Design, synthesis, and biological evaluation of novel benzimidazole derivatives as sphingosine kinase 1 inhibitor.

Sphingosine kinase 1 (SphK1) has emerged as an attractive drug target for different diseases. Recently, discovered SphK1 inhibitors have been recommended in cancer therapeutics; however, selectivity and potency are great challenges. In this study, a novel series of benzimidazoles was synthesized and evaluated as SphK1 inhibitors. Our design strategy is twofold: It aimed first to study the effect of replacing the 5-position of the benzimidazole ring with a polar carboxylic acid group on the SphK1-inhibitory activity and cytotoxicity. Our second aim was to optimize the structures of the benzimidazoles through the elongation of the chain. The enzyme inhibition potentials against all the synthesized compounds toward SphK1 were evaluated, and the results revealed that most of the studied compounds inhibited SphK1 effectively. The binding affinity of the benzimidazole derivatives toward SphK1 was measured by fluorescence binding and molecular docking. Compounds 33, 37, 39, 41, 42, 43, and 45 showed an appreciable binding affinity. Therefore, the SphK1-inhibitory potentials of compounds 33, 37, 39, 41, 42, 43, and 45 were studied and IC50 values were determined, to reveal high potency. The study showed that these compounds inhibited SphK1 with effective IC50 values. Among the studied compounds, compound 41 was the most effective one with the lowest IC50 value and a high cytotoxicity on a wide spectrum of cell lines. Molecular docking revealed that most of these compounds fit well into the ATP-binding site of SphK1 and form hydrogen bond interactions with catalytically important residues. Overall, the findings suggest the therapeutic potential of benzimidazoles in the clinical management of SphK1-associated diseases.

Part III: Novel checkpoint kinase 2 (Chk2) inhibitors; design, synthesis and biological evaluation of pyrimidine-benzimidazole conjugates.

Recently a dramatic development of the cancer drug discovery has been shown in the field of targeted cancer therapy. Checkpoint kinase 2 (Chk2) inhibitors offer a promising approach to enhance the effectiveness of cancer chemotherapy. Accordingly, in this study many pyrimidine-benzimidazole conjugates were designed and twelve feasible derivatives were selected to be synthesized to investigate their activity against Chk2 and subjected to study their antitumor activity alone and in combination with the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). The results indicated that the studied compounds inhibited Chk2 activity with high potency (IC50 = 5.56 nM - 46.20 nM). The studied candidates exhibited remarkable antitumor activity against MCF-7 (IG50 = 6.6  µM - 24.9 µM). Compounds 10a-c, 14 and 15 significantly potentiated the activity of the studied genotoxic drugs, whereas, compounds 9b and 20-23 antagonized their activity. Moreover, the combination of compound 10b with cisplatin revealed the best apoptotic effect as well as combination of compound 10b with doxorubicin led to complete arrest of the cell cycle at S phase where more than 40% of cells are in the S phase with no cells at G2/M. Structure-activity relationship was discussed on the basis of molecular modeling study using Molecular modeling Environment program (MOE).

Part II: New candidates of pyrazole-benzimidazole conjugates as checkpoint kinase 2 (Chk2) inhibitors.

The development of checkpoint kinase 2 (Chk2) inhibitors for the treatment of cancer has been an ongoing and attractive objective in drug discovery. In this study, twenty-one feasible pyrazole-benzimidazole conjugates were synthesized to study their effect against Chk2 activity using Checkpoint Kinase Assay. The antitumor activity of these compounds was investigated using SRB assay. A potentiation effect of the synthesized Chk2 inhibitors was also investigated using the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). In vivo Chk2 and antitumor activities of 8d as a single-agent, and in combination with doxorubicin, were evaluated in breast cancer bearing animals induced by N-methylnitrosourea. The effect of 8d alone and in combination with doxorubicin was also studied on cell-cycle phases of MCF-7 cells using flow cytometry analysis. The results revealed their potencies as Chk2 inhibitors with IC50 ranges from 9.95 to 65.07 nM. Generally the effect of cisplatin or doxorubicin was potentiated by the effect of most of the compounds that were studied. The in vivo results indicated that the combination of 8d and doxorubicin inhibited checkpoint kinase activity more than either doxorubicin or 8d alone. There was a positive correlation between checkpoint kinase inhibition and the improvement observed in histopathological features. Single dose treatment with doxorubicin or 8d produced S phase cell cycle arrest whereas their combination created cell cycle arrest at G2/M from 8% in case of doxorubicin to 51% in combination. Gold molecular modelling studies displayed a high correlation to the biological results.

Part I: Design, synthesis and biological evaluation of novel pyrazole-benzimidazole conjugates as checkpoint kinase 2 (Chk2) inhibitors with studying their activities alone and in combination with genotoxic drugs.

Activated checkpoint kinase 2 (Chk2) is a tumor suppressor as one of the main enzymes that affect the cell cycle. 2-Biarylbenzimidazoles are potent selective class of Chk2 inhibitors; the structure-based design was applied to synthesize a new series of this class with replacing the lateral aryl group by substituted pyrazoles. Ten pyrazole-benzimidazole conjugates from the best fifty candidates according to docking programs have been subjected to chemical synthesis in this study. The activities of the conjugates 5-14 as checkpoint kinase inhibitors and as antitumor alone and in combination with genotoxic drugs were evaluated. The effect of compounds 7 and 12 on cell-cycle phases was analyzed by flow cytometry analysis. Antitumor activity of compounds 7 and 12 as single-agents and in combinations with doxorubicin was assessed in breast cancer bearing animals induced by MNU. The Results indicated that compounds 5-14 inhibited Chk2 activity with high potency (IC50 52.8 nM-5.5 nM). The cytotoxicity of both cisplatin and doxorubicin were significantly potentiated by the most of the conjugates against MCF-7 cell lines. Compounds 7 and 12 and their combinations with doxorubicin induced the cell cycle arrest in MCF-7 cells. Moreover, compound 7 exhibited marked higher antitumor activity as a single agent in animals than it's combination with doxorubicin or doxorubicin alone. The combination of compound 12 with doxorubicin was greatly effective on animal than their single-dose treatment. In conclusion, pyrazole-benzimidazole conjugates are highly active Chk2 inhibitors that have anticancer activity and potentiate activity of genotoxic anticancer therapies and deserve further evaluations.

Synthesis of (benzimidazol-2-yl)aniline derivatives as glycogen phosphorylase inhibitors.

A series of (benzimidazol-2-yl)-aniline (1) derivatives has been synthesized and evaluated as glycogen phosphorylase (GP) inhibitors. Kinetics studies revealed that compounds displaying a lateral heterocyclic residue with several heteroatoms (series 3 and 5) exhibited modest inhibitory properties with IC50 values in the 400-600µM range. Arylsulfonyl derivatives 7 (Ar: phenyl) and 9 (Ar: o-nitrophenyl) of 1 exhibited the highest activity (series 2) among the studied compounds (IC50 324µM and 357µM, respectively) with stronger effect than the p-tolyl analogue 8.

New benzimidazoles and their antitumor effects with Aurora A kinase and KSP inhibitory activities.

A newly synthesized series of anticancer compounds comprising thiazolo[3,2-a]pyrimidine derivatives 6a-q bearing a benzimidazole moiety was produced via a one-pot reaction of N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-2-cyanoacetamide 5 with 2-aminothiazole and an appropriate aromatic aldehyde. Compound 7 was obtained via the reaction of 4-(1H-benzo[d]imidazol-2yl)benzenamide 1 with carbon disulphide and methyl iodide in the presence of concentrated aqueous solution of NaOH, then treated with o-phenylenediamine to give N-(4-1H-benzo[d]imidazol-2-yl)phenyl)-1H-benzo[d]imidazol-2-amine 8. The structures of the newly synthesized compounds were confirmed by analytical and spectroscopic measurements (IR, MS, and (1) H NMR). The synthesized products were screened and studied for their in vitro antitumor activity against three human cancer cell lines (namely colorectal cancer cell line HCT116, human liver cancer cell line HepG2, and human ovarian cancer cell line A2780) and their Aurora A kinase and KSP inhibitory activities. All newly synthesized compounds revealed marked results comparable with the standard drug CK0106023. The compounds 6e and 6k of the thiazolopyrimidine derivatives were the most active compounds when tested against the three cell lines in comparison with the standard drug CK0106023, and showed potent dual KSP and Aurora A kinase inhibition.

Design, synthesis and molecular docking study of novel quinoxalin-2(1H)-ones as anti-tumor active agents with inhibition of tyrosine kinase receptor and studying their cyclooxygenase-2 activity.

On continuation to our work, new quinoxalin-2(1H)-ones were synthesized to study their cytotoxic effect against HepG-2 and MCF-7 with their effect on the human tyrosine kinase (TRK). Compounds 12, 18, 15, 13, 11a, 20 and 16, respectively, were found to be more potent than cisplatin against HepG2 and selective to TRK. Also, compounds 12, 18, 20, 13, 14, and 22, respectively, exhibited decidedly activity against MCF-7 and selectivity against human TRK compared to cisplatin. A molecular docking study was also performed to gain comprehensive understanding into plausible binding modes and to conclude the structure activity relationships of the synthesized compounds. Moreover, anti-inflammatory activity was studied. Compounds 12, 15, 18 and 22 were found to be potent and selective against COX-2.

Design, synthesis and structure-activity relationship of novel quinoxaline derivatives as cancer chemopreventive agent by inhibition of tyrosine kinase receptor.

The cancer chemopreventive activity of quinoxaline derivatives 1-20 has been evaluated by studying the inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation. The quinoxaline derivatives 1-20 showed inhibitory effect on EBV-EA activation without cytotoxicity on Raji cells. All compounds exhibited dose dependent inhibitory activities, most of them showed significant activity at 1000 mol ratio/12-O-tetradecanoylphorbol-13-acetate (TPA). Compounds 7 and 9 exhibited stronger inhibitory effects on the EBV-EA activation than that of the representative control, oleanolic acid, at the highest measured concentration. In addition, compounds 7-10 showed potent and selective inhibition of human tyrosine kinase (TRK) in liver cancer HepG2 and breast cancer MCF-7 cell lines similar to the positive control, doxorubicin.

Synthesis and docking studies of novel antitumor benzimidazoles.

In this work, the benzimidazole-pyrrole conjugates 6a-h and benzimidazole-tetracycles conjugates 12-14 were prepared. The cytotoxicity of the compounds 3, 4a-h, 6a-h, 8, 10 and 12-14 was tested against lung cancer cell line A549. Compound 6b exhibited higher activity than the bis-benzoxazole natural product (UK-1), the standard. The tested 4g,h, 6a-h, 10 and 12-14 exhibited remarkable cytotoxicity activity against breast cancer cell line MCF-7 with higher activity than tamoxifen. Furthermore, compound 4h was found to be also more potent than doxurubicin. The antitumor promotion activity of synthesized compounds 4g,h, 6a-h, 10 and 12-14 has been estimated by studying their possible inhibitory effects on EBV-EA activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied compounds, the inhibitory activities of compounds 8, 13 and 14 demonstrated strong inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation without showing any cytotoxicity on the Raji cells and their effects being stronger than that of a representative control, oleanolic acid. Moreover, the molecular docking of the new compounds into plasminogen activator (uPA) receptor has been in correlation with the antitumor activity. All synthesized compounds 3, 4a-h, 6a-h, 8, 10 and 12-14 were docked into same groove of the binding site of the native co-crystalized (4-iodobenzo[b]thiophene-2-carboxamidine) ligand (PDB code:1c5x) for activity explaination. Compounds 4h, 6b and 13, giving the best docking results, were further studied to estimate their effect on the level of uPA using AssayMax human urokinase (uPA) ELISA kit. In case of A549 cell line, compound 6 exhibited similar activity to MMC, and for MCF-7 cell line, compound 4h exhibited similar activity to doxorubicin, in inhibiting the expression of uPA.

Novel benzimidazo[2,1-c][1,4]thiazinone derivatives with potent activity against HSV-1.

The synthesis of new 2-carboxymethylsulfanylmethyl-1H-benzimidazole and 1,3-dihydro-4H-benzo[4',5']imidazo[2,1-c][1,4]thiazine-4-one-8-carboxylic acid derivatives was investigated. The antiviral activity of compounds 1-14 was tested against the herpes simplex virus 1. Compounds 5 and 14 showed potent activity as they inhibited virus propagation by 94.7% and 91.3% at a dose of 50 µg, respectively. Compounds 5 and 14 showed higher potency than Acyclovir at doses of 20 µg and 50 µg.

Part I: Synthesis, cancer chemopreventive activity and molecular docking study of novel quinoxaline derivatives.

The reaction of o-phenylene diamine and ethyl oxamate is reinvestigated and led to 3-aminoquinoxalin-2(1H)-one rather than benzimidazole-2-carboxamide as was previously reported. The structure of the obtained quinoxaline has been confirmed by X-ray. The anti-tumor activity of synthesized quinoxalines 1-21 has been evaluated by studying their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied compounds 1-21, compounds 12, 8, 13, 18, 17 and 19, respectively, demonstrated strong inhibitory effects on the EBV-EA activation without showing any cytotoxicity and their effects being stronger than that of a representative control, oleanolic acid. Furthermore, compound 12 exhibited a remarkable inhibitory effect on skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. The result of the present investigation indicated that compound 12 might be valuable as a potent cancer chemopreventive agent. Moreover, the molecular docking into PTK (PDB: 1t46) has been done for lead optimization of the aforementioned compounds as potential PTK inhibitors.

Synthesis and antitumor activity of novel benzimidazole-5-carboxylic acid derivatives and their transition metal complexes as topoisomerease II inhibitors.

N-aminomethyl-1H-benzimidazole-5-carboxylic acid derivatives 2-5 and the ligand, 1-(5 (or 6-)-carboxy-1H-benzimidazol-2-ylmethyl)pyridinium chloride (6; H2L1) have been synthesized. New benzimidazole complexes 7-9 of the ligand 6; H2L1 with Cu2+, Co2 and Zn2+ were prepared. The growth-inhibitory against a panel of 21 human cancer cell lines of the synthesized compounds 1-9 was studied. Compounds 6-9 showed potent growth-inhibitory activity against the studied cell lines. The correlation coefficients according to COMPARE analysis of the National Cancer Institute screening protocol showed that the pattern of the growth-inhibitory effect of the compounds 6-9 was similar to that of etoposide and doxorubicin but different from that of SN-38 and cisplatin. The topoisomerase II inhibitory activity of the tested compounds 6-9 was studied. Compounds 6 and 8 inhibited topoisomerase II activity at 10 times lower concentration than etoposide in relaxation assay.

Novel antiviral benzofuran-transition metal complexes.

(5-(1H-benzo[d]imidazol-2-yl)-1H-pyrrol-3-yl)(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)methanone (4) and 3-(6-hydroxy-4,7-dimethoxybenzofuran-5-carbonyl)-6H-pyrimido[1,6-a]pyrimidine-6,8(7H)-dione (5) were synthesized by the reaction of 4,7-dimethoxy-5-oxo-5H-furo[3,2-g]chromene-6-carbaldehyde (1) with (1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride and 4-amino-2,6-dihydroxypyrimidine, respectively, via ROR in the presence of alcoholic KOH. The metal complexes 6-9 of compound 4; H(2)L(1) with (CuCl(2), FeCl(3), ZnCl(2), and LaCl(3)) and the metal complexes 10-13 of compound 5; H(2)L(2) with (CuCl(2), FeCl(3), CoCl(2) and LaCl(3)) were synthesized to form 1:1 or 1:2 (metal: ligand) complexes. The HIV inhibitory activity of all new compounds was tested. The EC(50) values showed that, all of tested compounds were more potent than Atevirdine. Moreover, the benzoimidazolylpyrrole derivative 4 (EC(50)=9x10(-6)muM) had higher therapeutic index than the standard. The HIV-1 RT inhibitory activity showed that all of the tested compounds showed significant potency but none of them showed higher activity than Atevirdine. The HCV NS3-4A protease inhibitor activity of the tested compounds revealed that the complex formation had great positive effect on the bioactivity, where the Fe-complex 7 was the most potent compound with higher therapeutic index than VX-950, the standard. Also, the cytotoxicity of the synthesized compounds on hepatocyte cell line, showed that Cu-complex 10 was the most potent compound with potency nearly to that of the standard.

Synthesis of potent antitumor and antiviral benzofuran derivatives.

A new series of potent antitumor and antiviral benzofuran derivatives was synthesized by the reaction of the furochromone-6-carboxaldehydes 1 and 2 with different heterocyclic amines to yield the benzofuran-5-carbonyl derivatives 4-11. The synthesized compounds 1, 3-11 were tested against twelve different human cancer cell lines and all of the compounds were more potent than the comparative standards. The HIV inhibitory activity of the tested compounds 1, 3-11 showed that they have higher potency than Atevirdine. Moreover, compound 6 was significantly potent with wider therapeutic index. The HIV-1 RT inhibitory activity showed that compounds 10, 11, 3 and 4 were notably potent but with lower therapeutic index than Atevirdine. The HCV NS3-4A protease inhibitor activity of the tested compounds revealed that they have weaker potency and less therapeutic index than VX-950, although compounds 1, 4, 9 and 6, respectively exhibited significant activity.

New transition metal ion complexes with benzimidazole-5-carboxylic acid hydrazides with antitumor activity.

Metal complexes of 2-methyl-1H-benzimidazole-5-carboxylic acid hydrazide (4a; L(1)) and its Schiff base 2-methyl-N-(propan-2-ylidene)-1H-benzimidazole-5-carbohydrazide (5a; L(2)) with transition metal ions e.g., copper, silver, nickel, iron and manganese were prepared. The complexes formed were 1:1 or 1:2 M:L complexes and have the structural formulae [Cu(L(1))Cl(H(2)O)]Cl x 3 H(2)O (6), [Ag(L(1))NO(3)(H(2)O)] (7), [Ni(L(1))Cl(2)(H(2)O)(2)] x H(2)O (8), [Fe(L(1))Cl(3)(H(2)O)] x 3 H(2)O (9) and [Mn(L(1))(2)Cl(H(2)O)]Cl x 3 H(2)O (10) for ligand L(1), and [Cu(L(2))Cl(2)(H(2)O)(2)] x H(2)O (11), [Ag(L(2))(2)]NO(3) x H(2)O (12), [Ni(L(2))(2)Cl(2)] x 5 H(2)O (13), [Fe(L(2))(2)Cl(2)]Cl x 2 H(2)O (14) and [Mn(L(2))Cl(2)(H(2)O)(2)] x H(2)O (15) for ligand L(2). The antitumor activity of the synthesized compounds has been studied. The silver complex 7 was found to display cytotoxicity (IC(50)=2 microM) against both human lung cancer cell line A549 and human breast cancer cell line MCF-7.