WO2006036743A2 - Halocombstatins and methods of synthesis thereof - Google Patents

Abstract

The invention relates to novel compounds denominated halocombstatins. The halocombstatins are derivatives of combretastatin A-3, and include compounds that exhibit cancer growth cell inhibition against a panel of human cancer cell lines and the murine P388 leukemia, as well as activity as inhibitors of tubulin polymerization and inhibitors of the binding of colchicine to tubulin.

Description

TITLE OF INVENTION [0001] Halocombstatins and Methods of Synthesis Thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0002] This application is based on and claims the priority to U.S. Patent

Application No. 10/948,926 filed on September 24, 2004 and to U.S. Provisional Patent Application No. 60/612,888 also filed on September 24, 2004, the disclosure of both which are incorporated by reference thereto in their entirety.

STATEMENTREGARDINGFEDERALLYSPONSEREDRESEARCH

[0003] Financial assistance for this invention was provided by the United

States Government, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Department of Health and Human Services Outstanding Investigator Grant Numbers CA44344-05-12; R01-CA90441-01; and ROl CA090441-03-041; the Arizona Disease Control Research Commission contract Number 9815; and private contributions. Thus, the United States Government may have certain rights in this invention.

FIELD OF THE INVENTION

[0004] This invention relates to novel compounds having utility in the treatment of cancer and/or as antimicrobials.

BACKGROUND OF THE INVENTION

[0005] Pharmaceutical agents to treat cancer and/or tumors are widely sought.

Antiangiogenesis agents are being pursued as a promising antitumor therapeutic agents. Combretastatin A-4 is one such antiangiogenesis agent. Studies have demonstrated that combretastatin A-4 disrupts the microtubules of human umbilical vein endothelial cells (HUVEC) in culture. It has also been shown that the tubulin-binding properties shown in cell-free systems are retained when the compound enters cells, and that tubulin binding is a significant component of biological activity. [0006] The African Bush Willow Combretum cajfrum has proved to be a very important source of cancer cell growth inhibitory constituents named combretastatins. The most potent of these constituents is combretastatin A-4 (Ia, "CA-4"), and its sodium phosphate derivative (Ib, "CA-4P") was advanced to Phase I human cancer clinical trials in 1998. (Rernick, S. C, et al, (1999) Phase I Pharmacokinetics Study of Single Dose Intravenous (IV) Combretastatin A-4 Prodrug (CA4P) in Patients (pts) with Advanced Cancer, Molecular Targets and Cancer Therapeutics Discovery, Development, and Clinical Validation, Proceedings of the AACR-NCI-EORTC International Congress, Washington, DC, #16, p. 4.) Overall results continue to be promising, and human cancer Phase II and combination Ib trials are currently underway.

[0007] Antivascular, antiangiogenesis and general antimetastatic activities of

CA4P as well as its synergistic utility in combination with other anticancer drugs, radioimmunotherapy and hyperthermia are all areas of active research interest, (see Griggs, J., et ah, Combretastatin A-4 Disrupts Neovascular Development in Non-Neoplastic Tissue, British J. of Cancer 2001, 84, 832-835; Folkman, J., Angiogenesis-Dependent Diseases, Seminars in Oncology 2001, 28, 536-542; Kruger, E. A. et al., Approaches to Preclinical Screening of Antiangiogenic Agents, Seminars in Oncology 2001, 28, 570-576; Jin, X., et al., Evaluation of Endostatin Antiangiogenesis Gene Therapy in vitro and in vivo, Cancer Gene Therapy 2001, 8, 982-989; Vacca, A., et al., Bone Marrow Angiogenesis in Patients with Active Multiple Myeloma, Seminars in Oncology 2001, 28, 543-550; Rajkumar, S. V., et al., Angiogenesis in Multiple Myeloma, Seminars in Oncology 2001, 28, 560-564; Griggs, J., et al, Potent Anti-metastatic Activity of Combretastatin A-4, Int. J. Oncol. 2001, 821-825; Pedley, R. B. et al, Eradication of Colorectal Xenografts by Combined Radioimmunotherapy and Combretastatin A-4 3-OPhosphate, Cancer Research 2001, 61, 4716-4722; Eikesdal, H. P., et al, Tumor Vasculature is Targeted by the Combination of Combretastatin A-4 and Hyperthermia, Radiotherapy and Oncology 2001, 61, 313-320.)

[0008] Several of the compounds of the present invention are particularly concerned with treatment of thyroid gland cancer. By 2002, some 20,000 people in the United States were diagnosed with carcinoma of the thyroid gland; of these the distribution was about 80% papillary and 14% follicular differentiated carcinomas derived from follicular epithelial cells producing thyroid hormone. Of the remaining thyroid malignancies, about 4% were medullary carcinoma (neuroendocrine) and 2% of the exceptionally aggressive anaplastic carcinoma (median survival 4-5 months and a near 100% lethal outcome). Significantly, the incidence of both follicular and anaplastic carcinomas are elevated in geographic areas of iodine deficiency. Radiation exposure represents the most general risk factor for thyroid cancer. In addition, excess production of the pituitary hormone thyroid- stimulating hormone (THS), which is very important in regulating thyroid gland growth and function, may be important in the etiology of thyroid cancer. Previously used clinical treatments for thyroid cancer include surgery, suppression of THS, ¹³¹I-radiotherapy, and anticancer drugs. But in 2002, another 1,300 victims of thyroid cancer in the U.S. died, emphasizing the great need for more routinely effective anticancer drugs.

SUMMARY OF THE INVENTION

[0009] The present invention relates to novel compounds constituting modifications of combretastatin A-3 (3a) and its phosphate prodrug (3b), wherein the 3- hydroxy group or the 3 -hydroxy and 5 -hydroxy groups are replaced with a halide. Representative halides are fluorine, chlorine, bromine and iodine. Salts of the novel compounds are also disclosed herein. Also described herein are phosphate ester derivatives of the 3-fluoro, 3-chloro, 3-bromo and 3-iodo-stilbenes.

Compounds of the invention comprise:

wherein X is F, Cl, Br or I;

wherein X is F, Cl, Br or I, and R is a metal cation such as Na, Li, K, Cs, Rb, Ca, Mg or is morpholine, piperidine, glycine-OCH₃, tryptophan-OCH₃ OrNH(CH₂OH)₃; and

wherein X is F, Cl, Br or I, and Z is a metal cation such as Na, Li, K, Cs, Rb, Ca₅ Mg or is morpholine, piperidine, glycine-OCH₃, tryptophan-OCH₃ or NH(CH₂OH)₃.

[0010] Several of the compounds of the invention exhibit greatly enhanced

(>10-100x) cancer cell growth inhibition, as compared to prior art combretastatin compounds such as CA-4 and CA-3, against a panel of human cancer cell lines and the murine P388 leukemia. The iodo compounds appear to show particular promise in the treatment of thyroid cancers. The compounds of the present invention exhibit inhibitor activity of tubulin polymerization and binding of colchicine to tubulin. In addition, several of the compounds exhibit antimicrobial properties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows the structural formulas of several prior art compounds.

[0012] Figure 2 shows the reaction scheme for synthesizing some of the compounds of the present invention, including structural formulas for the compounds of the invention.

[0013] Figure 3 shows a continuation of the reaction scheme of Figure 2.

[0014] Figure 4 shows the reaction scheme for synthesizing some of the compounds of the present invention, including structural formulas for the compounds of the invention.

[0015] Figure 5 shows photographs of results of the cord formation assay.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The concept of antiangiogenesis as a therapeutic approach for the treatment of cancer, particularly tumors, is being actively pursued as a promising strategy. The compound combretastatin A-4 has previously been demonstrated to disrupt the microtubules of human umbilical vein endothelial cells (HUVEC) in culture. Those studies confirmed that the tubulin-binding properties shown in cell-free systems are retained when the compound enters cells, and that tubulin binding is a significant component of the biological activity.

[0017] Thus, an object of the present invention is to provide new compounds that may be useful as tubulin binding agents. [0018] A further object of the invention is to provide compounds that possess antiangiogenesis properties.

[0019] Yet another object of the invention is to provide compounds for use as therapeutic agents for the treatment of mammals, including humans, afflicted with cancer, particularly tumors.

[0020] Still a further object of the invention is to provide compounds for use as antimicrobials.

[0021] Results and Discussion

[0022] Preparation of the stilbenes of the present invention was accomplished as described in detail herein. The reaction sequence was initiated by protection of isovanillin as the tert-butyldiphenylsilyl ether 4. Benzaldehyde 4 was reduced using sodium borohydride to benzyl alcohol 5, followed by conversion to phosphonium bromide 6. Condensation of Wittig intermediate 6 with the respective halo-aldehyde using n-butyllithium in THF led to silyl group protected stilbenes 7-10. Subsequent deprotection (Figure 3 a) with tetrabutylammonium fluoride afforded 3-halo-stilbenes 11-14. The Z isomers 11a, 13a and 14a were phosphorylated using dibenzylphosphite, diisopropylethyl-amine, N,N- dimethylamino-pyridine and carbon tetrachloride in acetonitrile to provide bisbenzyl phosphates 15-17. Debenzylation of phosphate esters 15-17 was achieved using trimethylsilybromide followed by the corresponding base to produce phosphates 18-20. (See Pettit, G. R., et ah, Antineoplastic Agents 440. Asymmetric Synthesis and Evaluation of the Combretastatin A-I SAR Probes (1S,25) and (li?,2i?)-l-2-Dihydroxy-l-(2',3'-dihydroxy-4'- methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)- ethane, J. Nat. Prod. 2000, 63, 969-974; Pettit, G. R., et ah, Antineoplastic Agents 460. Synthesis of Combretastatin A-2 Prodrugs, Anticancer Drug Design 2001, 16, 185-194; Pettit, G. R., et a Antineoplastic Agents 463. Synthesis of Combretastatin A-3 Diphosphates, Anticancer Drug Design 2000, 15, 397-404.; Ladd, D. L., et ah; A New Synthesis of 3-Fluoroveratrole and Z-Fluoro-3,4 Dimethoxy Benzaldahyde, Synth. Commun. 1985, 15, 61).

[0023] Compared to the related combretastatins, the new halo-stilbenes or halocombstatins shown in Table I as compounds 11a through 20a and Table Ia as compounds , all exhibited very strong inhibition of cancer cell growth. The three stilbenes (11a, 13a, 14a) converted to phosphate salts all retained strong activity and demonstrated markedly better aqueous solubility than their 3-halo-stilbene precursors. The E geometrical isomers evaluated appeared in vitro to be much less effective as inhibitors of cancer cell growth.

[0024] The ability of the iodo-stilbene series to inhibit cancer cell growth was specifically examined and compared to combretastatin A-3 and combretastatin A-4 (Table Ia). The iodo-stilbenes converted to the phosphate salts all retained strong activity and, as expected, demonstrated markedly better aqueous solubility (Table Ia) than the iodo-stilbene precursors.

[0025] In a separate study (see Table 7) employing two lines of human anaplastic thyroid carcinoma, KAT-4 and SW1736, the potential for increased specificity was evaluated for some of the new compounds. Diiodocombstatin 22a proved to be slightly more inhibitory than iodocombstatin (14a, Table 7). The potassium phosphates 20c and 24c produced similar activities. Although the growth of the human thyroid carcinoma KAT-4 cells was significantly reduced, SWl 736 cells appeared somehow resistant to these compounds and, in general, no increased specificity was noted. Antiangiogenesis as a therapeutic approach is now actively pursued as a promising antitumor strategy.

[0026] Because of their potent cytotoxicity, the four halocombstatins (Ha,

12a, 13a, and 14a) were compared to combretastatin A-4 (Ia) for inhibitory effects on tubulin polymerization and on the binding of [³H] colchicine to tubulin. The results of this comparison are shown in Table H These experiments demonstrate that the five compounds are essentially identical in their apparent interactions with tubulin. The four halocombretastatins inhibited the polymerization reaction with IC₅₀ values of 1.5-1.6 μM:M, versus an IC₅₀ value of 1.8 μM :M for CA4 (Ia). The minor differences between the compounds were within experimental error as indicated by the standard deviations.

[0027] Similarly, all four cw-stilbenes were highly potent inhibitors of the colchicine binding assay. When present at a concentration one fifth of that of [³H] colchicine but equimolar to the tubulin concentration, binding of the radio labeled ligand was inhibited by 75-89% (note that the lowest and highest inhibitory effects were observed with stilbenes Ha and 13 a, which were the two compounds that displayed the greatest inhibitory effects in the polymerization assay). In an earlier study, combretastatin A-3 (3a), with a hydroxyl substituent instead of the methoxy group or a halogen at position C-3 in the A ring, was found to be about half as active as CA4 (Ia) as an inhibitor of tubulin assembly, about one fifth as active as an inhibitor of colchicine binding to tubulin, and about one seventh as active as an inhibitor of cell growth. (See Lin, C. M., et al, Interactions of Tubulin with Potent Natural and Synthetic Analogs of the Antimitotic Agent Combretastatin: A Structure-activity Study, MoI Pharmacol, 1988, 34, 200-208). A related finding is that elimination of the C-3 substituent entirely, by replacing it with a hydrogen atom, results in about a 7-fold reduction in inhibitory effect on polymerization and complete loss of cytotoxic activity. (See Cushman, M., et al, Synthesis and Evaluation of (Z)-l-(4-methoxyphenyl)-2-(3,4,5- trimethoxyphenyl)ethane as Potential Cytotoxic and Antimitotic Agents, J. Med. Chem. 1992, 35, 2293-2306.)

[0028] Thus, while not intending to be bound by this theory, it appears the optimal activity observed with CA4 (Ia) and the novel halocombstatins of the present invention requires a C-3 substituent of some size, where the fluorine atom may represent a minimum. Therefore, it seems unlikely that the predominant effect of the substituent results from direct enhancement of the interaction of ligand with protein. The A-ring substituents most likely cause the active czs-stilbenes to assume with greater probability a conformation that favors the drug-tubulin interaction. (See Hamel, E.; Evaluation of Antimitotic Agents by Quantitative Comparisons of Their Effects on the Polymerization of Purified Tubulin, Cell Biochem. Biophys., In Press.)

[0029] Tubulin polymerization was evaluated by turbidimetry at 350 nm using

Beckman DU7400/7500 spectrophotometers as described in detail elsewhere. (See National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A. Wayne, PA: NCCLS, 1997.) Varying concentrations of drug were preincubated with 10 μM :M (10 mg/mL) purified tubulin (See Hamel, E., et al, Separation of Active Tubulin and Microtubule-associated Proteins by Ultracentrifugation and Isolation of a Component Causing the Formation of Microtubule Bundles, Biochemistry 1984, 23, 4173-4184). Samples were chilled on ice, GTP (0.4 mM) was added, and polymerization was followed at 30°C. The parameter measured was extent of the reaction after 20 minutes. Colchicine binding was measured as described in detail previously. Reaction mixtures contained 1.0 μM :M tubulin, 5.0 :M [³H]colchicine (from Dupont), and inhibitor at 1.0 μM :M. Incubation was for 10 minutes at 37°C.

[0030] The inventors have also demonstrated the ability of halocombstatins

11a and 12a to disrupt microtubules in human umbilical vein endothelial cells (HUVEC). HUVECs were isolated according to methods know to one of skill in the art (see Jaffe, E. A. et ah, Culture of Human Endothelial Cells Derived From Umbilical Veins. Identification by Morphologic and Immunologic Criteria, J. Clin. Invest. 1973, 52, 2754-2756.)

[0031] In a further detailed series of experiments, compound 11a

(fluorocombstatin) was further evaluated against HUVECs in vitro. These cells showed significant sensitivity to the fluorocombstatin (lla): ED50 0.00025 μg/mL. Cords length as well as junction numbers were markedly reduced at both 0.01 and 0.001 μg/mL compared to untreated controls. Such activity against endothelial cells is significant, as endothelial cells are known to play a central role in the angiogenic process. For both the mono and diiodocombstatins (14a and 22a), cord lengths as well as junction numbers were markedly reduced at 0.001 μg/ml (see Table III and Figure 5B and 5D) and similar to control at 0.0001 μg/ml (Figure 5C and 5E) compared to untreated controls (Figure 5A). At 0.001 μg/ml, 20c (Figure 5F) showed a slightly larger reduction in the size of the cords compared to 14c (Figure 5G). Such inhibitory activity against the HUVEC endothelial cells is of considerable interest as these cells play a central role in the angiogenic process.

[0032] The halocombstatins of the present invention appear to also have antimicrobial properties. More specifically, they appear to have antifungal and/or antibacterial properties. Antimicrobial evaluation of the halocombstatins involved susceptibility testing performed by the reference broth microdilution assay. The antimicrobial activities of the halocombstatins were very similar, targeting Gram-positive bacteria and the pathogenic fungi Cryptococcus neoformans, and results are shown in Table IV. The sodium phosphate derivative (16a) of fluorocombstatin (Ha) did not retain significant antimicrobial activity.

[0033] Similarly, the inventors have previously shown that combretastatin A-3 but not its sodium phosphate prodrug inhibited growth of the pathogenic fungus Cryptococcus neoformans. (See Pettit, G. R., et al., Antineoplastic Agents 463. Synthesis of Combretastatin A-3 Diphosphates, Anticancer Drug Design 2000, 15, 397-404.) To determine the antimicrobial activity of the present compounds, susceptibility testing was performed by the reference broth microdilution assay. (See National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard M7-A5. Wayne, PA: NCCLS, 2000. National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A. Wayne, PA: NCCLS, 1997.) The antimicrobial activities of the halocombretastatins of the present invention were very similar, targeting Gram-positive bacteria and Cryptococcus neoformans. This is illustrated in further detail in Table IV. Also in Table V it is shown that two of the diiodocombstatins, 22a and 22b, were active against M. luteus, and diiodocombstatins 24a- 24h were very active against N. gonorrhoeae (Table V). Iodocombstatins 2Od, 2Og, and 2Oh were also active against N. gonorrhoeae, and iodocombstatin 14a had marginal activity against the pathogenic yeast Cryptococcus neoformans (Table V). Thus, as can be seen, several of the novel compounds of the present invention appear to have potential as antimicrobial agents, such as antifungals and antibacterials.

[0034] A selection of our iodo and diiodocombstatins were evaluated against human umbilical vein endothelial cells (HUVEC) in vitro (Table VI). These cells showed significant sensitivity to all of these compounds. The most active iodocombstatin 14a: EDs₀ 0.000040 μg/ml was more active than the diiodocombstatin 22a and a similar pattern was observed between phosphates 20c and 20a.

[0035] Table VII shows the inhibition values of our iodo and diiodocombstatins on Human Anaplastic Thyroid Carcinoma cell lines. The inhibition values (GI₅₀) are expressed in μm/ml.

[0036] Experimental Section

[0037] Materials and Methods. All solvents (ether refers to diethyl ether) and reagents were obtained from commercial sources (Acros Organics, Sigma- Aldrich Co., Alfa Aesar, City Chemicals or Lancaster Synthesis, Inc.). The 3-iodo-4,5- dimethoxybenzaldehyde was purchased from Lancaster Synthesis. Solvents were redistilled. Solvent extracts of aqueous solutions were dried over anhydrous magnesium sulfate. Gravity column chromatography was performed using silica gel from VWR Scientific 70-230 mesh) or from Merck (230-400 mesh). Analtech silica gel GHLF plates were employed for TLC.

[0038] AU melting points were determined with an electrochemical digital melting point apparatus, Model 9100 or IA-9200, and are uncorrected. All ¹H- and ¹³C-NMR spectra were recorded employing Varian Gemini 300 MHz or Varian Unity 400 or 500 MHz instruments with CDCl₃ (tetramethylsilane internal reference) as solvent unless otherwise noted. The ³¹P-NMR spectra were obtained in CDCl₃ or D₂O solution with 85% H₃PO₄ as an external standard employing a 400 MHz or Unity 500 MHz instrument. Chemical shifts are reported in ppm downfield from tramethylsilane as an internal standard in CDCl₃ or where noted in D₂O. High resolution mass spectra were obtained with a Kratos Ms-50 instrument (Midwest Center for Mass Spectroscopy, University of Nebraska-Lincoln) or in the Cancer Research Institute at Arizona State University with a Jeol LCmate instrument. Elemental analyses were determined by Galbraith Laboratories, Inc., Knoxville, TN.

[0039] General Procedure for Synthesis of Dimethoxyhalobenzaldehydes.

[0040] 3-Fluoro-4,5-dimethoxybenzaldehyde. To a stirred solution prepared from 100 mL of DMF and 5-fluorovanillin (lit 1.0 g, 5.88 mmol). After 15 minutes, iodomethane was added, and stirring at room temperature continued for 16 hours. The reaction was terminated by the addition of water, the mixture was extracted with hexane (3 x 100 ML), and solvents were removed in vacuo. Purification by flash chromatography on a column of silica gel using hexane-ethyl acetate (4:1) as eluent afforded a colorless solid (Ig, 93% yield); mp 51-53 °C (Lit¹⁷ mp 52-53°C) ^l H-NMR (300 MHz, CDCl₃) δ 3.94 (s, 3H), 4.05 (s, 3H), 7.24 (s, IH), 7.26 (s, IH), 9.82 (s, IH).

[0041] 3-Chloro-4,5-dimethoxybenzaldehyde. The preceding reaction was repeated with 5-chlorovanillin (1Og, 54 mmol) to give this compound, which was isolated as set forth in the preceding experiment to afford a colorless solid (10.4 g, 97% yield); mp 88-90 °C (Lit¹⁷ mp 87-89⁰C); ^l H-NMR (300 MHz, CDCl₃) δ 3.95 (s, 3H), 3.96 (s, 3H), 7.36 (d, J= 1.5 Hz, IH), 7.50 (d, J= 1.5 Hz, IH), 9.85 (s, IH).

[0042] 3-Bromo-4,5-dimethoxybenzaldehyde. The experiment was repeated with 5 -bromo vanillin (10 g, 43.3 mmol) as described for the preceding aldehydes to give compound (6) which was separated by flash chromatography on a column of silica gel using hexane-ethyl acetate (9:1) as eluent to afford a colorless solid (8 g, 75% yield); mp 64-65°C; ¹H-NMR (300 MHz, CDCl₃) δ 3.94 (s, 3H), 3.95 (s, 3H), 7.40 (d, IH, J= 1.8 Hz), 7.65 (d, IH, J= 1.8 Hz), 9.85 (s, IH).

[0043] 3,5-diiodo-4-methoxybenzaIdehyde. A solution of 3,5 diiodo-4- hydroxybenzaldehyde (5g, 13.37 mmol) in anhydrous DMF (50 ml) was cooled to 0°C and sodium hydride (0.64 g, 16 mmol, 60 % dispersion in mineral oil) was slowly added. Iodomethane was then added and stirring was continued at rt in the dark for 19 hrs. The reaction was terminated by the addition of water (50 mL) and extracted into EtOAc-hexane (1:1, 3 x 50 mL). The combined organic extract was dried, filtered and concentrated. The residue was separated by column chromatography on silica gel using EtOAc-hexane (1:9) as eluent mp 121-123 °C, (lit¹¹ mp 124 ⁰C) to yield a colorless solid (4.2g, 80%) which was recrystallized from hexane to give colorless crystals: ¹H-NMR (300 MHz, CDCl₃) δ 3.93 (s, 3 H), 8.27 (s, 2 H), 9.81 (s, 1 H); and ¹³C-NMR (75 MHz, CDCl₃) δ 60.35, 90.72, 140.65, 140.71, 187.51, 187.56. Anal calcd for C₈H₆I₂O₂ C₅ 24.77; H, 1.56. Found C, 24.86; H, 1.58%.

[0044] 3-Iodo-4,5-dimethoxybenzaldehyde was obtained from Sigma-

Aldrich Chemical Company.

[0045] 3-0-tert-ButlydiphenyIsiloxy-4- methoxybenzyltriphenylphosphonium bromide (6). To 400 mL of dry dichloromethane was added benzyl alcohol 5 (84 g, 214 mmol) (Pettit, G. et al., Antineoplastic Agents 463, Synthesis of Combretastatin A-3 Diphosphates. Anticancer Drug Design 2000, 15, 397-404) and phosphorous tribromide (10 mL, 106 mmol, 0.5 eq). The reaction mixture was allowed to stir for 16 hours, and was terminated by the addition of 10% NaHCO₃, and the product was extracted with dichloromethane. The solvent was removed (in vacuo), the resulting benzyl bromide was dissolved in 500 mL of toluene, and triphenylphosphine (62 g, 236 mmol, 1.1 eq) was added. The mixture was heated at reflux for 1 hour and stirred at RT for 15 hours. The precipitate was collected and triturated with ether to afford 132 g of phosphonium salt, in 86% yield; ¹H-NMR (300 MHz CD₃OD) δl.00 (s,9H), 3.51 (s,3H), 4.69 (d, 2H, J =17.4 Hz), 6.34 (dt, IH, J = 2.4, 8.1 Hz), 6.59 34 (d, IH, J = 8.1 Hz), 6.65 34 (t, IH, J = 2.4 Hz); and ¹³C NMR (75 MHz CD₃OD) δ 20.47, 27.07, 55.60, 102.20, 113.15, 118.48, 119.60, 123.43, 126.56, 126.85, 128.17, 128.74, 131.07, 131.12, 133.91, 135.10, 135.23, 136.17, 136.55, 146.55, 152.76.

[0046] General Procedure for the Stilbene Syntheses.

[0047] 3-Fluoro-4,4',5-trimethoxy-3^f-0-tert-butyldiphenylsilyl-Z-stilbene

(7a). To a mixture of phosphonium salt 6 (4.7 g, 6.5 mmol) and tetrahydrofuran (25 ml, cooled to -78°C) was added ra-BuLi (2.6 mL, 2.5 M, 6.5 mmol, over 5 minutes), followed by stirring for one hour. Next, 3-fluoro-4,5-dimethoxybenzaldehyde (Ig, 5.4 mmol) in tetrahydrofuran (10 ml) was added (dropwise) over 30 minutes. The mixture was allowed to warm to room temperature, and stirring continued for 16 hours. The reaction was terminated by the addition of water (50 mL), the product was extracted with ethyl acetate, solvents were removed in vacuo, and the residue (1:1 E/Z, 75% yield) obtained was subjected to flash chromatography on silica gel using hexane-ethyl acetate (9:1) as eluent to afford Z-stilbene 7a (Ig, 34%) as a clear oil; ¹H-NMR (300 MHz, CDCl₃) δ 1.07 (s, 9H), 3.46 (s, 3H), 3.65 (s, 3H), 3.90 (s, 3H), 6.24 (d, IH, J= 12 Hz), 6.33 (d, IH, J= 12 Hz), 6.56 (m, 2H), 6.72 (m, 3H), 7.35 (m, 6H), 7.70 (m, 4H); and ¹³C NMR (75 MHz, CDCl₃) δ 19.75, 26.65, 55.07, 55.99, 61.43, 108.26, 108.28, 109.40, 109.55, 111.74, 120.83, 122.42, 127.36, 127.46, 127.48, 127.70, 129.37, 129.50, 129.63, 130.32, 132.59, 132.66, 133.57, 134.77, 135.27, 135.83, 135.95, 144.74, 149.88, 152.91, 152.95, 154.59, 156.53; HRMS (calcd for C₃₃H₃₆FO₄Si) [M+H]⁺ 543.2368, found 543.2372.

[0048] Further elution gave the E-isomer 7b (1.2 g, 41% yield): ¹H-NMR

(300 MHz, CDCl₃) δ 1.19 (s, 9H), 3.58 (s, 3H), 3.92 (s, 3H), 3.95 (s, 3H), 6.48 (d, IH, J = 15.9 Hz), 6.76 (d, IH, J= 8.7 Hz), 6.77 (d, IH, J= 16.5 Hz), 6.8 (d, IH, J= 1.5 Hz), 6.92 (d, IH, J= 2.1 Hz), 6.97 (dd, IH, J = 1.8, 8.4 Hz), 7.42 (m, 6H), 7.78 (m, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 19.76, 26.62, 55.20, 56.13, 61.39, 105.40, 106.45, 106.75 112.62, 117.62, 120.57, 125.26, 127.48, 128.52, 129.58, 139.68, 133.15, 133.28, 133.59, 135.35, 145.14, 150.52, 153.52. HRMS calcd for . C₃₃H₃₆FO₄Si [M+H]⁺ 543.2368, found 543.2392.

[0049] 3-Chloro-4,4',5-trimethoxy-3'-0-tert-butyl-diphenylsilyl-Z-stilbene

(8a). The experimental procedure noted above for 7a was repeated with 3-Chloro-4,5- dimethoxybenzaldehyde (2.8 g, 14 mmol) to yield the Z-isomer 8a (1.6 g, 21%) as a clear oil: ¹H-NMR (300 MHz, CDCl₃) δ 1.07 (s, 9H), 3.46 (s, 3H), 3.60 (s, 3H), 3.84 (s, 3H), 6.24 (d, IH, J= 12 Hz), 6.34 (d, IH, J= 12 Hz), 6.59 (s, IH, J= 7.5 Hz), 6.66 (s, IH), 6.73 (s, IH),

6.73 (d, IH₅ J= 9 Hz) 6.81 (s, IH), 7.33 (m, 6H), 7.65 (dd, 4H, J= 6.67, 1.2 Hz), and ¹³C NMR (125 MHz, CDCl₃) δ 19.76, 26.66, 55.11, 55.82, 60.71, 111.35, 111.75, 120.86, 122.40, 122.46, 127.13, 127.38, 127.85, 129.32, 129.51, 130.27, 133.60, 133.81, 135.27, 144.22, 144.75, 149.91, 153.12; HRMS calcd for . C₃₃H₃₆ClO₄SiCl 561.2042 [M+H]⁺ , found 561.2449, Cl 559.2071 [M+H]⁺; found 559.1996.

[0050] Continued elution of the chromatographic column led to the isolation of E-stilbene 8b (4.9 g, 62% yield) as a clear oil; ¹H-NMR (300 MHz, CDCl₃) δ 1.14 (s, 9H), 3.56 (s, 3H), 3.86 (s, 3H), 3.90 (s, 3H), 6.44 (d, IH, J= 16.5 Hz), 6.74 (d, IH, J= 16.5 Hz),

6.74 (s, IH), 6.82 (d, IH, J= 1.5 Hz), 6.87 (d, IH, J= 1.8 Hz), 6.94 (dd, IH, J= 8.1, 2.1 Hz), 6.99 (d, IH, J = 1.5 Hz), 7.40 (m, 6H), 7.75 (m, 4H); and ¹³C NMR (75 MHz, CDCl₃) δ 19.82, 26.67, 55.30, 55.09, 60.78, 108.43, 112.13, 117.71, 119.78, 120.59, 124.97, 127.53, 128.78, 129.61, 129.73, 133.65, 134.31, 135.41, 144.55, 150.61, 153.74.

[0051] 3-Bromo-4,4',5-trimethoxy-3'-0-tert-butyl-diphenylsilyl-Z-stiIbene

(9a). To 100 mL of THF was added phosphonium salt 6 (25.7 g, 36 mmol) and the solution cooled to -78°C. Once the temperature reached -78°C, n-BuLi (14.4 mL, 2.5 M, 36 mmol) was added over 5 minutes followed by stirring for one hour. Then the bromo-benzaldehyde (8 g, 33 mmol, in 100 mL THF) was added dropwise over 30 minutes. The mixture was allowed to warm to room temperature and stirring continued for 16 hours. The reaction was then terminated by the addition of water (50 mL), product was extracted with ethyl acetate, solvents were removed in vacuo, and the residue was separated by column chromatography to yield 4.2 g 9a (Z-stilbene), 2:1, E:Z (65% overall yield); HRMS (M + Na) + 625.1364, (M + Na)+2 627.1338; IR 2962, 1730, 1510, 1267, 908, 735, 650 cm ¹; ¹H NMR (300 MHz, CDCl₃) δ 1.07 (s, 9H), 3.45 (s, 3H), 3.58 (s, 3H), 3.82 (s, 3H), 6.97 (d, IH, J= 1.5 Hz), 6.23 (d, IH, J= 12 Hz), 6.32 (d, IH, J = 12 Hz), 6.52 (d, IH, J= 8.1 Hz), 6.71 (dd, IH, J= 1.5 Hz, J= 8.1 Hz), 7.57 (d, IH, J= 1.5 Hz), 7.32 (m, 6H), 7.65 (dd, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 152.87, 149.79, 145.16, 144.66, 135.17, 134.37, 133.52, 130.40, 129.43, 129.24, 127.30, 126.90, 125.16, 122.40, 120.79, 117.18, 112.01, 111.71, 94.38, 60.61, 55.81, 55.15, 21.10.

[0052] Further elution of the chromatogram led to isolation of 8.1 g of the E- isomer 9b: IR 2934, 2859, 1710, 1510, 1275, 908, 732, 650 cm ¹; ¹H-NMR (300 MHz, CDCl₃) δ 1.14 (s, 9H), 3.54 (s, 3H), 3.84 (s, 3H), 3.88 (s, 3H),), 6.46 (d, IH, J= 12 Hz), 6.76 (d, IH, J= 12 Hz), 6.71 (d, IH, J= 8.1 Hz), 6.85 (d, IH, J= 2.1 Hz), 6.87 (d, IH, J= 2.1), 6.94 (dd, IH, J= 8.4 Hz, J= 2.4 Hz), 7.15 (d, IH, J = 2.4) 7.38 (m, 6H), 7.74 (dd, 4H); and ¹³C-NMR (75 MHz, CDCl₃) δ 19.76, 26.65, 55.25, 56.03, 60.59, 109.18, 109.85, 112.13, 117.70, 120.57, 122.59, 124.79, 127.48, 128.80, 129.58, 133.64, 134.91, 135.35, 145.16, 150.57, 153.58.

[0053] 3-Iodo-4,4',5-trimethoxy-3'O-tert-butyl-diphenyl-Z-stilbene (10a).

A gradient column chromatogram from 0-3% ethyl acetate in hexane afforded Z-stilbene 10a (1.4 g) in 21% yield mp 122-124⁰C: HRMS, found: [M+H]⁺ 651.1474. C₃₃H₃₆O₄Si requires [M+Hf, 651.1427; ¹H-NMR (300 MHz, CDCl₃) δ 1.07 (s, 9H), 3.45 (s, 3H), 3.55 (s, 3H), 3.79 (s, 3H), 6.21 (d, IH, J = 12 Hz), 6.31 (d, IH, J = 12 Hz), 6.59 (d, IH, J = 7.8 Hz), 6.72 (s, 2H), 6.77 (dd, IH, J = 7.8, 1.5 Hz), 7.19 (d, IH, J = 1.8 Hz), 7.32 (m, 6H), 7.64 (d, 4H, J = 7.5 Hz); and ¹³C NMR (75 MHz, CDCl₃) δ 19.68, 26.62, 55.05, 55.56, 60.33, 91.94, 111.72, 113.09, 120.78, 122.43, 126.73, 127.33, 129.32, 130.28, 130.93, 133.54, 135.17, 144.70, 149.82, 151.82.

[0054] General Procedure for Cleavage of the Silyl Ether Protecting group.

[0055] 3-Fluoro-4,4',5-trimethoxy-3 '-hydroxy-Zrstilbene

(Ha, Fluorcombstatin). A solution prepared from Z-isomer 7a (2.4 g, 4.4 mmol), tetrahydrofuran (50ml) and IM tetrabutylammonium fluoride (4.5 ml, 4.5 mmol) was stirred fro 3 hours. The reaction was terminated by the addition of water (50ml), the mixture was extracted with ethyl acetate and the solvents were removed in vacuo. Separation by flash chromatography using: 1:4 ethyl acetate-hexane as eluent provided Z-stilbene (Ha) (1.12 g, 83%) as a colorless solid, which was recrystallized from ethyl acetate-hexane: mp 93-94°C; (300 MHz, CDCl₃) δ 3.67 (s, 3H), 3.87 (s, 3H), 3.90 (s, 3H), 5.30 (bs, IH), 6.35 (d, J = 12 Hz, IH)₃ 6.48 (d, J= 12 Hz, IH), 6.61 (d, J= 2.4 Hz, IH), 6.64 (d, J= 1.8 Hz, IH), 6.72 (d, J = 8.4 Hz, IH), 6.75 (dd, J= 1.5, 8.4 Hz, 2H), 6.86 (d, J= 1.5 Hz, IH); ¹³C NMR (75 MHz, CDCl₃) δ 55.87, 56.01, 61.39, 108.24, 109.37, 109.57, 110.32, 114.83, 120.88, 127.72, 130.03, 130.14, 132.38, 132.48, 135.81, 135.95, 145.15, 145.78, 152.85, 152.88, 154.22, 156.65; and ¹⁹F NMR (CDCl₃) δ -11.32 (d, J = 12.8 Hz, IF). HRMS calcd for C₁₇H₁₈FO₄ 305.1189 [M+H]⁺.

[0056] 3-Fluoro-4,4',5-trimethoxy-3'-hydroxy-E-stilbene (lib). Cleavage of silyl ester 7b (150mg, 0.27 mmol) was performed as described for the synthesis of Ha. Separation by flash chromatography on silica using ethyl acetate-hexane (3:7) afforded a colorless solid Hb (75 mg, 88% yield): mp 86-87°C; ¹H NMR (300 MHz, CDCl₃) δ 3.87 (s, 3H), 3.90 (s, 3H), 3.93 (s, 3H), 5.75 (bs, IH), 6.77-6.86 (m, 5H), 6.93 (m, IH), 6.86 (d, J = 1.8 Hz, IH). ¹³C NMR (75 MHz, CDCl₃) δ 55.87, 56.18, 61.39, 100.64, 105.68, 106.51, 106.79, 107.55, 110.63, 111.76, 119.30, 125.80, 127.61, 128.62, 129.53, 130.56, 133.13, 133.23, 134.73, 136.41, 145.78, 146.59, 153.57, 154.40, 157.64.

[0057] 3-Chloro-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene (12a).

Deprotection of silyl ester 8a (1.5g, 2.7 mmol) was conducted as summarized for the synthesis of Ha. Separation by flash chromatography on silica using ethyl acetate-hexane (3:7) gave compound 12a (754 mg, 89%). Recrystallization from hexane gave a white solid; mp 105-106°C; ¹H-NMR (300 MHz, CDCl₃) δ 3.65 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 5.52 (s, IH), 6.36 (d, IH, J= 12.3 Hz), 6.49 (d, IH, J= 12 Hz), 6.75 (m, 3H), 6.88 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 55.86, 55.94, 60.76, 110.37, 111.40, 114.86, 114.94, 121.05, 122.51, 127.60, 127.92, 130.15, 130.43, 133.74, 144.36, 145.31, 145.92, 153.21.

[0058] 3-Chloro-4,4',5-trimethoxy-3'-hydroxy-E-stilbene 5 (12b). Column chromatography (elution with 7:3 hexane-ethyl acetate) afforded a colorless solid, E-isomer 12b, mp 138-140°C, in 79% yield: ¹H-NMR (300 MHz, CDCl₃) δ 3.87 (s, 3H), 3.88 (s, 3H), 3.90 (s, 3H), 5.69 (bs, IH), 6.79 (d, IH, J= 15.9 Hz), 6.81 (d, IH, J= 8.4 Hz), 6.90 (d, IH, J = 1.5 Hz), 6.90 (d, IH, J= 15.9 Hz), 6.94 (dd, IH, J= 8.1, 1.5 Hz), 7.08 (dd, IH, J= 1.8 Hz), 7.11 (d, IH, J= 2.1 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 55.93, 56.06, 60.74, 100.66, 108.66, 110.65, 111.77, 119.38, 119.79, 125.49, 128.41, 128.85, 130.59, 134.24, 144.65, 145.79, 146.61, 153.78. HRMS calcd for C₁₇H₁₇ClO₄ 321.0894 [M+H]⁺ , found 321.0893. Anal. Calcd for C₁₇H₁₇ClO₄ C, H.

[0059] 3-Bromo-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene (13a). The silyl ester cleavage reaction for 9a (4g, 6.6 mmol) was completed as described for the synthesis of phenol 11a. Isolation by flash chromatography on silica gel using ethyl acetate-hexane (1:4) gave compound 13a (2.22 g of 92%). Recrystallization from hexane afforded a colorless solid: mp 108-109⁰C; HRMS calcd for Ci₇H₁₇BrO₄ 364.0303, found [M⁺²] 366.0287; ¹H NMR (300 MHz, CDCl₃) δ 3.63 (s, 3H) 3.84 (s, 3H), 3.86 (s, 3H), 6.34 (d, IH, J= 12 Hz), 6.49 (d, IH, J= 12 Hz), 6.73 (d, IH, J= 8.4 Hz), 6.77 (dd, IH, J= 8.7, 1.8 Hz), 6.79 (d, IH, J= 1.8 Hz), 6.86 (d, IH, J= 1.5 Hz), 7.04 (d, IH, J= 1.5 Hz). ¹³C NMR (75 MHz, CDCl₃) δ 55.77, 55.88, 60.56, 110.45, 112.13, 114.98, 117.18, 121.01, 125.28, 127.33, 130.07, 130.43, 134.37, 145.32, 146.02, 153.03. IR 3539, 3441, 3011, 2939, 2839, 1554, 1510, 1273, 1047, 908, 732 cm^"1. HRMS calcd for C₁₇H₁₇O₄ ⁸¹Br. 366.0287.

[0060] 3-Bromo-4,4',5-trimethoxy-3'-hydroxy-E-stilbene (13b). By the same procedure used to obtain phenol 13a, silyl ester 9b was converted to E phenol 13b, and isolated by flash chromatography on silica gel with ethyl acetate-hexane (3:7) to give E- isomer 13b (0.14 g, 81%). Recrystallization from hexane gave colorless solid, mp 152- 154°C; ¹H NMR (300 MHz, CDCl₃) δ 3.86 (s, 3H), 3.89 (s, 3H), 3.90 (s, 3H), 6.80 (d, IH, J = 15.9 Hz), 7.38(d, IH, J= 15.9 Hz), 6.82 (d, IH, J= 8.4 Hz), 6.96 (dd, IH, J= 8.4, 2.4 Hz), 6.88 (s, IH), 7.11 (d, IH, J = 1.8 Hz), 7.25 (d, IH, J = 1.5 Hz); and ¹³C NMR (75 MHz, CDCl₃) δ 56.00, 56.11, 60.58, 94.36, 109.32, 110.60, 11.72, 117.81, 119.32, 122.58, 125.29, 128.82, 130.53, 134.81, 145.60, 145.70, 146.50, 153.53.

[0061] 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene (14a). The silyl ester cleavage reaction for 10a was completed as described for the phenol 11a. The crude product was separated by column chromatography using 1 :4 ethyl acetate-hexane as eluent to give 1.38 g of Z-isomer 14a in 81% yield: mp 92-94°C: HRMS calc for C₁₇H₁₈O₄Si found (M+H)⁺ 413.0250. ¹H NMR (300 MHz, CDCl₃) δ 3.61 (s, 3H), 3.81 (s, 3H), 3.84 (s, 3H), 6.32 (d, IH, J = 12 Hz), 6.34 (s, IH), 6.56 (d, IH, J = 12 Hz), 6.75 (s, IH), 6.83 (d, IH, J = 1.8 Hz), 6.85 (s, 3H), 7.25 (d, IH, J = 1.5 Hz); and ¹³C NMR (75 MHz, CDCl₃) δ 55.56, 55.82, 60.33, 91.78, 110.50, 113.11, 115.00, 120.91, 126.96, 129.94, 130.28, 135.93 145.29, 146.10, 147.67, 151.79. IR 3543, 3011, 2937, 2841, 1510, 1273, 1001, 908, 732 cm⁴. [0062] 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-E-stilbene (14b). Separation by column chromatography (30% ethyl acetate-hexane as eluent) gave 0.29 g of E-isomer 14b in 98% yield: mp 111-113⁰C; ¹H NMR (300 MHz, CDCl₃) δ 3.84 (s, 3H), 3.87 (s, 3H), 3.88 (s, 3H), 5.85 (bs, IH), 6.77 (d, IH, J = 16.5 Hz), 6.89 (d, IH, J = 16.5 Hz), 6.82 (s, IH), 6.96 (s, IH), 6.93 (d, IH, J = 2.4 Hz), 7.11 (d, IH, J = 1.5 Hz), 7.46 (d, IH, J = 1.5 Hz); and ¹³C NMR (75 MHz, CDCl₃) δ 55.85, 60.41, 92.56, 110.40, 110.63, 111.77, 119.28, 124.97, 128.36, 128.70, 130.15, 135.71, 145.71, 146.56, 148.11, 152.44.

[0063] Dibenzyl (Z)-3-fluoro-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate

(15). Z-stilbene 11a (1.1 g, 3.6 mmol) in 20 mL of acetonitrile (20 mL) and 3.5 niL (36 mmol) of carbon tetrachloride was cooled to -10⁰C, and stirred for 10 minutes. Then DIPEA (1.3 mL, 7.4 mmol), immediately followed by DMAP (44 mg, 0.36 mmol), were added. After 1 minute dibenzyl phosphite (1.2 mL, 5.4 mmol) was added over 5 minutes, and the mixture was stirred for an additional 3 hours at -10°C. The reaction was terminated by the addition of 0.5 M KH₂PO₄, the mixture was extracted with ethyl acetate, solvents were removed in vacuo, and the product was isolated by column chromatography (1:1 elution with ethyl acetate-hexane) to yield 1.5 g of phosphate in 74% yield: b.p. dec. 280°C (0.01 mmHg); ¹H-NMR (500 MHz, CDCl₃) δ 3.65 (s, 3H), 3.77 (s, 3H), 3.87 (s, 3H), 5.12 (s, 2H), 5.14 (s, 2H), 6.38 (d, IH, J = 12 Hz), 6.43 (d, IH, J = 12 Hz), 6.57 (s, IH) 6.62 (dd, IH, J = 1.5, 11.5 Hz), 6.78 (d, IH, J = 8.5 Hz), 7.03 (d, IH, J = 8.5 Hz), 7.12 (s, IH), 7.82 (m, 10H); ¹³C NMR (125 MHz, CDCl₃) δ; and ³¹P NMR (162 MHz CDCl₃) δ -7.84 (s).

[0064] Dibenzyl (3-bromo-4,4',5-trimethoxy-Z-stiIbene 3'-0-phosphate

(16). The preceding reaction (see Compound 15) was repeated with Z-stilbene 13a (1 g, 2.7 mmol) to afford 1.6 g of phosphate 16 in 94% yield: b.p. dec. 271⁰C (0.01 mmHg); ¹H-NMR (300 MHz, CDCl₃) δ 3.59 (s, 3H), 3.76 (s, 3H), 3.79 (s, 3H), 5.11 (s, 2H), 5.13 (s, 2H), 6.37 (d, IH, J = 12.4 Hz), 6.43 (d, IH, J = 12 Hz), 6.72 (d, IH, J = 1.5 Hz), 6.78 (d, IH, J = 8.4 Hz), 7.02 (d, IH, J = 8.2 Hz), 7.03 (d, IH, J = 2.4 Hz), 7.10 (d, IH, J = 1.8 Hz), 7.28 (m, 10H); ¹³C NMR (75 MHz, CDCl₃) δ 55.77, 55.88, 60.51, 65.59, 69.67, 69.73, 111.83, 112.22, 117.23, 121.96, 121.99, 125.04, 126.32, 126.75, 127.30, 127.69, 127.77, 127.88, 128.31, 128.35, 129.46, 129.47, 133.93, 135.44, 135.51, 139.30, 139.38, 145.30, 149.77, 149.82, 152.99; and ³¹P NMR (162 MHz CDCl₃) δ -7.84 (s).

[0065] Dibenzyl 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-0-phosphate (17).

The phosphorylation reaction used to obtain phosphate 15 was repeated with Z-stilbene 14a (2.39 g, 0.95 mmol) to obtain 0.55 g of Z-stilbene 17 in 86% yield as a colorless oil: b.p. dec. 274⁰C (0.01 mmHg); HRMS calc for C₃₁H₃₁PIO₇ [M-HH]⁺, 673.0852; found [M+H]⁺, 673.0808. ¹H-NMR (300 MHz, CDCl₃) δ 3.51 (s, 3H), 3.65 (s, 3H), 3.72 (s, 3H), 5.04 (s, 2H), 5.06 (s, 2H), 6.36 (d, IH, J = 9 Hz), 6.42 (d, IH, J = 9 Hz), 6.77 (d, IH, J = 1.2 Hz), 6.89 (d, IH, J = 6 Hz), 7.02 (d, IH, J = 6 Hz), 7.01 (s, IH), 7.19 (d, IH, J = 1.2 Hz), 7.28-7.35 (m, 10H); and ¹³C-NMR (75 MHz, CDCl₃) δ 56.20, 56.50, 60.73, 71.18, 71.24, 92.73, 113.72, 114.23, 122.58, 122.61, 128.10, 128.93, 129.50, 129.56, 130.18, 130.85, 130.86, 131.87, 136.26, 136.66, 136.73, 140.32, 140.39, 149.12, 151.21, 151.25, 153.26.

[0066] A solution of cis stilbene 9a (0.68 g, 1.64 mmol) in acetonitrile (7 mL) was cooled to -10 ⁰C. Carbon tetrachloride (1.6 mL, 16.4 mmol) was added and the mixture was stirred for 10 min, at -10 ⁰C in the dark. Next, diisopropylamine (0.57 mL, 3.28 mmol), immediately followed by DMAP (20mg, cat) were added. After 1 min, dibenzylphosphite (0.44 mL, 1.96 mmol) was added, and the mixture was stirred for 20 mins at -10 °C. The reaction was terminated by the addition of 0.5 m KH₂PO₄ (7 mL) and extracted into EtOAc (3 x 15 mL), and the extract solution dried, filtered and concentrated. The oily residue was separated by column chromatography using 4:1 hexane-EtOAc as eluent to yield 0.94 g, 86% of a pure oil: Anal, calcd for C₃₁H₃₁IO₇P C, 55.37; H, 4.50. Found C, 55.37; H, 4.64%.

[0067] General Procedure for Synthesis of Phosphate Cation Derivatives

[0068] Method A. Each of the metal cation containing salts were obtained by the procedure outlined below for preparing sodium salt 19a. The metal counter ions were introduced by treatment of the phosphoric acid with either the corresponding hydroxide (e.g., potassium, lithium) or acetate (e.g. magnesium).

[0069] Sodium 3-bromo-4,4',-5-trimethoxy-Z-stilbene 3'-0-phosphate

(19a). To a solution of dibenzyl phosphate 16 (0.28 g, 0.45 mmol) in dry dichloromethane (10 mL) was added trimethylsilylbromide (125 μL, 0.95 mmol). The reaction mixture was stirred for 30 minutes under argon, and the reaction was terminated by the addition of methanol (20 mL). Following removal of solvents (in vacuo), the free phosphoric acid was dissolved in ethanol (10 mL) and sodium methoxide (49 mg, 0.9 mmol) were added to the residue. After the reaction mixture was stirred for 30 minutes, the precipitate was collected and washed with ether to provide sodium salt 19a (0.17 g) as a colorless solid: m.p. 196- 197⁰C; ¹H-NMR (300 MHz, D₂O) δ 3.53 (s, 3H), 3.68 (s, 3H), 3.70 (s, 3H), 6.52 (d, IH, J = 12 Hz), 6.72 (d, IH, J= 12 Hz), 6.75 (s, IH), 6.77 (s, IH), 6.79 (s, IH), 7.01 (s, IH)₅ 7.15 (s, IH). [0070] Sodium 3-fluoro-4,4',5-trimethoxy-Z-stilbene 3'-0-phosphate

(18a). m.p. 200-202°C; ¹H-NMR (300 MHz, D₂O) δ 3.52 (s, 3H), 3.67 (s, 3H), 3.68 (s, 3H), 6.52 (d, IH, J= 12 Hz), 6.71 (d, IH, J= 12 Hz), 6.72 (s, IH), 6.78 (s, IH)₅ 6.79 (s, IH), 7.03 (s, IH), 7.16 (s, IH).

[0071] Lithium 3-bromo-4,4',5-trimethoxy-Z-stilbene 3'-0-phosphate

(19b). m.p. 265-268°C (dec); ¹H NMR (300 MHz, D₂O) δ 3.53 (s, 3H), 3.66 (s, 3H), 3.69 (s, 3H), 6.35 (d, IH, J= 12 Hz), 6.52 (d, IH, J= 12 Hz), 6.70 (s, 2H), 6.81 (d, IH, J= 1.5 Hz), 7.01 (d, IH₅ J= 1.5 Hz)₅ 7.23 (s, IH).

[0072] Potassium 3-bromo-4,4',5-trimethoxy-Z-stilbene 3'-0-phosphate

(19c). m.p. 230-233°C (dec); ¹H-NMR (300 MHz₅ D₂O) δ 3.53 (s, 3H)₅ 3.66 (s, 3H), 3.69 (s, 3H), 6.35 (d, IH₅ J= 12 Hz)₅ 6.52 (d, IH₅ J= 12 Hz)₅ 6.70 (s, 2H)₅ 6.81 (d, IH, J= 1.5 Hz), 7.01 (d, IH₅ J= 1.5 Hz)₅ 7.23 (s, IH).

[0073] Cesium 3-bromo-4,4',5-trimethoxy-phenyl-Z-stilbene 3'-O- phosphate (19d). m.p. 233-235°C; ¹H-NMR (300 MHz, DMSO) δ 3.51 (s, 3H), 3.62 (s, 3H), 3.65 (s, 3H)₅ 6.38 (d₅ IH₅ J= 12 Hz)₅ 6.50 (d, IH, J= 12 Hz), 6.71 (s, IH), 6.83 (d, IH₅ J= 1.5 Hz)₅ 7.03 (d, 2H₅ J= 1.5 Hz)₅ 7.23 (s, IH).

[0074] Rubidium 3-bromo-4,4',5-trimethoxy-Z-stilbene 3^?-0-phosphate

(19e). m.p. 204-206°C; ¹H-NMR (300 MHz, DMSO) δ 3.50 (s, 3H)₅ 3.64 (s, 3H), 3.66 (s, 3H), 6.35 (d, IH, J= 12 Hz)₅ 6.52 (d, IH₅ J= 12 Hz)₅ 6.68 (s, 2H)₅ 6.80 (d, 2H₅ J= 1.5 Hz)₅ 7.00 (d, 2H, J= 1.5 Hz), 7.25 (s, IH).

[0075] Calcium 3-bromo-4,4',5-trimethoxy-Z-stilbene 3'-0-phosphate

(19T). m.p. 245-248°C (dec); ¹H-NMR (300 MHz₅ DMSO) δ 3.53 (s₅ 3H), 3.69 (s₅ 3H)₅ 3.70 (s, 3H)₅ 6.33 (d₅ IH, J= 12 Hz)₅ 6.50 (d, IH, J= 12 Hz), 6.71 (s, 2H)₅ 6.81 (d, 2H, J= 1.5 Hz)₅ 7.99 (d₅ 2H, J= 1.5 Hz)₅ 7.23 (s, IH).

[0076] Magnesium 3-bromo-4,4',5-trimethoxy-Z-stilbene 3'-0-phosphate

(19g). m.p. 280-285⁰C (dec); ¹H-NMR (300 MHz₅ DMSO) δ 3.50 (s, 3H) 3.60 (s, 3H), 3.65 (s, 3H), 6.33 (d, IH, J= 12 Hz)₅ 6.50 (d₅ IH₅ J= 12 Hz)₅ 6.68 (s, 2H), 6.79 (d, 2H₅ J = 1.5 Hz), 7.00 (d₅ 2H, J= 1.5 Hz), 7.21 (s, IH).

[0077] Sodium 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-0-phosphate (20a). m.p. 194-195°C, ¹H-NMR (300 MHz, D₂O) δ 3.50 (s, 3H), 3.67 (s, 3H)₅ 3.68 (s, 3H), 6.50 (d, IH, J= 12 Hz), 6.70 (d, IH₅ J= 12 Hz), 6.72 (s, IH), 6.77 (s, IH)₅ 6.79 (s, IH)₅ 7.01 (s₅ IH), 7.13 (s, IH). [0078] Method B The potassium salt 18c (approximately 30mg) was dissolved in de-ionized water (ImL) and applied to a Dowex-50w (HCR- W2) resin column (amine or amino acid) and developed by water. The eluent was concentrated by freeze drying to give the required compound.

[0079] 3-Iodo-4,4',5-trimethoxy-3'-0-tert-butyldiphenylsilyl-Z-stiIbene

(10a) and 3-Iodo-4,4',5-trimethoxy-3'-0-tert-butyldiphenylsilyl-E-stilbene (10b).

[0080] Method A. Phosphonium bromide 6 (3.67 g, 5.13 mmol) was dissolved in DCM at O⁰C. Sodium hydride (60% dispersion in mineral oil, 0.41 g, 10.2 mmol) was added and the mixture turned orange. Next, 3-iodo-4,5-dimethoxybenzaldehyde (1 g, 3.42 mmol) was added and stirring was continued for 21 hrs. The reaction was terminated by adding water (50 mL) and extracted with DCM (3 x 50 niL), which was dried, filtered and concentrated. The oil obtained was subjected to flash chromatography on silica gel with the eluent 0-3% ethyl acetate in hexane to afford z-stilbene 10a (0.86 g, 39%) which crystallized as a colorless solid from hexane: mp 122-124 °C: ¹H-NMR (300 MHz, CDCl₃) δ 1.07 (s, 9 H), 3.45 (s, 3 H), 3.55 (s, 3 H), 3.79 (s, 3 H), 6.21 (d, 1 H, J = 12 Hz), 6.31 (d, 1 H, J = 12 Hz), 6.59 (d, I HJ = 7.8 Hz), 6.72 (s, 2 H), 6.77 (dd, I HJ = 7.8, 1.5 Hz), 7.19 (d, 1 H, J = 1.8 Hz), 7.40-7.20 (m, 6 H), 7.64 (d, 4H, J = 7.5 Hz); ¹³C-NMR (75 MHz, CDCl₃) δ 19.68, 26.62, 55.05, 55.56, 60.33, 91.94, 111.72, 113.09, 120.78, 122.43, 126.73, 127.33, 129.32, 130.28, 130.93, 133.54, 135.17, 144.70, 149.82, 151.82; HRMS calcd for C₃₃H₃₆IO₄Si 651.1428 [M+H]⁺, found 651.1474; Anal, calcd for C₃₃H₃₅IO₄Si C, 60.92; H, 5.45. Found, C, 60.79; H, 5.67%.

[0081] Further elution gave E-stilbene 10b (0.96 g, 43%) that crystallized from hexane as a colorless solid; mp 98-99°C; ¹H-NMR (300 MHz, CDCl₃) δ 1.14 (s, 9H), 3.55 (s, 3H), 3.82 (s, 3H), 3.82 (s, 3H), 3.89 (s, 3H), 6.43 (d, IH, J = 15.9 Hz), 6.71-6.76 (m, 2H), 6.86-6.95 (m, 3H), 7.33-7.42 (m, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ 19.81, 26.67, 55.28, 60.50, 92.65, 110.22, 112.11, 117.70, 120.56, 124.58, 127.52, 128.45, 128.68, 129.60, 129.77, 133.64, 135.38, 135.82, 145.18, 148.13, 150.56, 152.49; HRMS calcd for C₃₃H₃₆IO₄Si 651.1428 [M+H]⁺, found 651.1400; Anal, calcd for C₃₃H₃₅IO₄Si, C, 60.92; H, 5.42, found C, 60.88; H, 5.63%.

[0082] Method B. Butyllithium (4.5 mL, 11.3 mmol) was added to a stirred and cooled (-7O⁰C) suspension of phosphonium bromide 6 in dry THF (100 mL). The solution was stirred for 30 min at -7O⁰C then 6 hours at room temperature. Water (50 mL) was added and the reaction mixture was extracted with EtOAc (3 x 100 mL), the extract dried, filtered and concentrated. The oil obtained was subjected to flash chromatography on silica eluent 0-3% ethyl acetate in hexane to afford Z-stilbene 10a (1.4 g, 21%) as a colorless solid: mp 122-124 ⁰C.

[0083] 3,5-diiodo-4,4'-dimethoxy-3'-0-tert-butyl-diphenylsilyl-z-stilbene

(25a) and 3,5-diiodo-4,4'-dimethoxy-3'-0-tert-butyl-diphenylsilyl-E-stilbene (25b).

[0084] Method A. Phosphonium bromide 6 (2.77 g, 3.87 mmol) (8) was dissolved in DCM at 0 °C. When sodium hydride (60% dispersion in mineral oil, 0.31 g, 7.7 mmol) was added, the mixture turned orange. Aldehyde (1.0 g, 2.57 mmol) was added and stirring was continued for 7.5 hrs. The reaction was terminated by adding water (50 mL) and extracted with DCM (3 x 50 mL). The organic extract was dried, filtered and concentrated. The oily residue was subjected to flash chromatography on silica gel using hexane as eluent to give an isomeric mixture of the title compounds (71% yield, 1.35g). Further elution gave E-isomer (0.10 g, 5%) as a colorless oil in pure form: ¹H-NMR (300 MHz, CDCl₃) δ 1.14 (s, 9H), 3.56 (s, 3H), 3.84 (s, 3H), 6.33(d, 1 H, J 15.9 Hz), 6.72 (d, 1 H, J 8.4 Hz), 6.73 (d, 1 H, J 15.9 Hz), 6.72 (d, 1 H, J 8.4 Hz, ArH), 6.85 (d, IH, J 2.1 Hz), 6.92 (dd, IH, J 1.8 Hz and J 8.4 Hz), 7.34-7.46 (m, 6H) and 7.72-7.75 (m, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ 19.82, 26.69, 55.30, 60.77, 90.59, 112.09, 117.73, 120.83, 122.47, 127.55, 129.38, 129.65, 129.99, 133.58, 135.40, 137.15, 137.73, 145.22, 150.84 and 157.55; and HRMS calcd for C₃₂H₃₃I₂O₃Si 747.0289 [M+H]⁺, found 747.0442.

[0085] Method B. Butyllithium (0.6 mL, 1.47 mmol) was added to a stirred and cooled (-10 °C) suspension of phosphonium bromide 6 (l.Olg, 1.4 mmol) in dry THF (80 mL). The orange-red solution was stirred for 10 minutes at room temperature. Aldehyde (0.50 g, 1.33 mmol) was added and the reaction mixture color changed from red to yellow. Stirring was continued at room temperature for 10 minutes, ice water (100 mL) was added and the mixture extracted with EtOAc (3 x 100 mL). The extract was washed with water (100 mL), dried, filtered and concentrated. The resulting oil was partially separated by flash chromatography on silica gel using hexane-EtOAc (100:1) as eluent to give an isomeric mixture in a ratio approximately 1:1.9, (cis:trans, 0.90 g, 90%).

[0086] 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-z-stilbene (14a). To a solution of silyl ether 7a (1.30 g, 1.99 mmol) in THF was added tetrabutylammonium fluoride (2.2 mL, 2.2 mmol). The mixture was stirred under Ar in the dark for 10 min. and the reaction was terminated by the addition of water (5 mL), the product was extracted with EtOAc (3 x 15 mL), and the extract dried, filtered and concentrated. The crude product was separated by silica gel column chromatography using 1 :4 ethyl acetate-hexane as eluent to give stilbene 14a (0.70 g, 85%) as colorless solid: mp 92-94 ⁰C; IR 3543, 3011, 2937, 2841, 1510, 1273, 1001, 908, 732 cm^'1; ¹H-NMR (300 MHz, CDCl₃) δ 3.61 (s, 3 H), 3.81 (s, 3 H), 3.84 (s, 3 H), 6.32 (d, 1 H, J = 12 Hz), 6.34 (s, 1 H), 6.56 (d, 1 H, J = 12 Hz), 6.75 (s, 1 H), 6.83 (d, I HJ = 1.8 Hz), 6.85 (s, 3 H), 7.25 (d, 1 H, J = 1.5 Hz); ¹³C-NMR (75 MHz, CDCl₃) δ 55.56, 55.82, 60.33, 91.78, 110.50, 113.11, 115.00, 120.91, 126.96, 129.94, 130.28, 135.93 145.29, 146.10, 147.67, 151.79; HRMS calcd for C₁₇Hi₈IO₄ 413.0259 [M+H]⁺, found 413.0250. Anal, calcd for C₁₇H₁₇IO₄ C, 49.53; H, 4.16. Found C, 49.38; H, 4.24%.

[0087] 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-E-stilbene (9b). The trans isomer 14b (0.29 g, 98%) was obtained from silyl ether 10b (0.46 g, 0.7 mmol) as described above for the synthesis of the cis isomer 14a. Separation by column chromatography (7:3 hexane-ethyl acetate as eluent) gave E-isomer 14b (0.29g, 98%) as colorless solid: mp 111 — 113 °C; ¹H-NMR (300 MHz, CDCl₃) δ 3.84 (s, 3 H), 3.87 (s, 3 H), 3.88 (s, 3 H), 5.85 (bs, 1 H), 6.77 (d, I HJ = 16.5 Hz), 6.89 (d, 1 H, J = 16.5 Hz), 6.82 (s, 1 H), 6.96 (s, 1 H), 6.93 (d, 1 H, J = 2.4 Hz), 7.11 (d, 1 H, J = 1.5 Hz), 7.46 (d, 1 H, J = 1.5 Hz); ¹³C-NMR (75 MHz, CDCl₃) δ 55.85, 60.41, 92.56, 110.40, 110.63, 111.77, 119.28, 124.97, 128.36, 128.70, 130.15, 135.71, 145.71, 146.56, 148.11, 152.44; HRMS calcd for C₁₇H₁₈IO₄ 413.0257 [M+H]⁺, found 413.0250. Anal, calcd for C₁₇H₁₇IO₄ C, 49.53; H, 4.16. Found, C, 49.38; H, 4.24%.

[0088] 3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-z-stilbene (22a) and 3,5- diiodo-4,4'-dimethoxy-3'-hydroxy-E-stilbene (22b).

[0089] These stilbenes were obtained from the z and E silyl ether mixture

21ab (1.35 g, 1.81 mmol) as described above for the synthesis of cis-isomer 14a. The oily mixture was separated by column chromatography with 2:1 hexane-EtOAc as eluent to provide cis-isomer 22a as an oil (0.45 g, 49%): ¹H-NMR (300 MHz, CDCl₃) δ 3.85 (s, 3 H), 3.89 (s, 3 H), 5.54 (s, 1 H), 6.26 (d, 1 H, J=12 Hz), 6.49 (d, 1 H, J=12 Hz), 6.74 (s, 2H), 6.82 (s, 1 H) and 7.67 (s, 2 H); ⁷⁵C-NMR (125 MHz, CDCl₃) δ 55.98, 60.73, 89.98, 110.46, 114.87, 120.98, 125.08, 29.47, 131.57, 137.37, 139.96, 145.42, 146.21, 157.50. HRMS calcd for C₁₆H₁₅I₂O₃ 508.9113 [M+H]⁺, found 508.9111. Anal, calcd for C₁₆H₁₄I₂O₃ C, 37.82; H, 2.78. Found, C, 37.80; H, 2.83. [0090] Further elution led to the E-stilbene 22b (0.46 g, 50% yield) as a colorless solid which was crystallized from hexane: mp 127-129 °C; ¹H-NMR (300 MHz, CDCl₃) δ 3.86 (s, 3 H), 3.91 (s, 3 H), 5.62 (s, 1 H), 6.71 (d, 1 H, J=16.5 Hz), 6.83 (d, 1 H, J=8.1 Hz), 6.90 (d, 1 H, J=17.1 Hz), 6.95 (d, 1 H, J=8.4 Hz), 7.10 (d, 1 H, J=2.4 Hz) and 7.85 (s, 2 H, H-2); ¹³C-NMR (75 MHz, CDCl₃) δ 55.51, 60.30, 90.17, 100.17, 100.21, 110.18, 111.35, 119.17, 122.56, 129.63, 129.81, 136.82, 137.19, 146.34 and 157.25; HRMS calcd for C₁₆H₁₅I₂O₃ 508.9113 [M+H]⁺, found 508.9119. Anal, calcd for C₁₆H₁₄I₂O₃ C, 37.82; H, 2.78. Found, C, 38.01; H, 2.91.

[0091] 3,5-diiodo-4,4'-dimethoxy-3'-acetyI-z-stilbene (22c).

[0092] An appropriate phenol 22a (0.45 g) was dissolved in pyridine (3 mL) - acetic anhydride (170 μL) and stirred for 2 hrs. The mixture was concentrated under reduced pressure from toluene (3 * 10 mL). The residue was diluted with EtOAc (30 mL), washed successively with water (10 mL), NaHCO₃ (10% aq. sol., 10 mL), dried, and the solution filtered and concentrated. The acetate was further purified by flash chromatography on silica using 1:24 hexane-EtOAc:hexane as eluent to afford acetate 22c (0.20 g, 41%) as a colorless solid: recrystallized from hexane mp 121-122 ⁰C; ¹H-NMR (300 MHz, CDCl₃) δ 2.29 (s, 3 H), 3.83 (s, 3 H), 3.85 (s, 3 H), 6.29 (d, 1 H, J = 12 Hz), 6.48 (d, 1 H, J = 12 Hz), 6.85 (d, 1 H, J = 8.7 Hz), 6.93 (d, I HJ = 2.43), 7.06 (d, 1 H, J = 1.5), 7.09 (d, 1 H, J = 2.4 Hz) and 7.67 (s, 2 H); ¹³C-NMR (125 MHz, CDCl₃) δ 20.66, 55.94, 60.72, 90.11, 112.16, 123.25, 125.41, 127.47, 128.85, 130.64, 137.10, 139.54, 139.89, 150.69, 157.67 and 168.79; HRMS calcd for C₁₉H₂₀I₂O₅ 582.9479 [M+CH₃OH]⁺, found 582.9482; Anal, calcd for C₁₈H₁₆I₂O₄ C, 39.30; H, 2.93. Found C, 39.30; H, 3.13%.

[0093] 3-iodo-4,4',5-trimethoxy-3'-acetyl-Z-stilbene (14c).

[0094] An appropriate phenol (0.1 g, 0.24 mmol) was dissolved in 3 mL anhydrous pyridine. Acetic anhydride (50 μL, 0.51 mmol) was added with cat DMAP. The mixture was stirred for 90 minutes. The reaction was terminated by the addition of 5 mL CH₃OH. The mixture was diluted with toluene and concentrated under reduced pressure. It was purified on flash chromatography on silica gel using EtOAc :hexane (1:9) as eluent to give a white solid (0.1 mg, 91%). The solid was crystallized from hexane: mp 103-104°C; ¹H-NMR (300 MHz, CDCl₃) δ 2.27 (s, 3H), 3.61 (s, 3H), 3.81 (s, 6H), 6.38 (d, IH, J = 12 Hz), 6.48 (d, IH, J = 12 Hz), 6.77 (d, IH, J = 1.8 Hz), 6.83 (d, IH, J = 8.4 Hz), 6.96 (d, IH, J = 1.5 Hz), 7.09 (dd, IH, J = 8.4 Hz, J = 2.4 Hz), and 7.26 (s, IH); ¹³C-NMR (125 MHz, CDCl₃) δ 20.61, 55.67, 55.93, 60.44, 92.07, 112.07, 112.92, 123.17, 127.63, 127.74, 129.39, 129.65, 103.97, 134.96, 139.49, 147.99, 150.39, 152.05 and 168.81; HRMS calcd for C₁₉H₂₀IO₅ 455.0355 [M+H]⁺, found 455.0356. Anal, calcd for C₁₉H₁₉IO₅ C, 50.24; H, 4.22. Found, C, 49.67; H, 4.18 %.

[0095] Dibenzyl 3,5-diiodo-4,4'-dimethoxy-Z-stiIbene 3'-0-phosphate (23).

[0096] An appropriate dibenzyl phosphate (0.38 g, 55% yield) was obtained

(0.46 g, 0.91 mmol) as described above for the synthesis of iodide 10a. Colorless oil: bp dec 220 ⁰C; ¹H-NMR (300 MHz, CDCl₃) δ 3.78 (s, 3 H), 3.81 (s, 6 H), 5.13 (s, 2 H), 5.16 (s, 2 H), 6.28 (d, 1 H, JΞ12 HZ), 6.42 (d, 1 H, J 12 Hz), 6.78 (d, 1 H, J 9 Hz), 7.00 (d, 1 H, J 8.7 Hz), 7.07 (s, 1 H), 7.33 (s, 10 H) and 7.64 (s, 2 H); ¹³C-NMR (100 MHz, CDCl₃) δ 55.96, 60.71, 69.83, 69.89, 90.15, 112.40, 122.23, 122.26, 125.60, 126.20, 126.21, 127.93, 128.49, 128.55, 130.66, 137.12, 139.92 and 157.68; HRMS calcd for C₃₀H₂₈I₂O₆P 768.9713 [M+H]⁺, found 768.9699; ³¹P-NMR (162 MHz, CDCl₃) δ -5.51.

[0097] General procedures for syntheses of the phosphoric acids and derivatives.

[0098] Method A. Each of the metal cation phosphate salts was obtained by the procedure outlined herein for preparing the potassium salt 20c, except for the metal counterions introduced by treatment of the phosphoric acid using either lithium hydroxide or sodium methoxide.

[0099] Method B. Dowex-50W (2g) (HCR-W2) was placed in a column and washed successively with CH₃OH (50 mL), 1 N HCl (until pH 1), water (until pH 7), base/amine/amino acid (until pH 7-14) and water (until pH 7). The column was recycled. The potassium salt or its corresponding diiodo phosphate salt (about 25 mg) was dissolved in de-ionized water (1 mL) and applied to a Dowex-50W (HCR- W2) resin column (bearing the appropriate amine or amino acid methyl ester) and developed with approximately 40 mL of water. The eluent was concentrated by freeze drying to give the required cation derivative.

[00100] Method C. Amino Acid Methyl Esters. The amino acid methyl ester hydrochloride was neutralized in CH₃OH solution by adding potassium carbonate. Ether was added to precipitate the potassium chloride and the solution was filtered and concentrated. The amino acid methyl ester residue was then applied to the Dowex-50W (HCR-W2) resin column as described in Method B. [00101] Potassium 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-0-phosphate

(20c).

[00102] Trimethylbromosilatie (277 μL, 1.8 mmol) was added to a cooled

(0⁰C) solution of phosphate 9a in DCM (40 mL). After stirring for 90 minutes, sodium thiosulfate (10% aq., 10 mL) was added and the mixture was stirred for an additional 1 minute. The phases were separated and the aqueous phase extracted with DCM (20 mL), followed by EtOAc (2 x 20 mL). The combined organic extracts were dried, filtered and concentrated to afford the phosphoric acid intermediate as a clear oil. After drying (high vacuum) for 1 hour, the oil was dissolved in CH₃OH (10 mL), cooled to 0°C, and KOH (1.8 mL, 1 M sol. in CH₃OH) was added. The mixture was stirred for 20 minutes, the precipitate was collected and triturated with ether to afford the potassium salt as a colorless solid: mp 197-198 ⁰C (dec); ¹H-NMR (300 MHz, D₂O) δ 3.51 (s, 3 H), 3.64 (s, 3 H), 3.71 (s, 3 H), 6.33 (d, 1 H, J = 12 Hz), 6.51 (d, 1 H, J = 12 Hz), 6.70 (s, 2 H), 6.84 (s, 1 H) and 7.22 (s, 2 H); and ³¹P-NMR (162 MHz, D₂O) δ 0.94.

[00103] Sodium 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-0-phosphate (20a).

[00104] Isolated as a colorless solid: mp 194-195 ⁰C (dec); ¹H-NMR (300

MHz, D₂O) δ 3.50 (s, 3 H), 3.67 (s, 3 H), 3.68 (s, 3 H), 6.50 (d, 1 H, J = 12 Hz), 6.70 (d, 1 H, J = 12 Hz), 6.72 (s, 1 H), 6.77 (s, 1 H), 6.79 (s, 1 H), 7.01 (s, 1 H) and 7.13 (s, 1 H).

[00105] Lithium 3-iodo-4,4',5-trimethoxy-z-stiIbene 3'-0-phosphate (lie).

[00106] Discovered as a colorless solid: mp 245-275 °C (dec); ¹H-NMR (400

MHz, D₂O) δ 3.50 (s, 3 H), 3.62 (s, 3 H), 3.66 (s, 3 H), 6.33 (d, 1 H, J = 12 Hz), 6.49 (d, 1 H, J = 12 Hz), 6.70 (s, 2 H), 6.83 (s, 1 H), 7.20 (s, 1 H) and 7.22 (s, 1 H).

[00107] Morpholine 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-0-phosphate

(2Od).

[00108] Another colorless oil: ¹H-NMR (300 MHz , D₂O) δ 3.11-3.15 (m, 8

H), 3.50 (s, 3 H), 3.63 (s, 3 H), 3.68 (s, 3 H), 3.77-3.81 (m, 8 H), 6.33 (d, 1 H, J 12 Hz), 6.50 (d, 1 H, J 12 Hz), 6.73 (s, 2 H), 6.82 (s, 1 H), 7.18 (s, 1 H) and 7.20 (s, 1 H).

[00109] Piperidene 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-0-phosphate

(2Oe). [00110] Colorless oil: ¹H-NMR (300 MHz , D₂O) δ 1.51 (m, 4 H), 1.62 (m, 8

H), 3.00 (t, 8 H, J = 6 Hz), 3.51 (s, 3 H), 3.63 (s, 3 H), 3.67 (s, 3 H), 6.34 (d, I HJ = 12.6 Hz), 6.51 (d, 1 H, J = 12.6 Hz), 6.72 (s, 2 H), 6.83 (s, 1 H) and 7.21 (s, 1 H).

[00111] Glycine-O-Me 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-0-phosphate

(2Of).

[00112] Obtained as a colorless solid: mp 74-78 °C; ¹H-NMR (300 MHz ,

D₂O) δ 3.48 (s, 3 H), 3.61 (s, 3 H), 3.67 (s, 3 H)₅ 3.68 (s, 3 H), 3.76 (s, 2 H), 6.30 (d, 1 H, J = 12 Hz), 6.46 (d, 1 H, J = 12 Hz), 6.69-6.77 (m, 3 H), 7.10 (s, 1 H) and 7.16 (s, 1 H).

[00113] Tryptophan-O-Me 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-O- phosphate (2Og).

[00114] Colorless solid: mp 108-112 °C; ¹H-NMR (300 MHz , DMSO) δ 3.19

(d, 2 H, J = 6.3 Hz), 3.56 (s, 3 H), 3.61 (s, 3 H), 3.66 (s, 3 H), 3.70 (s, 3 H), 4.09 (t, 1 H, J = 6 Hz), 6.35 (d, 1 H, J = 12 Hz), 6.47 (d, 1 H, J = 12 Hz), 6.81-6.85 (m, 2 H), 6.98 (t, I HJ = 7.2 Hz), 7.07 (t, 1 H, J = 8.1 Hz), 7.18 (s, 1 H), 7.22 (s, 1 H), 7.34 (d, 1 H, J = 8.1 Hz), 7.40 (s, 1 H) and 7.46 (d, I HJ = 7.2 Hz).

[00115] Tris 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-0-phosphate (2Oh).

[00116] Colorless solid: mp 75-81 °C; ¹H-NMR (300 MHz , DMSO) δ 3.42 (s,

9 H), 3.57 (s, 3 H), 3.67 (s, 3 H), 3.70 (s, 3 H), 6.35 (d, 1 H, J = 12 Hz), 6.48 (d, 1 H, J = 12 Hz), 6.76 (d, I HJ = 8.4 Hz), 6.81 (d, 1 HJ = 8.7 Hz), 6.92 (s, 1 H), 7.22 (s, 1 H) and 7.42 (s, 1 H).

[00117] Potassium 3,5-diiodo-4,4'dimethoxy-z-stilbene 3'-0-phosphate

(24a).

[00118] Phosphate (0.20 g, 80%) was obtained from appropriate ester 9c (0.29 g, 0.38 mmol) as described above for the synthesis of 20c, except the phosphoric acid was insoluble in EtOAc and DCM, so the aqueous phase was extracted with butyl alcohol (3 x 25 rnL). The potassium salt was a colorless solid: mp 210-215 °C (dec); ¹H-NMR (300 MHz, D₂O) δ 3.69 (s, 6 H), 6.27 (d, 1 HJ = 12 Hz), 6.49 (d, 1 HJ = 12 Hz), 6.64 (s, 2 H), 7.20 (s, 1 H) and 7.62 (s, 2 H); ³¹P-NMR (162 MHz, D₂O) δ 0.973.

[00119] Sodium 3,5-diiodo-4,4'dimethoxy-z-stilbene 3'-0-phosphate (24b). [00120] Obtained as a colorless solid: mp 215-234 ⁰C (dec); ¹H-NMR (300

MHz, D₂O) δ 3.69 (s, 3 H), 3.72 (s, 3 H), 6.29 (d, 1 H, J = 12 Hz), 6.49 (d, 1 H, J = 12 Hz), 6.69 (s, 2 H), 7.20 (s, 1 H) and 7.64 (s, 2 H).

[00121] Lithium 3,5-diiodo-4,4'dimethoxy-z-stilbene 3'-0-phosphate (24c).

[00122] A colorless solid melting at 250-270 °C (dec); ¹H-NMR (300 MHz₅

D₂O) δ 3.68 (s, 3 H), 3.71 (s, 3 H), 6.28 (d, 1 H, J = 12 Hz), 6.49 (d, 1 H, J = 12 Hz), 6.68 (s, 2 H), 7.19 (s, 1 H) and 7.64 (s, 2 H). ³¹P NMR (162 MHz, D₂O) δ 0.96.

[00123] Morpholine 3,5-diiodo-4,4'dimethoxy-z-stilbene 3'-0-phosphate

(24d).

[00124] Colorless waxy solid; mp 75-80 °C; ¹H-NMR (300 MHz, DMSO) δ

2.96-2.99 (m, 8 H), 3.74-3.77 (m, 8 H), 3.82 (s, 3 H), 3.83 (s, 3 H), 6.43 (d, 1 H, J = 12.5 Hz), 6.60 (d, 1 H, J = 12.5 Hz), 6.86 (d, 1 H, J = 8.2 Hz), 6.93 (d, 1 H, J = 8.2 Hz), 7.49 (s, 1 H) and 7.78 (s, 2 H).

[00125] Piperidine 3,5-diiodo-4,4'dimethoxy-z-stilbene 3^f-0-phosphate

(24e).

[00126] Isolated as a colorless oil; ¹H-NMR (300 MHz, DMSO) δ 1.51 (br s,

12 H), 2.79-2.81 (m, 8 H), 3.70 (s, 3 H), 3.72 (s, 3 H), 6.31 (d, 1 H, J = 12 Hz), 6.49 (d, 1 H, J = 12 Hz), 6.73 (d, I H₁ J= 8.4 Hz), 6.80 (d, 1 H, J = 8.4 Hz), 7.40 (s, 1 H) and 7.61 (s, 1 H).

[00127] Glycine-OMe 3,5-diiodo-4,4'dimethoxy-z-stilbene 3'-O-phosphate

(24f).

[00128] Colorless solid; mp 90-97 ⁰C; ¹H-NMR (300 MHz, DMSO) δ 3.61 (s,

4 H), 3.68 (s, 6 H), 3.70 (s, 3 H), 3.72 (s, 3 H), 6.31 (d, 1 H, J = 12 Hz), 6.49 (d, 1 H, J = 12 Hz), 6.72 (d, 1 H, J = 9.6 Hz), 6.80 (d, 1 H, J = 8.1 Hz), 7.37 (s, 1 H) and 7.67 (s, 1 H).

[00129] Tryptophan-OMe 3,5-diiodo-4,4'dimethoxy-z-stilbene 3'-O- phosphate (24g).

[00130] Collected as a colorless solid; melting at 125-130 °C; ¹H-NMR (300

MHz, DMSO) δ 3.34 (d, I HJ = 6.5 Hz), 3.36 (d, 1 H, J = 6.5 Hz), 3.66 (s, 3 H), 3.70 (s, 3 H), 3.72 (s, 3 H), 4.32 (t, 1 H, J = 6.5 Hz), 6.31 (d, 1 H, J = 12 Hz), 6.48 (d, 1 H, J = 12 Hz), 6.78-6.81 (m, 2 H), 7.01 (s, 1 H), 7.05 (t, 1 H, J = 7 Hz), 7.13 (t, 1 H, J = 7 Hz), 7.39 (d, 1 H, J = 7.5 Hz), 7.47 (d, 1 H, J = 8 Hz) and 7.60 (s, 1 H).

[00131] Tris 3,5-diiodo-4,4'dimethoxy-z-stilbene 3'-0-phosphate (24h). [00132] Colorless solid; mp 115-120 ⁰C; ¹H-NMR (300 MHz, DMSO) δ 3.34

(s, 18 H), 3.69 (s, 3 H), 3.71 (s, 3 H), 6.30 (d, 1 H, J = 12 Hz)₃ 6.47 (d, 1 H, J = 12 Hz), 6.70 (d, 1 H, J = 8.1 Hz), 6.78 (d, 1 H, J = 8.1 Hz), 7.37 (s, 1 H) and 7.67 (s, 2 H).

[00133] Cancer Cell Line Procedures

[00134] Inhibition of human cancer cell growth was assessed using the

National Cancer Institute's standard sulforhodamine B assay. After 48 hours, the plates were fixed with trichloracetic acid, stained with sulforhodamine B and read with an automated microplate reader. A growth inhibition of 50% (GI₅₀ or the drug concentration causing a 50% reduction in the net protein increase) was calculated from optical density data with Immunosoft software. Inhibition of the mouse leukemia P388 cells was assessed in a 10% horse serum/Fisher medium solution for 24 hours, followed by a 48 hour incubation with serial dilutions of the compounds. Cell growth inhibition (ED₅₀) was then calculated using a Zl Beckman/Coulter particle counter.

[00135] Tubulin Evaluations: Tubulin polymerization was evaluated by turbidirnetry at 35 ntn using Beckman DU7400/7500 spectrophotometers as known to one of skill in the art. Varying concentrations of the compound were preincubated with lOμM. Incubation was for 10 minutes at 37°C.

[00136] Antiangiogenesis

[00137] HUVEC Procedures

[00138] In vitro Matrigel antiangiogenesis assays were implemented according to the Developmental Therapeutics Program NCI/NIH protocols known to one of skill in the art. Matrigel, a basement membrane matrix, was obtained from BD Biosciences. Growth inhibition and cord formation assays were conducted using human umbilical vein endothelial cells obtained from GlycoTech. HUVEC cells were grown in EGM-2 medium.

[00139] Cord formation Assay

[00140] An aliquot of sixty microliters was placed in each well of an ice-cold

96-well plate. The plates were then left for 15 minutes at room temperature, then incubated for 30 minutes at 37°C to permit the matrigel to polymerize. Meanwhile, HUVEC cells were harvested and diluted to a concentration of 2xlO⁵ cells/ml. A solution of lOOμL containing the compounds to be tested was added next. After 24 hours incubation, pictures were taken for each concentration using an inverted Nikon Diaphot microscope and DlOO digital camera. Drug effect was assessed, compared to untreated controls, by measuring the length of cords formed and number of junctions.

[00141] The standard sulforhodamine B assay (see Cancer Cell Line

Procedures above) was used to evaluate results using HUVEC cells. IC_5O or ED₅₀ (drug concentration causing 50% inhibition) was calculated from the plotted data.

ADMINISTRATION

[00142] Dosages

[00143] The dosage to be administered to humans and other animals requiring treatment will depend upon the identity of the neoplastic disease or microbial infection; the type of host involved, including its age, health and weight; the kind of concurrent treatment, if any; the frequency of treatment and therapeutic ratio. Hereinafter are described various possible dosages and methods of administration, with the understanding that the following are intended to be illustrative only, and that the actual dosages to be administered, and methods of administration or delivery may vary therefrom. The proper dosages and administration forms and methods may be determined by one of skill in the art.

[00144] Illustratively, anticipated dosage levels of the administered active ingredients may be in the following ranges: intravenous, 0.1 to about 200 mg/kg; intramuscular, 1 to about 500 mg/kg; orally, 5 to about 1000 mg/kg; intranasal instillation, 5 to about 1000 mg/kg; and aerosol, 5 to about 1000 mg/k of host body weight.

[00145] Expressed in terms of concentration, an active ingredient can be present in the compositions of the present invention for localized use about the cutis, intranasally, pharyngolaryngeally, bronchially, intravaginally, rectally, or ocularly in concentration of from about 0.01 to about 50% w/w of the composition; preferably about 1 to about 20% w/w of the composition; and for parenteral use in a concentration of from about 0.05 to about 50% w/v of the composition and preferably from about 5 to about 20% w/v.

[00146] The compositions of the present invention are intended to be presented for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, suppositories, sterile parenteral solutions or suspensions, sterile non- parenteral solutions of suspensions, and oral solutions or suspensions and the like, containing suitable quantities of an active ingredient. Other dosage forms known in the art may be used.

[00147] For oral administration either solid or fluid unit dosage forms may be prepared. [00148] Powders may be prepared by comminuting the active ingredient to a suitably fine size and mixing with a similarly comminuted diluent. The diluent can be an edible carbohydrate material such as lactose or starch. Advantageously, a sweetening agent or sugar is present as well as a flavoring oil.

[00149] Capsules may be produced by preparing a powder mixture as hereinbefore described and filling into formed gelatin sheaths. Advantageously, as an adjuvant to the filling operation, a lubricant such as talc, magnesium stearate, calcium stearate and the like is added to the powder mixture before the filling operation.

[00150] Soft gelatin capsules may be prepared by machine encapsulation of a slurry of active ingredients with an acceptable vegetable oil, light liquid petrolatum or other inert oil or triglyceride or other pharmaceutically acceptable carrier.

[00151] Tablets may be made by preparing a powder mixture, granulating or slugging, adding a lubricant and pressing into tablets. The powder mixture may be prepared by mixing an active ingredient, suitably comminuted, with a diluent or base such as starch, lactose, kaolin, dicalcium phosphate and the like. The powder mixture can be granulated by wetting with a binder such as corn syrup, gelatin solution, methylcellulose solution or acacia mucilage and forcing through a screen. As an alternative to granulating, the powder mixture may be slugged, i.e., run through the tablet machine and the resulting imperfectly formed tablets broken into pieces (slugs). The slugs can be lubricated to prevent sticking to the tablet-forming dies by means of the addition of stearic acid, a stearic salt, talc or mineral oil. The lubricated mixture is then compressed into tablets.

[00152] Advantageously, for protection of the tablet itself and/or to ease swallowing, the tablet can be provided with a pharmaceutically acceptable coating such as a sealing coat or enteric coat of shellac, a coating of sugar and methylcellulose and polish coating of carnauba wax.

[00153] Fluid unit dosage forms for oral administration such as in syrups, elixirs and suspensions may be prepared wherein each teaspoonful of composition contains a predetermined amount of an active ingredient for administration.

[00154] The water-soluble forms may be dissolved in an aqueous vehicle together with sugar, flavoring agents and preservatives to form a syrup. An elixir is prepared by using a hydroalcoholic vehicle with suitable sweeteners together with a flavoring agent. Suspensions may be prepared of the insoluble forms with a suitable vehicle with the aid of a pharmaceutically acceptable suspending agent such as acacia, tragacanth, methylcellulose and the like.

[00155] For parenteral administration, fluid unit dosage forms may be prepared utilizing an active ingredient and a sterile vehicle, for example, water. The active ingredient, depending on the form and concentration used, can be either suspended or dissolved in the vehicle, hi preparing solutions the water-soluble active ingredient can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampule and sealing. Advantageously, adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle. Parenteral suspensions may be prepared in substantially the same manner except that an active ingredient is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The active ingredient may be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a pharmaceutically acceptable surfactant or wetting agent may be included in the composition to facilitate uniform distribution of the active ingredient.

[00156] In addition to oral and parenteral administration, the rectal and vaginal routes can be utilized. An active ingredient can be administered by means of a suppository. A vehicle which has a melting point at about body temperature or one that is readily soluble can be utilized. For example, cocoa butter and various polyethylene glycols (Carbowaxes) can serve as the vehicle.

[00157] For intranasal installation, a fluid unit dosage form may be prepared utilizing an active ingredient and a suitable pharmaceutical vehicle, such as purified water, a dry powder, can be formulated when insufflation is the administration of choice.

[00158] For use as aerosols, the active ingredients may be packaged in a pressurized aerosol container together with a gaseous or liquefied propellant, for example, dichlorodifluoromethane, carbon dioxide, nitrogen, propane, and the like, with the usual adjuvants such as cosolvents and wetting agents, as may be necessary or desirable.

[00159] The term "unit dosage form" as used in the specification and claims refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitation inherent in the art of compounding such an active material for therapeutic use in humans, as disclosed in this specification, these being features of the present invention. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, troches, suppositories, powder packets, wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampules, vials, segregated multiples of any of the foregoing, and other forms as herein described.

[00160] The active ingredients to be employed as antineoplastic agents may be prepared in such unit dosage form with the employment of pharmaceutical materials which themselves are available in the art and can be prepared by established procedures. The following preparations are illustrative of the preparation of the unit dosage forms of the present invention, and not as a limitation thereof. Shown in the following are examples of dosage forms for the compounds of the present invention, in which the notation "active ingredient" signifies the compounds described herein.

COMPOSITION "A"

Hard-Gelatin Capsules

[00161] One thousand two-piece hard gelatin capsules for oral use, each capsule containing 200 mg of an active ingredient may be prepared from the following types and amounts of ingredients:

Active ingredient, micronized 20O g

Corn Starch 20 g

Talc 2O g

Magnesium stearate 2 g

[00162] The active ingredient, finely divided by means of an air micronizer, is added to the other finely powdered ingredients, mixed thoroughly and then encapsulated in the usual manner.

[00163] Using the procedure above, capsules may be similarly prepared containing an active ingredient in 50, 250 and 500 nig amounts by substituting 50 g, 250 g and 500 g of an active ingredient for the 200 g used above.

COMPOSITION "B"

Soft Gelatin Capsules [00164] One-piece soft gelatin capsules for oral use, each containing 200 mg of an active ingredient, finely divided by means of an air micronizer, may prepared by first suspending the compound in 0.5 ml of corn oil to render the material capsulatable and then encapsulating in the above manner.

COMPOSITION "C"

Tablets

[00165] One thousand tablets, each containing 200 mg of an active ingredient, may be prepared from the following types and amounts of ingredients:

Active ingredient, micromzed 20O g

Lactose 300 g

Corn starch 5O g

Magnesium stearate 4 g

Light liquid petrolatum 5 g

[00166] The active ingredient, finely divided by means of an air micronizer, is added to the other ingredients and then thoroughly mixed and slugged. The slugs are broken down by forcing them through a Number Sixteen screen. The resulting granules are then compressed into tablets, each tablet containing 200 mg of the active ingredient.

[00167] Using the procedure above, tablets may similarly prepared containing an active ingredient in 250 mg and 100 mg amounts by substituting 250 g and 100 g of an active ingredient for the 200 g used above.

COMPOSITION "D"

Oral Suspension

[00168] One liter of an aqueous suspension for oral use, containing in each teaspoonful (5 ml) dose, 50 mg of an active ingredient, may be prepared from the following types and amounts of ingredients:

Active ingredient, micronized 1O g

Citric acid 2 g

Benzoic acid I g

Sucrose 79O g

Tragacanth 5 g

Lemon Oil 2 g

Deionized water, q.s. 1000 ml [00169] The citric acid, benzoic acid, sucrose, tragacanth and lemon oil are dispersed in sufficient water to make 850 ml of suspension. The active ingredient, finely divided by means of an air micronizer, is stirred into the syrup unit uniformly distributed. Sufficient water is added to make 1000 ml.

COMPOSITION "E"

Parenteral Product

[00170] A sterile aqueous suspension for parenteral injection, containing 30 mg of an active ingredient in each milliliter for treating a neoplastic disease, may be prepared from the following types and amounts of ingredients:

Active ingredient, micronized 30 g

POLYSORBATE 80 5 g

Methylparaben 2.5 g

Propylparaben 0.17 g

Water for injection, q. s. 1000 ml.

[00171] AU the ingredients, except the active ingredient, are dissolved in the water and the solution sterilized by filtration. To the sterile solution is added the sterilized active ingredient, finely divided by means of an air micronizer, and the final suspension is filled into sterile vials and the vials sealed.

COMPOSITION "F"

Suppository, Rectal and Vaginal

[00172] One thousand suppositories, each weighing 2.5 g and containing 200 mg of an active ingredient may be prepared from the following types and amounts of ingredients:

Active ingredient, micronized 15 g

Propylene glycol 15O g

Polyethylene glycol #4000, q.s. 2,500 g

[00173] The active ingredient is finely divided by means of an air micronizer and added to the propylene glycol and the mixture passed through a colloid mill until uniformly dispersed. The polyethylene glycol is melted and the propylene glycol dispersion is added slowly with stirring. The suspension is poured into unchilled molds at 40 ⁰C. The composition is allowed to cool and solidify and then removed from the mold and each suppository foil wrapped.

COMPOSITION "G"

Intranasal Suspension

[00174] One liter of a sterile aqueous suspension for intranasal instillation, containing 20 mg of an active ingredient in each milliliter, may be prepared from the following types and amounts of ingredients:

Active ingredient, micronized 15 g

POLYSORBATE 80 5 g

Methylparaben 2.5 g

Propylparaben 0.17 g

Deionized water, q.s. 1000 ml.

[00175] All the ingredients, except the active ingredient, are dissolved in the water and the solution sterilized by filtration. To the sterile solution is added the sterilized active ingredient, finely divided by means of an air micronizer, and the final suspension is aseptically filled into sterile containers.

COMPOSITION "H"

Powder

[00176] Five grams of active ingredient in bulk form is finely divided by means of an air micronizer. The micronized powder is placed in a shaker-type container.

COMPOSITION "I"

Oral Powder

[00177] One hundred grams of an active ingredient in bulk form may be finely divided by means of an air micronizer. The micronized powder is divided into individual doses of 200 mg and packaged.

COMPOSITION "J"

Insulation [00178] One hundred grams of an active ingredient in bulk form is finely divided by means of an air micronizer.

[00179] It is of course understood that such modifications, alterations and adaptations as will readily occur to the artisan confronted with this disclosure are intended within the spirit of the present invention.

Table I. Human cancer cell line inhibition (GI₅₀ μg/mL) and murine P388 lymphocytic leukemia inhibitory activity (EDsoμg/ml) of halocombstatins and other compounds.

Compound Leukemia Pancreas- Breast adn CNS Lung-NSC Colon Prostate P388 a BXPC-3 MCF-7 SF268 NCI-H460 KM20L2 DU-145

Ia 0.0003 0.39 - O.001 0.0006 0.061 0.0008

Ib 0.0004 - - 0.036 0.029 0.034 -

2a 0.251 4.4 - - 0.74 0.061 0.17

2b <0.01 1.5 0.024 0.036 0.038 0.53 0.034

3a 0.257 2.3 0.49 0.0083 0.19 1.2 0.0043

3b 0.305 2.8 0.92 0.052 0.45 3.5 0.048

11a <0.01 0.016 <0.01 <0.01 <0.01 1.1 <0.01 lib 0.253 2.2 0.051 0.35 0.18 0.53 0.18

12a <0.01 0.043 <0.001 O.001 O.001 0.15 O.001

12b 0.027 0.59 0.041 0.048 0.034 1.4 0.038

13a <0.01 0.16 O.001 <0.001 O.001 0.086 O.001

13b 0.0174 1.6 0.14 0.18 0.15 1.2 0.13

14a <0.01 0.11 0.00022 0.00035 0.00019 0.15 0.00052

14b 0.189 2.7 0.18 0.55 0.21 1.7 0.27

18a 0.0298 0.59 0.0044 0.0051 0.0094 1.5 0.0036

19a <0.01 0.093 0.0041 0.0034 0.0028 0.23 0.0046

19b <0.01 0.13 0.0039 0.0030 0.0026 0.11 0.0066

19c <0.01 0.20 0.0035 0.0032 0.0029 0.24 0.0028

19d <0.01 0.15 0.0044 0.0064 0.0066 0.48 0.0079

19e <0.01 0.56 0.043 0.023 0.041 2.6 0.042

19f 0.288 O.001 0.0022 0.0022 0.0068 0.37 0.0063

19g <0.01 0.074 0.0045 0.0053 0.0039 0.27 0.0045

19h <0.01 0.17 0.0049 0.0067 0.0047 0.45 0.0049

19i 2.22 >10 3.2 4.1 2.9 >10 2.8

20a <0.01 0.47 0.012 0.0052 0.0031 0.37 0.0078

Table Ia. Solubilities of some of the synthetic modifications, human cancer cell line growth inhibition (GI₅₀ μg/niL) and murine P388 lymphocytic leukemia inhibitory activity (ED₅₀μg/ml).

Compound Solubility³ Leukemia Pancreas Breast CNS Lung-NSC Colon Prostate P388 BXPC-3 MCF-7 SF268 NCI-H460 KM20L2 DU-145 (mg/mL)

Ia - 0.0003 0.39 - <0.001 0.0006 0.061 0.0048

Ib - 0.0004 — - 0.036 0.029 0.034 —

3a - 0.26 2.3 0.49 0.0083 0.19 1.2 0.0043

11a - 0.0020 0.745 0.0027 0.0016 0.0032 >1 0.019

14a - 0.0020 0.048 0.00022 0.00018 0.00029 0.328 0.00018

14b - 0.189 2.7 0.18 0.55 0.21 1.7 0.27

22a 0.0028 0.038 0.0027 0.0036 0.0034 0.15 0.0021

22b - >10 3.0 0.94 3.3 3.4 >10 5.8

22c ~ 0.0089 0.040 0.00053 0.0023 0.0032 0.075 0.0020

14c ~ 0.022 0.080 O.0001 0.0002 0.00031 0.16 0.00026

20c 14 0.0021 0.381 0.0064 0.0057 0.0043 >1 0.0038

20a 2 0.0020 0.469 0.018 0.018 0.017 >1 0.011 lie >2.4 0.017 0.490 0.0038 0.0040 0.0039 >1 0.0043

2Od — 0.0032 0.21 0.0047 0.0037 0.0036 0.24 0.0026

2Oe >4 0.0026 0.32 0.0065 0.0044 0.0036 0.51 0.0029

2Of >2 0.0026 0.16 0.0044 0.0033 0.0031 0.32 0.0021

2Og — 0.0022 0.26 0.035 0.0097 0.0034 0.59 0.0030

2Oh ~ 0.0029 0.37 0.0048 0.0043 0.0040 0.40 0.0047

24a 22 0.0034 0.44 0.050 0.053 0.046 >1 0.028

24b 2 0.030 >1 0.066 0.051 0.327 >1 0.242

24c >4 0.021 0.37 0.051 0.050 0.050 >1 0.032

24d 0.014 0.35 0.066 0.054 0.033 >1 0.028

24e — 0.011 0.33 0.070 0.041 0.025 >1 0.025

24f 0.011 0.36 0.10 0.054 0.030 >1 0.023

24g - 0.017 0.37 0.22 0.086 0.033 >1 0.026

24h 0.026 0.33 0.047 0.040 0.025 0.94 0.021

^aSolubility values were obtained using 1 niL D₂O at 25⁰C Key to Table Ia

Ia = combretastatin A-4

Ib = sodium combretastatin A-4 phosphate Key to Table Ia (cont'd)

3a = combretastatin A3

Ha = fluorocombstatin

14a=3-Iodo-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene

14b=3-Iodo-4,4',5-trimethoxy-3'-hydroxy-E-stilbene

22a=3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-Z-stilbene

22b=3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-E-stilbene

22c=3,5-diiodo-4,4'-dimethoxy-3'-acetyl-Z-stilbene

14c=3-iodo-4,4',5-trimethoxy-3'acetyl-Z-stilbene

20c = Potassium 3-iodo, 4,4',5 trimethoxy-Z-stilbene 3'-O-phosphate

20a = Sodium 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate lie = Lithium 3 iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate

2Od = Morpholine 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate

2Oe = Piperidine 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate

2Of = Glycine-O-Me-3-iodo, 4,4',5-trimethoxy-Z-stilbene-3'-O-phosphate

2Og = Tryptophan-O-Me-3-iodo-4,4',5-trimetnoxy-Z-stilbene 3'-O-phosphate

2Oh = Tris-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate

24a = Potassium 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate

24b = Sodium 3,5-diiodo-4,4'-dimethoxy-Z-stilbene 3'-O-phosphate

24c = Lithium 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate

24d = Morpholine 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate

24e = Piperidine 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'~O-phosphate

24f = Glycine-O-Me 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate

24g = Tryptophan-OMe-3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate

24h = Tris 3,5 diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate

Table II. Inhibition of tubulin polymerization and binding of [³H] colchicine to tubulin by halocombstatins

Inhibition of polymerization Inhibition of colchicine binding

Compound IC₅₀ (μM) ± S.D. % inhibition ± S. D.

Ia 1.8 ±0.2 81 ±3

11a 1.5 ±0.2 75 ±6

12a 1.6 ±0.3 85 ±4

13a 1.5 ±0.2 89±2

14a 1.6 ±0.2 84±7

Table III. Length of Cords Formed, Number of Junctions and Relative Percent Growth

Table IV. Antimicrobial activities of halocombstatins and other compounds

Range of minimum inhibitory concentration (μg/ml)

Compound Microorganism 11a lib 12a 14a 14b 13a 13b 18a 20a

Cryptococcus neoformans 64 64 64 32-64 64 * * * *

Candida albicans * * * * * * * * *

Staphylococcus aureus * 32-64 * * 8-64 * * * *

Streptococcus pneumoniae 64 64 32-64 64 * * * * *

Enterococcus faecalis * * * * * * * * *

Micrococcus luteus 32-64 16-32 32 16-32 4-8 32-64 * * *

Escherichia coli * * * * * * * * *

Enterobacter cloacae * * * * * * * *

Stenotrophomonas maltophilia * * * * * * * * *

Neisseria gonorrhoeae 32 8-16 16 16-32 4-16 32-64 * 16 16-32

* = no inhibition at 64 μ.g/ml

Table V. Antimicrobial activities of iodocombstatins

Range of MIC (μg/ml) Compound

Microorganism ATCC or Ha 14a 22a 22b 22c 20c 20a Hc 2Od 2Oe 2Of 2Og 2Oh 24a 24b 24c 24d 24e 24f 24g 24h_

(Presque

Isle)#

Cryptococcus 90112 * 64 neoformans

Candida 90028 * * albicans

Staphylococcus 29213 * aureus

Streptococcus 6303 * * pneumoniae

Enterococcus 29212 * * faecalis

Micrococcus (456) * * 4-16 2-4 luteus

Escherichia 25922 * coli

Enterobacter 13047 * * cloacae

Stenotrophomon 13637 * * as maltophilia

Neisseria 49226 64 * 16-32 * 4-8 32-64 <0.5-4 32-64 <0.5-2 <0.5 <0.5 <0.5-l <0.5 <0.5-2 gonorrhoeae

Key for Table V

Ha = Fluorocombstatin

14a = 3-iodo-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene

22a = 3,5-diiodo-4,4'-dimethoxy-3' hydroxy-Z-stilbene

22b = 3,5-diiodo-4,4'-dimethoxy-3' hydroxy-E-stilbene

22c = 3,5-diiodo-4,4'-dimethoxy-3'-acetyl-Z-stilbene

20c = Potassium 3 iodo-4,4'5-trimethoxy-Z-stilbene 3'-O-phosphate

Key for Table V (cont'd)

20a = Sodium 3 iodo-4,4',5 trimethoxy-Z-stilbene-3'-O-phosphate

Hc = Lithium-3-iodo-4,4'5 trimethoxy-Z-stilbene 3'0-phosphate

2Od = Morpholine 3 iodo-4,4'5-trimethoxy-Z-stilbene-3'-O phosphate

2Oe = Piperidene 3-iodo-4,4',5-trimethoxy-Z-stilbene-3'O phosphate

20f= Glycine-O-Me-3-iodo-4,4^I,5-trimethoxy-Z-stilbene-3'-O-phosphate

2Og = Tryptophan-O-Me-3'-iodo-4,4',5 trimethoxy-Z-stilbene 3'-O-phosphate

2Oh = Tris-3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate

24a = Potassium 3,5 diiodo-4,4¹ dimethoxy-Z-stilbene 3'-O-phosphate

24b = Sodium 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate

24c = Lithium 3,5-diiodo-4, 4' dimethoxy-Z-stilbene 3¹O phosphate

24d = Morpholine 3,5 diiodo-4,4' dimethoxy-Z-stilbene 3-O-phosphate

24e = Piperdine 3,5 diiodo-4,4' dimethoxy-Z-stilbene 3'0-phosphate

24f = Glycine O Me 3,5-diiodo-4,4' dimethoxy-Z-stilbene-3'-O-phosphate

24g = Tryptophan-0 Me 3,5 diiodo 4,4' dimethoxy-Z-stilbene-3'-O-phosphate

24h = Tris 3,5-diodo-4,4' methoxy-Z-stilbene 3'0-phosphate £

Table VL Human Umbilical Vein Endothelial CeU (HUVEC)

Inhibition Values (GI₅₀) expressed in μg/mL.

Table VII. Human Anaplastic Thyroid Carcinoma Cell Line Inhibition Values (GI₅₀) expressed in μg/mL.

Claims

CLAIMSWhat is claimed is:

1. A compound having a structure as follows:

wherein X is F, Cl, Br or I.

2. A compound having a structure as follows:

wherein X is F, Cl, Br or I, and R is a metal cation such as Na, Li, K, Cs, Rb, Ca, Mg or is morpholine, piperidine, glycine-OCH₃, tryptophan-OCH₃ OrNH(CH₂OH)₃.

3. A compound having a structure as follows:

wherein X is F, Cl, Br or I, and Z is a metal cation such as Na, Li, K, Cs, Rb, Ca, Mg or is morpholine, piperidine, glycine-OCH₃, tryptophan-OCH₃ OrNH(CH₂OH)₃.

4. The compound of claim 1 wherein X=F and the compound is in the Z configuration.

5. The compound of claim 1 wherein X=I and the compound is in the Z configuration.

6. The compound of claim 2 wherein X=F.

7. The compound of claim 2 wherein X=I.

8. The compound of claim 3 wherein X=I.

9. A method for treating cancer comprising administering to a human or mammal an effective amount of a compound of claim 1.

10. A method for treating cancer comprising administering to a human or mammal an effective amount of a compound of claim 2.

11. A method for treating cancer comprising administering to a human or mammal an effective amount of a compound of claim 3.

12. The method of claim 9 wherein the cancer is thyroid cancer and in the compound X=I.

13. The method of claim 10 wherein the cancer is thyroid cancer and in the compound X=I.

14. The method of claim 11 wherein the cancer is thyroid cancer and in the compound X=I.

15. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier therefore.

16. A pharmaceutical composition comprising a compound of claim 2 and a pharmaceutically acceptable carrier therefore.

17. A pharmaceutical composition comprising a compound of claim 3 and a pharmaceutically acceptable carrier therefore.