Design and synthesis of two new steroid derivatives with biological activity on heart failure via the M2-muscarinic receptor activation

Journal Pre-proofs Design and Synthesis of two new steroid derivatives with biological activity on heart failure via the M2-muscarinic receptor activation Figueroa-Valverde Lauro, Lopez-Ramos Maria, Lopez-Gutierrez Tomas, Diaz Cedillo Francisco, Garcia-Martinez Rolando, Rosas-Nexticapa Marcela, Mateu-Armand Virginia, Garcimarero-Espino E. Alejandra, Ortiz-Ake Yazmin PII: DOI: Reference:

S0039-128X(20)30045-3 https://doi.org/10.1016/j.steroids.2020.108620 STE 108620

To appear in:

Steroids

Received Date: Revised Date: Accepted Date:

10 December 2019 21 February 2020 26 February 2020

Please cite this article as: Lauro, F-V., Maria, L-R., Tomas, L-G., Cedillo Francisco, D., Rolando, G-M., Marcela, R-N., Virginia, M-A., Alejandra, G.E., Yazmin, O-A., Design and Synthesis of two new steroid derivatives with biological activity on heart failure via the M2-muscarinic receptor activation, Steroids (2020), doi: https://doi.org/ 10.1016/j.steroids.2020.108620

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Design and Synthesis of two new steroid derivatives with biological activity on heart failure via the M2-muscarinic receptor activation Figueroa-Valverde Lauroa*, Lopez-Ramos Mariaa, Lopez-Gutierrez Tomasa, Diaz Cedillo Franciscob, Garcia-Martinez Rolandoa, Rosas-Nexticapa Marcelac*, Mateu-Armand Virginiac, Garcimarero-Espino E. Alejandrac, Ortiz-Ake Yazmina aLaboratory

of Pharmaco-Chemistry at the Faculty of Chemical Biological Sciences of the

University Autonomous of Campeche, Av. Agustín Melgar s/n, Col Buenavista C.P.24039 Campeche Cam., México. bEscuela

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, aFacultad

de Nutrición, Universidad Veracruzana. Médicos y Odontólogos s/n, 91010,

Xalapa, Veracruz. México. aFacultad

de Medicina, Universidad Veracruzana. Médicos y Odontólogos s/n, 91010,

Xalapa, Veracruz. México.

*Corresponding author e-mail address: *[email protected]; [email protected]

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Abstract. Several drugs have been prepared to treat of heart failure using some protocols which require dangerous reagents and specific conditions. The aim of this study was to synthesize a series of steroid derivatives (compounds 2 to 18) using some chemical strategies. The biological activity of steroid derivatives against heart failure was evaluated using an ischemia/reperfusion model. In addition, the effect exerted by compounds 4 or 5 on left ventricular pressure was evaluated in the absence or presence of yohimbine, butaxamine and methoctramine. The results showed that 1) both compounds 4 or 5 significantly decrease the heart failure (translated as infarct area) compared with the compounds 2, 3 and 6-18. In addition, the compound 4 and 5 decreased the left ventricular pressure in a dose-dependent manner and this effect was significantly inhibited in the presence of methoctramine (p = 005). In conclusion, the compounds 4 or 5 decrease both the infarct area and left ventricular pressure via M2-muscarinic receptor activation. Keywords. Steroid, heart failure, left ventricular pressure, methoctramine

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1. Introduction Heart failure is a major cause of cardiovascular mortality and morbidity, among patients older than 65 years old [1-3]; there are several risk factors which may contribute to development this clinical pathology such as hypertension [4], obesity [5], diabetes, smocking [6] and others. It is important to mention that some drugs have been used for treatment of heart failure such as enanpril [7], nesiritide [8], digoxin [9], spironolactone [10], milrinone [11], dobutamine [12], and levosimendan [13]; however, some these drugs can produce secondary effects such as hypotension [14], arrhythmias [15], and hyponatremia [16]. In the search of new alternative therapeutics, some drugs have been developed for treat of heart failure; for example, the synthesis of an amidino-hydrazone derivative which showed positive inotropic activity through ATPase inhibition using an isolated rat heart. [17]. In addition, a report showed the preparation of a trichloro-acetamidine analogue which exerted positive inotropic activity via ATPase-inhibition in a calf heart model [18]. Other study showed the synthesis of a thiazinone derivative with negative inotropic activity through cytochrome-P450 inhibition using an in vitro model [19]. On the other hand, some reports have shown that several steroid derivatives may induce effects on heart failure; for example, a study showed that compound 3-(2Aminoethoxyimino)androstane-6,17-dione exerts positive inotropic activity in a heart dog model [20]. Other data indicate that a furosemide-pregnenolone derivative may induce a positive inotropic effect in an isolated rat heart model [21]. In addition, a study showed that a steroid derivative (F90927) may increase contraction force via L-type calcium channels activation in an isolated atria model [22]. Furthermore, a study showed that an estradiolderivative exerts a positive inotropic activity via L-type calcium channels activation in an isolated rat heart model [23].

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Additionally, a report indicated that k-strophanthidin (steroid derivative) increase the contraction force via Na+,K+-ATPase inhibition on guinea pig hearts. All these data indicate that several steroid derivatives can exert inotropic activity on heart; however, the molecular mechanism and the site of action is not very clear. Analyzing these data, in this study, a series of steroid derivatives were prepared to evaluate their biological activity against heart failure using an ischemia/reperfusion model.

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2. Material and Methods 2.1 General methods 2-nitroestrone (compound 1) was prepared using a previously method reported [57]. In addition, the other reagents used in this investigation were acquired from Sigma-Aldrich Co., Ltd. The melting point for compounds was evaluated on an Electrothermal (900 model). Infrared spectra (IR) were evaluated with a Thermo Scientific iSOFT-IR spectrometer.1H and 13C NMR spectra were recorded using a Varian VXR300/5 FT NMR spectrometer at 300 MHz in CDCl3 using TMS as internal standard. EIMS spectra were obtained with a Finnigan Trace Gas Chromatography Polaris Q-Spectrometer. Elementary analysis data were acquired from a Perkin Elmer Ser. II CHNS/02400 elemental analyzer. 2.2

Chemical Synthesis 13-Methyl-2-nitro-17-oxo-7,8,9,11,12,13,14,15,16,17-decahy-

dro-6H-cyclopenta[a]phenanthrene-3-carbaldehyde (2) In a round bottom flask (10 ml), 2-nitroestrone (100 mg, 0.58 mmol) and 5 ml of dimethyl sulfoxide were stirred at room temperature for 72 h. Then, the solvent was evaporated under reduced pressure and following the product was purified through crystallization using the methanol:water (4:1) system; yielding 64% of product; m.p. 68-70 oC; IR (Vmax, cm-1) 1726, 1722 and 1622: 1H NMR (300 MHz, CDCl3-d) δH: 0.90 (s, 3H), 1.201.92 (m, 7H), 2.08-2.84 (m, 7H), 3.02-8.02 (m, 3H), 10.80 (s, 1H) ppm. 13C NMR (300 Hz, CDCl3) C: 13.82, 21.70, 25.50, 27.52, 29.60, 31.02, 35.02, 37.20, 46.40, 48.32, 50.10, 122.04, 126.22, 126.44, 145.40,

150.84,

151.75, 194.22, 219.70 ppm. EI-MS m/z: 327.14. Anal. Calcd. for

C19H21NO4: C, 69.71; H, 6.47; N, 4.28; O, 19.55. Found: C, 69.68; H, 6.44.

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2.3 Preparation of alkyne-steroid derivatives (compound 3-6) 17-(3-Amino-phenylethynyl)-17-hydroxy-13-methyl-2-nitro7,8,9,11,12,13,14,15,16,17decahydro-6H-cyclopenta[a]phenanthrene-3-carbaldehyde (3) In a round bottom flask (10 ml), compound 2 (200 mg, 0.61 mmol), ethynylaniline (80 µl, 0.71), sodium hydroxide (15 mg, 0.37 mmol) and 5 ml of methanol were stirred to reflux temperature for 24 h. Then, the solvent was evaporated under reduced pressure and following the product was purified through crystallization using the methanol:hexane:water (4:1:1) system; yielding 64% of product; m.p. 58-60 oC; IR (Vmax, cm-1) 3402, 2192, 1722 and 1622: 1H NMR (300 MHz, CDCl3-d) δH: 0.86 (s, 3H), 1.12-1.50 (m, 4H), 1.66-1.86 (m, 4H), 2.08-2.40 (m 4H), 2.842.92 (m, 3H), 4.90 (broad, 3H), 6.66-7.08 (m, 4H), 7.80-7.92 (m, 2H), 10.80 (s, 1H) ppm. 13C

NMR (300 Hz, CDCl3) C: 12.32, 23.70, 26.90, 28.00, 29.62, 34.90, 36.76, 37.84,

44.96, 47.94, 52.80, 81.72, 85.14, 91.12, 116.32, 12.22, 121.44, 122.72, 126.14, 126.24, 126.50, 129.02, 145.52, 148.26, 151.34, 151.74, 194.24 ppm. EI-MS m/z: 444.20. Anal. Calcd. for C27H28N2O4: C, 72.95; H, 6.35; N, 6.30; O, 14.40. Found: C, 72.92; H, 6.31. 17-(6-Chloro-hex-1-ynyl)-17-hydroxy-13-methyl-2-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-carbaldehyde (4) In a round bottom flask (10 ml), compound 2 (200 mg, 0.61 mmol), 6-Chloro-hex-1-yne (80 µl, 0.66 mmol), sodium hydroxide (15 mg, 0.37 mmol) and 5 ml of methanol were stirred at reflux for 24 h. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 55% of product; m.p. 92-94 oC; IR (Vmax, cm-1) 3402, 2190, 1722 and 1622: 1H NMR (300 MHz, CDCl3-d) δH: 0.90 (s, 3H), 1.12-1.70 (m, 6H), 1.72 (m, 2H), 1.84-1.87 (m, 2H), 1.92 (m, 2H), 2.10-2.11 (m, 2H), 2.20 (m, 2H), 2.32-2.90 (m, 5H), 3.56 (m, 2H), 5.72 (broad, 1H), 7.807.92 (m, 2H), 10.80 (s, 1H) ppm. 13C NMR (300 Hz, CDCl3) C: 12.32, 19.20, 23.70, 26.30, 6

26.90, 28.00, 29.60, 31.40, 34.92, 36.60, 37.84, 44.96, 45.22, 48.10, 52.82, 80.10, 81.82, 83.40, 122.72, 126.22, 126.49, 145.53, 151.34, 151.72, 194.22 ppm. EI-MS m/z: 443.18. Anal. Calcd. for C25H30ClNO4: C, 67.63; H, 6.81; Cl, 7.99; N, 3.15; O, 14.42. Found: C, 67.50; H, 6.78. 6-(3-Formyl-17-hydroxy-13-methyl-2-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-6Hcyclopenta[a]phenan-thren-17-yl)hex-5-ynoic acid (5) In a round bottom flask (10 ml), compound 2 (200 mg, 0.61 mmol), 5-hexynoic acid (80 µl, 0.72 mmol), sodium hydroxide (15 mg, 0.37 mmol) and 5 ml of methanol were stirred at reflux for 24 h. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:hexane:water (4:2:1) system; yielding 39% of product; m.p. 70-72 oC IR (Vmax, cm-1) 2192, 1722, 1622 and 1612: 1H NMR (300 MHz, CDCl3-d) δH: 0.90 (m, 3H), 1.12-1.52 (m, 4H), 1.66 (m, 2H), 1.68-2.12 (m, 6H), 2.22 (m, 2H), 2.30-2.40 (m, 2H), 2.48 (m, 2H), 2.82-7.80 (m, 4H), 7.90 (broad, 2H), 7.92 (m, 1H), 10.80 (s, 1H) ppm. 13C NMR (300 Hz, CDCl3) C: 12.32, 18.96, 22.52, 23.70, 26.88, 28.00, 29.60, 33.02, 34.90, 36.62, 37.84, 44.96, 48.10, 52.80, 78.12, 80.10, 83.40, 122.72, 126.22, 126.44, 145.52, 151.34, 151.73, 178.40, 194.22 ppm. EI-MS m/z: 439.19. Anal. Calcd. for C25H29NO6: C, 68.32; H, 6.65; N, 3.19; O, 21.84. Found: C, 68.28; H, 6.62. 17-Hydroxy-17-(6-hydroxy-hex-1-ynyl)-13-methyl-2-nitro7,8,9,11,12,13,14,15,16,17decahydro-6H-cyclopenta[a]phe- nanthrene-3-carbaldehyde (6) In a round bottom flask (10 ml), compound 2 (200 mg, 0.61 mmol), Hex-5-yn-1-ol (80 µl, 0.72 mmol), and sodium hydroxide (15 mg, 0.37 mmol) in 5 ml of ethanol was stirring for 24 h to reflux. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 56%

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of product; m.p. 65-67 oC; IR (Vmax, cm-1) 3402, 2192, 1720 and 1622: 1H NMR (300 MHz, CDCl3-d) δH: 0.90 (s, 3H), 1.12-1.52 (m, 4H), 1.58-1.60 (m, 4H), 1.66-2.12 (m, 6H), 2.18 (m, 2H), 2.30-2.90 (m, 5H), 3.66 (m 2H), 3.80 (broad, 2H), 7.80-7.92 (m, 2H), 10.80 (s, 1H) ppm.

13C

NMR (300 Hz, CDCl3) C: 12.32, 18.86, 23.70, 25.52, 26.86, 28.00, 29.60,

31.82, 34.90, 36.62, 37.87, 44.96, 48.11, 52.82, 62.06, 80.12, 80.72, 83.40, 122.72, 126.23, 126.47, 145.54, 151.34, 151.72, 194.22 ppm. EI-MS m/z: 425.22. Anal. Calcd. for C25H31NO5: C, 70.57; H, 7.34; N, 3.19; O, 18.80. Found: C, 68.28; H, 6.62. 2.4 Synthesis of carbonitrile-steroid derivatives (compounds 7-10) 17-(3-Amino-phenylethynyl)-17-hydroxy-13-methyl-2-nitro-7,8,9,11,12,13,14,15,16, 17decahydro-6H-cyclopenta[a]phenanthrene-3-carbonitrile (7) In a round bottom flask (10 ml), compound 3 (200 mg, 0.45 mmol), hydroxylamine hydrochloride (60 mg, 0.86 mmol), and 5 ml of dimethyl sulfoxide were stirred reflux for 48 h. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 48% of product; m.p. 102104 oC; IR (Vmax, cm-1) 3302, 2192, 2252, and 1622: 1H NMR (300 MHz, CDCl3-d) δH: 0.82 (s, 3H), 1.12-1.88 (m, 8H), 2.08-2.92 (m, 7H), 4.92 (broad, 3H), 6.66-7.08 (m, 4H), 7.627.92 (m, 2H) ppm. 13C NMR (300 Hz, CDCl3) C: 12.32, 23.66, 26.88, 28.00, 32.12, 34.90, 36.74, 37.84, 44.96, 47.96 52.84, 81.72, 85.15, 91.56, 106.20, 116.32, 117.27, 121.22, 121.44, 124.55, 126.14, 128.66, 129.02, 144.42, 145.73, 148.22, 148.28 ppm. EIMS m/z: 441.20. Anal. Calcd. for C27H27N3O3: C, 73.45; H, 6.16; N, 9.52; O, 10.87. Found: C, 73.42; H, 6.12.

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17-(6-Chloro-hex-1-ynyl)-17-hydroxy-13-methyl-2-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenan- threne-3-carbonitrile (8) In a round bottom flask (10 ml), compound 4 (200 mg, 0.45 mmol), hydroxylamine hydrochloride (60 mg, 0.86 mmol), and 5 ml of dimethyl sulfoxide were stirred reflux for 48 h. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 62% of product; m.p. 112114 oC; IR (Vmax, cm-1) 3400, 2192, 21252 and 1622: 1H NMR (300 MHz, CDCl3d) δH: 0.90 (m, 3H), 1.12-1.70 (m, 6H), 1.72 (m, 2H), 1.82-1.86 (m, 2H), 1.92 (m, 2H), 2.102.12 (m, 2H), 2.20 (m, 2H), 2.30-2.92 (m, 5H), 3.56 (m, 2H, J = 1.00 Hz), 5.70 (broad, 1H), 7.60-7.94 (m, 2H) ppm. 13C NMR (300 Hz, CDCl3) C: 12.32, 19.22, 23.72, 26.30, 26.89, 28.00, 31.43, 32.12, 34.90, 36.56, 37.84, 44.96, 45.22, 48.10, 52.80, 80.12, 81.82, 83.40, 106.22, 117.26, 124.55, 128.65, 144.40, 145.74, 148.24, ppm. EI-MS m/z: 440.18. Anal. Calcd. for C25H29ClN2O3: C, 68.09; H, 6.33; Cl, 8.04; N, 6.35; O, 10.88. Found: C, 68.06; H, 6.30. 6-(3-Cyano-17-hydroxy-13-methyl-2-nitro-7,8,9,11,12,13,14,15,16,17-decahydro-6Hcy-clopenta[a]phenanthren-17-yl)hex-5-ynoic acid (9) In a round bottom flask (10 ml), compound 5 (200 mg, 0.45 mmol), hydroxylamine hydrochloride (60 mg, 0.86 mmol), and 5 ml of dimethyl sulfoxide were stirred reflux for 48 h. Then, the solvent was evaporated under reduced pressure and following the product was purified through crystallization using the methanol:water (4:1) system; yielding 45% of product; m.p. 122-124 oC; IR (Vmax, cm-1) 3400, 2250, 2192, 1622 and 1612: 1H NMR (300 MHz, CDCl3-d) δH: 0.90 (s, 3H), 1.12-1.52 (m, 4H), 1.66 (m, 2H), 1.68-2.10 (m, 6H), 2.22 (m, 2H), 2.30-2.40 (m, 2H), 2.48 (m, 2H), 2.88-7.60 (m, 4H), 7.90 (broad, 2H), 7.94 (m, 1H)

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ppm. 13C NMR (300 Hz, CDCl3) C: 12.32, 18.96, 22.52, 23.68, 26.86, 28.00, 32.10, 33.02, 34.90, 36.60, 37.84, 44.96, 48.10, 52.80, 78.12, 80.10, 83.40, 106.22, 117.26, 124.56, 128.64,

144.40, 145.72,

148.22, 178.40 ppm. EI-MS m/z: 436.19. Anal. Calcd. for

C25H28N2O5: C, 68.79; H, 6.47; N, 6.42; O, 18.33. Found: C, 68.76; H, 6.44. 17-Hydroxy-17-(6-hydroxy-hex-1-ynyl)-13-methyl-2-nitro-7,8,9,11,12,13,14,15,16,17decahydro-6Hcyclopenta[a]phenanthrene-3-carbonitrile (10) In a round bottom flask (10 ml), compound 6 (200 mg, 0.47 mmol), hydroxylamine hydrochloride (60 mg, 0.86 mmol), and 5 ml of dimethyl sulfoxide were stirred reflux for 48 h. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 62% of product; m.p. 133-135 oC; IR (Vmax, cm-1) 3402, 2250, 2190 and 1620: 1H NMR (300 MHz, CDCl3d) δH: 0.90 (s, 3H), 1.12-1.52 (m, 4H), 1.56-1.58 (m, 4H), 1.66-2.10 (m, 6H), 2.18 (m, 2H), 2.30-2.90 (m, 5H), 3.64 (m, 2H), 3.80 (broad, 2H), 7.60-7.94 (m, 2H) ppm. 13C NMR (300 Hz, CDCl3) C: 12.32, 18.84, 23.66, 25.52, 26.88, 28.00, 31.82, 32.10, 34.90, 36.60, 37.84, 44.96, 48.12, 52.80, 62.09, 80.08, 80.72, 83.42, 106.20, 117.26, 124.55, 128.64, 144.42, 145.74, 148.22 ppm. EI-MS m/z: 422.22. Anal. Calcd. for C25H30N2O4: C, 71.07; H, 7.16; N, 6.63; O, 15.15. Found: C, 71.04; H, 7.12. 2.5 Preparation of ether-steroid derivatives 17-(3-Amino-phenylethynyl)-17-hydroxy-13-methyl-2-(1-phenyl-but-3-enyloxy)-7,8,9, 11,12,13,14,15,16,17-decahydro-6Hcyclopenta[a]phenanthrene-3-carbonitrile (11) In a round bottom flask (10 ml), compound 7 (200 mg, 0.45 mmol), 4-phenyl-1-buten-4-ol (80 µl, 0.53 mmol), potassium carbonate anhydrous (50 mg, 0.36 mmol) and 5 ml of dimethyl sulfoxide were stirred at room temperature for 72 h. Then, the solvent was evaporated under

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reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 63% of product; m.p. 144-146 oC, IR (Vmax, cm-1) 3400, 3302, 2250 and 1150: 1H NMR (300 MHz, CDCl3-d) δH: ppm. 0.82 (s, 3H), 1.12-1.86 (m, 8H), 2.06-2.10 (m, 2H), 2.14 (m, 1H), 2.30 (m, 1H), 2.36 (m, 1H), 2.38-2.90 (m, 4H), 4.66 (m, 1H), 4.92 (broad, 3H), 5.16 (d, 1H, J = 1.50 Hz), 5.20-5.90 (m, 2H), 6.66-7.06 (m, 4H), 7.12 (m, 1H), 7.22-7.38 (m, 3H), 7.42 (m, 1H), 7.44 (m, 2H) ppm. 13C NMR (300 Hz, CDCl3) C: 12.32, 23.66, 26.89, 28.00, 32.10, 34.90, 36.72, 37.84, 44.20, 45.40, 47.92, 52.80, 81.34, 81.72, 85.16, 91.55, 97.94, 104.88, 114.44, 116.32, 118.60, 121.22, 121.44, 126.12, 127.76, 127.90, 128.16, 128.40, 129.02, 130.34, 138.97, 141.72, 143.72, 148.26, 160.60 ppm. EI-MS m/z: 542.29. Anal. Calcd. for C37H38N2O2: C, 81.88; H, 7.06; N, 5.16; O, 5.90. Found: C, 81.85; H, 7.02. 17-(6-Chloro-hex-1-ynyl)-17-hydroxy-13-methyl-2-(1-phenylbut-3-enyloxy)-7,8,9,11, 12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-carbonitrile (12) In a round bottom flask (10 ml), compound 8 (200 mg, 0.45 mmol), 4-phenyl-1-buten-4-ol (80 µl, 0.53 mmol), potassium carbonate anhydrous (50 mg, 0.36 mmol) and 5 ml of dimethyl sulfoxide was stirring to room temperature. Then, the solvent was evaporated under reduced pressure and following the product was purified through crystallization using the methanol:water (4:2) system; yielding 54% of product; m.p. 143-145 oC; IR (Vmax, cm-1) 3402, 2250, 2192 and 1150: 1H NMR (300 MHz, CDCl3-d) δH: 0.90 (s, 3H), 1.12-1.70 (m, 6H), 1.72 (m, 2H), 1.82-1.86 (m, 2H), 1.92 (m, 2H), 2.10-2.12 (m, 2H), 2.16 (m, 1H), 2.20 (m, 2H), 2.30 (m, 1H), 2.36 (m, 1H), 2.40-2.90 (m, 4H), 3.56 (m, 2H), 4.66 (m, 1H), 5.165.20 (m, 2H), 5.72 (broad, 1H), 5.88 (d, 1H, J = 1.50 Hz), 7.12 (m, 1H), 7.22-7.40 (m, 3H), 7.42 (m, 1H), 7.46 (m, 2H) ppm. 13C NMR (300 Hz, CDCl3) C: 12.32, 19.22, 23.70, 26.30,

11

26.86, 28.00, 31.40, 32.10, 34.91, 36.54, 37.87, 44.20, 45.22, 45.42, 48.12, 52.80, 69.86, 79.00, 80.12, 81.34, 81.83, 83.42, 97.93, 104.84, 114.42, 118.56, 127.80, 127.90, 128.16, 128.40, 130.32, 138.96, 141.72, 143.70, 160.60 ppm. EI-MS m/z: 541.27. Anal. Calcd. for C35H40ClNO2: C, 77.54; H, 7.44; Cl, 6.54; N, 2.58; O, 5.90. Found: C, 77.50; H, 7.41. 6-[3-Cyano-17-hydroxy-13-methyl-2-(1-phenyl-but-3-enyl-oxy)-7,8,9,11,12,13,14,15, 16,17-decahydro-6H-cyclopenta[a] phenanthren-17-yl]-hex-5-ynoic acid (13) In a round bottom flask (10 ml), compound 9 (200 mg, 0.46 mmol), 4-phenyl-1-buten-4-ol (80 µl, 0.53 mmol), potasium carbonate anhydrous (50 mg, 0.36 mmol) and 5 ml of dimethyl sulfoxide was stirring to room temperature. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 64% of product; m.p. 122-124 oC; IR (Vmax, cm-1) 3402, 2252, 2190, 1622 and 1152: 1H NMR (300 MHz, CDCl3-d) δH: 0.92 (s, 3H), 1.12-1.52 (m, 4H), 1.66 (m, 2H), 1.68-2.10 (m, 6H), 2.16 (m, 1H), 2.22 (m, 2H), 2.30 (m, 1H), 2.36 (m, 1H), 2.40-2.44 (m, 2H), 2.48 (m, 2H), 2.88-2.90 (m, 2H), 4.66 (m, 1H), 5.16-5.20 (m, 2H), 5.90 (d, 1H, J = 1.90 Hz), 7.12 (m, 1H), 7.22-7.40 (m, 3H), 7.42 (m, 1H), 7.44 (m, 2H), 7.90 (broad, 2H) ppm. 13C NMR (300 Hz, CDCl3) δC: 12.32, 18.96, 22.52, 23.66, 26.88, 28.00, 32.12, 33.02, 34.90, 36.56, 37.88, 44.22, 45.42, 48.12, 52.81, 78.12, 80.10, 81.34, 83.40, 97.92, 104.86, 114.42, 118.60, 127.76, 127.92, 128.16, 128.38, 130.32, 138.94, 141.72, 143.70, 160.60, 178.40 ppm. EI-MS m/z: 537.28. Anal. Calcd. for C35H39NO4: C, 78.18; H, 7.31; N, 2.60; O, 11.90. Found: C, 78.15; H, 7.28. 17-Hydroxy-17-(6-hydroxy-hex-1-ynyl)-13-methyl-2-(1-phenyl-but-3-enyloxy)-7,8,9, 11,12,13,14,15,16,17-decahydro-6Hcyclopenta[a]phenanthrene-3-carbonitrile (14) In a round bottom flask (10 ml), compound 10 (200 mg, 0.47 mmol), 4-phenyl-1-buten-4-ol (80 µl, 0.53 mmol), potassium carbonate anhydrous (50 mg, 0.36 mmol) and 5 ml of dimethyl 12

sulfoxide was stirring to room temperature. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:2) system; yielding 46% of product; m.p. 154-156 oC; IR (Vmax, cm-1) 3402, 2250, 2192, and 1152: 1H NMR (300 MHz, CDCl3-d) δH: 0.90 (s, 3H), 1.12-1.52 (m, 4H), 1.56-1.58 (m, 4H), 1.66-2.16 (m, 7H), 2.18 (m, 2H), 2.30 (m, 1H), 2.36 (m, 1H), 2.40-2.90 (m, 4H), 3.64 (m, 2H), 3.80 (broad, 2H), 4.68 (m, 1H), 5.16-5.20 (m, 2H), 5.90 (d, 1H, J = 1.50 Hz), 7.12 (m, 1H), 7.207.40 (m, 3H), 7.42 (m, 1H), 7.44 (m, 2H) ppm. 13C NMR (300 Hz, CDCl3) δC: 12.32, 18.84, 23.70, 25.52, 26.88, 28.00, 31.82, 32.10, 34.90, 36.59, 37.84, 44.20, 45.40, 48.10, 52.80, 62.09, 80.10, 80.72, 81.34, 83.40, 97.92, 104.84, 114.42, 118.60, 127.74, 127.92, 128.14, 128.39, 130.32, 138.96, 141.72, 143.70, 160.60 ppm. EI-MS m/z: 523.30. Anal. Calcd. for C35H41NO3: C, 80.27; H, 7.89; N, 2.67; O, 9.17. Found: C, 80.24; H, 7.86. 2.6 Preparation of azahexacyclo derivatives (5S)-6-[2-(3-aminophenyl)ethynyl]-5-methyl-21-phenyl-22oxa-17-azahexacyclo[11.11. 0.02,10.05,9.015,23.016,19]tetracosa1(24),13,15(23),16-tetraen-6-ol (15) In a round bottom flask (10 ml), compound 11 (200 mg, 0.36 mmol), Copper(II) chloride anhydrous (0.70 mg, 0.52 mmol) and 5 ml of methanol were stirred at room temperature for 72 h. Then, the solvent was evaporated under reduced pressure and following the product was purified through crystallization using the methanol:water (4:1) system;yielding 60% of product; m.p. 178180 oC; IR (Vmax, cm-1) 3400, 3302, 2192 and 1152: 1H NMR (300 MHz, CDCl3-d) δH: 0.82 (s, 3H), 1.12 (m,1H), 1.20 (m, 1H), 1.26-1.52 (m, 3H), 1.62 (m, 1H), 1.682.98 (m, 11H), 3.36 (m, 1H), 3.50-3.74 (m, 2H), 4.92 (broad, 3H), 5.18 (m, 1H), 6.667.06 (m, 5H), 7.34-7.60 (m, 5H), 7.66 (m, 1H) ppm. 13C NMR (300 Hz, CDCl3) δC: 12.82, 22.82, 27.06, 27.60, 29.32, 32.90, 34.46, 35.16, 39.04, 39.22, 45.40, 47.04, 49.52, 50.05, 78.94, 13

82.20, 85.14, 91.56, 111.40,

116.30,

116.34,

121.22, 121.44, 126.10, 126.96,

127.90,128.50, 128.82, 128.87, 129.02, 139.44, 142.64, 148.26, 149.60, 189.25 ppm. EIMS m/z: 542.29. Anal. Calcd. for C37H38N2O2: C, 81.88; H, 7.06; N, 5.16; O, 5.90. Found: C, 81.85; H, 7.02. (5S)-6-(6-chlorohex-1-yn-1-yl)-5-methyl-21-phenyl-22-oxa17-azahexacyclo[11.11.0. 02,10.05,9.015,23.016,19]tetracosa-1- (24),13,15(23),16-tetraen-6-ol (16) In a round bottom flask (10 ml), compound 12 (200 mg, 0.37 mmol), Copper(II) chloride anhydrous (0.70 mg, 0.52 mmol) and 5 ml of methanol were stirred at room temperature for 72 h. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 64% of product; m.p. 165-167 oC; IR (Vmax, cm-1) 3402, 2190 and 1152: 1H NMR (300 MHz, CDCl3d) δH: 0.90 (s, 3H), 1.12 (m, 1H), 1.20 (m, 1H), 1.26-1.52 (m, 3H), 1.62 (m, 1H), 1.68-1.70 (m, 2H), 1.72 (m, 2H), 1.82-1.86 (m, 2H), 1.92 (m, 2H), 2.10-2.12 (m, 2H), 2.20 (m, 2H), 2.30-3.00 (m, 5H), 3.36 (m, 1H), 3.48 (m, 1H), 3.58 (m, 2H), 3.74 (m, 1H), 5.18 (m, 1H), 5.72 (broad, 1H), 6.76 (m, 1H), 7.347.62 (m, 5H), 7.66 (m, 1H) ppm. 13C NMR (300 Hz, CDCl3) δC: 12.82, 19.20, 22.82, 26.30, 27.10, 27.62, 29.30, 31.40, 32.92, 34.50, 35.16, 38.90, 39.24, 45.22, 45.40, 47.22, 49.46, 50.04, 78.94, 80.64, 81.82, 83.40, 111.40, 116.34, 126.96, 127.90, 128.52, 128.82, 128.87, 139.40, 142.60, 149.60, 189.25 ppm. EIMS m/z: 541.27. Anal. Calcd. for C35H40ClNO2: C, 77.54; H, 7.44; Cl, 6.54; N, 2.58; O, 5.90. Found: C, 77.50; H, 7.41. 6-[(5S)-6-hydroxy-5-methyl-21-phenyl-22-oxa-17-azahexacyclo[11.11.0.02,10.05,9.015,23. 016,19]tetracosa-1(24),13,15(23),16tetraen-6-yl]hex-5-ynoic acid (17) In a round bottom flask (10 ml), compound 13 (200 mg, 0.37 mmol), Copper(II) chloride anhydrous (0.70 mg, 0.52 mmol) and 5 ml of methanol were stirred at room temperature for 14

72 h. Then, the solvent was evaporated under reduced pressure and following the product was purified via crystallization using the methanol:water (4:1) system; yielding 64% of product; m.p. 121-123 oC; IR (Vmax, cm-1) 3402, 2190, 1620 and 1150: 1H NMR (300 MHz, CDCl3-d) δH: 0.90 (s, 3H), 1.12-1.52 (m, 5H), 1.62 (m, 1H), 1.66 (m, 2H), 1.68-2.10 (m, 6H), 2.22 (m, 2H), 2.30-2.44 (m, 3H), 2.48 (m, 2H), 2.96-2.98 (m, 2H), 3.36 (m, 1H), 3.48-3.74 (m, 2H), 5.16 (m, 1H), 6.74 (m, 1H), 7.34-7.62 (m, 5H), 7.66 (m, 1H), 7.90 (broad, 2H) ppm. 13C

NMR (300 Hz, CDCl3) δC: 12.82, 18.96, 22.52, 22.82, 27.09, 27.60, 29.32, 32.90,

33.02, 34.44, 35.16, 38.90, 39.24, 45.42, 47.22, 49.46, 50.07, 78.12, 78.90, 80.62, 83.40, 111.40, 116.32, 126.94, 127.90, 128.50, 128.80, 128.84, 139.40, 142.62, 149.60, 178.40, 189.24 ppm. EI-MS m/z: 537.28. Anal. Calcd. for C35H39NO4: C, 78.18; H, 7.31; N, 2.60; O, 11.90. Found: C, 78.14; H, 7.28. (5S)-6-(6-hydroxyhex-1-yn-1-yl)-5-methyl-21-phenyl-22-oxa17-azahexacyclo[11.11.0. 02,10.05,9.015,23.016,19]tetracosa-1(24), 13,15(23),16-tetraen-6-ol (18) In a round bottom flask (10 ml), compound 14 (200 mg, 0.38 mmol), Copper(II) chloride anhydrous (0.70 mg, 0.52 mmol) and 5 ml of methanol were stirred at room temperature for 72 h. Then, the solvent was evaporated under reduced pressure and following the product was purified through crystallization using the methanol:water (4:2) system; yielding 47% of product; m.p. 187-189 oC; IR (Vmax, cm-1) 3400, 2192 and 1152: 1H NMR (300 MHz, CDCl3d) δH: 0.90 (s, 3H), 1.12-1.52 (m, 5H), 1.561.58 (m, 4H), 1.62 (m, 1H), 1.68-2.10 (m, 6H), 2.16 (m, 2H), 2.30-3.00 (m, 5H), 3.36-3.50 (m, 2H), 3.64 (m, 2H), 3.74 (m, 1H), 3.80 (broad, 2H), 5.16 (m, 1H), 6.74 (m, 1H), 7.34-7.62 (m, 5H), 7.66 (m, 1H) ppm. 13C NMR (300 Hz, CDCl3) C: 12.82, 18.84, 22.80, 25.52, 27.09, 27.56, 29.32, 31.82, 32.92, 34.50, 35.12, 38.90, 39.24, 45.42, 47.22, 49.50, 50.04, 62.10, 78.90, 80.62, 80.72, 83.40, 111.40,

15

116.32, 126.96, 127.90, 128.50, 128.82, 128.87, 139.40, 142.62, 149.60, 189.22 ppm. EIMS m/z: 523.30. Anal. Calcd. for C35H41NO3: C, 80.27; H, 7.89; N, 2.67; O, 9.17. Found: C, 80.24; H, 7.86. 2.7 Biological Methods All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal care and use Committee of University Autonomous of Campeche (no. PI-420/12) and were in accordance with the Guide for the Care and Use of Laboratory Animals [52]. Male Wistar rats, weighing 200-250 g, were obtained from University Autonomous of Campeche. It is important to mention that all drugs were dissolved in methanol and different dilutions were obtained using Krebs-Henseleit solution (≤0.01%, v/v). 2.8 Experimental Design Briefly, the male rat (200-250 g) was anesthetized by injecting them with pentobarbital at a dose rate of 50 mg/Kg body weight. Then the chest was opened, and a loose ligature passed through the ascending aorta. The heart was then rapidly removed and immersed in ice cold physiologic saline solution. The heart was trimmed of non-cardiac tissue and retrograde perfused via a non-circulating perfusion system at a constant flow rate. The perfusion medium was the Krebs-Henseleit solution (pH = 7.4, 37 oC) composed of (mmol) 117.8, NaCl; 6, KCl; 1.75, CaCl2; 1.2, NaHPO4; 1.2, MgSO4; 24.2, NaHCO3; 5, glucose; 7 and 5, sodium pyruvate. In addition, the solution was actively bubbled with a mixture of O2/CO2 (95%:5%). The coronary flow was adjusted with a variable speed peristaltic pump. An initial perfusion rate of 15 mL/min for 5 min; then, was followed by a 15 min equilibration period at a perfusion rate of 10 mL/min. All experimental measurements were done after this equilibration period. 16

2.9 Perfusion Pressure To evaluate the biological activity of drugs against perfusion pressure, a pressure transducer was used which was connected to the chamber where the hearts were mounted. The results were entered into a computerized data capture system (Biopac) for their evaluation. 2.10 First Stage Biological activity induced by 2-nitroestrone and compounds 2-18 was evaluated using an ischemia/reperfusion model. After 15 minutes of equilibration time, the hearts were subjected to ischemia for 40 minutes by turning off the perfusion system [55]. Then, the system was restarted, and the hearts were re-perfused by 40 minutes with Krebs-Henseleit solution. The hearts were randomly divided into 19 major treatment groups (Table 1) with n = 9. The areas of the normal left ventricle no-risk region, area at risk, and infarct region were determined using a previous method reported [56]. Total area at risk was expressed as the percentage of the left ventricle. 2.11 Second Stage Biological activity induced by either of the compound 4 or 5 on left ventricular pressure via α2-adrenergic receptor activation. Intracoronary boluses (50 μL) of either compounds 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the left ventricular pressure was evaluated. The dose response curve (control) was repeated in the presence of yohimbine at a concentration of 1 nM (duration of the preincubation with yohimbine was for a period of 10 min). Effects exerted by of either of the compounds 4 or 5 on left ventricular pressure through the β2-adrenergic receptor activation. Intracoronary boluses (50 μL) of either compound 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the left ventricular pressure was evaluated. The dose-response curve (control) was repeated in the presence of 17

butaxamine* at a concentration of 1 nM (duration of the preincubation with butaxamine was for a period of 10 min). *The dose of butaxamine has been administered using a previously method reported [55]. Biological activity induced by either of the compound 4 or 5 on left ventricular pressure via M2-muscarinic receptor activation. Intracoronary boluses (50 μL) of either carbachol or compounds 4 and 5 [0.001 to 100 nM] were administered and the corresponding effect on the left ventricular pressure was evaluated. The dose-response curve (control) of either compounds 4 or 5 was repeated in the presence of methoctramine§ at a concentration of 1 nM (duration of the preincubation with methoctramine was for a period of 10 min). §The dose of methoctramine has been administered using a previously method reported [55]. Effects exerted by either of the compound 4 or 5 on left ventricular pressure through nitric oxide synthase activation. Intracoronary boluses (50 μL) of either compound 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the left ventricular pressure was evaluated. The dose-response curve (control) was repeated in the presence of L-NAME¥ at a concentration of 1 nM (duration of the preincubation with L-NAME was for a period of 10 min). ¥The dose of L-NAME (Nω-Nitro-L-arginine methyl ester) has been administered using a previously method reported [55]. 2.12 Statistical Analysis The obtained values are expressed as average ± SE. The results were put under analysis of variance (ANOVA) with the Bonferroni correction factor using the SPSS 12.0 program [57]. The differences were considered significant when was equal or smaller than 0.05.

18

3. Results and Discussion The aim of this study was to synthetize a series of estrone derivatives to evaluate their inotropic activity using an ischemia/reperfusion model as follows: 3.1 Synthesis of an aldehyde-steroid derivative The first stage was achieved by preparation of an aldehyde-steroid derivative (2); it is important to mention that several aldehyde analogs have prepared using some reagents such as PdCl2(MeCN)2 [25], I2/CuCl2 [26], POCl3 [27], TiCl4 [28], HgO [29], Au/Al2O3 [30]. In this study, the compound 2 was prepared using a previously method reported for synthesis of carbaldehyde derivatives [31]; in this way, 2-nitroestrone reacted with dimethyl sulfoxide in mild conditions to form 2 (Scheme1). The 1H NMR spectrum for compound 2 displayed some signals at 0.90 ppm for methyl group; at 1.20-8.02 ppm for steroid moiety; at 10.80 ppm for aldehyde group. In addition, the 13C NMR spectra showed some bands at 13.82 ppm for methyl group; at 21.70-151.75 ppm for steroid moiety; at 194.22 ppm for aldehyde group; at 219.70 ppm for ketone group. Additionally, the mass spectrum (m/z) from compound 2 was found to 327.14. 3.2 Preparation of alkyne-alcohol steroid derivatives Several methods for preparation of some alkyn-alcohol derivatives use different reagents such as disulfide-oxazolidine [32], Ti(O-i-Pr)4-BINOL complex [33], chiral diaminecoordinated tin(II) triflate [34], P(PhCH2NCH2CH2)3N [35] and others; however some of these reagents are difficult to handle and require special conditions. Analyzing these data, in this study, the compound 3 was prepared via reaction of 2 with 3-ethynylaniline in the presence of sodium hydroxide (Scheme 2). 1H NMR spectrum for compound showed several bands to 0.86 ppm for methyl group; at 1.12-2.92 and 6.66-7.92 ppm for steroid moiety; at 4.90 ppm for both hydroxyl and amino groups; at 10.80 ppm for aldehyde group.

13C

NMR 19

spectra showed several signals at 12.32 ppm for methyl group; at 23.70-81.72, 122.72, 126.24-126.50, 145.52 and 151.34-151.74 ppm for steroid moiety; at 116.32-121.44, 126.14 and 129.02 and 148.26 ppm for phenyl group; at 194.24 ppm for aldehyde group. Finally, the mass spectrum (m/z) from compound 3 was found to 444.20. On the other hand, a second alkyne-alcohol-steroid derivative (compound 4) was prepared from 2 and 6-Chloro-hex-1-yne (Scheme 2). The results showed several signals involved in the 1H NMR spectrum for compound 4 at 0.90 ppm for methyl group; at 1.12-1.70, 1.841.87, 2.10-2.11, 2.32-2.90 and 7.807.92 ppm for steroid moiety; at 1.72, 1.92, 2.20 and 3.56 ppm for methylene groups bound to both alkyne group and chloride; at 5.72 ppm for hydroxyl group; at 10.80 ppm for aldehyde group.

13C

NMR spectra showed several signals at 12.30

ppm for methyl group; at 19.20, 26.30, 31.40 and 45.22 ppm for methylene groups bound to both alkyne group and chloride; at 23.70, 26.90-29.60, 34.32-44.96, 48.10-80.10 and 122.72151.72 ppm for steroid moiety; at 81.82-83.40 ppm for alkyne group; at 194.22. Additionally, the mass spectrum (m/z) from compound 4 was found to 443.18. In the third stage, the compound 5 was prepared via reaction of 2 with hex-5-ynoic acid (Scheme 2). The 1H NMR spectrum for compound 5 showed several bands to 0.86 ppm for methyl group; at 1.12-1.52, 1.68-2.12, 2.30-2.40, 2.82-7.80 and 7.92 ppm for steroid moiety; at 1.66, 2.22 and 2.48 ppm for methylene groups bound to both alkyne and carboxyl groups; at 7.90 ppm for both hydroxyl and carboxyl groups; at 10.80 ppm for aldehyde group.

13C

NMR spectra showed several signals at 12.32 ppm for methyl group; at 23.70-29.60, 34.9052.80, 80.10 and 122.72151.72 ppm for steroid moiety; at 78.12 and 83.40 ppm for alkyne group; at 178.40 ppm for carboxyl group; at 194.22 for aldehyde group. In addition, the mass spectrum (m/z) from compound 5 was found to 439.19.

20

Finally, the compound 6 was synthesized from 2 and hex-5-yn1-ol (Scheme 2). The 1H NMR spectrum for compound 6 showed several bands to 0.90 ppm for methyl group; at 1.12-1.52, 1.66-2.12, 2.30-2.90 and 7.80-7.92 ppm for steroid moiety; at 1.58-1.60, 2.18 and 3.66 ppm for methylene bound to both alkyne and hydroxyl groups; at 3.80 ppm for both hydroxyl groups; at 10.80 ppm for aldehyde group. 13C NMR spectra showed several signals at 12.32 ppm for methyl group; at 18-86, 25.52, 31.82 and 62.06 ppm for methylene groups bound to both alkyne and hydroxyl groups; at 23.70, 26.86-29.60, 34.90-52.82, 80.12 and 122.72151.72 ppm for steroid moiety; at 80.72-83.40 ppm for alkyne group; at 194.22 ppm for aldehyde group. Finally, the mass spectrum (m/z) from compound 6 was found to 425.22. 3.3 Synthesis of carbonitrile derivatives. There are several reports on the synthesis of some carbonitrile analogs using different reagents such as pyridine [35], hydrogen peroxide [36], CuI/PPh3 [37], POCl3 [38], thiourea derivative [39], NH2OH/DMSO [40] and others. Analyzing these data in this study the compounds 3-6 reacted with hydroxylamine in the presence of dimethyl sulfoxide (DMSO) to form some steroid-carbonitrile derivatives (compounds 7-10; Scheme 3 and 4). The results showed several signals of 1H NMR spectrum for compound 7 at 0.82 ppm for methyl group; at 1.12-2.92 and 7.62-7.92 ppm for steroid moiety; at 4.90 ppm for both hydroxyl and amino groups; at 6.66-7.08 ppm for phenyl group. 13C NMR spectra showed several signals at 12.32 ppm for methyl group; at 23.66-81.72, 106.20, 124.55, 128.66 and 144.42-148.22 ppm for steroid moiety; at 85.15-91.56 ppm for alkyne group; at 116.32, 121.22-121.24, 126.14, 129.02 and 148.22 ppm for phenyl group; at 117.27 ppm for nitrile group. In addition, the mass spectrum (m/z) from compound 7 was found to 421.20. Other data showed several signals involved in the 1H NMR spectrum for compound 8 (Scheme 3) at 0.90 ppm for methyl group; at 1.12-1.70, 1.82-1.86, 2.10-2.12, 2.30-2.92 and 21

7.607.94 ppm for steroid moiety; at 1.72, 1.92, 2.20 and 3.56 ppm for methylene groups bound to both alkyne and chloride group; at 5.70 ppm got hydroxyl group.

13C

NMR spectra

showed several signals at 12.32 ppm for methyl group; at 19.22, 26.30, 31.43 and 45.22 ppm for methylene bound to both alkyne group and chloride; at 23.72, 26.89-28.00, 32.12-44.96, 48.10-80.11, 106.22 and 124.55148.24 ppm for steroid moiety; at 83.43 ppm for alkyne group; at 117.26 ppm for carbonitrile group. Additionally, the mass spectrum (m/z) from compound 8 was found to 440.18. On the other hand, the 1H NMR spectrum for compound 9 showed several bands at 0.90 ppm for methyl group; at 1.12-1.52, 1.68-2.10, 2.30-2.40, 2.88-7.60 and 7.94 ppm for steroid moiety; 1.66, 2.22 and 2.48 ppm for methylene groups bound to alkyne and carboxyl groups; at 7.90 ppm for hydroxyl groups.

13C

NMR spectra showed several signals at 12.32 ppm for

methyl group; at 18.96-22.52 and 33.02 ppm for methylene groups bound to alkyne and carboxyl groups; at 23.88-32.10, 34.90-52.82, 80.10, 106.22 and 124.56-148.22 ppm for steroid moiety; at 78.12 and 83.40 ppm for alkyne group; 117.26 ppm for carbonitrile group; at 178.40 ppm for carboxyl group. In addition, the mass spectrum (m/z) from compound 9 was found to 436.19. Finally, the 1H NMR spectrum for compound 10 showed several bands at 0.90 ppm for methyl group; at 1.121.52, 1.66-2.10, 2.30-2.90 and 7.60-7.94 ppm for steroid moiety; at 1.56-1.58, 2.18 and 3.64 ppm for methylene groups bound to both alkyne and hydroxyl groups; at 3.80 ppm for hydroxyl groups.

13C

NMR spectra showed several signals at 12.32

ppm for methyl group; at 18.84, 25.52, 31.82 and 62.09 ppm for methylene groups bound to both alkyne and hydroxyl groups; at 23.66-26.88-28.00, 32.10-52.80, 80.08, 106.20 and 124.55148.22 ppm for steroid moiety; at 80.72 and 83.42 ppm for alkyne group; at 117.26

22

ppm for carbonitrile group. Additionally, the mass spectrum (m/z) from compound 10 was found to 422.22. 3.4 Preparation of ether-steroid derivatives Several ether derivatives have been prepared using some reagents such as aluminum oxide [41], zinc chloride [42], 2,2′dibromo-4,4′dinitrobenzophenone [43], and others. In this study, a previously method reported [44] was used to synthesize four ether-steroid derivatives (compounds 11 to 14) via displacement of the nitro group involved in the chemical structure of either the compounds 7 to 10 with 4-phenyl-1-buten4-ol in the presence of dimethyl sulfoxide and mild conditions (Scheme 5). The 1H NMR spectrum for 11 showed several signals at 0.82 ppm for methyl group; at 1.12-2.10, 2.30, 2.382.90, 7.16 and 7.42 ppm for steroid moiety; at 2.14, 2.36 and 4.66 ppm for methylene groups bound to both alkene and ether groups; at 4.92 ppm for both amino and hydroxyl groups; at 5.16-5.90 ppm for alkene group; at 6.66-7.06 ppm for phenyl group bound to amino group; at 7.22-7.38 and 7.44 ppm for phenyl bound to methylene group. 13C NMR spectra showed several signals at 12.32 ppm for methyl group; at 23.66-37.84, 45.40-52.80, 86.72, 97.94-104.88, 128.16, 130.34, 143.72 and 160.60 ppm for steroid moiety; at 44.20 and 81.34 ppm for methylene groups bound to both alkene and ether groups; at 85.16-91.55 ppm for alkyne group; at 144.44-141.72 ppm for alkene group; at 116.32, 121.22-126.12, 129.02 and 148.26 ppm for phenyl bound to amino group; at 127.26-127.90, 128.40 and 138.97 ppm for phenyl bound to methylene group. Finally, the mass spectrum (m/z) from compound 11 was found to 542.29. On the other hand, the 1H NMR spectrum for 12 showed several signals at 0.90 ppm for methyl group; at 1.121.70, 1.82-1.86, 2.102.12, 2.30, 2.40-2.90, 7.12 and 7.42 ppm for steroid moiety; at 1.72, 1.92 and 3.56 ppm for methylene groups bound to both alkyne group and chloride; at 2.20, 2.36 and 4.66 ppm for methylene groups bound to both alkene and ether 23

groups; at 5.16-5.20 and 5.88 ppm for alkene group; at 5.72 ppm for hydroxyl groups at 7.227.40 and 7.46 ppm for phenyl group.

13C

NMR spectra showed several signals at 12.32 ppm

for methyl group; at 19.22, 26.30, 31.40 and 45.22 ppm for methylene groups bound to both alkyne group and chloride; at 23.70, 26.86-28.00, 32.10-37.87, 45.42-80.12, 97.93-104.84, 128.16, 130.32, 143.70-160.60 ppm for steroid moiety; at 44.20 and 81.34 ppm for methylene groups bound to both ether and alkene groups; at 81.83-83.42 ppm for alkyne group; at 114.47 and 141.72 ppm for alkene group; at 118.56 ppm for carbonitrile group; at 127.80127.90, 128.40 and 138.96 ppm for phenyl group. Additionally, the mass spectrum (m/z) from compound 12 was found to 541.22. Other data showed several bands involved in the 1H NMR spectrum for 13 at 0.92 ppm for methyl group; at 1.12-1.52, 1.68-2.10, 2.30, 2.40-2.44, 2.88-2.90, 7.12 and 7.42 ppm for steroid moiety; at 1.66-2.22 and 2.48 ppm for methylene groups bound to both alkyne and carboxyl groups; at 2.16, 2.36 and 4.66 for methylene groups bound to both ether and alkene groups; at 5.16-5.90 ppm for alkene group; at 7.22-7.40 and 7.44 ppm for phenyl group; at 7.90 ppm for both hydroxyl and carboxyl group. 13C NMR spectra showed several signals at 12.32 ppm for methyl group; at 18.96-22.52 and 33.02 ppm for methylene groups bound to both alkyne and carboxyl groups; at 23.66-32.12, 34.90-37.88, 45.42-52.81, 80.10, 97.92104.86, 128.16, 130.32 and 143.70-160.60 ppm for steroid moiety; at 44.20 and 81.34 ppm for methylene groups bound to both alkyne and ether groups; at 78.12 and 83.40 ppm for alkyne group; at114.42 and 141.72 ppm for alkene group; at 118.60 ppm for carbonitrile group; at 127.76-127.92, 128.38 and 138.94 ppm for phenyl group; at 178.40 ppm for carboxyl group. In addition, the mass spectrum (m/z) from compound 13 was found to 537.28.

24

Finally, the 1H NMR spectrum for 14 showed several signals at 0.90 ppm for methyl group; at 1.12-1.52, 1.662.16, 2.30, 2.40-2.90, 7.12 and 7.42 ppm for steroid moiety; at 1.56-1.58, 2.18 and 3.64 ppm for methylene groups bound to both alkyne and hydroxyl groups; at 2.36 and 4.68 ppm for methylene groups bound to both alkyne and ether groups; at 3.80 ppm for both hydroxyl groups; at 5.16-5.90 ppm for alkene group; at 7.20-7.40 and 7.44 ppm for phenyl group.

13C

NMR spectra showed several signals at 12.32 ppm for methyl group; at

18.84, 25.52, 31.82, 62.09 and 81.34 ppm for methylene groups bound to both alkyne and hydroxyl groups; at 23.70, 26.88-28.00, 33.10-37.84, 45.40-52.80, 80.10, 97.92104.84, 128.14, 130.32 and 143.70-160.60 ppm for steroid moiety; at 44.20 for methylene group bound to both alkene and ether groups; at 80.72 and 83.40 for alkyne group; at 114.40 and 141.72 for alkene group; at 118.60 ppm for carbonitrile group; at 127.74-127.92, 128.39 and 138.96 ppm for phenyl group. Additionally, the mass spectrum (m/z) from compound 14 was found to 523.30. 3.5 Preparation of azahexacyclo derivatives. Several protocols have been used for the synthesis of azahexacyclo derivatives which use some reagents such as benzylamine [45], heptylamine [46], N-benzyl-4,5,6,7tetrafluoroisoindole [47], Pd/C [48], NH4OAc [49] and others. In this investigation, either the compounds 15 to 18 were formed via internal reaction between nitrile and alkene groups involved in the chemical structure of compounds 11 to 18 using Copper(II) as catalyst (Scheme 6). The 1H NMR spectrum for 15 showed several signals at 0.82 ppm for methyl group; at 1.12, 1.26-1.52, 1.682.98 and 7.66 ppm for steroid moiety; at 1.20, 1.62, 3.16 and 5.18 ppm for 2,3,4,5-tetrahydro-oxepine ring; at 3.50-3.74 ppm for 2,3-dihydro-azete; at 4.92 ppm for both hydroxyl and amino group; at 6.66-7.06 ppm for phenyl bound to amino group; at 7.34-7.60 ppm for phenyl group bound to 2,3,4,5-tetrahydrooxepine ring.

13C

NMR 25

spectra showed several signals at 12.82 ppm for methyl group; at 22.82-32.90, 39.04-49.52, 82.20, 111.40, 116.34, 126.96, 128.82, 142.64 and 149.60 ppm for steroid moiety; at 34.4635.16, 78.94 and 189.25 ppm for 2,3,4,5tetrahydro-oxepine ring; at 50.05 and 189.25 ppm for 2,3dihydro-azete; at 85.14-91.56 ppm for alkyne group; at 116.30, 121.22-126.10 and 129.02 ppm for phenyl group bound to amino; at 127.90-128.50, 128.87 and 139.44 ppm for phenyl group bound to for 2,3,4,5-tetrahydro-oxepine ring. In addition, the mass spectrum (m/z) from compound 15 was found to 542.29. Other data showed several bands involved in the 1H NMR spectrum for 16 at 0.90 ppm for methyl group; at 1.12, 1.26-1.52, 1.68-1.70, 1.82-1.86, 2.10-2.12, 2.30-3.00, 6.76 and 7.66 ppm; at 1.20, 1.62, 3.36 and 5.18 ppm for 2,3,4,5-tetrahydro-oxepine ring; at 1.72, 1.92, 2.20 and 3.58 ppm for methylene groups bound to both alkyne group and chloride; at 3.48 and 3.74 ppm for 2,3-dihydro-azete; at 5.72 ppm for hydroxyl group; at 7.34-7.62 ppm for phenyl group.

13C

NMR spectra showed several signals at 12.82 ppm for methyl group; at 19.20,

26.30, 31.40 and 45.22 ppm for methylene groups bound to both alkyne group and chloride; at 22.82, 27.10-29.30, 32.92, 38.90-39.24, 45.40-49.46, 80.64, 111.40-126.96, 128.82 and 142.60-149.60 ppm for steroid moiety; at 34.50-35.16 and 78.94 for 2,3,4,5-tetrahydrooxepine ring; at 50.04 and 189.25 ppm for 2,3-dihydro-azete; at 81.8283.40 ppm for alkyne group; at 127.90-128.52 and 128.87139.40 ppm for phenyl group. Additionally, the mass spectrum (m/z) from compound 16 was found to 541.27. On the other hand, the 1H NMR spectrum for 17 showed several signals at 0.82 ppm for methyl group; at 1.121.52, 1.68-2.10, 2.30-2.44, 2.96-2.98, 6.74 and 7.66 ppm for steroid moiety; at 1.62, 3.36 and 5.16 ppm for 2,3,4,5-tetrahydro-oxepine ring; at 1.66, 2.22 and 2.48 ppm for methylene groups bound to both alkyne and carboxyl groups; at 3.48-3.78 ppm for 2,3-dihydro-azete ring; at 7.34-7.66 ppm for phenyl group; at 7.90 ppm for carboxyl and 26

hydroxyl groups. 13C NMR spectra showed several signals at 12,82 ppm for methyl group; at 18.96-22.52 and 33.02 ppm for methylene groups bound to both carboxyl and alkyne groups; at 22.82, 32.90, 38.90-49.46, 80.62, 111.40-126.94, 128.80 and 142.62149.60 ppm for steroid moiety; at 34.44-35.16 and 78.90 ppm for 2,3,4,5-tetrahydro-oxepine ring; at 50.07 and 189.24 ppm for 2,3-dihydro-azete ring; at 78.92 and 83.40 ppm for alkyne group; at 127.90-128.50 and 128.84-139.40 ppm for phenyl group; at 178.90 ppm for carboxyl group. In addition, the mass spectrum (m/z) from compound 17 was found to 537.28. Finally, the 1H NMR spectrum for 18 showed several signals at 0.90 ppm for methyl group; at 1.12-1.52, 1.682.10, 2.30-3.00, 6.74 and 7.66 ppm for steroid moiety; at 1.561.58, 2.16 and 3.64 ppm for methylene groups bound to alkyne and hydroxyl group; at 1.62 and 3.16 ppm for 2,3,4,5-tetrahydrooxepine ring; at 3.26-3.50 and 3.74 ppm for 2,3-dihydro-azete; at 3.80 for both hydroxyl groups; at 7.34-7.62 ppm for phenyl group. 13C NMR spectra showed several signals at 12.82 ppm for methyl group; at 18.84, 25.52, 31.82 and 62.10 ppm for methylene groups bound to both alkyne and hydroxyl groups; at 22.80, 27.09-29.32, 32.92, 38.90-49.50, 80.62, 111.40-126.96, 128.82, 142.62-149.60 ppm for steroid moiety; at 34.5035.12 and 78.90 ppm for 2,3,4,5-tetrahydro-oxepine ring; at 50.04 and 189.22 ppm for 2,3dihydro-azete; at 80.72-83.40 ppm for alkyne group; at 127.90-128.50 and 128.87-139.40 ppm for phenyl group. Finally, the mass spectrum (m/z) from compound 18 was found to 523.30. 3.6 Biological activity In this study, the biological activity of compounds 1 to 18 against myocardial injury was evaluated using an ischemia/reperfusion model. In this way, the hearts were subjected to ischemia/reperfusion and treated in the absence (received vehicle only; Krebs-Henseleit solution) or presence of following compounds 1 to 18 at a dose of 0.001 nM before ischemia 27

period (for 10 minutes) and during the entire period of reperfusion (30 minutes). It is important to mention that the dose of 0.001 nM administered in this study was used based on other types of studies that show that some steroids may have biological activity against heart failure at this dose [21].The results showed that both compounds 4 and 5 can significantly reduce (p = 0.05) the infarct size which was expressed as a percentage of the area at risk compared with the compounds 1-3 and 6-18 and vehicle-treated hearts (Schemes 7 and 8). This phenomenon may be conditioned by the interaction of the different functional groups involved in the chemical structure of both compounds 4 or 5 with some biomolecules. Analyzing these data and other studies which indicate that some adrenergic drugs exerts beneficial effects in patients with heart failure [50, 51]; in this investigation, the biological activity exerted by either compounds 4 or 5 against left ventricular pressure in the presence or absence of yohimbine (α2 receptor antagonist) [51] or butaxamine (β2 receptor antagonist) [52] was evaluated. The results showed that both compounds 4 or 5 decrease the left ventricular pressure (Scheme 9) in a dose-manner dependent and these effects was not inhibited in the presence of yohimbine (Scheme 10) or butaxamine (Scheme 11), These data suggest that molecular mechanism was not via the adrenergic system. In the search for the molecular mechanism involved in the biological activity (translated as negative inotropic activity) exerted by the compounds 4 or 5 on left ventricular pressure and analyzing some reports which suggest that heart failure is related to an increase in the density of M2 muscarinic receptors [53, 54]. In this investigation, the negative inotropic activity of either compounds 4 or 5 was evaluated using carbachol as control. The results showed that carbachol and both compounds 4 and 5 decrease the left ventricular pressure in a dosedependent manner. This phenomenon suggest that both compounds 4 and 5 can exert their effect on left ventricular pressure via cholinergic receptors activation. To evaluate this 28

hypothesis, the effect exerted by either compounds 4 or 5 was evaluated in the absence or presence of methoctramine (M2-muscarinic receptor antagonist) [55]. The results showed that negative inotropic activity of either compounds 4 or 5 was significantly inhibited (p = 0.05) with methoctramine (Scheme 12); these data suggest that effect exerted by either 4 or 5 was via M2-muscarinic receptor activation. Analyzing these data and other reports which indicate that M2-muscarinic receptor may induce changes in the levels of nitric oxide [56], in this study, the effect exerted by either compounds 4 or 5 in the presence or absence of

L-

NAME was evaluated. The results showed (Scheme 13) that both compounds 4 and 5 decreases the left ventricular pressure in a dose-dependent manner; however, this effect was significantly inhibited (p = 0.05) by L-NAME. These data suggest that the negative inotropic activity exerted by both compounds 4 or 5 implies an indirect of nitric oxide synthase activation. 4. Conclusions In this study, is reported a facile synthesis of a series of steroid derivatives (2 to 18) using some chemical strategies. Furthermore, the results of biological evaluation that exert both compounds 4 and 5 against heart failure indicated that either 4 or 5 can decrease both the infarct area and left ventricular pressure via M2-muscarinic receptor activation and resulting in nitric oxide synthase activation.

Acknowledgements None

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[44] T. Takekoshi, T. Synthesis of high performance aromatic polymersvianucleophilic nitro displacement reaction. Polymer J. 19 (1987) 191–202. [45] L. Figueroa, F. Díaz, M. Rosas, E. García, E. Pool, M. López, F. Rodríguez, Design and synthesis of some carbamazepine derivatives using several strategies, Lett. Org. Chem. 12 (2015) 394–401. [46] T. Sasaki, S. Eguchi, T. Kiriyama, O. Hiroaki, Studies on hetero-cage compounds—VI: Transannular cyclizations in pentacyclo [6.2. 1.0. 2, 70.4, 1005, 9] undecan-3, 6-dione system.Tetrahedron. 30 (1974) 2707-2712. [47] L. Young, W. Geldenhuys, O. Domingo, S. Malan, C. Van der Schyf, Synthesis and Biological Evaluation of Pentacycloundecylamines and Triquinylamines as Voltage‐Gated Calcium Channel Blockers, Archiv. Der Pharm. 349 (2016) 252-267. [48] R. Warrener, J. Malpass, D. Butler, G. Sun, Isoindole Cycloadditions. Part III: The Synthesis of" Windscreen Wiper" and Other N-Bridged Cavity Systems, Struct. Chem. 12 (2001) 291-304. [49] O. Onajole, Y. Coovadia, H. Kruger, G. Maguire, M. Pillay, T. Govender, Novel polycyclic ‘cage’-1, 2-diamines as potential anti-tuberculosis agents, Eur. J. Med Chem. 54 (2012) 1-9. [50] A. Levov, Y. Le-An, A. Komarova, V. Strokina, A. Soldatenkov, V. Khrustalev, Russian J. Org. Chem 44 (2008) 456-461. [51] J. Gozo, R. Yebes, Hemodynamic effects of isoxsuprine in cardiac failure, Chest. 86 (1984) 736-740. [52] T. Quinn, Ventricular tachycardia-like complexes in acute myocardial infarction, Chest. 88 (1985) 644.

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[53] A. Kumar, A. Joshi, S. Starling, β-Blockers: A systematic review, J. Chem. 3 (2011) 3247. [54] M. Wilkinson, A. Giles, J. Armour, R. Cardinal, Ventricular, but not atrial, M2muscarinic receptors increase in the canine pacing-overdrive model of heart failure, Canadian J. Cardiol. 12 (1996) 71-76. [55] E. Giraldo, R. Micheletti, E. Montagna, A. Giachetti, M. Vigano, H. Ladinsky, C. Melchiorre, Binding and functional characterization of the cardioselective muscarinic antagonist methoctramine, J. Pharmacol. Exp. Ther. 244 (1988) 1016-1020. [56] L. Sterin‐Borda, A. Echagüe, C. Leiros, A. Genaro, E. Borda, Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor‐inotropic response, British J. Pharmacol. 115 (1995) 1525-1531. [57] A. Cornelis, P. Laszlo, P. Pennetreau, Clay-supported reagents. 5. Nitration of estrone into 2-nitroestrone by clay-supported ferric nitrate, J. Org. Chem. 48 (1983) 4771-4772.

36

O

O

O–

O–

N+

N+

i

O

O

HO

1

O

2

Scheme 1. Synthesis of 2-nitroestrone carbaldehyde (2). Conditions and regents: i = dimethyl sulfoxide, reflux.

37

O

OH

O

HO

–

N+ O

5 NH 2

O Cl

OH

iv

O O–

OH

O– N+

O–

N+

iii

ii

O

O

N+ O

3 2

O

O

4 O

v

OH

HO

O– N+ O

6 O

Scheme 2. Preparation of alkyne-steroid derivatives (compounds 3 to 6). Conditions and regents: ii = 3-ethynylaniline, reflux; iii = 6-Chloro-hex-1-yne, reflux; iv = Hex-5-ynoic acid, reflux; v = Hex-5-yn-1-ol, reflux.

38

NH 2

NH 2

OH

OH

O–

O– N+

N+

vi

O

O

3

7

N

O

Cl

Cl

OH

OH

O–

O–

vi

N+

N+

O

O

4

8

N

O

O

O

OH

HO

OH

HO

O–

O–

vi

N+

N+ O

O

5

9

N

O

OH

OH

HO

HO

O–

O–

vi

N+

N+ O

O

6

N

10

O

Scheme 3. Preparation of carbonitrile-steroid derivatives (compounds 7 to 10). Conditions and regents: vi = hydroxilamine hydrochloride, dimethyl sulfoxide, reflux.

39

H

O

O

HO

O

O2N

+ 2H+

- 1H+

H

NH2OH

O

O2 N

O2N

O2N

O2N

Cl

S

S

S

O2N OH

Cl

HO

N

HO

NH

O2N

- H2O

H N

O H

H O

O

N H

OH2

O

H

OH S

Cl

O2 N

O2N

O2 N

O2N

+ H+ O

O O

N

S H

N

O

N

N

H

S H

O

O

- H2O H

O2N

O2N

O

+ O

N

S

O S

C S

N H

Scheme 4. Reaction mechanism involved in the formation of a carbonitrile-steroid derivative.

40

NH 2 NH2 OH OH

O– O N+

vii

O

11

N

7

N

Cl Cl

OH OH

O– O

vii

N+ O

12

N

8

N

O O

OH OH

HO

HO

O– O N+

vii

O

13

N

9

N

OH OH

HO

HO

O– O

vii

N+ O

N N

14

10

Scheme 5. Preparation of ether-steroid derivatives (compounds 11 to 14). Conditions and regents: vii = 4-phenyl-1-buten-4-ol, dimethyl sulfoxide/K2CO3, room temperature.

41

NH2

OH

NH2

OH O

viii O

15 N

11

N

Cl

Cl

OH

OH O

viii O

16 N

12

N

O

O

OH

OH

HO

OH

HO

HO O

viii O

17 N

13

N

OH

HO O

viii O

18 N N

14

Scheme 6. Preparation of steroid-azahexacyclo derivatives (15-18). Conditions and regents: viii = Copper(II), room temperature.

42

25

INFARCT AREA (%)

20

15

10

5

0 Control C-1

C-2

C-3

C-4

C-5

C-6

C-7

C-8

C-9

COMPOUNDS Scheme 7. Effect exerted by the control (without treatment) and compounds 1 to 9 [0.001 nM] against ischemia/reperfusion injury translated as infarct area. The results showed that compounds 4 and 5 decreased the area de infarct (p = 0.05) compared with control conditions. Each bar represents the mean ± S.E. of 9 experiments.

43

30

INFARCT AREA (%)

25

20

15

10

5

0 Control C-10

C-11

C-12

C-13

C-14

C-15

C-16

C-17

C-18

COMPOUNDS Scheme 8. Effect exerted by the control (with treatment) and compounds 10 to 18 [0.001 nM] against ischemia/reperfusion injury translated as infarct area. The results showed that there was no significant difference in the effect exerted by any of the compounds (10 to 18) on the area of infarction compared to the control conditions. Each bar represents the mean ± SE of 9 experiments.

44

Scheme 9. Biological activity of compounds 4 (left) or 5 (right) on left ventricular pressure at a dose of 0.001 to 100 nM. The results showed that effect exerted by either compounds 4 or 5 decrease the left ventricular pressure in a dose-dependent manner.

45

Compound 4 Compound 5 Compound 4 + Yohimbine [1nM]

46

Compound 5 + Yohimbine [1NM]

44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM] Scheme 10. Biological activity exerted by either compounds 4 or 5 in the absence or presence of yohimbine on LVP. Intracoronary boluses (50 μl) of 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the LVP was determined. The results showed that compounds 4 or 5 decrease the LVP in a dependent-dose manner and this effect was not inhibited in the presence of yohimbine. Each bar represents the mean ± SE of 9 experiments. LVP = left ventricular pressure.

46

Compound 4 Compoun 5 Compound 4 + Butaxamine [1nM] Compound 5 + Butaxamine [1nM]

48 46 44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM] Scheme 11. Effects induced by either compounds 4 or 5 in the absence or presence of butaxamine on LVP. Intracoronary boluses (50 μl) of 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the LVP was determined. The results showed that compounds 4 or 5 decrease the LVP in a dependent-dose manner and this effect was not inhibited in the presence of butaxamine. Each bar represents the mean ± SE of 9 experiments. LVP = left ventricular pressure.

47

Compound 4 Carbachol Compound 5 Compound 4 + Methoctramine [1 nM] Compound 5 + Methoctramine [1 nM]

48 46 44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM] Scheme 12. Effects induced by either charbachol or compounds 4 and 5 on LVP. Intracoronary boluses (50 μl) of carbachol or 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the LVP was determined. The results showed that either carbachol or compounds 4 and 5 decrease the LVP in a dependent dose manner. Other results showed that biological activity of 4 and 5 was significantly inhibited (p = 0.05) in the presence of methoctramine. Each bar represents the mean ± SE of 9 experiments. LVP = left ventricular pressure.

48

Compound 4 Compound 5 Compound 4 + L-NAME [1 nM] Compound 5 + L-NAME [1 nM]

48 46 44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM] Scheme 13. Effects exerted by either compounds 4 or 5 in the absence or presence of LNAME on LVP. Intracoronary boluses (50 μl) of 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the LVP was determined. The results showed that both compounds 4 or 5 increases the LVP in a dependent dose manner and this effect was significantly inhibited (p = 0.05) in the presence of L-NAME. Each bar represents the mean ± SE of 9 experiments. LVP = left ventricular pressure; L-NAME (Nω-Nitro-L-arginine methyl ester).

49

Table 1. Experimental design: Hearts were subjected to ischemia/reperfusion and treated with in absence (control; received vehicle only, Krebs-Henseleit solution) or in the presence of compounds C-1 to C-18 (at a dose of 0.001 nM). n = 9 Treatment Groups Control I Compound 1 II Compound 2 III Compound 3 IV Compound 4 V Compound 5 VI Compound 6 VII Compound 7 VIII Compound 8 IX Compound 9 X Compound 10 XI Compound 11 XII Compound 12 XIII Compound 13 XIV Compound 14 XV Compound 15 XVI Compound 16 XVII Compound 17 XVIII Compound 18 XIX

O

O

O–

O–

N+

N+

i

O

O

HO

1

O

2

Scheme 1. Synthesis of 2-nitroestrone carbaldehyde (2). Conditions and regents: i = dimethyl sulfoxide, reflux.

50

O

OH

HO

O– N+ O

5 NH 2

O Cl

OH

iv

O O–

OH

O– N+

O– N

N+

iii

ii

O

O

+

O

3 2

O

O

4 O

v

OH

HO

O– N+ O

6 O

Scheme 2. Preparation of alkyne-steroid derivatives (compounds 3 to 6). Conditions and regents: ii = 3-ethynylaniline, reflux; iii = 6-Chloro-hex-1-yne, reflux; iv = Hex-5-ynoic acid, reflux; v = Hex-5-yn-1-ol, reflux.

51

NH 2

NH 2

OH

OH

O–

O– N+

N+

vi

O

O

3

7

N

O

Cl

Cl

OH

OH

O–

O

–

vi

N+

N+

O

O

4

8

N

O

O

O

OH

HO

OH

HO

O–

O–

vi

N+

N+ O

O

5

9

N

O

OH

OH

HO O

O–

vi

N+

HO

–

N+ O

O

6

N

10

O

Scheme 3. Preparation of carbonitrile-steroid derivatives (compounds 7 to 10). Conditions and regents: vi = hydroxilamine hydrochloride, dimethyl sulfoxide, reflux.

52

H

O

O

HO

O

O2N

+ 2H+

- 1H+

H

NH2OH

O

O2 N

O2N

O2N

O2N

Cl

S

S

S

O2N OH

Cl

HO

N

NH

O2N

- H2O

H N

HO

O H

H

H

OH2

O

O

N

O

H

OH S

Cl

O2 N

O2N

O2 N

O2N

+ H+ O

O O

N

S H

N

O

N

N

H

S H

O

O

- H2O H

O2N

O2N

O

+ O

N

S

O S

C S

N H

Scheme 4. Reaction mechanism involved in the formation of a carbonitrile-steroid derivative.

53

NH 2 NH2 OH OH

O– O N+

vii

O

11

N

7

N

Cl Cl

OH OH

O– O

vii

N+ O

12

N

8

N

O O

OH OH

HO

HO

O– O N+

vii

O

13

N

9

N

OH OH

HO

HO

O– O

vii

N+ O

N N

14

10

Scheme 5. Preparation of ether-steroid derivatives (compounds 11 to 14). Conditions and regents: vii = 4-phenyl-1-buten-4-ol, dimethyl sulfoxide/K2CO3, room temperature.

54

NH2

OH

NH2

OH O

viii O

15 N

11

N

Cl

Cl

OH

OH O

viii O

16 N

12

N

O

O

OH

OH

HO

OH

HO

HO O

viii O

17 N

13

N

OH

HO O

viii O

18 N N

14

Scheme 6. Preparation of steroid-azahexacyclo derivatives (15-18). Conditions and regents: viii = Copper(II), room temperature.

55

25

INFARCT AREA (%)

20

15

10

5

0 Control C-1

C-2

C-3

C-4

C-5

C-6

C-7

C-8

C-9

COMPOUNDS Scheme 7. Effect exerted by the control (without treatment) and compounds 1 to 9 [0.001 nM] against ischemia/reperfusion injury translated as infarct area. The results showed that compounds 4 and 5 decreased the area de infarct (p = 0.05) compared with control conditions. Each bar represents the mean ± S.E. of 9 experiments.

56

30

INFARCT AREA (%)

25

20

15

10

5

0 Control C-10

C-11

C-12

C-13

C-14

C-15

C-16

C-17

C-18

COMPOUNDS Scheme 8. Effect exerted by the control (with treatment) and compounds 10 to 18 [0.001 nM] against ischemia/reperfusion injury translated as infarct area. The results showed that there was no significant difference in the effect exerted by any of the compounds (10 to 18) on the area of infarction compared to the control conditions. Each bar represents the mean ± SE of 9 experiments.

57

Scheme 9. Biological activity of compounds 4 (left) or 5 (right) on left ventricular pressure at a dose of 0.001 to 100 nM. The results showed that effect exerted by either compounds 4 or 5 decrease the left ventricular pressure in a dose-dependent manner.

58

Compound 4 Compound 5 Compound 4 + Yohimbine [1nM]

46

Compound 5 + Yohimbine [1NM]

44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM] Scheme 10. Biological activity exerted by either compounds 4 or 5 in the absence or presence of yohimbine on LVP. Intracoronary boluses (50 μl) of 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the LVP was determined. The results showed that compounds 4 or 5 decrease the LVP in a dependent-dose manner and this effect was not inhibited in the presence of yohimbine. Each bar represents the mean ± SE of 9 experiments. LVP = left ventricular pressure.

59

Compound 4 Compoun 5 Compound 4 + Butaxamine [1nM] Compound 5 + Butaxamine [1nM]

48 46 44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM] Scheme 11. Effects induced by either compounds 4 or 5 in the absence or presence of butaxamine on LVP. Intracoronary boluses (50 μl) of 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the LVP was determined. The results showed that compounds 4 or 5 decrease the LVP in a dependent-dose manner and this effect was not inhibited in the presence of butaxamine. Each bar represents the mean ± SE of 9 experiments. LVP = left ventricular pressure.

60

Compound 4 Carbachol Compound 5 Compound 4 + Methoctramine [1 nM] Compound 5 + Methoctramine [1 nM]

48 46 44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM] Scheme 12. Effects induced by either charbachol or compounds 4 and 5 on LVP. Intracoronary boluses (50 μl) of carbachol or 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the LVP was determined. The results showed that either carbachol or compounds 4 and 5 decrease the LVP in a dependent dose manner. Other results showed that biological activity of 4 and 5 was significantly inhibited (p = 0.05) in the presence of methoctramine. Each bar represents the mean ± SE of 9 experiments. LVP = left ventricular pressure.

61

Compound 4 Compound 5 Compound 4 + L-NAME [1 nM] Compound 5 + L-NAME [1 nM]

48 46 44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM]

Scheme 13. Effects exerted by either compounds 4 or 5 in the absence or presence of LNAME on LVP. Intracoronary boluses (50 μl) of 4 or 5 [0.001 to 100 nM] were administered and the corresponding effect on the LVP was determined. The results showed that both compounds 4 or 5 increases the LVP in a dependent dose manner and this effect was significantly inhibited (p = 0.05) in the presence of L-NAME. Each bar represents the mean ± SE of 9 experiments. LVP = left ventricular pressure; L-NAME (Nω-Nitro-L-arginine methyl ester).

62

Table 1. Experimental design: Hearts were subjected to ischemia/reperfusion and treated with in absence (control; received vehicle only, Krebs-Henseleit solution) or in the presence of compounds C-1 to C-18 (at a dose of 0.001 nM). n = 9 Treatment Groups Control I Compound 1 II Compound 2 III Compound 3 IV Compound 4 V Compound 5 VI Compound 6 VII Compound 7 VIII Compound 8 IX Compound 9 X Compound 10 XI Compound 11 XII Compound 12 XIII Compound 13 XIV Compound 14 XV Compound 15 XVI Compound 16 XVII Compound 17 XVIII Compound 18 XIX

GRAPHICAL ABSTRACT Design and Synthesis of two new steroid derivatives with biological activity on heart failure via the M2muscarinic receptor activation *Figueroa-Valverde Lauro, Lopez-Ramos Maria, Lopez-Gutierrez Tomas, Diaz Cedillo Francisco, GarciaMartinez Rolando, Rosas-Nexticapa Marcela, Mateu-Armand Virginia, Garcimarero-Espino E. Alejandra, Ortiz-Ake Yazmin. *Laboratory of Pharmaco-Chemistry at the Faculty of Chemical Biological Sciences of the University Autonomous of Campeche,

O Cl O OH OH

HO

O– O–

O–

2 stages

N+

2 stages

O

N+ O

N+ O HO

1 4

5 O

O

63

Compound 4 Carbachol Compound 5 Compound 4 + Methoctramine [1 nM] Compound 5 + Methoctramine [1 nM]

48 46 44

LVP (mm Hg)

42 40 38 36 34 32 30 28 0.0001

0.001

0.01

0.1

1

10

100

1000

Log DOSE [nM]

Highlights



The aim of this study was to synthesize two steroid derivatives (4 or 5) to evaluate their biological activity on infarct area and left ventricular pressure using an ischemia/reperfusion model.



The results showed that either the compounds 4 or 5 significantly decrease the heart failure (translated as infarct area) and left ventricular pressure compared with control conditions.



In conclusion, the biological activity of compounds 4 or 5 exerted on left ventricular pressure was via M2-muscarinic receptor activation

64