined by Galbraith Laboratories, Inc., Knoxville, TN.

S^-Methylenedioxy-S^-dimethoxy-S^nitro-stilbene, Z and E isomers (6a and 6b). The phosphonium bromide 4 (20.1 g, 39.6 mmol) was placed in a flame dried flask under Ar and suspended in THF (300 ml). After being stirred for 45 minutes at room temperature, the solution was cooled to 0°C, and n-butyllithium (15.9 ml, 39.8 mmol) was added. This resulted in the reaction mixture turning a deep red color. Stirring continued for 4 hours at room temperature. 3-Nitro-4-methoxy-benzaldehyde (5, 7.20 g, 3.97 mmol) was dissolved in THF (100 ml), and the solution was added dropwise to the reacting mixture via

13 an addition funnel. The solution color turned from deep red to yellow/green. After being stirred 18 hours, the reaction mixture was cooled to 0°C The reaction was terminated with ethyl acetate, and the solution filtered and concentrated under vacuum to yield a dark green oil. The product was separated by gravity column chromatography on silica gel (4:1, hexane: ethyl acetate) and recrystallized (hexane-acetone) to yield the Z-stilbene 6a (4.09 g, 31%) as a yellow/green solid: m.p. 109-110°C; R_f 0.29 (4:1, hexane:ethyl acetate); 1H-NMR (400 MHz, CDC1₃) δ 3.78 (3H, s, OCH₃), 3.94 (3H, s, OCH₃), 5.96 (2H, s, -CH₂-), 6.41 (3H, m, vinyl H, 2 x ArH), 6.54 (1H, d, J= 12.4 Hz, vinyl H), 6.94 (1H, d, J= 8.4 Hz, ArH), 7.42 (1H, dd, J= 8.8, 2.0 Hz, ArH), 7.76 (1H, d, J= 2.0 Hz, ArH); ¹³C-NMR (400 MHz, CDC1₃) δ

151.7, 148.9, 143.6, 139.6, 134.9, 134.5, 131.1, 130.7, 129.7, 126.6, 126.0, 113.2, 108.5,

102.8, 101.5, 56.5, 56.5; HRMS calcd for Cι₇Hι₆NO₆ [M+H]⁺ 330.0978, found 330.0947. Anal. (Cι₇Hι₅NO₆) C, H, N.

The E-stilbene 6b was isolated from the aforementioned column and recrystallized (hexane-acetone) as an orange solid (2.90 g, 22%): m.p. 165.5-167°C; R_f 0.18 (4:1, hexane:ethyl acetate); 1H-NMR (500 MHz, CDC1₃) δ 3.92 (3H, s, OCH₃), 3.94 (3H, s, OCH₃), 5.97 (2H, s, -CH₂-), 6.62 (1H, s, ArH), 6.70 (1H, s, ArH), 6.81 (1H, d, J = 16 Hz, vinyl H), 6.90 (1H, d, J= 16 Hz, vinyl H), 7.03 (1H, d, J= 9.0 Hz, ArH), 7.58 (1H, dd, J = 9.0, 2.0 Hz, ArH), 7.92 (1H, d, J = 2.0 Hz, ArH); ¹³C-NMR (500 MHz, CDC1₃) δ 152.0, 149.3, 143.6, 139.7, 135.4, 131.7, 131.6, 130.3, 129.2, 124.4, 122.9, 113.7, 107.1, 101.6, 99.8, 56.6, 56.6; HRMS calcd for Cι₇Hι₆NO₆ [M+H]⁺ 330.0978, found 330.0869. Anal. (C₁₇H₁₅NO₆) C, H, N.

Photochemical isomerization of E-stilbene 6b to Z-stilbene (6a). To a stirred solution of the E-stilbene 6b (2.9 g, 8.8 mmol) in benzene (550 ml) was added benzil (9.5 g, 45 mmol, 5.1 eq). After flushing the reaction flask with Ar, the reaction mixture was stirred overnight. The mixture was then irradiated with a 254 nm UV lamp for 5 hours. Upon

14 removal of the benzene in vacuo, the product was separated by gravity column chromatography (4:1, hexane:ethyl acetate) to afford unreacted starting material 6b (0.92 g, 32%) and the desired Z-stilbene 6a (1.8 g, 62%).

S^-Methylenedioxy-S^-dimethoxy-S^amino-Z-stilbene (lc). To a stirred solution of nitrostilbene 6a (1.4 g, 4.3 mmol) in acetic acid (350 ml) was added zinc dust (60 g, <10 μm diameter). After 1.5 hours the solution was filtered under vacuum through celite, and the filtrate was concentrataed under vacuum. The product was separated by flash column chromatography (4:1, hexane:ethyl acetate) and recrystallized (~9:1, hexane:ethyl acetate) to afford colorless crystals lc (1.0 g, 77%): m.p. 93.5-94.5°C; R 0.17 (4:1, hexane:ethyl acetate); 1H-NMR (300 MHz, CDC1₃) δ 3.75 (3H, s, OCH₃), 3.84 (3H, s, OCH₃), 4.25-4.45 (2H, br, NH₂), 5.93 (2H, s, -CH₂-), 6.34 (1H, d, J= 12.0 Hz, vinyl H), 6.41 (1H, d, J= 12.0 Hz, vinyl H), 6.48 (1H, s, ArH), 6.51 (1H, s, ArH), 6.68 (2H, m, 2 x ArH), 6.72 (1H, s, ArH); ¹³C-NMR (400 MHz, CDC1₃) δ 148.5, 146.6, 143.2, 135.5, 134.1, 132.0, 130.0, 129.6, 129.2, 119.5, 115.4, 110.1, 108.3, 103.0, 101.3, 56.3, 55.4; HRMS calcd for Cι₇Hι₈NO₄ [M+H]⁺ 300.1236, found 330.1250. Anal. (Cι₇Hι₇NO₄) C, H, N.

S^-Methylenedioxy-S^-dimethoxy-S'-amino-Z-stilbene hydrochloride (Id). To a stirred solution of amine lc (40 mg, 0.13 mmol) in ethyl acetate (1 ml) was added ethereal HCI (1 M) in excess. A white solid immediately formed, and this was collected and washed with ethyl acetate followed by ether to yield a colorless powder Id (45 mg, quantitative): m.p. 179.5-181°C Anal. (Cι₇H₁₈NO₄Cl) C, H, N.

X-Ray Crystal Structure Determination of Amine lc. Amine lc: A single, plate- shaped X-ray sample (-0.40 x 0.10 x 0.10 mm) was obtained by cleavage from a pale-yellow crystalline cluster grown from a hexane-ethyl acetate solution and mounted on the tip of a glass fiber. An initial set of cell constants was calculated from reflections collected from three sets of 60 frames at 298(2) °K on a Bruker 6000 diffractometer. Cell parameters

15 indicated a monoclinic space group. Subsequent data collection, using 15 second scans/frame and 0.396° steps in ω, was conducted in such a manner as to completely survey a complete hemisphere of reflections. This resulted in >93% coverage of the total reflections possible to a resolution of 0.83 A. A total of 7211 reflections were harvested from the total data collection and final cell constants were calculated from a set of 332 strong, unique reflections from these data. Subsequent statistical analysis of the complete reflection data set using the XPREP¹ program indicated the space group was P2ι. Crystal Data: Cι₇Hι₇NιO₄, a = 11.5714(3) A, b = 5.3425(2) A, c = 12.5632(3) A, β = 105.605 (1)° V = 748.03(4) A³, λ = (Cu Kα) = 1.54178 A, μ(Cu Kα) = 0.783 mm^"1, Pc = 1.329 g cm^"3 for Z = 2 and M_r = 299.32, F(000) = 316.

After data reduction, merging of equivalent reflections and rejection of systematic absences, 2310 unique, observed reflections remained (Rj_nt = 0.1867) and these were used in the subsequent structure solution and refinement. An absorption correction was applied to the data with SADBS. Blessing R. H., An Empirical Corrrection for Absorption Anisotropy, Acta Crystolagraphic, 1995, A51, 33-38. Direct methods structure determination and refinement were accomplished with the SHELXTL NT ver.V5.10 suite of programs. "SHELXTL-NT- version 5.10 (1997)", an integrated suite of programs for the determination of crystal structures from diffraction data, is available from Bruker AXS, Inc., Madison, Wisconsin 53719, USA. This package includes, among others, XPREP (an automatic space group determination program), SHELXS (a structure solution program via Patterson or direct methods), and SHELXL (structure refinement software). All non-hydrogen atoms for amine lc were located using the default settings of that program. Hydrogen atom coordinates were calculated at optimum positions and forced to ride the atom to which they were attached. Anisotropic refinement of the model shown in Figure 1 resulted in a final residual value of 0.0778 for observed data (0.0878 for all data). The difference Fourier map showed

16 insignificant residual electron density, the largest difference peak and hole being +0.324 and - 0.286 e/A³, respectively. Final bond distances and angles were all within acceptable limits. Unless otherwise noted, the following general procedure was employed for synthesis of the Fmoc-protected amino acid amides of amine lc.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(S-Trt-Nα-Fmoc-L-Cys)-amido-Z-stilbene (7a). To a stirred mixture of amine lc (57 mg, 0.19 mmol), S-Trt-Nα-Fmoc-L-Cys (0.15 g, 0.26 mmol, 1.4 eq), and PyBrOP (127 mg, 0.27 mmol, 1.4 eq) in DCM (1 ml) at 0°C under Ar was added DIPEA (0.075 ml, 0.43 mmol, 2.3 eq). The reaction mixture was stirred for 1 hour at room temperature. Ethyl acetate was added, and the solvents were removed in vacuo, leaving a white foam. The product was obtained by flash column chromatography (8:1, DCM:ethyl acetate) as a white foam 7a (0.14 g, 85%): R_f 0.81 (8:1, DCM:ethyl acetate); [α]²² _D -11.5° (c 0.87, CHC1₃); 1H-NMR (500 MHz, CDC1₃) δ 2.71 (IH, m), 2.80 (IH, m), 3.71 (3H, s, OCH₃), 3.72 (3H, s, OCH₃), 3.93 (IH, m), 4.21 (IH, t, J = 7.0 Hz, Fmoc), 4.38 (2H, d, J= 7.0, Fmoc), 5.88 (2H, s, -CH₂-), 6.39 (IH, d, J= 12.0 Hz, vinyl H), 6.44 (IH, s, ArH) 6.44 (IH, d, J= 12.5 Hz, vinyl H), 6.47 (IH, s, ArH), 6.66 (IH, d, J= 9.0 Hz, ArH), 6.98 (IH, dd, J= 8.0, 1.5 Hz, ArH), 7.27 (2H, m, Fmoc), 7.36 (2H, m, Fmoc), 7.58 (2H, d, J = 6.5 Hz, Fmoc), 7.74 (2H, m, Fmoc), 8.23 (IH, d, J= 2.0 Hz, ArH); ¹³C-NMR (500 MHz, CDC1₃) δ 167.8, 155.8, 148.5, 147.1, 147.1, 144.3, 143.7, 143.6, 143.3, 141.3, 134.3, 131.8, 130.0, 129.5, 129.3, 129.0, 128.1, 127.9, 127.8, 127.1, 126.9, 125.0, 124.6, 120.8, 120.7, 120.0, 120.0, 109.6, 108.4, 103.0, 101.3, 67.4, 67.1, 56.3, 55.7, 53.4, 47.1, 33.9; Anal.

(C₅₄Ϊ-₄₆N₂O₇S) C, H, N.

Synthesis of 3'-L-Cys-amide-Z-stilbene 3 a provides the general procedure (except for use of trifluoroacetic acid (TFA) and triethylsilane (TES) with Cys Trt cleavage) for cleavage of the Fmoc-amino acid protecting group.

17 3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(L-Cys)-amido-Z-stilbene (3a). To a stirred solution of S^L-Cys-amido-Z-stilbene (7a, 42 mg, 0.048 mmol) in DCM (1 ml) was added TES (0.5 ml) and TFA (1.5 ml). The reaction mixture was stirred for 20 minutes, and the solution was concentrated under vacuum. The residue was dissolved in DCM (1 ml), and tris-(2-aminoethyl)-amine (TAEA, 0.5 ml) was added. Fifteen minutes later DCM (10 ml) was added, and the mixture was washed with brine (10 ml). The organic solvent was removed in vacuo to yield an oil which was subjected to gravity column chromatography (8:1, DCM:ethyl acetate followed by 9:1, DCM:CH₃OH). The product (3a) was obtained as a colorless foam (5.0 mg, 26%): R_f 0.87 (9:1, DCM-CH₃OH); [α]²⁸ _D -97.3° (c 0.44, CHC1₃); 1H-NMR (300 MHz, CDC1₃) δ 2.90 (IH, m), 3.36 (IH, m), 3.72 (3H, s, OCH₃), 3.83 (IH, m), 3.86 (3H, s, OCH₃), 5.91 (2H, s, -CH₂-), 6.38 (IH, d, J = 12.3 Hz, vinyl H), 6.45 (IH, s, ArH), 6.47 (IH, s, ArH), 6.48 (IH, d, J= 12.6 Hz, vinyl H), 6.72 (IH, d, J= 8.7 Hz, ArH), 6.98 (IH, dd, J= 8.7, 1.8 Hz, ArH), 8.33 (IH, d, J= 1.5 Hz, ArH), 9.90 (IH, s); ¹³C-NMR (500 MHz, CDC1₃) δ 170.9, 148.5, 147.6, 143.3, 134.2, 131.9, 130.0, 129.4, 128.8, 127.1, 124.3, 120.4, 109.7, 108.4, 103.0, 101.3, 56.5, 56.3, 55.8, 38.2; Anal. (C₂₀H₂₂N₂O₅S) C, H, N.

S^-Methylenedioxy-S^^dimethoxy-S^CNα-Fmoc-L-G^-amido-Z-stilbene (7b). Following the general procedure, to Nα-Fmoc-Gly (82 mg, 0.28 mmol, 1.9 eq) in DCM (1 ml) at 0°C under Ar was added DIPEA (0.075 ml, 0.43 mmol, 2.9 eq). Next, PyBrOP (0.123 g, 0.26 mmol, 1.8 eq) and amine lc (44 mg, 0.15 mmol) were added. The product was crystallized from hexane-ethyl acetate (75 mg, 88%): m.p. 67-69°C (dec); R_f 0.40 (8:1, DCM-ethyl acetate); Anal. (C₃₄H₃₀N₂O₇) C, H, N.

S^-Methylenedioxy-S^-dimethoxy-S^L-Gly^amido-Z-stilbene (3b). Following the general procedure, amide 7b (75 mg, 0.13 mmol) in chloroform (6 ml) was treated with TAEA (0.4 ml, 2.7 mmol, 21 eq). After the reaction mixture was stirred for 15 minutes,

18 additional TAEA (0.4 ml, 2.7 mmol, 21 eq) was added. The product was obtained as a colorless oil 3b (38 mg, 83%): R_f 0.38 (9:1, DCM-CH₃OH); HRMS calcd for Cι₉H₂ιN₂O₅ [M+H]⁺ 357.1450, found 357.1489. Anal. (Cι₉H₂₀N₂O₅*0.5H₂O) C, H; N: calcd, 7.67; found 8.19.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(Nα-Fmoc-L-Phe)-amido-Z-stilbene (7c). Amine lc (0.16 g, 0.53 mmol) was mixed with Nα-Fmoc-Phe (0.31 g, 0.80 mmol, 1.5 eq) in DCM (3 ml), DIPEA (0.23 ml, 1.3 mmol, 2.5 eq) and PyBrOP (0.36 g, 0.77 mmol, 1.5 eq). After 40 minutes, a white precipitate was collected and recrystallized from DCM-ethyl acetate to afford the product as a colorless solid (7c, 270 mg, 75%): m.p. 85-87°C; Rf 0.42 (2:1, hexane:ethyl acetate); [α]²⁵ _D -29.4° (c 1.00, CHC1₃); Anal. (C₄ιH₃₆N₂O₇O.5H₂O) C, H, N.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(L-Phe)-amido-Z-stilbene (3c). Cleavage of amide 7c (0.12 g, 0.18 mmol) was achieved in chloroform (3 ml) and TAEA (0.60 ml, 4.0 mmol, 22 eq). After 20 minutes, DCM (12 ml) was added to the reaction mixture, and the solvent was successively washed with brine (4 ml) and phosphate buffer (pH 5.5, 2 x 6 ml). The organic phase was concentrated under vacuum, and the residue was subjected to gravity column chromatography (1:1, hexane:DCM followed by 95:5, DCM:CH₃OH) to yield a colorless oil 3c (74 mg, 94%): R_f 0.76 (95:5, DCM:CH₃OH); [α]²⁵ -67.0 (c 1.00, CHC1₃); Anal. (C₂₆H₂₆N₂O₅) C, H, N.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(O-Bu^t-Nα-Fmoc-L-Ser)-amido-Z-stilbene (7d). To a stirred mixture of lc (0.13 g, 0.43 mmol) and O-Bu'-Nα-Fmoc-Ser (0.25 g, 0.66 mmol, 1.5 eq) in DCM (2 ml) at 0°C under Ar was added DIPEA (0.180 ml, 1.0 mml, 2.3 eq) followed by PyBrOP (0.29 g, 0.63 mmol, 1.5 eq). The mixture was stirred at room temperature for 1 hour. It was then washed with aqueous citric acid (10% by weight), dried with magnesium sulfate, and concentrated under vacuum. DCM and ethyl acetate were

19 added to the residue, and the resulting off-white precipitate was collected. The solvents were removed in vacuo, and the residue was subjected to gravity column chromatography (1:1, DCM: ethyl acetate) to yield a pale yellow oil. The product was precipitated from hexane- ethyl acetate to afford a colorless powder 7d (0.21 g, 73%): m.p. 107.5-109.5°C; R_f 0.80 (1:1 DCM-ethyl acetate); [α]²⁴ _D -3.4 (c 1.01, CHC1₃); Anal. (C₃₉H₄₀N₂O₈) C, H, N.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(L-Ser)-amido-Z-stilbene (3d). To a stirred solution of 7d (110 mg, 0.16 mmol) in DCM (1 ml) at 0°C under Ar was added trifluoracetic acid (1 ml). The mixture immediately turned a magenta color. After being stirred for 1 hour, the solution was concentrated under vacuum. The residue was taken up in DCM (2 ml). TAEA (1 ml) was added, and stirring continued for 10 minutes. DCM (15 ml) was added, and the solution was washed successively with brine (6 ml) and phosphate buffer (pH 5.5, 15 ml). After back extracting the phosphate buffer with DCM, the organic extracts were concentrated under vacuum and subjected to gravity column chromatography (1:1, DCM:ethyl acetate followed by 95:5, DCM:CH₃OH) to afford an oil 3d (25 mg, 41%): R_f 0.33 (95:5, DCM-CH₃OH); [α]²⁶ _D -8.6° (c 0.70, CHC1₃); Anal. (C₂₀H₂₂N₂O₆O.5H₂O) C, H, N.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(N-Boc-Nα-Fmoc-L-Trp)-amido-Z- stilbene (7e). Following the general method, amine lc (51 mg, 0.17 mmol), Nα-Fmoc- Trp(Boc) (0.13 g, 0.25 mmol, 1.5 eq), PyBrOP (0.12 g, 0.26 mmol, 1.5 eq) in DCM (1 ml) and DIPEA (0.075 ml, 0.43 mmol, 2.5 eq) were used to obtain amide 7e as a colorless solid (0.13 g, 93%, reprecipitated from hexane-ethyl acetate): m.p. 118-120°C; R_f 0.71 (8:1, DCM- ethyl acetate); [α]²² _D -22.8° (c 0.65, CHC1₃); Anal. (C₄₈H₄₅N₃O₉) N; C, H: calcd, 71.36, 5.61; found, 71.84, 6.03.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(L-Trp)-amido-Z-stilbene (3e). Amide 7e (90 mg, 0.11 mmol), DCM (0.2 ml) and TFA (1.8 ml) were mixed and stirred for 1 hour at

20 0°C and then condensed in vacuo. Using the general Fmoc deprotection procedure, the residue was taken up in DCM (2 ml), treated with TAEA (1 ml), and stirred for 20 minutes to yield an oil (3e, 11 mg, 20%): R_f 0.31 (8:1, DCM-ethyl acetate); [α]²⁹ _D -87.5° (c 0.44, CHC1₃); HRMS calcd for C₂₈H₂₈N₃O₅ [M+H]⁺ 486.2029, found 486.2008. Anal. (C₂₈H₂₇N₃O₅*H₂O) H, N; C: calcd, 66.79; found 66.31.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(O-Bu^t-Nα-Fmoc-L-Tyr)-amido-Z- stilbene (7f). Amine lc (0.13 g, 0.43 mmol), O-Bu'-Nα-Fmoc-Tyr (0.29 g, 0.63 mmol, 1.5 eq), DCM (2 ml) DIPEA (0.180 ml, 1.0 mmol, 2.3 eq) and PyBrOP (0.29 g, 0.63 mmol, 1.5 eq) were mixed to yield stilbene 7f. The product was isolated by gravity column chromatography (1:1, DCM:ethyl acetate) as a slightly yellow oil. Stilbene 7f was then precipitated from hexane-ethyl acetate as a colorless powder (7f, 0.29 g, 91%): m.p. 95-97°C; R_f 0.78 (1:1, DCM-ethyl acetate); [α]²⁴ _D -9.2 (c 1.15, CHC1₃); Anal. (C₄₅H₄₄N₂O₈) C, H, N.

S^-Methylenedioxy-S^-dimethoxy-S^^-Tyr^amido-Z-stilbene (3f). To a stirred solution of 7f (0.63 g, 0.85 mmol) in DCM (4 ml) was added TFA (4 ml). The mixture was stirred for 20 minutes. Solvent was removed under vacuum, and the mixture was subjected to gravity column chromatography (4:1, DCM:ethyl acetate). The phenol obtained was dissolved in DCM (4.5 ml) to which TAEA (2.7 ml) was added. The mixture was stirred for 20 minutes, washed with brine, and dried with magnesium sulfate. Solvent was removed under vacuum, and the product was purified by gravity column chromatography (4:1, DCM:ethyl acetate followed by 9:1, DCM:CH₃OH). The product was obtained as a colorless oil (3f, 0.35 g, 90%): R_f 0.65 (9:1, DCM-CH₃OH); [α]²⁴ _D -94.9° (c 1.02, CH₂C1₂); Anal. (C₂₆H₂₆N₂O₆-H₂O) C, H, N.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(Nα-Fmoc-L-Val)-amido-Z-stilbene (7g). To a stirred mixture of amine lc (49 mg, 0.16 mmol), Nα-Fmoc-Val (93 mg, 0.27 mmol, 1.7 eq), and PyBrOP (114 mg, 0.25 mmol, 1.5 eq) in DCM (1 ml at 0°C under Ar) was added

21 DIPEA (0.075 ml, 0.43 mmol, 2.6 eq). The mixture was stirred for 1 hour at room temperature, and the solvent was removed under vacuum to yield an oil. The product was precipitated from ether and collected as a colorless solid 7g (93 mg, 91%): m.p. 203-204.5°C; R_f 0.28 (2:1, n-hexane: ethyl acetate); [α]²³ -36.6° (c 1.01, CHC1₃); Anal. (C₃₇H₃₆N₂O₇Η₂O) C, H, N.

3,4-Methylenedioxy-5,4¹-dimethoxy-3¹-(L-Val)-amido-Z-stilbene (3g). Following the general method amide 7g (93 mg, 0.15 mmol) in DCM (5 ml) and TAEA (0.75 ml, 5.0 mmol, 33 eq) were mixed to yield an oil, which was subjected to gravity column chromatography (4:1, DCM:ethyl acetate). The product was obtained as a colorless oil (3g, 59 mg, 98%): R_f 0.26 (4:1, DCM-ethyl acetate); [α]²⁵ -39.5° (c 0.43, CHC1₃); Anal. (C₂₂H₂₆N₂O₅ ^«0.5 H₂O) C, H, N.

Assays for evaluation of tubulin polymerization and the binding of [3H] colchicine to tubulin were performed as described previously. Verdier-Pinard, P., et al, Structure-activity Analysis of the Interaction of Curacin A, the Potent Colchicine Site Antimitotic Agent, with Tubulin and Effects of Analogs on the Growth of MCF-7 Breast Cancer cells, Mol Pharmacol, 1998, 53, 62-76. The mitotic index of drug-treated MCF-7 cells was obtained by examining cells grown on Lab-Tek II Chamber Slides obtained from Nalge Nunc International. The cells were maintained at 37°C and 5% CO₂ in RPMI medium supplemented with 10% fetal bovine serum and 2 mM glutamine. Cells were seeded at 5000/chamber the day before exposure to drugs and grown an additional 12 hours (final dimethylsulfoxide concentration, 1%). The cells were fixed in a solution containing 8% formaldehyde, 50 mM 1,4-piperzineethanesulfonate (pH 6.9 with NaOH), 5 mM MgCl₂, and 5% dimethylsulfoxide for 45 min. The slide was washed twice with phosphate-buffered saline (pH 7.4), and the DNA was fluorescently labeled with 2.5 μM 4',6-diamidino-2- phenylindole. Coverslips were mounted on the Citifluor AF1 antifade agent obtained from

22 Marivac, Ltd. The slides were examined with a Nikon E800 epifluorescence microscope equipped with an appropriate filter, and cells with condensed chromosomes were quantitated. The following Tables illustrate testing of the compounds of the invention.

Table 1 Human cancer cell line GI₅o (μg/ml) and murine P388 lymphocytic leukemia cell line inhibitory activity ED₅0 (μg/ml) of the 3 ^substituted stilbenes.

23 Table 2 Effects of combretastatin A-2 derivatives on tubulin and on mitosis in MCF-7 breast cancer cells

Compound Inhibition of tubulin assembly" Inhibition of colchicine binding^b Mitotic index⁰

(IC₅₀, μM, ± SD) (% inhibition) (% mitotic cells ± SD)

Inhibitor concentration (μM)

2 5 20 100

Combretastatin A-4 2.1 ± 0.1 96 98 57 ± 6

Combretastatin A-2 4.0 ± 0.2 79 92 57 ± 4 lc 3.1 ± 0.05 84 94 48 ± 5

Id 2.9 ± 0.05 87 95 47 ± 6

3a > 40 15 42 ± 4

3b > 40 31 66 52 ± 7

3c > 40 16 52 ± 4

3d > 40 22 64 35 ± 7

3e > 40 16 57 ± 12

3f > 40 19 44 ± 1

3g > 40 14 48 ± 1

7b > 40 8 56 ± 11

"Reaction mixtures contained 10 μM tubulin (1.0 mg/mL) and varying drug concentrations. A drug-tubulin preincubation in the absence of GTP preceded the assembly reaction. Verdier-Pinard, P., Lai, J.-Y., Yoo, H.-D., Yu, J., Marquez, B., Nagle, D. G., Nambu, M., White, J. D., Falck, J. R., Gerwick, W. H., Day, B. W., and Hamel, E. Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells. Mol. Pharmacol., 1998, 53, 62- 76. Reaction mixtures contained tubulin at 1.0 μM, [³H]colchicines at 5.0 μM, and the inhibitory compounds at the indicated concentrations. Verdier-Pinard, P., Lai, J.-Y., Yoo, H.-D., Yu, J., Marquez, B., Nagle, D. G., Nambu, M., White, J. D., Falck, J. R., Gerwick, W. H., Day, B. W., and Hamel, E. Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells. Mol. Pharmacol., 1998, 53, 62-76.

°MCF-7 cells were treated for 12 h with ten times the GI₅o concentrations shown in Table II, except that the concentrations of combretastatins A-4 and A-2 and of 7b were 50 nM, 1.0 μM, and 1.0 μM, respectively. Cells with condensed chromosomes were quantitated as mitotic cells. The mitotic index in untreated cells was 3.6%. See text for further details.

24

Claims

CLAIMS What we claim is:

1. A compound having the formula

wherein R is selected from the group consisting of NHRi, wherein Ri selected from the group consisting of H, Cl , or an amino acid, and pharmaceutically acceptable salts thereof.

2. A compound having the formula

wherein R is selected from the group consisting of NH , NH₃C1, NH-Cys, NH-Gly, NH- Phe, NH-Ser, NH-Trp, NH-Tyr, and NH-Val, and pharmaceutically acceptable salts thereof.

3. The compound of claim 2, wherein R is selected from the group consisting of NH₂, NH₃CI, NH-Gly and NH-Tyr.

4. The compound of claim 2, wherein R= NH .

5. The compound of claim 2, wherein R= NH₃C1.

25

6. The compound of claim 2, wherein R=NH-Gly.

7. The compound of claim 2, wherein R=NH-Tyr.

8. A method for treating neoplastic disease comprising administering to a human subject a pharmaceutically effective amount of one or more of the compounds of claim 1.

9. A method for treating neoplastic disease comprising administering to a human subject a pharmaceutically effective amount of one or more of the compounds of claim 2.

10. A method for treating neoplastic disease comprising administering to a human subject a pharmaceutically effective amount of one or more of the compounds of claim 3.

10. A pharmaceutical composition comprising one of the compounds of claim 1.

11. A pharmaceutical composition comprising one of the compounds of claim 2.

13. A pharmaceutical composition comprising one of the compounds of claim 3.

26