Antineoplastic Agents. 548. Synthesis of Iodo- and Diiodocombstatin Phosphate Prodrugs

Abstract

Toward the objective of designing a structurally modified analogue of the combretastatin A-4 phosphate prodrug (1b) with the potential for increased specificity toward thyroid carcinoma, synthesis of a series of iodocombstatin phosphate (11a-h) and diiodocombstatin phosphate prodrugs (12a-h) has been accomplished. The diiodo series was obtained via 8a and 9c from condensation of 4 and 6, and the iodo sequence involved a parallel pathway. Both series of iodocombstatins were found to display significant to powerful inhibition of the growth of a panel of human cancer cell lines and of the murine P388 lymphocytic leukemia cell line. Of the diiodo series, 12a was also found to markedly inhibit growth of the pediatric neuroblastoma, and monoiodocombstatin 9a strongly inhibited HUVEC growth. Overall, the strongest activity was found against the breast, CNS, leukemia, lung, and prostate cancer cell lines and the least activity against the pancreas and colon lines. Parallel biological investigations of tubulin interaction, antiangiogenesis, and antimicrobial effects were also conducted.

From our initial discoveries over 30 years ago of the combretastatins, which are constituents of the African tree Combretum caffrum (Combretaceae), knowledge of their chemistry, biology, and medical potential has continued to advance.² Preclinical and clinical developments over the last twelve years have been rapidly accelerating, especially for combretastatin A-4 (CA4, 1a), in the form of the phosphate prodrug CA4P (1b), and combretastatin A-1 (CA1, 1c) as promising cancer vascular disrupting and ophthalmology drugs.^2,3 In turn, these encouraging developments have stimulated a variety of efforts devoted to synthesis and biological evaluation of combretastatin structural modifications, related primarily to CA4 (1a) and CA1 (1c). Recent reports include SAR studies that provide varying levels of cancer cell growth inhibition.⁴

For 2011, the estimated incidence of thyroid cancer in the United States is at about 48,000, including 1,740 deaths.^5a Most thyroid cancers are well differentiated papillary (about 80%) and follicular (about 14%) carcinomas. Both types of tumor cells are believed to be derived from follicular epithelial cells that produce thyroid hormone. Of the remaining thyroid malignancies, about 4% are medullary carcinoma (neuroendocrine) and about 2% are the exceptionally aggressive anaplastic carcinoma (median survival of 4-5 months and a near-100% lethal outcome).^2b,5b Significantly, the incidence of both follicular and anaplastic carcinomas is increased in populations residing in 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 (TSH), important in the regulation of thyroid gland growth and function, may be involved in the etiology of thyroid cancer. Previously used clinical treatments^5c,d for thyroid cancer include surgery, suppression of TSH, ¹³¹I-radiotherapy,^5e and anticancer drugs. Thyroid cancer is the fastest increasing (240% increase in the past 30 years) cancer type in both men and women in the United States; most of the increase in incidence of papillary carcinomas is probably due to earlier detection,^5f but the need for new and effective anticancer drugs, especially for the more aggressive carcinomas, is clear. Early in the human cancer phase I clinical trials of CA4 (1a), as the sodium phosphate prodrug 1b (CA4P, fosbretabulin), evidence for activity against otherwise refractory anaplastic thyroid carcinoma was observed, and this was further supported in a phase II clinical trial.^2b,6,7 The pioneering clinical studies of Remick and colleagues^2b,7a have led via phase II clinical trials of CA4P against anaplastic thyroid carcinoma to the current phase III level.^5b

The most active thyroid hormone components are triiodothyronine (2a) and its tetraiodo-derivative thyroxine (2b). Both are derived from 3,5-diiodotyrosine, with release controlled by TSH. Consequently, the focus of the present investigation was evaluation of iodo modifications of CA4P (1b), with the ultimate goal of obtaining enhanced concentration of drug in the thyroid carcinoma tissue. Of the continuing CA4 (1a) structural modification studies,⁴ those involving halogen substitutions have been primarily concerned with fluoro, chloro, bromo, and iodo derivatives.^4a,8 Our objective here was the synthesis and initial evaluation of the 3-iodo- and 3,5-diiodostilbene CA4 modifications, herein designated iodocombstatin (3a) and diiodocombstatin (3b), and of their phosphate prodrug modifications.

RESULTS AND DISCUSSION

The initial intermediates were synthesized by Wittig reaction of compound 4 with 3-iodo-4,5-dimethoxybenzaldehyde (5) and with 3,5-diiodo-4-methoxybenzaldehyde (6) to provide iodostilbenes 7 and 8, respectively, as Z/E mixtures (Scheme 1). The isomers were separated by silica gel column chromatography (CC), and subsequent deprotection with tetrabutylammonium fluoride (TBAF) afforded the corresponding phenols 9a-d. Iodocombstatins 9a and 9c were further characterized as their 3-acetyl derivatives 9e and 9f. The Z-isomers 9a and 9c were treated with dibenzylphosphite and CCl₄ in the presence of diisopropylethylamine (DIPEA) and N,N-dimethylaminopyridine (DMAP) to provide bisbenzylphosphates 10a and 10b, from which the benzyl ester protecting groups were cleaved with bromotrimethylsilane (TMSBr).^3b,8b,9 Subsequent treatment of the resulting phosphoric acids with the appropriate cation precursor readily afforded prodrugs 11a-h and 12a-h. In this sequence, the debenzylation step proved to be the most challenging: in order to minimize conversion of the Z- to E-geometry, presumably due to available bromonium ion, cooling (0 °C) was required, as well as an aqueous sodium thiosulfate treatment. Once the potassium salts of the prodrugs were in hand, a selection of alkali metal, ammonium and amino acid salts of the phosphates were synthesized by ion exchange chromatography to complete the 11a-h and 12a-h prodrug series.

Scheme 1.

The ability of the iodostilbenes to inhibit cancer cell growth was examined and compared to the activities of combretastatin A-3 (3c),¹⁰ fluorcombstatin (3d),^8b and combretastatin A-4 (1a) (Table 1). The iodostilbene phosphate salts all retained strong activity and, as expected, demonstrated markedly better aqueous solubility (Table 1) than the iodostilbene precursors (9a-d). Compound 12a was also tested against three pediatric neuroblastoma cell lines (GI₅₀ in μg/mL: SK-N-SH, 0.0075; SK-N-AS, 0.002; IMR-32, 0.081).

Table I.

Human Cancer Cell Line Growth Inhibition (GI₅₀ μg/mL) and Murine P388 Lymphocytic Leukemia Inhibitory Activities (ED50 μg/mL)

compound	solubilitya (mg/mL)	leukemia P388	pancreas BXPC-3	breast MCF-7	CNS SF-268	lung- NSC NCI- H460	colon KM20L2	prostate DU-145
1a	--	0.0003	0.39	-	<0.001	0.0006	0.061	0.0008
1b	--	0.0004	0.23	--	0.036	0.029	0.034	0.0072
3c	--	0.26	2.3	0.49	0.0083	0.19	1.2	0.0043
3d	--	0.0020	0.745	0.0027	0.0016	0.0032	>1	0.0019
9a	--	0.0020	0.048	0.00022	0.00018	0.00029	0.328	0.00018
9c	--	0.0028	0.038	0.0027	0.0036	0.0034	0.15	0.0021
9b	--	0.189	2.7	0.18	0.55	0.21	1.7	0.27
9d	--	> 10	3.0	0.94	3.3	3.4	> 10	5.8
11a	14	0.0021	0.381	0.0064	0.0057	0.0043	0.41	0.0038
11b	2	0.0020	0.469	0.018	0.018	0.017	>1	0.011
11c	≥ 2.4	0.017	0.490	0.0038	0.0040	0.0039	>1	0.0043
11d	--	0.0032	0.21	0.0047	0.0037	0.0036	0.24	0.0026
11e	≥ 4	0.0026	0.32	0.0065	0.0044	0.0036	0.51	0.0029
11f	≥ 2	0.0026	0.16	0.0044	0.0033	0.0031	0.32	0.0021
11g	--	0.0022	0.26	0.035	0.0097	0.0034	0.59	0.0030
11h	--	0.0029	0.37	0.0048	0.0043	0.0040	0.40	0.0047
12a	22	0.0034	0.44	0.050	0.053	0.046	>1	0.028
12b	2	0.030	>1	0.066	0.051	0.327	>1	0.242
12c	≥ 4	0.021	0.37	0.051	0.050	0.050	>1	0.032
12d	--	0.014	0.35	0.066	0.054	0.033	>1	0.028
12e	--	0.011	0.33	0.070	0.041	0.025	>1	0.025
12f	--	0.011	0.36	0.10	0.054	0.030	>1	0.023
12g	--	0.017	0.37	0.22	0.086	0.033	>1	0.026
12h	--	0.026	0.33	0.047	0.040	0.025	0.94	0.021

^aSolubility values represent solution in 1 mL of H₂O at 25 °C.

Against two cancer cell lines long believed to be human anaplastic thyroid carcinoma cells (KAT-4 and SW1736), we had earlier evaluated the potential for increased specificity by the new iodocombstatins. Diiodocombstatin 9c proved to be more inhibitory than iodocombstatin (9a, Table 2), but both strongly inhibited growth of the ATC line KAT-4. However, phenols 9a and 9c were 100× less active against the SW1736 line. The potassium phosphates 11a and 12a as expected proved to be less inhibitory (10×). While growth of the KAT-4 cells was significantly reduced by phosphates 11a and 12a, the SW1736 cells proved to be resistant. It has since been reported that KAT-4 is one of 12 redundant cell lines of 40 presumptive human thyroid cancer cell lines tested for cross-contamination and is likely not of thyroid origin.^11a Thus the KAT-4 cancer cell line data, while interesting, are not directly significant in respect to thyroid cancer but are a further indication of the cancer cell growth inhibitory activity of these compounds.

Table 2.

Human Anaplastic Thyroid Carcinoma Cell Line Inhibition Values (GI₅₀ μg/mL)

compound	KAT-4	SW1736
9a	0.089	2.2
9c	0.039–0.063	1.2
11a	0.37–0.43	> 10
12a	0.38–0.44	> 10

Antiangiogenesis is now actively pursued as a promising antitumor strategy.^11b Iodo- (9a) and diiodocombstatin (9b), as well as phosphate prodrugs 11a,b and 12a,b, were evaluated against human umbilical vein endothelial cells (HUVEC) in vitro (Table 3). These cells showed significant sensitivity to the new compounds. Iodocombstatin (9a) was the most active, with an ED₅₀ value of 4 × 10⁻⁵ μg/mL, followed by diiodocombstatin (9c). A similar pattern was observed with phosphates 11a and 12a. With both the mono- and diiodocombstatins, 9a and 9c, cord lengths as well as junction numbers were markedly reduced at 0.001 μg/mL (see Table 4 and Figure 1B,D) but were similar to the untreated control (Figure 1A) at 0.0001 μg/mL (Figure 1C,E). At 0.001 μg/mL, 11a (Figure 1F) showed a slightly larger reduction in the size of the cords as compared to 12a (Figure 1G). Such inhibitory activity against the HUVECs is of considerable interest since endothelial cells play a central role in the angiogenic process. Moreover, combretastatin derivatives have been shown to be involved in vascular targeting therapy by destroying the existing vasculatures of a tumor, specifically staunching blood flow and inhibiting tumor growth through the disruption of the tubulin cytoskeleton of endothelial cells, which leads to thrombosis of the vasculature (see Table 5).¹²

Table 3.

Human Umbilical Vein Endothelial Cell (HUVEC) Inhibition Values (GI₅₀ μg/mL)

compound	HUVEC
9a	0.000040
9c	0.00028
11a	0.00025
11b	0.00035
12a	0.0049
12b	0.051

Table 4.

Length of Cords Formed, Number of Junctions, and Relative Percent Growth of Cells

	drug conc. (μg/mL)	lengths of cordsa	junction no.b	rel.% growthc
9a	0.01	−	−	14
	0.001	+	+	14
	0.0001	++(+)	++(+)	18
	0.00001			90
9c	0.01	−	−	4
	0.001	+	(+)	8
	0.0001	+++	+++	84
	0.00001			87
11a	0.01			1
	0.001	++	++(+)	10
	0.0001	+++	+++	77
	0.00001	+++	+++	95
11b	0.1	−	−	7
	0.01	−	−	14
	0.001	+	+	5
	0.0001			104
12a	0.1	−	−	15
	0.01	++(+)	++(+)	33
	0.001	+++	+++	88
	0.0001			96
12b	1	−	−	−7
	0.1	+	(+)	−2
	0.01	++(+)	++(+)	> 100
	0.001			> 100

^aLengths of cords: −, no cords; +, small; +(+), 25% control; ++, 50% control; ++(+), 75% control; +++, same as control.

^bNumber of junctions: −, no junctions; +, few; +(+), 25% control; ++, 50% control; +(+), 75% control; +++, same as control.

^cRelative to control.

Figure 1. Treated human umbilical vein endothelial cells.^a.

^aMorphological appearance of the cords in HUVEC cells. 1A, untreated control; 1B, after exposure to 9a (0.001 μg/mL for 24 h); 1C, after exposure to 9a (0.0001 μg /mL for 24 h); 1D, after exposure to 9c (0.001 μg /mL for 24 h); 1E, after exposure to 9c (0.0001 μg /mL for 24 h); 1F, after exposure to 11a (0.001 μg μg /mL for 24 h); 1G, after exposure to 12a (0.001 μg /mL for 24 h).

Table 5.

Inhibitory Effects of Iodocombstatins on Tubulin Assembly and on Colchicine Binding to Tubulin in Comparison with Those of Combretastatin A-4 (1a)

	inhibition of tubulin assembly	inhibition of colchicine binding to tubulin
		1.0 μM compound	10 μM compound
	IC50 (μM) ± SD	% inhibition ± SD
1a	2.1 ± 0.03	81 ± 5	99 ± 0.9
1b	> 40a	--	--
9a	2.5 ± 0.04	78 ± 8	100 ± 0.8
9c	1.5 ± 0.3	95 ± 0.8	100 ± 0.3
11b	> 40	--	--
12b	> 40	--	--

^aData from previous evaluations, not repeated in current studies.

The mono (9a) and diiodo (9b) analogs of CA4 (1a) and their sodium phosphate derivatives (11b and 12b, respectively) were also evaluated and compared to 1a for inhibitory effects on tubulin (experiments were performed contemporaneously, Table 5). Similar IC₅₀ values were obtained for the nonphosphorylated compounds, with 9a appearing to be somewhat less inhibitory than 1a, and 9c more active than 1a. The two phosphorylated derivatives 11b and 12b had little or no inhibitory effect on tubulin assembly, as had been observed previously with CA4P (1b).

Combretastatin A-4 (1a) is a potent inhibitor of the binding of [³H]colchicine to tubulin. In our standard assay with 1.0 μM tubulin and 5.0 μM [³H]colchicine, the binding of the radiolabel to the protein has routinely been 95-100% inhibited by 5.0 μM 1a and 75-85% inhibited when 1a was present in the reaction mixture at 1.0 μM, equimolar with the tubulin.¹³ In the current experiments, this finding was obtained again (Table 5). We compared 9a and 9c with 1a as inhibitors of colchicine binding in the standard reaction conditions. While 9a was identical to 1a in its inhibitory effect, the diiodo 9c was more potent. Virtually complete inhibition of colchicine binding occurred with 9c at 1 μM, making this compound the most potent inhibitor of colchicine binding we have yet examined.

Combretastatin A-4 (1a) and the prodrug CA4P (1b) were previously shown to have marginal antimicrobial activity (minimum inhibitory concentration [MIC] = 25-100 μg/disk) against the Gram positive opportunist Micrococcus luteus and the Gram negative pathogen Neisseria gonorrhoeae.¹³ The new CA4 analogues 9a,c, as well as 9b, and phosphate salts 11a-h and 12a-h were tested against a panel of bacteria and fungi. Phenols 9a and 9c were active against M. luteus (MIC: 2-4 and 4-16 μg/mL, respectively), and the diiodocombstatin phosphates 12a-12h were very active against N. gonorrhoeae (MIC in μg/mL: 12a, <0.5-4; 12b, 32-64; 12c, <0.5-2; 12d, <0.5; 12e, <0.5; 12f, <0.5-1; 12g, <0.5; 12h, <0.5-2). Iodocombstatins 11d, 11g, and 11h were also active against N. gonorrhoeae (MIC in μg/mL: 11d, 16-32; 11g, 4-8; 11h, 32-64), and iodocombstatin 9a had marginal activity against the pathogenic yeast Cryptococcus neoformans (MIC: 64 μg/mL).

From all evidence now in hand, diiodocombstatin (9c) appears especially suitable for further preclinical development. The very powerful biological properties of the 3-iodo- and especially the 3,5-diodo-comstatins may be the result, in part, of increased halogen-protein bonding, as suggested by Ley and colleagues.^4a 3,5-Diiodocombstatin (9c) and derived prodrugs offer promising candidates for further preclinical development.

EXPERIMENTAL SECTION

General Experimental Methods

All solvents were redistilled, and ether refers to diethyl ether. Isovanillin, 3-iodo-4,5-dimethoxybenzaldehyde, anhydrous CH₂Cl₃, anhydrous toluene, tetrabutylammonium fluoride [1.0 M solution in tetrahydrofuran (THF)], dibenzyl phosphate, 4-dimethylaminopyridine (DMAP), CCl₄, Dowex-50W (HCR-W2) and l-trypotophan methyl ester hydrochloride were obtained from Sigma-Aldrich Chemical Company. 3,5-Diiodo-4-hydroxybenzaldehyde was obtained from Lancaster Synthesis Inc, and diisopropylethylamine (DIPEA) from Avocado (Alfa Aesar, Ward Hill, MA). The remaining reagents were purchased from Acros Organics (Fisher Scientific, Pittsburg, PA). All reactions were performed under an argon atmosphere, protected from bright light to avoid the potential for photochemical side reactions. The reactions and products were monitored by TLC using Analtech silica gel GHLF Uniplates visualized under long-wave and short-wave UV irradiation and stained by dipping into phosphomolybic acid in EtOH followed by heating. All organic extracts of aqueous solutions were dried over anhydrous magnesium sulfate. Where appropriate, the crude products were separated by column chromatography, using flash (230-400 mesh ASTM) silica gel from E. Merck.

Melting points were measured with an electrothermal digital melting point apparatus and are uncorrected. All ¹H and ¹³C NMR spectra were obtained using Varian Gemini 300 MHz or Varian Unity 400 or 500 MHz instruments with CDCl₃ (TMS 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 Varian Unity 400 MHz or 500 MHz instrument. Elemental analyses were performed by Galbraith Laboratories, Inc. Mass spectra were obtained in our institute with a JEOL LCmate instrument.

3,5-Diiodo-4-methoxybenzaldehyde (6)

A solution of 3,5-diiodo-4-hydroxybenzaldehyde (5 g, 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 H₂O (50 mL), and the mixture was extracted with EtOAc-hexane (1:1, 3 × 50 mL). The combined organic extract was filtered and concentrated in vacuo. The residue was separated by CC on silica gel using EtOAc-hexane (1:9) as eluent. The product was a colorless solid (4.2 g, 80%) that crystallized from hexane: mp 121-123 °C (lit.¹⁵ mp 124 °C); ¹H NMR (CDCl₃, 300 MHz) δ 3.93 (s, 3 H), 8.27 (s, 2 H), 9.81 (s, 1 H); ¹³C NMR (CDCl₃, 75 MHz) δ 60.35, 90.72, 140.65, 140.71, 187.51, 187.56; anal. C 24.86, H 1.58%, calcd for C₈H₆I₂O₂, C 24.77, H 1.56%.

3-Iodo-4,4′,5-trimethoxy-3′-O-tert-butyldiphenylsilyl-Z-stilbene (7a) and 3-iodo-4,4′,5-trimethoxy-3′-O-tert-butyldiphenylsilyl-E-stilbene (7b)

Method A

Phosphonium bromide 4¹⁰ (3.67 g, 5.13 mmol) was dissolved in DCM at 0 °C. 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 (5, 1 g, 3.42 mmol) was added, and stirring was continued for 21 h. The reaction was terminated by addition of H₂O (50 mL), and the mixture was extracted with DCM (3 × 50 mL). The organic phase was filtered and concentrated. The oil obtained was subjected to flash chromatography on silica gel and eluted with 0-3% EtOAc in hexane to afford Z-stilbene 7a (0.86 g, 39%), which crystallized as a colorless solid from hexane: mp 122-124 °C: ¹H NMR (CDCl₃, 300 MHz) 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, 1 H, J = 7.8 Hz), 6.72 (s, 2 H), 6.77 (dd, 1 H, J = 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 (CDCl₃, 75 MHz) 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 m/z 651.1474 [M + H]⁺ (calcd for C₃₃H₃₆IO₄Si, 651.1428); anal. C, 60.79; H, 5.67%, calcd for C₃₃H₃₅IO₄Si, C 60.92, H 5.45%.

In a parallel series of Wittig reactions evaluating a variety of solvents (CH₂Cl₂, EtOAc, toluene, and CHCl₃), temperature (-70 °C to 0 °C to rt), time (2.5-9.25 h), and base (NaH, BuLa, KOH/18-crown-6), production of the Z-isomer ranged from 42 to 52% yields as judged by NMR analyses. Further elution gave E-stilbene 7b (0.96 g, 43%), which crystallized from hexane as a colorless solid; mp 98-99 °C; ¹H NMR (CDCl₃, 300 MHz) δ 1.14 (s, 9 H), 3.55 (s, 3 H), 3.82 (s, 3 H), 3.82 (s, 3 H), 3.89 (s, 3 H), 6.43 (d, 1 H, J = 15.9 Hz), 6.71-6.76 (m, 2 H), 6.86-6.95 (m, 3 H), 7.33-7.42 (m, 6 H); ¹³C NMR (CDCl₃, 100 MHz) 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 m/z 651.1400 [M + H]⁺ (calcd for C₃₃H₃₆IO₄Si, 651.1428); anal. C 60.88, H 5.63%, calcd for C₃₃H₃₅IO₄Si, C 60.92, H 5.42%.

Method B

Butyllithium (4.5 mL, 11.3 mmol) was added to a stirred and cooled (−70 °C) suspension of phosphonium bromide 4 in dry THF (100 mL). The solution was stirred for 30 min at −70°C and then for 6 h at rt. Water (50 mL) was added, and the reaction mixture was extracted with EtOAc (3 × 100 mL). The organic phase was filtered and concentrated to an oil that was subjected to flash chromatography on silica and eluted with 0-3% EtOAc in hexane to afford Z-stilbene 7a (1.4 g, 21%) as a colorless solid: mp 122-124 °C.

3,5-Diiodo-4,4′-dimethoxy-3′-O-tert-butyldiphenylsilyl-Z-stilbene (8a) and 3,5-diiodo-4,4′-dimethoxy- 3′-O-tert-butyldiphenylsilyl-E-stilbene (8b)

Method A

To a solution of phosphonium bromide 4 (2.77 g, 3.87 mmol) (8) in DCM at 0 °C was added sodium hydride (60% dispersion in mineral oil, 0.31 g, 7.7 mmol), and the mixture turned orange. After addition of aldehyde 6 (1.0 g, 2.57 mmol), stirring was continued for 7.5 hrs. The reaction was terminated by addition of H₂O (50 mL) and extracted with DCM (3 × 50 mL). The organic extract was filtered and concentrated. The oily residue was flash chromatographed on silica gel using hexane as eluent to give an isomeric mixture of 8a and 8b (71% yield, 1.35g). Further elution gave the E-isomer 8b (0.10 g, 5%) as a colorless oil: ¹H NMR (CDCl₃, 300 MHz) δ 1.14 (s, 9 H), 3.56 (s, 3 H), 3.84 (s, 3 H), 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, 1H, J 2.1 Hz), 6.92 (dd, 1 H, J 1.8 Hz and J 8.4 Hz), 7.34-7.46 (m, 6 H), 7.72-7.75 (m, 6 H); ¹³C NMR (CDCl₃, 100 MHz) 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, 157.55; HRMS m/z 747.0442 [M + H]⁺ (calcd for C₃₂H₃₃I₂O₃Si, 747.0289).

Method B

Butyllithium (0.6 mL, 1.47 mmol) was added to a stirred and cooled (-10 °C) suspension of phosphonium bromide 4 (1.01g, 1.4 mmol) in dry THF (80 mL). The orange-red solution was stirred for 10 min at rt. Aldehyde 6 (0.50 g, 1.33 mmol) was added, and the reaction mixture color changed from red to yellow. Stirring was continued at rt for 10 min before addition of ice water (100 mL) and extraction of the the mixture with EtOAc (3 × 100 mL). The organic phase was washed with water (100 mL), 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 of approximately 1:1.9 (Z:E, 0.90 g, 90%).

3-Iodo-4,4′,5-trimethoxy-3′-hydroxy-Z-stilbene (9a)

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 H₂O (5 mL). The product was extracted with EtOAc (3 × 15 mL), and the organic phase was filtered and concentrated. The crude product was separated by silica gel CC using 1:4 EtOAc-hexane to give stilbene 9a (0.70 g, 85%) as a colorless solid: mp 92-94 °C; IR (film) ν_max 3543, 3011, 2937, 2841, 1510, 1273, 1001, 908, 732 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) 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, 1 H, J = 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 m/z 413.0250 [M + H]⁺ (calcd for C₁₇H₁₈IO₄, 413.0259); anal. C 49.38, H 4.24%, calcd for C₁₇H₁₇IO₄, C 49.53, H 4.16%.

3-Iodo-4,4′,5-trimethoxy-3′-hydroxy-E-stilbene (9b)

The E-isomer 9b (0.29 g, 98%) was obtained from silyl ether 7b (0.46 g, 0.7 mmol) as described above for the synthesis of the Z-isomer 9a. Separation by CC (7:3 hexane-EtOAc as eluent) gave E-isomer 9b (0.29g, 98%) as a colorless solid: mp 111-113 °C; ¹H NMR (CDCl₃, 300 MHz) δ 3.84 (s, 3 H), 3.87 (s, 3 H), 3.88 (s, 3 H), 5.85 (bs, 1 H), 6.77 (d, 1 H, J = 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 (CDCl₃, 75 MHz) 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 m/z 413.0250 [M + H]⁺ (calcd for C₁₇H₁₈IO₄, 413.0257); anal. C 49.38, H 4.24%, calcd for C₁₇H₁₇IO₄, C 49.53, H 4.16%.

3,5-Diiodo-4,4′-dimethoxy-3′-hydroxy-Z-stilbene (9c) and 3,5-diiodo-4,4′-dimethoxy-3′-hydroxy-E-stilbene (9d)

Stilbenes 9c and 9d were obtained from a mixture of silyl ethers 8a and 8b (1.35 g, 1.81 mmol) as described above for the synthesis of Z-isomer 9a. The oily mixture was separated by CC with 2:1 hexane-EtOAc as eluent to provide Z-isomer 9c as an oil (0.45 g, 49%): ¹H NMR (CDCl₃, 300 MHz) 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); ¹³C NMR (CDCl₃, 125 MHz) δ 55.98, 60.73, 89.98, 110.46, 114.87, 120.98, 125.08, 129.47, 131.57, 137.37, 139.96, 145.42, 146.21, 157.50. HRMS m/z 508.9111 [M + H]⁺ (calcd for C₁₆H₁₅I₂O₃, 508.9113); anal. C 37.80, H 2.83%, calcd for C₁₆H₁₄I₂O₃, C 37.82, H 2.78%.

Further elution led to the E-stilbene 9d (0.46 g, 50% yield) as a colorless solid, which crystallized from hexane: mp 127-129 °C; ¹H NMR (CDCl₃, 300 MHz) 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), 7.85 (s, 2 H, H-2); ¹³C NMR (CDCl₃, 75 MHz) 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, 157.25: HRMS m/z 508.9119 [M + H]⁺ (calcd for C₁₆H₁₅I₂O₃, 508.9113); anal. C 38.01, H 2.91%, calcd for C₁₆H₁₄I₂O₃, C 37.82, H 2.78%.

3-Iodo-4,4′,5-trimethoxy-3′-acetyl-Z-stilbene (9e)

Phenol 9a (0.45 g) was dissolved in pyridine (3 mL)-acetic anhydride (170 μL), and the mixture was stirred for 2 h. The mixture was concentrated under reduced pressure from toluene (3 × 10 mL). The residue was diluted with EtOAc (30 mL), and the solution was washed successively with H₂O (10 mL) and NaHCO₃ (10% aq. sol., 10 mL), filtered, and concentrated. Flash chromatography on silica using 1:24 EtOAc-hexane yielded acetate 9e (0.20 g, 41%) as a colorless solid that crystallized from hexane: mp 121-122 °C; ¹H NMR (CDCl₃, 300 MHz) 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, 1 H, J = 2.43), 7.06 (d, 1 H, J = 1.5 Hz), 7.09 (d, 1 H, J = 2.4 Hz), 7.67 (s, 2 H); ¹³C NMR (CDCl₃, 125 MHz) 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, 168.79; HRMS m/z 582.9482 [M + CH₃OH]⁺ (calcd for C₁₉H₂₀I₂O₅, 582.9479); anal. C 39.30, H 3.13%, calcd for C₁₈H₁₆I₂O₄, C 39.30, H 2.93%

3,5-Diiodo-4,4′-dimethoxy-3′-acetyl-Z-stilbene (9f)

To a solution of phenol 9c (0.1 g, 0.24 mmol) in 3 mL of anhydrous pyridine was added acetic anhydride (50 μL, 0.51 mmol) and a catalytic amount of DMAP. The mixture was stirred for 90 min, and the reaction was terminated by the addition of CH₃OH (5 mL). Toluene was added and the solution was concentrated under reduced pressure prior to flash chromatography on silica gel. EtOAc-hexane (1:9) as eluent provided a colorless solid (0.1 mg, 91%) that crystallized from hexane: mp 103-104 °C; ¹H NMR (CDCl₃, 300 MHz) δ 2.27 (s, 3 H), 3.61 (s, 3 H), 3.81 (s, 6 H), 6.38 (d, 1 H, J = 12 Hz), 6.48 (d, 1 H, J = 12 Hz), 6.77 (d, 1 H, J = 1.8 Hz), 6.83 (d, 1 H, J = 8.4 Hz), 6.96 (d, 1 H, J = 1.5 Hz), 7.09 (dd, 1 H, J = 8.4 Hz, J = 2.4 Hz), 7.26 (s, 1 H); ¹³C NMR (CDCl₃, 125 MHz) 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, 168.81: HRMS m/z 455.0356 [M + H]⁺ (calcd for C₁₉H₂₀IO₅, 455.0355); anal. C 49.67, H 4.18 %, calcd for C₁₉H₁₉IO₅, C 50.24, H, 4.22%.

Dibenzyl 3-iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (10a)

A solution of Z-stilbene 9a (0.68 g, 1.64 mmol) in acetonitrile (7 mL) was cooled to −10 °C before the addition of CCl₄ (1.6 mL, 16.4 mmol), and the mixture was stirred for 10 min at −10 °C in the dark. Diisopropylamine (0.57 mL, 3.28 mmol), and DMAP (20 mg, cat) were added in rapid succession. After 1 min, dibenzylphosphite (0.44 mL, 1.96 mmol) was added, and the mixture was stirred for 20 min at −10 °C. The reaction was terminated by the addition of 0.5 M KH₂PO₄ (7 mL) and extracted with EtOAc (3 × 15 mL). The organic phase was filtered and concentrated. The oily residue was separated by CC using 4:1 hexane-EtOAc as eluent to yield 0.94 g (86%) of a pure oil: bp (dec) 274 °C (0.01 mm Hg); ¹H NMR (CDCl₃, 300 MHz) δ 3.51 (s, 3 H), 3.65 (s, 3 H), 3.72 (s, 3 H), 5.04 (s, 2 H), 5.06 (s, 2 H), 6.36 (d, 1 H, J = 9 Hz), 6.42 (d, 1 H, J = 9 Hz), 6.77 (d, 1 H, J = 1.2 Hz), 6.89 (d, 1 H, J = 6 Hz), 7.02 (d, 1 H, J = 6 Hz), 7.01 (s, 1 H), 7.19 (d, 1 H, J = 1.2 Hz), 7.28-7.35 (m, 10 H); ¹³C NMR (CDCl₃, 75 MHz) 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: HRMS m/z 673.0808 [M + H]⁺ (calcd for C₃₁H₃₁IO₇P, 673.0852); anal. C 55.37, H 4.64%, calcd for C₃₁H₃₀IO₇P, C 55.37, H 4.50%.

Dibenzyl 3,5-diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate (10b)

Dibenzyl phosphate 10b (0.38 g, 55% yield) was obtained from 9c (0.46 g, 0.91 mmol), by the method described above for the synthesis of monoiodide 10a, as a colorless oil: bp (dec) 220 °C; (0.01 mm Hg), ¹H NMR (CDCl₃, 300 MHz) 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, J 12 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), 7.64 (s, 2 H); ¹³C NMR (CDCl₃, 100 MHz) 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, 157.68; ³¹P NMR (CDCl₃,162 MHz) δ −5.51; HRMS m/z 768.9699 [M + H]⁺ (calcd for C₃₀H₂₈I₂O₆P, 768.9713).

General Procedures for Syntheses of the Phosphoric Acids and Derivatives

Method A

Each of the metal cation phosphate salts was obtained by the procedure outlined below for preparing the potassium salt 11a, except for the metal counterions introduced by treatment of the phosphoric acid using either LiOH or NaOMe.

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 H₂O (until pH 7). The column was recycled. The potassium salt 11a or 12a (about 25 mg) was dissolved in deionized H₂O (1 mL) and applied to a Dowex-50W (HCR-W2) resin column (bearing the appropriate amine or amino acid methyl ester) and eluted with approximately 40 mL of H₂O. The eluent was concentrated by freeze drying to give the required cation derivative.

Method C. Amino Acid Methyl Esters

The amino acid methyl ester hydrochloride was neutralized in CH₃OH solution by addition of 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.

Potassium 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (11a)

Trimethylbromosilane (277 μL, 1.8 mmol) was added to a cooled (0 °C) solution of benzyl phosphate 10a in DCM (40 mL), and the mixture was stirred for 90 min before addition of sodium thiosulfate (10% aq., 10 mL). After an additional 1 min stirring, the phases were separated and the aqueous phase was extracted successively with DCM (20 mL) and EtOAc (2 × 20 mL). The combined organic extracts were filtered and concentrated to afford the phosphoric acid intermediate as a clear oil. After drying (high vacuum) for 1 h, the oil was dissolved in CH₃OH (10 mL) with cooling to 0 °C, KOH (1.8 mL, 1 N sol. in CH₃OH) was added, and the mixture was stirred for 20 min. The precipitate was collected and triturated with ether to afford the potassium salt as a colorless solid: mp 197-198 °C (dec); ¹H NMR (D₂O, 300 MHz) 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), 7.22 (s, 2 H); ³¹P NMR (D₂O, 162 MHz) δ 0.94.

Sodium 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (11b)

colorless solid: mp 194-195 °C (dec); ¹H NMR (D₂O, 300 MHz) 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).

Lithium 3-iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (11c)

colorless solid: mp 245-275 °C (dec); ¹H NMR (D₂O, 400 MHz) 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), 7.22 (s, 1 H).

Morpholine 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (11d)

colorless oil: ¹H NMR (D₂O, 300 MHz) 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), 7.20 (s, 1 H).

Piperidine 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (11e)

colorless oil: ¹H NMR (D₂O, 300 MHz) 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, 1 H, J = 12.6 Hz), 6.51 (d, 1 H, J = 12.6 Hz), 6.72 (s, 2 H), 6.83 (s, 1 H), 7.21 (s, 1 H).

Glycine-OMe 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (11f)

colorless solid: mp 74-78 °C; ¹H NMR (D₂O, 300 MHz) δ 3.48 (s, 3 H), 3.61 (s, 3 H), 3.67 (s, 3 H), 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), 7.16 (s, 1 H).

Tryptophan-OMe 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (11g)

colorless solid: mp 108-112 °C; ¹H NMR (DMSO, 300 MHz) 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, 1 H, J = 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), 7.46 (d, 1 H, J = 7.2 Hz).

Tris 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate (11h)

colorless solid: mp 75-81 °C; ¹H NMR (DMSO, 300 MHz) 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, 1 H, J = 8.4 Hz), 6.81 (d, 1 H, J = 8.7 Hz), 6.92 (s, 1 H), 7.22 (s, 1 H), 7.42 (s, 1 H).

Potassium 3,5-Diiodo-4,4′dimethoxy-Z-stilbene 3′-O-phosphate (12a)

Phosphate 12a (0.20 g, 80%) was obtained from ester 10b (0.29 g, 0.38 mmol) as described above for the synthesis of 11a, except that the aqueous phase was extracted with butyl alcohol (3 × 25 mL) because the phosphoric acid was sparingly soluble in EtOAc and DCM. The potassium salt was a colorless solid: mp 210-215 °C (dec); ¹H NMR (D₂O, 300 MHz) δ 3.69 (s, 6 H), 6.27 (d, 1 H, J = 12 Hz), 6.49 (d, 1 H, J = 12 Hz), 6.64 (s, 2 H), 7.20 (s, 1 H) and 7.62 (s, 2 H); ³¹P NMR (D₂O, 162 MHz) δ 0.973.

Sodium 3,5-Diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate (12b)

colorless solid: mp 215-234 °C (dec); ¹H NMR (D₂O, 300 MHz) 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).

Lithium 3,5-Diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate (12c)

colorless solid: 250-270 °C (dec); ¹H NMR (D₂O, 300 MHz) 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), 7.64 (s, 2 H); ³¹P NMR (D₂O, 162 MHz) δ 0.96.

Morpholine 3,5-Diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate (12d)

colorless waxy solid: mp 75-80 °C; ¹H NMR (DMSO, 300 MHz) 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), 7.78 (s, 2 H).

Piperdine 3,5-Diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate (12e)

colorless oil: ¹H NMR (DMSO, 300 MHz) 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, 1 H, J = 8.4 Hz), 6.80 (d, 1 H, J = 8.4 Hz), 7.40 (s, 1 H), 7.61 (s, 1 H).

Glycine-OMe 3,5-Diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate (12f)

colorless solid: mp 90-97 °C; ¹H NMR (DMSO, 300 MHz) 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), 7.67 (s, 1 H).

Tryptophan-OMe 3,5-Diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate (12g)

colorless solid: mp 125-130 °C; ¹H NMR (DMSO, 300 MHz) δ 3.34 (d, 1 H, J = 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), 7.60 (s, 1 H).

Tris 3,5-Diiodo-4,4′dimethoxy-Z-stilbene 3′-O-phosphate (12h)

colorless solid; mp 115-120 °C; ¹H NMR (DMSO, 300 MHz) δ 3.34 (s, 18 H), 3.69 (s, 3 H), 3.71 (s, 3 H), 6.30 (d, 1 H, J = 12 Hz), 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), 7.67 (s, 2 H).

Tubulin Evaluation Procedures

The methods used to measure both inhibition of tubulin assembly¹⁶ and of [³H]colchicine binding to tubulin¹² have been described in detail elsewhere. In brief, the assembly reaction was evaluated using purified tubulin at 10 μM in 0.8 M monosodium glutamate (pH 6.6 with HCl), varying compound concentrations, 0.4 mM GTP, and 4% (v/v) dimethyl sulfoxide (compound solvent). Extent of assembly after 20 min at 30 °C was measured. In the colchicine binding assay, the tubulin concentration was 1.0 μM, the [³H]colchicine concentration 5.0 μM, and compound concentrations as indicated. Additional reaction components are detailed in reference 13. Incubation was for 10 min at 37 °C, and the tubulin-[³H] colchicine complex was separated from unbound colchicine by retention of the complex by DEAE-cellulose filters.

Cancer Cell Line Procedures

Inhibition of human cancer cell growth was assessed using the National Cancer Institute’s standard sulforhodamine B assay as previously described.¹⁷ Briefly, cells in a 5% fetal bovine serum/RPMI1640 medium were inoculated in 96-well plates and incubated for 24 hrs. Serial dilutions of the compounds were then added. After 48 h, the plates were fixed with trichloroacetic 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.

Mouse leukemia P388 cells¹⁸ were incubated for 24 hrs in a 10% horse serum/Fisher medium followed by a 48-h incubation with serial dilutions of the compounds. Cell growth inhibition (ED₅₀) was then calculated using a Z1 Beckman/Coulter particle counter.

Antiangiogenesis Assays

In vitro Matrigel antiangiogenesis assays were conducted according to the Developmental Therapeutics Program, NCI protocols.¹⁹ Growth inhibition and cord formation assays were performed using HUVECs purchased from GlycoTech. HUVECs were grown in EGM-2 medium (Cambrex). The standard sulforhodamine B assay (see above) was used to evaluate the activity against HUVECs. IC₅₀ values (drug concentration causing 50% inhibition) were calculated from the plotted data.

Cord Formation Assay

Matrigel, a basement membrane matrix, was purchased from BD Biosciences. An aliquot of 60 μL was placed in each well of an ice-cold 96-well plate. The plates were left for 15 min at rt and incubated for 30 min at 37 °C to permit the Matrigel to polymerize. Meanwhile, HUVECs were harvested and diluted to a concentration of 2 × 10⁵ cells/mL, and 100 μL of this preparation was added to each well. A second solution of 100 μL containing the compound to be tested was added. After a 24-h incubation, images were taken at each concentration using an inverted Nikon Diaphot microscope and D100 digital camera. Drug effect was assessed, compared to untreated controls, by measuring the length of cords formed and the number of junctions.

Antimicrobial Susceptibility Testing

The stilbenes (9a-c, 11a-h, 12a-h) were evaluated against the bacteria Stenotrophomonas maltophilia ATCC 13637, Micrococcus luteus Presque Isle 456, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 6303, and Neisseria gonorrhoeae ATCC 49226 and the fungi Candida albicans ATCC 90028 and Cryptococcus neoformans ATCC 90112, according to established broth microdilution susceptibility assays.^20,21 Each substance was reconstituted in a small volume of sterile DMSO and diluted in the appropriate medium immediately prior to susceptibility experiments. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of compound that inhibited all visible growth of the test organism (optically clear). Assays were repeated on separate days.