Synthesis and antiproliferative activity of novel 4-azasteroidal-17-hydrazone derivatives

Abstract

A new series of 4-azasteroidal-17-hydrazone derivatives have been synthesized from androstenedione. Their structures were characterized by analysis and spectroscopic data. The antiproliferative activity of synthesized compounds against three cancer cells (human lung adenocarcinoma, human oesophageal cervical cancer, human gastric adenocarcinoma) and a normal cell line (human gastric mucosal) was investigated. The studies show that the compound bearing a naphthyl group displayed the same antiproliferative activity in vitro against tested cells as cis-platin did (a positive control). Most of the compounds show very weak toxicity towards normal human gastric mucosal cell line.

4-azasteroidal -17-hydrazone derivatives have been synthesized. The title compounds were screened for their preliminary anticancer activity against 446 (human lung adenocarcinoma cell line), Eca-109 (human esophageal cervical cancer cell line), AGS (human gastric adenocarcinoma cell line) and a normal cell GES-1(human gastric mucosal).

Introduction

Some steroidal compounds have been used as traditional medicines, such as antibacterial and hormone kind medication. Besides the naturally occurring substances, the majority of steroidal drugs are semi-synthetic compounds.^1–4 Modification of steroidal compounds by introduction of heteroatom or replacement of carbon atoms by a heteroatom in steroids often affects the chemical properties of the steroidal molecule and results in alterations of biological activities.^5–8 Nitrogen-containing steroid derivatives are common clinical drugs in cancer chemotherapy. It has been reported that 4-azasteroidal analogues may lead to compounds having anticancer activity, cytotoxicity, antibacterial and 5α-reductase inhibitor activity.^9–13

Hydrazone compounds and their derivatives are a class of substances displaying antibacterial, antiviral, antitumor or other biological activities.^14,15 T Zoltan et al.^16–18 have reported some synthetic work of 4-azasteroidal-17-hydrazone. However, the study of hydrazine derivatives with respect to antiproliferative activity has few reports. In this article, novel 4-azasteroidal-17-hydrazone derivatives from the commercially available androstenedione were designed and synthesized. Furthermore, the antiproliferative activity of the compounds against 446 (human lung adenocarcinoma cell line), Eca-109 (human oesophageal cervical cancer cell line), AGS (human gastric adenocarcinoma cell line) and a normal cell line (GES-1) were evaluated in vitro.

Scheme 1 outlines the synthetic procedures of compounds 5a–g. Starting from androstenedione (1), ring A was oxidatively cleared by treatment with sodium periodate and potassium permanganate to give 5,17-dioxo-A-nor-3,5-secoandrostan-3-oic acid (2), which was treated with methylamine to afford 4-methyl-4-aza-5-androstene-3,17-dione (3) in 51.1% yield.^19,20 Compound 3 was converted to the corresponding 3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone (4) via reaction with hydrazine hydrate in anhydrous ethanol. Compound 4 reacted with appropriate aromatic aldehydes to give 4-azasteroidal-17-hydrazone derivatives 5a–g. Their structures were confirmed by ¹H nuclear magnetic resonance (NMR), ¹³C NMR and elemental analysis. As a representative analysis of compound N-p-chlorine-benzylidene-3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone (5a) in the ¹H NMR and ¹³C NMR spectra, the resonances showing CH=N at 8.26 ppm (s) and 156.20 ppm demonstrate the formation of the 17=N–N=CH bond in 5a, while the doublet at 7.69 and 7.38 corresponds to protons of the phenyl ring. The ¹³C NMR spectrum of phenyl appears at 136.52, 133.14, 129.22 and 128.96 ppm. Three singlets at 3.14, 1.10 and 1.01 were attributed to protons of N-CH₃, 18-CH₃ and 19-CH₃, respectively. The structure of compound 5a was further confirmed by elementary analyses. Structures of all derivatives were ascertained similarly.

Scheme 1.

Synthesis of compounds 5a–g.

To further investigate the configuration of the 17-hydrazone derivatives, 5c and 5f were subjected to two-dimensional (2D) NMR measurements. Since the nuclear Overhauser effect (NOE) cross-peaks between the protons that are closer than 0.4 nm in space will be observed in nuclear Overhauser effect spectroscopy (NOESY) and the relative intensities of these cross-peaks depend on the spaces between the corresponding protons. As shown in Figures 1 and 2, the NOESY spectra of 5c and 5f show clear NOE cross-peaks of H (18-CH₃, 0.93 ppm) and H (phenyl, 7.71 ppm or naphthyl, 9.06 and 7.97 ppm). Meanwhile, the NOE effect observed between H (18-CH₃) and H (CH=N, 8.28 ppm) indicates an imino group in 17-hydrazone derivatives only possible in the E-isomer (see Figures 1–3).

Figure 1.

NOESY spectrum of (a) 5c and (b) 5f.

Figure 2.

Partially enlarged NOESY spectrum of 5f.

Figure 3.

Example of NOE relationships of compound 5c.

To evaluate the antiproliferative activity of the compounds, we determined their IC₅₀ values on 446 (human lung adenocarcinoma cell line), Eca-109 (human oesophageal cervical cancer cell line) and AGS (human gastric adenocarcinoma cell line) cancer cells and one normal cell line (human gastric mucosal cell GES-1) using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetazolium) assay. MTS is a compound that can be taken up by viable cells and reduced by a mitochondrial dehydrogenase forming a formazan product in living cells. The absorbance of the formazan product at 492 nm is in linear proportion to cell numbers. The results are summarized as IC₅₀ values in μmol L⁻¹ in Table 1.

Table 1.

In vitro ^a antiproliferative activities (IC₅₀ in μmol L⁻¹) of 4-azasteroidal-17-hydrazone derivatives.

Substrate	R	No.	446	Eca-109	AGS	GES-1
		5a	89.7	81.9	ND	96.7
		5b	ND	44.6	ND	95.4
		5c	51.3	69.3	71.3	>100
		5d	>100	56.8	ND	90.1
		5e	90.1	43.5	>100	>100
		5f	40.1	28.8	34.2	>100
		5g	35.5	>100	50.4	89.8
cis-platin			44.2	30.4	36.7	98.9

ND: not determined.

a

Data represent the mean values of three independent determinations.

From Table 1, it is clear that some of the compounds show moderate to good cytotoxicity against 446, Eca-109 and AGS cancer cell lines and very weak toxicity towards the GES-1 normal cell line. In general, all the compounds were more active against the oesophageal cervical cancer cell line (Eca-109) than the other two cell lines. Compound 5e showed an IC₅₀ value of 43.5 μM against the Eca-109 cell line and exhibited weak inhibitory activity on 446 and AGS. Compound 5b with a Br group in the para-substituted benzene ring displayed an IC₅₀ value of 44.6 μM against Eca-109, while it showed significantly lower antiproliferative activity against the 446 and AGS cells. Substituents Cl, OCH₃ and CH₃ present in the benzene ring of compounds 5a, 5c and 5d showed weak effects, which can clearly be seen from Table 1. It was evident from the data that the introduction of group (either electron-donating group or electron-withdrawing group) on the phenyl ring resulted in a marked decrease against Eca-109 in potency compared to 5e. Replacement of the phenyl with thienyl (compound 5g) displayed strong cell growth inhibitory activity against 446. However, this compound exhibited weak inhibitory activity on the cell lines Eca-109 and AGS. In particular, compound 5f bearing a naphthyl group was the most potent molecule. It not only inhibited the proliferation of 446, Eca-109 and AGS cells with the IC₅₀ values of 40.1, 28.8 and 34.2 μM, respectively, but also showed very weak toxicity towards the normal cell line GES-1.

The information obtained from the studies is valuable for the design of novel steroidal chemotherapeutic drugs. Our findings provide new evidence showing the relationship between the chemical structure and biological function.

Experimental

All chemicals and solvents were of analytical grade and solvents were purified by general methods before being used. Melting points (m.p.) were determined on an X4 apparatus and were uncorrected. Infrared (IR) spectra were determined with KBr pellets on an FTS-135 spectrophotometer. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 500 NMR spectrometer (Bruker Instruments Inc., Billerica, MA, USA) at either 500 MHz (¹H NMR) or 125 MHz (¹³C NMR). Electrospray ionization mass spectrometry (ESI-MS) was performed on an LCQ Advantage MAX spectrometer. Elementary analyses were performed by a vario ELsuperuser.

5,17-Dioxo-A-nor-3,5-secoandrostan-3-oic acid (2)

A mixture of androstenedione 1 (15.0 g, 51.7 mmol) in tert-butanol (450 mL), anhydrous sodium carbonate (6.7 g, 63.7 mmol) and H₂O (20 mL) was heated to reflux, and a solution of sodium periodate (66.4 g, 310.3 mmol) and potassium permanganate (0.5 g, 3.1 mmol) in hot water (250 mL) was added dropwise to the refluxing mixture. Then the reaction mixture was refluxed for another 5 h. All inorganic material was filtered off and washed with water and methylene chloride. The filtrate was acidified with dilute hydrochloric acid and extracted with dichloromethane (150 mL × 3). The organic layer was washed with brine, dried over anhydrous sodium sulphate and evaporated under reduced pressure. Compound 2 (13.0 g) was obtained as pale yellow solid, which was used directly without further purification. IR (KBr), γ_max: 3050–3500, 1736, 1703 cm⁻¹; ESI-MS m/z: 307.3 (M + H)⁺; ¹H NMR (400 MHz, CDCl₃) δ: 10.05 (br, 1H, –COOH), 1.02 (s, 3H, 19-CH₃), 0.81 (s, 3H, 18-CH₃). ²⁰

4-Methyl-4-aza-5-androstene-3,17-dione (3)

A mixture of 2 (13.0 g, 42.5 mmol) and methylamine water solution (45 mL, 40%) in ethylene glycol (90 mL) was stirred over night at room temperature. The reaction mixture was refluxed for 7 h, cooled to room temperature, added with water (50 mL) and stirred for 1 h. The mixture was acided with dilute hydrochloric acid and extracted with dichloromethane (70 mL × 3). The organic layer was washed with brine, dried over anhydrous sodium sulphate and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel and eluted with petroleum ether/ethyl acetate to afford compound 3 (6.5 g, 51.1%) as a white solid. m.p. 161–162 °C (lit. ²⁰ 162–167.5 °C); ESI-MS m/z: 302.4 (M + H)⁺; ¹H NMR (500 MHz, CDCl₃) δ: 5.08 (br, s, 1H, 6-H), 3.14 (s, 3H, 4N-CH₃), 1.15 (s, 3H, 19-CH₃), 0.93 (s, 3H, 18-CH₃).

3-Oxo-4-methyl-4-aza-5-androstene-17-hydrazone (4)

Compound 3 (6.5 g, 21.6 mmol) was dissolved in 40 mL ethanol. The mixture was heated to 45 °C, and 5.1 mL of 85% hydrazine hydrate was added. The solution was stirred for 4 h until no starting material was observed. The reaction was evaporated under reduced pressure. The solid was recrystallized using MeOH as a solvent. Compound 4 was obtained as a white solid (3.5 g, 51.6%) after drying. m.p. 215–217 °C; ESI-MS m/z: 316.1 (M + H)⁺; ¹H NMR (500 MHz, dimethyl sulfoxide (DMSO)) δ: 5.34 (s, 2H, –NH₂), 5.04 (br, s, 1H, 6-H), 3.01 (s, 3H, 4N-CH₃), 1.00 (s, 3H, 19-CH₃), 0.79 (s, 3H, 18-CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 167.81, 154.43, 131.61, 104.55, 50.18, 46.07, 41.32, 40.42, 35.13, 32.13, 30.34, 30.20, 29.87, 27.18, 25.43, 23.27, 18.91, 17.16, 16.79; Anal. calcd for C₁₉H₂₉N₃O: C, 72.34; H, 9.27; N, 13.32; found: C, 72.45; H, 9.4661; N, 13.54.

General procedure for the synthesis of compounds (5a–g)

To a stirred solution of compound 4 (0.2 g, 0.63 mmol) in 20 mL of anhydrous ethanol, aromatic aldehyde (0.65 mmol) was added. The mixture was heated to 50 °C and stirred continually at the temperature until no starting material was present. The solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel and eluted with petroleum ether/ethyl acetate to afford compounds 5a–g.

(22E)-N-p-chlorine-benzylidene-3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone (5a): Yield 73.6%; m.p. 175–176 °C; ESI-MS m/z: 438.1 (M + H)⁺; ¹H NMR (500 MHz, CDCl₃) δ: 8.26 (s, 1H, CH=N), 7.68 (d, J = 8.5 Hz, 2H, Ph), 7.37 (d, J = 8.5 Hz, 2H, Ph), 5.06 (br, s, 1H, 6-H), 3.14 (s, 3H, 4N-CH₃), 1.10 (s, 3H, 19-CH₃), 1.01 (s, 3H, 18-CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 182.79, 168.40, 156.20, 144.45, 136.52, 133.14, 129.22, 128.96, 104.00, 53.34, 49.12, 44.37, 35.54, 33.65, 31.75, 31.20, 30.62, 30.13, 28.90, 27.99, 23.27, 20.61, 18.91, 16.59; Anal. calcd for C₂₆H₃₂ClN₃O: C, 71.30; H, 7.36; N, 9.59; found: C, 71.45; H, 7.61; N, 9.67.

(22E)-N-p-bromine-benzylidene-3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone (5b): Yield 75.2%; m.p. 166–167 °C; ESI-MS m/z: 382.3 (M + H)⁺; ¹H NMR (500 MHz, CDCl₃) δ: 8.24 (s, 1H, CH=N), 7.61 (d, J = 8.5 Hz, 2H, Ph), 7.53 (d, J = 8.5 Hz, 2H, Ph), 5.06 (br, s, 1H, 6-H), 3.14 (s, 3H, 4N-CH₃), 1.10 (s, 3H, 19-CH₃), 1.01 (s, 3H, 18-CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 182.80, 168.39, 156.27, 144.43, 133.55, 131.91, 129.43, 124.93, 104.00, 53.32, 49.10, 44.37, 35.53, 33.63, 31.74, 31.20, 30.61, 30.12, 28.90, 27.99, 23.26, 20.60, 18.91, 16.58; Anal. calcd for C₂₆H₃₂BrN₃O: C, 64.73; H, 6.69; N, 8.71; found: C, 64.92; H, 6.89; N, 8.65.

(22E)-N-p-methoxyl-benzylidene-3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone(5c): Yield 58.4%; m.p. 181–182 °C; ESI-MS m/z: 456.3 (M + Na)⁺; ¹H NMR (500 MHz, DMSO) δ: 8.28 (s, 1H, CH=N), 7.71 (d, J = 8.5 Hz, 2H, Ph), 7.00 (d, J = 9.0 Hz, 2H, Ph), 5.06 (br, s, 1H, 6-H), 3.81 (s, 3H, Ph-OCH₃), 3.02 (s, 3H, 4N-CH₃), 1.03 (s, 3H, 19-CH₃), 0.93 (s, 3H, 18-CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 182.07, 168.39, 161.66, 157.26, 144.44, 129.70, 127.43, 114.13, 104.05, 55.38, 53.39, 49.13, 44.27, 35.64, 33.73, 31.75, 31.20, 30.62, 30.15, 28.92, 27.96, 23.30, 20.63, 18.93, 16.62; Anal. calcd for C₂₇H₃₅N₃O₂: C, 74.79; H, 8.14; N, 9.69; found: C, 74.89; H, 8.40; N, 9.78.

(22E)-N-p-methyl-benzylidene-3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone (5d): Yield 64.2%; m.p. 198–199 °C; ESI-MS m/z: 418.4 (M + H)⁺; ¹H NMR (500 MHz, DMSO) δ: 8.28 (1H, s, CH=N), 7.66 (d, J = 8.0 Hz, 2H, Ph), 7.26 (d, J = 8.0 Hz, 2H, Ph), 5.06 (br, s, 1H, 6-H), 3.02 (s, 3H, 4N-CH₃), 2.35 (s, 3H, Ph-CH₃), 1.03 (s, 3H, 19-CH₃), 0.94 (s, 3H, 18-CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 182.03, 168.42, 157.45, 144.46, 140.95, 131.97, 129.41, 128.07, 104.06, 53.38, 49.15, 44.29, 35.55, 33.72, 31.76, 31.22, 30.64, 30.16, 28.93, 27.94, 23.31, 21.58, 20.64, 18.93; Anal. calcd for C₂₇H₃₅N₃O: C, 77.66; H, 8.45; N, 10.06; found: C, 77.48; H, 8.59; N, 10.28.

(22E)-N-benzylidene-3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone (5e): Yield 52.3%; m.p. >250 °C; ESI-MS m/z: 404.3 (M + H)⁺; ¹H NMR (500 MHz, DMSO) δ: 8.31 (s, 1H, CH=N), 7.76–7.79 (m, 2H, Ph), 7.45–7.47 (m, 3H, Ph), 5.07 (br, s, 1H, 6-H), 3.02 (s, 3H, 4N-CH₃), 1.03 (s, 3H, 19-CH₃), 0.95 (s, 3H, 18-CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 182.28, 168.02, 158.86, 144.56, 140.46, 128.61, 123.96, 121.59, 104.38, 53.37, 49.10, 44.38, 35.56, 33.63, 31.34, 30.90, 30.61, 30.12, 28.90, 27.93, 23.26, 20.58, 18.91, 16.58; Anal. calcd for C₂₆H₃₃N₃O: C, 77.38; H, 8.24; N, 10.41; found: C, 77.52; H, 8.36; N, 10.50.

(22E)-N-naphthylmethylene-3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone (5f): Yield 68.7%; m.p. 227–229 °C; ESI-MS m/z: 454.3 (M + H)⁺; ¹H NMR (500 MHz, DMSO) δ: 9.06 (d, J = 8.5Hz, 1H, naphth), 8.90 (s, 1H, CH=N), 8.05 (d, J = 8.5Hz, 1H, naphth), 7.97 (d, J = 7.0 Hz, 1H, naphth), 7.58–7.66 (m, 3H, naphth), 5.07–5.08 (br, s, 1H, 6-H), 3.03 (s, 3H, 4N-CH₃), 1.05 (s, 3H, 19-CH₃), 1.00 (s, 3H, 18-CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 182.80, 168.43, 158.03, 144.47, 133.94, 131.26, 130.20, 129.38, 128.72, 127.23, 126.15, 125.31, 125.02, 104.05, 53.44, 49.15, 44.37, 35.67, 33.76, 31.17, 29.73, 28.94, 28.44, 23.36, 20.67, 18.95, 16.69; Anal. calcd for C₃₀H₃₅N₃O: C, 79.43; H, 7.78; N, 9.26; found: C, 79.58; H, 7.86; N, 9.30.

(22E)-N-thenylidene-3-oxo-4-methyl-4-aza-5-androstene-17-hydrazone (5g): Yield 71.5%; m.p. >250 °C; ESI-MS m/z: 410.2 (M + H)⁺; ¹H NMR (500 MHz, DMSO) δ: 8.51 (s, 1H, CH=N), 7.69 (d, J = 4.5 Hz, 1H, thienyl), 7.51 (m, 1H, thienyl), 7.15–7.17 (m, 1H, thienyl), 5.06–5.07 (br, s, 1H, 6-H), 3.02 (s, 3H, 4N-CH₃), 1.03 (s, 3H, 19-CH₃), 0.93 (s, 3H, 18-CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 182.92, 168.41, 151.71, 144.44, 131.03, 129.04, 127.54, 104.06, 53.38, 49.12, 44.34, 35.54, 33.68, 31.75, 30.62, 30.14, 28.93, 28.03, 23.27, 20.62, 18.93, 18.59; Anal. calcd for C₂₄H₃₁N₃OS: C, 70.38; H, 7.63; N, 10.26; found: C, 70.42; H, 7.41; N, 10.38.

Biological activity

Cell viability assay

The anticancer potency of test compounds was measured using the MTS assay. The cells (1 × 10⁴) were seeded in 96-well plates and cultured for 24 h, followed by treatment with different concentrations of the test compound for a range of durations for the different experiments. A volume 20 μL of MTS solution was added to each well and incubated for 3 h at 37 °C, following which the supernatant was removed and 150 μL DMSO was added. The absorbance was recorded using a microplate reader (Titertek Multiskan; Flow Laboratories, North Ryde, Austria) at a wavelength of 492 nm. All experiments were performed six times. The cell growth inhibition rate was calculated using the following formula: 1 − OD_experiment/OD_control. The IC₅₀ values were calculated as the concentration of drug yielding 50% cell survival.^21,22