Synthesis and biological evaluation of 3-(azolylmethyl)-1H-indoles and 3-(α-azolylbenzyl)-1H-indoles as selective aromatase inhibitors

Abstract

This present study identifies a number of azolyl-substituted indoles as potent inhibitors of aromatase. In the sub-series of 3-(azolylmethyl)-1H-indoles, four imidazole derivatives and their triazole analogues were tested. Imidazole derivatives 11 and 14 in which the benzyl moiety was substituted by 2-chloro and 4-cyano groups, respectively, were the most active, with IC₅₀ values ranging between 0.054 and 0.050 μM. In the other sub-series, eight 3-(α-azolylbenzyl)-1H-indoles were prepared and tested. Compound 30, the N-ethyl imidazole derivative, proved to be an aromatase inhibitor, showing an IC₅₀ value of 0.052 μM. All target compounds were further evaluated against 17α-hydroxylase/C17,20-lyase to determine their selectivity profile.

Keywords::

• Indole
• azoles
• inhibitor
• aromatase
• 17α-hydroxylase/C17,20-lyase
• breast cancer
• selective

Introduction

Estrogens not only play a key role in normal expression of secondary sexual characteristics, and establishment and maintenance of pregnancy but are also involved in the natural history of breast cancer. A high proportion of breast cancer tumors are dependent upon estrogens for growth and respond to therapeutic measures designed to deplete circulating estrogens. Aromatase, a cytochrome P450 enzyme (P450 arom), catalyzes the biosynthesis of these estrogens from androgens. Activity of aromatase results in aromatization of the A ring of androgens with the concomitant loss of the C₁₈ angular methyl group. So, the inhibition of the enzyme becomes a logical aim in breast cancer hormonotherapy [1,2].

The structurally diverse group of aromatase inhibitors are classically categorized into two major group: steroidal and non-steroidal derivatives; they appear to be different on their ability to interact with the enzyme [3,4]. A great number of steroidal compounds structurally related to the natural substrates (androstenedione or testosterone) have been developped. 4-Hydroxyandrostenedione (4-OHA) [5,6] was the first aromatase steroidal inhibitor to become available, and had high enzyme selectivity. But it had to be administered as a parental formulation due to its poor oral bioavailability. Other steroidal inhibitors, in addition to 4-OHA, have been also shown to cause inactivation of aromatase 7-11. One of these, exemestane [11], has proved to be orally effective in postmenopausal women and reached the market in 1999. Steroidal inhibitors bind either very tightly or irreversibly to aromatase and then cause its inactivation [6,12].

Among the non-steroidal inhibitors, the most studied compounds were aminoglutethimide [AG, 3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione] [13], 4-substituted anilines 14-16, imidazole antifungals and analogs[17,18]. AG, the first commercially available non-steroidal inhibitor, was launched in 1981. However, the use of AG [19] has been limited due to (i) its lack of specificity - AG inhibits other cytochrome P450 enzymes such as P450scc and, P450c11 - and (ii) its intrinsic toxicity - main side-effects are lethargy, dizziness and skin rashes. Investigations of many research teams have resulted notably in the discovery of several heterocyclic azoles 20-23 such as fadrozole, anastrozole, letrozole, vorozole, YM-511 and MPV-2213ad (see Figure 1 for structures).

Figure 1 Structures of nonsteroidal inhibitors.

As shown in Table I, a major advance in potency is observed for these new non-steroidal aromatase inhibitors. The selectivity profile is also increased for fadrozole [24], vorozole [5], letrozole [26], anastrozole [27], YM-511 [28] and MPV-2213ad [29]. About their mechanism of action, it can be noted that they perturb the catalytic properties of the heme prosthetic group of aromatase [30].

Table I.  Inhibition of aromatase by major nonsteroidal inhibitors.

compound	P450 arom inhibition [23] IC50 (nM)	material
aminoglutethimide	3,800.000	Human placenta
MPV-2213ad	0,180.000	Human placenta
anastrozole	14.600	Human placenta
fadrozole	6.910	Human placenta
letrozole	0.390	Human placenta
vorozole	1.400	Rat granulosa cells
YM-511	0.125	Human placenta

In previous works 31-33, we initially prepared and evaluated 3-azolylmethylindoles and 3-(α-azolylbenzyl)indoles as potential aromatase inhibitors. 1-4-Fluorobenzyl-3-(1H-imidazol-1-ylmethyl)-1H-indole ML60, 1-ethyl-3-[(1H-imidazol-1-yl)(4-fluorophenyl)methyl]-1H-indole PI19 and 1-ethyl-3-[(1H-imidazol-1-yl)(4-fluorophenyl)methyl]-2-methyl-1H-indole CLE36 (see Figure 2 for structures) proved to be the most potent agents in inhibiting aromatization of androgens (androstenedione and, testosterone).

Figure 2 Structures and aromatase inhibitory activities of compounds ML60, PI19 and CLE36.

The present paper describes further structural optimizations in azolylmethyl-substituted indole series, namely the development of 1-substituted-3-(azolylmethyl)-1H-indoles and 1-substituted-3-(α-azolylbenzyl)-1H-indoles. Synthesis and structure-activity studies on the in vitro activity (inhibition of P450 arom) of compounds 11–18 and 30–37 are described in this paper. The most potent compounds were further evaluated for selectivity (inhibition of 17α-hydroxylase/C17,20-lyase).

Materials and methods

Chemistry

Instrumentation

Chemicals were purchased from Sigma-Aldrich. Compounds 3 and 20 were prepared according to reported methods [34,33]. Evaporations of final product solutions were done under vacuum with a rotatory evaporator. Column chromatography (CC) was carried out by using silica gel (silica gel 60, 63–200 mesh, E. Merck). Melting points were determined on a Tottoli-Büchi apparatus and are uncorrected. IR spectra (KBr or NaCl) were recorded as either neat (liquid or oil) or a KBr pellet (solids) on a Perkin-Elmer Paragon 1000 PC spectrophotometer. ¹H NMR spectra were recorded on a Bruker AC 250 (250 MHz) spectrometer, using DMSO d₆ as solvent. Chemical shifts are expressed in ppm (δ) downfield from internal TMS. Coupling constants J are expressed in Hz. Signals of protons exchangeable with D₂O are denoted by an asterisk (*). EI MS spectra were obtained on a Hewlett-Packard HP 5989A spectrometer (250°C, 70 eV).

Synthesis of N-substituted 1H-3-indolecarbaldehydes 3–6

A mixture of 1H-indole-3-carbaldehyde 2 (3.4 mmol) and K₂CO₃ (17 mmol) in dry acetone (15 mL) was stirred at room temperarure for 1 h. The corresponding benzyl chloride (4.1 mmol) was added and the reaction mixture was heated to reflux for 5 h. After filtration, the filtrate was evaporated. Water was added to the residue and the solution was extracted three times with CH₂Cl₂. The combined organic layers were dried (Na₂SO₄) and the solvent was evaporated under reduced pressure. The crude product was purified by trituration to give the target compound.

1-(2-Chlorobenzyl)-1H-3-indolecarbaldehyde (3)

This compound was prepared from precursor 2. Yield: 80%, brown crystals, mp 90–91°C (isopropyl ether). IR (KBr, cm^{− 1}) 1656 (CO); ¹H NMR (DMSO d₆) δ 5.68 (s, 2H, CH₂), 7.0 (dd, J = 7.5, 1.6 Hz, 1H, H-6′), 7.30–7.44 (m, 4H, H-5, H-6, H-4′, H-5′), 7.54–7.61 (m, 2H, H-7, H-3′), 8.16–8.21 (m, 1H, H-4), 8.41 (s, 1H, H-2), 9.99 (s, 1H, CHO).

1-(2-Fluorobenzyl)-1H-3-indolecarbaldehyde (4)

This compound was prepared from precursor 2. Yield: 88%, brown crystals, mp 95–97°C (diethyl ether). IR (KBr, cm^{− 1}) 1665 (CO); ¹H NMR (DMSO d₆) δ 5.65 (s, 2H, CH₂), 7.63–7.66 (s, 1H, H-7), 7.17–7.46 (m, 6H, H-5, H-6, H-3′, H-4′, H-5′, H-6′), 8.15-8.18 (m, 1H, H-4), 8.44 (s, 1H, H-2), 9.99 (s, 1H, CHO).

1-(3-Fluorobenzyl)-1H-3-indolecarbaldehyde (5)

This compound was prepared from precursor 2. Yield: 87%, brown solid, mp 105–107°C (isopropyl ether). IR (KBr, cm^{− 1}) 1664 (CO); ¹H NMR (DMSO d₆) δ 5.61 (s, 2H, CH₂), 7.14–7.35 (m, 5H, H-5, H-6, H-2′, H-4′, H-6′), 7.42 (ddd, J = 7.9, 7.9, 6.1 Hz, 1H, H-5′), 7.62–7.66 (m, 1H, H-7), 8.14–8.18 (m, 1H, H-4), 8.53 (s, 1H, H-2), 9.99 (s, 1H, CHO).

1-(4-Cyanobenzyl)-1H-3-indolecarbaldehyde (6)

This compound was prepared from precursor 2. Yield: 85%, brown solid, mp 153–155°C (isopropyl ether). IR (KBr, cm^{− 1}) 2230 (CN), 1658 (CO); ¹H NMR (DMSO d₆) δ 5.71 (s, 2H, CH₂), 7.26–7.34 (m, 2H, H-5, H-6), 7.47 (d, J = 8.2 Hz, 2H, H-2′, H-6′), 7.56–7.60 (m, 1H, H-7), 7.86 (d, J = 8.2 Hz, 2H, H-3′, H-5′), 8.15–8.18 (m, 1H, H-4), 8.54 (s, 1H, H-2), 9.99 (s, 1H, CHO).

Synthesis of 3-aroylindoles 21–23

To a magnetically stirred suspension of AlCl₃ (10 mmol) in CH₂Cl₂ (20 mL) at 25°C was added 4-fluorobenzoyl chloride (10 mmol) and the mixture was stirred at the same temperature for 1 h. A solution of 5-bromo-1H-indole 19 or 5-bromo-1-ethyl-1H-indole 20 (10 mmol) in CH₂Cl₂ (10 mL) was added dropwise to the mixture at 25°C. After stirring for 24 h at 25°C, the reaction mixture was poured into ethyl acetate and ice water. The organic layer was separated, washed with water, and dried over Na₂SO₄. After evaporation of solvent, the crystalline residue was recrystallized in the appropriate solvent.

5-Bromo-3-(4-fluorobenzoyl)-1H-indole (21)

This compound was prepared from precursor 19. Yield: 45%, fine white crystals, mp 250°C (ethyl acetate, decomposition). IR (KBr, cm^{− 1}) 3157 (NH), 1596 (CO); ¹H NMR (DMSO d₆) δ 7.40 (dd, J = 8.9, 8.9 Hz, 2H, H-3″, H-5″), 7.44 (dd, J = 1.7, 8.6 Hz, 1H, H-6), 7.54 (d, J = 8.6 Hz, 1H, H-7), 7.92 (dd, J = 5.7, 8.7 Hz, 2H, H-2″, H-6″), 8.09 (d, J = 3.1 Hz, 1H, H-2), 8.43 (d, J = 1.7 Hz, 1H, H-4), 12.35 (s*, 1H, NH).

5-Bromo-1-ethyl-3-(4-fluorobenzoyl)-1H-indole (22)

This compound was prepared from precursor 20. Yield: 80%, brown crystals, mp 155–157°C (diisopropyl ether/MeOH). IR (KBr, cm^{− 1}) 1616 (CO); ¹H NMR (DMSO d₆) δ 1.42 (t, J = 7.2 Hz, 3H, CH₃), 4.36 (q, J = 7.2 Hz, 2H, CH₂), 7.42 (dd, J = 8.8, 8.8 Hz, 2H, H-3″, H-5″), 7.50 (dd, J = 2.0, 8.7 Hz, 1H, H-6), 7.69 (d, J = 8.7 Hz, 1H, H-7), 7.93 (dd, J = 5.6, 8.8 Hz, 2H, H-2″, H-6″), 8.19 (s, 1H, H-2), 8.45 (d, J = 2.0 Hz, 1H, H-4).

5-Bromo-3-(2,4-dichlorobenzoyl)-1-ethyl-1H-indole (23)

This compound was prepared from precursor 20. Yield: 91%, pink solid, mp 147–149°C (diisopropyl ether/MeOH). IR (KBr, cm^{− 1}) 1630 (CO). ¹H NMR (DMSO d₆) δ 1.57 (t, J = 7.2 Hz, 3H, CH₃), 4.48 (q, J = 7.2 Hz, 2H, CH₂), 7.60 (dd, J = 1.8, 8.9 Hz, 1H, H-6), 7.62 (dd, J = 1.8, 8.2 Hz, 1H, H-5″), 7.68 (d, J = 8.2 Hz, 1H, H-6″), 7.72 (d, J = 8.9 Hz, 1H, H-7), 7.74 (d, J = 1.8 Hz, 1H, H-3″), 7.96 (s, 1H, H-2), 8.64 (d, J = 1.8 Hz, 1H, H-4).

Synthesis of N-substituted 3-aroylindoles 24 and 25

A mixture of 5-bromo-3-(4-fluorobenzoyl)-1H-indole 21 (2.50 mmol) and Cs₂CO₃ (5 mmol) in acetonitrile (15 mL) was heated to reflux for 2 h. 2-Chlorobenzyl chloride or 4-fluorobenzyl chloride (2.80 mmol) was added and the reaction mixture was heated to reflux for 1 h. Then, it was filtered and the filtrate was evaporated. To the residue was added water and the solution was extracted three times with CH₂Cl₂. The combined organic layers were dried (Na₂SO₄) and the solvent was evaporated under reduced pressure. The crude product was triturated from isopropyl ether to give 24 or 25.

5-Bromo-1-(2-chlorobenzyl)-3-(4-fluorobenzoyl)-1H-indole (24)

This compound was prepared from precursor 21. Yield: 98%, brown solid (isopropyl ether/CH₂Cl₂), mp 150–151°C. IR (KBr, cm^{− 1}) 1632 (CO); ¹H NMR (DMSO d₆) δ 5.70 (s, 2H, CH₂), 6.83 (d, J = 7.3 Hz, 1H, H-6′), 7.25–7.38 (m, 2H, H-4′, H-5′), 7.37–7.52 (m, 4H, H-6, H-7, H-3″, H-5″), 7.56 (d, J = 7.6 Hz, 1H, H-3′), 7.94 (dd, J = 5.8 Hz, 8.4 Hz, 2H, H-2″, H-6″), 8.33 (s, 1H, H-2), 8.48 (s, 1H, H-4).

5-Bromo-3-(4-fluorobenzoyl)-1-(4-fluorobenzyl)-1H-indole (25)

This compound was prepared from precursor 21. Yield: 93%, white solid, mp 150–152°C (isopropyl ether). IR (KBr, cm^{− 1}) 1629 (CO). ¹H NMR (DMSO d₆) δ 5.58 (s, 2H, CH₂), 7.19 (dd, J = 8.8, 8.8 Hz, 2H, H-3′, H-5′), 7.38–7.43 (m, 2H, H-2′, H-6′), 7.43 (dd, J = 8.8, 8.8 Hz, 2H, H-3″, H-5″), 7.45 (dd, J = 1.9, 8.8 Hz, 1H, H-6), 7.59 (d, J = 8.8 Hz, 1H, H-7), 7.95 (dd, J = 5.6, 8.8 Hz, 2H, H-2″, H-6″), 8.42 (s, 1H, H-2), 8.45 (d, J = 1.9 Hz, 1H, H-4).

Synthesis of carbinol derivatives 7–10 and 26–29

A solution of sodium borohydride (12.6 mmol) in methanol (10 mL) was added dropwise to a solution of the appropriate indole-3-carbaldehyde 3–6 or ketone derivative 22–25 (4.2 mmol) in methanol (10 mL). The reaction mixture was stirred at room temperature for 1 h. Water (30 mL) was added and the solution was extracted three times with diethyl ether. The combined organic layers were dried (Na₂SO₄) and the solvent was carefully evaporated leaving the desired crude product.

1-(2-Chlorobenzyl)-3-hydroxymethyl-1H-indole (7)

This compound was prepared from precursor 3. Yield: 82%, white solid, mp 85–87°C (isopropyl ether). IR (KBr, cm^{− 1}) 3380 (OH); ¹H NMR (DMSO d₆) δ 4.70 (d, J = 5.4 Hz, 2H, CH₂), 4.89 (t*, J = 5.4 Hz, 1H, OH), 5.51 (s, 1H, CH₂), 6.76 (dd, J = 7.5, 1.6 Hz, 1H, H-6′), 7.05–7.18 (m, 2H, H-5, H-6), 7.25 (ddd, J = 7.5, 7.5, 1.3 Hz, 1H, H-5′), 7.31–7.41 (m, 3H, H-2, H-7, H-4′), 7.55 (dd, J = 7.8, 1.3 Hz, 1H, H-3′), 7.68 (m, 1H, H-4).

1-(2-Fluorobenzyl)-3-hydroxymethyl-1H-indole (8)

This compound was prepared from precursor 4. Yield: 88%, yellow oil. IR (NaCl, cm^{− 1}) 3390 (OH). ¹H NMR (DMSO d₆) δ 4.70 (d, J = 5.4 Hz, 2H, CH₂), 4.91 (t*, J = 5.4 Hz, 1H, OH), 5.47 (s, 2H, CH₂), 7.05–7.38 (m, 6H, H-5, H-6, H-3′, H-4′, H-5′, H-6′), 7.39 (s, 1H, H-2), 7.46–7.52 (m, 1H, H-7), 7.66–7.69 (m, 1H, H-4).

1-(3-Fluorobenzyl)-3-hydroxymethyl-1H-indole (9)

This compound was prepared from precursor 5. Yield: 66%, white solid, mp 63–65°C (isopropyl and diethyl ethers, cyclohexane). IR (KBr, cm^{− 1}) 3388 (OH); ¹H NMR (DMSO d₆) δ 4.68 (d, J = 5.4 Hz, 2H, CH₂), 4.86 (t*, J = 5.4 Hz, 1H, OH), 5.43 (s, 2H, CH₂), 7.03–7.17 (m, 5H, H-5, H-6, H-2′, H-4′, H-6′), 7.38 (ddd, J = 8.0, 8.0, 6.1 Hz, 1H, H-5′), 7.45 (s, 1H, H-2), 7.45–7.48 (m, 1H, H-7), 7.64–7.67 (m, 1H, H-4).

1-(4-Cyanobenzyl)-3-hydroxymethyl-1H-indole (10)

This compound was prepared from precursor 6. Yield: 93%, white solid, mp 60–62°C (isopropyl ether). IR (KBr, cm^{− 1}) 3560 (OH), 2220 (CN); ¹H NMR (DMSO d₆) δ 4.68 (d, J = 5.4 Hz, 2H, CH₂), 4.88 (t*, J = 5.4 Hz, 1H, OH), 5.53 (s, 2H, CH₂), 7.03–7.16 (m, 2H, H-5, H-6), 7.35 (d, J = 8.2 Hz, 2H, H-2′, H-6′), 7.42 (d, J = 7.9 Hz, 1H, H-7), 7.45 (s, 1H, H-2), 7.66 (d, J = 7.4 Hz, 1H, H-4), 7.81 (d, J = 8.2 Hz, 2H, H-3′, H-5′).

5-Bromo-1-ethyl-3-[(4-fluorophenyl)(hydroxy)methyl]-1H-indole (26)

This compound was prepared from precursor 22. Yield: 88%, white solid, mp 162–164°C (isopropyl ether). IR (KBr, cm^{− 1}) 3164 (OH). ¹H NMR (DMSO d₆) δ 1.34 (t, J = 7.2 Hz, 3H, CH₃), 4.18 (q, J = 7.2 Hz, 2H, CH₂), 5.80 (d*, J = 4.5 Hz, 1H, OH), 5.97 (d, J = 4.5 Hz, 1H, CH), 7.18 (dd, J = 8.8, 8.8 Hz, 2H, H-3″, H-5″), 7.24 (dd, J = 1.9, 8.6 Hz, 1H, H-6), 7.25 (s, 1H, H-2), 7.45 (d, J = 8.6 Hz, 1H, H-7), 7.50 (dd, J = 5.7, 8.8 Hz, 2H, H-2″, H-6″), 7.66 (d, J = 1.9 Hz, 1H, H-4).

5-Bromo-3-[(2,4-dichlorophenyl)(hydroxy)methyl]-1-ethyl-1H-indole (27)

This compound was prepared from precursor 23. Yield: 78%, white solid, mp 120–122°C (isopropyl ether). IR (KBr, cm^{− 1}) 3232 (OH). ¹H NMR (DMSO d₆) δ 0.85 (t, J = 7.0 Hz, 3H, CH₃), 4.16 (q, J = 7.0 Hz, 2H, CH₂), 6.01 (d*, J = 4.9 Hz, 1H, OH), 6.20 (d, J = 4.9 Hz, 1H, CH), 7.06 (s, 1H, H-2), 7.28 (dd, J = 1.9, 8.7 Hz, 1H, H-6), 7.47 (d, J = 8.7 Hz, 1H, H-7), 7.55 (dd, J = 8.3 Hz, 1H, H-5″), 7.60 (d, J = 2.1 Hz, 1H, H-3″), 7.78 (d, J = 1.9 Hz, 1H, H-4), 7.84 (d, J = 8.3 Hz, 1H, H-6″).

5-Bromo-1-(2-chlorobenzyl)-3-[(4-fluorophenyl)(hydroxy)methyl]-1H-indole (28)

This compound was prepared from precursor 24. Yield: 93%, brown solid, mp 101–103°C (isopropyl ether). IR (KBr, cm^{− 1}) 3274 (OH); ¹H NMR (DMSO d₆) δ 5.51 (s, 2H, CH₂), 5.85 (d*, J = 4.5 Hz, 1H, OH), 5.99 (d, J = 4.5 Hz, 1H, CH), 6.75 (d, J = 7.3 Hz, 1H, H-6′), 7.15-7.55 (m, 8H, H-2, H-6, H-7, H-3′, H-4′, H-5′, H-3″, H-5″), 7.50 (dd, J = 5.9, 7.2 Hz, 2H, H-2″, H-6″), 7.67 (d, J = 1.7 Hz, 1H, H-4).

5-Bromo-1-(4-fluorobenzyl)-3-[(4-fluorophenyl)(hydroxy)methyl]-1H-indole (29)

This compound was prepared from precursor 25. Yield: 95%, white solid, mp 125–127°C (cyclohexane). IR (KBr, cm^{− 1}) 3114 (OH); ¹H NMR (DMSO d₆) δ 5.40 (s, 2H, CH₂), 5.85 (d*, J = 4.5 Hz, 1H, OH), 5.99 (d, J = 4.5 Hz, 1H, CH), 7.14–7.24 (m, 5H, H-6, H-3′, H-5′, H-3″, H-5″), 7.28 (dd, J = 5.9 Hz, 8.6 Hz, 2H, H-2′, H-6′), 7.38 (s, 1H, H-2), 7.45 (d, J = 8.8 Hz, 1H, H-7), 7.51 (dd, J = 5.8, 8.5 Hz, 2H, H-2″, H-6″), 7.67 (d, J = 1.7 Hz, 1H, H-4).

Synthesis of imidazole derivatives 11–14, 30, 31, 34 and 35

A solution of the appropriate carbinol derivative 7–10 or 26–29 (3.70 mmol) and CDI (3.70 mmol) in dry THF (20 mL) was stirred at room temperature for 3 h. The reaction mixture was partitioned between H₂O and diethyl ether and extracted three times with diethyl ether. The combined organic layers were dried (Na₂SO₄) and concentrated. The residue was purified by CC (CH₂Cl₂:EtOH = 19:1).

1-(2-Chlorobenzyl)-3-(1H-imidazol-1-ylmethyl)-1H-indole (11)

This compound was prepared from precursor 7. Yield: 42%, white solid, mp 45–47°C (isopropyl ether). ¹H NMR (DMSO d₆) δ 5.38 (s, 2H, CH₂), 5.54 (s, 2H, CH₂), 6.80 (dd, J = 7.5, 1.8 Hz, 1H, H-6′), 6.88 (s, 1H, H_imide), 7.06–7.20 (m, 2H, H-5, H-6), 7.20 (s, 1H, H_imide), 7.28 (m, 1H, H-5′), 7.35 (m, 1H, H-4′), 7.43 (d, J = 8.0 Hz, 1H, H-7), 7.55 (dd, J = 7.8, 1.3 Hz, 1H, H-3′), 7.59 (s, 1H, H-2), 7.62 (d, J = 7.7 Hz, 1H, H-4), 7.80 (s, 1H, H_imide).

1-(2-Fluorobenzyl)-3-(1H-imidazol-1-ylmethyl)-1H-indole (12)

This compound was prepared from precursor 8. Yield: 42%, white solid, mp 65–68°C (isopropyl ether). ¹H NMR (DMSO d₆) δ 5.38 (s, 2H, CH₂), 5.51 (s, 2H, CH₂), 6.91 (s, 1H, H_imide), 7.06–7.41 (m, 6H, H-5, H-6, H-3′, H-4′, H-5′, H-6′), 7.21 (s, 1H, H_imide), 7.53 (d, J = 8.1 Hz, 1H, H-7), 7.61 (s, 1H, H-2), 7.63 (d, J = 7.7 Hz, 1H, H-4), 7.82 (s, 1H, H_imide).

1-(3-Fluorobenzyl)-3-(1H-imidazol-1-ylmethyl)-1H-indole (13)

This compound was prepared from precursor 9. Yield: 92%, white solid, mp 62–65°C (cyclohexane). ¹H NMR (DMSO d₆) δ 5.37 (s, 2H, CH₂), 5.47 (s, 2H, CH₂), 6.88 (s, 1H, H_imide), 7.04–7.16 (m, 5H, H-5, H-6, H-2′, H-4′, H-6′), 7.20 (s, 1H, H_imide), 7.34–7.43 (m, 1H, H-5′), 7.49 (d, J = 8.2 Hz, 1H, H-7), 7.60 (d, J = 7.7 Hz, 1H, H-4), 7.66 (s, 1H, H-2), 7.81 (s, 1H, H_imide); IR (KBr, cm^{− 1}) 1086.

1-(4-Cyanobenzyl)-3-(1H-imidazol-1-ylmethyl)-1H-indole (14)

This compound was prepared from precursor 10. Yield: 60%, white solid, mp 102–104°C (isopropyl ether). IR (KBr, cm^{− 1}) 2229 (CN); ¹H NMR (DMSO d₆) δ 5.38 (s, 2H, CH₂), 5.57 (s, 2H, CH₂), 6.89 (s, 1H, H_imide), 7.04–7.16 (m, 2H, H-5, H-6), 7.21 (s, 1H, H_imide), 7.36 (d, J = 8.2 Hz, 2H, H-2′, H-6′), 7.45 (d, J = 8.1 Hz, 1H, H-7), 7.60 (d, J = 7.6 Hz, 1H, H-4), 7.65 (s, 1H, H-2), 7.81 (s, 1H, H_imide), 7.83 (d, J = 8.2 Hz, 2H, H-3′, H-5′).

5-Bromo-1-ethyl-3-[(4-fluorophenyl)(1H-imidazol-1-yl)methyl]-1H-indole (30)

This compound was prepared from precursor 26. Yield: 71%, white solid, mp 143–145°C (isopropyl ether). ¹H NMR (DMSO d₆) δ 1.33 (t, J = 7.2 Hz, 3H, CH₃), 4.22 (q, J = 7.2 Hz, 2H, CH₂), 6.97 (s, 1H, H_imide), 7.12 (s, 1H, CH), 7.14 (s, 1H, H-2), 7.20 (s, 1H, H_imide), 7.24–7.30 (m, 2H, H-3″, H-5″), 7.30–7.34 (m, 4H, H-4, H-6, H-2″, H-6″), 7.54 (d, J = 8.6 Hz, 1H, H-7), 7.75 (s, 1H, H_imide).

5-Bromo-3-[(2,4-dichlorophenyl)(1H-imidazol-1-yl)methyl]-1-ethyl-1H-indole (31)

This compound was prepared from precursor 27. Yield: 66%, white solid, mp 138–140°C (isopropyl ether/EtOH). ¹H NMR (DMSO d₆) δ 1.32 (t, J = 7.1 Hz, 3H, CH₃), 4.21 (q, J = 7.1 Hz, 2H, CH₂), 6.91 (d, J = 8.4 Hz, 1H, H-6″), 6.99 (s, 1H, H_imide), 7.07 (s, 1H, H-2), 7.15 (s, 1H, H_imide), 7.27 (s, 1H, CH), 7.34 (dd, J = 8.8, 8.8 Hz, 1H, H-6), 7.49 (d, J = 1.7 Hz, 1H, H-4), 7.52 (dd, J = 2.1, 8.4 Hz, 1H, H-5″), 7.56 (d, J = 8.8 Hz, 1H, H-7), 7.71 (s, 1H, H_imide), 7.75 (d, J = 2.1 Hz, 1H, H-3″).

5-Bromo-1-(2-chlorobenzyl)-3-[(4-fluorophenyl)(1H-imidazol-1-yl)methyl]-1H-indole (34)

This compound was prepared from precursor 28. Yield: 71%, white crystals, mp 145°C (isopropyl ether/MeOH, decom position). ¹H NMR (base) (DMSO d₆) δ 5.55 (s, 2H, CH₂), 6.70 (d, J = 7.1 Hz, 1H, H6′), 6.99 (s, 1H, H_imide), 7.21 (s, 1H, H_imide), 7.17–7.42 (m, 11H, H-2, H-4, H-6, H-7, H-4′, H-5′, H-2″, H-3″, H-5″, H-6″, CH), 7.53 (d, J = 7.6 Hz, 1H, H-3′), 7.77 (s, 1H, H_imide).

5-Bromo-1-(4-fluorobenzyl)-3-[(4-fluorophenyl)(1H-imidazol-1-yl)methyl]-1H-indole (35)

This compound was prepared from precursor 29. Yield: 80%, white solid, mp 140–142°C (cyclohexane/diethyl ether). ¹H NMR (DMSO d₆) δ 5.50 (s, 2H, CH₂), 6.99 (s, 1H, H_imide), 7.15–7.33 (m, 12H, H-2, H-4, H-6, H-2′, H-3′, H-5′, H-6′, H-2″ H-3″ H-5″ H-6″, CH), 7.21 (s, 1H, H_imide), 7.47 (d, J = 8.7 Hz, 1H, H-7), 7.77 (s, 1H, H_imide).

Synthesis of triazole derivatives 15–18, 32, 33, 36 and 37

Thionyl chloride (11.8 mmol) was dropped into an ice-cooled solution of 1H-1,2,4-triazole (47.2 mmol) in dry acetonitrile (30 mL). The mixture was stirred at room temperature for 1 h, then filtered. This solution was added dropwise to a solution of the appropriate carbinol derivative 7–10 or 26–29 (3.0 mmol) in dry acetonitrile (10 mL). After addition, the mixture was stirred at room temperature for 2 h, then filtered and concentrated. The residue was dissolved in CH₂Cl₂. The organic solution was washed with brine, dried over Na₂SO₄, filtered, and evaporated to provide the crude product. This mixture was purified by CC (CH₂Cl₂:EtOH = 19:1). In the case of compounds 15 and 16, appropriate eluates were collected and evaporated to give the corresponding regioisomers.

1-(2-Chlorobenzyl)-3-(1H-1,2,4-triazol-1-ylmethyl)-1H-indole (15a)

This compound was prepared from precursor 7. Yield: 74%, pale yellow crystals, mp 65–66°C (cyclohexane/CH₂Cl₂). ¹H NMR (DMSO d₆) δ 5.54 (s, 2H, CH₂), 5.60 (s, 2H, CH₂), 6.81 (d, J = 7.5 Hz, 1H, H-6′), 7.07–7.12 (m, 1H, H-5), 7.15–7.20 (m, 1H, H-6), 7.24–7.30 (m, 1H, H-5′), 7.33–7.38 (m, 1H, H-4′), 7.43 (d, J = 8.1 Hz, 1H, H-7), 7.55 (d, J = 7.8 Hz, 1H, H-3′), 7.60 (s, 1H, H-2), 7.67 (d, J = 7.7 Hz, 1H, H-4), 7.97 (s, 1H, H_triazole), 8.64 (s, 1H, H_triazole).

1-(2-Chlorobenzyl)-3-(4H-1,2,4-triazol-4-ylmethyl)-1H-indole (15b)

This compound was prepared from precursor 7. Yield: 6%, white solid, mp 130°C (diisopropyl ether). ¹H NMR (DMSO d₆) δ 5.47 (s, 1H, CH₂), 5.55 (s, 1H, CH₂), 6.83 (d, J = 7.1 Hz, 1H, H-6′), 7.08–7.22 (m, 2H, H-5, H-6), 7.25–7.31 (m, 1H, H-5′), 7.33–7.39 (m, 1H, H-4′), 7.44 (d, J = 8.1 Hz, 1H, H-7), 7.55 (d, J = 7.8 Hz, 1H, H-3′), 7.62 (s, 1H, H-2), 7.66 (d, J = 7.6 Hz, 1H, H-4), 8.62 (s, 2H, H_triazole).

1-(2-Fluorobenzyl)-3-(1H-1,2,4-triazol-1-ylmethyl)-1H-indole (16a)

This compound was prepared from precursor 8. Yield: 29%, brown solid, mp 121–123°C (diisopropyl ether/CH₂Cl₂). ¹H NMR (DMSO d₆) δ 5.50 (s, 2H, CH₂), 5.58 (s, 2H, CH₂), 7.05–7.42 (m, 6H, H-5, H-6, H-3′, H-4′, H-5′, H-6′), 7.52 (d, J = 8.2 Hz, 1H, H-7), 7.61 (s, 1H, H-2), 7.65 (d, J = 7.7 Hz, 1H, H-4), 7.96 (s, 1H, H_triazole), 8.64 (s, 1H, H_triazole).

1-(2-Fluorobenzyl)-3-(4H-1,2,4-triazol-4-ylmethyl)-1H-indole (16b)

This compound was prepared from precursor 8. Yield: 24%, yellow crystals, mp 118–120°C (diisopropyl ether/CH₂Cl₂). ¹H NMR (DMSO d₆) δ 5.46 (s, 2H, CH₂), 5.51 (s, 2H, CH₂), 7.07–7.39 (m, 6H, H-5, H-6, H-3′, H-4′, H-5′, H-6′), 7.53 (d, J = 8.0 Hz, 1H, H-7), 7.64 (s, 1H, H-2), 7.65 (d, J = 7.8 Hz, 1H, H-4), 8.62 (s, 2H, H_triazole).

1-(3-Fluorobenzyl)-3-(1H-1,2,4-triazol-1-ylmethyl)-1H-indole (17)

This compound was prepared from precursor 9. Yield: 65%, white solid, mp 135–137°C (cyclohexane). ¹H NMR (DMSO d₆) δ 5.48 (s, 2H, CH₂), 5.59 (s, 2H, CH₂), 7.05–7.20 (m, 5H, H-5, H-6, H-2′, H-4′, H-6′), 7.35–7.43 (m, 1H, H-5′), 7.50 (d, 1H, H-7, J = 8.0 Hz), 7.65 (d, 1H, H-4, J = 7.60 Hz), 7.68 (s, 1H, H-2), 7.96 (s, 1H, H_triazole), 8.65 (s, 1H, H_triazole).

1-(4-Cyanobenzyl)-3-(1H-1,2,4-triazol-1-ylmethyl)-1H-indole (18)

This compound was prepared from precursor 10. Yield: 63%, white solid, mp 104–106°C (isopropyl ether). IR (KBr, cm^{− 1}) 2226; ¹H NMR (DMSO d₆) δ 5.58 (s, 2H, CH₂), 5.60 (s, 2H, CH₂), 7.08–7.17 (m, 2H, H-5, H-6), 7.37 (d, J = 8.1 Hz, 2H, H-2′, H-6′), 7.45 (d, J = 7.9 Hz, 1H, H-7), 7.64–7.68 (m, 2H, H-2, H-4), 7.83 (d, J = 8.1 Hz, 2H, H3′, H-5′), 7.97 (s, 1H, H_triazole), 8.65 (s, 1H, H_triazole).

5-Bromo-1-ethyl-3-[(4-fluorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (32)

This compound was prepared from precursor 26. Yield: 67%, white solid, mp 126–128°C (isopropyl ether/EtOH). ¹H NMR (DMSO d₆) δ 1.33 (t, J = 7.20 Hz, 3H, CH₃), 4.22 (q, J = 7.20 Hz, 2H, CH₂), 7.22–7.27 (m, 3H, H-2, H-3″, H-5″), 7.28–7.35 (m, 2H, H-6, CH), 7.37–7.42 (m, 2H, H-2″, H-6″), 7.43 (d, J = 1.50 Hz, 1H, H-4), 7.54 (d, J = 8.70 Hz, 1H, H-7), 8.08 (s, 1H, H_triazole), 8.67 (s, 1H, H_triazole).

5-Bromo-3-[(2,4-dichlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-ethyl-1H-indole (33)

This compound was prepared from precursor 27. Yield: 30%, white solid, mp 124–126°C (acetonitrile). ¹H NMR (DMSO d₆) δ 1.33 (t, J = 7.2 Hz, 3H, CH₃), 4.22 (q, J = 7.2 Hz, 2H, CH₂), 7.18 (s, 1H, H-2), 7.22 (d, J = 8.5 Hz, 1H, H-6″), 7.33 (dd, J = 1.8, 8.7 Hz, 1H, H-6), 7.48 (s, 1H, CH), 7.49–7.57 (m, 3H, H-4, H-7, H-5″), 7.76 (d, J = 2.0 Hz, 1H, H-3″), 8.11 (s, 1H, H_triazole), 8.69 (s, 1H, H_triazole).

5-Bromo-1-(2-chlorobenzyl)-3-[(4-fluorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (36)

This compound was prepared from precursor 28. Yield: 70%, pale yellow crystals, mp 145–147°C (MeOH/CH₂Cl₂). ¹H NMR (DMSO d₆) δ 5.56 (s, 2H, CH₂), 6.73 (d, J = 6.5 Hz, 1H, H-6′), 7.23-7.47 (m, 11H, H-2, H-4, H-6, H-7, H-4′, H-5′, H-2″, H-3″, H-5″, H-6″, CH), 7.53 (d, J = 7.6 Hz, 1H, H-3′), 8.10 (s, 1H, H_triazole), 8.71 (s, 1H, H_triazole).

5-Bromo-1-(4-fluorobenzyl)-3-[(4-fluorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (37)

This compound was prepared from precursor 29. Yield: 85%, white solid, mp 163–165°C (cyclohexane). ¹H NMR (DMSO d₆) δ 5.45 (s, 2H, CH₂), 7.17 (dd, J = 8.7, 8.7 Hz, 2H, H-3′, H-5′), 7.24–7.30 (m, 4H, H-2′, H-6′, H-3″, H-5″), 7.28 (dd, J = 1.8, 8.7 Hz, 1H, H-6), 7.38–7.45 (m, 5H, H-2, H-4, H-2″, H-6″, CH), 7.48 (d, J = 8.6 Hz, 1H, H-7), 8.09 (s, 1H, H_triazole), 8.69 (s, 1H, H_triazole).

Pharmacology

Inhibition of human placental aromatase (in Vitro)

The compounds were tested for aromatase inhibitory activity according to the procedure of Thompson and Siiteri [35]. The microsomal fraction of freshly delivered human term placenta provided the source of the aromatase enzyme and [1β,2β-³H]testosterone was used as substrate [36].

Inhibition of rat testicular P450 17α (in Vitro)

Rat testicular microsomes were used as source of the enzyme, and nonlabeled progesterone served as substrate. The separation of the steroids was accomplished using the procedure recently described by Sergejew and Hartmann [37].

Results and discussion

Chemistry

3-(Azolylmethyl)-1H-indoles incorporating differently substituted R groups were synthezised from indole-3-carbaldehyde 2 (see Scheme 1). N-Substitution of indole-3-carbaldehyde 2 with benzyl chlorides, using potassium carbonate in acetone, afforded the N-substituted indole derivatives 3–6. Reduction of the carbonyl group was carried out by using sodium borohydride [38] in methanol and provided 7–10. The synthesis of imidazolyl-substituted compounds 11–14 was achieved by the direct reaction of the carbinols 7–10 with 1,1′-carbonyldiimidazole (CDI) in THF [39], via decarboxylation of the carbamate intermediate. The triazolyl-substituted compounds 15–18 were prepared by the direct reaction of the corresponding carbinols with sulfinylditriazole (SDT) [40], which was readily generated from triazole and SOCl₂ in acetonitrile.

Scheme 1 Synthetic route to 3-azolylmethyl-1H-indoles 11–14. Reagents: (a) see reference [34]; (b) K₂CO₃, acetone, RCl; (c) NaBH₄, methanol; (d) CDI, THF; (e) SDT, CH₃CN.

3-(α-Azolylbenzyl)-1H-indoles were obtained by two synthetic routes as depicted in Scheme 2. A Friedel Crafts procedure [41,42], using AlCl₃ catalysis, was employed to give the N-ethyl-3-aroylindole derivatives 22 and 23. A direct acylation of 5-bromo-1H-indole 19 was attempted, with 4-fluorobenzoyl chloride; it gave 3-acylindole derivative 21, in moderate yield. N-Substitution of 21 was achieved with benzyl chlorides, using Cs₂CO₃ in acetonitrile, and gave 3-aroyl-1-benzyl-1H-indoles 24 and 25 in excellent yields. Then the corresponding alcohols 28 and 29 were obtained by using NaBH₄ in methanol. Introduction of the imidazole and triazole moieties was achieved with CDI and SDT, leading to compounds 30–37.

Scheme 2 Synthetic route to 3-(azolylbenzyl)-1H-indoles 30–37. Reagents: (a) see reference [33]; (b) AlCl₃, CH₂Cl₂, ArCOCl; (c) CsCO₃, CH₃CN, R₁Cl; (d) NaBH₄, methanol; (e) CDI, THF; (f) SDT, CH₃CN.

Biological results

The ability of compounds under investigation to inhibit aromatase was investigated. The IC₅₀ values and the potencies of the compounds, relative to AG, are given in Tables II and III. The reference standard used was aminoglutethimide, with an IC₅₀ value of 18.5 μM.

Table II.  Inhibition of P450 arom and P450 17α by 3-(azolylmethyl)-1H-indoles 11–18.

			P450 arom		P450 17α
compound	R	Het	IC50 (μM)^a	RP^b	% inhibition^c
11	2-chlorobenzyl	imidazol-1-yl	0.054	342.6	62.8
12	2-fluorobenzyl	imidazol-1-yl	0.124	149.2	55.5
13	3-fluorobenzyl	imidazol-1-yl	0.082	225.6	70.2
14	4-cyanobenzyl	imidazol-1-yl	0.050	370.0	15.4
15a	2-chlorobenzyl	triazol-1-yl	0.173	106.9	9.3
15b	2-chlorobenzyl	triazol-4-yl	3.500	5.3	0.0
16a	2-fluorobenzyl	triazol-1-yl	0.420	44.0	0.0
16b	2-fluorobenzyl	triazol-4-yl	7.220	2.6	0.5
17	3-fluorobenzyl	triazol-1-yl	0.307	60.3	14.4
18	4-cyanobenzyl	triazol-1-yl	0.141	131.2	1.8

^a IC₅₀ is the concentration of inhibitor required to give 50% inhibition. Concentration of testosterone: 2.5 μM. The given values are mean values of at least three experiments. The deviations were within ± 5%.

^b Relative potency, calculated from the IC₅₀ values and related to AG (IC₅₀ of AG: 18.5 μM).

^c Concentration of progesterone: 25 μM. Concentration of inhibitor: 2.5 μM. All values are the mean of at least 2 determinations.

Table III.  Inhibition of P450 arom and P450 17α by 3-(α-azolylbenzyl)-1H-indoles 30–37.

compound	R	X	Het	IC50 (μM)^a	RP^b	% inhibition^c
30	ethyl	4-F	imidazol-1-yl	0.052	357.1	3.7
31	ethyl	2,4-diCl	imidazol-1-yl	0.333	55.6	0.7
32^d	2-chlorobenzyl	4-F	imidazol-1-yl	0.964	19.2	1.6
33	4-fluorobenzyl	4-F	imidazol-1-yl	0.623	29.7	0.0
34	ethyl	4-F	triazol-1-yl	0.980	18.9	0.9
35	ethyl	2,4-diCl	triazol-1-yl	4.400	4.2	0.0
36	2-chlorobenzyl	4-F	triazol-1-yl	< 25	-	0.0
37	4-fluorobenzyl	4-F	triazol-1-yl	7.1	2.6	1.0

^a IC₅₀ is the concentration of inhibitor required to give 50% inhibition. Concentration of testosterone: 2.5 μM. The given values are mean values of at least three experiments. The deviations were within ± 5%.

^b Relative potency, calculated from the IC₅₀ values and related to AG (IC₅₀ of AG: 18.5 μM).

^c Concentration of progesterone: 25 μM. Concentration of inhibitor: 2.5 μM. All values are the mean of at least 2 determinations.

^d Tested as nitrate.

In the sub-series of 3-(azolylmethyl)-1H-indoles, all imidazole derivatives 11–14 were more active than their triazole analogues 15–18. Triazol-4-yl derivatives 15b and 16b showed the lowest inhibitory potencies (IC₅₀ > 3.5 μM). The presence of a chloro substituent in the ortho position of the N-benzyl moiety gave a positive result (compare compound 11 with respect to compound 12). The presence of a fluorine atom at position 3 of the N-benzyl substituent improved the antiaromatase activity: compound 13 is 1.5 times more active than parent compound 12. Imidazole derivative 14 in which the benzyl moiety was substituted by a 4-cyano group, was the most active, with an IC₅₀ value of 0.050 μM. All compounds of this sub-series did not exhibit any significant inhibitory activity on rat testicular P450 17α. The highest percentage inhibition value found was 70 with compound 13 at the concentration of 2.5 μM. IC₅₀ value was only calculated in the case of percentage inhibition value superior to 85%.

Table III shows the in vitro data of the antiaromatase activity of 3-(α-azolylbenzyl)-1H-indoles 30–37. The N-ethyl derivative 30 showed an IC₅₀ value of 0.052 μM and this imidazole derivative was the best inhibitor of this sub-series. Further structure-activity study regarding the presence of a 2,4-dichlorophenyl substituent did not improve activity (compound 30 compared with 31). The replacement of the N-ethyl chain of compound 30 by the N-halobenzyl moiety (2-chloro or 4-fluoro) gave negative results for compounds 32 and 33. The triazole derivative 34 was 19 times less active than its imidazole analogue 30. Other triazole analogues 35–37 were less active than parent molecules 31–33. As found with the first sub-series, all 3-(α-azolylbenzyl)-1H-indoles 30–37 were poor P450 17α inhibitors, with no significant percentage inhibition values ( < 3.7).

In order to evaluate the in vivo efficacy, the effect of select 3-(azolylmethyl)-1H-indoles and 3-(α-azolylbenzyl)-1H-indoles on the androgen-stimulated uterine growth will be determined. In juvenile female rats, androstedione treatment strongly stimulates uterine weight. This effect is due to ovarian aromatization of the androgen and can be dose-dependently antagonized by aromatase inhibitors such as compounds 14 and 30. Thus the antiuterotrophic activity could give further biological data to continue our research. Furthermore we are doing molecular modelling studies on compound 14 and 30 to better understand their interactions in the active site of P450 arom and then to be able to design new non steroidal aromatase inhibitors.