Antineoplastic Agents 515. Synthesis of Human Cancer Cell Growth Inhibitors Derived From 3,4-Methylenedioxy-5,4′-dimethoxy-3′-amino-Z-stilbene

Abstract

Further structure-activity relationship (SAR) exploration of 3,4-methylenedioxy-5,4′-dimethoxy-3′-amino-Z-stilbene (1a) derivatives resulted in the efficient synthesis of tyrosine amide hydrochloride 9, two tyrosine amide phosphate prodrugs (3a and 6), and sodium aspartate amide 11. Two additional cancer cell growth inhibitors (14 and 16) were synthesized by employing peptide coupling between amine 1a and the Dap residue of dolastatin 10 (4a) to yield amide 14 followed by Dov-Val-Dil (15) to yield peptide 16. The latter represents a combination of stilbene 1a with the des-Doe tetrapeptide unit of the powerful tubulin assembly inhibitor dolastatin 10. Peptide 16 was examined for potential binding to tubulin in the vinca and/or colchicine regions and found to perform primarily as a relative of dolastatin 10. Amide 14 had anticryptococcal and antibacterial activities.

We recently completed syntheses of 3'-amino derivatives (1a–b, 1d–j) of combretastatin A-2 (2c)¹, encouraged by the remarkable success of the potent cancer vascular targeting^2–4 that occurs with combretastatin A-4 phosphate prodrug (CA4P) 2h.^5–10 Subsequently, we undertook the synthesis and initial anticancer evaluation of two phosphate derivatives (3a,b) of tyrosine stilbene amide 1i. Attachment of the phosphate group at the 3'-phenol position has been investigated in detail in the combretastatin A and B series.^1,11–13 In the present study we chose to use the 4"-hydroxyl group of tyrosine amide 1i for attachment of a phosphate group (3a) as well as obtaining a diphosphoryl derivative (3b) by phosphorylating tyrosine amide 1i at both the amine¹⁴ and hydroxyl groups. A parallel important objective of this research involved employing a tetrapeptide segment of dolastatin 10 (4a) for bonding to the 3′-amino group (1b). Dolastatin 10 (D-10, 4a), isolated^15–17 from the Indian Ocean sea hare Dolabella auricularia, has continued to progress through preclinical¹⁸ and clinical development as an impressive antineoplastic agent.^19–22 Dolastatin 10 (4a) and various synthetic derivatives also have specific antifungal activity against Cryptococcus neoformans.^23,24 Previous SAR studies have confirmed that D-10 (4a) inhibits microtubule assembly by binding to the β-tubulin subunit near the vinca site. SAR studies have shown the des-Doe tetrapeptide unit (4b, Dov-Val-Dil-Dap) to be necessary for potent anticancer activity.^25,26 We now also report synthesis of the amide representing coupling of amine 1a to Dov-Val-Dil-Dap (4b).

Results and Discussion

Our synthesis of the tyrosine stilbene amide 1i²⁷ presented the opportunity to develop two additional phosphate prodrugs in the combretastatin series. Accordingly, the 4"-phenol and α-amino groups were phosphorylated in one step using dibenzyl phosphite (1i to 5) (Scheme 1). Debenzylation of the resulting phosphate diesters (5) was achieved using bromotrimethylsilane to afford the free acid (3b) which was subsequently converted to sodium salt 6 using sodium methoxide in methanol.^11,12 Phosphorylation solely at the 4"-hydroxyl position was accomplished via the coupling of the 3'-amino stilbene 1a to O-tert-butyl-N^α-Z-L-Tyr using PyBroP as the coupling reagent to provide amide 7 (Scheme 2).²⁸ tert-Butyl removal using TFA in DCM followed by phosphorylation afforded the dibenzyl ester 8. Debenzylation at the ester, and benzyloxycarbonyl removal at the amine, were achieved simultaneously with the in situ generation of trimethylsilyl iodide (TMSI) from the reaction of sodium iodide and chlorotrimethylsilane in acetonitrile.^29,30 This method resulted in the immediate precipitation of the phosphate prodrug 3a. Surprisingly, both phosphoric acids and the corresponding sodium salts proved to be sparingly soluble in water. In retrospect, the physical properties of 6 might correspond better to a betaine structure arising from enolization of the amide group in 3b with a concurrent shift of the adjacent phosphate to give a phosphate ester (see Figure 1). This structure would account for the trisodium salt to give 6 and the lower aqueous solubility.

Scheme 1.

Scheme 2.

Figure 1.

We further investigated the possibility of making a soluble amide derivative first by making the hydrochloride salt of 1i (Scheme 3), and second by coupling amine 1a to aspartic acid and converting to the β-aspartate sodium salt 11 (Scheme 4). Amide formation was accomplished using PyBroP followed by removal of the β-tert-butyl protecting group in the presence of triethylsilane.³¹ The resulting carboxylic acid was subsequently treated with sodium methoxide in methanol yielding sodium salt 11. Both the hydrochloride salt 9 and the sodium salt 11 were essentially insoluble (<0.1 mg/mL) in water. Although we found earlier that the combretastatin A-2 phosphate prodrug (2d) has reduced aqueous solubility when compared to its combretastatin A-1 (2b) and A-3 counterparts (2f),^11,12 we were surprised to find that our new CA2 modifications (1b, 3a, 6, 9, and 11) exhibited only sparing water solubility, presumably owing to the hydrophobicity created by the stilbene. Interestingly, addition of the phosphate group to the tyrosine amide 1i to yield 3a resulted in decreased anticancer activity (Table 1). Since the cancer cell line results obtained for each of the substances recorded in Table 1 were obtained in solution, presumably the sparing water solubility did not significantly influence the cell line results. However, the reduced ability to hydrogen bond in vitro could be important.

Scheme 3.

Scheme 4.

Table 1.

Human cancer cell line GI₅₀ (µg/mL) and murine P388 lymphocytic leukemia cell line inhibitory activity ED₅₀ (µg/mL) of the 3′-substituted stilbenes.

Compound	Leukemia P388	Pancreas BXPC-3	Breast MCF-7	CNS SF-295	Lung-NSC NCI-H460	Colon KM20L2	Prostate DU145	Mean
1a	0.00189	0.0071	0.0047	0.023	0.0050	0.022	0.028	0.013
3a	>10	>1	>1	>1	>1	>1	>1	-
5	>10	-	-	-	-	-	-	-
6	0.270	18.6	1.3	>1	>1	>1	>1	-
7	0.380	0.049	0.36	0.44	0.41	1.8	0.39	0.61
8	0.133	>10	>10	>10	>10	>10	>10	-
9	0.0329	0.064	0.041	0.064	0.096	0.034	0.080	0.059
10	0.385	3.4	2.4	1.7	3.8	2.3	3.0	2.4
11	0.361	0.38	0.51	0.25	0.30	0.19	0.33	0.33
13	0.225	0.58	0.53	0.75	0.52	1.9	0.38	0.70
14	0.0263	0.23	0.054	0.062	0.083	0.26	0.070	0.11
16	<0.01	0.048	0.0031	0.025	0.033	0.020	0.032	-

The des-Doe tetrapeptide unit (4b) of the potent anticancer drug dolastatin 10 (D-10, 4a) isolated in 1984¹⁵ was chosen for coupling with amine 1a. By replacing the Doe portion of D-10 with the amine 1a, we planned to synthesize a stilbene peptide with the possibility of binding to the colchicine and/or vinca regions of tubulin. The tripeptide Dov-Val-Dil TFA salt (15) and Boc-Dap (12) were obtained via published methods.^32,33 PyBroP coupling of amine 1a to Boc-Dap (12) followed by Boc deprotection with TFA in DCM afforded compound 14 (Scheme 5). Further coupling resulted in des-Doe D-10 stilbene amide 16. The cancer cell inhibition activity of the potentially dual functioning anticancer agent 16 was notable (Table 1), as was the broad spectrum antimicrobial action of amide 14. The latter substance (14) also inhibited the growth of the pathogenic fungus Cryptococcus neoformans (MIC = 64 µg/ml), the pathogenic bacterium Neisseria gonorrhoeae ATCC 49226 (MIC = 16 µg/ml), and the opportunistic bacteria Streptococcus pneumoniae ATCC 6303 (MIC = 8–16 µg/ml) and Enterococcus faecalis ATCC 29212 (MIC = 32–64 µg/ml). Prodrug 3a inhibited N. gonorrhoeae (MIC = 0.125 µg/ml) and E. faecalis (MIC = 16 µg/ml), and prodrug 6 inhibited N. gonorrhoeae (MIC = 4 µg/ml). Stilbenes 8 and 13 inhibited E. faecalis (MIC = 64 µg/ml) or C. neoformans (MIC = 64 µg/ml), respectively.

Scheme 5.

Previous work showed that D-10 (4a) is an exceptionally cytotoxic antimitotic agent and that it inhibits tubulin assembly by binding tightly in the vinca domain of tubulin (its inhibition of vinblastine binding to tubulin, although extensive, is noncompetitive).^34,35 In contrast, while combretastatin A-4 (2g) is quite potent for a colchicine site drug, it is about 100-fold less cytotoxic than D-10 (4a), and the stilbene inhibits tubulin assembly by binding avidly to the colchicine site in a competitive fashion.^36,37 These findings were confirmed in the studies presented in Table 2. Note the similar inhibitory effects of 4a and 2g on tubulin assembly, their contrasting effects on inhibition of binding of radiolabeled vinblastine and colchicine, and the greater than 100-fold enhanced inhibition of growth of the MCF-7 cells by 4a relative to 2g.

Table 2.

Effects of Peptide 16 and Related Compounds on Tubulin Assembly, Binding of Colchicine and Vinblastine to Tubulin, and the Growth and Mitotic Index of MCF-7 Breast Cancer Cells

Compound	Inhibition of tubulin assembly IC50 (µM) ±SDa	Inhibition of colchicine binding % Inhibition ± SDa		Inhibition of vinblastine binding % Inhibition ± SDa		Inhibition of growth of MCF-7 breast cancer cells IC50 (nM) ± SDa	Mitotic index of MCF-7 cells % Mitosesb
		2 µMc	50 µMc	10 µMc	80 µMc
16	1.3 ± 0.04		30 ± 2	48 ± 3	87 ± 2	17 ± 7	57
Dov-Val-Dil-Dap (4b)	1.4 ± 0.03		5.2 ± 3	16 ± 6	68 ± 0.4	36 ± 10	56
1b	3.4 ± 0.5d	85 ± 3		0	0	68 ± 30	65
Dolastatin 10 (4a)	0.70 ± 0.06		13 ± 5	96 ± 0.7		0.083 ± 0.06	39
4b + 1be						14 ± 2	52
Combretastatin A-4 (2g)	1.8 ± 0.3	98 ± 0.4		0	0	11 ± 10	46

^aSD, Standard deviation.

^bThe mitotic indices were determined following growth of the cells for 16 h at 10 times the IC₅₀ concentration. Without drug, 2.9% of the cells were in mitosis.

^cInhibitor concentrations.

^dCompound 1a had a nearly identical IC₅₀ value (4.3 ± 0.5 µM).

^eEquimolar concentrations of compounds 4b and 1b were present in all cell culture mixtures.

Previous SAR studies with D-10²⁴ (4a) structural modifications had shown that loss of its C-terminal unit Doe had a significant effect on cell growth, and a greater effect on inhibition of ligand binding to tubulin than on inhibition of tubulin assembly. Thus, Dov-Val-Dil-Dap (4b) is only 2-fold less active than 4a as an assembly inhibitor, but at least 6-fold less active as an inhibitor of MCF-7 cell growth (Table 2). These SAR studies had also shown that replacement of the Doe residue with a variety of aromatic derivatives could restore some or all of the activity of D-10 (4a) in both the cytological and biochemical assays.^23,24

SAR studies with structural manipulation of combretastatin A4^27,36 (2g) revealed that relatively minor effects on cytotoxicity and tubulin interactions were observed when a methylenedioxy bridge replaced two vicinal methoxy groups in the A ring or an amino group replaced the hydroxyl group in the B ring. These findings are demonstrated again in Table 2, where amine hydrochloride 1b, incorporating both these changes, is compared with CA4 2g. In addition, we found that a variety of amino acid amides formed from amine 1a had sharply reduced activity in the tubulin-based biochemical assays but retained the amine’s cytotoxic activity.²⁷ Since these compounds caused a marked increase in the mitotic index of drug-treated cells, the most reasonable explanation was that the amine 1a was regenerated by either extracellular or intracellular hydrolysis of the amides.

When the dolastatin 10-combretastatin amine hybrid 16 was evaluated, it was found to be active in all the biochemical assays (Table 2), although its effect on the binding of [³H]colchicine was relatively weak, concordant with the weak activities observed previously with the amino acid amides of 1a.²⁷ In contrast, compound peptide 16 was about half as active as D-10 (4a) as an inhibitor of both tubulin assembly and [³H]vinblastine binding to tubulin. We therefore conclude that at the biochemical level, peptide 16 is acting primarily as a D-10 (4a) analog.

The cytotoxic properties of peptide 16, however, are more difficult to unravel. Using MCF-7 cells for a detailed comparison, we found that peptide 16 was about twice as active as Dov-Val-Dil-Dap (4b) and 4 times as active as hydrochloride 1b, but only 1/200^th as active as D-10 (4a). Further, when 4b and 1b were mixed in equimolar amounts, as would occur if 16 were completely hydrolyzed, an IC₅₀ value for inhibition of the growth of the MCF-7 cells was obtained that was almost identical to that obtained with peptide 16 (14 vs. 17 nM). Since the IC₅₀ values obtained for 16, 4b, and 1b are in the same range, it is impossible to determine whether the cytotoxicity of 16 derives from its acting primarily as a relatively weak D-10 (4a) analog or primarily as a relatively potent combretastatin A-4 (2g) analog. The similarity in IC₅₀ values obtained with 16 and with the mixture of its components 4b and 1b indicates that it, like the previously studied amides,²⁷ undergoes extracellular or intracellular hydrolysis. Finally, the similarity in IC₅₀ values for 16, 4b, 1b, and the 4b + 1b mixture indicates that there is little, if any, synergistic in vitro cytotoxic effect obtained from having 4b and 1b conjoined in a single molecule.

In conclusion, although initial attempts to synthesize a very water soluble derivative from the 3,4-methylenedioxy-5,4′-dimethoxy-3′-amido-Z-stilbene tyrosine amide and aspartate amide series proved unrewarding and will require a different approach, three new stilbene amides (9, 14, and 16) strongly inhibiting a mini-panel of human cancer cell lines were discovered. Most importantly, the preliminary biological results from the potentially multi-targeting compounds (amide 14 and peptide 16) merit further investigation.

Experimental Section

Materials and Methods

Ether refers to diethyl ether and Ar to argon gas. Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), O^β-tert-butyl-N^α-Boc-L-aspartic acid, and O-tert-butyl-N^α-Z-L-tyrosine were obtained from Calbiochem-Novabiochem Corporation (San Diego, CA). Diisopropylethylamine (DIPEA), anhydrous methanol, sodium methoxide, triethylsilane (TES), and trifluoroacetic acid (TFA) were obtained from Acros Organics (Fisher Scientific, Pittsburg, PA). All other reagents were purchased from Sigma-Aldrich Chemical Company (Milwaukee, WI).

Reactions were monitored by TLC using Analtech silica gel GHLF Uniplates visualized under short-wave UV irradiation. Solvent extracts of aqueous solutions were dried over anhydrous MgSO₄ or Na₂SO₄. Where appropriate, the crude products were separated by column chromatography on flash (230–400 mesh ASTM) silica from E. Merck, gravity (70–230 mesh ASTM) silica from E. Merck, or Sephadex LH-20.

Melting points are uncorrected and were determined employing an Electrothermal 9100 apparatus. Optical rotations were measured using a Perkin-Elmer 241 polarimeter. The [α]_D-values are given in 10⁻¹ deg cm² g⁻¹. The ¹H and ¹³C NMR spectra were recorded employing Varian Gemini 300, Varian Unity 400, or Varian Unity 500 instruments using a deuterated solvent and were referenced either to TMS or the solvent. HRMS data were recorded with a JEOL LCmate mass spectrometer. Elemental analyses were determined by Galbraith Laboratories, Inc., Knoxville, TN.

3,4-Methylenedioxy-5,4′-dimethoxy-3′-[O,N^α-di(bis-benzylphosphoryl)-L-Tyr]-amido-Z-stilbene (5)

To a stirred solution of amine 1i²⁷ (71 mg, 0.15 mmol) in acetonitrile (1 mL) at −10 EC in an acetone/ice bath, under Ar, was added CCl₄ (0.15 mL, 1.6 mmol, 11 eq). Ten minutes later DIPEA (0.12 mL, 0.66 mmol, 4.4 eq) and 4-dimethylaminopyridine (3.8 mg, 0.031 mmol, 0.21 eq) were added, followed 1 min later by the dropwise addition of dibenzylphosphite (0.11 mL, 0.47 mmol, 3.1 eq). After 1 h, the reaction was warmed to rt and aqueous 0.5 M KH₂PO₄ was added. The mixture was extracted with EtOAc (3 × 10 mL), and the combined extracts were washed with brine (15 mL) and H₂O (15 mL). Upon drying and condensing in vacuo, the product was separated by gravity column chromatography (4:1, DCM-EtOAc) to afford a colorless oil 5 (97 mg, 65%): R_f 0.12 (4:1, DCM:EtOAc); [α]²⁵_D −45.1E (c 0.72, CHCl₃); P-NMR (400 MHz, CDCl₃) δ 5.693, −8.113; ¹H-NMR (400 MHz, CDCl₃) δ 2.89 (1H, m, -CH₂-), 3.18 (1H, m, -CH₂-), 3.56 (3H, s, OCH₃), 3.71 (3H, s, OCH₃), 4.05 (1H, m, alpha H), 4.96 (8H, m, 4 × -CH₂-), 5.87 (2H, s, -OCH₂O-), 6.39 (1H, d, J = 12.0 Hz, vinyl H), 6.45 (3H, m, vinyl H, 2 × ArH), 6.59 (1H, d, J = 8.4 Hz, ArH), 6.97 (1H, dd, J = 8.4, 1.6 Hz, ArH), 6.99 (2H, d, J = 8.4 Hz, 2 × ArH), 7.03 (2H, d, J = 8.8 Hz, 2 × ArH), 7.29 (20 H, m, 20 × ArH), 8.27 (1H, d, J = 1.6, ArH), 8.36 (1H, s); and HRMS calcd for C₅₄H₅₃N₂O₁₂P₂ [M+H]⁺ 983.3074, found 983.3115. Anal. calcd for C₅₄H₅₂N₂O₁₂P₂; C, 65.98; H, 5.33; N, 2.85. Found: C, 65.51; H, 5.55; N, 2.84.

Sodium 3,4-methylenedioxy-5,4′-dimethoxy-3′-(L-Tyr)-amido-Z-stilbene 3′-O,N^α-diphosphate (6)

To a stirred solution of benzyl phosphate 5 (76 mg, 0.077 mmol) in DCM (1 mL) at 0 EC, under Ar, was added bromotrimethylsilane (35 µL, 0.33 mmol, 4.3 eq). The reaction mixture was stirred for 30 min and concentrated under vacuum. The residue was dissolved in EtOH (1 mL), and NaOMe (20 mg, 0.36 mmol, 4.7 eq) was added, yielding a precipitate that was collected and washed with EtOAc and ether. The product (6) was obtained as a colorless powder (52 mg, 95%); m.p. (dec.) 188–190 EC; [α]²⁴_D −30.6E (c 0.72, DMSO); P-NMR (400 MHz, DMSO) δ 6.262, 0.585; ¹³C-NMR (300 MHz, DMSO) δ 143.8, 132.3, 130.4, 130.2, 129.7, 129.4, 127.4, 127.2, 126.3, 122.1, 119.7, 116.5, 115.1, 111.0, 106.8, 101.2, 99.5, 56.6, 56.2, 55.9; ¹H-NMR (400 MHz, DMSO) δ 2.63 (1H, m, -CH₂-), 3.10 (1H, m, -CH₂-), 3.30 (1H, d, J = 8.8 Hz), 3.56 (1H, m), 3.86 (3H, s, OCH₃), 3.87 (3H, s, OCH₃), 6.00 (2H, s, -OCH₂O-), 6.55 (1H, d, J = 0.8 Hz, ArH), 6.99 (8H, m, 2 × vinyl H, 6 × ArH), 7.11 (1H, d, J = 8.4 Hz, ArH), 7.23 (1H, dd, J = 6.8, 1.6 Hz, ArH), 10.12 (1H, br s). Anal. calcd for C₂₆H₂₅N₂Na₃O₁₂P₂·3H₂O: C, 42.06; H, 4.21; N, 3.77. Found: C, 41.68; H, 4.65; N, 3.45.

3,4-Methylenedioxy-5,4′-dimethoxy-3′-(O-tert-butyl-N^α-Z-L-Tyr)-amido-Z-stilbene (7)

To a stirred mixture of amine 1a (0.19 g, 0.63 mmol), O-tert-butyl-N^α-Z-L-Tyr (0.45 g, 1.2 mmol, 1.9 eq), and PyBroP (0.57 g, 1.2 mmol, 1.9 eq) in DCM (2 mL) at 0 EC under Ar was added DIPEA (0.55 mL, 3.2 mmol, 5.1 eq). The reaction mixture was stirred for 45 min at rt and concentrated under vacuum. The product was obtained by gravity column chromatography (4:1, DCM:EtOAc) as a colorless oil (7, 0.38 g, 93%); R_f 0.74 (4:1, DCM:EtOAc); [α]²⁴_D +1.9E (c 1.04, CHCl₃); ¹H-NMR (300 MHz, CDCl₃) δ 1.30 (9H, s, tBu), 3.05 (2H, m, -CH₂-), 3.63 (3H, s, OCH₃), 3.68 (3H, s, OCH₃), 4.47 (1H, m, alpha H), 5.05 (2H, d, J = 2.4 Hz, -CH₂-), 5.60 (1H, br), 5.85 (2H, s, -OCH₂O-), 6.33 (1H, d, J = 12.0 Hz, vinyl H), 6.40 (3H, m, vinyl H, 2 × ArH), 6.59 (1H, d, J = 9.0 Hz, ArH), 6.83 (2H, m, ArH), 6.91 (1H, dd, J = 8.1, 1.5 Hz, ArH), 7.05 (2H, d, J = 8.4 Hz, 2 × ArH), 7.26 (5H, m, 5 × ArH), 7.92 (1H, s), 8.22 (1H, d, J = 2.1 Hz, ArH); and HRMS calcd for C₃₈H₄₁O₈N₂ [M+H]⁺ 653.2863, found 653.2876. Anal. calcd for C₃₈H₄₀N₂O₈: C, 69.92; H, 6.18; N, 4.29. Found: C, 69.45; H, 6.37; N, 4.21.

3,4-Methylenedioxy-5,4′-dimethoxy-3′-(O-bis-benzylphosphoryl-N^α-Z-L-Tyr)-amido-Z-stilbene (8)

To a stirred solution of amide 7 (0.26 g, 0.40 mmol) in DCM (2 mL) was added TFA (2 mL). The reaction mixture was stirred for 20 min, the solution was concentrated under vacuum, and the free phenol obtained by gravity column chromatography (4:1, DCM:EtOAc) was placed in acetonitrile (4 mL) under Ar. The mixture was cooled to −10 EC, and CCl₄ (0.21 ml, 2.1 mmol, 5.3 eq) was added. Ten minutes later DIPEA (0.16 mL, 0.89 mmol, 2.2 eq) and 4-dimethylaminopyridine (5 mg, 0.041 mmol, 0.10 eq) were added, followed 1 min later by the dropwise addition of dibenzyl phosphite (0.15 mL, 0.65 mmol, 1.6 eq). After 2 h the reaction mixture was warmed to rt, and aqueous 0.5 M KH₂PO₄ (16 mL) added. The same procedure given for phosphate 5 was followed to obtain the product 8 as a colorless oil (0.24 g, 71%); R_f 0.60 (4:1, DCM:EtOAc); [α]²³_D −5.1E (c 0.25, CHCl₃); P-NMR (400 MHz, CDCl₃) δ −8.095; ¹H-NMR (MHz, CDCl₃) δ 3.08 (2H, m, -CH₂-), 3.66 (1H, s, OCH₃), 3.72 (1H, s, OCH₃), 4.51 (1H, m, alpha H), 5.08 (2H, d, J = 0.9 Hz, -CH₂-), 5.11 (4H, m, 2 × -CH₂-), 5.44 (1H, br d, J = 7.2 Hz), 5.90 (2H, s, -OCH₂O-), 6.39 (1H, d, J = 12.0 Hz, vinyl H), 6.46 (3H, m, vinyl H, 2 × ArH), 6.62 (1H, d, J = 8.1 Hz, ArH), 6.98 (1H, dd, J = 8.4, 1.8 Hz, ArH), 7.04 (2H, d, J = 8.1 Hz, 2 × ArH), 7.14 (2H, d, J = 8.1 Hz, 2 × ArH), 7.31 (15H, m, 15 × ArH), 7.90 (1H, s), 8.24 (1H, d, J = 2.4 Hz, ArH); and HRMS calcd for C₄₈H₄₆N₂O₁₁P [M+H]⁺ 857.2840, found 857.2853. Anal. calcd for C₄₈H₄₅N₂O₁₁P; C, 67.28; H, 5.29; N, 3.27. Found: C, 66.89; H, 5.27; N, 3.17.

3,4-Methylenedioxy-5,4′-dimethoxy-3′-(O-phosphoryl-N^α-L-Tyr)-amido-Z-stilbene (3a)

To a stirred solution of benzyl ester 8 (98 mg, 0.11 mmol) in acetonitrile under Ar was added sodium iodide (60 mg, 0.40 mmol, 3.6 eq), followed by chlorotrimethylsilane (51 µL, 0.41 mmol, 3.7 eq). A white precipitate formed while the reaction mixture was stirred for 20 min. Aqueous (1%) sodium thiosulfate (0.5 mL) was added to the mixture before the precipitate was collected and washed with ethyl acetate, H₂O, and acetone. The product was obtained as a colorless amorphous solid (3a, 35 mg, 58%): m.p. (dec.) 175–177 EC; [α]²⁵_D −6.3E (c 0.35, DMSO); P-NMR (400 MHz, DMSO) δ −2.043; ¹³C-NMR (400 MHz, DMSO) δ 170.6, 156.1, 149.0, 148.8, 143.3, 136.9, 134.2, 132.3, 129.7, 129.6, 128.9, 128.3, 127.7, 127.2, 127.0, 126.4, 122.8, 119.5, 118.9, 111.3, 106.8, 101.2, 99.5, 65.4, 57.0, 56.2, 36.4; ¹H-NMR (400 MHz, DMSO) δ 2.78 (2H, m, -CH₂-), 3.06 (1H, m, -CH₂-), 3.82 (3H, s, OCH₃), 3.86 (3H, s, OCH₃), 4.49 (1H, m, alpha H), 5.98 (2H, s, -OCH₂O-), 6.89 (1H, d, J = 12.8 Hz, vinyl H), 6.94 (1H, s, ArH), 7.07 (2H, m, vinyl H, ArH), 7.22 (1H, d, J = 6.4 Hz, ArH), 7.28 (2H, d, J = 5.6, 2 × ArH), 7.32 (2H, d, J = 5.6, 2 × ArH), 7.74 (1H, d, J = 6.4, ArH), 8.22 (1H, s), 9.25 (1H, s).

3,4-Methylenedioxy-5,4′-dimethoxy-3′-(N^α-L-Tyr)-amido-Z-stilbene hydrochloride (9)

To a stirred solution of amine 1i (27 mg, mmol) in ethyl acetate (1 mL) was added ethereal HCl (1 M) in excess. A white solid immediately formed, the solvent was removed in vacuo, and the resulting solid was recrystallized from ethanol-ethyl acetate to yield an off white powder (9, 29 mg, quantitative): m.p. 169–170.5 EC; [α]²⁵_D 82.5E (c 0.73, CH₃OH); ¹H-NMR (300 MHz, CDCl₃) δ 3.07 (2H, m, -CH₂-), 3.70 (3H, s, OCH₃), 3.79 (3H, s, OCH₃), 4.27 (1H, m, alpha H), 5.93 (2H, s, -OCH₂O-), 6.37 (1H, d, J = 1.2 Hz, ArH), 6.42 (1H, d, J = 12.0 Hz, vinyl H), 6.46 (1H, d, J = 12.6 Hz, vinyl H), 6.49 (1H, d, J = 1.2 Hz, ArH), 6.75 (2H, d, J = 8.4 Hz, 2 × ArH), 6.90 (1H, d, J = 8.1 Hz, ArH), 7.03 (1H, dd, J = 8.1, 2.1 Hz, ArH), 7.09 (2H, d, J = 8.7 Hz, 2 × ArH), 7.92 (1H, d, J = 1.5 Hz, ArH); Anal. calcd for C₂₆H₂₆ClN₂O₆: C, 59.32; H, 5.75; N, 5.33. Found: C, 59.53; H, 5.56; H, 5.26.

3,4-Methylenedioxy-5,4′-dimethoxy-3′-(O^β-tert-butyl-N^α-Boc-L-Asp)-amido-Z-stilbene (10)

To a stirred mixture of amine 1a (0.13 g, 0.43 mmol), O^β-tert-butyl-N^α-Boc-L-Asp (0.22 g, 0.76 mmol, 1.8 eq), and PyBroP (0.35 g, 0.76 mmol, 1.8 eq) in DCM (3 mL) at 0 EC under Ar was added DIPEA (0.21 mL, 1.2 mmol, 2.8 eq). The reaction mixture was stirred for 75 min at rt, and the solvent was removed in vacuo. The product was obtained by flash column chromatography (1:1, n-hexane:acetone) as an oil (10, 0.22 g, 88%): R_f 0.64 (1:1, n-hexane-acetone); [α]²⁴_D −13.0E (c 1.42, CHCl₃); ¹H-NMR (300 MHz, CDCl₃) δ 1.44 (9H, s, tBu), 1.49 (9H, s, tBu), 2.88 (2H, m, -CH₂-), 3.73 (3H, s, OCH₃), 3.84 (3H, s, OCH₃), 4.59 (1H, m, alpha H), 5.81 (1H, br), 5.92 (2H, s, -OCH₂O-), 6.38 (1H, d, J = 12.6 Hz, vinyl H), 6.44 (1H, s, ArH), 6.46 (1H, d, J = 12.6 Hz, vinyl H), 6.47 (1H, s, ArH), 6.70 (1H, d, J = 8.1 Hz, ArH), 6.97 (1H, dd, J = 8.4, 1.8 Hz, ArH), 8.28 (1H, d, J = 2.4 Hz, ArH), 8.76 (1H, s); and HRMS calcd for C₃₀H₃₉N₂O₉ [M+H]⁺ 571.2655, found 571.2617. Anal. calcd for C₃₀H₃₈N₂O₉: C, 63.14; H, 6.71; N, 4.91. Found: C, 62.64; H, 7.00; N, 4.89.

Sodium 3,4-methylenedioxy-5,4′-dimethoxy-3′-(N^α-L-Asp)-amido-Z-stilbene (11)

To t-butyl ester 10 (0.13 g, 0.23 mmol) in DCM (1.5 mL) was added a mixture of TFA (0.71 mL, 9.6 mmol, 42 eq) and TES (0.30 mL, 1.8 mmol, 7.8 eq), and stirring was continued for 4 h under Ar. The solvents were removed in vacuo, and the TFA salt, obtained by Sephadex LH-20 column chromatography (solvent, MeOH), was placed in methanol (1 mL). Sodium methoxide (0.22 mg, 0.41 mmol, 1.8 eq) was added to the reaction mixture, and the white precipitate that formed was collected and reprecipitated from DCM-CH₃OH to give the product 11 as a colorless solid (62 mg, 62%): m.p. 168–170 EC; [α]²⁵_D +14.0E (c 1.18, CH₃OH); ¹H-NMR (300 MHz, CD₃OD) δ 2.62 (1H, m, -CH₂-), 2.77 (1H, m, -CH₂-), 2.78 (2H, br), 3.69 (3H, s, OCH₃), 3.87 (3H, s, OCH₃), 4.29 (1H, m, alpha H), 5.87 (2H, s, -OCH₂O-) 6.36 (1H, d, J = 1.2 Hz, ArH), 6.40 (1H, d, J = 12.0 Hz, vinyl H), 6.45 (1H, d, J = 12.3 Hz, vinyl H), 6.50 (1H, d, J = 0.9 Hz, ArH), 6.91 (1H, d, J = 8.4 Hz, ArH), 7.02 (1H, dd, J = 8.4, 1.8 Hz, ArH), 7.99 (1H, d, J = 2.4 Hz, ArH), 8.52 (1H, s); Anal. calcd for C₂₁H₂₁N₂NaO₇·H₂O: C, 55.51; H, 5.10; N, 6.16. Found: C, 55.81; H, 5.70; N, 6.16.

3,4-Methylenedioxy-5,4′-dimethoxy-3′-(N^α-Boc-L-Dap)-amido-Z-stilbene (13)

To a stirred mixture of amine 1a (51 mg, 0.17 mmol), N^α-Boc-L-Dap 12 (52 mg, 0.18 mmol, 1.1 eq),^32,33 and PyBroP (87 mg, 0.19 mmol, 1.1 eq) at 0 EC under Ar was added DIPEA (65 µL, 0.37 mmol, 2.2 eq). The reaction mixture was stirred for 1.5 h at rt. DCM (10 mL) was added, and the mixture was washed with aqueous citric acid (10% by wt, 10 mL). The organic layer was dried with MgSO₄ and concentrated in vacuo, and the residue was subjected to gravity column chromatography (8:1, DCM-EtOAc), resulting in the product 13 as a colorless oil (40 mg, 41%): R_f 0.24 (8:1, DCM:EtOAc); [α]²⁴_D −48.5E (c 1.2, CHCl₃); ¹³C-NMR (500 MHz, CDCl₃) δ 148.5, 146.9, 143.3, 134.2, 131.9, 130.1, 129.5, 128.8, 127.6, 123.8, 120.8, 109.6, 108.5, 103.0, 101.3, 58.7, 56.3, 55.8, 28.5; ¹H-NMR (300 MHz, CDCl₃) δ 1.30 (3H, d, J = 6.9 Hz, CH₃), 1.46 (9H, s, Boc), 1.72 (1H, m, Pro), 1.90 (3H, m, Pro), 2.62 (1H, m), 3.23 (1H, m), 3.44 (1H, m), 3.49 (3H, s, OCH₃), 3.58 (1H, m), 3.74 (3H, s, OCH₃) 3.85 (3H, s, OCH₃), 3.90 (1H, m), 5.92 (2H, s, -OCH₂-), 6.38 (1H, d, J = 12.3 Hz, vinyl H), 6.45 (1H, s, ArH), 6.46 (1H, d, J = 12.0 Hz, vinyl H), 6.47 (1H, s, ArH), 6.69 (1H, d, J = 8.7, ArH), 6.96 (1H, dd, J = 8.4, 1.8 Hz, ArH), 8.30 (1H, d, J = 2.4, ArH), 8.41 (1H, br); and HRMS calcd for C₃₁H₄₁N₂O₈ [M+H]⁺ 569.2863, found 569.2884. Anal. calcd for C₃₁H₄₀N₂O₈: C, 65.48; H, 7.09; N, 4.93. Found: C, 64.92; H, 7.42; N, 4.97.

3,4-Methylenedioxy-5,4′-dimethoxy-3′-(N^α-L-Dap)-amido-Z-stilbene TFA salt (14)

To a stirred solution of amide 13 (0.15 g, 0.26 mmol) in DCM (0.65 mL) at 0 EC was added TFA (0.20 mL, 2.6 mmol, 10 eq). The reaction mixture was stirred for 20 min at rt. The product was obtained by gravity column chromatography (20:1, DCM:MeOH) as an oil (14, 69 mg, 46%): R_f 0.54 (8:1, DCM:MeOH); [α]²⁴_D −61.9E (c 0.88, CHCl₃; ¹³C-NMR (300 MHz, CDCl₃) δ 170.5, 148.1, 146.9, 142.8, 131.3, 129.6, 128.6, 126.3, 124.3, 120.5, 109.3, 108.1, 102.4, 100.8, 100.3, 80.2, 60.5, 58.6, 55.8, 55.3, 44.8, 41.5, 24.1, 23.4, 12.1; ¹H-NMR (300 MHz, CDCl₃) δ 1.27 (3H, d, J = 6.9, CH₃), 1.98 (4H, m), 2.94 (1H, m), 3.33 (2H, m), 3.57 (3H, s, OCH₃), 3.73 (1H, s, OCH₃), 3.78 (1H, m), 3.85 (3H, s, OCH₃), 3.89 (1H, m), 5.91 (2H, s, -OCH₂O-), 6.38 (1H, d, J = 12.6, vinyl H), 6.43 (3H, m, vinyl H, 2 × ArH), 6.73 (1H, d, J = 9.0, ArH), 7.00 (1H, dd, J = 8.4, 1.8 Hz, ArH), 8.15 (1H, d, J = 1.8 Hz, ArH), 8.35 (1H, s); and HRMS calcd for C₂₆H₃₃N₂O₆ [M+H]⁺ 469.2339, found 469.2374. Anal. calcd for C₃₀H₃₄F₆N₂O₁₀·2H₂O: C, 49.18; H, 5.23; N, 3.82. Found: C, 49.48; H, 5.23; N, 4.09.

3,4-Methylenedioxy-5,4′-dimethoxy-3′-(Dov-Val-Dil-Dap)-amido-Z-stilbene (16)

To a stirred mixture of amide 14 (41 mg, 0.070 mmol), Dov-Val-Dil TFA salt 15 (62 mg, 0.11 mmol, 1.6 eq) and PyBroP (50 mg, 0.11 mmol, 1.6 eq) in DCM (0.5 mL) at 0 EC under Ar was added DIPEA (91 µL, 0.52 mmol, 7.4 eq). The reaction mixture was stirred 14 h at rt, and the solvent was removed in vacuo. The product was obtained by gravity column chromatography (1:1, n-hexane:acetone) as a colorless oil (16, 30 mg, 48%): R_f 0.34 (1:1, n-hexane-acetone); [α]²³_D −62E (c 1.05, CHCl₃); ¹H-NMR (300 MHz, CDCl₃) δ 0.79 (3H, t, J = 6.9 Hz, CH₃), 0.91 (3H, d, J = 6.6 Hz, CH₃), 0.94 (3H, d, J = 6.6 Hz), 0.94 (3H, d, J = 6.3 Hz, CH₃), 1.00 (3H, d, J = 6.6 Hz, CH₃), 1.01 (3H, d, J = 6.3 Hz, CH₃), 1.32 (3H, d, J = 6.9 Hz, CH₃), 1.35 (2H, m), 1.80 (1H, m), 2.0 (3H, m), 2.31 (6H, s, 2 × NCH₃), 2.34 (4H, m), 2.62 (1H, m), 3.00 (3H, s, NCH₃), 3.05 (1H, m) 3.08 (1H, s), 3.20 (1H, m), 3.29 (1H, m), 3.32 (3H, OCH₃), 3.34 (1H, m), 3.44 (1H, m), 3.45 (3H, s, OCH₃), 3.72 (3H, s. OCH₃), 3.84 (3H, s, OCH₃), 4.06 (1H, m), 4.12 (1H, m), 4.20 (1H, m), 4.79 (1H, m), 5.90 (2H, s, -OCH₂O-), 6.36 (1H, d, J = 12.0 Hz, vinyl H), 6.44 (3H, m, vinyl H, 2 × ArH), 6.68 (1H, d, J = 8.7 Hz, ArH), 6.94 (1H, dd, J = 8.1, 1.5 Hz, ArH), 8.27 (1H, d, J = 1.8 Hz), 8.33 (1H, s); and HRMS calcd for C₄₈H₇₄N₅O₁₀ [M+H]⁺ 880.5435, found 880.5426. Anal. calcd for C₄₈H₇₃N₅O₁₀: C, 65.50; H, 8.36; N, 7.96. Found: C, 65.42; H, 8.74; N, 7.59.

Tubulin and Cancer Cell Procedures

Bovine brain tubulin was prepared³⁸ and the tubulin assembly³⁹ and colchicine binding⁴⁰ assays were performed as described previously. In the assembly assay, reaction mixtures contained 1.0 mg/mL (10 µM) tubulin and varying concentrations of potential inhibitors. In the colchicine binding assay reaction mixtures contained 0.1 mg/mL (1.0 µM) tubulin, 5.0 µM [³H]colchicine, and potential inhibitor as indicated. The vinblastine binding assay was performed as described previously for GTP binding⁴¹ by centrifugal gel filtration, except that a Beckman Allegra 6KR centrifuge equipped with a GH-3.8A swinging bucket rotor was used and the syringe-columns were centrifuged at 2,000 rpm. Reaction mixtures contained 0.5 mg/mL (5.0 µM) tubulin, 5 µM [³H]vinblastine, potential inhibitor as indicated, 0.5 mM MgCl₂, 0.1 M 4-morpholineethanesulfonate (1.0 M stock solution adjusted to pH 6.9 with NaOH, and 4% (v/v) dimethyl sulfoxide. Incubation (30 min) and centrifugal gel filtration were performed at room temperature (20–22 °C).

Cytotoxicity assays were performed by the sulforhodamine B method, in which inhibition of formation of cellular protein is measured.⁴² The mitotic index of MCF-7 breast cancer cells was determined as described previously,²⁷ except that cells were incubated in the presence of drug for 16 h prior to addition of the DNA stain.

Antimicrobial Evaluation

Compounds were screened in broth microdilution assays according to National Committee for Clinical Laboratory Standards.^43,44 Assays were repeated at least twice on separate days.