Cytotoxic Effects of Newly Synthesized Heterocyclic Candidates Containing Nicotinonitrile and Pyrazole Moieties on Hepatocellular and Cervical Carcinomas

In this study, a series of newly synthesized substituted pyridine 9, 11–18, naphthpyridine derivative 10 and substituted pyrazolopyridines 19–23 by using cycnopyridone 8 as a starting material. Some of the synthesized candidates are evaluated as anticancer agents against different cancer cell lines. In vitro cytotoxic activities against hepatocellular and cervical carcinoma cell lines were evaluated using standard MTT assay. Different synthesized compounds exhibited potential in vitro cytotoxic activities against both HepG2 and HeLa cell lines. Furthermore, compared to standard positive control drugs, compounds 13 and 19 showed the most potent cytotoxic effect with IC50 values of 8.78 ± 0.7, 5.16 ± 0.4 μg/mL, and 15.32 ± 1.2 and 4.26 ± 0.3 μg/mL for HepG2 and HeLa cells, respectively.


Introduction
Multicomponent reactions (MCR) "in which three or more starting materials react to form a product" play a significant role in the synthesis of heterocyclic compounds with pharmaceutical and chemical importance [1]. Several nicotinonitriles have been constructed via (MCR) and showed antitumor [2], antimicrobial [3], and antioxidant [4] activities. Also nicotinonitriles have been utilized as a scaffold for the synthesis of heterocyclic compounds containing a pyridine moiety with antimicrobial and antiviral activities [5]. A series of nicotinonitriles 1-3 ( Figure 1) and have been synthesized and anti-proliferative [6], anti-Alzheimer's [7], and anti-inflammatory [8] activities. The pyrazole moiety is both pharmacologically and medicinally significant [9]. A series of pyrazoles 4-7 ( Figure 2) has been reported as anti-inflammatory activity by Bekhit et al. [10], they observed that the synthesized pyrazoles showed more anti-inflammatory activity than the standard indomethacin [11]. Trisubstituted pyrazoles have been constructed by Christodoulou et al. (2010) [11] and evaluated as anti-angiogenic agents; these derivatives showed a potent anti-angiogenic efficacy and moreover inhibited the growth of Mammary gland breast cancer (MCF-7) and cervical carcinoma (Hela) [12]. Recently novel derivatives of pyrazoles 5,6 have been prepared as antimicrobial [13] and anticonvulsant [14] agents. The pyrazole 7 has been prepared by Bonesi et al. (2010) [15] and showed effective Angiotensin -1-Converting Enzyme (ACE) inhibitor activity [15]. Based on the previous facts about the importance of pyrazoles and nicotinonitriles in medicinal chemistry, we have herein synthesized of some novel heterocyclic candidates containing nicotinonitrile and pyrazole moieties and tested their anticancer activity.

Chemistry
The nicotinonitriles were obtained by two different ways, from the reaction of chalcone with ethylcyanoacetate, ammonium acetate and drops of piperidine as a base and from one pot four components reaction of methylketone, aldehyde, ethylcyanoacetate, ammonium acetate and drops of piperidine as a base [15]. In prolongation of our work in the synthesis of heterocyclic compounds and evaluation of their medicinal importance [16][17][18][19][20][21][22][23][24][25][26][27] and based on the literature survey about the The pyrazole moiety is both pharmacologically and medicinally significant [9]. A series of pyrazoles 4-7 ( Figure 2) has been reported as anti-inflammatory activity by Bekhit et al. [10], they observed that the synthesized pyrazoles showed more anti-inflammatory activity than the standard indomethacin [11]. Trisubstituted pyrazoles have been constructed by Christodoulou et al. (2010) [11] and evaluated as anti-angiogenic agents; these derivatives showed a potent anti-angiogenic efficacy and moreover inhibited the growth of Mammary gland breast cancer (MCF-7) and cervical carcinoma (Hela) [12]. Recently novel derivatives of pyrazoles 5,6 have been prepared as antimicrobial [13] and anticonvulsant [14] agents. The pyrazole 7 has been prepared by Bonesi et al. (2010) [15] and showed effective Angiotensin -1-Converting Enzyme (ACE) inhibitor activity [15]. The pyrazole moiety is both pharmacologically and medicinally significant [9]. A series of pyrazoles 4-7 ( Figure 2) has been reported as anti-inflammatory activity by Bekhit et al. [10], they observed that the synthesized pyrazoles showed more anti-inflammatory activity than the standard indomethacin [11]. Trisubstituted pyrazoles have been constructed by Christodoulou et al. (2010) [11] and evaluated as anti-angiogenic agents; these derivatives showed a potent anti-angiogenic efficacy and moreover inhibited the growth of Mammary gland breast cancer (MCF-7) and cervical carcinoma (Hela) [12]. Recently novel derivatives of pyrazoles 5,6 have been prepared as antimicrobial [13] and anticonvulsant [14] agents. The pyrazole 7 has been prepared by Bonesi et al. (2010) [15] and showed effective Angiotensin -1-Converting Enzyme (ACE) inhibitor activity [15]. Based on the previous facts about the importance of pyrazoles and nicotinonitriles in medicinal chemistry, we have herein synthesized of some novel heterocyclic candidates containing nicotinonitrile and pyrazole moieties and tested their anticancer activity.

Chemistry
The nicotinonitriles were obtained by two different ways, from the reaction of chalcone with ethylcyanoacetate, ammonium acetate and drops of piperidine as a base and from one pot four components reaction of methylketone, aldehyde, ethylcyanoacetate, ammonium acetate and drops of piperidine as a base [15]. In prolongation of our work in the synthesis of heterocyclic compounds and evaluation of their medicinal importance [16][17][18][19][20][21][22][23][24][25][26][27] and based on the literature survey about the Based on the previous facts about the importance of pyrazoles and nicotinonitriles in medicinal chemistry, we have herein synthesized of some novel heterocyclic candidates containing nicotinonitrile and pyrazole moieties and tested their anticancer activity.

Scheme 1.
Synthesis of compound 8 as starting material.
The structure of the nicotinonitrile 8 has been confirmed from its spectral data. IR spectrum showing absorption frequencies at ν 3159 cm -1 , 2220 cm -1 and ν 1647 cm -1 for OH, C≡N and C=N groups, respectively. Also, 1 H-NMR spectrum of the assigned compound displayed signals at δ 12.89 ppm (disappeared with D2O) corresponding to acidic OH. A compelling evidence for the structure of 8 was provided by 13 C-NMR spectrum that showed a singlet signal at δ 149.8, 139.3 and 139.3 ppm for C-OH, C=N and C≡N groups respectively. Mass spectra of 8 showed [M + ] at m/z (%) 482 (22). Treatment of 8 with ethylchloroacetate afforded compound 9, which was hydrazinolysis with NH2NH2 to give the corresponding cyclized product 10.
Remediation of the nicotinonitrile derivative 8 with malononitrile in the presence of few drops of piperidine afforded 1,8-naphthyridine-3-carbonitrile derivative 11. Chlorination of 8 by a mixture of (POCl3/PCl5) afforded 2-chloronicotinonitrile derivative 12, which was reacted with malono nitrile as a carbon nucleophile gave the nicotinonitrile derivative 13. Reaction of 12 with primary and secondary amines, namely, o-aminothiophenol, morpholine, 1-methylpiperazine and hydrazine hydrate gave novel nicotinonitriles 14, 15a, b and 16 (Scheme 2). The mechanism formation route of compound 11 has been shown in Figure 3. The structure of the nicotinonitrile 8 has been confirmed from its spectral data. IR spectrum showing absorption frequencies at ν 3159 cm −1 , 2220 cm −1 and ν 1647 cm −1 for OH, C≡N and C=N groups, respectively. Also, 1 H-NMR spectrum of the assigned compound displayed signals at δ 12.89 ppm (disappeared with D 2 O) corresponding to acidic OH. A compelling evidence for the structure of 8 was provided by 13 C-NMR spectrum that showed a singlet signal at δ 149.8, 139.3 and 139.3 ppm for C-OH, C=N and C≡N groups respectively. Mass spectra of 8 showed [M + ] at m/z (%) 482 (22). Treatment of 8 with ethylchloroacetate afforded compound 9, which was hydrazinolysis with NH 2 NH 2 to give the corresponding cyclized product 10.
Remediation of the nicotinonitrile derivative 8 with malononitrile in the presence of few drops of piperidine afforded 1,8-naphthyridine-3-carbonitrile derivative 11. Chlorination of 8 by a mixture of (POCl 3 /PCl 5 ) afforded 2-chloronicotinonitrile derivative 12, which was reacted with malono nitrile as a carbon nucleophile gave the nicotinonitrile derivative 13. Reaction of 12 with primary and secondary amines, namely, o-aminothiophenol, morpholine, 1-methylpiperazine and hydrazine hydrate gave novel nicotinonitriles 14, 15a, b and 16 (Scheme 2). The mechanism formation route of compound 11 has been shown in Figure 3.     Treatment of 16 with 4-chlorobenzaldehyde and/or tetrachlorophthalic anhydride in the presence of acetic acid afforded the cyclized 19 followed by condensation to give the Schiff`s base 21 and tetra chloroisoindoline 22, respectively. The structures of 21 and 22 were confirmed chemically by condensation of compound 19 with 4-chlorobenzaldehyde and/or tetrachlorophthalic anhydride to provide compounds 21 and 22, respectively. Treatment of hydrazinyl derivative 16 with CS2 in the presence of alcoholic KOH provided thioxotriazolo pyridine derivative 23 (Scheme 3).

Cytotoxic Activity
The newly synthesized compounds were screened for their anticancer potentials against hepatocellular carcinoma HepG2 and cervical carcinoma HeLa. The cytotoxicity of the compounds was determined using MTT assay and DOX as a positive control [28][29][30][31].
The cytotoxic activities of the novel synthesized compounds 8-23 were estimated and the obtained results are presented in Figure 4. In general, it can be seen that all synthesized compounds exhibited cytotoxic activities against both tested cancer cell lines. Moreover, it can be seen that both cells reacted in a dose-dependent manner toward the applied concentrations. Additionally, both tested cell lines varied in their response toward different synthesized compounds. Furthermore, based on the IC 50 values (  16 showed IC 50 value of 33.45 ± 2.3 and 10.37 ± 0.9 µg/mL against HepG2 and HeLa cells, respectively. Also, Cpd. 22 showed IC 50 of 26.64 ± 1.9 and 9.33 ± 0.8 µg/mL for HepG2 and HeLa cells, respectively. On the other hand, Cpd. 17 showed strong activity towards HepG2 cells (IC 50 20.00 ± 1.7 µg/mL) and moderate activity towards HeLa cells (IC 50 35.58 ± 2.6 µg/mL). Finally, Cpds. 9, 10, 11, 12, 15a, b, 17, 20, 21 and 23 showed activities ranging from moderate to non-cytotoxic, with IC 50 values ranging from 24.83 ± 1.8 to >100 µg/mL.

Discussion
During current work, multi-component reaction strategy was used to synthesize of compound 8, which was used as a building block for preparing 16 new derivatives. The cytotoxic potential of the new prepared compounds has been evaluated against HepG2 and HeLa cells. Results obtained showed potential cytotoxic activities against both cell lines. Compounds 13 and 19 showed the most cytotoxic effects (IC 50 8.78 ± 0.7 and 5.16 ± 0.4 µg/mL, for HepG2 cells, and 15.32 ± 1.2 and 4.26 ± 0.3 µg/mL for HeLa cells, respectively). Also, results showed that both tested cell lines varied in their response toward different synthesized compounds. This can be attributed to the inherent differences in both cell lines in terms of membrane structure and organization, hence different cell lines react differently towards different compounds [32][33][34][35].
Different activities of the prepared compounds may be attributed to the structure-activity relationship of these compounds. It can be seen that conversion of Cpd. 12 to 13, 14 and 16, 18, 19 and 22 altered the cytotoxicity from weak to moderate and strong activity towards two cell lines. This explained due to the introduction of two more nitrile groups, which significantly increased the activity. Compound 14 exhibited very strong activity due to the entity of the SH and NH groups, which may be added to any unsaturated group in DNA (thia or aza Michael addition) or the formation of hydrogen bonds with either one of the nucleo-bases of the DNA, thus causing DNA damage. Furthermore, the cytotoxicity of Cpd. 16 may be due to the intermolecular hydrogen bonding of NH and NH 2 groups with DNA moieties. Additionally, conversion of Cpd. 16 to 18, 19 and 22 increased their cytotoxic activities against both cell lines. Introducing thiophene ring increases the cytotoxic effect of Cpd. 18 beside the effect of the pyrazole ring and the trifluoromethyl group. Additionally, introducing pyrazole ring bearing NH 2 group to Cpd. 16 increases the cytotoxic effect of Cpd. 19 to very strong effect against both cell lines. The introduction of chloroiso-indoline-1,3-dione increases the cytotoxic effects of Cpd. 22. The chloro-group, with more electron withdrawing properties, may be the crucial for tumor cell inhibition beside the effect of the isoindoline-1,3-dioneas moderate cytokine inhibitor in cancer cells.

Chemistry
"Melting points reported are inaccurate. IR spectra were registered on Shimadzu FT-IR 8300 E (Shimadzu Corporation, Kyoto, Japan) spectrophotometer using the (KBr) disk technique. 1 H-NMR spectra were determined on a Varian Spectrophotometer at 400 MHz using (TMS) as an internal reference and DMSO-d 6 as solvent using (TMS) as internal standard. All chemical shifts (δ) are uttered in ppm. The mass spectra were determined using (MP) model MS-5988 and Shimadzu single focusing mass spectrophotometer (70 eV). Elemental analysis was investigated by Elemental analyzer Vario EL III". A solution of 16 (4.9 g, 0.01 mol) in a mixture of AcOH/Ac 2 O (10 mL) or in glacial AcOH (10 mL) was refluxed for 2 h, poured on ice/water, filtered off and crystallized from EtOH/dioxane to give 19 and 20, respectively. Also, refluxing of 19 (0.5 g, 0.01 mol) in acetic anhydride (7 mL) afforded compound 20.

MTT Assay
Cytotoxic assay depends on the formation of purple formazan crystals by the action of dehydrogenase in living cells. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, antibiotic solution (100 units/mL penicillin, 100 µg/mL streptomycin) at 37 • C in a 5% CO2 incubator. Cells were seeded in a 96-well plate (10 4 cells/well), and the plates were incubated for 48 h. Afterwards, cells were exposed to variable concentrations of prepared derivatives and incubation proceeded for further 24 h. After treatment, 20 µL of MTT solution (5 mg/mL) was added and incubated for 4 h. DMSO (100 µL/well) is added and the developed color density was measured at 570 nm using a plate reader (ELx 800, BioTek, Winuski, VT, USA). Relative cell viability was calculated as (Atreated/Auntreated) ×100 [36,37]. Results were compared with doxorubicin as a positive control.