Synthesis of Some Polysubstituted Nicotinonitriles and Derived Pyrido [ 2 , 3-d ] pyrimidines as In Vitro Cytotoxic and Antimicrobial Candidates

1Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia 2Department of Chemistry, Faculty of Science, University of Alexandria, Alexandria, Egypt 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, P.O. Box 80260, Jeddah 21589, Saudi Arabia 4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521, Egypt


Introduction
Over the past two decades, several reports have discussed the growing number of cancerous diseases and the prominent role displayed by chemotherapy as the most fruitful treatment for many disseminated types of tumors.This has resulted in the activation of much research targeting the discovery of prominent lead structures that would be beneficial in styling novel antitumor chemotherapeutic agents [1][2][3].Additionally, the dilemma of multi-drug resistant (MDR) bacteria arising from the misuse of traditional antimicrobial agents has been extensively studied [4].In addition, mycotic invasions became a complicated problem especially in patients who experience immunotherapy, organ transplantation, carcinomas, or AIDS [5].Therefore, there is a genuine demand for finding out new agents endowed with antimicrobial activity that acts with nonconventional mechanisms.Patients who undergo cancer chemotherapy are clearly liable to microbial infections owing to the subsidiary reduction of immunity.Concomitant use of several drugs for treating cancer joined with microbial invasions might cause additional complications in patients with malfunctioned kidneys.Consequently, the idea of administering one drug with dual action could be beneficial from both curative and economic viewpoints.In recent years, cyanopyridines and some of their fusedring systems such as quinolines and pyridopyrimidines have been under focus as potential chemotherapeutic candidates because of their reported distinctive antimicrobial [6][7][8], antitubercular [9], and antiviral [10] activities, in addition to the well documented antineoplastic [11], antiproliferative [12], and cytotoxic [13] potentials.Furthermore, several pyridinecontaining compounds were reported to possess dual anticancer and antimicrobial activities [14][15][16][17].
During our current interest in exploring new lead structures that possess chemotherapeutic potential, much attention was focused on the antimicrobial and anticancer activities of some pyridine derivatives [18][19][20][21][22][23][24], particularly those possessing the 4,6-disubstituted-2-aminopyridin-3-carbonitrile (nicotinonitriles) scaffold which revealed favourable broad spectrum anticancer and/or antimicrobial efficacy.The collected results persuaded the design and synthesis of novel polysubstituted nicotinonitriles and some derived pyrido [2,3-d]pyrimidines incorporating different functional groups that are claimed to account for the bioactivity of related antitumor and antimicrobial candidates.Additionally, the substituents encountered in these derivatives were chosen so as to affect the electronic environment of the compounds, which would synergistically influence the targeted biological activities, in an attempt to obtain novel candidates with both anticancer and antimicrobial potentials.

Experimental
2.1.Chemistry.Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected.The infrared (IR) spectra were recorded on Shimadzu FT-IR 8400S infrared spectrophotometer using the KBr pellet technique. 1H and 13 C NMR spectra were recorded on a Bruker DPX-400 FT NMR spectrometer using Tetramethylsilane (TMS) as the internal standard and DMSO- 6 as a solvent (Chemical shifts in , ppm).Splitting patterns were designated as follows: s: singlet; t: triplet; q: quartet; m: multiplet.The Electron Impact Ionization Mass Spectra were recorded on a Nermag R10-10C at 70 ev.1000 Ex spectrometer.Elemental analyses were performed on a 2400 Perkin Elmer Series 2 analyzer and the values found were within ±0.4% of the theoretical values.Follow-up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) and the spots were detected by exposure to UV-lamp at  254.(1)(2)(3)(4)(5).A one-pot mixture of the appropriate aromatic aldehyde (10 mmol), the ketone (10 mmol), malononitrile (0.66 g, 10 mmol), and ammonium acetate (6.2 g, 80 mmol) in absolute ethanol (50 mL) was refluxed for 4-5 h.The reaction mixture was allowed to attain room temperature and the resulting precipitate was filtered, washed with water, dried, and recrystallized from ethanol.

In Vitro MTT Cytotoxicity
Assay.The synthesized compounds were investigated for their in vitro cytotoxic effect via the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [25,26] against a panel of three human tumor cell lines, namely, Caucasian breast adenocarcinoma MCF7, hepatocellular carcinoma HepG2, and colon carcinoma HT29 and a normal nontransformed human foreskin fibroblast Hs27 cell line.The procedures were done in a sterile area using a laminar flow cabinet biosafety class II level (Baker, SG403INT, Stanford, ME, USA).Cells were batch-cultured for 10 days and then seeded at concentration of 10 × 10 3 cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37 ∘ C for 24 h under 5% CO 2 using a water jacketed carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA).Media was aspirated, fresh medium (without serum) was added, and cells were incubated either alone (negative control) or with different concentrations of the test compounds to give a final concentration of 100-50-25-12.5-6.25-3.125-1.56-0.78g/mL.DMSO was employed as a vehicle for dissolution of the tested compounds and its final concentration on the cells was less than 0.2%.Cells were suspended in RPMI 1640 medium (for HepG2 and HT29 cell lines) and DMEM (for MCF 7 cell line), 1% antibiotic-antimycotic mixture (10,000 IU/mL Penicillin Potassium, 10,000 g/mL Streptomycin Sulphate, and 25 g/mL Amphotericin B), and 1% L-Glutamine in 96-well flat bottom microplate at 37 ∘ C under 5% CO 2 .After 24 h of incubation, the medium was aspirated and 40 L of MTT salt (2.5 g/mL) was added to each well and incubated for further 4 h at 37 ∘ C under 5% CO 2 .To stop the reaction and dissolve the formed crystals, 200 L of 10% sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37 ∘ C. The absorbance was then measured using a microplate multiwell reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595 nm and a reference wavelength of 620 nm.A statistical significance was tested between samples and negative control (cells with vehicle) using independent t-test by SPSS 11 program.The results are presented in Table 1 as LC 50 (M) which is the lethal concentration of the compound which causes death of 50% of the cells in 24 h.

In Vitro Antibacterial and Antifungal Activities.
All of the newly synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), and Micrococcus luteus (ATCC 21881) as examples of Gram-positive bacteria and Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumonia (clinical isolate) as examples of Gram-negative bacteria.They were also evaluated for their in vitro antifungal potential against Candida albicans (ATCC 10231) and Aspergillus niger (recultured) fungal strains.Each 100 mL of sterile molten agar (at 45 ∘ C) received 1 mL of 6 h-broth culture and then the seeded agar was poured into sterile Petri dishes.Cups (8 mm in diameter) were cut in the agar.Each cup received 0.1 mL of the 1 mg/mL solution of the test compounds.The plates were then incubated at 37 ∘ C for 24 h or, in case of C. albicans, for 48 h.A control using DMSO without the test compound was included for each organism.Ampicillin trihydrate and Clotrimazole were used as reference drugs.The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the discs in mm.The minimal inhibitory concentrations (MIC) of the most active compounds were measured using the twofold serial broth dilution method [27].The test organisms were grown in their suitable broth: 24 h for bacteria and 48 h for fungi at 37 ∘ C. Twofold serial dilutions of solutions of the test compounds were prepared [28] using 200, 100, 50, 25, 12.5, and 6.25 g/mL.The tubes were then inoculated with the test organisms; each 5 mL received 0.1 mL of the above inoculum and was incubated at 37 ∘ C for 48 h.Then, the tubes were observed for the presence or absence of microbial growth.

Results and Discussion
3.1.Chemistry.The pathways adopted for the preparation of the intermediate and target compounds are illustrated in Scheme 1. Preparation of the key intermediates 2-amino-4,6-disubstituted nicotinonitriles 1-5 was performed according to Hantzsch-type synthesis via multicomponent onepot cyclocondensation of the appropriate aromatic aldehyde (piperonal or thiophene-2-carbaldehyde) with the substituted acetophenone, malononitrile, and ammonium acetate as a nitrogen source.The reaction passes through a twostep procedure that involved the formation of chalcones via Claisen-Schmidt condensation, followed by cyclocondensation with malononitrile and ammonium acetate.When compounds 1-5 were heated with either formic acid or acetic anhydride, the targeted 5,7-disubstituted pyrido [2,3d]pyrimidine-4(3H)-ones 6-10 and their 2-methyl analogs 11-15, respectively, were successfully obtained.The IR spectra of the latter compounds showed the absence of the CN group absorption and the appearance of new sharp absorption bands at 1656-1648 cm −1 attributed to the newly formed C=O groups at position 4, beside the NH absorption bands at 3320-3259 cm −1 .Meanwhile, the Reacting compounds 1-5 with the phenyl isothiocyanate in pyridine medium led to the formation of the substituted dihydropyrido [2,3-d]pyrimidine-2(1H)-thiones 16-20.Their IR spectra showed new bands at 1642-1635 cm −1 and at 1204-1188 cm −1 corresponding to the C=N moiety and to the C=S group, respectively.Their 13 C NMR spectral data were characterized by the presence of two signals at  179.3-181.2and  164.4-164.8ppm for the C=S and C=NH groups, respectively.However, condensing the starting compounds 2-4 with benzoyl isothiocyanate in acetone afforded the corresponding thioureido derivatives 21-23.Their IR spectra revealed the CN absorption bands at 2226-2215 cm −1 , in addition to the C=O and C=S bands at 1665-1661 and 1174-1166 cm −1 , respectively, whereas their 13 C NMR spectra exhibited two characteristic signals at  168.1-169.3 and 179.9-180.4ppm for the CO and CS carbons, respectively.

In Vitro MTT Cytotoxicity Assay.
All the newly synthesized compounds were evaluated for their in vitro cytotoxic effect via the standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method [25,26] against a panel of three human tumor cell lines, namely, colon carcinoma HT29, hepatocellular carcinoma HePG2, and Caucasian breast adenocarcinoma MCF7.The results are presented in Table 1 as LC 50 (M) which is the lethal concentration of the compound that causes death of 50% of the cells in 24 h.
The obtained data revealed that the three tested human tumor cell lines exhibited variable degree of sensitivity profiles towards nine of the tested compounds, namely, 21, 22, 23, 30, 32, 33, 34, 36, and 37, whereas the other twenty-six compounds were either marginally active or even totally inactive.Regarding the activity against the human colon carcinoma HT29, this cell line proved to be very sensitive to all the nine active compounds.In particular, it revealed distinctive sensitivity towards compounds 33, 34, and 37 (LC 50 25.2,28.8, and 26.9 M, resp.) even higher than doxorubicin (LC 50 40.0M), the reference standard cytotoxic agent utilized in this assay.Meanwhile, compounds 22 and 36 (LC 50 46.7 and 40.4 M, resp.) were nearly equipotent to doxorubicin (LC 50 40.0M), whereas compounds 21 and 23 (LC 50 70.5 and 62.2 M, resp.)showed moderate cytotoxic potential against the same cell line.Shifting to the hepatocellular carcinoma HepG2, this cell line showed mild to weak sensitivity towards seven of the tested analogs with LC 50 range 64.6-111.3M, when compared to doxorubicin (LC 50 3.0 M).Among these, the highest activity was displayed by compounds 33, 34, and 37 (LC 50 64.4,70.1, and 71.2 M, resp.).On the other hand, the human breast cancer MCF 7 emerged as the least sensitive among the cell lines tested as its growth was affected by the presence of only six test compounds.However, a remarkable growth inhibition potential was shown by analogs 33, 34, and 37 as evidenced from their LC 50 values (LC 50 6.4,7.9, and 8.91 M, resp.), which represents about 40-60% of the activity of doxorubicin (LC 50 4.0 M).
Further interpretation of the results revealed that compounds 33, 34, 36, and 37 showed considerable broad spectrum cytotoxic activity against the three tested human tumor cell lines.In particular, compounds 33, 34, and 37 proved to be the most active members in this study with special effectiveness against both the colon carcinoma HT29 (almost twice as active as doxorubicin; LC 50 25.2,28.8, and 26.9 versus 40 M, resp.) and human breast cancer MCF 7 (about 40-60% of the activity of doxorubicin; LC 50 6.4,7.9, and 8.91 versus 4.0 M, resp.).
A close examination of the structures of the active compounds showed that the nature of substituents (X and/or R), together with ring entity (mono-or bicyclic), seemed to influence the cytotoxic activity.In this context, compounds substituted with the 4-bromo-or 4-methoxyphenyl moities (X = Br and OCH 3 ) together with the benzo[d] [1,3]dioxol-5-yl counterpart (22, 30, 33, and 34) were in favour of better cytotoxic activity, when compared with their 2-thienyl congeners (23, 32, 36, and 37), as revealed from their LC 50 values in Table 1.Moreover, the bicyclic pyrido [2,3d]pyrimidines proved to be more active than the monocyclic nicotinonitriles.In this view, although the starting nicoti- On the other hand, a remarkable improvement in both cytotoxic spectrum and potency was observed by thioalkylation of the 2-thione function of compounds 28-32 either with a methyl or ethyl group, where four potentially active compounds 33, 34, 36, and 37 were obtained.Among these, the analogs compounds 33 (X = Br; R = 3,4- and 37 (X = OCH 3 ; R = 2-thienyl; R 2 = C 2 H 5 ) stemmed as the most active members in this study.

In Vitro Differential Cytotoxicity Effect.
Owing to the known common side effects often induced by chemotherapeutic agents exemplified by the possible destruction of normal cells, it was thought worthwhile to evaluate the in vitro differential cytotoxic effect of the most active analogs 33, 34, 36, and 37 on the nontransformed human foreskin fibroblast Hs27 cell line, under the same previously mentioned MTT assay experimental conditions.
Considering the obvious selective inhibitory potential displayed by the analogs (33, 34, 36, and 37) on the growth of the human breast MCF7 and colon HT29 cancer cell lines, the results revealed that the potential cytotoxic activity exhibited by 33, 34, and 37 (LC 50 values range 6.4-28.8M) was confirmed by their clear differential cytotoxic behavior, showing a marginal inhibitory effect on the growth of the nontransformed human foreskin fibroblast Hs27 cell line (LC 50 values > 200 M), whereas a lower differential cytotoxic profile was shown by analog 36 against the tested normal cell line (LC 50 > 200 M), when compared with its LC 50 values against the human breast MCF7 and colon HT29 cancer cell lines (22.8 and 40.4 M, resp.)(Table 1). ) and Aspergillus niger (recultured) fungal strains.Agardiffusion method was used for determination of preliminary antibacterial and antifungal activity.Ampicillin trihydrate (antibiotic) and Clotrimazole (antifungal) were used as reference drugs [27].Dimethylsulfoxide (DMSO) was used as a blank and showed no antimicrobial activity.The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the discs in mm.The minimum inhibitory concentration (MIC, g/mL) was determined for compounds that showed significant growth inhibition zones (≥13 mm) using the twofold serial dilution method [29].

In Vitro
As revealed from MIC data recorded in Table 2, nineteen of the newly synthesized compounds displayed variable inhibitory effects on the growth of the tested Gram-positive and Gram-negative microorganisms with pronounced activity against S. aureus and E. coli bacterial strains.In addition, some members exhibited moderate antifungal activity against C. albicans, whereas all the tested compounds lacked antifungal activity against Aspergillus niger.
Among the tested Gram-positive bacterial strains, two organisms, namely, S. aureus and B. subtilis, showed relatively high sensitivity towards the tested compounds.Concerning the activity against Gram-positive bacteria, compound 33 (MIC 6.25 g/mL) was equipotent to ampicillin against S. aureus; meanwhile analogs 30, 34, and 36 (MIC 12.5 g/mL) and 18, 20, 35, and 37 (MIC 25 g/mL) showed 50% and 25% of the activity of ampicillin, respectively.With regard to the activity against B. subtilis, compounds 33 and 34 (MIC 25 g/mL) exhibited half the activity of ampicillin (MIC 12.5 g/mL).M. luteus was the least sensitive Gram-positive microorganism towards the majority of the tested compounds, except analog 33 which showed 50% of the activity of ampicillin (MIC 25 versus 12.5 g/mL).On the other hand, investigation of anti-Gram-negative efficacy of the active compounds revealed that three analogs 30, 33, and 34 were able to produce a distinctive growth inhibitory profile against E. coli being equipotent to ampicillin (MIC 6.25 g/mL), whereas compounds 31, 36, and 37 (MIC 12.5 g/mL) were 50% less active than ampicillin against the same organism.Moreover, the tested P. aeruginosa strain showed high sensitivity towards compound 33 (MIC 12.5 g/mL) which was equiactive with ampicillin, whereas the rest of the active compounds revealed moderate to weak activity against the same strain (MIC range 25-200 g/mL).Shifting to the antifungal potential, ten compounds 18, 20, 29-31, and 33-37 were able to display appreciable growth inhibitory activity against C. albicans (MIC values 12.5-100 g/mL, resp.) when compared with Clotrimazole (MIC 6.25 g/mL), the standard antifungal agent utilized in this assay.Among these, analog 33 exhibited the best antifungal activity (MIC 12.5 g/mL) that represents 50% of the standard.It is to be noted that all the tested compounds lacked any antifungal activity against Aspergillus niger.Structurally, the nineteen active compounds represent two variations: the substituted nicotinonitriles and the bicyclic pyrido [2,3-d]pyrimidines.In general, relatively better antibacterial potential and spectrum were mostly confined to the derivatives comprising the bromine (X = Br) and the benzo[d] [1,3]dioxol-5-yl groups, when compared with their methoxylated (X = OCH 3 ) and 2-thienyl congeners.Besides, better antimicrobial activity was associated with the bicyclic pyrido [2,3-d]pyrimidines when compared with the monocyclic nicotinonitrile precursors (Table 2).In this context, while the key nicotinonitrile precursors 3-5 showed mild antimicrobial activity, yet condensation of the 2-amino group with benzoyl isothiocyanate afforded three thiocarbamoyl benzamide derivatives 21-23 which revealed a slight improvement in the antimicrobial spectrum although not in efficacy.Annelation of nicotinonitriles 1-5 into the pyrido [2,3-d]pyrimidine-4(3H)-ones 6-10 resulted in complete abolishment of activity, whereas introducing a methyl group at C 2 to the latter ring system furnished two 2-methyl-pyrido [2,3-d]pyrimidine-4(3H)-one derivatives 13 and 15 which were noticeably less active than their parent compounds 3 and 5. Furthermore, reaction of starting nicotinonitriles 1-5 with phenyl isothiocyanate gave rise to three active compounds 18-20, among which analog 18 (X =Br; R = 3,4-(OCH 2 O)C 6 H 3 ) showed relatively better antimicrobial spectrum and potential.Although cyclization of nicotinonitriles 1-5 with urea into the corresponding pyrido [2,3-d]pyrimidine-2(1H)-ones 24-27 led to total loss of activity, yet isosteric replacement of the 2-one with a 2thione functionality yielded three significantly active broad spectrum compounds 29-31, among which analog 30 (X = Br; R = 3,4-(OCH 2 O)C 6 H 3 ) was the most active being equipotent to ampicillin against E. coli, together with a mild antifungal activity.Finally, thioalkylation of the 2-thione function of 28-32 afforded five methyl-or ethylthio derivatives 33-37 with a great enhancement in both antimicrobial spectrum and potential.In particular, compound 33 (X = Br; R = 3,4-(OCH 2 O)C 6 H 3 ; R 2 = CH 3 ) proved to be equipotent to ampicillin against S. aureus, E. coli, and P. aeruginosa, together with a noticeable antifungal activity against C. albicans, whereas analog 34 (X = Br; R = 3,4-(OCH 2 O)C 6 H 3 ; R 2 = C 2 H 5 ) was equiactive with ampicillin against E. coli.

Conclusions
Thirty-seven polysubstituted pyridines and some derived bicyclic pyrido [2,3-d]pyrimidine ring systems supported with various chemotherapeutically active functionalities were successfully synthesized, characterized, and evaluated for their biological activity as cytotoxic and/or antimicrobial agents.Globally, the data obtained from both the cytotoxic and antimicrobial assays suggested that higher biological activities were confined mainly to compounds comprising the 4-bromo, 4-methoxy, benzo[d] [1,3]dioxol-5-yl substituents, and the bicyclic pyrido [2,3-d]pyrimidines rather than the monocyclic nicotinonitriles.
Regarding the cytotoxic activity, nine compounds were able to show variable cytotoxic efficiency, among which alkylthio analogs 33, 34, and 37 proved to be the most active members with considerable broad spectrum cytotoxic activity against the three tested human tumor cell lines and particular effectiveness against both the colon carcinoma HT29 (almost twice as active as doxorubicin; LC 50 25.2,28.8, and 26.9 versus 40 M, resp.) and human breast cancer MCF 7 (about 40-60% of the activity of doxorubicin; LC 50 6.4,7.9, and 8.91 versus 4.0 M, resp.).In addition, the same three analogs showed a clear differential cytotoxic profile as they exhibited a marginal inhibitory effect on the growth of the normal nontransformed human foreskin fibroblast Hs27 cell line (LC 50 values > 200 M).
On the other hand, the results of the in vitro antimicrobial screening revealed that nineteen compounds were able to exhibit significant antibacterial potential against both Grampositive and Gram-negative bacteria, together with mild to moderate antifungal activities.The pyrido [2,3-d]pyrimidine-2(1H)-thione 30 in connection with its alkylthio derivatives 33 and 34 stemmed as the most active antimicrobial members being equipotent to ampicillin against S. aureus, E. coli, and P. aeruginosa, together with a noticeable antifungal activity against C. albicans.Globally, the distinctive cytotoxic and antimicrobial behavior displayed by alkylthio derivatives 33 and 34 are concordant with several literature findings that emphasized the role of thioethers in enhancing the antimicrobial and antitumor activities [28,30,31].This makes such type of compounds a fruitful matrix for further development of more potent and selective anticancer and/or antimicrobial agents.In particular, compounds 33 and 34 could be considered as possible dual antimicrobialanticancer candidates that deserve further investigation and derivatization in order to explore the scope and limitation of their biological activities.

Table 1 :
Cytotoxic effects LC 50 (M) a of the active compounds on three human tumor and nontransformed cell lines and using the MTT assay.LC 50 : lethal concentration of the compound which causes death of 50% of cells in 24 h; b totally inactive against this cell line; c NT: not tested; a d Doxorubicin: positive control cytotoxic agent.
1H-NMR spectra of compounds 11-15 showed new singlets at  2.28-2.36ppmdue to the newly introduced CH 3 group, whereas their13C NMR spectral data exhibited new singlets at  20.2-21.1 ppm due to the new CH 3 group and the CO signals at  168.8-173.2ppm.

Table 2 :
Minimal inhibitory concentrations (MIC; g/mL) of the tested compounds.