Synthesis and antibacterial activity evaluation of two androgen derivatives

Steroids 93 (2015) 8–15

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Synthesis and antibacterial activity evaluation of two androgen derivatives Figueroa-Valverde Lauro a,⇑, Díaz-Cedillo Francisco b, García-Cervera Elodia a, Pool-Gómez Eduardo a, López-Ramos Maria a, Rosas-Nexticapa Marcela c, Hau-Heredia Lenin a, Sarabia-Alcocer Bety d a Laboratory of Pharmaco-Chemistry, Faculty of Chemical Biological Sciences, University Autonomous of Campeche, Av. Agustín Melgar s/n, Col Buenavista, C.P. 24039 Campeche Cam., Mexico b Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, Prol. Carpio y Plan de Ayala s/n, Col. Santo Tomas, México, D.F. C.P. 11340, Mexico c Facultad de Nutrición, Universidad Veracruzana, Médicos y Odontologos s/n C.P. 91010, Unidad del Bosque, Xalapa, Veracruz, Mexico d Faculty of Medicine, University Autonomous of Campeche, Av. PatricioTrueba de Regil s/n, Col. Lindavista, C.P. 24090 Campeche Cam., Mexico

a r t i c l e

i n f o

Article history: Received 25 April 2014 Received in revised form 18 August 2014 Accepted 7 September 2014 Available online 3 November 2014 Keywords: Androgen derivative Testosterone Boric acid Bacteria

a b s t r a c t In this study two androgen derivatives were synthesized using several strategies; the first stage an azasteroid derivative (3) was developed by the reaction of a testosterone derivative (1) with thiourea (2) in presence of hydrogen chloride. The second step, involves the synthesis of an amino-steroid derivative (4) by the reaction of 1 with 2 using boric acid as catalyst. The third stage was achieved by the preparation of an aminoaza-androgen derivative (6) by the reaction of 3 with ethylenediamine using boric acid as catalyst. In addition, the compound 6 was made reacting with dihydrotestosterone to form a new androgen derivative (7) in presence of boric acid. The following step was achieved by the reaction of 7 with chloroacetyl chloride to synthesize an azetidinone-androgen derivative (8) using triethylamine as catalyst. Additionally, a thiourea-androgen derivative (9) was synthetized by the reaction of 4 with dihydrotestosterone using boric acid as catalyst. Finally, the compound 9 was made reacting with chloroacetyl chloride in presence of triethylamine to synthesize a new azetidinone-androgen derivative (10). On the other hand, antibacterial activity of compounds synthesized was evaluated on Gram negative (Escherichia coli and Vibrio cholerae) and Gram positive (Staphylococos aureus) bacteria. The results indicate that only the compound 3 and 8 decrease the growth bacterial of E. coli and V. cholerae. Nevertheless, growth bacterial of S. aureus was not inhibited by these compounds. These data indicate that antibacterial activity exerted by the compounds 3 and 8 depend of their structure chemical in comparison with the controls and other androgen derivatives that are involved in this study Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Since several years ago, some steroid derivatives have been developed to evaluate its biological activity; for example, a series of antibacterial steroid-derivatives were synthesized by the reaction of steroidal thiosemicarbazones with 2,3-dichloroquinoxalines [1]. Other data showed the reaction of 16-(bis-methylsulfanylmethylene)-3-hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-one with 2-aminophenylamine to form the compound 3b-hydroxy-10 -methyl-16-(benzimidazol-20 -ylidino)androstan-17-one which exert antibacterial activity on bacteria negative [2]. In addition, other reports show the synthesis of steroid-thiosemicarbazone conjugates and their antibacterial activity ⇑ Corresponding author. Tel.: +52 (981) 8119800x3070105. E-mail address: [email protected] (F.-V. Lauro). http://dx.doi.org/10.1016/j.steroids.2014.09.003 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

exerted on Gram positive and Gram negative bacteria [3]. Other study showed the synthesis of an antibacterial steroid-derivative (cholest-5-en-3-one semicarbazone) by the reaction of cholest-5en-3-one with semicarbazone hydrochloride [4]. Additionally, other report indicates the preparation of antibacterial cationicsteroids by conjugating tripeptides with derivatives of cholic acid [5]. Recently, was synthesized an antibacterial-steroid derivative (pregnenolone–vitamin B1 conjugate) by the reaction of hemisuccinate of pregnenolone and vitamin-B1 [6]. Also, some x-pyridinium alkylethers-steroid derivatives were synthesized as antimicrobial agents by the reaction of 3-hydroxy-estra-1,3,5(10)-triene-17-one and 1-hydroxy-4-methyl-estra-1,3,5(10)-triene-17-one with x,x0 dibromoalkanes/pyridine [7]. Other data indicate the synthesis of an antibacterial steroid (dihydrotestosterone–ciprofloxacin conjugate) via the reaction of a ciprofloxacin derivative with dihydrotestosterone [8]. All these experimental results show several

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procedures which are available for synthesis of several antibacterial steroid-derivatives; nevertheless, expensive reagents and special conditions are required. Therefore, in this study two androgen derivatives were synthesized using several strategies. It is noteworthy that antibacterial activity of these androgen derivatives was evaluated in vitro on a bacteria model.

1-[(2-amino-ethylamino)phenyl-methyl]-naphta-len-2-ol with androsterone using as catalyst boric acid [16]. Therefore, in this study the synthesis of the compound 4 was developed by the reaction of 17b-[(tert-butyldimethylsilyl)oxy]androst-4-en-3-one with thiourea to form the compound 4 using boric acid as catalyst. The results indicate that 1H NMR spectrum of 4 showed signals at 0.06–0.82 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 0.90–1.02, 1.06–1.10 and 1.30–6.00 ppm for steroid moiety; at 1.04 and 1.22 ppm for methyl groups bound to steroid nucleus; at 6.90 ppm for both amino groups. The 13C NMR spectra displays chemical shifts at 4.50, 17.98 and 25.44 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 11.22 and 17.69 ppm for methyl groups bound to steroid nucleus; at 20.90–23.50 and 30.40–162.26 ppm for steroid moiety; at 185.26 ppm for thiourea group. Finally, the presence of 4 was further confirmed from mass spectrum which showed a molecular ion at 460.26 m/z. The third step was achieved by the reaction of 3 with ethylenediamine to form the compound 6 (N-17-[3-(tert-butyl-dimethylsilanyloxyl)-5b,3a-dimethyl-octahydro-indeno [4,5-d]10,12-diazatricyclo[7.3.1.01,68]tridec-7-en-9-yli-dene]-ethane-1,2-diamine) using boric acid (Scheme 2). It is noteworthy that the fragment bound to both amino groups of compound 6, has a free amino group which can react with other type of compounds with specific functional groups. Also, this fragment can serve as a spacer arm with other molecules to decrease some steric hindrance when their pharmacological activity will be assessed in any biological model. The results indicate that 1H NMR spectrum of 6 showed signals at 0.06 and 0.87 ppm for methyl groups bound to steroid nucleus; at 0.68 and 0.82 ppm for methyl groups bound to steroid nucleus; at 0.94–2.36, 3.50 and 4.65 ppm for steroid moiety; at 3.18 and 3.70 ppm for both methylene groups bound to both amine groups; at 3.90 ppm for both amino groups. The 13C NMR spectra displays chemical shifts at 4.50, 17.96 and 25.46 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 11.30 and 13.58; at 21.50, 27.90–40.10, 43.32–51.90 and 61.16–130.16 ppm for steroid moiety; at 41.30 and 52.48 ppm for methylene groups bound to both amino groups; at 156.60 ppm for imino group.

2. Results and discussion In this study several straightforward routes are reported for synthesis of two androgen derivatives using strategies different; the first stage was achieved by the synthesis of 3-(terbuthyldimethyl-silanyloxyl)-5b,3a-dimethyl-octahydro-indeno[4,5-d]10, 12-diazatri-cycle[7.3.1.01,611]tridecan-9-one (3). It is important to mention that there are many procedures for preparation of several aza-steroid derivatives; nevertheless, despite its wide scope, have some drawbacks; for example, several agents used have limited stability and their preparation requires special conditions [9–13]. Therefore 3 was synthesized by the reaction of 17b-[(tert-butyldimethylsilyl)oxy]androst-4-en-3-one with thiourea in presence of hydrogen chloride (Scheme 1). The 1H NMR spectrum of 3 shows signals at 0.06–0.84 ppm for methyl groups involved the tert-butyldimethylsilane fragment; at 0.67 and 0.82 ppm for methyl groups bound to steroid nucleus; at 0.90–5.20 ppm for steroid moiety; at 9.80 ppm for both amino groups. The 13C NMR spectra displays chemical shifts at 4.50, 17.98 and 25.44 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 11.18 and 13.60 ppm for methyl groups bound to steroid nucleus; at 21.44– 23.50 and 27.90–134.98 ppm for steroid moiety; at 156.74 ppm for carbon bound to both amino groups. Finally, the presence of 3 was further confirmed from mass spectrum which showed a molecular ion at 460.20 m/z. The second step was achieved by the synthesis of an imine group involved in the compound 4 (Scheme 1). It is important to mention, that there are several procedures for the synthesis of imines which are described in the literature [14,15]. For example, the synthesis of imine derivatives by the reaction of the compound

CH 3 CH 3

CH 3

H C CH

3 3

H C 3

Si

O

CH

CH

O

3

CH 3

Si

CH 3

CH 3

CH

3

3

S

+

CH 3

i

H2N

CH 3

NH2 2 O

HN 1

NH

3

ii S CH 3

CH 3

H C 3

O

CH 3

Si

CH 3

CH 3

CH 3

S H2N

N 4

Scheme 1. Synthesis of 3 by the reaction of 17b-[(tert-butyldimethylsilyl)oxy]androst-4-en-3-one (1) with thiourea (2) in 6 presence of hydrogen chloride (i). In addition, the compound 4 was synthetized by the reaction of 1 with 2 using boric acid as catalyst (ii).

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F.-V. Lauro et al. / Steroids 93 (2015) 8–15 CH 3 CH 3

CH 3

H C

CH

3

3

H C 3

O

CH 3

Si

Si

CH 3

O

CH CH

3

CH 3

CH

3

3

CH 3 CH 3

+

iii

H2N NH2

HN

NH

3

5

6 HN

NH

S

N NH2

CH 3

CH 3

H C 3

iv

O

CH 3

Si

CH 3

CH 3

CH 3

HO H C 3

HN

NH

7

H C 3

N

N

Scheme 2. Synthesis of the compound 6 by the reaction of 3 with ethylenediamine (5) using boric acid as catalyst (iii). In addition, the compound 7 was synthetized by the reaction of dihydrotestosterone (iv) with the compound 6 using boric acid as catalyst.

Finally, the presence of 6 was further confirmed from mass spectrum which showed a molecular ion at 486.32 m/z. The fourth stage was achieved by the reaction of 6 with dihydrotestosterone to form the compound 7 (10,13-dimethyl-3-[2[3 (tert-butyl-dimethyl-silanyloxyl)-5b,3a-dimethylocta-hydro-indeno [4,5-d]10,12-diaza-tricyclo[7.3.1.01,6]tridec-7-en-9-ylideneamino]ethyl-imino]-hexadecahydro-cyclopenta[a]phenanthren-17-ol) using boric acid (Scheme 2). The results indicate that 1H NMR spectrum of 7 showed signals at 0.06 and 0.86 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 0.67–0.85 and 0.90 ppm for methyl groups bound to steroid nucleus; at 0.95–2.34, 3.53, 3.64 and 4.60 ppm for steroid moiety; at 3.50 and 3.60 ppm for methylene groups bound to both imino groups; at 4.42 ppm for both hydroxyl and amino groups. The 13C NMR spectra displays chemical shifts at 4.50 and 25.48 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 11.08 and 11.36–13.60 ppm for methyl groups bound to steroid nucleus; at 20.76–23.96, 27.90–48.80, 51.00–51.90 and 53.20–153.32 ppm for steroid moiety; at 50.00 and 52.00 ppm for methylene groups bound to both imino groups; at 156.60 for imino group bound to both amino groups. Finally, the presence of 7 was further confirmed from mass spectrum which showed a molecular ion at 758.52 m/z. On the other hand, the fifth stage was achieved by the reaction of 7 with chloroacetyl chloride to form chloroazetidinone groups involved in the compound 8 using triethylamine as catalyst (Scheme 3). It is important to mention that this method has been previously reported for other type of compounds with imino groups involved in its structure chemical, which react with chloroacetyl chloride to form chloroazetidinones groups [17]. The 1H NMR spectrum of 8 showed signals at 0.06 and 0.87 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 0.58–0.66, 0.78 and 0.86 ppm for methyl groups bound to steroid nucleus; at 0.72, 0.83, 0.90–3.54, 4.20 and 4.82 ppm for steroid moiety; at 4.08 ppm for methylene group of chloroacetic acid; at 4.48 and 5.10–5.40 for both azetidinone rings; at 5.64 ppm for

methylene group bound to both amino groups, at 8.46 ppm for both amino groups. The 13C NMR spectra displays chemical shifts at 4.50 and 25.50 ppm for methyl groups involved in the tertbutyldimethylsilane fragment; at 11.30–14.30 ppm for methyl groups bound to steroid nucleus; at 17.96 for carbon bound to methyl groups involved in the tert-butyldimethylsilane fragment; at 20.56–24.50, 27.90–39.27, 43.32–52.54, 60.60, 66.50 and 81.60–137.12 ppm for steroid moiety; at 57.70–59.12, 64.30 and 69.74 ppm for both azetidinone rings; at 78.90 ppm for methylene bound to both amino groups; at 167.56–171.68 ppm for ketone groups; at 168.00 ppm for methylene group of chloroacetic acid. Finally, the presence of 8 was further confirmed from mass spectrum which showed a molecular ion at 986.50 m/z. The eighth stage (Scheme 4) was achieved by the reaction of 4 with dihydrotestosterone using boric acid as catalyst to form 1[17-(tert-butyldimethyl-silanyoxy)-10,13-dimethyl 1,2,6,7,8,9,10, 11,12,13,14,15,16,17-11-tetradecahydro-cyclopenta[a]phenanthren-3-ylide]-3-(17-hydroxy-10,13-dimethyl-hexadecahydrocyclopenta[a]phenanthren-3-ylidene)thio-urea (9). The 1H NMR spectrum of 9 showed signals at 0.06 and 0.86 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 0.78, 0.90, 1.04 and 1.22 ppm for methyl groups bound to steroid nucleus; 0.94–1.02, 1.06–1.10 and 1.24–6.20 ppm for steroids moiety. The 13C NMR spectra displays chemical shifts at 4.50 and 17.98 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 11.00, 11.30, 11.48, 17.70 and 25.38 ppm for methyl groups bound to steroid nucleus; at 20.76–23.50 and 27.30–165.86 ppm for steroid moiety; at 193.44 ppm for carbon of thiourea group. In addition, the presence of 9 was further confirmed from mass spectrum which showed a molecular ion at 732.50 m/z. Finally the compound 10 was synthesized by the reaction of dihydrotestosterone with 9 with chloroacetyl chloride in presence of triethylamine (Scheme 4). The 1H NMR spectrum of 10 showed signals at 0.06 and 0.86 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 0.76, 0.80, 1.02 and

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F.-V. Lauro et al. / Steroids 93 (2015) 8–15 CH 3

CH 3

H C 3

O

CH

Si

CH 3

CH

3

3

CH 3

HO H C 3

HN

NH

7

H C 3

N

N

CH 3

CH 3

H C 3

iv

O

CH 3

Si

CH 3

CH 3

CH 3

Cl

HN

NH

O N

N

O Cl

CH 3

8

CH 3

O Cl

O

Scheme 3. Synthesis of the compound 8 by the reaction of 7 with chloroacetyl chloride in presence of triethylamine (iv).

1.20 ppm for methyl groups bound to steroid nucleus; at 0.78, 0.88–0.94, 1.25–3.60 and 4.80–5.50 ppm for steroid nucleus; at 4.10 ppm for methylene group of chloroacetic acid; at 4.70– 4.78 ppm for both azetidinone rings. The 13C NMR spectra displays chemical shifts at 4.50 and 25.50 ppm for methyl groups involved in the tert-butyldimethylsilane fragment; at 11.30–12.26 and 19.40 ppm for methyl groups bound to steroid nucleus; at 17.96 ppm for carbon bound to methyl groups involved in the tert-butyldimethylsilane fragment; at 20.80–25.20, 27.70–36.93, 42.50–52.44 and 65.10–142.02 ppm for steroid moiety; at 64.10– 64.40 ppm for both azetidinone rings; at 40.80 ppm for methylene group of chloroacetic acid; at 168.00 ppm for ester group; at 163.38–164.12 ppm for ketone groups; at 194.28 ppm for carbon of thiourea group. Finally, the presence of 10 was further confirmed from mass spectrum which showed a molecular ion at 960.40 m/z. 2.1. Biological activity In order to evaluate the possibility of that compound synthesized may have biological; in this study its antibacterial activity (minimal inhibitory concentration, MC) on Gram negative (Escherichia coli and V. cholerae in) and Gram positive (S. aureus) bacteria was evaluated. The results showed in the Schemes 5 and 6 indicate that only the compounds 3 (MIC = 2.05  103 mmol) and 8 (MIC = 1.01  103 mmol) have antibacterial activity on E. coli and

V. cholerae in a dose manner dependent; nevertheless, this effect was different in comparison with the controls (cefotaxime, MIC = 2.62  104 mmol; gentamicin, MIC = 1.29  104 mmol; and ciprofloxacin, MIC = 1.88  104 mmol). Other experimental data obtained (Scheme 6) showed that bacterial growth of Vibrio cholera was inhibited by the compounds 3 (MIC = 2.05  103 mmol) and 8 (MIC = 1.01  103 mmol), cefotaxime (MIC = 2.62  104 mmol), gentamicin (MIC = 2.62  104 mmol) and ciprofloxacin (MIC = 3.77  104 mmol). It is important to mention that compounds 3 and 8 have not antibacterial activity on S. aureus. All these data indicate that antibacterial activity exerted by the compounds 3 and 8 on Gram negative bacteria depend of their structure chemical in comparison with the controls and other androgen derivatives that are involved in this study. This phenomenon may involve the interaction of compounds 3 and 8 with some components of the bacterial cell, which may result in disturbance of bacterial growth and induce cell death, through perturbation of membrane bacterial. In this sense, the intramolecular interaction of compounds 3 and 8 could be via divalent cations (Mg2+ and Ca2+) involved in the membrane, consequently resulting a substantial increase the permeability of the outer membrane of Gram negative as happening with other type of antibacterial agents [5,20]. In conclusion, in this study new androgens were synthesized using several strategies, which provide some advantages such as simple procedure and ease of workup in comparison with other

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F.-V. Lauro et al. / Steroids 93 (2015) 8–15 CH 3

CH 3

H C 3

O

CH

Si

CH 3

3

CH 3

CH 3

S H2N

4

N

CH 3

CH 3

H C 3

v

Si

O

CH 3

CH 3

CH 3

HO

CH 3

H C 3

S H C 3

N

O Cl

9

N

H C 3

CH 3

O

vi

H C 3

CH 3

Si

H C 3

O

CH 3

H C 3

H C 3

H C 3

S N N

Cl O

O

10 Cl

Scheme 4. Synthesis of the compound 9 by the reaction of 4 with dihydrotestosterone using boric acid as catalyst (v). In addition, the compound 10 was synthetized by the reaction of 9 with chloroacetyl chloride in presence of triethylamine (vi).

0.0025 0.0025

0.0020 0.0015

MIC [mmol]

MIC [mmol]

0.0020

0.0010

0.0015

0.0010

0.0005 0.0005

Scheme 5. Antibacterial activity induced by two androgen derivatives (compound 3 and 8) and controls (cefotaxime, CEFOT; gentamicin, GENT; and ciprofloxacin, CIPROF) on E. coli. Experimental data showed that E. coli was susceptibly to CEFOT (MIC = 2.62  104 mmol), GENT (MIC = 1.29  104 mmol) and CIPROF (MIC = 1.88  104 mmol). In addition, in presence of the compounds 3 (2.05  103 mmol) and 8 (1.01  103 mmol) the bacterial growth of this microorganism was inhibit. Each bar represents the mean ± S.E. of 9 experiments. MIC = Minimal inhibitory concentration.

M O C

M O

PO

PO

U

U

N

N

D

D

8

3

F O R IP C

G

EN

T

T O EF

C

DRUGS

0.0000

C

PO U N D

8

3 C O M

C O M PO U N D

C IP R O F

G EN T

C EF O T

0.0000

DRUGS Scheme 6. Effect exerted by two androgen derivatives (compound 3, and 8) and controls (cefotaxime, CEFOT; gentamicin, GENT; and ciprofloxacin, CIPROF) on V. cholerae. The results showed that bacterial growth of V. cholera in presence of CEFOT (MIC = 2.62  104 mmol), GENT (MIC = 2.62  104 mmol), CIPROF (3.77  103 mmol), compounds 3 (2.05  103 mmol) and compound 8 (1.01  103 mmol) was inhibited. Each bar represents the mean ± S.E. of 9 experiments. MIC = Minimal inhibitory concentration.

F.-V. Lauro et al. / Steroids 93 (2015) 8–15

techniques involved in the synthesis of other steroid derivatives. In addition, two of the androgen derivatives exert antibacterial activity on E. coli and V. cholerae.

3. Experimental The compound 1 (17b-[(t-butyldimethylsilyl)oxy]androst-4-en3-one) was synthesized with previously methods reported [18,19]. The other compounds evaluated in this study were purchased from Sigma–Aldrich Co., Ltd. The melting point for the androgen derivatives was determined on an Electrothermal (900 model). Infrared spectra (IR) were recorded using KBr pellets on a Perkin Elmer Lambda 40 spectrometer. 1H and 13C NMR (nuclear magnetic resonance) spectra were recorded on a Varian VXR-300/5 FT NMR spectrometer at 300 and 75.4 MHz in CDCl3 using TMS as internal standard. EIMS (electron impact mass spectroscopy) spectra were obtained with a Finnigan Trace Gas Chromatography Polaris Q Spectrometer. Elementary analysis data were acquired from a Perkin Elmer Ser.II CHNS/0 2400 elemental analyzer.

3.1. 3-(terbuthyl-dimethyl-silanyloxyl)-5b,3a-Dimethyl-octahydroindeno[4,5-d]10,12-diaza-tricyclo[7.3.1.01,6]tridecan-9-thione (3) A solution of 1 (100 mg, 0.24 mmol), thiourea (38 mg, 0.5 mmol), hydrogen chloride (0.5 ml) and 10 ml of methanol was stirred for 72 h to room temperature. The reaction mixture was evaporated to dryness under reduced pressure. After the mixture was diluted with water and extracted with chloroform. The organic phase was evaporated to dryness under reduced pressure, the residue was purified by crystallization from methanol:water (3:1) yielding 48% of product, m.p. 78–79 °C; IR (tmax, cm1): 3450, 1092; 1H NMR (300 MHz, CDCl3) dH: 0.06 (s, 6H), 0.66 (s, 3H), 0.82 (s, 3H), 0.84 (s, 9H), 0.90–1.36 (m, 5H), 1.40–1.66 (m, 6H), 1.78–1.94 (m, 4H), 2.20–2.32 (m, 2H), 2.50–2.72 (m, 2H), 3.50–5.20 (m, 2H), 9.80 (broad, 2H) ppm. 13C NMR (75.4 MHz, CDCl3) dC: 4.50, 11.18, 13.60, 17.98, 21.44, 23.50, 25.44, 27.90, 29.50, 31.05, 32.99, 34.70, 36.62, 38.36, 38.40, 39.80, 43.32, 48.80,51.90, 58.15, 81.44, 108.70, 134.98, 156.74 ppm. EI-MS m/z: 460.20 (M+8). Anal. Calcd. for C26H44N2OSSi: C, 67.77; H, 9.62; N, 6.08; O, 3.47; S, 6.96; Si, 6.10. Found: C, 67.72; H, 9.60, N, 6.04

3.2. [17-(tert-Butyl-dimethyl-silanyloxy)-10,13-dimethyl 1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydrocyclopenta[a]phenanthren-3-ylidene]-thiourea (4) A solution of 1 (100 mg, 0.24 mmol), thiourea (38 mg, 0.5 mmol), boric acid (90 mg, 1.45 mmol) and 10 ml of methanol was stirred for 72 h to room temperature. The reaction mixture was evaporated to dryness under reduced pressure. After the mixture was diluted with water and extracted with chloroform. The organic phase was evaporated to dryness under reduced pressure, the residue was purified by crystallization from methanol:water (3:1) yielding 85% of product, m.p. 117–119 °C; IR (tmax, cm1): 3380, 3322, 1090; 1HNMR (300 MHz, CDCl3) dH: 0.06 (s, 6H), 0.82 (s, 9H), 0.90–1.02 (m, 2H), 1.04 (s, 3H), 1.06–1.10 (m, 2H), 1.22 (s, 3H), 1.30–1.42 (m, 2H), 1.56–1.64(m, 5H), 1.76–1.92 (m, 4H), 2.20–2.40 (m, 4H), 3.50 (m,1H), 6.00 (d, 1H, 1.84 J = 6.0 Hz) 6.90 (broad, 2H) ppm. 13CNMR (75.4 MHz, CDCl3) dC: 4.50, 11.22, 17.69, 17.98, 20.90, 22.66, 23.50, 25.44, 30.40, 31.05, 31.10, 31.71, 35.26, 35.35, 37.08, 43.32, 50.50, 52.29, 81.68, 122.50, 155.36, 165.26, 185.26 ppm. EI-MS m/z: 460.26 (M+8). Anal. Calcd. for C26H44N2OSSi: C, 67.77; H, 9.62; N, 6.08; O, 3.47; S, 6.96; Si, 6.10. Found: C, 67.73; H, 9.60; N, 6.04.

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3.3. N-17-[3-(tert-butyl-dimethyl-silanyloxyl)-5b,3a-dimethyloctahydro-indeno[4,5-d]10,12-diaza-tricyclo[7.3.1.01,6]tridec-7-en9-ylidene]-ethane-1,2-diamine (6) A solution of 3 (100 mg, 0.22 mmol), ethylenediamine (100 ll, 1.50 mmol) and boric acid (90 mg, 1.45 mmol) in methanol (10 ml) was stirred for 72 h to room temperature. The reaction mixture was evaporated to a smaller volume. After the mixture was diluted with water and extracted with chloroform. The organic phase was evaporated to dryness under reduced pressure, the residue was purified by crystallization from methanol:water (3:1) yielding 52% of product, m.p. 124 °C; IR (tmax, cm1): 3446, 3382, 3338, 1092; 1H NMR (300 MHz, CDCl3) dH: 0.06 (s, 6H), 0.68 (s, 3H), 0.82 (s, 3H), 0.87 (s, 9H), 0.94–1.06 (m, 2H), 1.30–1 1.52 (m, 7H), 1.60–1.86 (m, 6H), 1.96 (m, 1H), 2.12–2.36 (m, 4H), 3.18 (t, 2H, J = 6.44 Hz), 3.50 (m, 1H), 3.70 (t, 2H, J = 6.44 Hz), 3.90 (broad, 4H), 4.62 (m, 1H) ppm. 13CNMR (75.4 MHz, CDCl3) dC: 4.50, 11.30, 13.58, 17.96, 21.50, 23.46, 25.46, 27.90, 29.79, 31.00, 34.68, 35.14, 36.62, 38.37, 40.10, 41.30, 43.32, 48.74, 51.90, 52.48, 61.16, 81.44, 108.08, 130.16, 156.60 ppm. EI-MS m/z: 486.32 (M+12). Anal. Calcd. for C28H50N4OSi: C, 69.08; H, 10.35; N, 11.51; O, 3.29; Si, 5.77. Found: C, 69.04; H, 10.32, N, 11.50. 3.4. 10,13-Dimethyl-3-[2[3(tert-butyl-dimethyl-silanyloxyl)-5b,3adimethyloctahydro-indeno[4,5-d]10,12-diaza-tricyclo[7.3.1.01,6]tridec-7-en-9-ylideneamino]-ethylimino]-hexa decahydrocyclopenta[a]phenanthren-17-ol (7) A solution of 6 (100 mg, 0.20 mmol), dihydrotestosterone (58 mg, 0.20 mmol) and boric acid (90 mg, 1.45 mmol) in 10 ml of methanol was stirred for 72 h to room temperature. The reaction mixture was evaporated to a smaller volume. After the mixture was diluted with water and extracted with chloroform. The organic phase was evaporated to dryness under reduced pressure, the residue was purified by crystallization from methanol:water (3:1) yielding 66% of product, m.p. 160–162 °C; IR (tmax, cm1): 3442, 3336, 1094; 1H NMR(300 MHz, CDCl3) dH: 0.06 (s, 6H), 0.67 (s, 3H), 0.85 (s, 3H), 0.86 (s, 9H), 0.90 (s, 3H), 0.95–1.08 (m, 6H), 1.16–1.23 (m, 2H), 1.32–1.38 (m 7H), 1.40–1.52 (m, 6H), 1.60– 1.70 (m, 8H), 1.72–1.96 (m, 5H), 2.10–2.34 (m, 7H), 3.50 (t, 2H, J = 6.44 Hz), 3.53 (m, 1H), 3.60 (t, 2H, J = 6.44 Hz), 3.64 (m, 1H), 4.42 (broad, 3H), 4.60 (m, 1H) ppm. 13C NMR (75.4 MHz, CDCl3) dC: 4.50, 11.08, 11.36, 11.40, 13.60, 18.00, 20.76, 21.51, 23.40, 23.50, 23.96, 25.48, 27.90, 28.67, 29.79, 30.55, 31.05, 31.40, 34.76, 35.14, 35.50, 36.25, 36.69, 36.70, 36.80, 37.71, 38.37, 40.13, 43.00, 43.32, 47.89, 48.80, 50.00, 51.00, 51.90, 52.00, 53.20, 61.24, 81.67, 81.81, 108.10, 130.47, 153.32, 156.60 ppm. EI-MS m/z: 758.52 (M+10). Anal. Calcd. for C47H78N4O2Si: C, 74.35; H, 10.36; N, 7.38; O, 4.21; Si, 3.70. Found: C, 74.32; H, 10.34, N, 7.35. ´ S,170 S)-1-((100 R,130 S,170 S)-170 -((tert-Butyldimethylsilyl)3.5. (100 S,13 oxy)-3-chloro-100 ,130 -dimethyl-4-oxo 10 ,20 ,60 ,70 ,80 ,90 ,100 ,110 ,120 ,130 ,140 , 150 ,160 ,170 -tetradecahydro spiro[azetidine-2,30 -cyclopenta[a]phenanthrene]-1-carbonothioyl)-3-chloro-100 ,130 -dimethyl-4-oxohexadecahydrospiro[azetidine-2,30 -cyclopenta[a]phenanthren]-170 -yl 2chloroacetate (8) A solution of 7 (100 mg, 0.13 mmol), triethylamine (70 lL, 0.50 mmol) and chloroacetyl chloride (70 lL, 0.88 mmol) in 10 mL of methanol was stirred for 72 h to room temperature. The reaction mixture was evaporated to a smaller volume. After the mixture was diluted with water and extracted with chloroform. The organic phase was evaporated to dryness under reduced pressure, the residue was purified by crystallization from methanol:water (3:1) yielding 40% of product, m.p. 148–150 °C; IR

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F.-V. Lauro et al. / Steroids 93 (2015) 8–15

(tmax, cm1): 3446, 1716, 1180; 1H NMR (300 MHz, CDCl3) dH: 0.06 (s, 6H), 0.58 (s, 3H), 0.66 (s, 3H), 72 (m, 1H), 0.78 (s, 3H), 0.83 (m, 1H), 0.86 (s, 3H), 0.87 (s, 9H), 0.90–0.96 (m, 2H), 1.04–1.38 (m, 10H),1.40–1.46 (m, 5H), 1.52–1.68 (m, 10H), 1.76–1.88 (m, 5H), 2.00–2.38 (m, 7H), 3.50–3.54(m, 2H), 4.08 (m, 2H), 4.20 (m, 1H), 4.48 (m, 1H), 4.82 (m, 1H), 5.10–5.40 (m, 3H), 5.64 (m, 1H), 8.46 (broad, 2H) ppm. 13C NMR (75.4 MHz, CDCl3) dC: 4.50, 11.30, 12.13, 13.60, 14.30, 17.96, 20.56, 21.50, 23.46, 24.50, 25.50, 27.90, 28.25, 28.52, 28.70, 30.02, 30.34, 31.05, 33.26, 34.66, 35.10, 35.30, 35.60, 36.20, 36.60, 38.30, 38.50, 39.27, 40.80, 43.32, 43.50, 44.20, 48.82, 50.90, 51.92, 52.24, 57.70, 59.12, 60.60, 64.30, 66.50, 69.74, 78.90, 78.96, 81.60, 84.60, 105.00, 137.12, 167.56, 168.00, 171.68, ppm. EI-MS m/z: 986.50 (M+12). Anal. Calcd. for C53H81Cl3N4O5Si: C, 64.39; H, 8.26; Cl, 10.76; N, 5.67; O, 8.09; Si, 2.84. Found: C, 64.36; H, 8.24; N, 5.64. 3.6. 1-[17-(tert-Butyl-dimethyl-silanyoxy)-10,13-dimethyl-1,2,6,7,8,9, 10,11,12,13,14,15,16,17-tetradecahydro-cyclopenta[a]phenanthren3-ylidene]-3-(17-hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-3-ylidene)thiourea (9) A solution of 4 (100 mg, 0.20 mmol), dihydrotestosterone (58 mg, 0.20 mmol) and boric acid (90 mg, 1.45 mmol) in 10 ml of methanol was stirred for 72 h to room temperature. The reaction mixture was evaporated to a smaller volume. After the mixture was diluted with water and extracted with chloroform. The organic phase was evaporated to dryness under reduced pressure, the residue was purified by crystallization from methanol:water (3:1) yielding 48% of product, m.p. 112–114 °C; IR (tmax, cm1): 3402, 3336, 1088; 1H NMR (300 MHz, CDCl3) dH: 0.06 (s, 6H), 0.78 (s, 3H), 0.86 (s, 9H), 0.90 (s, 3H), 0.94–1.02 (m, 4H), 1.04 (s, 3H), 1.06–1.10 (m, 4H), 1.22 (s; 3H), 1.24–1.36 (m, 5H), 1.40 1.58 (m, 5H), 1.60–1.66 (m, 7H), 1.70–1.88 (m, 5H), 1.94–1.98 (m, 4H), 2.12–2.38 (m, 7H), 3.52–3.60 (m, 2H), 6.00 (d, 1H, J = 0.70 Hz), 6.20 (broad, 1H) ppm. 13C NMR (75.4 MHz, CDCl3) dC: 4.50, 11.00, 11.30, 11.48, 17.70, 18.00, 20.76, 20.94, 22.26, 22.50, 23.38, 23.50, 25.38, 27.30, 29.66, 30.50, 30.60, 30.82, 31.00, 31.30, 31.68, 31.90, 35.20, 35.30, 35.50, 36.22, 36.76, 37.02, 42.88, 42.98, 43.30, 50.48, 51.04, 52.20, 53.14, 81.60, 81.78, 122.36, 155.48, 163.38, 165.86, 193.44 ppm. EI-MS m/z: 732.50 (M+12). Anal. Calcd. for C45H72N2O2SSi: C, 73.71; H, 9.90; N, 3.82; 1 O, 4.36; S, 4.37; Si, 3.83. Found: C, 73.70; H, 9.88; N, 3.80. 3.7. (100 S,130 S,170 S)-1-((100 R,130 S,170 S)-170 -((tert-Butyldimethlsilyl)oxy)-3-chloro-100 ,130 -dimethyl-4-oxo 10 ,20 ,60 ,70 ,80 ,90 ,100 ,110 ,120 ,130 ,140 , 150 ,160 ,170 ,tetradecahydrospiro [azetidine-2,30 -cyclopenta[a]phenanthrene]-1-carbonothioyl)-3-chloro-100 ,130 -dime-thyl-4-oxohexadecahydrospiro[a]phenanthren]-170 -yl 2-chloroacetate (10) A solution of 9 (100 mg, 0.14 mmol), dihydrotestosterone (40 mg, 0.14 mmol) and boric acid (90 mg, 1.45 mmol) in 10 ml of methanol was stirred for 72 h to room temperature. The reaction mixture was evaporated to a smaller volume. After the mixture was diluted with water and extracted with chloroform. The organic phase was evaporated to dryness under reduced pressure, the residue was purified by crystallization from methanol:water (3:1) yielding 76% of product, m.p. 120–122 °C; IR (tmax, cm1): 1712, 1182, 1096; 1H NMR (300 MHz, CDCl3) dH: 0.06 (s, 3H), 0.76 (m, 3H), 0.78 (m, 1H), 0.80 (s, 3H), 0.86 (s, 9H), 0.88–0.94 (m, 4H), 1.02 (s, 3H), 1.06–1.15 (m, 3H), 1.20 (s, 3H), 1.25–1.38 (m, 4H), 1.40–1.50 (m, 5H), 1.52–1.62 (m, 6H), 1.74–2.20 (m, 9H), 2.22– 2.30 (m, 4H), 2.40–3.60 (m, 6H), 4.10 (t, 2H, J = 14.70 Hz), 4.70– 4.78 (m, 2H), 4.80–5.50 (m, 2H) ppm.13C NMR (75.4 MHz, CDCl3) dC: 4.50, 11.30, 12.00, 12.26, 17.96, 19.40, 20.80,21.00, 23.46, 23.70, 25.20, 25.50, 27.70, 28.50, 28.87, 30.52, 31.10, 31.75, 32.40, 32.80, 33.90, 34.00, 34.60, 36.30, 36.36, 36.50, 36.66,

36.93, 40.80, 42.50, 42.60, 43.30, 49.60, 51.45, 52.29, 52.44, 64.10, 73.30, 81.68, 81.74, 120.70, 142.02, 163.38, 164.12, 168.00, 194.28 ppm. EI-MS m/z: 960.40 (M+10). Anal. Calcd. for C51H75Cl3N2O5SSi: C, 63.63; H, 7.85; Cl, 11.05; N, 2.91; O, 8.31; S, 3.33; Si, 2.95. Found: C, 63.60; H, 7.82; N, 2.88. 4. Antimicrobial activity The evaluation of antimicrobial effect of the different compounds on the bacterial species was made by a previously method described [8]. The bacterial species were incubated on McConkey (E. coli and Vibrio cholarae) and Staphylococcus 110 (S. aureus) agars for 24 h at 37 °C. After such time, it was be determined whether growth had taken place or not. In addition, a series of tubes were prepared, the first of which contained 2 ml of culture medium (tripticase soye) at double concentration and the remainder (11 tubes), contained the same quantity of medium at single concentrations. From the first tube (double concentration) an aliquot of 2 ml of the studied compound (1 mg/ml) was added and stirred, from this tube an aliquot of 2 ml was taken and added to the following tube (simple concentration) and the process was successively repeated until the last 2 ml of dissolution had been used up. After this process, each tube was inoculated with 0.1 ml of the bacterial suspension, whose concentration corresponded to Mc-Farland scale (9  108 cells/ml) and all the tubes were incubated at 37 °C for 24 h. Subsequently, a loop was taken from each of them and inoculated into the appropriate cultures for different bacterial organisms, and were incubated for 24 h at 37 °C. After such time, the minimum inhibitory concentration (MIC) was evaluated to consider the antimicrobial effect of the different compounds. In order to discard the effect of methanol (solvent) on the bacterial species studied, a series of the same number of tubes was prepared in parallel, to which 2 ml of methanol at 60% was added to the first and corresponding successive dilutions were added in the same way as before. In addition a control series was also performed using distilled water to pH 7.0. Acknowledgement We are grateful to Gloria Velazquez-Zea for financial support. References [1] Khan S, Saleem K, Khan Z. Synthesis, characterization and in vitro antibacterial activity of new steroidal thiazoloquinoxalines. Eur J Med Chem 2007;42:103–8. [2] Abdelhalim M, El-Saidi M, Rabie S, Elmegeed G. Synthesis of novel steroidal heterocyclic derivatives as antibacterial agents. Steroids 2007;72:459–65. [3] Khan S, Yusuf M. Synthesis, spectral studies and in vitro antibacterial activity of steroidal thiosemicarbazone and their palladium (Pd (II)) complexes. Eur J Med Chem 2009;44:2270–4. [4] Khan S. Synthesis, characterization and in vitro antibacterial activity of new steroidal 5-en-3-oxazolo and thiazoloquinoxaline. Eur J Med Chem 2008;43:2040–4. [5] Ding B, Taotofa U, Orsak T, Chadwell T, Savage P. Synthesis and characterization of peptide-cationic steroid antibiotic conjugates. Org Lett 2004;6:3433–6. [6] Figueroa-Valverde L, Díaz-Cedillo F, Ceballos-Reyes G, López-Ramos M. Synthesis and antibacterial activity of pregnenolone-vitamin B1 conjugate. J Mex Chem Soc 2008;52:130–5. [7] Lange C, Holzhey N, Schönecker B, Beckert R, Möllmann U, Dahse H. xPyridiniumalkylethers of steroidal phenols: new compounds with potent antibacterial and antiproliferative activities. Bioorg Med Chem 2004;12:3357–62. [8] Figueroa-Valverde L, Díaz-Cedillo F, Camacho-Luis A, López Ramos M, Garcia Cervera M. Synthesis of a dihydrotestosterone–ciprofloxacin conjugate: relationship between descriptors log P, p, Rm, and Vm and its antibacterial activity in S. aureus and E. coli. Monatsh Chem 2010;141:373–80. [9] Oumzil K, Ouali I, Santelli M. First total synthesis of (±)-3-aza-11-oxa1,3.5(10)-trieno steroids. Steroids 2006;71:886–94. [10] Oualia I, Rocheblave L. Recent advances in azasteroids chemistry. Steroids 2008;73:375–407. [11] Marson C, Pinka J, Smith C. Synthesis of first monoaromatic B-ring 13azasteroid ring system by sequential angular annulation. Tetrahedron 2003;59:10019–23.

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