Introduction

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Interest in the metabolites of estrogen has grown in the last decade, because it has been appreciated that a number of the actions of this sex hormone may be caused by biologically active metabolites. For example, the glucuronides of estrogen and its metabolites cause cholestatic jaundice (Zhu et al., 1996), whereas the catecholestrogens are implicated in the association between estrogen and increased incidence of certain tumors (Zhu and Conney, 1998). The cardioprotective effects of estrogen have been ascribed to an interaction between estrogen and the high-affinity estrogen receptor (ER); recent evidence suggests that the cardioprotective effects of estrogen are mediated largely, if not exclusively, via the ER subtype (Brouchet et al., 2001; Hodgin et al., 2001). Alternative lower affinity receptors that bind estrogen have been described, but these do not seem to interact with physiological levels of estrogen, opening up the possibility that a related ligand binds and activates these receptors under physiological conditions (Eriksson et al., 1980). 2-Methoxyestradiol (2-MEO, compound 1; Table 1), once regarded as an inactive metabolite of estrogen, has been identified as an inhibitor of the proliferation of a variety of cell types and as an antiangiogenic and antitumor agent (Fotsis et al., 1994). Inhibition of smooth muscle proliferation by 2-MEO has given rise to suggestions that this or related compounds may have therapeutic applications in atherosclerosis (Nishigaki et al., 1995) and asthma (Stewart et al., 1999b), whereas regulatory effects of 2-MEO on lymphocytes and leukocytes provide a case for its use to treat rheumatoid arthritis (Josefsson and Tarkowski, 1997). Furthermore, cytotoxic and antiangiogenic actions of 2-MEO on various tumor cell lines indicate that this agent has potential as an antitumor agent (Lottering et al., 1992; Fotsis et al., 1994; Cushman et al., 1995; Hamel et al., 1996; Cushman et al., 1997).

2-MEO has been evaluated for its antitumor potential in murine models of tumor growth, including immunodeficient mice bearing human tumor xenografts. Oral doses of 50 to 150 mg/kg have shown efficacy against murine angiosarcoma (Arbiser et al., 1999), murine Meth A sarcoma, and B16 melanoma (Fotsis et al., 1994). Furthermore, in immunodeficient mice, the growth of human pancreatic carcinoma (Schumacher et al., 1999) and human breast carcinoma [MDA-MB-435 (Klauber et al., 1997)] are reduced as a result of treatment with 2-MEO.

The precise mechanisms underlying the various actions of 2-MEO have not been elucidated. 2-MEO inhibits the depolymerization of microtubules and also retards the formation of polymeric tubulin, giving characteristics that overlap with those of both paclitaxel (Taxol) and colchicine (D'Amato et al., 1994; Hamel et al., 1996; Wang et al., 2000). It seems likely that tubulin binding and consequent inhibition of mitotic spindle function explains part of the antiproliferative action of 2-MEO. However, we and others have described an inhibitory effect of 2-MEO on cell-cycle progression to S-phase through G₁, as inferred from the inhibition of DNA synthesis in smooth muscle and in some other cell types (Nishigaki et al., 1995; Stewart et al., 1999b, 2001). This effect does not seem to be explained by microtubule actions, because it is not mimicked by either paclitaxel or colchicine (T. Harris and A. G. Stewart, unpublished observations). The extent to which suppression of DNA synthesis rather than microtubule dysfunction explains the potentially desirable in vivo activities of 2-MEO is uncertain. However, it is clear that in most circumstances of the predicted therapeutic use of 2-MEO, affinity for high-affinity ERs would be undesirable.

In the present study, we have examined the ability of 2-MEO and a series of analogs to inhibit DNA synthesis in smooth muscle cells and to bind to high affinity ER in rat uterine cytosol. We then used the technique of comparative molecular field analysis (CoMFA) to generate models describing three-dimensional quantitative structure activity relationships (3D-QSARs) for these two activities. The marked differences between the respective models suggest that there are distinct structural requirements for DNA synthesis inhibition and ER binding. The models should be valuable for guiding the synthesis of compounds showing increased DNA synthesis inhibitory activity without significant affinity for ER.

	Compounds

Top Abstract Introduction Compounds Materials and Methods Results Discussion References

Compounds used in this study are shown in Tables 4, 5, and 6. The syntheses of compounds 1, 2, 4, 9 to 12, 26 (Cushman et al., 1995), 5, 18 (Cushman et al., 1997), 21, 22 (Verdier-Pinard et al., 2000), and 23 to 31 (Wang et al., 2000) have been described previously. Compounds 3, 16, 18, 19, and 34-39 were purchased from Sigma (St. Louis, MO). ICI 182,780 (17) was purchased from Sapphire Bioscience (Alexandria, NSW, Australia). The syntheses of additional new compounds used in this study are described below.

Synthesis of 3,17-O-Bis(methoxymethyl)estradiol (40). A solution of estradiol (16) (10.0 g, 36.7 mmol) in anhydrous tetrahydrofuran (THF, 80 ml) containing diisopropylethylamine (70 ml, 401 mmol) was stirred at 0°C under argon for 0.5 h. Methoxymethyl chloride (25 g, 310 mmol) was introduced and the resulting reaction mixture stirred at 60°C for 8 h. The reaction mixture was cooled to room temperature, poured into ice water (300 ml), and extracted with ethyl acetate (3 × 200 ml). The ethyl acetate layers were washed with saturated aqueous sodium bicarbonate (150 ml) and brine (2 × 100 ml), combined, dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue (silica gel 230-400 mesh, ethyl acetate/hexane 1:8 by volume) gave compound 40 as a colorless oil (11.7 g, 88.4%): ¹H NMR (CDCl₃)  7.20 (d, J = 8.5 Hz, 1 H), 6.82 (dd, J = 8.5 and 2.6 Hz, 1 H), 6.77 (d, J = 2.6 Hz, 1 H), 5.14 (s, 2 H), 4.65 (d, J = 1.2 Hz, 2 H), 3.61 (t, J = 8.3 Hz, 1 H), 3.47 (s, 3 H), 3.37 (s, 3 H), 2.83 (m, 2 H), 2.15 (m, 5 H), 1.85 (m, 1 H), 1.40 (m, 7 H), 0.81 (s, 3 H).

Synthesis of 2-Formyl-3,17-O-bis(methoxymethyl)estradiol (41). Sec-BuLi (1.3 M in cyclohexane, 100 ml, 130 mmol) was added dropwise to a solution of 40 (12.2 g, 33.8 mmol) in anhydrous THF (200 ml) at 78°C under argon and the resulting mixture was stirred at that temperature for 2.5 h. Anhydrous dimethylformamide (DMF; 20 ml, 255 mmol) was introduced dropwise and the resulting reaction mixture was stirred from 78°C to room temperature for 3 h. The reaction mixture was poured into an ice/water mixture (200 ml) and acetic acid (20 ml). The product was extracted with ethyl acetate (3 × 150 ml) and the ethyl acetate layers were washed with saturated aqueous sodium bicarbonate (150 ml) and brine (2 × 150 ml), combined, dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue (silica gel 230-400 mesh, ethyl acetate/hexane 1:6 by volume) gave compound 41 as a colorless oil (11 g, 87%), which solidified after standing at room temperature overnight: m.p. 89 to 92°C. ¹H NMR (CDCl₃)  10.46 (s, 1 H), 7.80 (s, 1 H), 6.94 (s, 1 H), 5.29 (s, 2 H), 4.83 (ABq, J = 7.2 Hz,  = 3.4 Hz, 2 H), 3.64 (t, J = 8.5 Hz, 1 H), 3.54 (s, 3 H), 3.40 (s, 3 H), 2.84 (m, 2 H), 2.42-1.15 (m, 13 H), 0.81 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 445 (MH⁺, 22), 389 (100). Analysis calculated for C₂₃H₃₂O₅: C, 71.11; H, 8.19. Found: C, 71.07; H, 8.48.

Synthesis of 2-Formylestradiol (42). A solution of compound 41 (370 mg, 0.95 mmol) and lithium tetrafluoroborate (1.8 g, 20 mmol) in acetonitrile (15 ml) containing water (1 ml) was heated at 72°C under nitrogen for 5 h. The reaction mixture was cooled to room temperature, poured into ice-cooled water (50 ml), and extracted with ethyl acetate (3 × 30 ml). The ethyl acetate layers were washed with brine (2 × 30 ml), dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue with 10% ethyl acetate in hexane gave the desired product 42 (200 mg, 70%), which was crystallized from ethanol to afford a white crystalline solid: m.p. 236 to 237°C literature m.p. 230-232°C (Cushman et al., 1995)]. ¹H NMR (CDCl₃)  9.91 (s, 1 H), 7.58 (s, 1 H), 6.66 (s, 1 H), 3.68 (t, J = 8.4 Hz, 1 H), 2.88 (m, 2 H), 2.40 (m, 1 H), 2.18 (m, 1 H), 2.10-1.15 (m, 11 H), 0.79 (s, 3 H).

Synthesis of 2-Hydroximinomethylestradiol (13). Under nitrogen, hydroxylamine hydrochloride (1.1 g, 16 mmol) was added to a solution of compound 42 (240 mg, 0.8 mmol) in pyridine (10 ml). The resulting mixture was stirred and heated at 90°C for 1 h. The pyridine was removed under reduced pressure at 30 to 35°C and the residue was dissolved in an ethyl acetate (60 ml) and water (50 ml) mixture. The ethyl acetate solution was washed with water (30 ml) and brine (30 ml), dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue on a silica gel column using 15% ethyl acetate in methylene chloride gave compound 13 (185 mg, 73%), which crystallized from ethyl acetate-methylene chloride to afford a white solid: m.p. 268 to 270°C. ¹H NMR (CCl₃D)  8.17 (s, 1 H), 7.13 (s, 1 H), 6.58 (s, 1 H), 4.86 (s, 2 H, OH), 3.66 (t, J = 8.3 Hz, 1 H), 2.81 (m, 2 H), 2.35-1.1 (m, 14 H), 0.77 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 316 (MH⁺, 100). Analysis calculated for C₁₉H₂₅O₃N: C, 72.35; H, 7.99; N, 4.44. Found: C, 72.18; H, 8.22; N, 4.35.

Synthesis of 2-Methoximinomethylestradiol (14). Methoxylamine hydrochloride (1.1 g, 16 mmol) was added to a solution of compound 42 (100 mg, 0.8 mmol) in pyridine (5 ml) under nitrogen. The resulting mixture was stirred and heated at 90°C for 2 h. The pyridine was removed under reduced pressure at 30 to 35°C and the residue was dissolved in ethyl acetate (50 ml) and water (50 ml). The ethyl acetate solution was washed with water (30 ml) and brine (30 ml), dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue on a silica gel column using 15% ethyl acetate in methylene chloride gave compound 14 (99 mg, 90%), which crystallized from ethyl acetate-methylene chloride to afford a white solid: m.p. 165 to 166°C; ¹H NMR (CDCl₃)  9.61 (s, 0.4 H), 8.11 (s, 0.6 H), 7.03 (s, 1 H), 6.71 (s, 1 H), 3.97 (s, 2 H, OH), 3.72 (t, J = 8.3 Hz, 1 H), 2.82 (m, 2 H), 2.35-1.1 (m, 16 H), 0.79 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 330 (MH⁺, 100), 312 (MH⁺-H₂O, 40). Analysis calculated for C₂₀H₂₇O₆N·1/6 H₂O: C, 72.26; H, 8.28; N, 4.21. Found: C, 72.13; H, 8.38; N, 4.19.

Synthesis of 2-Formylestradiol Hydrazone (15). Hydrazine dihydrochloride (594 mg, 16 mmol) was added to a solution of compound 42 (85 mg, 0.8 mmol) in pyridine (5 ml) under nitrogen. The resulting mixture was stirred and heated at 90°C for 2 h. The pyridine was removed under reduced pressure at 30 to 35°C and the residue was suspended in a mixture of ethyl acetate (50 ml) and saturated aqueous NaHCO₃ (50 ml). The yellow solid was collected, washed with water and ethyl acetate, and dried under vacuum. Recrystallization from THF-methylene chloride gave compound 15 (62 mg, 70%): m.p. 340°C. ¹H NMR (CDCl₃)  10.9 (s, 0.4 H), 8.89 (s, 0.6 H), 7.53 (s, 1 H), 6.65 (s, 1 H), 3.54 (t, J = 8.3 Hz, 1 H), 2.81 (m, 2 H), 2.35-1.1 (m, 17 H), 0.69 (s, 3 H).

Synthesis of 17-Acetoxy-2-ethoxy-3-hydroxy-6-hydroximinoestra-1,3,5(10)-triene (8). A solution of compound 43 (Cushman et al., 1997) (800 mg, 1.93 mmol) in pyridine (12 ml) was treated with hydroxylamine hydrochloride (1.07 g, 15.4 mmol). The resulting mixture was stirred and heated at 100°C for 30 min. The mixture was cooled to room temperature and then poured into ice water (150 ml). The compound was extracted with ethyl acetate (3 × 100 ml). The organic layers were washed with sodium bicarbonate (100 ml), water (2 × 100 ml), and brine (2 × 100 ml), dried over sodium sulfate and evaporated to dryness. Chromatography of the residue on a silica gel column using 30% ethyl acetate in hexane gave compound 8 (763 mg, 89%), which was crystallized from ethyl acetate-hexane to afford yellow crystals: m.p. 226 to 227°C. ¹H NMR (CDCl₃)  7.47 (s, 1 H), 6.75 (s, 1 H), 4.69 (t, J = 8.5 Hz, 1 H), 4.12 (q, J = 6.9 Hz, 2 H), 3.17 (dd, J = 4.3 and 18 Hz, 1 H), 2.40-1.20 (m, 14 H), 2.05 (s, 3 H), 1.43 (t, J = 6.9 Hz, 3 H), 0.85 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 388 (MH⁺, 100), 372 (MH-H₂O, 10), 328 (75). Analysis calculated for C₂₂H₂₉O₅N: C, 68.20; H, 7.54; N, 3.61. Found: C, 68.04; H, 7.62; N, 3.54.

Synthesis of B-Homo-6-aza-2-ethoxy-3,17-dihydroxyestra-1,3,5(10)-trien-6-one (44). A solution of compound 8 (700 mg, 1.63 mmol) in pyridine (10 ml) was treated with tosyl chloride (686 mg, 3.6 mmol) at room temperature. The resulting mixture was stirred for 2 h and the pyridine was removed in vacuo at ambient temperature to afford the crude oxime tosylate as a residue that was used without further purification. The residue was dissolved in a minimum amount of 40% chloroform in benzene (5 ml) and the material was applied to the top of a basic alumina column (30 × 3.5 cm). The column was eluted with chloroform-benzene (40 to 90% chloroform) and then allowed to stand overnight. The column was washed with methanol (200 ml) and 80% methanol in water (200 ml). The methanol eluate was collected and then evaporated to dryness. Chromatography of the residue on a silica gel column using ethyl acetate gave the ring-expanded lactam 44 (267 mg, 47%), which was crystallized from ethyl acetate to afford white crystals: m.p. 222 to 223°C. IR (KBr) 3336, 2931, 1661, 1514, 1469, 1440, 1371, 1320, 1243, 1119, 1046 cm^-1; ¹H NMR (CDCl₃)  7.31 (brs, 1 H), 6.74 (s, 1 H), 6.58 (s, 1 H), 4.12 (q, J = 6.7 Hz, 2 H), 3.75 (t, J = 8.5 Hz, 1 H), 2.41 (m, 2 H), 2.15-1.22 (m, 13 H), 1.44 (t, J = 7 Hz, 3 H), 0.82 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 346 (MH⁺, 100), 328 (MH-H₂O, 20). Analysis calculated for C₂₀H₂₇O₄N·1/3 H₂O: C, 68.35; H, 7.93; N, 3.98. Found: C, 68.25; H, 8.03; N, 3.82.

Synthesis of B-homo-6-aza-2-ethoxy-3,17-dihydroxyestra-1,3,5(10)-triene (45). A solution of borane in THF (1.0 M, 5.6 ml, 5.6 mmol) was added dropwise by syringe to a solution of compound 44 (97.8 mg, 0.28 mmol) in THF (5 ml) under argon at room temperature. The resulting solution was stirred for 6 h and then at gentle reflux for 4 h. The mixture was cooled and 6 N hydrochloric acid (2 ml) was added slowly through a pipette. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (60 ml). The ethyl acetate solution was washed with sodium bicarbonate (30 ml) and brine (2 × 20 ml), dried over sodium sulfate and evaporated to dryness. Chromatography of the residue on a silica gel column using 50% ethyl acetate in methylene chloride gave compound 45 (85 mg, 90%), which formed a white stable foam when it was evaporated from methylene chloride solution under reduced pressure. ¹H NMR (CDCl₃)  6.70 (s, 1 H), 6.28 (s, 1 H), 4.00 (q, J = 6.9 Hz, 2 H), 3.68 (t, J = 8.5 Hz, 1 H), 3.33 (t, J = 6.9 Hz, 1 H), 2.82 (t, J = 6.9 Hz, 1 H), 2.55 (dt, J = 8.5 and 2 Hz, 1 H), 1.20-2.20 (m, 15 H), 1.34 (t, J = 7.0 Hz, 3 H), 0.78 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 332 (MH⁺, 100), 314 (MH-H₂O, 60). Analysis calculated for C₂₀H₂₉O₃N·1/4 H₂O: C, 71.50; H, 8.85; N, 4.14. Found: C, 71.64; H, 8.90; N, 4.29.

Synthesis of B-Homo-6-aza-3,17,6a-triacetyl-2-ethoxyestra-1,3,5(10)-triene (46). Under nitrogen, acetic anhydride (1.83 g, 1.7 ml, 17.65 mmol) was added to a solution of compound 45 (146 mg, 0.44 mmol) in pyridine (5 ml). The resulting mixture was stirred at room temperature for 24 h and then poured into ice water (30 ml). The compounds were extracted with ethyl acetate (3 × 50 ml) and the organic layers were washed with water (30 ml), saturated aqueous sodium bicarbonate (30 ml) and brine (30 ml), dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue on a silica gel column using 33% ethyl acetate in methylene chloride gave compound 46 (169 mg, 84%), which formed a white stable foam when it was evaporated from ethyl acetate/hexane solution under reduced pressure: ¹H NMR (CDCl₃)  6.87 (s, 1 H), 6.78 (s, 1 H), 4.66 (t, J = 8.5 Hz, 1 H), 4.09 (q, J = 6.9 Hz, 2 H), 4.08 (t, J = 6.9 Hz, 1 H), 3.09 (m, 1 H), 2.46 (dt, J = 8.5 and 2 Hz, 1 H), 2.40 (s, 3 H), 2.28 (s, 3 H), 2.20-1.25 (m, 12 H), 1.93 (s, 3 H), 1.43 (t, J = 6.8 Hz, 3 H), 0.78 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 458 (MH⁺, 100), 398 (35). Analysis calculated for C₂₆H₃₅O₆N: C, 68.25; H, 7.71; N, 3.06. Found: C, 68.36; H, 7.90; N, 3.43.

Synthesis of B-homo-6-aza-6-acetyl-2-ethoxy-3,17-dihydroxyestra-1,3,5(10)-triene (47). A solution of compound 46 (150 mg, 0.33 mmol) in methanol (15 ml) was deoxygenated by bubbling a slow stream of nitrogen through it for 30 min. A similarly deoxygenated 1 M solution of sodium hydroxide in water (3.2 ml, 3.2 mmol) was added and the mixture was stirred at room temperature for 4.5 h. The reaction mixture was adjusted to pH 6-7 with acetic acid and the solvents were removed under reduced pressure. The residue was dissolved in ethyl acetate (60 ml) and the organic layer was washed with water (30 ml), saturated aqueous sodium bicarbonate (30 ml) and brine (30 ml), dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue on a silica gel column using ethyl acetate gave compound 47 (105 mg, 86%), which crystallized from ethyl acetate-hexane to afford white crystals: m.p. 189 to 191°C. ¹H NMR (CDCl₃)  6.77 (s, 1 H), 6.67 (s, 1 H), 4.09 (q, J = 6.9 Hz, 2 H), 4.08 (t, J = 6.9 Hz, 1 H), 3.71 (t, J = 8.5 Hz, 1 H), 3.03 (m, 1 H), 2.40 (dt, J = 8.5 and 2.0 Hz, 1 H), 2.12-1.15 (m, 14 H), 1.87 (s, 3 H), 1.43 (t, J = 7.2 Hz, 3 H), 0.78 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 374 (MH⁺, 100). Analysis calculated for C₂₂H₃₁O₄N·1/3 H₂O: C, 69.75; H, 8.41; N, 3.69. Found: C, 69.65; H, 8.25; N, 3.87.

Synthesis of B-Homo-6-aza-2-ethoxy-6a-ethyl-3,17-dihydroxyestra-1,3,5(10)-triene (32). A solution of borane in THF (1.0 M, 4.8 ml, 4.8 mmol) was added dropwise by syringe under argon at room temperature to a solution of compound 47 (90 mg, 0.24 mmol) in THF (5 ml), and the resulting solution was stirred for 4 h and then at gentle reflux for 4 h. The mixture was cooled to room temperature and 6 N hydrochloric acid (1 ml) was added slowly through a pipette. The solvent was removed under reduced pressure and the residue was neutralized with saturated aqueous sodium bicarbonate solution and then extracted with ethyl acetate (3 × 30 ml). The ethyl acetate layers were washed with brine (2 × 30 ml), dried over sodium sulfate and evaporated to dryness. Chromatography of the residue on a silica gel column using 20% ethyl acetate in methylene chloride gave compound 32 (70 mg, 81%), which was converted into a white stable foam when it was evaporated from ethyl acetate-hexane solution under reduced pressure: ¹H NMR (CDCl₃)  6.68 (s, 1 H), 6.51 (s, 1 H), 5.51 (s, 1 H), 4.07 (q, J = 6.9 Hz, 2 H), 3.78 (t, J = 8.5 Hz, 1 H), 3.38 (td, J = 11.5 and 2.2 Hz, 1 H), 3.20 (qd, J = 12.6 and 7.1 Hz, 1 H), 2.94 (qd, J = 11.7 and 6.8 Hz, 1 H), 2.75 (td, J = 11.5 and 2.2 Hz, 1 H), 2.55 (dt, J = 8.5 and 2 Hz, 1 H), 2.20-1.20 (m, 16 H), 1.42 (t, J= 6.4 Hz, 3 H), 0.84 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 360 (MH⁺, 100), 342 (MH-H₂O, 50). Analysis calculated for C₂₂H₃₃O₃N·1/6 H₂O: C, 72.96; H, 9.27; N, 3.86. Found: C, 72.97; H, 9.27; N, 3.85.

Synthesis of 3,17-Diacetoxy-2-ethoxyestra-1,3,5(10),6-tetraene-6-yl Triflate (48). A stirred solution of compound 43 (Cushman et al., 1997) (1.0 g, 2.4 mmol) and 2,6-di-tert-butyl-4-methylpyridine (743 mg, 3.6 mmol) in dry methylene chloride (48 ml) was treated with triflic anhydride (0.52 ml, 3.12 mmol) dropwise under argon at 0°C and stirring was continued for 3 h at 0°C. The mixture was poured into ice water (200 ml) and extracted with ethyl acetate (3 × 100 ml). The combined organic layer was washed with 2 N HCl (4 ml) in water (50 ml) and then brine (3 × 100 ml) until neutral, dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue on a silica gel column using 20% ethyl acetate in hexane gave compound 48 (1.15 g, 90%), which crystallized from ethyl acetate-hexane to afford white crystals: m.p. 148 to 150°C. ¹H NMR (CDCl₃)  7.03 (s, 1 H), 6.88 (s, 1 H), 5.78 (s, 1 H), 4.71 (t, J = 8.5 Hz, 1 H), 4.09 (q, J = 6.9 Hz, 2 H), 2.51-1.36 (m, 11 H), 2.29 (s, 3 H), 2.05 (s, 3 H), 1.35 (t, J = 6.0 Hz, 3 H), 0.82 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 547 (MH⁺, 100). Analysis calculated for C₂₅H₂₉O₈F₃S·1/2 ethyl acetate: C, 54.94; H, 5.63; F, 9.65. Found: C, 55.24; H, 5.39; F, 9.43.

Synthesis of 3,17-Diacetoxy-2-ethoxy-7-hydroxy-6-oxoestra-1,3,5(10)-triene (6). A solution of chromium trioxide (484 mg, 4.84 mmol) on 90% glacial acetic acid (10 ml) was added dropwise at 12 to 15°C to a solution of compound 43 (500 mg, 1.21 mmol) in glacial acetic acid (15 ml). The resulting mixture was stirred at 12 to 15°C for 3 h. The mixture was poured into an ice/water mixture (150 ml) and the product was extracted with ethyl acetate (3 × 100 ml). The combined organic payer was washed with water (2 × 100 ml), sodium bicarbonate (2 × 100 ml) and brine (2 × 100 ml), dried over sodium sulfate and evaporated to dryness. Chromatography of the residue on a silica gel column using 30% ethyl acetate in hexane gave compound 6 (185 mg, 36%), which was crystallized from ethyl acetate-hexane to afford yellow crystals: m.p. 188 to 190°C. ¹H NMR (CDCl₃)  7.75 (s, 1 H), 6.95 (s, 1 H), 4.75 (t, J = 8.5 Hz, 1 H), 4.10 (q, J = 6.9 Hz, 2 H), 2.71 (q, J = 4 Hz, 1H), 1.30-2.41 (m, 19 H), 0.82 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 431 (MH⁺, 100), 413 (MH-H₂O, 50). Analysis calculated for C₂₄H₃₀O₇: C, 66.96; H 7.02. Found: C, 66.92; H 6.90.

Synthesis of 3,17-Diacetoxy-2-ethoxyestra-1,3,5(10),6-tetraene (33). Under argon, 96% formic acid (0.31 ml, 8 mmol) was added to a stirred solution of compound 48 (1.1 g, 2.07 mmol), palladium acetate (18 mg, 0.08 mmol), triphenylphosphine (42 mg, 0.16 mmol), and triethylamine (1.68 ml, 12 mmol) in anhydrous DMF (8 ml). The mixture was stirred for 3 h at 60 to 62°C and then cooled to room temperature, diluted with brine (100 ml), and extracted with ethyl acetate (3 × 100 ml). The organic layers were washed with brine (3 × 100 ml), dried over sodium sulfate and evaporated to dryness. Chromatography of the residue on a silica gel column using 20% ethyl acetate in hexane provided compound 33 (0.75 g, 89%), which was crystallized from ethyl acetate-hexane to afford white crystals: m.p. 158 to 159°C. ¹H NMR (CDCl₃)  6.87 (s, 1 H), 6.71 (s, 1 H), 6.37 (d, J = 9.8 Hz, 1 H), 5.82 (d, J = 9.8 Hz, 1 H), 4.71 (t, J = 8.5 Hz, 1 H), 4.09 (q, J = 6.9 Hz, 2 H), 2.51-1.36 (m, 11 H), 2.28 (s, 3 H), 2.06 (s, 3 H), 1.37 (t, J= 6.9 Hz, 3 H), 0.82 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 399 (MH⁺, 100). Analysis calculated for C₂₄H₃₀O₅: C, 72.34; H, 7.59. Found: C, 72.45; H, 7.60.

Synthesis of 3,17-Diacetoxy-2-ethoxy-7-hydroxyestra-1,3,5(10)-trien-6-(m-chlorobenzoate) (49) and 3,17-Diacetoxy-2-ethoxy-7-hydroxyestra-1,3,5(10)-trien-6-(m-chlorobenzoate) (50). A solution of m-chloroperbenzoic acid (260 mg, 57 to 81%, 0.86-1.23 mmol) in methylene chloride (10 ml) was added dropwise to a stirred solution of compound 33 (343 mg, 0.86 mmol) in methylene chloride (20 ml) under nitrogen atmosphere at 0°C. The mixture was allowed to come to room temperature and stirred for 20 h. The mixture was poured into ice-cold saturated sodium bicarbonate (100 ml) and the mixture was extracted with methylene chloride (3 × 50 ml). The combined organic layer was washed with water (2 × 50 ml), sodium bicarbonate (2 × 50 ml) and brine (2 × 50 ml), dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue on a silica gel column using 25% ethyl acetate in hexane gave compound 49 (153 mg, 31%), which was crystallized from diethyl ether to afford a white solid: m.p. > 110°C (dec); ¹H NMR (CDCl₃)  8.08 (s, 1 H), 8.00 (d, J = 7.8 Hz, 1 H), 7.57 (d, J = 7.8 Hz, 1 H), 7.40 (t, J = 7.8 Hz, 1 H), 6.91 (s, 1 H), 6.87 (s, 1 H), 6.19 (d, J = 3.5 Hz, 1 H), 4.72 (t, J = 8.5 Hz, 1 H), 4.24 (d, J = 3.7 Hz, 1 H), 4.01 (q, J = 6.9 Hz, 2 H), 2.88 (bs, 1 H), 2.24 (s, 3 H), 2.04 (s, 3 H). 2.04-1.30 (m, 11 H), 1.36 (t, J = 6.9 Hz, 3 H), 0.83 (s, 3 H). Compound 50 (260 mg, 53%) was crystallized from ethyl acetate-hexane to afford a white solid: m.p. 140-145°C. ¹H NMR (CDCl₃)  8.0 (s, 1 H), 7.84 (d, J = 7.8 Hz, 1 H), 7.50 (d, J = 7.8 Hz, 1 H), 7.35 (t, J = 7.8 Hz, 1 H), 7.01(s, 1 H), 6.94 (s, 1 H), 5.90 (d, J = 2.2 Hz), 4.72 (t, J = 8.5 Hz, 1 H), 4.06 (q, J = 6.9 Hz, 2 H), 4.02 (d, J = 2.2 Hz, 1 H), 2.71 (dt, J = 4.2 and 2.1 Hz, 1 H), 2.24 (s, 3 H), 2.05 (s, 3 H), 2.04-1.30 (m, 11 H), 1.37 (t, J = 6.9 Hz, 3 H), 0.83 (s, 3 H).

Synthesis of 2-Ethoxy-3,6,7,17-tetrahydroxyestra-1,3,5(10)-triene (51). A solution of LiAlH₄ in THF (1.0 M, 5 ml, 5 mmol) was added to a solution of compound 49 (270 mg, 0.49 mmol) in THF (10 ml) and the resulting suspension was stirred at gentle reflux overnight (14 h). The reaction mixture was cooled and added cautiously into ice/water (50 ml), and then acidified to pH 1 with 3 M HCl. The mixture was extracted with ethyl acetate (3 × 60 ml). The organic layers were washed with water (50 ml), sodium bicarbonate (50 ml), and brine (50 ml), dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue on a silica gel column using 30% acetone in methylene chloride gave compound 51 (141 mg, 88%), which was crystallized from ethyl acetate-methylene chloride to afford white crystals: m.p. 231 to 232°C. ¹H NMR (CDCl₃)  7.05 (s, 1 H), 6.82 (s, 1 H), 4.53 (d, J = 3.7 Hz, 1 H), 4.08 (q, J = 6.9 Hz, 2 H), 3.93 (d, J = 3.8 Hz, 1 H), 3.69 (t, J = 8.5 Hz, 1 H), 2.95 (m, 2 H), 2.68 (dt, J = 7.6 Hz and 4.0 Hz, 1 H), 2.34 (dd, J = 10.0 and 3.2 Hz, 1 H), 2.14-1.20 (m, 11 H), 1.35 (t, J = 6.8 Hz, 3 H), 0.78 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 349 (MH⁺, 20), 331 (MH-H₂O, 100), 313 (MH⁺-2H₂O, 50). Analysis calculated for C₂₀H₂₈O₅·1/3 H₂O: C, 67.78; H, 8.15. Found: C, 67.76; H, 8.21.

Synthesis of 2-Ethoxy-3,6,7,17-tetrahydroxyestra-1,3,5(10)-triene (7). A solution of LiAlH₄ in THF (1.0 M, 5 ml, 5 mmol) was added to a solution of compound 50 (120 mg, 0.21 mmol) in THF (10 ml), and the resulting suspension was stirred at gentle reflux overnight (14 h). The reaction mixture was cooled and added cautiously into ice/water (50 ml) and then acidified to pH 1 with 3 M HCl. The mixture was extracted with ethyl acetate (3 × 60 ml). The organic layers were washed with water (50 ml), sodium bicarbonate (50 ml) and brine (50 ml), dried over sodium sulfate, and evaporated to dryness. Chromatography of the residue on a silica gel column using 30% acetone in methylene chloride gave compound 7 (48 mg, 66%), which was crystallized from ethyl acetate/diethyl ether to afford white crystals: m.p. 182 to 184°C. ¹H NMR (CDCl₃)  6.84 (s, 1 H), 6.78 (s, 1 H), 4.34 (bs, 1 H), 4.09 (q, J = 6.9 Hz, 2 H), 3.86 (bs, 1 H), 3.71 (t, J = 8.5 Hz, 1 H), 2.95 (bs, 3 H, OH), 2.56 (dt, J = 7.6 and 4.0 Hz, 1 H), 2.34 (dd, J = 9.9 and 3.2 Hz, 1 H), 2.14-1.20 (m, 11 H), 1.38 (t, J = 6.9 Hz, 3 H), 0.78 (s, 3 H); CIMS (isobutane) m/z (relative intensity) 349 (MH⁺, 18), 331 (MH-H₂O, 100), 313 (MH⁺-2H₂O, 50). Analysis calculated for C₂₀H₂₈O₅·1/3 H₂O: C, 67.78; H, 8.15. Found: C, 67.52; H, 8.21.

	Materials and Methods

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Melting points were determined in capillary tubes on a Mel-Temp apparatus and are uncorrected. Spectra were obtained as follows: ¹H NMR spectra were recorded on a Varian VXR-300S spectrometer (Palo Alto, CA) using tetramethylsilane as an internal standard; CIMS spectra were obtained on a Finnigan 4000 spectrometer (Thermo Finnigan, San Jose, CA); microanalyses were performed at the Purdue Microanalysis Laboratory. Flash column chromatography was performed on 60-µm silica gel from Scientific Adsorbents, Inc. (Atlanta, GA). Most chemicals and solvents were analytical grade and used without further purification. Commercial reagents were purchased from Aldrich Chemical Company (Milwaukee, WI).

Culture of Human Airway Smooth Muscle

Human airway smooth muscle was cultured from macroscopically normal bronchi (0.5- to 2-cm diameter) obtained from lung resection or heart-lung transplant specimens provided by the Alfred Hospital (Melbourne) according to methods published in detail previously (Fernandes et al., 1999; Stewart et al., 1999a). Approximately 0.1 g of smooth muscle was stripped from the wall of the bronchus for each cell culture. Dissected tissue was immersed in Dulbecco's modified Eagle's medium (DMEM; Flow Laboratories, Irvine, Scotland, UK), supplemented with 100 U/ml penicillin G (CSL Australia, Parkville, Victoria, Australia) and 100 µg/ml streptomycin (CSL Australia). The tissue was rinsed in phosphate-buffered saline (PBS; Oxoid, Basingstoke, Hampshire, England) and the airway smooth muscle was chopped into 2-mm³ pieces and digested for 2 h in DMEM containing elastase (0.5 mg/ml; Worthington Biochemical, Freehold, NJ) followed by a 12-h incubation in DMEM containing collagenase (1 mg/ml) (Worthington Biochemical), at 37°C with agitation. The resulting cell suspension was centrifuged and washed three times with PBS. After the last centrifugation step, the cells were resuspended in 25 ml of DMEM supplemented with L-glutamine (2 mM; Sigma), penicillin G (100 U/ml), streptomycin (100 µg/ml), amphotericin B (2 µg/ml; Wellcome, Stevenage, UK) and heat-inactivated FCS (10% v/v; CSL Australia) and seeded into 25-cm² culture flasks. The primary isolates were incubated for 7 to 14 days to reach confluence. Cells were harvested weekly by 10 min exposure to trypsin (0.5%; CSL Australia) and EDTA (1 mM in PBS; BDH, Kilsyth, Victoria, Australia) and passaged at a 1:3 ratio into 75-cm² flasks.

Inhibition of DNA Synthesis

Cells were subcultured into 24-well plates in a 1:3 split ratio at a density of approximately 1.5 × 10⁴ cells/cm² and grown to monolayer confluence in antimicrobial-supplemented DMEM containing 10% fetal calf serum, over a 72 to 96 h period (5% CO₂ in air, 37°C). Quiescence was induced 24 h before stimulation by removing the medium, washing with PBS and replacing the medium with serum-free DMEM (supplemented as described previously) to synchronize the cells in G₀ of the cell cycle. Cells were stimulated by the addition of thrombin [bovine plasma, 0.3 U/ml, prepared in PBS containing bovine serum albumin (0.25% w/v; Sigma). Monomed A (1% v/v; CSL Australia), a serum-free medium supplement containing insulin, transferrin, and selenium was added to all wells. 2-MEO and analogs were dissolved in 100% dimethyl sulfoxide (BDH, Poole, Dorset, UK) at a concentration of 10 mM and diluted serially in PBS, before addition of 100 µl to 1 ml of medium overlying airway smooth muscle, up to a maximal final analog concentration of 10 µM. This resulted in a final maximum concentration of 0.1% dimethyl sulfoxide, which was added to the corresponding control wells. Concentration-response relationships were constructed from an evaluation of the effects of the analogs (0.1 µM to 10 µM, in half-log increments) on thrombin-stimulated DNA synthesis. Individual responses were calculated by incubating cells with the analog agents for 24 h (5% CO₂ in air, 37°C) then pulsing the cells with [³H]thymidine (1 µCi/ml; Amersham, Little Chalfont, Buckinhamshire, UK) for 4 h to label newly synthesized DNA. Before harvest, the medium was removed and the cells were solubilized by the addition of 100 µl sodium hydroxide (1 M; Selby Biolab, Melbourne, Australia) to each well. Cellular DNA was immobilized onto glass fiber filters using a Packard Filtermate 196 Cell Harvester (Packard BioSciences, Melbourne, Australia). The filters were washed three times with 3 ml distilled H₂O and 1 ml ethanol (100%). Packard Microscint-40 (Packard) was added to dried filters and radioactivity was measured using a Packard Top Count Microplate Scintillation Counter.

ER Binding Assay

ER affinity of estradiol (16), its metabolites [2-hydroxyestradiol (19), 2-MEO (1)] and analogs were determined using rat uterine cytosol as a source of ER (Markaverich and Clark, 1979). Uteri (6-10 per batch) were isolated from Brown Norway rats (250-350 g), washed free from blood in saline, and homogenized in 10 mM Tris buffer [pH 7.4, 1.5 mM EDTA, 10% (w/v) glycerol, 1 mM phenylmethylsulfonylfluoride] using an Ultra Turrax homogenizer for 3 15-s bursts with intervals of 1 min on ice. For additional studies of ER in smooth muscle, cytosol from human airway smooth muscle was prepared from 16- × 175-cm² flasks of confluent cells grown as described, and serum-deprived for 24 h before harvest. The cells were washed 3 times with PBS before the addition of a total of 4 ml of the 10 mM Tris buffer described above. The cells were scraped from the plates and subjected to homogenization by 30 passes in a glass homogenizer. The airway smooth muscle and rat uterus preparations were then processed in the following manner. Nuclear material and cellular debris were removed by centrifugation at 750g at 4°C for 10 min (Sorvall RT7) and cytosol was prepared by centrifugation at 30,000g for 120 min at 4°C in a Beckman JM1 centrifuge. The cytosol was diluted to contain approximately 1 mg/ml protein (rat uterine extract) or 0.015 mg/ml (airway smooth muscle) determined using the Bradford method (Bio-Rad, Hercules, CA). Binding assays were carried out by overnight incubation at 4°C in 300 µl of the above described buffer: 200 µl of cytosol, 50 µl of displacer [10 mM estradiol (16) or buffer] and 50 µL of 0.2 nM [³H]estradiol. Separation of bound from free radioligand was achieved by the addition of 500 µl of dextran-coated charcoal (400 mg of dextran clinical grade C and 2 g of charcoal, Norit A in 100 ml of Tris buffer) and centrifugation at 4°C in a Sorval RT7 at 2000g for 10 min. Preliminary saturation analysis over the range 0.01 to 50 nM [³H]estradiol yielded a K_d and B_max values of 0.18 nM and 112 fmol/mg protein, respectively [n = 3, analysis using Prism (GraphPad Software, San Diego, CA)], indicating that the 0.2 nM concentration was suitable for displacement studies. Saturation analysis carried out on airway smooth muscle-derived cytosol revealed an affinity (K_D) of 0.53 nM and a much higher density of binding sites of 1071 fmol/mg protein. The very low yield of protein from large amounts of cultured airway smooth muscle made further analyses of ER binding in this cell type impractical. Analyses of 2-MEO (1) and analogs were initially carried out over the range 0.1 nM to 10 µM in decade increments and, after identification of the appropriate range, displacement of estradiol was determined using three to four concentrations per decade. The data were fitted to the Cheng and Prusoff equation using the K_d value of 0.18 nM (Prism) and the data are presented as pIC₅₀.

Cell Enumeration

Cells were seeded onto six-well plates at a density of 1.5 × 10⁴ cells cm², made quiescent as described previously, and then stimulated for 48 h with thrombin (0.3 U/ml). At the end of the 48 h incubation period, cells were detached from the culture plate by the addition of trypsin (0.5% w/v in PBS containing 1 mM EDTA), incubated for 5 min at ambient temperature in PBS containing 0.5% (w/v) trypan blue, washed twice (2% FCS in PBS), isolated by centrifugation (12,000g, 5 min) and resuspended in 200 µl 2% FCS in PBS for counting in a hemocytometer chamber.

Flow Cytometric Analyses of Cell Cycle Status

Cells seeded into 6-well plates at a density of 1.5 × 10⁴ cells cm² and made quiescent as described previously were then stimulated with thrombin (0.3 U/ml) for 48 h. Monomed A was added to all cells (including control cells) at the time of mitogen addition. Cells were detached from the culture plates by incubation with trypsin for 30 min at 37°C (0.5% w/v). The resulting suspension was washed in PBS twice before resuspension in 1 ml of 70% ethanol for storage for up to 3 weeks at 20°C. Before staining, cells were washed twice (2% FCS in PBS) to remove the ethanol. Fixed cells were stained with propidium iodide (50 µg/ml) in Triton X-100 (0.1% v/v) with RNase II (180 mU/ml). The cell suspension was passed through an 18-gauge needle to facilitate the separation of cell clumps. Cells were stored for 24 h before analysis. Cell cycle status was analyzed using a FACScan Instrument (BD, Franklin Lakes, NJ). Ten thousand events from each sample were counted and analyzed using a ModFitLT V2.0 analysis package (Verity Software House, Topsham, ME).

Statistical Analyses

All incubations for the [³H]thymidine incorporation assays were conducted in triplicate or quadruplicate. Experiments were carried out in at least three cell lines, each derived from at least three different individuals; results are expressed as the mean of these estimations. To minimize the influence of variability between tissue donors on comparisons of data, the response to thrombin in the presence of the antagonist was expressed as a percentage of the thrombin control in individual cultures (100%). The potency of the analogs was determined by fitting log concentration-response data using nonlinear regression to fit a sigmoidal relationship. The pIC₅₀ values represent the negative log of the concentrations that reduce the thrombin-stimulated DNA synthesis to 50% of the response in the absence of inhibitor. The binding data were analyzed by the Cheng and Prusoff equation (Prism) and are presented as pIC50 values that represent the log of the concentration of analog that displaces 50% of the specifically bound [³H]estradiol. The standard errors of these pIC50 values (Tables 4 to 6) did not exceed 5% of the mean value.

Molecular Modeling Studies

Molecular modeling studies were carried out using SYBYL software (version 6.4; Tripos Inc., St Louis, MO) running on a Silicon Graphics O2 workstation equipped with a 175-MHz R10000 processor (Silicon Graphics, Mountain View, CA).

Generation of Molecular Models. Molecular models of 2-MEO, its analogs, and other compounds used in this study were constructed from standard SYBYL fragments, and their geometry was optimized (Powell conjugate gradient minimization, termination at a gradient of <0.005 kcal mol¹ Ź) using the Tripos forcefield and Gasteiger-Marsili partial atomic charges (Gasteiger and Marsili, 1980). Lowest energy conformations of flexible sidechains were identified by applying the Systematic Search routine in SYBYL to all rotatable bonds (10° torsion angle increments of flexible bonds) with reminimization, as described above, of low-energy candidates. Spectroscopic data of compound 6 was consistent with a degree of keto-enol tautomerism at the 6- and 7-positions; the compound was thus modeled as the enediol.

Calculation of CoMFA Fields. 3D-QSAR relationships for the compounds were generated using the CoMFA routines in the QSAR option in Sybyl (Cramer et al., 1988). Compounds were first aligned (see below) and steric and electrostatic energies calculated for each molecule throughout a 2-Å grid, extending 4 Å beyond the van der Waals' volume of all molecules, using an sp³ carbon atom probe with a charge of +1 and a distance-dependent dielectric constant. The resultant matricesa row for each compound, a column of biological activities (either DNA synthesis inhibition or ER binding), and multiple columns for each molecule containing the molecular field strengths at each grid pointwere subjected to partial least-squares (PLS) regression analysis (see below) to derive 3D-QSAR models.

Three-Dimensional Alignments for CoMFA. 3D-QSAR models obtained by CoMFA techniques are sensitive to the spatial alignment of the molecules under study. To examine the effect of alignment, CoMFA models were generated for the following alignments (created by aligning molecules with 2-MEO using the Fit routine in Sybyl):

Steroid alignments.

1.	Aligned by overlaying the atoms corresponding to the three oxygen atoms in 2-MEO. These atoms are common to all analogs and, by analogy to the requirements for ER binding (Anstead et al., 1997), might therefore correspond to key pharmacophoric points.
2.	Aligned as in 1, except that the side chain in position R2 was oriented to match the conformation of this group in 2-MEO, except for compounds with an acetate group in this position. These compounds had their side chains adjusted to the conformation of the side chain in 2-MEO, followed by minimization, rather than in their strictly low energy conformations as in alignment 1. This alignment would be expected to reduce the likelihood that alternative side chain comformations (any of which might be reasonably accessible in a biological milieu) could introduce inappropriate structural variation in compounds.
3.	Aligned by fitting all common, nonhydrogen atoms. This alignment takes weight away from the positions of the oxygen atoms used in alignments 1 and 2 and places more emphasis on the positions of backbone atoms. Side chains were in their low energy conformations as in alignment 1. This alignment was thought to be worthy of examination, given that the estrogens are likely to be compounds that are well matched to the ER (Andrews et al., 1984).
4.	Aligned as in 3, except that side chain conformations were adjusted to match those of 2.

Nonsteroid alignments.

1.	4-OH-tamoxifen (37) was aligned by superimposing the centroids of two aromatic rings of 37 with the centroids of the A and D rings of 2-MEO and the aromatic O-substituents of 37 with the O-substituents of 2-MEO in positions R2 and R5. This alignment has previously been used in developing 3D-QSAR relationships for nonsteroidal ligands for the estrogen receptor (Sadler et al., 1998).
2.	Flavones (38 and 39) were aligned by superimposing the carbon atoms of the aromatic ring of the flavone  to the carbonyl group. The centroid of the other aromatic ring was fitted to the centroid of the steroid D-ring.

Partial Least Squares (PLS). PLS is a type of regression analysis particularly suited to situations such as CoMFA, where the independent variables (i.e., molecular field strengths) greatly outnumber the dependent variables (biological activities) (Wold, 1991). It uses a means of data reduction to transform the dependent and independent variables into a set of so-called principal components, which are mutually orthogonal descriptors from which the QSAR equations can subsequently be derived. The optimal number of components (after which the inclusion of additional components does not further improve the quality of the model) for the models for ER binding and DNA synthesis inhibition was determined in so-called cross-validated runs. In these runs, successive QSAR equations are generated in which one compound is omitted, and the model derived in the absence of this compound and used to predict the activity of the omitted compound. The process was repeated until equations had been derived with each compound omitted oncethe activity of each omitted compound was predictedgiving a set of n QSAR equations for n compounds (leave-one-out method). The final CoMFA models were then derived from non-cross-validated runs using the optimal number of components determined from the cross-validated runs. The quality of the QSAR equations generated during the cross-validated runs was measured by the term q² (the cross-validated r²), calculated thus: q² = (SD  PRESS) / SD, where SD is the variance of the biological activities, and PRESS is the sum of the squares of the differences between predicted and observed biological activities of each compound as it is left out of the analysis during the cross-validation process.

Optimization of CoMFA Models. The CoMFA implementation in Sybyl allows the user to optimize the model by altering several parameters.

1.	Choosing the cut-off value for the energy at a given grid point (grid points whose computed energy is greater than the cut-off value are assigned the cut-off value). The effect of cut-off on the quality and predictive capabilities of the CoMFA models was thus examined over a range of cut-off values (10-100 kcal/mol).
2.	Scaling the field strength data (implementing either CoMFA standard scaling, the default choice, which attempts to balance the potential influence of each field and column on the resulting QSAR by scaling the data to the overall field mean and standard deviation, or no scaling, which emphasizes variables with a greater numerical spread, typically those describing the steric field).
3.	Dropping electrostatic values at the steric maximum, (i.e., so that electrostatic field contributions from "inside" a molecule are not considered).

Presentation of CoMFA Models. The final optimized CoMFA models for DNA inhibition and ER binding obtained from non-cross-validated runs are presented as isocontour maps of extreme values of the product of the S.D. and coefficients of the 3D-QSAR equations derived by PLS analysis at the grid points, after scaling of this product from 0 to 100%. (The product of the S.D. and coefficient was also used to obtain the relative contributions of the steric and electrostatic fields.) Such plots indicate where in space differences in either the steric or electrostatic fields of the molecules in the data set correlate most with differences in biological activity (Cramer et al., 1988). Generally, contours corresponding to >85% and <15% of the signal gave readily interpretable isocontour maps (i.e., regions associated with the molecular fields of the datasets that correlated either positively or negatively with biological activity were represented by clearly defined, discrete polyhedra), except for the electrostatic field map for DNA synthesis inhibition, where contours of >80% and <20% were used to give a clearer indication of the position of electrostatically and sterically favorable substituents.

	Results

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Synthetic Chemistry

Prior work has demonstrated that the cytotoxicities and tubulin polymerization inhibitory potencies of 2-MEO analogs can be modulated by replacement of the 2-methoxyl group by other substituents (Cushman et al., 1995, 1997). Replacement of the 2-methoxyl group of 2-MEO by 2-ethoxyl and 2-(E)-1-propenyl substituents resulted in compounds that were more potent than estradiol itself (Cushman et al., 1995). In general, the optimal substituent in the 2-position proved to be a straight chain containing three atoms from the second row of the periodic table. To investigate this in more detail, the oxime (13), methoxime (14), and hydrazone (15) derivatives were prepared as outlined in Scheme 1. Treatment of estradiol (16) with methoxymethyl chloride in the presence of diisopropylethylamine afforded the bis(methoxymethyl) ether 40. Intermediate 40 was regioselectively lithiated in the 2-position with sec-butyllithium in THF, followed by formylation with DMF to yield the aldehyde 41 in high yield as a single regioisomer. Deprotection of the bis(methoxymethyl) ether 41 with lithium tetrafluoroborate in acetonitrile provided 2-formylestradiol (42) (Ireland and Varney, 1986). The oxime oxime (13), methoxime (14), and hydrazone (15) derivatives were then obtained by reaction of 42 with hydroxylamine, methoxylamine, and hydrazine, respectively.

View larger version (21K): [in this window] [in a new window]   Scheme 1.   Reagents and conditions: a, CH3OCH2Cl, i-Pr2EtN, THF, 60°C (8 h); b, (1) sec-BuLi, THF, 78°C (2.5 h), (2) DMF, 78 to 23°C (3 h); c, LiBF4, aq CH3CN, 72°C (5 h); d, H2NOH·HCl, pyridine, 90°C (1 h); e, H2NOCH3, pyridine, 90°C (2 h); f, H2NNH2·2HCl, pyridine, 90°C (2 h).

Certain B-ring expanded analogs of 2-ethoxyestradiol resemble paclitaxel in their ability to enhance tubulin polymerization and stabilize microtubules (Wang et al., 2000). We have therefore synthesized a variety of additional B-ring homologated analogs of 2-ethoxyestradiol, including the benzo[f]azepine derivative 32 (Scheme 2). Treatment of 2-ethoxy-3,17-diacetoxy-6-oxoestra-1,3,5(10)-triene (43) (Cushman et al., 1997) with hydroxylamine resulted in selective deacetylation of the phenolic acetate as well as oxime formation to afford 8. There are several reports of the use of oxime tosylates as substrates for Beckmann rearrangements (Tamura et al., 1973; Ciattini et al., 1990). These rearrangements are reported to occur when the oxime tosylate is passed through a column of acidic or basic alumina. Reaction of 8 with tosyl chloride in pyridine resulted in the formation of the oxime tosylate, which underwent the desired Beckmann rearrangement when passed through a column of basic alumina and eluted with a mixture of benzene and chloroform to afford the lactam 44. Diborane reduction of 44 yielded the expected benzo[f]azepine system 45. Reaction of 45 with acetic anhydride resulted in acetylation of both alcohols as well as the amine to give 46, which was hydrolyzed with sodium hydroxide to provide the diol 47. Diborane reduction of the amide present in 47 produced the desired product 32.

View larger version (14K): [in this window] [in a new window]   Scheme 2.   Reagents and conditions: a, H2NOH·HCl, pyridine, 100°C (30 min); b, (1) TsCl, pyridine, 23°C (2 h), (2) Al2O3, CHCl3-C6H6; c, B2H6, THF, 23°C (6 h) and reflux (4 h); d, Ac2O, pyridine, 23°C (12 h); e, Aq NaOH, MeOH, 23°C (4.5 h); f, B2H6, THF, 23°C (4 h) and reflux (4 h).

Oxidation of the B ring of 2-ethoxyestradiol has afforded a number of very potent cytotoxic tubulin polymerization inhibitors (Cushman et al., 1997). These included 2-ethoxy-6-oxoestradiol and the corresponding oxime. Methods have therefore been sought for oxidation of the B ring of 2-ethoxyestradiol. As shown in Scheme 3, treatment of 2-ethoxy-6-oxoestradiol diacetate (43) (Cushman et al., 1997) with triflic anhydride under basic conditions afforded the enol triflate 48, which was reduced in the presence of formic acid, triethylamine, triphenylphosphine, and palladium acetate in DMF to afford the cyclic alkene 33 (Ciattini et al., 1990). Instead of the expected 6,7-epoxide, oxidation of 33 with m-chloroperbenzoic acid gave compounds 49 and 50, which are the addition products of m-chloroperbenzoic acid to the 6,7-epoxide (Burdett et al., 1982). The stereochemical assignments are based on the larger axial-equatorial coupling observed between the C-6 and C-7 methine protons in 49 (J = 3.5 Hz) versus the smaller diequatorial coupling constant (J = 2.2 Hz) seen with 50 (Jackman and Sternhell, 1969). Lithium aluminum hydride reduction of 49 and 50 cleaved all of the esters, resulting in the tetraols 51 and 7. Oxidation of the starting material 43 with chromium trioxide in acetic acid gave the -hydroxyketone 6.

View larger version (18K): [in this window] [in a new window]   Scheme 3.   Reagents and conditions: a, (TfO)2O, 2,6-(t-Bu)-4-Me-pyridine, CH2Cl2, 0°C (3 h); b, CrO3, AcOH, 12-15°C (3 h); c, HCOOH, Et3N, Ph3P, Pd(Ac)2, DMF, 60 to 62°C (3 h); d, MCPBA, TsOH, CH2Cl2, 23°C (20 h); e, LiAlH4, THF, reflux (14 h).

2-MEO Effects on DNA Synthesis, Proliferation and Cell-Cycle Distribution of Human Airway Smooth Muscle Cells

2-MEO (0.3-10 µM) concentration-dependently inhibited ³H-thymidine incorporation in cultures of human airway smooth muscle cells (Table 1). Cell enumeration experiments revealed that 2-MEO (10 µM) prevented 0.3 U/ml thrombin-induced increase in cell number but had no effect on the number of nonviable cells after incubation for 48 h (Table 2). Flow cytometric analyses of cell-cycle distribution (Fig. 1) indicated that 2-MEO (10 µM) reduced the proportion of cells in S-phase and increased that in G₂/M (Table 3). In a separate series of experiments, the incubation of cells with thrombin (0.3 U/ml) increased cell number (×10⁴ well of a 6-well plate) from 29.8 ± 3.9 to 45.5 ± 8.5; this was significantly reduced (P < 0.05, paired t test) to 33.8 ± 6.7 and 32.0 ± 5.0, for 2-MEO (10 µM) and compound 5, respectively, without concomitant increase in the number of trypan blue staining (i.e., nonviable) cells.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Influence of thrombin (0.3 U/ml) on propidium iodide DNA profiles in human airway smooth muscle cells in culture and its modification by 2-MEO (10 µM). These profiles are representative of 12 others (see Table 2) and show that thrombin (B) increases the proportion of cells in S-phase over that observed in unstimulated cells (A). The thrombin-induced increase in cells in S-phase is prevented by 2-MEO (C), which also increases the proportion of cells in G2/M.

Inhibition of Human Airway Smooth Muscle DNA Synthesis by 2-MEO Analogs

The range of data obtained for DNA synthesis inhibition in cultures of human airway smooth muscle (Tables 4-6) was narrow, covering only one order of magnitude for active compounds. The most potent inhibitors were the 2-ethoxyestradiol derivatives 8 and 6, 2-(hydroximino)estradiol (13) and ICI 182,780 (17) (pIC₅₀ for DNA synthesis inhibition of 5.99, 5.97, 5.95, and 5.88, respectively), all being considerably more potent than 2-MEO itself (pIC₅₀ 5.21). Compound 6 also displays negligible ER binding potency (see below). Both 8 and 6 are acetylated at the 17 position (R5), although this feature is itself not sufficient for potent DNA synthesis inhibition, because other acetylated compounds show reduced activity (e.g., the diacetylated 33, pIC₅₀ 5.24). Two compounds, 3 and 36, stimulated rather than inhibited DNA synthesis; these compounds were not examined further by CoMFA.

Displacement of [³H]Estradiol from Rat Uterine ER

In contrast to the data obtained for DNA synthesis inhibition, ER binding affinities covered a much larger range of values (approximately 5 orders of magnitude; Tables 4-6). The most potent displacement activity was exhibited by 16, 34, and 19 (pIC₅₀ for ER binding of 9.89, 9.77, and 9.49, respectively). All compounds tested with a seven-membered B ring (Table 2) were devoid of ER binding, although most of these analogs retained DNA synthesis inhibition (23-31). Some compounds tested were potent inhibitors of DNA synthesis and ER binding (e.g., 13, pIC₅₀ for DNA synthesis inhibition of 5.95, pIC₅₀ for ER binding 9.16). Overall, however, there was no correlation between potency in the two biological assays (r² = 0.038 for the 23 compounds for which biological potency was determined in both assays).

Generation and Optimization of 3D-QSARs

Selection of Optimal Alignments. Table 7 shows the q² obtained for the preliminary CoMFA models obtained from the four different alignments for DNA synthesis inhibition and ER binding data. For DNA synthesis inhibition data, the q² values obtained were low, indicative of models of limited predictive values. Using the default cut-off (30 kcal/mol¹), alignment 4 gave the most predictive model (q² = 0.144); this alignment was thus used to generate optimized models. For ER binding, the highest q² values were obtained for alignments 1 and 3 (q² = 0.776 and 0.760, respectively). Optimized CoMFA plots were subsequently generated from alignment 3, because initial CoMFA plots obtained from alignment 1 were poorly defined, and difficult to interpret, even after preliminary attempts to optimize them (data not shown).

Optimization of CoMFA Models. Optimized CoMFA models were obtained from the preliminary models by systematically altering user-modifiable parameterscut-off energy, field-strength scaling, and dropping electrostatic values at the steric maximuman