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Design, synthesis, and biological activities of novel thiophene, pyrimidine, pyrazole, pyridine, coumarin and isoxazole: Dydrogesterone derivatives as antitumor agents

On the basis of our consideration to design and to develop antitumor activities of heterocyclic compound derivatives, especially in fused ring system, we refer to the possibility of the heterocyclic extension of one of the most important steroid compounds used as a medicinal drug. The reaction of dydrogesterone with each of the malononitrile or ethylcyanoacetate containing elemental sulfur afforded thiophene derivatives 1a,b. Also, dydrogesterone was reacted with a mixture of ethylcyanoacetate–hydrazine, ethylcyanoacetae–urea, or ethylcyanoacetate–thiourea to produce pyrazole derivative 4 and pyrimidine derivatives 5a,b. Thienopyrimidine derivatives 2a–d were introduced from the reaction of thiophene derivatives 1a,b with either phenylisothiocyanate or benzoylisothioyanate. Furthermore, compounds 1a,b were directed toward the reaction with ethylcyanoacetate to produce compounds 6a,b, and the last compounds 6a,b were directed toward cyclization to obtain thienopyridine derivatives 7a,b. In addition, compounds 6a,b were subjected to react with different carbonyl compounds, such as salicylaldehyde, cyclopentanone-elemental sulfur, malonaldehyde, and acetylacetone to produce coumarin derivatives 8a,b, fused thiophene derivatives 9a,b, and pyridine derivatives 10a–d. Isooxazole derivatives 12a,b were afforded through the reaction of compounds 6a,b with hydroxylaminehydrochloride. Finally, 2-pyridonederivatives 14a,b were obtained through the reaction of compounds 6a,b with benzoylacetonitrile. Conformation structure of the synthesized compounds was established by applying IR, H NMR, C NMR, and mass spectrometry, and their antitumor activity was examined. Some compounds showed promising growth inhibitory effects on the three different cell lines.


Introduction
Dydrogesterone is one of the most important nonacetylated pregnane [1] derivatives ( Figure 1); many medical uses of dydrogestrone were introduced in the treatment of either miscarriage threats or miscarriage prevention [2,3]. The oral treatment of dydrogesterone versus vaginal progesterone gel was applied in the luteal phase support [4]. Oral estradiol and dydrogesterone combination therapy in postmenopausal women was applied [5]. Dydrogesterone was used in the treatment of endometriosis, adenomyosis [6], and recurrent pregnancy loss, and it was also used for modulation of cytokine production [7]. Furthermore, it was used in the treatment of premenstrual syndrome [8] and for the prevention of myometrial contraction [9]. Also, dydrogesterone and its derivative act as selective enzyme regulators for breast cancer cell lines [10] and used for the treatment of dysmenorrhea [11]. Moreover, dydrogesterone has an effective role against abortion [12]. However, the combination of pregnane derivative with heterocyclic rings afforded biologically active properties; thus, pregnane containing imidazole, triazole rings, glycoside moiety, and piperazine ring has anticancer activity, cytotoxic activity [13], antioxidant activity [14], and antileukemic properties [15], respectively.

Chemicals and reagents
All the chemicals and reagents in this study were obtained from Sigma Aldrich (United Kingdom).

Instruments
The melting points of the synthesized dydrogesterone derivatives were measured in open capillaries, and they are uncorrected. Elemental analyses were performed on a Yanaco CHNS Corder elemental analyzer (Japan). IR spectra were estimated using KBr discs on a Pye Unicam SP-1000 spectrophotometer. 1   Either malononitrile (0.66 g, 0.01 mol) or ethylcyanoacetate (1.131 g, 0.01 mol) together with elemental sulfur (0.32 g, 0.01 mol) was added to a solution of dydrogesterone (3.124 g, 0.01 mol) in 1,4 dioxane (100 mL) containing catalytic amount of triethylamine (1.0 mL). The reaction mixture in each case was heated under reflux for 5 h and then poured onto ice/water mixture containing few drops of hydrochloric acid (HCl). The solid product formed was collected by filtration and recrystallized from absolute ethanol.

General procedure for the synthesis of compounds 6a,b
The solution dydrogesterone thiophene derivative 1a (1.177 g, 0.003 mol) or 1b (1.319 g, 0.003 mol) in absolute ethanol (70 mL) containing triethylamine (1 mL) and ethylcyanoacetate (0.339 g, 0.003 mol) was added. The reaction mixture was heated under reflux for 6 h, and the reaction was observed by TLC control, and then, the mixture poured onto ice/water containing few drops of HCl. The formed solid product in each case was filtered, dried, and recrystallized from the absolute ethanol.

Antitumor activity tests
All the reagents and chemicals, penicillin, streptomycin, doxorubicin, sulforhodamine B (SRB), and dimethyl sulfoxide (DMSO), used for antitumor activity tests were obtained from Sigma Chemical Co. (USA). Fetal bovine serum (FBS) and L-glutamine were purchased from Gibco Invitrogen Co. (UK). RPMI-1640 medium was obtained from Cambrex (USA).
The three human tumor cell lines that are used for the evaluation are as follows: SF-268 (CNS cancer), MCF-7 (breast adenocarcinoma), and NCI-H460 (non-small lung cancer cell). The SF-268, NCI-H460, and normal fibroblast cells (WI-38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt), and MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK).
The cell lines were grown as monolayers and were routinely maintained in RPMI-1640 medium supplemented with 5% heat-inactivated FBS, antibiotics (penicillin 100 U mL −1 and streptomycin 100 µg mL −1 ), and 2 mmol L −1 glutamine at 37°C in a humidified atmosphere containing 5% CO 2 . Exponentially growing cells were obtained by plating 1.5 × 105 cell mL −1 for SF-268 and MCF-7 and 0.75 × 104 cell mL −1 for NCI-H460, followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.
Tumor cell growth assay: The effects on the in vitro growth of human tumor cell lines were evaluated on the newly synthesized compounds 1a,b, 2a-d, 4-10a-d, 12a,b, and 14a,b according to the specified procedure by the National Cancer Institute (NCI, USA) [16] that uses the protein-binding dye sulforhodamine B to consider the cell growth. Exponentially growing cells in 96-well plates were exposed for 48 h to five serial concentrations of each synthesized compound starting from the maximum concentration of 150 µmol L −1 . After this exposure period, the adherent cells were fixed, washed, and stained. The bound stain was dissolved in dimethylsulfoxide, and then, the absorbance was measured at 492 nm in a plate reader (Power wave XS, Bio-Tec Instruments, USA). For each test compound and cell line, a dose-response curve was obtained and the growth inhibition of 50% (GI 50 ; corresponding to the concentration of each compound that inhibited 50% of the net cell growth) was calculated as described earlier [17]. Doxorubicin was used as a positive control test, and it was tested under the same condition.
Ethical approval: The conducted research is not related to either human or animal use.

Chemistry
In the framework of our ongoing attempts of our team to try to develop and synthesize some of the heterocyclic compound derivatives with interesting antitumor activities [18,19], here we designed the newly dydrogesterone heterocyclic extension compounds, such as thiophene, thienopyrimidine, pyrimidine, pyrazole, thienopyridine, coumarin, and isoxazole derivatives, Schemes 1-4. The reaction of dydrogesterone with either malononitrile or ethylcyanoacetate together with elemental sulfur to form 2-aminothiophene derivatives 1a,b, the structure of such compounds 1a,b was established using analytical and spectral data. Fused thiophene derivatives were afforded through the reaction of the latter compounds 1a,b with either isothiocyanate derivatives or ethylcyanoacetate to afford thienopyrimidine derivatives 2a-d and thienopyridine derivatives 7a,b, respectively. The structure of compounds 2a-d and 7a,b was established by applying analytical and spectral data; the compound 2a 1 H NMR spectrum indicated the presence one singlet at δ = 6.78 ppm because of the presence of 1H of thiophene ring, and multiplet at δ = 7.33-7.54 ppm indicates the presence of 5H of phenyl group, broad signal at δ = 8.68 ppm corresponding to 1H of SH group and one singlet, D 2 Oexchangeable at δ = 9.11 ppm because of the presence of 1H of NH group. In addition, the mass spectrum revealed m/z at 527 [M] + and m/z at 77 [C 6 H 5 ] + for the phenyl moiety. In addition, compounds 7a,b were obtained through the reaction of ethylcyanoacetate with compounds 1a,b to produce compounds 6a,b, which underwent cyclization afforded fused thiophene derivatives 7a,b. The structures of these compounds are confirmed using analytical and spectral data. Also, the reaction of ethylcyanoacetate with dydrogesterone in the presence of hydrazine, urea, or thiourea afforded either pyrazole derivative 4 or pyrimidine derivatives 5a,b. The structure of compound 5a was proved using analytical and spectral data; 1 6a,b, 7a,b, 8a,b and 9a,b. the 13 C NMR spectrum revealed two signals at δ = 119.8 and 121.6 ppm because of the presence of C]C group and three signals at δ = 130.8, 132.5, 139.2 ppm corresponding to the pyrimidine ring. A series of chemical reaction were acceptable of compounds 6a,b; thus, cyanoacetamidothiophene derivatives 6a,b were reacted with either salicylaldehyde or cyclopentanone and elemental sulfur to form coumarin derivatives 8a,b and cyclopenta[b]thiophene derivatives 9a,b, respectively. The structure of such compounds was confirmed according to the analytical and spectral data. Compound 8a was proved using the analytical and spectral data; 1 H NMR spectrum showed the presence of singlet at δ = 6.57 ppm because of the presence of 1H of coumarin ring, multiplet at δ = 7.28-7.41 ppm corresponding to 4H of benzene ring. In addition, IR spectrum of compound 8a detected the presence of stretching vibration bands of the CN group at υ max = 2,228 cm −1 (Schemes 1 and 2).
However, compounds 6a,b were directed toward the reaction with either 1,3-dicarbonyl compounds or hydroxylaminehydrochloride to produce pyridine derivatives 10a-d or isoxazole derivatives 12a,b through intermediate formation 11a,b, respectively (Scheme 3). The structure of compounds 10a-d and 12a,b was confirmed by the analytical and spectral data. The 1 H NMR spectrum of compound 10a showed the presence of multiplet at

Effect on the growth of human tumor cell lines and QSAR
The inhibitory effect of the newly synthesized compounds 1a,b-14a,b was evaluated on the in vitro growth of the three human tumor cell lines representing different tumor types, namely, breast adenocarcinoma (MCF-7), nonsmall cell lung cancer (NCI-H460), and CNS cancer (SF-268) after a continuous exposure for 48 h. All of the synthesized products 1a,b-14a,b were able to inhibit the growth of the tested human tumor cell lines in a dose-dependent manner. The results ( Table 1)  reference standard material (doxorubicin). In addition, phenyl thienopyrimidine derivative 2c showed the highest inhibitory effect against all the three tumor cell lines, (SF-268), (MCF-7), and (NCI-H460), corresponding to the remaining synthesized compounds. However, imino-thienopyrimidine derivatives 2a,b, hydroxypyrimidine derivatives 5a,b, cyanoacetamido thiophene derivatives 6a,b, and thienopyridine derivatives 10a-d showed good inhibitory effects against three cancer cell lines, (SF-268), (MCF-7), and (NCI-H460). Regarding the remaining, com- pounds 1a,b, 4, 7a,b, 8a,b, 9a,b, 12a,b, and 14a,b showed a moderate growth in the inhibitory effect.
Comparing thienopyrimidine derivatives 2a-d, it was found that compound 2d acts as the most effective one, that is may be because of the presence of benzoylpyrimidinone moiety. Also, comparing thienopyridine compound derivatives 10a-d, one can say that compound 10a is the most effective one in this group. It may be because of the presence of cyano group with the absence of the methyl group. Furthermore, on comparing compounds hydroxypyrimidine derivatives 5a,b and cyanoacetamido thiophene derivatives 6a,b, although these compounds nearly has the same good inhibitory effect against three different cell lines, (SF-268), (MCF-7), and (NCI-H460), it may be that they are containing hydroxypyrimidine and cyanoacetamidothiophene moieties, respectively, but it was clear that compound 5b is slightly more effective than the remaining compounds 5a,6a,b on three different tumor cell lines, (SF-268), (MCF-7), and (NCI-H460). It may be because of containing thiopyrimidine moiety. In addition, comparing compounds 1a,b, 4, 7a,b, 8a,b, 9a,b, 12a,b, and 14a,b, it was clear that they have a moderate inhibitory effect, but compound 1b showed that it is the most effective one in this groups. It may be because of the presence of both amino and ethylcarboxylate groups of thiophene moiety.
The results are presented in concentrations that were able to cause 50% of cell growth inhibition (GI 50 ) after a continuous exposure for 48 h and show means ± SEM of three independent experiments performed in duplicate.

Conclusion
In this article, the synthesized compounds of dydrogesterone heterocyclic extension such as thiophene, thienopyrimidine, pyrimidine, pyrazole, thienopyridine, coumarin, and isoxazole derivatives were investigated to detect the antitumor activity of such compounds against three different cell lines. Among the synthesized compounds, benzoyl thienopyrimidine derivative 2d showed the highest one effective against all the three different tumor cell lines, (SF-268), (MCF-7), and (NCI-H460), with respect to the reference standard material (doxorubicin). In addition, phenyl thienopyrimidine derivative 2c showed the highest inhibitory effect against all the three tumor cell lines, (SF-268), (MCF-7), and (NCI-H460), corresponding to the remaining synthesized compounds.