Potential Anticancer Agents: Design, Synthesis of New Pyrido[1,2-a]benzimidazoles and Related Derivatives Linked to Alkylating Fragments

The incentive of the present work has been primarily directed towards the design and synthesis of some novel pyrido[1,2-a]benzimidazoles with specific functionalities believed to have alkylation ability. This combination of pharmacological agents may enable synergistic anticancer effect. Nine compounds 5b, 13a, 13d, 13e, 14b, 14c, 15, 16, and 17 were selected by the National Cancer Institute (NCI), Bethseda, Maryland, USA to be evaluated for their in vitro antitumor activity. All the selected compounds were tested initially at a single dose (10 μM) in the full NCI 60 cell panel including leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancer cell lines. Majority of the test compounds exhibited moderate cytotoxic activity. The highest activity in all the investigated cancer cells was displayed by 14c against melanoma SK-MEL-5 cell line. This may be due to the impact of the lipophilic trifluoromethyl substitution on the biological activity profile.


Introduction
Cancer is a devastating affliction, the frequency of which is progressively increasing all over the world. Its occurrence is escalating rapidly and is a major cause in health complications [1]. The treatment approach dictates that the treatment of cancer is directed toward eradication of all cancer cells and this is attained by frenziedly discovery of new candidates of anticancer activity [2].
Previously, we have utilized pyrido[1,2-a]benzimidazole (PBI) as a privileged scaffold for the design of many PBI derivatives of potential cytotoxic activity [3][4][5][6][7][8]. In fact, this ring system is characterized by the presence of pyridine or 2-pyridone units which constitute a subject of great interest due to their extensive presence in the skeletal backbone of many biologically active compounds. They possess a wide variety of biological activities such as antiulcer [9], antidiabetic [9], antiinflammatory [10], anticoagulant [11], antiviral [12], antibacterial [13], antifungal [14] and anticancer activities [15]. Pyridine moiety is one of the building units of some tyrosine-kinase inhibitors; imatinib is used in the treatment of multiple cancers; whereas, sorafenib is used in the treatment of advanced renal and hepatocellular carcinoma [16]. The 2-pyridone unit is an integral part of some cytotoxic agents such as roquinimex which investigated as adjuvant therapy after bone marrow transplantation in chronic myelogenous leukemia [17] and diazaquinomycin A which demonstrates in vitro cytotoxicity against some tumor cell lines [18].
Among the investigated PBI series,NSC649900 [3], NSC682011 [4] and NSC699944 [5] (Figure 1) were identified by the NCI as promising candidates for further testing in an in vivo anticancer hollow fiber assay because of their good cytotoxic activity and subpanel disease selectivity especially against leukemic cell in the in vitro screen. In fact, the PBI backbone of these compounds demonstrates structural complementarity with the isosteric β-carboline and pyrido [2,3-a] indolizine (PI) which constitute the key scaffolds of many cytotoxic agents such as the β-carboline alkaloid harmine which is identified as a useful inhibitor of tumor development [19] and the antitumor antibiotic camptothecin [20].
Biochemical data suggests that camptothecin act as DNA topoisomerase I inhibitor. It possesses a novel mechanism of action involving the inhibition of DNA relaxation by DNA topoisomerase I, and more specifically the stabilization of a covalent binary complex formed between topoisomerase I and DNA [20]. In addition, it is proposed that the planar nature of camptothecin allows its intercalation between DNA base pairs at the site of single-strand cleavage [21]. For this reason, it may be worthy to study the possible interactions of NSC649900, NSC682011 and NSC699944 with topisomerase-I as a target enzyme because of the evident structural complementarity between PBI scaffold of these agents and pyrido [2,3-a]indolizine (PI) backbone of camptothecin ( Figure 2). Docking results revealed that NSC649900, NSC682011 and NSC699944 displayed arene-arene interactions with one or more amino acid residues similar to camptothecin.NSC649900 showed arene-arene interactions with DAC113 and TGPB11 residues, in addition to hydrogen bonding with ArgD364; whereas, NSC682011 displayed arene-arene interactions with TGPB11and ArgD364 amino acid residues. In addition, NSC699944 revealed arene-arene interactions with TGPB11 and DAC113 amino acid residues. The result indicates that the planar PBI scaffold contribute to binding to the main active sites similar to camptothecin and it is possible that these PBIs may intercalate between DNA base pairs of topo I-DNA complex through arene-arene interactions ( Figure 2). Inspired by these findings and in a continuation of our efforts to discover and explore new heterocyclic compounds of promising anticancer activities two series of PBIs comprising pyridine and 2-pyridone nuclei ( Figure 3) were designed to be synthesized and evaluated for their in vitro anticancer activity. Both series are designed with specific functionalities such as 2-hydoxyalkyl, 2-chloroalkyl, piperidino-and morpholino alkyl moieties at postion-2 through an aminomethylene spacer ( Figure 3, Scheme 1) or at position-1( Figure 3, Scheme 2). Other analogues comprising aliphatic amino and aryl amino moieties are proposed.

Experimental Chemistry
All reagents and solvents were purchased from commercial suppliers and were purified and dried when necessary by standard techniques. Melting points were determined in open glass capillaries using Stuart capillary melting point apparatus (Stuart Scientific Stone, Staffordshire, UK) and are uncorrected. IR spectra were recorded, for potassium bromide discs, ύ (cm -1 ), on Perkin Elmer 1430 spectrophotometer. 1 H-NMR spectra were determined either on a Bruker Avance spectrometer (400 MHz) at the microanalytical unit, Faculty of Science, Cairo University, or on Jeol (125 MHz) at the microanalytical unit, Faculty of Science, Alexandria University, using DMSO-d 6 as a solvent and TMS as internal standard. The chemical shifts are given in δ ppm values (s, singlet; d, doublet; t, triplet and m, multiplet). 13 C-NMR spectra were determined on Jeol (125 MHz), Faculty of Science, Alexandria University, using TMS as internal standard. Mass spectra were run on a Finnigan mass spectrometer model SSQ/7000 (70 eV), Faculty of Science, Cairo University. Microanalyses were performed at the microanalytical unit, Faculty of Science, Cairo University. The results of the microanalyses were within ± 0.4% of the calculated values. Follow-up of the reactions and checking the homogeneity of the compounds were made by ascending TLC run on silica gel G (Merck 60) coated glass plates. The spots were visualized by exposure to iodine vapor or UV lamp at λ 254 nm for few seconds.

-( 2 -H y d r o x y e t h y l ) a m i n o -3 -m e t h y l p y r i d o [ , 2 -a ] benzimidazole-4-carbonitrile (11):
A mixture of the chloro derivative (10) (4 mmol, 0.97 g) and ethanolamine, (12 mmoles, 0.73 g) in dioxane (20 ml) was heated at 60-80°C for 10 h. The reaction mixture was then poured into ice cold water. The product was filtered, washed with water, dried and crystallized from DMF/ ethanol.

Antitumor activity
Nine compounds 5b, 13a, 13d ,13e,14b, 14c, 15, 16, and 17 were selected by the National Cancer Institute (NCI), Bethseda, Maryland, USA to be evaluated for their in vitro antitumor activity. Effective onedose assay has been added to the NCI 60 Cell screen in order to increase compound throughput and reduce data turnaround time to suppliers while maintaining efficient identification of active compounds. All the selected compounds were tested initially at a single dose (10 µM) in the full NCI 60 cell panel including leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancer cell lines. The results are presented in Table 1 as growth (G%) and only compounds which satisfy pre-determined threshold inhibition criteria would progress to the five-dose screen [26][27][28][29].

Chemistry
The synthetic procedures implemented to obtain the newly synthesized compounds are demonstrated in Schemes 1 and 2.
In Scheme 1, the enamine tautomers of Schiff bases 2, 3, 6 and 8 were synthesized by reacting the aldehyde 1 [7] with the proper amine. 1 HNMR spectrum of compound 2 displayed a singlet at 1.50 ppm due to tert-butyl protons and a doublet at 8.35 ppm integrated for one proton attributed to the methine proton. It also revealed one D 2 O exchangeable doublet at 11.60 ppm characteristic for NH proton. Meanwhile, the 1 HNMR spectrum of compound 3 revealed one triplet at 3.69 ppm attributed to the four protons of the ethanolamine side chain and a D 2 O exchangeable triplet at 5.07 ppm due to OH proton. One doublet at 8.46 ppm integrated for one proton representing the methine proton. Also its 13 CNMR spectrum showed peaks at around 53 and 60 ppm corresponding to NCH 2 and OCH 2 moieties of the ethanolamine side chain. 1 HNMR spectrum of compound 6 revealed a singlet at 1.42 ppm due to the six protons of the two CH 3 groups of the side chain and a doublet at 3.50 ppm corresponding to the CH 2 -O protons. In addition, a D 2 O exchangeable triplet at 5.53 ppm due to OH proton was observed. The spectrum also showed one doublet at 8.31 ppm attributed to the methine proton and a doublet at 11.68 ppm characteristic for the NH proton. Additionally, 1 HNMR spectrum of compound 8 revealed a singlet at 1.36 ppm due to the CH 3 protons of the side chain and a D 2 O exchangeable triplet at 5.41 ppm due to two OH protons. Two doublets at 8.32 and 11.70 ppm, each integrated for one proton attributed to the methine and the NH protons, respectively were shown.
Chlorination of aliphatic OH group was achieved by heating the starting material 3, 6 and 8 in excess phosphorous oxychloride to give the corresponding chlorinated compounds 4, 7 and 9. The 1 HNMR spectrum of compound 4 lacked the D 2 O exchangeable triplet of the OH proton and revealed a doublet at 8.52 ppm attributed to the  methine proton. The spectrum also showed a multiplet at around 11.2 ppm characteristic for the NH proton. Furthermore, the 13 CNMR spectrum displayed peaks at 44.15 and 52.02 ppm attributed to NCH 2 and CH 2 Cl moieties of the side chain, respectively. Nucleophilic displacement of aliphatic chlorine atom by aliphatic amines was achieved by reacting a suspension of 4 and the proper amine in a mixture of absolute ethanol and dimethylformamide to furnish compounds 5a-c. their IR spectrum showed absorption bands between 3227-3420 cm -1 corresponding to the NH groups. 1 HNMR spectrum of compound 5a revealed a singlet at 1.50 ppm representing the nine protons of the tert-butyl group and one doublet at 8.36 ppm corresponding to the methine proton. A mulitplet at 11.61 ppm attributed to the enamine NH proton was also displayed whereas, 1 HNMR spectrum of compound 5b revealed two multiplets at 1.40-1.53 and 2.44-2.50 ppm corresponding to the piperidine protons. The spectrum also showed one doublet at 8.46 ppm integrated for one proton attributed to the methine proton. A mulitplet at 11.18 ppm attributed to the enamine NH proton was also displayed. Furthermore, the 1 HNMR spectrum of compound 5c showed two multiplets due to the morpholine protons, one doublet at 8.47 ppm attributed to the methine proton and a mulitplet at 11.20 ppm attributed to the enamine NH proton. 1 HNMR spectrum of the chlorinated compound 7 also lacked the D 2 O exchangeable triplet of the OH proton and revealed one doublet at 8.32 ppm characteristic for the methine proton. A D 2 O exchangeable doublet at 11.60 ppm attributed to the NH was also displayed. Moreover, 1 HNMR spectrum of compound 9 lacked the D 2 O exchangeable triplet of the two OH protons and revealed one doublet at 8.35 ppm integrated for one proton characteristic for the methine proton. A doublet at 11.63 ppm attributed to the NH proton was also displayed.
In Scheme 2, the following amines 11, 14a-c and 16 were prepared by reacting the chloro derivative 10 [7] with ethanolamine, substituted aryl amines and diethanolamine in dioxane at 60-80°C. 1 HNMR spectrum of compound 11 revealed two quartets at 3.55 and 3.76 ppm representing the four protons of ethanolamine moiety. The presence of both the OH proton and the NH proton was confirmed by the presence of two D 2 O exchangeable triplets at 5.01 and 7.24 ppm, respectively. While 1 HNMR spectrum of compound 14b showed a singlet at 3.80 ppm representing the OCH 3 protons and a doublet and a multiplet at 7.04 and around 7.3 ppm, respectively, indicating the presence of the 4-methoxyphenyl group. Also the 13 CNMR spectrum displayed signals at around 55.73, 115.36 and 126.27 ppm attributed to OCH 3 carbon and methoxyphenyl C 2,6 and C 3,5 respectively. In addition, peaks at 131.96 and 149.16 ppm corresponding to methoxyphenyl C 1 and C 4 were observed. 1 HNMR spectrum of compound 16 revealed multiplet at 3.36-3.69 ppm representing the eight protons of the diethanolamine group. The presence of both OH protons was confirmed by the presence of a D 2 O exchangeable triplet at 4.56ppm. The hydroxyl precursors 11 and 16 were chlorinated by heating in excess phosphorous oxychloride to produce compounds 12 and 17. Their IR spectra lacked the absorption bands attributed to the OH groups. In addition; their 1 HNMR spectra revealed disappearance of the D 2 O exchangeable OH proton signal confirming its replacement by the chloro group. 1 HNMR spectrum of compound 12 revealed a quartet at 3.85 ppm and a triplet at 3.98 ppm representing the four protons of the chloroethyl group. Its 13 CNMR spectrum showed peaks at 42.95 and 44.84 ppm corresponding to NCH 2 and CH 2 Cl, respectively. Scheme 2 also describes the reaction of chloro derivative 12 with dimethylamine, tert-butylamine, piperidine, morpholine and piperazine to give the corresponding amines 13 a-e, respectively. Their IR spectra showed absorption bands in the range between 3206 and 3536 cm -1 corresponding to the NH groups. 1 HNMR spectrum of compound 13a revealed a singlet at 2.30 ppm integrated for six protons indicating the presence of two CH 3 groups. The reaction of 1-Chloro-3methylpyridobenzimidazole-4-carbonitrile 10 [7] with dimethylamine in dioxane at room temperature led to compounds 15; its 1 HNMR spectrum revealed a singlet at 2.93 ppm representing six protons of the two CH 3 groups.

In vitro antitumor activity
Nine compounds 5b, 13a, 13d, 13e, 14b, 14c, 15, 16, and 17 were selected by NCI and tested initially at a single dose (10 µM) in the full NCI 60 cell line panel. The results are recorded as percentage growth (G%); for example, a value of (100%) means no growth inhibition. A value of (20%) would mean (80%) growth inhibition.

Structure Activity Relationship (SAR)
Compound 5b revealed a weak inhibitory effect against many cell lines from some types of cancer. This would indicate that the presence of 2-pyridone scaffold did not result in a significant improvement on the activity. Structural activity correlation revealed that the PBIs which lack the 2-pyridone unit but have instead a pyridine moiety showed variable cytotoxic activity. For instance, substitution at position-1 with N,N-dimethylaminoethylamino (13a), morpholinoethylamino (13d) and piperazinyl ethylamino (13e) did not let significant activity. On the other hand, the PBI which carry 4-methoxyphenylamino moiety (14b) exhibited weak inhibitory effects against several cell lines from non-small cell lung cancer, melanoma, renal and breast cancer and it demonstrated remarkable cytotoxic activity against one cell line from leukemia. It is worthy to mention that the highest anticancer activity was recorded for compounds 14c. Results revealed that replacement of the 4-methoxy (14b) with 3-trifluoromethyl group (14c) resulted in broad spectrum and variable degree of activity against many of the tested cell lines. In fact, this finding would indicate the impact of the lipophilic 3-trifluoromethylphenylamino substituents on the activity.
The presence of bis(2-hydroxyethyl)amino group (16) did not show any significant impact on the activity; whereas, the PBI (17) which carry an alkylating fragment, bis(2-choloroethyl)amino, displayed a remarkable inhibitory effect against colon cancer HCT-116 cell line and weak inhibitory effects against many cell lines from leukemia, melanoma, non-small lung cancer and colon, renal, prostate and breast cancer.

Conclusion
In conclusion, two series of benzimidazoles comprising pyridine and 2-pyridone nuclei together with various functionalities believed to have alkylation ability were synthesized. These series were designed as an example of a new molecular hybrids having anticancer activity. The anticancer activity results revealed that among the tested compounds, compound (14c) was found to possess promising anticancer activity and the most significant inhibition as revealed from the growth percentage (G%)was found against melanoma SK-MEL-5 (4.24%) and UACC-62 (11.90%), CNS cancer U251 (11.62%), colon cancer HT-29 (13.19%); and breast cancer BT-549 (19.39%) and T-47D (8.68%) cell lines. In fact, this finding together with the remarkable antineoplastic activity reported to the related PBI analogues (NSC682011 and NSC699944, Figure 1) would indicate the impact of the lipophilic 3-tripfluoromethylphenylamino and 4-fluorophenylamino substituents on the activity. Although, none of the screened compounds satisfied the threshold inhibition criteria to pass for evaluation in the full panel fivedose in vitro antitumor screen, The PBI 14c can be considered starting structure that merit further optimization in order to design more active lead compounds for further studies.