Efficient approach to novel 1α-triazolyl-5α-androstane derivatives as potent antiproliferative agents

Efficient approach to novel 1-triazolyl-5-androstane derivatives as potent antiproliferative agents Zalán Kádár,a Ádám Baji,a István Zupkó,b Tibor Bartók,c,d János Wölflinga and Éva Frank*,a 5

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Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X First published on the web Xth XXXXXXXXX 200X DOI: Stereoselective 1,4-Michael addition of azoimide to 17-acetoxy-5-adrost-1-en-3-one was carried out to furnish a 1-azido-3-ketone, which was reduced to give the 3- and 3-hydroxy epimers in a ratio of 5:2. The Cu(I)-catalyzed 1,3-dipolar cycloaddition of the major isomer to terminal alkynes afforded 1-triazolyl derivatives, which were deacetylated to the corresponding 3,17diols or oxidized to the analogous 3-ketones. However, the ability of the minor 1,3-azidoalcohol to undergo similar cyclization was found to be affected significantly by the steric bulk of the substituents on the alkyne reaction partner. All triazolyl compounds were tested in vitro on three malignant gynecological cell lines (HeLa, MCF7 and A2780).

1. Introduction OH

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The Huisgen 1,3-dipolar cycloaddition of organic azides to terminal alkynes has received considerable attention in recent years following the independent introduction of Cu(I) catalysis in 2002 by the research groups of Sharpless 1 and Meldal. 2 The presence of the catalyst dramatically improves both the rate and the regioselectivity of the reaction, leading exclusively to the 1,4-disubstituted 1,2,3-triazole, 3 and eliminating the need for elevated temperature and a prolonged reaction time. The catalytic version has further advantageous benefits, such as high yields of the desired products, tolerability to a variety of common parameters (functional groups, solvents, pH and temperature), the lack of sidereactions and insensitivity to the steric and electronic properties of the reactants. Consequently, such Cu(I)catalyzed azide-alkyne cycloaddition (CuAAC) commonly meets all the criteria for click chemistry 4 and has gained general application across a wide variety of disciplines, including preparative organic synthesis, 5 polymer and material science,6 dendrimer design, 7 chemical ligation 8 and combinatorial drug discovery. 9 The triazole formed during the reaction can mimic the atom placement and electronic properties of a peptide bond; however, it is essentially chemically inert against oxidation, reduction and hydrolytic conditions, and possesses a much stronger dipole moment than an amide bond. 10 Perhaps due in part to the structural mimicry, a number of diverse 1,2,3triazoles have been reported to exhibit varied biological activity, including anti-HIV, 11 antibacterial,12 antihistamine13 or cytostatic effects. 14 The main driving force toward the preparation of steroidal compounds at present is the development of novel derivatives with a biological target other than a hormone receptor and therefore the reduction or elimination of the undesirable hormonal activity. The synthetic tools for achievement of this goal are i) the synthesis of molecules lacking the functionalities necessary for effective binding to the hormone receptors; 15 ii) modification of the binding ability by chemical

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OH

H H O

H H

H O

H

H

H

methenolone

mesterolone

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OR N3

H H

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O

H

H

Fig. 1 1-Substituted steroids in the 5-androstane series

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transformation of the extant functional groups; 16 iii) steric hindrance of the substrate-receptor interaction by chemical substitution near the original groups; 17 iv) altering the primary stereostructure or the number of ring members; 18 and v) the design of heterocyclic derivatives that are not recognized by the receptor protein in consequence of their specific structure or the fact that their geometry differs from that of the natural hormones.19 The most frequent synthetic modifications are introduced at the positions adjacent to the existing C-3, C-17 or C-20 functional groups, where substitution is facilitated. Substitution at other positions of the sterane skeleton has proved to be more difficult, necessitating several reaction steps, and is therefore rarely applied. To the best of our knowledge, only a few 1-substituted derivatives have been synthetized to date, among them 1-methylated androstanes (methenolone and mesterolone) exerting anabolic rather than androgenic activity20 (Figure 1). The foregoing results led us to set out to introduce an azido group at the unconventional position 1 of the sterane skeleton and thereby to synthetize a mesterolone analog (Figure 1). The intermolecular ring closure of the steroidal azide with different terminal alkynes via CuAAC was also planned with a view to preparing novel 1-exo-triazolyl derivatives. Since some steroid triazoles are known to exert antiproliferative

OAc

OAc

H O

Table 1 CuAAC of steroidal trans azidoalcohol 4 with terminal alkynes

10

H O

H 1

H

H

N3

H

10

HO

O

H H 3

OAc N3

H H

HO

H

25

N N N

CuI (0.1 eq.) Ph3P (0.2 eq.) DIPEA (3 eq.) HO toluene 111 °C, 3 h

R

OAc H H

H

H 7ag

Triazole

Yielda (%)

6a

7a

93

2

6b

7b

93

3

6c

7c

92

4

6d

7d

93

5

6e

7e

96

6

6f

7f

93

7

6g

7g

97

Alkyne

1

R

H

KBH4 MeOH pH=68 20

H

Entry

H

1

H

4

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OAc N3

H H

2

NaN3 AcOH/THF

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OAc R C CH 6ag

H

H 5

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OAc N3

+

H

H

H HO

4

H

H

5

Scheme 1 Synthesis of 1-azides in the 5-androstane series 21

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activity, it was decided to screen all these compounds in vitro for their activities against a panel of three human cancer cell lines (HeLa, MCF7 and A2780). Although determination of their affinities for the androgen receptor did not fall within the scope of the present work, the steric bulk of the heterocyclic moiety at position 1 may interfere with the receptor binding.

2. Results and discussion 35

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2.1. Synthetic studies For the introduction of an azido group onto position 1 of the sterane skeleton, the starting material applied was 17acetoxy-5-adrost-1-en-3-one (2), which is readily available from stanolone acetate (1) in a two step-pathway, by bromination at C-2 and subsequent dehydrohalogenation 22 (Scheme 1). The 1,4-Michael addition of azoimide, 23 generated in situ from sodium azide and acetic acid, afforded 17-acetoxy-1-azido-5-androstan-3-one (3) in a yield of 67% after purification by flash chromatography. The stereoselective formation of the 1-azido derivative (3) is not surprising considering the steric bulk of the adjacent angular -methyl group on C-10. Since -substituted ketones such as 3 are often susceptible to elimination and undergo facile transformation to the corresponding enone, 24 azidoketone 3 was reduced with KBH 4 under pH-controlled conditions so as to avoid this adverse side-reaction. The 1H NMR spectrum of the reaction mixture indicated that the epimeric diols 4 and 5 were formed in a ratio of 5:2. After separation by column chromatography, the isomeric azidoalcohols were subjected to CuAAC with different terminal alkynes. Although CuAAC is generally not affected by the steric features of the alkyne and azide components, the trans and cis azidoalcohols 4 and 5 displayed considerably different behavior under similar reaction conditions. Accordingly, the opposite or same spatial

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Yields of purified isolated products.

orientation of the hydroxy group on C-3 with that of the azido function on C-1 was expected to have an influence on the intermolecular ring closures. During optimization of the reaction conditions for the CuAAC of 4 with phenylacetylene (6a), the best conversion was found to occur on the use of a catalytic amount of CuI as Cu(I) source, triphenylphosphane15 as stabilizing ligand to protect the Cu(I) from oxidation, and excess N,N-diisopropyl ethylamine as base to facilitate formation of the active copper acetylide complex, and to minimize side-reactions.25 Ring closure in refluxing toluene for 3 h furnished the corresponding phenyltriazolyl derivative 7a in excellent yield after purification (Table 1, entry 1). After determination of the optimal conditions, similar cycloadditions of 4 with different aryl- and cycloalkyl-substituted acetylenes (6bg) were performed, which resulted in steroidal 1-exo-triazolyl derivatives (7bg) in yields exceeding 90%, independently of the substituents on the alkyne dipolarophile (Table 1, entries 210). However, the reaction of the cis-azidoalcohol 5 with phenylacetylene 6a was not complete even within 5 h, and the purified product 8a was obtained in a yield of only 61% (Table 2, entry 1). Treatment of substrate 5 with benzoic acid propargyl ester 6h, containing the aromatic ring farther from the reaction center than in phenylacetylene 6a, resulted in the triazolyl derivative 8h in a higher isolated yield (83%) (Table 2, entry 2). The reaction of 6h with the acetylated azidoalcohol 9 proceeded similarly as for 8h, but an even lower conversion was observed on the use of phenylacetylene 6a (Table 2, entries 3 and 4). These results suggest that the

Table 2 CuAAC of steroidal cis azidoalcohols 5 and 9 with terminal alkynes R2 OAc R2 C CH 6a, 6h

N3

H H

R1O Ac2O py

Entry

H

H 5 R1= H 9 R1= Ac

CuI (0.1 eq.) Ph3P (0.2 eq.) DIPEA (3 eq.) R1O toluene 111 °C, 5 h

Azide/alkyne

R

N N N

OAc

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H H

H

H

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8a, 8h R1= H 10a, 10h R1= Ac

Triazole

Yielda (%) 50

1

5/6a

8a

61

2

5/6h

8h

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a

3

9/6a

10a

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4

9/6h

10h

86

60

Yields of purified isolated products.

of around 10.3 Hz. This assignment was confirmed by HSQC and HMBC measurements.

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Scheme 2 Synthesis of 3,17-diols and 3-keto derivatives of steroidal triazoles

intermolecular ring closure is significantly influenced by the OH group on C-3, spatially close to the azide dipole, and especially by the steric bulk of the alkyne substituent, which presumably causes a crowded transition state of the Cu(I)catalyzed process. The heterocyclic products (7ag, 8a and 8h) were deacetylated in alkaline methanol to the corresponding 3,17-diols (11ag) and 3,17-diols (12a and 12i), respectively (Scheme 2). In the course of the reaction of 8h, the side-chain of the triazolyl moiety was also hydrolyzed to give the rather polar hydroxymethyl-substituted derivative 12i. The 3-keto analogs (13ag) were also obtained by Jones oxidation, during which a slight formation of enone 2 was observed. The structures of all synthetized compounds were confirmed by 1H and 13C NMR measurements. The 1H NMR spectra of triazoles containing an aromatic moiety connected directly (7ad, 8a, 10a, 11ad, 12a, 13ad) or indirectly (8h, 10h) to the hetero ring revealed the signals of the incorporated aryl groups at 7.28.0 ppm as compared with the spectra of the starting azides (4, 5 and 9). The 5’-H singlet of the newly formed heterocycles was identified at 7.58.3 ppm for the aryl-substituted derivatives, and at 7.17.3 ppm for those containing cycloalkyl substituents (6eg, 11eg, 13eg). The most characteristic difference between the 1H spectra of the 3- (6ag, 11ag) and 3-OH compounds (8a, 8h, 12a, 12i) was the upfield shift of 3-H in the former group of derivatives, due to the magnetic anisotropic effect caused by the aromatic triazole ring cis to this proton. The influence of the heteroaromatic ring was also manifested in the higher chemical shift of the OH proton in 6ag and 11ag, which even in CDCl 3 appeared as a doublet with a coupling constant

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2.2. Pharmacological studies With the exceptions of the 3,17-diacetates (10a and 10h), the novel triazolyl derivatives (7ag, 8a, 8h, 11ag, 12a, 12i and 13ag) were subjected to in vitro pharmacological studies of their antiproliferative effects (Table 3). The activities were determined by using three malignant gynecological cell lines in the microplate-based MTT colorimetric assay, 26 in comparison with cisplatin as positive control. Although there is no generally accepted threshold for efficacy, a substance exhibiting less than 50% inhibition of cell growth at 30 M can not be considered a promising lead compound. Final concentrations not exceeding 30 M were therefore used in the in vitro assays. The structural diversity of the tested compounds suggested certain structure  activity relationships. With the only exception of 7g, the transhydroxytriazoles bearing an acetate at C-17 (7ag) did not exhibit substantial activity. Interestingly, the phenylsubstituted cis-hydroxytriazole 8a, bearing the 3-OH group and the hetero ring in the same spatial orientation, proved to be more potent than its trans counterpart 7a. The effect was similar for the triazole containing an ester side-chain (8h) instead of a phenyl group. Deacetylation of the 17-acetates 7ag did not cause any noteworthy effect for the arylsubstituted compounds (11ad), but appeared favorable for the cyclopentyl-substituted derivative 11f. Hydrolysis of 17acetates 8a and 8h resulted in less potent derivatives 12a and 12i. The compounds obtained by Jones oxidation (13ag) exerted outstanding cytotoxic activity on HeLa cells, characterized by IC 50 values between 1 and 2 µM, i. e. lower

Table 3 Calculated IC50 values of synthesized triazole derivatives IC50 (M) Compound HeLa MCF7 A2780 >30 >30 >30 7a >30 >30 >30 7b >30 15.33 11.30 7c 5.87 >30 7.40 7d >30 >30 >30 7e >30 >30 >30 7f 6.77 12.04 7.01 7g 13.55 20.51 11.83 8a 10.31 8.96 17.17 8h >30 >30 >30 11a 27.37 >30 15.33 11b >30 >30 22.96 11c >30 >30 13.79 11d >30 >30 >30 11e 13.89 15.87 16.48 11f 16.82 18.97 11.98 11g 27.83 >30 >30 12a >30 >30 >30 12i 1.22 26.24 11.22 13a 1.12 21.22 11.79 13b 1.13 21.33 12.32 13c 1.16 12.68 9.20 13d 1.64 15.96 11.68 13e 1.55 25.74 10.85 13f 1.40 20.33 11.81 13g 12.43 9.63 1.30 cisplatin

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than that of the reference cisplatin. On the other hand, the other two cell lines (and especially MCF7) seemed to be less sensitive to these structures. The selective, rather than the generally toxic behavior of this latter group of 1-triazolyl5-androstane derivatives could be regarded as a valuable feature, and this skeleton is therefore suitable for further lead finding research.

3. Conclusions In summary, a novel A-ring-modified steroidal 1,3azidoketone was prepared from stanolone acetate via a multistep stereoselective synthesis, and reduced to give a diastereomeric mixture of azidoalcohols. CuAAC of the trans and cis isomers with different terminal alkynes was achieved under optimized reaction conditions to give 1-exo-triazolyl derivatives in good to excellent yields. The reactions were affected significantly by the stereostructure of the steroidal azide component and especially by the steric bulk of the substituent in the acetylene dipolarophile. The synthetized 5androstane derivatives are of interest from a pharmacological aspect, since several analogs proved to exert marked in vitro cytotoxic activity. A certain selectivity against HeLa cells was also confirmed.

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4.2. 17-Acetoxy-1α-azido-5α-androstan-3-one (3)

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4. Experimental 4.1. General

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Melting points (mp) were determined on a Kofler block and are uncorrected. The reactions were monitored by TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm thick); solvent systems (ss): (A) CH 2Cl2, (B) CH 2Cl2/EtOAc (98:2 v/v), (C) CH2Cl2/EtOAc (95:5 v/v), (D) CH 2Cl2/EtOAc (90:10 v/v), (E) CH2Cl2/EtOAc (80:20 v/v), (F) CH 2Cl2/EtOAc (70:30 v/v),

(G) CH 2Cl2/EtOAc (50:50 v/v), (H) CH 2Cl2/EtOAc (40:60 v/v). The spots were detected by spraying with 5% phosphomolybdic acid in 50% aqueous phosphoric acid. The Rf values were determined for the spots observed by illumination at 254 and 365 nm. Flash chromatography: Merck silica gel 60, 40–63 μm. All solvents were distilled prior to use. Reagents and materials were obtained from commercial suppliers and were used without purification. Elementary analysis data were determined with a PerkinElmer CHN analyzer model 2400. NMR spectra were obtained at room temperature with a Bruker DRX 500 instrument. Chemical shifts are reported in ppm (δ scale), and coupling constants (J) in Hz. For the determination of multiplicities, the J-MOD pulse sequence was used. Automated flow injection analyses were performed by using an HPLC/MSD system. The system comprised an Agilent 1100 micro vacuum degasser, a quaternary pump, a micro-well plate autoinjector and a 1946A MSD equipped with an electrospray ion source (ESI) operated in positive ion mode. The ESI parameters were: nebulizing gas N 2, at 35 psi; drying gas N 2, at 350 °C and 12 L/min; capillary voltage (VCap) 3000 V; fragmentor voltage 70 V. The MSD was operated in scan mode with a mass range of m/z 60−620. Samples (0.2 μL) with automated needle wash were injected directly into the solvent flow (0.3 mL/min) of CH 3CN/H 2O 70:30 (v/v) supplemented with 0.1% formic acid. The system was controlled by Agilent LC/MSD Chemstation software.

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Compound 2 (10.0 g, 30.2 mmol) was dissolved in a mixture of THF (100 mL) and acetic acid (100 mL), and a solution of NaN3 (15.5 g, 0.20 mol) in water (40 mL) was poured into the organic phase. The reaction mixture was stirred at ambient temperature for 24 h, and then poured into saturated NaHCO 3 solution and extracted with CH 2Cl2 (3 x 50 mL). The combined organic layers were dried over Na2SO4 and evaporated in vacuo. Purification of the resulting crude product by flash chromatography with CH2Cl2 as eluent afforded 3 as a white solid (7.6 g, 67%), mp 127-129 °C, Rf = 0.38 (ss A); 1H NMR (500 MHz, CDCl 3): H = 0.80 (s, 3H, 18-H3), 0.86-0.96 (m, 2H), 1.05 (s, 3H, 19-H3), 1.12 (m, 1H), 1.25-1.53 (m, 9H), 1.61-1.69 (m, 2H), 1.76 (m, 1H), 2.03 (s, 3H, Ac-CH3), 2.13-2.22 (m, 3H), 2.58 (d, 1H, J = 18.8 Hz, 2H), 2.69 (dd, 1H, J = 18.8 Hz, J = 3.8 Hz, 2-H ), 3.97 (bs, 1H, 1-H), 4.60 (t, 1H, J = 8.5 Hz, 17-H); 13C NMR (125 MHz, CDCl3): C = 12.1 (C-18), 12.7 (C-19), 20.3 (CH 2), 21.1 (AcCH3), 23.5 (CH 2), 27.5 (CH2), 28.3 (CH2), 30.7 (CH 2), 35.2 (CH), 36.5 (CH 2), 39.2 (CH), 39.5 (C-10), 42.5 (CH 2), 42.6 (C-13), 44.3 (CH 2), 47.4 (CH), 50.4 (CH), 66.5 (C-1), 82.6 (C-17), 171.1 (Ac-CO), 207.8 (C-3); Anal. Calcd for C21H31N3O3 C, 67.53; H, 8.37; N, 11.25. Found: C, 67.35; H, 8.52; N, 11.45. 4.3. 17-Acetoxy-1α-azido-5α-androstan-3-ol (4) and -3α-ol (5) Compound 3 (7.0 g, 18.7 mmol) was dissolved in MeOH (100 mL), and KBH 4 (5.0 g, 89.1 mmol) was added in small

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portions. To maintain pH 6-8, the solution was repeatedly acidified as needed with MeOH/AcOH (1:1), using bromothymol blue as indicator. The mixture was stirred for 3 h, and after completion of the reaction, diluted with water and acidified with dilute HCl. The precipitate that formed was filtered off, washed with water and dried. The resulting epimeric azidoalcohols could be separated by column chromatography with 1% EtOAc/CH 2Cl2 as eluent, yielding 4 (4.53 g, 68%) and 5 (2.14 g, 27%). (4): mp 145-147 °C, R f = 0.55 (ss C); 1H NMR (500 MHz, CDCl 3): H = 0.80 (s, 3H, 18-H3), 0.83 (s, 3H, 19-H3), 0.88-0.96 (m, 1H), 1.09-1.50 (m, 11H), 1.61-1.75 (m, 4H), 1.88-1.93 (m, 1H), 2.02 (s, 3H, AcCH3), 2.08 (m, 1H), 2.12-2.19 (m, 1H), 2.90 (d, 1H, J = 9.4 Hz), 3.67 (bs, 1H, 1-H), 3.94 (m, 1H, 3-H), 4.60 (t, 1H, J = 8.4 Hz, 17-H); 13C NMR (125 MHz, CDCl 3): C = 12.2 (C18), 12.5 (C-19), 19.9 (CH 2), 21.1 (Ac-CH3), 23.4 (CH 2), 27.5 (CH2), 28.1 (CH2), 31.0 (CH 2), 32.1 (CH 2), 32.5 (CH), 35.3 (CH), 35.8 (CH 2), 36.5 (CH2), 40.1 (C-10), 42.6 (C-13), 48.1 (CH), 50.7 (CH), 65.0 and 66.5 (C-1 and C-3), 82.6 (C-17), 171.1 (Ac-CO); Anal. Calcd for C 21H33N3O3 C, 67.17; H, 8.86; N, 11.19. Found: C, 67.05; H, 9.05; N, 11.36. (5): mp 141-143 °C, Rf = 0.35 (ss C); 1H NMR (500 MHz, CDCl 3): H = 0.77 (s, 3H, 18-H3), 0.85 (s, 3H, 19-H3), 0.87-0.92 (m, 1H), 1.06-1.39 (m, 8H), 1.42-1.50 (m, 3H), 1.58-1.64 (m, 3H), 1.67-1.74 (m, 3H), 2.02 (s, 3H, Ac-CH3), 2.10-2.19 (m, 2H), 3.70 (bs, 1H, 1-H), 3.91 (m, 1H, 3-H), 4.58 (t, 1H, J = 8.4 Hz, 17-H); 13C NMR (125 MHz, CDCl 3): C = 12.1 (C-18), 13.2 (C-19), 20.1 (CH 2), 21.1 (Ac-CH3), 23.5 (CH2), 27.5 (CH 2), 28.3 (CH 2), 31.0 (CH 2), 34.6 (CH 2), 35.2 (CH), 36.5 (CH 2), 37.7 (CH 2), 37.8 (CH), 39.2 (C-10), 42.6 (C-13), 47.7 (CH), 50.5 (CH), 65.6 and 66.2 (C-1 and C-3), 82.7 (C-17), 171.1 (Ac-CO); Anal. Calcd for C 21H33N3O3 C, 67.17; H, 8.86; N, 11.19. Found: C, 67.03; H, 9.02; N, 11.40.

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4.4. General procedure for the preparation of triazoles 7a–g 35

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To a solution of 17-acetoxy-1α-azido-5α-androstan-3-ol (4) (300 mg, 0.79 mmol) in toluene (10 mL) were added Ph3P (41 mg, 0.16 mmol), CuI (15 mg, 0.08 mmol) and DIPEA (0.40 mL, 2.4 mmol). Finally the appropriate terminal alkyne (6ag, 1.1 eq) was added to the reaction mixture, which was then refluxed for 3 h, allowed to cool and evaporated in vacuo. The resulting crude product was purified by column chromatography. 4.4.1. 17-Acetoxy-1α-[4′-phenyl-1′H-1′,2′,3′-triazol-1′-yl]-5αandrostan-3-ol (7a) Alkyne: phenylacetylene (6a, 0.09 mL). After purification with CH2Cl2/EtOAc (90:10) as eluent, 7a was obtained as a white solid (355 mg, 93%), mp 266-268 °C, Rf = 0.32 (ss D); 1 H NMR (500 MHz, CDCl 3): H = 0.22 (m, 1H), 0.76 (s, 3H, 18-H3), 0.82-0.89 (m, 3H), 1.06 (s, 3H, 19-H3), 1.19-1.29 (m, 2H), 1.34-1.44 (m, 4H), 1.49-1.80 (m, 6H), 1.98 (s, 3H, AcCH3), 2.05 (m, 2H), 2.34-2.42 (m, 2H), 4.01 (bs, 1H, 3-H), 4.43 (t, 1H, J = 8.4 Hz, 17-H), 4.58 (d, 1H, J = 5.2 Hz, 1-H), 5.19 (bs, 1H, 3-OH), 7.34 (t, 1H, J = 7.3 Hz, 4″-H), 7.43 (t, 2H, J = 7.3 Hz, 3″-H and 5″-H), 7.80 (d, 2H, J = 7.3 Hz, 2″-H and 6″-H), 7.91 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl 3): C = 12.4 (C-18), 13.4 (C-19), 21.1 (Ac-CH3), 21.3 (CH2), 23.3 (CH 2), 27.4 (CH 2), 28.7 (CH 2), 30.4 (CH 2), 32.9 (CH),

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33.2 (CH2), 35.8 (CH), 36.3 (2C, 2 × CH 2), 40.4 (C-10), 42.6 (C-13), 47.5 (CH), 50.5 (CH), 63.8 and 64.0 (C-1 and C-3), 82.4 (C-17), 122.3 (C-5′), 125.8 (2C, C-2″ and C-6″), 128.3 (C-4″), 128.9 (2C, C-3″ and C-5″), 130.2 (C-1″), 146.7 (C-4′), 170.9 (Ac-CO); ESI-MS: 478 [M+H] +; Anal. Calcd for C29H39N3O3 C, 72.92; H, 8.23; N, 8.80. Found: C, 73.03; H, 8.39; N, 8.98. 4.4.2. 17-Acetoxy-1α-[4′-(4″-tolyl)-1′H-1′,2′,3′-triazol-1′-yl]5α-androstan-3-ol (7b) Alkyne: 4-tolylacetylene (6b, 0.09 mL). After purification with CH2Cl2/EtOAc (90:10) as eluent, 7b was obtained as a white solid (362 mg, 93%), mp 274-275 °C, Rf = 0.41 (ss E); 1 H NMR (500 MHz, CDCl 3): H = 0.20 (m, 1H), 0.75 (s, 3H, 18-H3), 0.81-0.88 (m, 3H), 1.05 (s, 3H, 19-H3), 1.18-1.26 (m, 2H), 1.36-1.44 (m, 4H), 1.50-1.79 (m, 6H), 1.98 (s, 3H, AcCH3), 2.04 (m, 2H), 2.36 (m, 2H), 2.38 (s, 3H, 4″-CH3), 4.00 (bs, 1H, 3-H), 4.42 (t, 1H, J = 8.3 Hz, 17-H), 4.55 (d, 1H, J = 4.9 Hz, 1-H), 5.29 (d, 1H, J = 10.2 Hz, 3-OH), 7.23 (d, 2H, J = 7.7 Hz, 3″-H and 5″-H), 7.69 (d, 2H, J = 7.7 Hz, 2″-H and 6″-H), 7.85 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl3): C = 12.4 (C-18), 13.3 (C-19), 21.1 (Ac-CH3), 21.2 (4”-CH3), 21.3 (CH2), 23.3 (CH2), 27.4 (CH 2), 28.6 (CH 2), 30.3 (CH2), 32.8 (CH), 33.2 (CH 2), 35.8 (CH), 36.2 (CH 2), 36.3 (CH 2), 40.4 (C-10), 42.6 (C-13), 47.5 (CH), 50.5 (CH), 63.7 and 64.0 (C-1 and C-3), 82.4 (C-17), 122.0 (C-5′), 125.6 (2C, C-2″ and C6″), 127.3 (C-1″), 129.6 (2C, C-3″ and C-5″), 138.2 (C-4″), 146.7 (C-4′), 170.9 (Ac-CO); ESI-MS: 492 [M+H] +; Anal. Calcd for C 30H41N3O3 C, 73.29; H, 8.41; N, 8.55. Found: C, 73.43; H, 8.28; N, 8.62. 4.4.3. 17-Acetoxy-1α-[4′-(4″-ethylphenyl)-1′H-1′,2′,3′triazol-1′-yl]-5α-androstan-3-ol (7c) Alkyne: 4-ethylphenylacetylene (6c, 0.12 mL). After purification with CH2Cl2/EtOAc (80:20) as eluent, 7c was obtained as a white solid (372 mg, 92%), mp 253-255 °C, Rf = 0.27 (ss D); 1H NMR (500 MHz, CDCl3): H = 0.19 (m, 1H), 0.75 (s, 3H, 18-H3), 0.80-0.89 (m, 3H), 1.05 (s, 3H, 19-H3), 1.20 (m, 2H), 1.26 (t, 3H, J = 7.6 Hz, 4″-CH2CH3), 1.36-1.43 (m, 4H), 1.51-1.80 (m, 6H), 1.98 (s, 3H, Ac-CH3), 2.04 (m, 2H), 2.37 (m, 2H), 2.67 (q, 2H, J = 7.6 Hz, 4″-CH2CH3), 4.00 (bs, 1H, 3-H), 4.42 (t, 1H, J = 8.4 Hz, 17-H), 4.55 (d, 1H, J = 5.4 Hz, 1-H), 5.30 (d, 1H, J = 10.3 Hz, 3-OH), 7.26 (d, 2H, J = 7.7 Hz, 3″-H and 5″-H), 7.71 (d, 2H, J = 7.7 Hz, 2″-H and 6″-H), 7.89 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl3): C = 12.4 (C-18), 13.3 (C-19), 15.5 (4″-CH2CH3), 21.0 (Ac-CH3), 21.3 (CH2), 23.3 (CH 2), 27.4 (CH 2), 28.6 (2C, 2 × CH 2), 30.3 (CH2), 32.8 (CH), 33.1 (CH 2), 35.8 (CH), 36.3 (2C, 2 × CH 2), 40.4 (C-10), 42.6 (C-13), 47.5 (CH), 50.5 (CH), 63.7 and 64.0 (C-1 and C-3), 82.4 (C-17), 122.0 (C-5′), 125.7 (2C, C-2″ and C-6″), 127.6 (C-1″), 128.4 (2C, C-3″ and C-5″), 144.6 (C-4″), 146.7 (C-4′), 170.9 (Ac-CO); ESI-MS: 506 [M+H] +; Anal. Calcd for C 31H43N3O3 C, 73.63; H, 8.57; N, 8.31. Found: C, 73.51; H, 8.75; N, 8.46. 4.4.4. 17-Acetoxy-1α-[4′-(4″-tert-butylphenyl)-1′H-1′,2′,3′triazol-1′-yl]-5α-androstan-3-ol (7d) Alkyne: 4-tert-butylphenylacetylene (6d, 0.16 mL). After purification with CH2Cl2/EtOAc (80:20) as eluent, 7d was

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obtained as a white solid (397 mg, 93%), mp 271-273 °C, Rf = 0.29 (ss E); 1H NMR (500 MHz, CDCl 3): H = 0.19 (m, 1H), 0.75 (s, 3H, 18-H3), 0.79-0.89 (m, 3H), 1.05 (s, 3H, 19-H3), 1.20 (m, 2H), 1.34 (s, 9H, 4″-tBu-CH3), 1.36-1.43 (m, 4H), 1.51-1.79 (m, 6H), 1.98 (s, 3H, Ac-CH3), 2.04 (m, 2H), 2.37 (m, 2H), 4.00 (bs, 1H, 3-H), 4.43 (t, 1H, J = 8.4 Hz, 17-H), 4.56 (d, 1H, J = 5.4 Hz, 1-H), 5.32 (d, 1H, J = 10.3 Hz, 3OH), 7.45 (d, 2H, J = 7.9 Hz, 3″-H and 5″-H), 7.73 (d, 2H, J = 7.9 Hz, 2″-H and 6″-H), 7.87 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl 3): C = 12.4 (C-18), 13.3 (C-19), 21.1 (Ac-CH3), 21.3 (CH 2), 23.3 (CH 2), 27.4 (CH 2), 28.6 (CH 2), 30.4 (CH2), 31.3 (3C, 4″-tBu-CH3), 32.8 (CH), 33.2 (CH 2), 34.7 (4″-tBuC), 35.8 (CH), 36.3 (2C, 2 × CH 2), 40.4 (C-10), 42.6 (C-13), 47.5 (CH), 50.5 (CH), 63.7 and 63.9 (C-1 and C-3), 82.4 (C17), 122.1 (C-5′), 125.5 (2C) and 125.8 (2C): (C-2″, C-3″, C5″ and C-6″), 127.3 (C-1″), 146.6 (C-4′), 151.4 (C-4″), 170.9 (Ac-CO); ESI-MS: 534 [M+H] +; Anal. Calcd for C 33H47N3O3 C, 74.26; H, 8.88; N, 7.87. Found: C, 74.45; H, 8.81; N, 7.99. 4.4.5. 17-Acetoxy-1α-[4′-cyclopropyl-1′H-1′,2′,3′-triazol-1′yl]-5α-androstan-3-ol (7e) Alkyne: cyclopropylacetylene (6e, 0.07 mL). After purification with CH2Cl2/EtOAc (80:20) as eluent, 7e was obtained as a white solid (338 mg, 96%), mp 237-239 °C, Rf = 0.29 (ss E); 1H NMR (500 MHz, CDCl 3): H = 0.01 (m, 1H), 0.75 (s, 3H, 18-H3), 0.77-0.90 (m, 5H), 0.96 (m, 2H), 1.01 (s, 3H, 19-H3), 1.14-1.27 (m, 2H), 1.31-1.47 (m, 5H), 1.54 (m, 2H), 1.62-1.75 (m, 3H), 1.90-1.97 (m, 2H), 2.00 (s, 3H, AcCH3), 2.06 (m, 1H), 2.33 (m, 2H), 3.94 (bs, 1H, 3-H), 4.38 (d, 1H, J = 5.3 Hz, 1-H), 4.50 (t, 1H, J = 8.5 Hz, 17-H), 5.54 (d, 1H, J = 10.9 Hz, 3-OH), 7.28 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl 3): C = 6.5 (C-1″), 7.7 and 8.2 (C-2″ and C-3″), 12.4 (C-18), 13.3 (C-19), 21.1 (Ac-CH3), 21.3 (CH2), 23.2 (CH2), 27.3 (CH2), 28.6 (CH 2), 30.3 (CH 2), 32.7 (CH), 33.0 (CH2), 35.8 (CH), 36.4 (2C, 2 × CH 2), 40.4 (C-10), 42.7 (C13), 47.4 (CH), 50.6 (CH), 63.4 and 63.9 (C-1 and C-3), 82.4 (C-17), 122.7 (C-5′), 149.0 (C-4′), 171.0 (Ac-CO); ESI-MS: 442 [M+H] +; Anal. Calcd for C 26H39N3O3 C, 70.71; H, 8.90; N, 9.52. Found: C, 70.81; H, 8.71; N, 9.66.

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4.5. 3α,17-Diacetoxy-1α-azido-5α-androstane (9)

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4.4.6. 17-Acetoxy-1α-[4′-cyclopentyl-1′H-1′,2′,3′-triazol-1′yl]-5α-androstan-3-ol (7f) Alkyne: cyclopentylacetylene (6f, 0.10 mL). After purification with CH2Cl2/EtOAc (80:20) as eluent, 7f was obtained as a white solid (349 mg, 93%), mp 265-267 °C, Rf = 0.30 (ss D); 1 H NMR (500 MHz, CDCl 3): H = 0.04 (m, 1H), 0.75 (s, 3H, 18-H3), 0.79-0.86 (m, 3H), 1.02 (s, 3H, 19-H3), 1.15-1.46 (m, 8H), 1.51-1.77 (m, 11H), 2.00 (s, 3H, Ac-CH3), 2.09 (m, 3H), 2.34 (m, 2H), 3.17 (m, 1H), 3.95 (m, 1H, 3-H), 4.40 (d, 1H, J = 5.6 Hz, 1-H), 4.46 (t, 1H, J = 8.5 Hz, 17-H), 5.70 (d, 1H, J = 11.2 Hz, 3-OH), 7.29 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl3): C = 12.4 (C-18), 13.3 (C-19), 21.1 (Ac-CH3), 21.4 (CH2), 23.3 (CH2), 25.1 (CH 2), 27.4 (CH 2), 28.6 (CH2), 30.3 (CH2), 32.7 (CH), 33.0 (CH 2), 33.2 (CH2), 33.3 (CH 2), 35.8 (CH), 36.4 (3C, 3 × CH 2), 36.5 (CH), 40.4 (C-10), 42.6 (C13), 47.5 (CH), 50.7 (CH), 63.4 and 63.9 (C-1 and C-3), 82.5 (C-17), 122.8 (C-5′), 151.5 (C-4′), 171.1 (Ac-CO); ESI-MS: 470 [M+H] +; Anal. Calcd for C 28H43N3O3 C, 71.61; H, 9.23; N, 8.95. Found: C, 71.72; H, 9.36; N, 8.87.

4.4.7. 17-Acetoxy-1α-[4′-cyclohexyl-1′H-1′,2′,3′-triazol-1′yl]-5α-androstan-3-ol (7g) Alkyne: cyclohexylacetylene (6g, 0.11 mL). After purification with CH2Cl2/EtOAc (80:20) as eluent, 7g was obtained as a white solid (375 mg, 97%), mp 243-245 °C, Rf = 0.28 (ss D); 1 H NMR (500 MHz, CDCl 3): H = 0.04 (m, 1H), 0.76 (s, 3H, 18-H3), 0.79-0.86 (m, 3H), 1.02 (s, 3H, 19-H3), 1.15-1.45 (m, 13H), 1.51-1.79 (m, 8H), 2.00 (s, 3H, Ac-CH3), 2.04 (m, 3H), 2.35 (m, 2H), 2.75 (m, 1H), 3.95 (m, 1H, 3-H), 4.40 (d, 1H, J = 5.5 Hz, 1-H), 4.46 (t, 1H, J = 8.5 Hz, 17-H), 5.71 (d, 1H, J = 11.2 Hz, 3-OH), 7.27 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl3): C = 12.4 (C-18), 13.3 (C-19), 21.1 (Ac-CH3), 21.4 (CH2), 25.9 (CH2), 26.0 (CH 2), 27.4 (CH 2), 28.6 (CH2), 30.3 (CH2), 32.7 (CH), 33.0 (2C, 2 × CH 2), 33.1 (CH 2), 33.3 (CH2), 35.0 (CH), 35.8 (CH), 36.4 (3C, 3 × CH 2), 40.5 (C-10), 42.6 (C-13), 47.5 (CH), 50.7 (CH), 63.5 and 63.9 (C-1 and C-3), 82.5 (C-17), 122.5 (C-5′), 152.4 (C-4′), 171.1 (Ac-CO); ESIMS: 484 [M+H] +; Anal. Calcd for C 29H45N3O3 C, 72.01; H, 9.38; N, 8.69. Found: C, 71.89; H, 9.47; N, 8.84.

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Compound 5 (1.0 g, 2.7 mmol) was dissolved in a mixture of pyridine (30 mL) and Ac 2O (15 mL), and the solution was stirred at room temperature for 4 h. It was then poured onto a mixture of ice and H2SO4 (25 mL), diluted with water and extracted with CH 2Cl 2 (3 x 50 mL). The combined organic layers were washed with water, dried over Na 2SO4 and evaporated in vacuo. The resulting crude product was purified by flash chromatography with CH 2Cl2 as eluent to give 9 (978 mg, 88%), mp 88-91 °C, Rf = 0.23 (ss A); 1H NMR (500 MHz, CDCl3): H = 0.77 (s, 3H, 18-H3), 0.87 (s, 3H, 19-H3), 0.880.93 (m, 1H), 1.06-1.12 (m, 1H), 1.17-1.69 (m, 14H), 1.73 (m, 1H), 1.78-1.83 (m, 1H), 2.01 (s, 3H, Ac-CH3), 2.02 (s, 3H, Ac-CH3), 2.11-2.20 (m, 2H), 3.72 (bs, 1H, 1-H), 4.59 (t, 1H, J = 8.4 Hz, 17-H), 4.96 (m, 1H, 3-H); 13C NMR (125 MHz, CDCl3): C = 12.1 (C-18), 13.2 (C-19), 20.1 (CH 2), 21.1 (AcCH3), 21.3 (Ac-CH3), 23.5 (CH 2), 27.5 (CH 2), 28.2 (CH 2), 30.9 (CH 2), 31.2 (CH 2), 33.5 (CH 2), 35.2 (CH), 36.5 (CH 2), 37.5 (CH), 39.3 (C-10), 42.6 (C-13), 47.6 (CH), 50.5 (CH), 65.4 and 69.1 (C-1 and C-3), 82.7 (C-17), 170.3 (Ac-CO), 171.1 (Ac-CO); Anal. Calcd for C 23H35N3O4 C, 66.16; H, 8.45; N, 10.06. Found: C, 66.37; H, 8.29; N, 10.19. 4.6. General procedure for the preparation of triazoles 8a, 8h, 10a and 10h

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To a solution of 17-acetoxy-1α-azido-5α-androstan-3α-ol (5) (300 mg, 0.79 mmol) or 3α,17-diacetoxy-1α-azido-5αandrostane (9) (300 mg, 0.72 mmol) in toluene (10 mL) were added Ph 3P (41 mg, 0.16 mmol), CuI (15 mg, 0.08 mmol) and DIPEA (0.40 mL, 2.4 mmol). Finally, the appropriate terminal alkyne (6a or 6h, 1.1 eq) was added to the reaction mixture, which was then refluxed for 5 h, allowed to cool and evaporated in vacuo. The resulting crude product was purified by column chromatography. 4.6.1. 17-Acetoxy-1α-[4′-phenyl-1′H-1′,2′,3′-triazol-1′-yl]-5αandrostan-3α-ol (8a)

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Alkyne: phenylacetylene (6a, 0.09 mL). After purification with CH2Cl2/EtOAc (80:20) as eluent, 8a was obtained as a white solid (233 mg, 61%), mp 182-184 °C, Rf = 0.29 (ss E); 1 H NMR (500 MHz, CDCl 3): H = 0.37 (m, 1H), 0.75 (s, 3H, 18-H3), 0.80-0.91 (m, 3H), 1.11 (s, 3H, 19-H3), 1.18-1.33 (m, 2H), 1.34-1.47 (m, 5H), 1.49-1.62 (m, 4H), 1.94 (m, 1H), 1.97 (s, 3H, Ac-CH3), 1.99-2.01 (m, 3H), 2.47 (m, 1H), 4.42 (t, 1H, J = 8.3 Hz, 17-H), 4.60-4.65 (m, 2H, 1-H and 3-H), 7.32 (t, 1H, J = 7.3 Hz, 4″-H), 7.41 (t, 2H, J = 7.3 Hz, 3″-H and 5″H), 7.71 (s, 1H, 5′-H), 7.78 (d, 2H, J = 7.3 Hz, 2″-H and 6″H); 13C NMR (125 MHz, CDCl3): C = 12.3 (C-18), 13.9 (C19), 21.1 (Ac-CH3), 21.3 (CH 2), 23.3 (CH2), 27.4 (CH 2), 28.8 (CH2), 30.6 (CH 2), 35.7 (CH), 36.4 (2C, 2 × CH 2), 37.8 (CH 2), 38.0 (CH), 40.1 (C-10), 42.6 (C-13), 47.4 (CH), 50.3 (CH), 65.5 and 65.7 (C-1 and C-3), 82.4 (C-17), 121.4 (C-5′), 125.5 (2C, C-2″ and C-6″), 128.2 (C-4″), 128.9 (2C, C-3″ and C-5″), 130.2 (C-1″), 146.0 (C-4′), 171.0 (Ac-CO); ESI-MS: 478 [M+H] +; Anal. Calcd for C29 H39N3O3 C, 72.92; H, 8.23; N, 8.80. Found: C, 73.06; H, 8.10; N, 8.93.

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4.6.2. 17-Acetoxy-1α-[4′-(O-benzoyl)hydroxymethyl-1′H1′,2′,3′-triazol-1′-yl]-5α-androstan-3α-ol (8h) Alkyne: benzoic acid propargyl ester (6h, 0.13 mL). After purification with CH2Cl2/EtOAc (80:20) as eluent, 8h was obtained as a white solid (351 mg, 83%), mp 226-228 °C, R f = 0.26 (ss F); 1H NMR (500 MHz, CDCl 3): H = 0.15 (m, 1H), 0.71 (s, 3H, 18-H3), 0.76 (m, 3H), 1.08 (s, 3H, 19-H3), 1.141.36 (m, 6H), 1.45-1.55 (m, 4H), 1.85 (m, 1H), 1.99 (s, 3H, Ac-CH3), 2.03 (m, 3H), 2.35 (m, 1H), 2.53 (m, 1H), 4.44 (m, 1H, 17-H), 4.53 (m, 1H, 3-H), 4.61 (bs, 1H, 1-H), 5.44 (s, 2H, O-CH2), 7.41 (t, 2H, J = 7.3 Hz, 3”-H and 5”-H), 7.53 (t, 1H, J = 7.2 Hz, 4″-H), 7.67 (s, 1H, 5′-H), 8.01 (d, 2H, J = 7.2 Hz, 2″-H and 6″-H); 13C NMR (125 MHz, CDCl 3): C = 12.3 (C18), 13.8 (C-19), 21.1 (Ac-CH3), 21.2 (CH 2), 23.3 (CH 2), 27.4 (CH2), 28.7 (CH 2), 30.5 (CH 2), 35.6 (CH), 36.3 (2C, 2 × CH 2), 37.6 (CH 2), 37.9 (CH), 40.1 (C-10), 42.5 (C-13), 47.5 (CH), 50.3 (CH), 57.9 (O-CH2), 65.6 (2C, C-1 and C-3), 82.5 (C17), 126.1 (C-5′), 128.4 (2C, C-3″ and C-5″), 129.7 (2C, C-2″ and C-6″), 133.2 (C-4″), 141.3 (2C, C-1″ and C-4′), 166.4 (C=O), 170.8 (Ac-CO); ESI-MS: 536 [M+H] +; Anal. Calcd for C31H41N3O5 C, 69.51; H, 7.71; N, 7.84. Found: C, 69.68; H, 7.87; N, 8.03. 4.6.3. 3α,17-Diacetoxy-1α-[4′-phenyl-1′H-1′,2′,3′-triazol-1′yl]-5α-androstane (10a) Alkyne: phenylacetylene (6a, 0.08 mL). After purification with CH2Cl2/EtOAc (90:10) as eluent, 10a was obtained as a white solid (112 mg, 30%), mp 158-160 °C, Rf = 0.32 (ss B); 1 H NMR (500 MHz, CDCl 3): H = 0.36 (m, 1H), 0.75 (s, 3H, 18-H3), 0.79-0.91 (m, 3H), 1.14 (s, 3H, 19-H3), 1.19-1.31 (m, 2H), 1.35-1.46 (m, 5H), 1.49-1.63 (m, 4H), 1.97 (s, 3H, AcCH3), 1.99 (s, 3H, Ac-CH3), 2.02-2.20 (m, 4H), 2.41 (m, 1H), 4.43 (t, 1H, J = 8.6 Hz, 17-H), 4.77 (bs, 1H, 1-H), 5.52 (m, 1H, 3-H), 7.33 (t, 1H, J = 7.6 Hz, 4″-H), 7.42 (t, 2H, J = 7.6 Hz, 3″-H and 5″-H), 7.77 (s, 1H, 5′-H), 7.83 (d, 2H, J = 7.6 Hz, 2″-H and 6″-H); 13C NMR (125 MHz, CDCl 3): C = 12.3 (C-18), 13.9 (C-19), 21.0 (Ac-CH3), 21.1 (CH2), 21.2 (AcCH3), 23.3 (CH 2), 27.4 (CH2), 28.6 (CH2), 30.4 (CH 2), 32.7 (CH2), 33.4 (CH2), 35.7 (CH), 36.3 (CH 2), 37.7 (CH), 40.1

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(C-10), 42.6 (C-13), 47.4 (CH), 50.3 (CH), 64.9 and 69.2 (C-1 and C-3), 82.4 (C-17), 120.8 (C-5′), 125.7 (2C, C-2″ and C6″), 128.1 (C-4″), 128.8 (2C, C-3″ and C-5″), 130.5 (C-1″), 146.5 (C-4′), 170.0 and 170.9 (2 × Ac-CO); ESI-MS: 520 [M+H] +; Anal. Calcd for C31H41N3O4 C, 71.65; H, 7.95; N, 8.09. Found: C, 71.83; H, 7.79; N, 8.36. 4.6.4. 3α,17-Diacetoxy-1α-[4′-(O-benzoyl)hydroxymethyl1′H-1′2′3′-triazol-1′-yl]-5α-androstane (10h) Alkyne: benzoic acid propargyl ester (6h, 0.12 mL). After purification with CH2Cl2/EtOAc (90:10) as eluent, 10h was obtained as a white solid (357 mg, 86%), mp 261-263 °C, Rf = 0.38 (ss C); 1H NMR (500 MHz, CDCl 3): H = 0.12 (m, 1H), 0.71 (s, 3H, 18-H3), 0.75 (m, 3H), 1.09 (s, 3H, 19-H3), 1.131.56 (m, 11H), 1.97 (s, 3H, Ac-CH3), 1.99 (s, 3H, Ac-CH3), 2.01-2.14 (m, 4H), 2.37 (m, 1H), 4.28 (t, 1H, J = 8.5 Hz, 17H), 4.67 (bs, 1H, 1-H), 5.42-5.52 (m, 3H, O-CH2 and 3-H), 7.41 (t, 2H, J = 7.4 Hz, 3″-H and 5″-H), 7.53 (t, 1H, J = 7.4 Hz, 4″-H), 7.71 (s, 1H, 5′-H), 8.02 (d, 2H, J = 7.4 Hz, 2″-H and 6″-H); 13C NMR (125 MHz, CDCl 3): C = 12.3 (C-18), 13.8 (C-19), 21.0 (CH2), 21.1 (Ac-CH3), 21.2 (Ac-CH3), 23.3 (CH2), 27.4 (CH2), 28.5 (CH 2), 30.3 (CH 2), 32.6 (CH2), 33.4 (CH2), 35.6 (CH), 36.2 (CH 2), 37.5 (CH 2), 40.1 (C-10), 42.4 (C-13), 47.4 (CH), 50.3 (CH), 58.0 (O-CH2), 64.9 and 69.1 (C-1 and C-3), 82.4 (C-17), 125.8 (C-5′), 128.4 (2C, C-3″ and C-5″), 129.7 (2C, C-2″ and C-6″), 133.1 (C-4”), 141.5 (2C, C1″ and C-4′), 166.4 (C=O), 170.0 and 170.8 (2 × Ac-CO); ESI-MS: 578 [M+H] +; Anal. Calcd for C33H43N3O6 C, 68.61; H, 7.50; N, 7.27. Found: C, 68.73; H, 7.65; N, 7.58.

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4.7. General procedure for the preparation of 11a–g, 12a and 12i

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Compound 7a–g, 8a, or 8h (120 mg) was dissolved in MeOH (10 mL), and KOH (50 mg, 0.89 mmol) was added. The solution was stirred at room temperature for 24 h, diluted with water and acidified with dilute HCl. The precipitate that formed was filtered off, washed with water and dried.

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4.7.1. 1α-[4′-Phenyl-1′H-1′,2′,3′-triazol-1′-yl]-5α-androstane3,17-diol (11a) Substrate: 7a (0.25 mmol). 11a was obtained as a white solid (105 mg, 96%), mp 149-151 °C, Rf = 0.22 (ss F); 1H NMR (500 MHz, CDCl 3): H = 0.18 (m, 1H), 0.73 (s, 3H, 18-H3), 0.76-0.84 (m, 3H), 1.08 (s, 3H, 19-H3), 1.13-1.28 (m, 2H), 1.31-1.81 (m, 10H), 1.97 (m, 1H), 2.06 (m, 1H), 2.34-2.45 (m, 2H), 3.48 (t, 1H, J = 8.4 Hz, 17-H), 4.04 (bs, 1H, 3-H), 4.59 (d, 1H, J = 5.6 Hz, 1-H), 7.36 (t, 1H, J = 7.5 Hz, 4″-H), 7.45 (t, 2H, J = 7.6 Hz, 3″-H and 5″-H), 7.82 (d, 2H, J = 7.6 Hz, 2″-H and 6″-H), 7.94 (s, 1H, 5′-H); ESI-MS: 436 [M+H] +; Anal. Calcd for C 27H37N3O2 C, 74.45; H, 8.56; N, 9.65. Found: C, 74.59; H, 8.44; N, 9.82.

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4.7.2. 1α-[4′-(4″-Tolyl)-1′H-1′,2′,3′-triazol-1′-yl]-5αandrostane-3,17-diol (11b) Substrate: 7b (0.24 mmol). 11b was obtained as a white solid (101 mg, 94%), mp 161-163 °C, Rf = 0.28 (ss F); 1H NMR (500 MHz, CDCl 3): H = 0.17 (m, 1H), 0.72 (s, 3H, 18-H3), 0.75-0.83 (m, 3H), 1.07 (s, 3H, 19-H3), 1.14-1.60 (m, 12H), 1.95 (m, 1H), 2.06 (m, 1H), 2.38 (m, 2H), 2.39 (s, 3H, 4”-

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CH3), 3.46 (m, 1H, 17-H), 4.02 (m, 1H, 3-H), 4.56 (d, 1H, J = 5.5 Hz, 1-H), 5.25 (d, 1H, J = 10.5 Hz, 3-OH), 7.25 (d, 2H, J = 7.9 Hz, 3″-H and 5″-H), 7.71 (d, 2H, J = 7.9 Hz, 2″-H and 6″-H), 7.86 (s, 1H, 5′-H); ESI-MS: 450 [M+H] +; Anal. Calcd for C28H39N3O2 C, 74.80; H, 8.74; N, 9.35. Found: C, 74.96; H, 8.92; N, 9.22. 4.7.3. 1α-[4′-(4″-Ethylphenyl)-1′H-1′,2′,3′-triazol-1′-yl]-5αandrostane-3-diol (11c) Substrate: 7c (0.24 mmol). 11c was obtained as a white solid (104 mg, 93%), mp 154-157 °C, Rf = 0.34 (ss F); 1H NMR (500 MHz, CDCl 3): H = 0.17 (m, 1H), 0.72 (s, 3H, 18-H3), 0.75-0.84 (m, 3H), 1.07 (s, 3H, 19-H3), 1.14-1.22 (m, 2H), 1.27 (t, 3H, J = 7.6 Hz, 4″-CH2CH3), 1.34-1.58 (m, 7H), 1.691.81 (m, 3H), 1.95 (m, 1H), 2.06 (m, 1H), 2.39 (m, 2H), 2.68 (q, 2H, J = 7.6 Hz, 4”-CH2CH3), 3.47 (m, 1H, 17-H), 4.02 (m, 1H, 3-H), 4.57 (d, 1H, J = 5.4 Hz, 1-H), 5.24 (d, 1H, J = 10.5 Hz, 3-OH), 7.27 (d, 2H, J = 8.0 Hz, 3″-H and 5″-H), 7.73 (d, 2H, J = 8.0 Hz, 2″-H and 6″-H), 7.87 (s, 1H, 5′-H); ESI-MS: 464 [M+H] +; Anal. Calcd for C 29H41N3O2 C, 75.12; H, 8.91; N, 9.06. Found: C, 75.24; H, 9.06; N, 9.28. 4.7.4. 1α-[4′-(4″-tert-Butylphenyl)-1′H-1′,2,′3′-triazol-1′-yl]5α-androstane-3-diol (11d) Substrate: 7d (0.22 mmol). 11d was obtained as a white solid (103 mg, 95%), mp 170-172 °C, Rf = 0.38 (ss F); 1H NMR (500 MHz, CDCl 3): H = 0.16 (m, 1H), 0.72 (s, 3H, 18-H3), 0.75-0.84 (m, 3H), 1.07 (s, 3H, 19-H3), 1.14-1.28 (m, 2H), 1.35 (s, 9H, 4”-tBu-CH3), 1.38-1.57 (m, 7H), 1.69-1.81 (m, 3H), 1.96 (m, 1H), 2.07 (m, 1H), 2.39 (m, 2H), 3.47 (m, 1H, 17-H), 4.02 (m, 1H, 3-H), 4.57 (d, 1H, J = 5.4 Hz, 1-H), 5.25 (d, 1H, J = 10.5 Hz, 3-OH), 7.47 (d, 2H, J = 8.1 Hz, 3″-H and 5″-H), 7.76 (d, 2H, J = 8.1 Hz, 2″-H and 6″-H), 7.88 (s, 1H, 5′-H); ESI-MS: 492 [M+H] +; Anal. Calcd for C 31H45N3O2 C, 75.72; H, 9.22; N, 8.55. Found: C, 75.59; H, 9.04; N, 8.77.

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4.7.6. 1α-[4′-Cyclopentyl-1′H-1′,2′,3′-triazol-1′-yl]-5αandrostane-3-diol (11f) Substrate: 7f (0.26 mmol). 11f was obtained as a white solid (102 mg, 92%), mp 255-256 °C, Rf = 0.24 (ss G); 1H NMR (500 MHz, CDCl 3): H = 0.02 (m, 1H), 0.68 (m, 1H), 0.72 (s, 3H, 18-H3), 0.75-0.84 (m, 2H), 1.03 (s, 3H, 19-H3), 1.15-1.45 (m, 9H), 1.47-1.77 (m, 9H), 2.00 (m, 2H), 2.10 (m, 2H), 2.35 (m, 2H), 3.19 (m, 1H), 3.51 (m, 1H, 17-H), 3.97 (m, 1H, 3-H), 4.41 (d, 1H, J = 5.2 Hz, 1-H), 5.72 (d, 1H, J = 11.3 Hz, 3OH), 7.31 (s, 1H, 5′-H); ESI-MS: 428 [M+H] +; Anal. Calcd

4.7.7. 1α-[4′-Cyclohexyl-1′H-1′,2′,3′-triazol-1′-yl]-5αandrostane-3-diol (11g) Substrate: 7g (0.25 mmol). 11g was obtained as a white solid (105 mg, 95%), mp 244-246 °C, Rf = 0.31 (ss G); 1H NMR (500 MHz, CDCl 3): H = 0.01 (m, 1H), 0.68 (m, 1H), 0.72 (s, 3H, 18-H3), 0.74-0.84 (m, 2H), 1.03 (s, 3H, 19-H3), 1.16-1.58 (m, 14H), 1.66-1.81 (m, 6H), 1.94-2.03 (m, 4H), 2.35 (m, 2H), 2.78 (m, 1H), 3.51 (m, 1H, 17-H), 3.96 (m, 1H, 3-H), 4.41 (d, 1H, J = 5.2 Hz, 1-H), 5.73 (d, 1H, J = 11.3 Hz, 3-OH), 7.29 (s, 1H, 5′-H); ESI-MS: 442 [M+H] +; Anal. Calcd for C27H43N3O2 C, 73.43; H, 9.81; N, 9.51. Found: C, 73.64; H, 9.67; N, 9.32.

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4.7.8. 1α-[4′-Phenyl-1′H-1′,2′,3′-triazol-1′-yl]-5α-androstane3α,17-diol (12a)

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4.7.5. 1α-[4′-Cyclopropyl-1′H-1′,2′,3′-triazol-1′-yl]-5αandrostane-3-diol (11e) Substrate: 7e (0.27 mmol). 11e was obtained as a white solid (101 mg, 94%), mp 257-260 °C, Rf = 0.22 (ss H); 1H NMR (500 MHz, CDCl 3): H = 0.07 (m, 1H), 0.72 (s, 3H, 18-H3), 0.74-0.88 (m, 4H), 0.97 (m, 2H), 1.03 (s, 3H, 19-H3), 1.151.24 (m, 2H), 1.32-1.44 (m, 6H), 1.47-1.59 (m, 2H), 1.66-1.78 (m, 3H), 1.93-2.03 (m, 3H), 2.35 (m, 2H), 3.54 (m, 1H, 17-H), 3.96 (m, 1H, 3-H), 4.40 (d, 1H, J = 5.8 Hz, 1-H), 5.50 (d, 1H, J = 11.1 Hz, 3-OH), 7.29 (s, 1H, 5′-H); ESI-MS: 400 [M+H] +; Anal. Calcd for C 24H37N3O2 C, 72.14; H, 9.33; N, 10.52. Found: C, 72.33; H, 9.15; N, 10.69.

for C26H41N3O2 C, 73.03; H, 9.66; N, 9.83. Found: C, 73.21; H, 9.82; N, 9.62.

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Substrate: 8a (0.25 mmol). 12a was obtained as a white solid (104 mg, 95%), mp 265-267 °C, Rf = 0.38 (ss G); 1H NMR (500 MHz, MeOD): H = 0.26 (m, 1H), 0.62 (m, 1H), 0.70 (s, 3H, 18-H3), 0.76 (m, 2H), 1.14 (s, 3H, 19-H3), 1.18 (m, 1H), 1.27-1.54 (m, 8H), 1.60 (m, 2H), 1.69 (m, 1H), 1.79-1.89 (m, 2H), 1.96 (m, 1H), 2.07 (m, 1H), 2.44 (m, 1H), 3.37 (t, 1H, J = 8.6 Hz, 17-H), 4.40 (m, 1H, 3-H), 4.59 (m, 2H, 1-H and OH), 7.33 (t, 1H, J = 7.4 Hz, 4″-H), 7.43 (t, 2H, J = 7.4 Hz, 3″-H and 5″-H), 7.82 (d, 2H, J = 7.4 Hz, 2″-H and 6″-H), 8.39 (s, 1H, 5′-H); ESI-MS: 436 [M+H]+; Anal. Calcd for C27H37N3O2 C, 74.45; H, 8.56; N, 9.65. Found: C, 74.62; H, 8.73; N, 9.92. 4.7.9. 1α-[4′-(O-Benzoyl)hydroxymethyl-1′H-1′,2′,3′-triazol1′-yl]-5α-androstane-3α,17-diol (12i) Substrate: 8h (0.22 mmol). 12h was obtained as a white solid (74 mg, 85%), mp 267-269 °C; 1H NMR (500 MHz, MeOD): H = 0.21 (m, 1H), 0.69 (s, 3H, 18-H3), 0.70 (m, 3H), 1.12 (s, 3H, 19-H3), 1.17 (m, 1H), 1.27-1.88 (m, 14H), 2.03 (m, 1H), 2.35 (m, 1H), 3.41 (m, 1H, 17-H), 4.30 (m, 1H, 3-H), 4.56 (bs, 1H, 1-H), 4.66 (s, 2H, O-CH2), 7.96 (s, 1H, 5′-H); 13C NMR (125 MHz, MeOD): C = 11.9 (C-18), 14.3 (C-19), 22.3 (CH2), 24.2 (CH2), 30.0 (CH 2), 30.6 (CH 2), 32.1 (CH2), 37.3 (CH), 37.6 (2C, 2 × CH 2), 38.5 (CH 2), 39.6 (CH), 41.3 (C-10), 44.2 (C-13), 49.4 (CH), 52.3 (CH), 56.5 (O-CH2), 66.6 and 66.8 (C-1 and C-3), 82.3 (C-17), 126.0 (C-5′), 147.7 (C-4′); ESI-MS: 390 [M+H] +; Anal. Calcd for C22H35N3O3 C, 67.83; H, 9.06; N, 10.79. Found: C, 67.94; H, 9.19; N, 10.71.

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4.8. General procedure for the preparation of triazoles 13ag

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Compound 7ag (200 mg) was dissolved in acetone (10 mL) and Jones reagent (0.5 mL) was dropped into the reaction mixture, which was then stirred at room temperature for 20 min., and diluted with water. The precipitate that formed was filtered off and dried, and the crude product was purified by column chromatography. The by-product (2) could generally be isolated, in yields of 19-28%. 4.8.1. 17-Acetoxy-1α-[4′-phenyl-1′H-1′,2′,3′-triazol-1′-yl]-5αandrostan-3-one (13a) Eluent: CH2Cl2/EtOAc (98:2), yielding 2 (32 mg, 23%) and

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13a as a white solid (144 mg, 72%), mp 195-197 °C, Rf = 0.44 (ss D); 1H NMR (500 MHz, CDCl 3): H = 0.40 (m, 1H), 0.71 (m, 1H), 0.82 (s, 3H, 18-H3), 0.94 (m, 1H), 1.12 (m, 1H), 1.26 (s, 3H, 19-H3), 1.30 (m, 3H), 1.43-1.61 (m, 5H), 1.80 (m, 1H), 1.95 (m, 1H), 2.02 (s, 3H, Ac-CH3), 2.05-2.19 (m, 2H), 2.34 (m, 1H), 2.52 (m, 1H), 2.69 (m, 1H), 2.97 (m, 1H), 4.53 (t, 1H, J = 8.2 Hz, 17-H), 5.02 (d, 1H, J = 4.5 Hz, 1-H), 7.34 (t, 1H, J = 7.2 Hz, 4″-H), 7.43 (t, 2H, J = 7.1 Hz, 3″-H and 5″H), 7.63 (s, 1H, 5′-H), 7.82 (d, 2H, J = 7.1 Hz, 2″-H and 6″H); 13C NMR (125 MHz, CDCl3): C = 12.4 (C-18), 13.8 (C19), 21.1 (Ac-CH3), 21.2 (CH 2), 23.3 (CH2), 27.4 (CH 2), 28.3 (CH2), 30.1 (CH2), 35.6 (CH), 36.4 (CH 2), 38.2 (CH), 40.1 (C-10), 42.8 (C-13), 43.1 (CH 2), 44.0 (CH 2), 47.5 (CH), 50.3 (CH), 64.6 (C-1), 82.3 (C-17), 119.8 (C-5′), 125.7 (2C, C-2″ and C-6″), 128.3 (C-4″), 128.9 (2C, C-3″ and C-5″), 130.2 (C1″), 147.1 (C-4′), 171.0 (Ac-CO), 206.6 (C-3); ESI-MS: 476 [M+H] +; Anal. Calcd for C29 H37N3O3 C, 73.23; H, 7.84; N, 8.83. Found: C, 73.44; H, 7.73; N, 8.99. 4.8.2. 17-Acetoxy-1α-[4′-(4″-tolyl)-1′H-1′,2′,3′-triazol-1′-yl]5α-androstan-3-one (13b) Eluent: CH2Cl2/EtOAc (95:5), yielding 2 (35 mg, 26%) and 13b as a white solid (139 mg, 69%), mp 213-215 °C, Rf = 0.35 (ss C); 1H NMR (500 MHz, CDCl 3): H = 0.38 (m, 1H), 0.70 (m, 1H), 0.81 (s, 3H, 18-H3), 0.87-0.96 (m, 1H), 1.10 (m, 1H), 1.24 (s, 3H, 19-H3), 1.30 (m, 3H), 1.42-1.59 (m, 5H), 1.78 (m, 1H), 1.91 (m, 1H), 2.01 (s, 3H, Ac-CH3), 2.07 (m, 1H), 2.16 (m, 1H), 2.30 (m, 1H), 2.37 (s, 3H, 4″-CH3), 2.48 (m, 1H), 2.66 (m, 1H), 2.95 (m, 1H), 4.51 (t, 1H, J = 8.2 Hz, 17-H), 5.00 (d, 1H, J = 5.0 Hz, 1-H), 7.22 (d, 2H, J = 7.7 Hz, 3″-H and 5″-H), 7.58 (s, 1H, 5′-H), 7.69 (d, 2H, J = 7.7 Hz, 2″-H and 6″-H); 13C NMR (125 MHz, CDCl 3): C = 12.3 (C-18), 13.7 (C-19), 21.1 (Ac-CH3), 21.1 (CH2), 21.2 (4”-CH3), 23.3 (CH2), 27.4 (CH2), 28.3 (CH 2), 30.0 (CH 2), 35.5 (CH), 36.4 (CH2), 38.1 (CH), 40.0 (C-10), 42.7 (C-13), 43.0 (CH 2), 43.9 (CH2), 47.4 (CH), 50.3 (CH), 64.5 (C-1), 82.3 (C-17), 119-5 (C-5′), 125.6 (2C, C-2″ and C-6″), 127.4 (C-1″), 129.5 (2C, C3″ and C-5″), 138.1 (C-4″), 147.0 (C-4′), 170.9 (Ac-CO), 206.6 (C-3); ESI-MS: 490 [M+H] +; Anal. Calcd for C30H39N3O3 C, 73.59; H, 8.03; N, 8.58. Found: C, 73.72; H, 8.20; N, 8.48. 4.8.3. 17-Acetoxy-1α-[4′-(4″-ethylphenyl)-1′H-1′,2′,3′triazol-1′-yl]-5α-androstan-3-one (13c) Eluent: CH2Cl2/EtOAc (95:5), yielding 2 (37 mg, 28%) and 13c as a white solid (135 mg, 68%), mp 188-191 °C, Rf = 0.62 (ss C); 1H NMR (500 MHz, CDCl 3): H = 0.38 (m, 1H), 0.70 (m, 1H), 0.81 (s, 3H, 18-H3), 0.91 (m, 1H), 1.10 (m, 1H), 1.24 (t, 3H, J = 7.5 Hz, 4″-CH2CH3), 1.25 (s, 3H, 19-H3), 1.30 (m, 3H), 1.42-1.60 (m, 6H), 1.78 (m, 1H), 1.93 (m, 1H), 2.02 (s, 3H, Ac-CH3), 2.07 (m, 1H), 2.16 (m, 1H), 2.32 (m, 1H), 2.51 (m, 1H), 2.67 (q, 2H, J = 7.5 Hz, 4″-CH2CH3), 2.95 (m, 1H), 4.52 (t, 1H, J = 8.3 Hz, 17-H), 5.00 (d, 1H, J = 5.0 Hz, 1-H), 7.25 (d, 2H, J = 7.7 Hz, 3″-H and 5″-H), 7.59 (s, 1H, 5′-H), 7.72 (d, 2H, J = 7.7 Hz, 2″-H and 6″-H); 13C NMR (125 MHz, CDCl3): C = 12.3 (C-18), 13.7 (C-19), 15.5 (4″-CH2CH3), 21.1 (Ac-CH3), 21.1 (CH 2), 23.3 (CH 2), 27.4 (CH2), 28.3 (CH2), 28.7 (CH2), 30.0 (CH 2), 35.6 (CH), 36.4 (CH 2), 38.1 (CH), 40.0 (C-10), 42.7 (C-13), 43.0 (CH 2), 43.9 (CH 2), 47.4

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(CH), 50.3 (CH), 64.5 (C-1), 82.3 (C-17), 119.5 (C-5′), 125.7 (2C, C-2″ and C-6″), 127.6 (C-1″), 128.4 (2C, C-3″ and C-5″), 144.5 (C-4″), 147.1 (C-4′), 170.9 (Ac-CO), 206.6 (C-3); ESIMS: 504 [M+H] +; Anal. Calcd for C 31H41N3O3 C, 73.92; H, 8.20; N, 8.34. Found: C, 73.77; H, 8.31; N, 8.58.

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4.8.4. 17-Acetoxy-1α-[4′-(4″-tert-butylphenyl)-1′H-1′,2′,3′triazol-1′-yl]-5α-androstan-3-one (13d) Eluent: CH2Cl2/EtOAc (95:5), yielding 2 (23 mg, 19%) and 13d as a white solid (149 mg, 75%), mp 213-216 °C, Rf = 0.64 (ss C); 1H NMR (500 MHz, CDCl 3): H = 0.37 (m, 1H), 0.70 (m, 1H), 0.81 (s, 3H, 18-H3), 0.91 (m, 1H), 1.10 (m, 1H), 1.25 (s, 3H, 19-H3), 1.30 (m, 3H), 1.34 (s, 9H, 4″-tBu-CH3), 1.421.60 (m, 6H), 1.78 (m, 1H), 1.93 (m, 1H), 2.02 (s, 3H, AcCH3), 2.07 (m, 1H), 2.16 (m, 1H), 2.32 (m, 1H), 2.51 (m, 1H), 2.95 (m, 1H), 4.52 (t, 1H, J = 8.4 Hz, 17-H), 5.00 (d, 1H, J = 5.0 Hz, 1-H), 7.44 (d, 2H, J = 8.2 Hz, 3″-H and 5″-H), 7.61 (s, 1H, 5′-H), 7.74 (d, 2H, J = 8.2 Hz, 2″-H and 6″-H); 13C NMR (125 MHz, CDCl 3): C = 12.3 (C-18), 13.7 (C-19), 21.0 (AcCH3), 21.1 (CH 2), 23.3 (CH2), 27.4 (CH2), 28.3 (CH 2), 30.0 (CH2), 31.2 (3C, 4″-tBu-CH3), 34.7 (4″-tBu-C), 35.6 (CH), 36.4 (CH 2), 38.1 (CH), 40.4 (C-10), 42.7 (C-13), 43.0 (CH 2), 43.9 (CH2), 47.5 (CH), 50.3 (CH), 64.5 (C-1), 82.3 (C-17), 119.5 (C-5′), 125.4 (2C) and 125.8 (2C): (C-2″, C-3″, C-5″ and C-6″), 127.4 (C-1″), 147.0 (C-4′), 151.4 (C-4″), 171.0 (Ac-CO), 206.5 (C-3); ESI-MS: 532 [M+H] +; Anal. Calcd for C33H45N3O3 C, 74.54; H, 8.53; N, 7.90. Found: C, 74.70; H, 8.36; N, 8.03. 4.8.5. 17-Acetoxy-1α-[4′-cyclopropyl-1′H-1′,2′,3′-triazol-1′yl]-5α-androstan-3-one (13e) Eluent: CH2Cl2/EtOAc (80:20), yielding 2 (34 mg, 24%) and 13e as a white solid (141 mg, 71%), mp 176-179 °C, Rf = 0.47 (ss E); 1H NMR (500 MHz, CDCl 3): H = 0.30 (m, 1H), 0.72 (m, 1H), 0.80 (s, 3H, 18-H3), 0.82-0.89 (m, 2H), 0.90-0.97 (m, 3H), 1.08 (m, 1H), 1.20 (s, 3H, 19-H3), 1.23-1.33 (m, 2H), 1.39-1.60 (m, 6H), 1.73 (m, 1H), 1.83-1.93 (m, 2H), 2.02 (s, 3H, Ac-CH3), 2.05-2.16 (m, 2H), 2.28 (dd, 1H, J = 16.6 Hz, J = 13.0 Hz), 2.45 (m, 1H), 2.58 (d, 1H, J = 16.8 Hz), 2.89 (dd, 1H, J = 16.8 Hz, J = 6.1 Hz), 4.55 (t, 1H, J = 8.3 Hz, 17-H), 4.88 (d, 1H, J = 5.0 Hz, 1-H), 7.11 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl3): C = 6.6 (C-1″), 7.8 and 8.0 (C-2″ and C3″), 12.3 (C-18), 13.7 (C-19), 21.0 (CH 2), 21.1 (Ac-CH3), 23.3 (CH 2), 27.4 (CH 2), 28.3 (CH 2), 30.0 (CH 2), 35.5 (CH), 36.4 (CH 2), 38.1 (CH), 39.9 (C-10), 42.7 (C-13), 43.1 (CH 2), 43.9 (CH2), 47.4 (CH), 50.4 (CH), 64.3 (C-1), 82.3 (C-17), 120.2 (C-5′), 149.2 (C-4′), 171.0 (Ac-CO), 206.7 (C-3); ESIMS: 440 [M+H] +; Anal. Calcd for C 26H37N3O3 C, 71.04; H, 8.48; N, 9.56. Found: C, 71.31; H, 8.32; N, 9.74. 4.8.6. 17-Acetoxy-1α-[4′-cyclopentyl-1′H-1′,2′,3′-triazol-1′yl]-5α-androstan-3-one (13f) Eluent: CH2Cl2/EtOAc (95:5), yielding 2 (34 mg, 24%) and 13f as a white solid (147 mg, 73%), mp 194-196 °C, Rf = 0.53 (ss E); 1H NMR (500 MHz, CDCl 3): H = 0.27 (m, 1H), 0.71 (m, 1H), 0.80 (s, 3H, 18-H3), 0.90 (m, 1H), 1.03 (m, 1H), 1.21 (s, 3H, 19-H3), 1.27 (m, 3H), 1.40-1.84 (m, 13H), 2.02 (s, 3H, Ac-CH3), 2.07-2.18 (m, 4H), 2.28 (dd, 1H, J = 16.5 Hz, J = 13,0 Hz), 2.45 (m, 1H), 2.60 (d, 1H, J = 16.8 Hz), 2.89 (dd,

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1H, J = 16.8 Hz, J = 6.0 Hz), 3.14 (m, 1H), 4.53 (t, 1H, J = 8.4 Hz, 17-H), 4.88 (d, 1H, J = 5.4 Hz, 1-H), 7.12 (s, 1H, 5′H); 13C NMR (125 MHz, CDCl3): C = 12.4 (C-18), 13.7 (C19), 21.0 (CH2), 21.1 (Ac-CH3), 23.3 (CH2), 25.1 (CH 2), 27.4 (CH2), 28.2 (CH2), 30.1 (CH 2), 33.1 (CH 2), 33.3 (CH2), 35.5 (CH), 36.4 (2C, 2 × CH 2), 36.6 (CH), 38.0 (CH), 39.9 (C-10), 42.7 (C-13), 43.0 (CH 2), 43.9 (CH 2), 47.5 (CH), 50.5 (CH), 64.2 (C-1), 82.4 (C-17), 120.1 (C-5′), 151.8 (C-4′), 171.0 (AcCO), 206.7 (C-3); ESI-MS: 468 [M+H] +; Anal. Calcd for C28H41N3O3 C, 71.91; H, 8.84; N, 8.99. Found: C, 72.04; H, 9.05; N, 9.25. 4.8.7. 17-Acetoxy-1α-[4′-cyclohexyl-1′H-1′,2′,3′-triazol-1′yl]-5α-androstan-3-one (13g) Eluent: CH2Cl2/EtOAc (80:20), yielding 2 (34 mg, 24%) and 13g as a white solid (142 mg, 72%), mp 202-204 °C, Rf = 0.42 (ss D); 1H NMR (500 MHz, CDCl 3): H = 0.25 (m, 1H), 0.70 (m, 1H), 0.80 (s, 3H, 18-H3), 0.90 (m, 1H), 1.21 (s, 3H, 19H3), 1.25-1.60 (m, 14H), 1.69-1.84 (m, 5H), 2.02 (s, 3H, AcCH3), 2.08-2.18 (m, 4H), 2.28 (dd, 1H, J = 16.5 Hz, J = 12.9 Hz), 2.46 (m, 1H), 2.62 (d, 1H, J = 16.8 Hz), 2.73 (m, 1H), 2.89 (dd, 1H, J = 16.8 Hz, J = 6.0 Hz), 4.53 (t, 1H, J = 8.2 Hz, 17-H), 4.88 (d, 1H, J = 5.2 Hz, 1-H), 7.10 (s, 1H, 5′-H); 13C NMR (125 MHz, CDCl3): C = 12.4 (C-18), 13.7 (C-19), 21.1 (CH2), 21.2 (Ac-CH3), 23.3 (CH 2), 26.0 (3C, 3 × CH 2), 27.4 (CH2), 28.3 (CH 2), 30.1 (CH 2), 32.9 (2C, 2 × CH 2), 35.1 (CH), 35.5 (CH), 36.5 (CH 2), 38.0 (CH), 40.0 (C-10), 42.7 (C-13), 43.0 (CH 2), 43.9 (CH 2), 47.5 (CH), 50.5 (CH), 64.2 (C-1), 82.4 (C-17), 119.8 (C-5′), 152.8 (C-4′), 171.1 (Ac-CO), 206.7 (C-3); ESI-MS: 482 [M+H] +; Anal. Calcd for C 29H43N3O3 C, 72.31; H, 9.00; N, 8.72. Found: C, 72.47; H, 9.19; N, 8.55. 4.9. Determination of antiproliferative activities

60

wells. Cisplatin was used as positive control. Stock solutions of the tested substances (10 mM) were prepared with DMSO. The DMSO content of the medium did not have any significant effect on the cell proliferation.

Acknowledgements 65

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This work was supported by the New Hungary Development Plan (TÁMOP 4.2.1/B-09/1/KONV-2010-0005) and the Hungarian Scientific Research Fund (OTKA PD72403 and K101659). The project named ”TÁMOP-4.2.1/B-09/1/KONV2010-0005 – Creating the Center of Excellence at the University of Szeged” is supported by the European Union and co-financed by the European Regional Fund. The authors thank Mrs Irén Forgó (University of Szeged, Hungary) for technical support.

Notes and references Department of Organic Chemistry, University of Szeged, Dóm tér 8., H6720 Szeged, Hungary. Fax: +36-62-544200; Tel: +36-62-544275; Email: [email protected] b Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6., H-6720 Szeged, Hungary. c Faculty of Engineering, University of Szeged, Moszkvai krt. 5-7, H-6725 Szeged, Hungary. d Fumizol Ltd., Moszkvai krt. 5-7, H-6725 Szeged, Hungary a 75

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2. 3.

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Cytotoxic effects were measured in vitro on three human cell lines of gynecological origin (ECACC, Salisbury, UK): HeLa (cervix adenocarcinoma), A2780 (ovarian carcinoma) and MCF7 (breast adenocarcinoma). The cells were cultivated in minimal essential medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 1% non-essential amino acids and an antibiotic-antimycotic mixture. Near-confluent cancer cells were seeded onto a 96-well microplate (5000 cells/well) and, after overnight standing, new medium (200 μL) containing the tested compound was added. The highest concentration was 30 M. After incubation for 72 h at 37 ºC in humidified air containing 5% CO 2, the living cells were assayed by the addition of 5 mg/mL MTT solution (20 μL). MTT was converted by intact mitochondrial reductase and precipitated as blue crystals during a 4-h contact period. The medium was then removed and the precipitated formazan crystals were dissolved in DMSO (100 μL) during a 60-min period of shaking at 25 ºC. Finally, the reduced MTT was assayed at 545 nm, using a microplate reader; wells with untreated cells were utilized as controls.26 Sigmoidal dose  response curves were fitted to the determined data and the IC50 values (the concentration at which the extent of cell proliferation was half that of the untreated control) were calculated by means of GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA). All in vitro experiments were carried out on two microplates with at least five parallel

5. 95

6. 7. 100

8. 105

9. 10. 110

11.

115

12.

120

13.

V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. Int. Ed., 2002, 41, 2596-2599. C. W. Tornøe, C. Christiensen, M. Meldal, J. Org. Chem., 2002, 67, 3057-3064. M. Meldal, C. W. Tornøe, Chem. Rev., 2008, 108, 2952-3015; V. D. Bock, H. Hiemstra, J. H. van Maarseveen, Eur. J. Org. Chem., 2006, 51-68; P. Appukkuttan, W. Dehaen, V. V. Fokin, E. van der Eycken, Org. Lett., 2004, 6, 4223-4225. H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed., 2001, 40, 2004-2021. J. Andersen, S. Bolvig, X. Liang, Synlett, 2005, 2941-2947; A. K. Feldman, B. Colasson, V. V. Fokin, Org. Lett., 2004, 6, 38973899; X. Han, Tetrahedron Lett., 2007, 48, 2845-2849. W. H. Binder, R. Sachsenhofer, Macromol. Rapid Commun., 2007, 28, 15-54; J. F. Lutz, Angew. Chem. Int. Ed., 2007, 46, 1018-1025. P. Wu, M. Malkoch, J. N. Hunt, R. Vestberg, E. Kaltgrad, M. G. Finn, V. V. Fokin, K. B. Sharpless, C. J. Hawker, Chem. Commun., 2005, 5775-5777; D. T. S. Rijkers, G. W. van Esse, R. Merkx, A. J. Brouwer, H. J. F. Jacobs, R. J. Pieters, R. M. J. Liskamp, Chem. Commun., 2005, 4581-4583. D. S. Y. Yeo, R. Srinivasan, G. Y. J. Chen, S. Q. Yao, Chem.-Eur. J., 2004, 10, 4664-4672; M. Köhn, R. Breinbauer, Angew. Chem. Int. Ed., 2004, 43, 3106-3116. A. Brik, C. Y. Wu, C. H. Wong, Org. Biomol. Chem., 2006, 4, 1446-1457. H. C. Kolb, K. B. Sharpless, Drug Discov. Today, 2003, 8, 11281137; W. S. Horne, M. K. Yadav, C. D. Stout, M. R. Ghadiri, J. Am. Chem. Soc., 2004, 126, 15366-15367; N. G. Angelo, P. S. Arora, J. Am. Chem. Soc., 2005, 127, 17134-17135. R. Alvarez, S. Velazquez, A. San-Felix, S. Aquaro, E. De Clercq, C.-F. Perno, A. Karlsson, J. Balzarini, M. J. Carmarasa, J. Med. Chem., 1994, 37, 4185-4194. M. J. Genin, D. A. Allwine, D. J. Anderson, M. R. Barbachyn, D. E. Emmert, S. A. Garmon, D. R. Graber, K. C. Grega, J. B. Hester, D. K. Hutchinson, J. Morris, R. J. Reischer, C. W. Ford, G. E. Zurenko, J. C. Hamel, R. D. Schaadt, D. Stapert, B. H. Yagi, J. Med. Chem., 2000, 43, 953-970. D. R. Buckle, C. J. M. Rockell, H. Smith, B. A. Spicer, J. Med. Chem., 1986, 29, 2262-2267.

14.

15. 5

16. 17. 18. 10

19. 20. 15

Y. S. Sanghvi, B. K. Bhattacharya, G. D. Kini, S. S. Matsumoto, S. B. Larson, W. B. Jolley, R. J. Robins, G. R. Revankar, J. Med. Chem., 1990, 33, 336-344. É. Frank, J. Molnár, I. Zupkó, Z. Kádár, J. Wölfling, Steroids, 2011, doi:10.1016/j.steroids.2011.05.002. G. M. Anstead, K. E. Carlson, J. A. Katzenellenbogen, Steroids, 1997, 62, 268-303. A. O. Mueck, H. Seeger, Steroids, 2010, 75, 625-631. J. Wölfling, E. Mernyák, É. Frank, G. Falkay, Á. Márki, R. Minorics, Gy. Schneider, Steroids, 2003, 68, 277-288. É. Frank, Z. Mucsi, I. Zupkó, B. Réthy, G. Falkay, Gy. Schneider, J. Wölfling, J. Am. Chem. Soc., 2009, 131, 3894-3904. A. G. Fragkaki, Y. S. Angelis, M. Koupparis, A. TsantiliKakoulidou, G. Kokotos, C. Georgakopoulos, Steroids, 2009, 74, 172-197.

21.

20

22. 23. 24. 25.

25

26.

A. H. Banday, S. A. Shameem, B. D. Gupta, H. M. S. Kumar, Steroids, 2010, 75, 801-804; Z. Kádár, D. Kovács, É. Frank, Gy. Schneider, J. Huber, I. Zupkó, T. Bartók, J. Wölfling, Molecules, 2011, 16, 4786-4806. H. Zhang, Z. Qiu, Steroids, 2006, 71, 1088-1090. I. Cerny, V. Pouzar, O. Lapcik, R. Hampl, Collect. Czech. Chem. Commun., 1997, 62, 1931-1939. J. N. Marx, Tetrahedron, 1983, 39, 1529-1531. L. L. Rossi, A. Basu, Bioorg. Med. Chem. Lett., 2005, 15, 35963599; R. E. Looper, D. Pizzirani, S. L. Schreiber, Org. Lett., 2006, 8, 2063-2066; J. Tejler, F. Skogman, H. Leffler, U. J. Nilsson, Carbohydr. Res., 2007, 342, 1869-1875. T. Mosmann, J. Immunol. Methods, 1983, 65, 55-63.