The Synthesis, Characterization, Cytotoxic Activity Assessment and Structure-Activity Relationship of 4-Aryl-6-(2,5-dichlorothiophen-3-yl)-2-methoxypyridine-3-carbonitriles

A series of 2-methoxypyridine-3-carbonitrile (5a–i)-bearing aryl substituents were successfully synthesized in good yields by the condensation of chalcones (4a–i) with malononitrile in basic medium. The condensation process, in most cases, offers a route to a variety of methoxypyridine derivatives (6a–g) as side products in poor yields. All new compounds were fully characterized using different spectroscopic methods. Mass ESI-HMRS measurements were also performed. Furthermore, these compounds were screened for their in vitro cytotoxicity activities against three cancer cell lines; namely, those of the liver (line HepG2), prostate (line DU145) and breast (line MBA-MB-231). The cytotoxicity assessment revealed that compounds 5d, 5g, 5h and 5i exhibit promising antiproliferative effects (IC50 1–5 µM) against those three cancer cell lines.


Synthesis and Characterization
The derivative 3-acetyl-2,5-dichlorothiophene, 2, was prepared following a published procedure [20] by Friedel-Crafts acylation of 2,5-dichlorthiophene, 1, in carbon disulfide (CS 2 ). The condensation of 3-acetyl-2,5-dichloro-thiophene (2) with different aromatic aldehydes, 3a-i, in the presence of potassium hydroxide, afforded chalcones 4a-i (Scheme 1). The targeted pyridine-3-carbonitrile derivatives, 5a-i were obtained by the reaction of chalcones, 4a-i, with malononitrile using methanolic solution of sodium hydroxide in good yields. In some cases, decyanation of pyridine-3-carbonitrile derivatives, 5a-i, provided the pyridine compounds 6a-g in poor yields; see Scheme 1. Spectroscopic data ( 1 H-NMR, 13 C-NMR, COSY, HSQC and HMBC) and mass ESI-HMRS measurements support the proposed structures for compounds 5a-i and 6a-g. The formation of 2-methoxypyridine-3-carbonitrile products 5a-i, Scheme 2, was assumed to take place via conjugated addition of the malononitrile anion to the β-carbon of the corresponding chalcone, followed by methanol attack to C≡N group, leading to the imine A which tautomerizes to amine derivative B. The intermolecular dehydrative cyclization of amine B furnished the dihydropyridine C which oxidizes (aromatization) to the desired 5a-i product (Scheme 2) [21][22][23][24]. The decyanation of 5a-i is believed to proceed through the hydrolysis of the nitrile derivative to the corresponding carboxylic acid followed by the decarboxylation to form pyridines 6a-g (Scheme 2). Scheme 1. Synthesis of pyridine derivatives (5 and 6).
The formation of 2-methoxypyridine-3-carbonitrile products 5a-i, Scheme 2, was assumed to take place via conjugated addition of the malononitrile anion to the β-carbon of the corresponding chalcone, followed by methanol attack to C≡N group, leading to the imine A which tautomerizes to amine derivative B. The intermolecular dehydrative cyclization of amine B furnished the dihydropyridine C which oxidizes (aromatization) to the desired 5a-i product (Scheme 2) [21][22][23][24]. The decyanation of 5a-i is believed to proceed through the hydrolysis of the nitrile derivative to the corresponding carboxylic acid followed by the decarboxylation to form pyridines 6a-g (Scheme 2  All new compounds were characterized by 1 H and 13 C-NMR, and 2D-NMR (See Supplementary Materials). In the 1 H-NMR spectra of compounds 5a-i, the methoxy protons (OCH3-2) appeared at δ 4. 16-4.20. The singlet signals which resonated at 7.58-7.67 and 7.40-7.43 were assigned to pyridine (H-5) and thiophene (H-4′) protons, respectively. This was supported by HMBC and COSY correlations. In addition, the 1 H-NMR spectra of compounds 6a-g exhibit two singlets at 7.41-7.43 and 4.04-4.08 attributed to H-4′ of thiophene and the methoxy protons (OCH3-2), respectively. The two mutually-coupled doublet signals at δ 7.66-7.72 and 6.89-6.97, respectively, could be assigned to H-5 and H-3 protons, respectively, based on their coupling constants (J = 1.2-1. 7 Hz), and the chemical shifts of carbons to which they are attached (C-5, δ 113.9-117.0; C-3, δ 107.3-110.3).
The 13 C-NMR spectra of compounds 5a-i and 6a-g are very similar, with one significant exception that the chemical shifts of C-3 in the series 5a-i, appeared at 95.9 ppm instead of the 107.3 ppm for compounds 6a-g. Finally, all structural assignments are well-matched with the highresolution HSESIMS data and further confirmed by a combination of COSY, HSQC and HMBC experiments (See Supplementary Materials). A complete analysis of all NMR data is listed in the experimental section.

Cytotoxicity Properties
All compounds tested, 5a-i and 6a-g, were subjected to different concentrations for cytotoxicity screening against three human cancer cell lines, namely, HepG2 (liver), DU145 (prostate) and MBA-MB-231 (breast) along with standard 5-fluorouracil. The cytotoxicity scanning results showed variable degrees of toxicity on all the cancer cell lines (Table 1).

Scheme 2.
Proposed mechanism for the formation of pyridine derivatives (5 and 6).
The 13 C-NMR spectra of compounds 5a-i and 6a-g are very similar, with one significant exception that the chemical shifts of C-3 in the series 5a-i, appeared at 95.9 ppm instead of the 107.3 ppm for compounds 6a-g. Finally, all structural assignments are well-matched with the high-resolution HSESIMS data and further confirmed by a combination of COSY, HSQC and HMBC experiments (See Supplementary Materials). A complete analysis of all NMR data is listed in the experimental section.

Cytotoxicity Properties
All compounds tested, 5a-i and 6a-g, were subjected to different concentrations for cytotoxicity screening against three human cancer cell lines, namely, HepG2 (liver), DU145 (prostate) and MBA-MB-231 (breast) along with standard 5-fluorouracil. The cytotoxicity scanning results showed variable degrees of toxicity on all the cancer cell lines (Table 1). In general, the cytotoxicity results, based on IC 50 values for the compounds we tested, displayed considerable effects (<100 µM) against all cancer cell lines, but 5a, 5b and 5c did not (>100 µM). Among compounds tested, 5i exhibited the most promising anticancer activity with a considerably broad spectrum of cytotoxic activity against all three human cancer cell lines. In tissue-specific toxicity, compounds 5d, 5h and 5i showed very strong cytotoxic activity against HepG2 cell lines, all having IC 50 values of 1.53 µM; comparatively, 5-fluorouracil had an IC 50 value of 1.65 µM. Both compounds 5h and 5i demonstrated potent cytotoxic effects on DU145 cell lines with respect to the cytotoxicity of the 5-fluorouracil. Compound 5i was the most potent cytotoxic agent against MBA-MB-231 cell lines, with an IC 50 value of 1.38 µM for each.
On the other hand, compounds 6a-g displayed mild to moderate cytotoxic activity with IC 50 values in the range of 10.34-64.59 µM against all of the cancer cell lines. In addition, the selectivity indexes of compounds were determined in this study. All compounds were tested on normal human fibroblast cell lines. The IC 50 values of compounds 5d, 5g, 5h and 5i were found to be high in normal human fibroblast cell lines (Table 1), suggesting their high selectivity for cancer cells over normal cells. These results may provide some important justification for further development as anticancer agents.
The activity was increased in the 5d derivative compared to the chloro-5c one. This leads to the conclusion that relatively larger substituents are valuable for good activity. Introducing a methoxy group at position 2 of the phenyl ring of 5f, increases the activity. Therefore, the presence of a hydrophilic substituent on the phenyl ring will increase the activity. To explore the influence of electron withdrawing groups, a nitro group was introduced at positions 3 and 4 for 5g and 5h, respectively. Surprisingly, these two compounds showed very potent inhibitions against cell lines between 1 and 5 µM. Consequently, the nitro group can be considered an interesting moiety. In addition, compound 5i with bromo and methoxy substitutions exhibited a similar activity to 5h.
In conclusion, the structural requirements of pyridine-3-carbonitrile derivatives to enhance their cytotoxic activity against tested cell lines are as follows: (a) the nitrile substituent at position 3 on the pyridine ring is essential for activity and as a basic pharmacophore; (b) the nitro, methoxy and chloro substituents on the benzene ring are useful for the enhancement of activity (Figure 2). Such substituents were selected to offer variable lipophilic, electronic and steric environments in order to influence the biological activity being targeted; said specificity is believed to be responsible for the biological significance of some anticancer agents [11].
Molecules 2019, 24, 4072 6 of 12 substituents on the benzene ring are useful for the enhancement of activity ( Figure 2). Such substituents were selected to offer variable lipophilic, electronic and steric environments in order to influence the biological activity being targeted; said specificity is believed to be responsible for the biological significance of some anticancer agents [11].

Materials
The starting material, 3-acetyl-2,5-dichlorothiophene (2) was prepared as described in the previously-reported procedure [20]. The chalcone compounds, 4a-i, were synthesized according to literature's methods [25]. All reagents were purchased from Fluka and used as purchased. Solvents were dried and distilled according to standard protocols.

Instrumentation
1 H (500 MHz) and 13 C (125 MHz) NMR spectra were recorded in CDCl3 used as an internal standard at 300 K on Bruker spectrometers (Bruker Biospin Mri GmbH, Ettlingen, Germany). Chemical shifts (δ) are given in parts per million (ppm) and were determined from the center of the respective coupling pattern (s: singlet; d: doublet; dd: doublet of d; t: triplet). ESI-HMRS measurements were performed on an LTQ-FT mass spectrometer (Thermo Fisher Scientific, Schwerte, Germany). All reactions were monitored using thin layer chromatography (TLC) coated with silica gel (60 F254, Merck, Darmstadt, Germany). Pure compounds were obtained by preparative TLC plates using a CHCl3/pentane (30:70) mixture as an eluent.

Materials
The starting material, 3-acetyl-2,5-dichlorothiophene (2) was prepared as described in the previously-reported procedure [20]. The chalcone compounds, 4a-i, were synthesized according to literature's methods [25]. All reagents were purchased from Fluka and used as purchased. Solvents were dried and distilled according to standard protocols.

Instrumentation
1 H (500 MHz) and 13 C (125 MHz) NMR spectra were recorded in CDCl 3 used as an internal standard at 300 K on Bruker spectrometers (Bruker Biospin Mri GmbH, Ettlingen, Germany). Chemical shifts (δ) are given in parts per million (ppm) and were determined from the center of the respective coupling pattern (s: singlet; d: doublet; dd: doublet of d; t: triplet). ESI-HMRS measurements were performed on an LTQ-FT mass spectrometer (Thermo Fisher Scientific, Schwerte, Germany). All reactions were monitored using thin layer chromatography (TLC) coated with silica gel (60 F 254 , Merck, Darmstadt, Germany). Pure compounds were obtained by preparative TLC plates using a CHCl 3 /pentane (30:70) mixture as an eluent.

Synthesis
Synthesis of chalcones 4a-i, went according to the reported procedure [19]. A solution of 3-acetyl-2,5-dichlorothiophene, 2, in MeOH (10 mL) was added dropwise to an equimolar mixture of the corresponding aldehydes 3a-I and KOH in MeOH (50 mL). After the addition was completed, the reaction mixture was stirred at RT for 10 h. The precipitate formed was filtered off, washed with MeOH and dried without any further purification.

Cell Culture and Maintenance
Three different cancer cell lines; namely, DU145 derived from prostate cancer cells (American Type Culture Collection No. HTB-81), HepG2 derived from human liver cancer cells (American Type Culture Collection No. HB-8065), MBA-MB-231 derived from breast cancer cells (American Type Culture Collection No. HTB-26) and normal human fibroblasts (HSF1184) were grown and cultivated according to our previously described method [18]. Briefly, cells were seeded in Dulbecco s modified Eagle's media (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, 100 mg/mL streptomycin and 2 mM glutamine. The cells were maintained in a humidified atmosphere with 5% CO 2 at 37 • C for 8 days, with subsequent media renewal. Then, cells were plated in a 96-well flat bottom plate at a concentration of 104 cells/well in fresh complete growth medium for 24 h and maintained in 37 • C in a 5% CO 2 -humidified incubator.

Cytotoxicity Assay
Cells in 96-well flat bottom plates were first serum-starved for 4 h by replacing cultured media with fresh medium (without serum). All synthesized compounds were properly dissolved in dimethyl sulfoxide (DMSO) to produce a stock concentration of 0.1 M. Cells were incubated with a range of different concentrations of the compounds we were testing (0-1000 µM) to give a final volume of 300 µL per well. In comparison, cells were incubated either alone with DMSO (negative control) or with 5-fluorouracil as a positive control. DMSO was used as a vehicle for the dissolution of the tested compounds and its final concentration in cultured media was less than 0.1%. Triplicate wells were designed for each individual dose. The compounds we synthesized were evaluated for in-vitro cytotoxicity properties via the standard 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay accordingly to our previously-reported work [2]. The normal human fibroblast line (HSF1184) was used as a standard reference for the measurement of selectivity indexes (SIs) relative to cancer cell lines. The IC 50 values and SIs were calculated accordingly.

Conclusions
In conclusion, a series of 2-methoxypyridine-3-carbonitrile bearing aryl substituents were synthesized, in most cases in good yields, by the condensation of chalcones with malononitrile in basic medium. Although decarbonylation byproducts were observed in poor yields, they offer a route to a variety of methoxypyridine derivatives. All new compounds were fully characterized using different spectroscopic methods. Furthermore, these compounds were screened for their in-vitro cytotoxicity activities against three cancer cell lines; namely, those of the liver (line HepG2), prostate (line DU145) and breast (line MBA-MB-231). Although the IC 50 values are not very impressive, their selectivity with respect to normal cells is, in some cases, very high. Remarkably, the decarbonylated substrates show less cytotoxicity on normal cells than in cancer cells. The cytotoxicity assessments revealed that compounds 5d, 5g, 5h and 5i exhibit promising antiproliferative effects (IC 50 1-5 µM) against tested cancer cell lines.