Transcription
Mistry and Singh Future Journal of Pharmaceutical Sciences(2022) e Journal ofPharmaceutical SciencesOpen AccessRESEARCHSynthesis and in vitro antimicrobial activityof new steroidal hydrazone derivativesShailesh Mistry*and Akhilesh Kumar SinghAbstractBackground: For many years, various drugs have been used for the treatment of infectious diseases but some bacterial microorganisms have induced resistance to several drugs. In a search of new antimicrobial agents, a series of newsteroidal hydrazones were designed and synthesized.Result: The structures of the compounds were established based on the spectral data. The in vitro antimicrobialactivity of some newly synthesized compounds against bacteria and fungi was studied.Conclusion: New compounds showed better or similar antimicrobial activity. Designing more efficient steroidalhydrazones from ketosteroid based on the current study may successfully lead to the development of antimicrobialagent.Keywords: Androstene, Estrane, Hydralazine hydrochloride, Hydrazone, Antimicrobial activityGraphical abstract*Correspondence: [email protected] Institute of Applied Science, Parul University, P.O. Limbda, Ta.Waghodiya, Vadodara, Gujarat 391760, India The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, whichpermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit lineto the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
Mistry and Singh F uture Journal of Pharmaceutical Sciences(2022) 8:7BackgroundHydrazones are synthesized by condensation of aldehyde/ketone with hydrazine. Hydrazones are also synthesized by coupling reaction of aryl diazonium saltswith active hydrogen compounds [1]. Hydrazone havegained great importance due to their diverse biologicalproperties including antibacterial and antifungal [2],anticonvulsant [3], anti-inflammatory [4], antimalarial[5] and antituberculosis [6] activities. When they areused as intermediates, coupling products can be synthesized by using the active hydrogen component ofazomethine group [7].Searching for new molecules in the field of steroid willnever end. Researchers are always been interested to doresearch on steroid due to its particular biological andpharmacological action. The Steroidal drugs have beenwidely used in traditional medicines. The versatile activity of androstene and estrane series indicates that thesemolecules could be a key starting material for developinga new drug. Particularly, this invention relates to therapeutically valuable steroids of androstene and estraneseries having hydrazone function. Steroidal hydrazoneshave received extensive attention of scientists becausethey exhibit some biological activities such as antifungal,antibacterial, antiproliferative, antituberculosis, antiviraland anticancer [8–16].Structural modification of steroids requires great synthetic effort and still a vivacious area of research. Steroidal ring modification and incorporation of heteroatomor replacing one or more carbon atoms in steroidal molecule may improves its biological activities have beenresearched and reported [17–26]. About preparationof steroidal derivatives, introduction of methyl group ata certain position of steroids may significantly changeFig. 1 Steroidal hydrazone of androstene and estranePage 2 of 10their bioactivities [27]. The investigation of new steroidalderivatives has been given great attention. Hydralazineplays an important role as antihypertensive drug and soldunder the brand name Apresolin. Hydralazine belongs tothe hydrazinophthalazine class of drugs [28]. Hydralazine derivatives have wide applications in the treatmentof diseases such as tuberculosis, mental disorder [29].Hydralazine can be used as antimicrobial, antihypertensive, antimalarial and antitumoral agents [30–32].Steroidal hydrazone containing nitrogen atom has beensynthesized with the aim of improving selectivity. However steroidal hydrazones with hydralazine hydrochloridewere rarely reported. We decided to further explore theantimicrobial properties of steroidal hydrazone by synthesizing new analogs with suitable structural modifications (Fig. 1).MethodsAll the chemicals were used as received from commercialsources. All reaction progress were monitored by thinlayer chromatography (TLC) analysis using silica gel 60 F254 TLC plates. The melting point was determined ona Veego-matic melting point apparatus. IR spectra wererecorded using potassium bromide disks on a ShimadzoIR Affinity 1S. The wave numbers are given in cm 1. 1Hand C13 NMR spectra were recorded on Bruker AvanceII spectrophotometer at 400 and 300 MHz and 100 and75 MHz, respectively, with tetramethylsilane as an internal reference; the chemical shifts were measured in ppmwith respect to the solvent. Mass spectra were recordedon TSQ Quantum and water make Acquity model UPLCconnected with SQ detector (Single Quadra pole) software Mass Lynx (401) instrument equipped with electro
Mistry and Singh F uture Journal of Pharmaceutical Sciences(2022) 8:7Page 3 of 10Scheme 1 Reagents and conditions (i) methanol, potassium acetate, 2 (hydralazine hydrochloride), reflux, 6 hScheme 2 Reagents and conditions (i) chloranil, tert. butanol, reflux, 5 h; (ii) tert. butanol, potassium tert. butoxide, methyl iodide, reflux,4 h; (iii)methanol, potassium acetate, 2 (hydralazine hydrochloride), reflux, 6 hspray ionization (ESI) ion source. Measurements weretaken in positive (MS ) ion mode.ExperimentalGeneral procedure for the preparation of steroidalhydrazone (3–8, 10, 11, 15, 16, 18, 20 and 22)Ketosteroid (1, 9A, 9B, 14A, 14B, 17A, 19 and 21)(Schemes 1, 2, 4, 5, 6, 7) (2.5 mmol) and hydralazinehydrochloride (2) (2.6 mmol) with Potassium acetate(2.6 mmol) in Methanol (25 ml) was refluxed for 6 h.After the end of the reaction (monitored by TLC), themixture was allowed to cool and added water (25 ml).On stirring, the precipitate was formed and collectedby filtration. This solid was purified from Methanol(10 ml) and dried at 45–50 C to afford the corresponding compounds as yellow solid.Synthetic procedure of 9A (Δ6‑testosterone)To a solution of Testosterone (9) (2.0 g) in tert. butanol(20 ml) was added chloranil (1.6 g) and the reactionmixture was heated to 80 C for 5 h. After cooling, thereaction mixture was poured into 10% Na2CO3, solution and the products were extracted with methylenedichloride. The extracts were washed with water driedover anhydrous sodium sulfate and the solvent wasevaporated to afford crude crystals. Recrystallizationfrom acetone gave (1.0 g) 9A.Synthetic procedure of 9B5 g of Testosterone (9) in tert. butanol (50 ml) wasstirred under nitrogen and charged potassium tert.butoxide (5 g) in the mixture. Stirred the reaction massuntil clear solution obtained. Added drop wise solution
Mistry and Singh F uture Journal of Pharmaceutical Sciences(2022) 8:7of methyl iodide (7.0 ml) in to the reaction mass at25–30 C. Reaction mass allowed to stirred for 4 h(monitored by TLC) at 25–30 C. Water (100 ml) wasadded, the tert-butanol removed in vacuum and cooledthe suspended mass and filtered. Recrystallizationfrom acetone to give compound (2.5 g) 9B. MS (ESI )m/z: Calculated for C 21H32O2 [M H] 316.48; found,317.31.Method for the preparation of compound 10 and 11according to the general procedureSee Scheme 2.Synthetic procedure of (13)Estrone (12) (0.75 g, 2.8 mol) and hydralazine hydrochloride (2) (0.58 g, 2.9 mol) with Potassium acetate(0.28 g, 2.8 mol) in Tetrahydrofuran (25 ml) was refluxedfor 8–10 h. After the end of the reaction (monitored byTLC), Distilled off Tetrahydrofuran under reduced pressure and yellowish oily mass allowed to cool and addedMethanol (15 ml). On stirring, the precipitate was formedPage 4 of 10and collected by filtration and washed with water (20 ml).This solid was purified from Methanol (10 ml) and driedat 45–50 C to afford the corresponding compounds (13)as yellow solid (Scheme 3).Synthetic procedure of 14A (4‑methyl nandrolone) and 14BTo a solution of Nandrolone (14) in tert. butanol wasadded potassium tert-butoxide with stirring underinert atmosphere by nitrogen blanketing. The solutionof methyl iodide (3.8 ml, 61.04 mmol) in tert-butanol(18 ml) was added drop wise over a period of 30 min. andthe resulting mixture was refluxed for 30 min (monitoredby TLC). After cooling, the reaction mixture was acidified with 1 M HCl and the solvent was evaporated. Thecrude product was extracted with Methylene dichloride,the organic layer was washed with N aHSO3 and water,dried with anhydrous sodium sulfate and distilled out solvent under vacuum to give crude product which was separated by flash chromatography over short silica columneluting with 30% ethyl acetate in n-Hexane to give whitesolid of 4-methyl-androst-4-en-3-one-17β-ol (14A) MS(ESI ) m/z: Calculated for C19H28O2 [M H] 288.42;Scheme 3 Reagents and conditions (i) methanol, potassium acetate, 2 (hydralazine hydrochloride), reflux, 6 hScheme 4 Reagents and conditions (i) tert-butanol, potassium tert-butoxide, methyl iodide, reflux, 1 h; (ii) methanol, potassium acetate, 2(hydralazine hydrochloride), reflux, 6 h
Mistry and Singh F uture Journal of Pharmaceutical Sciences(2022) 8:7Page 5 of 10found, 289.16 and 4,4-dimethyl-androst-5-en-3-one17β-ol(14B) MS (ESI ) m/z: Calculated for C20H30O2[M H] 302.45; found, 303.21.Synthetic procedure of 17A (Δ6‑norethisterone) accordingto the procedure 9A and method for the preparationof compound 18 according to the general procedureMethod for the preparation of compound 15 and 16according to the general procedureMethod for the preparation of compound 20 accordingto the general procedureSee Scheme 4.See Scheme 5.See Scheme 6.Scheme 5 Reagents and conditions (i) Chloranil, tert-butanol, reflux, 5 h; (ii) methanol, potassium acetate (2) hydralazine hydrochloride, reflux, 6 hScheme 6 Reagents and conditions (i) methanol, potassium acetate (2) hydralazine hydrochloride, reflux, 6 hScheme 7 Agents and conditions (i) methanol, potassium acetate, (2) hydralazine hydrochloride, reflux, 6 h
Mistry and Singh F uture Journal of Pharmaceutical Sciences(2022) 8:7Method for the preparation of compound 22 accordingto the general procedureSee Scheme androstene‑17β‑ol(3) Yellow solid, 0.63 g, Yield 67%; mp: 190 C Dec.;IR (KBr, cm 1): 1582 (C C), 1605(C N), 2934 (CH),3387(NH), 3410(OH); 1H NMR (400 MHz C DCl3) δ,ppm: 0.76(s, 3H, –CH3), 1.17(s, 3H, –CH3), 3.62(s, 1H,–CH), 5.70(s, 1H, –CH) 7.51(m, 1H, Ar–H), 7.67(m,2H, Ar–H), 7.86(s, 1H, Ar–H), 8.30 (m, 1H,Ar–H); 13CNMR (100 MHz C DCl3): 11.0, 17.4, 20.6, 23.3, 30.3, 31.5,32.8, 33.9, 35.6, 35.7, 36.4, 42.8, 50.4, 53.9, 81.4, 123.7,125.8, 126.9, 127.3, 128.7, 131.4, 131.7, 137.5, 146.9,152.0, 161.4; MS (ESI ) m/z: calculated for C27H34N4O[M H] 430.27; found, or‑4‑androstene‑17β‑ol (4) Yellow solid, 0.70 g, Yield 62.5%; mp: 200 C;IR (KBr, cm 1): 1590(C C), 1612(C N), 2912(CH),3352(NH), 3409 (OH); 1H NMR (400 MHz C DCl3) δ,ppm: 0.76(s, 3H, –CH3), 3.61(s, 1H, –CH), 5.78(s, 1H, –CH), 7.71(m, 1H, Ar–H), 7.81(m, 2H, Ar–H), 8.29(s, 1H,Ar–H), 8.51(m, 1H, Ar–H); 13C NMR (100 MHz C DCl3):11.1, 23.1, 26.1, 26.5, 30.3, 30.6, 35.4, 36.4, 40.4, 42.5,43.0, 49.5, 49.7, 81.5, 122.9, 124.4, 126.2, 127.5, 128.1,131.2, 137.6, 145.8, 166.9; MS (ESI ) m/z: Calculated forC26H32N4O [M H] 416.26; found, azono)‑19‑nor‑4‑androstene‑17β‑ol (5) Yellow solid, 0.65 g, Yield 60%; mp:165–167 C; IR (KBr, c m 1): 1585(C C), 1615(C N),2956(CH), 3271( C–H), 3331(OH); 1H NMR (400 MHz CDCl3) δ, ppm: 0.97(s, 3H, –CH3), 2.86(s, 1H, –CH),5.73(s, 1H, –CH), 7.75(m, 1H, Ar–H), 7.92(m, 2H,Ar–H), 8.30(s, 1H, Ar–H), 8.45(m, 1H, Ar–H); 13C NMR(100 MHz CDCl3): 11.9, 23.1, 26.4, 26.8, 30.8, 32.6, 35.6,36.7, 39.0, 41.2, 42.7, 47.0, 49.3, 74.3, 79.8, 87.6, 124.7,125.9, 126.4, 127.3, 129.1, 131.9, 132.7, 135.5, 141.7,158.5, 165.8; MS (ESI ) m/z: Calculated for C28H32N4O[M H] 440.58; found, ��17β‑ol (6) Yellow solid, 0.73 g,Yield 67%; mp: 200 C; IR (KBr, cm 1): 1580(C C),1608(C N), 2934(CH), 3251 ( C–H), 3404(OH); 1HNMR (400 MHz CDCl3) δ, ppm: 0.75(d, 3H, J 7.2 Hz,–CH3), 0.90(s, 3H, –CH3), 2.56(s, 1H, –CH), 5.82(s, 1H, –CH), 7.75(m, 1H, Ar–H), 7.92(m, 2H, Ar–H), 8.33(s, 1H,Ar–H), 8.69(m, 1H, Ar–H); 13C NMR (100 MHz C DCl3):12.6, 12.8, 22.2, 26.7, 30.6, 32.3, 36.6, 38.7, 42.0, 43.2,Page 6 of 1043.4 45.9, 46.9, 74.1, 78.6, 87.3, 124.1, 125.2, 126.7, 127.6,128.9, 131.5, 132.8, 135.9, 141.3, 157.9, 165.0; MS (ESI )m/z: Calculated for C29H34N4O [M H] 454.27; ol (7) Yellow solid, 0.75 g, Yield 66%;mp: 210 C; IR (KBr, c m 1): 1587(C C), 1610(C N),2927(CH), 3362(NH), 3410(OH); 1H NMR (400 MHz CDCl3) δ, ppm: 0.73(d, 3H, J 8 Hz, –CH3), 0.90(s,3H,–CH3), 3.61(s,1H,–CH), 5.72(s,1H,–CH), 7.71(m,1H,Ar–H), 7.97(m,2H,Ar–H), 8.35(s,1H,Ar–H), 8.67(m,1H,Ar–H); 13C NMR (100 M HzCDCl3): 11.8, 12.9, 22.5, 26.9,30.4, 32.9, 36.9, 38.4, 42.1, 43.3, 43.9, 45.4, 46.3, 81.6,123.9, 125.3, 126.5, 127.8, 128.3, 131.7, 132.6, 135.4,141.2, 156.9, 163.7; MS (ESI ) m/z: calculated forC27H34N4O [M H] 430.27; found, 431.22. Anal. Calc.C:75.31, H:7.96, N:13.01; found C:75.24, H:7.59, azono)‑19‑nor‑4‑androstene‑17β‑ol (8) Yellow solid, 0.77 g, Yield 71.9%;mp: 200 C; IR (KBr, c m 1): 1575(C C), 1609(C N),2923(CH), 3393(OH); 1H NMR (400 MHz CDCl3) δ,ppm: 0.81(t, 3H, J 7.1 Hz, –CH3), 1.20(m, 2H, –CH2),3.48(s,1H,–CH), 5.73(s,1H,–CH), 7.76(m, 1H, Ar–H),7.93(m, 2H, Ar–H), 8.33(s, 1H, Ar–H), 8.49(m, 1H, Ar–H);13C NMR (100 MHz CDCl3): 8.9, 18.6, 21.5, 26.0,28.0, 30.4, 35.2, 35.8, 38.7, 40.5, 42.2, 47.5, 48.6, 50.2,80.2, 123.6, 124.1, 125.9, 127.0, 128.2, 131.4, 137.3,145.2, 168.5; MS (ESI ) m/z: Calculated for C27H34N4O[M H] 430.59; found, ta‑4,6‑dien‑17β‑ol(10) Yellow solid, 0.66 g, Yield 58.9%; mp: 200 C;IR(KBr, cm 1):1587(C C), 1606(C N), 2933(CH),3381(NH), 3456(OH); 1H NMR (400 MHz C DCl3) δ,ppm: 0.74(s, 3H, –CH3), 0.94(s, 3H, –CH3), 3.55(t, 1H,J 16 Hz, –CH), 5.70(s, 1H, –CH), 5.98(s, 1H, –CH),7.34–7.38(m, 1H, Ar–H), 7.49–7.53(m, 2H, Ar–H),7.68 (s, 1H, Ar–H), 8.25–8.30(m, 1H, Ar–H); 13C NMR(100 MHz CDCl3): 11.0, 16.8, 20.5, 22.3, 23.0, 30.3, 33.2,36.1, 36.5, 37.2, 43.7, 48.7, 50.9, 81.3, 124.1, 124.2, 125.9,127.2, 127.5, 128.6, 131.5, 131.6, 133.8, 137.8, 146.5, 152.0, 161.6; MS (ESI ) m/z: Calculated for C27H32N4O[M H] 428.26; found, 429. 4. Anal. Calc. C:75.67,H:7.53, N:13.07; found (C:74.29, H:8.15, ��‑dimethyl‑5‑androstene‑17β‑ol (11) Yellow solid, 0.6 g, Yield 55.0%; mp:208–210 C; IR (KBr, c m 1): 1584(C C), 1607(C N),2931(CH), 3367(NH), 3426 (OH); 1H NMR (400 MHz CDCl3) δ, ppm: 0.72(s,3H,–CH3), 0.96(s, 3H, –CH3),1.22(s, 6H, –CH3), 3.67(d, 1H, J 8.03 Hz, –CH),
Mistry and Singh F uture Journal of Pharmaceutical Sciences(2022) 8:75.63(m,1H,–CH), 7.65(m, 1H, Ar–H), 7.87(m, 2H, Ar–H),8.11(s, 1H, Ar–H), 8.39(m, 1H, Ar–H); 13C NMR(100 MHz CDCl3): 12.0, 18.7, 20.8, 23.1, 25.4, 28.4, 30.1,31.3, 39.2, 42.4, 48.7, 50.2, 81.5, 124.2, 126.1, 126.5, 127.3,128.1, 144.6, 149.5, 163.2; MS (ESI ) m/z: calculated forC29H38N4O [M H] 458.64; found, �(phthalazine‑1yl‑hydrazono) (13) Yellow solid, 0.75 g, Yield 66%; mp: 167–169 C; IR (KBr, c m 1): 1585(C C), 1602, 1649(C N),2935(CH), 3055(aromatic CH), 3383(NH); 1H NMR(400 MHz DMSO) δ, ppm: 0.98(s, 3H, –CH3), 6.51(d, 1H,J 4 Hz, Ar–H, estrone), 6.57(d, 1H, J 4 Hz, estrone),7.14(d, 1H, J 8.48 Hz, Ar–H, estrone), 7.70 (m, 3H,Ar–H, hydralazine), 8.00(s, 1H, Ar–H, hydralazine),8.23(s, 1H, Ar–H, hydralazine), 9.08(s, 1H, –OH), 11.21(s,1H, –NH); 13C NMR (100 MHz DMSO): 16.9, 25.9, 27.2,29.1, 30.7, 35.1, 38.1,43.8, 44.3, 52.1, 112.86, 114.9, 115.9,123.3, 126.2, 126.8, 131.4, 136.5, 137.1, 145.7, 168.0, 176.6;MS (ESI ) m/z: Calculated for C26H28N4O [M H] 412.53; found, 413.4. Anal. Calc. C:75.70, H:6.84, N:13.58;found (C:74.35, H:6.72, methyl‑19‑nor‑4‑androstene‑17β‑ol (15) Yellow solid, 0.4 g, Yield 36%;mp: 200 C; IR (KBr, c m 1): 1579(C C), 1607(C N),2932(CH), 3366(NH), 3415(OH); 1HNMR (400 MHz CDCl3) δ, ppm: 0.93(s, 3H, –CH3), 1.22(s, 3H, –CH3),3.53(s, 1H, –CH), 7.71(m, 1H, Ar–H), 7.87(m, 2H,Ar–H), 8.18(s, 1H, Ar–H), 8.51(m, 5H, Ar–H); 13C NMR(100 MHz CDCl3):11.3, 18.0, 23.3, 24.7, 26.1, 26.5, 29.7,30.1, 30.6, 35.4, 36.4, 40.4, 42.5, 43.0, 49.5, 49.7, 81.5,124.4, 129.2, 138.4, 166.9; MS (ESI ) m/z: Calculated forC27H34N4O [M H] 430.27; found, l (16) Yellow solid, 0.6 g, Yield66.6%; mp: 109–108 C; IR (KBr, cm 1): 1588(C C),1606(C N), 2954(CH), 3371(NH), 3456(OH); 1H NMR(400 MHz CDCl3) δ, ppm: 0.97(s, 3H, –CH3), 1.21 (s, 6H,–CH3), 3.57(s, 1H, –CH), 5.56 (d, 1H, J 7.1 Hz, –CH),7.51(m, 1H, Ar–H), 7.71(m, 2H, Ar–H), 8.21(s,1H,Ar–H),8.41(m, 1H, Ar–H); 13C NMR (100 MHz CDCl3): 13.6,21.4, 23.9, 24.7, 27.4, 30.7, 31.1, 31.8, 32.1, 33.8, 37.1, 38.4,44.2, 48.9, 80.8, 119.9, 124.3, 124.6, 125.5, 127.0, 127.4,128.3, 131.2, 131.8, 133.4, 137.9, 146.6, 152.3, 162.3; MS(ESI ) m/z: calculated for C28H38N4O [M H] 446.63;found, azono)‑estr‑4,6‑dien‑17β‑ol (18) Yellow solid, 0.55 g, Yield 74%; mp:167–169 C; IR (KBr, c m 1):1582(C C), 1619(C N),2977(CH), 3322(NH), 3401 (OH); 1H NMR (400 MHzPage 7 of 10 CDCl3) δ, ppm: 0.95(s,3H,–CH3), 2.86(s, 1H, CH), 5.80(d,1H, J 10.7 Hz, CH), 5.92(s, 1H, –CH), 6.23(d, 1H,J 10.7 Hz, –CH), 7.60(m,1H,Ar–H), 7.79(m,2H,Ar–H),8.28(s,1H,Ar–H), 8.43(m,1H,Ar–H); 13C NMR (100 MHz CDCl3): 12.0, 22.5, 25.1, 26.9, 32.3, 37.8, 38.7, 40.8, 41.9,45.7, 47.3, 48.7, 74.3, 80.4, 87.0, 124.4, 125.6, 126.9,127.5,128.8, 131.6, 132.1, 136.5, 141.5, 158.9, 161.8; MS (ESI )m/z: Calculated for C28H30N4O [M H] ; 438.56; found,439.49.3‑( Phthal a z in‑1yl‑hy dra z ono)‑1α‑methyl‑5α‑ androstan‑17β‑ol (20) Yellow solid, 0.67 g, Yield61%; mp: 200 C; IR (KBr, cm 1):1586(C C),11629(C N), 2930(CH), 3392(OH); H NMR (300 MHz .79(m,11H,Mesterolone), 3.58 (t, 1H, J 9.0 Hz, –CH), 7.40–7.45(m, 1H, Ar–H), 7.56–7.59(m, 2H, Ar–H),7.72(d, 1H, J 6 Hz, Ar–H), 8.30–8.35 (m, 1H, Ar–H);13CNMR (75 MHz CDCl3): 11.3, 14.0, 14.8, 20.0, 23.5, 28.7,30.5, 30.9, 31.3, 35.6, 36.7, 38.6, 38.9, 39.2, 39.6, 40.6,43.1, 48.7, 48.9, 51.0, 81.8, 124.1, 126.0, 127.2, 131.4,131.6, 137.4, 146.4, 166.8; MS (ESI ) m/z: Calculated forC28H38N4O [M H] 446.63; found, biphenyl‑2‑carbonitrile(22) Yellow solid, 0.50 g, Yield 76.9%; mp: 200 C; IR (KBr, cm 1): 1586(C C), 1602(C N), 1648, 2223(C N),2937(CH), 3064(CH aromatic), 3398(NH); 1H NMR(400 MHz CDCl3) δ, ppm: 1.1(s, 3H, –CH3), 5.16(s, 2H, –OCH2–), 6.80–8.85(m, 20H, Ar–H);13C NMR (100 MHz CDCl3):17.0, 25.1, 27.3, 29.1, 30.7, 35.1, 38.1, 43.8, 44.3,52.1, 112.86, 114.9, 115.9, 123.3, 126.2, 126.8, 131.4, 136.5,137.1, 137.8, 141.4, 145.7, 149.7, 154.0, 167.1, 183.5; MS(ESI ) m/z: calculated for C40H37N5O [M H] 603.75;found, 604.86.DiscussionChemistryThe literature survey was done by focusing on steroidalhydrazone where ketosteroid used as a starting material. Allah HMF synthesized phthalazinohydrazone of17α-methyltestosterone [33]. Rasras et al. [34] conveyed the efficient procedure for the synthesis of novelhydrazide–hydrazone of cholic acid and tested them forantibacterial activity. Mohareb et al. reported synthesis ofhydrazide‑hydrazone, pyrazole, pyridine, thiazole, thiophene derivatives and their cytotoxicity evaluations [35].A method conveyed by Nadaria et al. [36, 37] for the synthesis and biological activity of hydrazone of 5α-steroidsand synthesis and cytotoxicity of epiandrosteronehydrazones. Jaben et al. [38] specified the synthesis of
Mistry and Singh F uture Journal of Pharmaceutical Sciences(2022) 8:7anticancer agents of progesterone and testosterone. Zickovic et al. [39] synthesized steroidal thiosemicarbazonesand evaluated their cytotoxic activity.The structural chemistry of these steroidal hydrazonesinvolves the condensation of hydralazine hydrochlorideat C3 and C17 of ketosteroid. The reaction is catalyzed bypotassium acetate in methanol as solvent. Androgen andestrogen scaffold used as ketosteroid for the synthesis oftitle compounds. The synthesis of steroidal hydrazoneswas developed without chromatographic purification(column/flash chromatography).The structures of the synthesized compounds (3–8,10, 11, 15, 16, 18, 20 and 22) were established using 1H,13C-NMR and mass spectral data. In 1H NMR spectrum of steroidal hydrazones (3–8, 10, 11, 15, 16, 18,20 and 22) singlet signals of 4-CH3, 4,4′-CH3, 18-CH3and 19-CH3 groups were present at δ 1.22 ppm, 0.76–0.96 ppm and 0.81 ppm and doublet signal of 7-CH3group was present at δ 0.73–0.75 ppm. Aromaticprotons of hydralazine were noted in the interval atδ7.5–8.5 ppm. In the 13C NMR spectra of steroidalhydrazones (5, 6 and 18) peaks of CH carbon existence at around δ 79.0 ppm and –C at around 87 ppm.Signals of C N bond at 161.6 ppm and 168.0 ppm. TheC17 peaks of steroidal hydrazones (3–8, 10, 11, 15, 16,18, 20 and 22) were observed at around δ 81 ppm. In1H NMR spectrum of steroidal hydrazones (3–8, 11,15, 16 and 18) and singlet signal of –CH were presentat around δ 2.48–3.61 ppm. Where as in compound 10and 20, –CH gave triplet at δ 3.55 and 3.58 ppm. In themass spectral analysis [M H] of 9A, 14A, 14B, 10, 13and 20 matched with theoretical values.In the 1H NMR spectra (in DMSO) of steroidal hydrazone (13) singlet signals of angular 18-CH3 group waspresent at δ 0.98 ppm. The signals of aromatic protonswere present in the range of δ 6.57–7.14 ppm. The singles of aromatic protons of hydralazine were present inthe range of δ7.70–8.23 ppm. Singlet signal of the protonof –OH group at δ 9.08 ppm. The protons of the –NHat δ 11.21 ppm. The IR spectrum of the steroidal hydrazone (13) contained absorption bands the NH– groupat 3358 cm 1, C N bond at 1649 cm 1, bands at 1602,1585 cm 1for –C C–. The infrared spectra of steroidalhydrazones (3–8, 10, 11, 15, 16, 18, 20 and 22) showedthe NH-band in the range of 3381–3209 c m 1.Biological activityIn vitro antimicrobial activityWe have selected compounds 3 (testosterone), 6 (iso tibolone), 7 (7-methyl nandrolone), 10 (δ6-testosterone), 11(4,4-dimethyl steroid) and 18 (δ6-norethisterone) for theantimicrobial screening based on the steroidal skeleton.Page 8 of 10Table 1 Antibacterial activity of steroidal hydrazonesCompoundsE. coliP. aeruginosa S. aureus S. pyogenusMTCC 443 MTCC 1688MTCC 96 MTCC 442MIC (minimal inhibition concentration) .1Ampicillin286286Chloramphenicol loxacin31313131The difference in the structure of above compounds is thepresence of -CH3, Unsaturation and ethinyl group. Theposition of groups are encourages us that what will be theactivity of the selected compounds among the all.The in vitro antimicrobial activity of some of the synthesized compounds was accomplished by broth microdilution method [40]. It is one of the non-automatedin vitro bacterial susceptibility tests. This classic methodyields a quantitative result for the amount of antimicrobial agents that is needed to inhibit growth of specificmicroorganisms. It is carried out in tubes. Mueller–Hinton broth was used as nutrient medium to grow anddilute the compound suspension for the test bacteria andSabouraud Dextrose broth used for fungal nutrition.Each synthesized drug was diluted obtaining 2000 µg /ml concentration, as a stock solution.Primary screen In primary screening 1000 micro/ml,500 micro/ml and 250 micro/ml concentrations of thesynthesized drugs were taken. The active synthesizeddrugs found in this primary screening were furthertested in a second set of dilution against all microorganisms.Secondary screen The drugs found active in primaryscreening were similarly diluted to obtain 200 micro/ml,100 micro/ml, 50 micro/ml, 25 micro/ml, 12.5 micro/ml, 6.250 micro/ml and concentrations.Reading result The highest dilution showing at least99% inhibition zone is taken as MIC. The result of thisis much affected by the size of the inoculum. The testmixture should contain 108 organism/ml. Inoculum sizefor test strain was adjusted to 1 08 CFU [Colony Forming Unit] per milliliter by comparing the turbidity. The
Mistry and Singh F uture Journal of Pharmaceutical Sciences(2022) 8:7strains employed for the activity were procured from[MTCC—Micro Type Culture Collection] Institute ofMicrobial Technology, Chandigarh.The compounds 3, 6, 7, 10, 11 and 18 were screenedfor their antibacterial activity against Escherichia coli(E. coli), Pseudomonas aeruginosa (P. aeruginosa),Staphylococcus aureus (S.aureus), Streptococcus pyo‑genes (S. pyogenes) and antifungal activity against Can‑dida albicans (C. albicans), Aspergillus niger (A. Niger)and Aspergillus clavatus (A. Clavatus). DMSO was usedas media to get desired concentration of compounds totest upon microbial strains. The lowest concentration,which showed no visible growth after spot subculturewas considered as MIC for each compound. The standard antibiotics used for comparison in the presentstudy were gentamycin, ampicillin, chloramphenicol,ciprofloxacin and norfloxacin for evaluating antibacterial activity while nystatin and griseofulvin for antifungal activity. The results are summarized in Tables 1 and2.From the antimicrobial data, steroidal hydrazones 3,7, 10 and 18 showed better activity (MIC 116–289 µM)against gram-positive bacteria Staphylococcus aureus (S.aureus) as compare to ampicillin (MIC 715 µM). Compounds 7 and 10 showed excellent activity (MIC 145and 116 µM) against gram-negative bacteria Escherichiacoli (E. coli) as compared to ampicillin (MIC 286 µM),compound 6 (MIC 55 µM) is found active against Strep‑tococcus pyogenes (S. pyogenes) as compare to chloramphenicol (MIC 154 µM) and ciprofloxacin (MIC 150 µM).Compound 6 (MIC 110 µM) and 11 (MIC 142 µM) exhibited powerful activity against Pseudomonas aeruginosa(P. aeruginosa) as compare to ampicillin (MIC 286 µM).Compound 6 showed equivalent potency against Escheri‑chia coli (E. coli) and Staphylococcus aureus (S. aureus) ascompared to ampicillin.Page 9 of 10Entire steroidal hydrazones (MIC 580–2319 µM)showed inferior activity against all gram-positive andgram-negative bacteria as compare to gentamycin (MIC0.10–2 µM) and norfloxacin (MIC 31 µM). Compound7 (MIC 580 µM) is found active against C. albicans ascompare to griseofulvin (MIC 1417 µM) and rest of thesteroidal hydrazones exhibited less potency than standard fungicidal nystatin and griseofulvin against Candidaalbicans, Aspergillus niger and Aspergillus clavatus.ConclusionsThe antimicrobial activities of steroidal hydrazone werestudied by the broth microdilution method. Compound6, 7 and 10 displayed excellent antibacterial activityamong the tested compounds due to bearing an ethinylat C-17 of compound 6, methyl at C-7 of compound 7.Compound 10 showed excellent antibacterial activitydue to the compounds bearing an additional –C C– inthe structure. Hence, these substituted steroidal skeletonconsidered for the development of the new antimicrobialagent.AcknowledgementsWe are thankful to D. K. Pharma, Powoi, Mumbai for generous gift of hydralazine hydrochloride.Authors’ contributionsSM contributed to synthesis, characterization and activity. AKS contributed toanalytical work. All authors have read and approved the manuscript.FundingNo funding was received for this work.Availability of data and materialsData and materials are available upon request.DeclarationsEthics approval and consent to participateNot applicable.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Table 2 Antifungal activity of steroidal hydrazonesCompoundsC. albicansA. nigerA. clavatusMTCC 227MTCC 282MTCC 1323MIC (minimal inhibition concentration) 64 1164111088108810881811372274 d: 1 June 2021 Accepted: 22 December 2021References1. Rollas S, Kucukguzel SG (2007) Biological activities of hydrazone derivatives. Molecules 12:1910–19392. Subhashini NJP, Janaki P, Bhadraiah B (2017) Synthesis of hydrazonederivatives of benzofuran and their antibacterial and antifungal activity.Russ J Gen Chem 87:2021–20263. Sridhar KS, Pandeya SN, Stables JP, Atmakuru R (2002) Anticonvulsantactivity of hydrazones, Schiff and Mannich bases of isatin derivatives. EurJ Pharm Sci 16:129–132
Mistry and Singh F uture Journal of Pharmaceutical 20.21.22.23.24.25.26.(2022) 8:7Debnatha U, Mukherjee S, Joardar N, Sinha BS, JanaMisra KAK (2019) Arylquinolinyl hydrazone d
Synthetic procedure of 14A (4‑methyl nandrolone) and 14B To a solution of Nandrolone (14) in tert. butanol was added potassium tert-butoxide with stirring under inert atmosphere by nitrogen blanketing. e solution of methyl iodide (3.8 ml, 61.04 mmol) in tert-butanol
