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Full Paper Synthesis, Biological Evaluation, and Docking Studies of New 2-Furylbenzimidazoles as Anti-Angiogenic Agents: Part II Ahmed Temirak1, Yasser M. Shaker1, Fatma A. F. Ragab2, Mamdouh M. Ali3, Salwa M. Soliman4, Jeremie Mortier4,5, Gerhard Wolber4, Hamed I. Ali6,7, and Hoda I. El Diwani1 1
2 3
4 5 6
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Division of Pharmaceutical and Drug Industries, Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, Cairo, Egypt Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Cairo University, Giza, Egypt Division of Genetic Engineering and Biotechnology, Department of Biochemistry, National Research Center, Cairo, Egypt Department of Pharmaceutical Chemistry, Institute of Pharmacy, Free University of Berlin, Berlin, Germany Department of Organic Chemistry, Free University of Berlin, Berlin, Germany Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Round Rock, TX, USA Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Helwan University, Cairo, Egypt
The 2-(5-methyl-2-furyl)-1H-benzimidazole moiety has shown promising activity against vascular endothelial growth factor (VEGF)-induced angiogenesis. In part I of this study, we have synthesized new analogs and tested their anti-angiogenic potentials. Here, we continue our previous study with different new analogs. Some compounds show promising cytotoxic activity against the human breast cancer cell line MCF-7, with IC50 in the range of 7.80–13.90 mg/mL, and exhibited remarkable in vitro inhibition against VEGF in the MCF-7 cancer cell line, with 95–98% of inhibition in comparison to tamoxifen as reference (IC50: 8.00 mg/mL, % of inhibition ¼ 98%). Additionally, a molecular docking study was carried out to gain insight into plausible binding modes and to understand the structure– activity relationships of the synthesized compounds. Keywords: Angiogenesis / Cytotoxicity / 2-(2-Furyl)-1H-benzimidazoles / Molecular modeling / Vascular endothelial growth factor (VEGF) Received: September 29, 2013; Revised: November 12, 2013; Accepted: November 15, 2013 DOI 10.1002/ardp.201300356
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Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Introduction Angiogenesis, which is the formation of new capillary blood vessels from preexisting ones [1], is a crucial process that promotes tumor growth, survival, and metastasis through supplying these proliferating tissues with oxygen and nutrients [2–4]. Angiogenesis is stimulated in tumor tissues through the production and secretion of angiogenic factors like vascular endothelial growth factor (VEGF) [5], plateletCorrespondence: Dr. Yasser M. Shaker, Division of Pharmaceutical and Drug Industries, Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, 12622 Cairo, Egypt. E-mail: [email protected], [email protected] Fax: þ20 2 3337093
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derived growth factor (PDGF) [6], basic fibroblast growth factor (bFGF) [7], endothelial growth factor (EGF) [8], and angiopoietin [9]. The VEGF signaling pathway that acts through the VEGF receptor 2 (VEGFR2) plays a key role in the regulation of tumor angiogenesis, in which the binding of VEGF to VEGFR2 leads to receptor dimerization, followed by the autophosphorylation of tyrosine residues in the intracellular kinase domain, resulting in potent mitogenic and chemotactic effects on endothelial cells [10]. The discovery of new tumor angiogenesis inhibitors targeting VEGFR has been the scope of many researchers during the past few years [11, 12]. New inhibitors have passed the clinical trials and are marketed as novel drugs targeting
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angiogenesis such as multikinase inhibitors; sunitinib and sorafenib and the monoclonal antibody bevacizumab. These agents have shown proven efficacy against many cancers that encourage researchers to move forward in this area [13–16]. There are many examples in the literature of 2-subsituted benzimidazoles as potential inhibitors for angiogenesis through targeting the VEGFR (Fig. 1). 4-(2-(4-Bromophenylamino)-1-methyl-1H-benzo[d]imidazol-5-yloxy)-N-methylpyridine2-carboxamide 1 was reported as a novel 2-arylbenzimidazole compound showing potential RAF kinase inhibition. It inhibits four RTKs (CSF1R kinase, VEGFR kinase, c-Kit, and PDGFR) [17]. Moreover, dovitinib (TKI-258) 2, a modified scaffold of 3benzimidazol-2-ylhydroquinolin-2-one has shown selective inhibition of FGFR and VEGFR’s. It exhibited promising efficacy against metastatic renal cell carcinoma in clinical trials (Fig. 1) [18, 19]. A series of 2-anilino-4-(benzimidazol-2-yl)pyrimidines 3 showed multikinase inhibition with antiproliferative activity toward four cancer-related protein kinases
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(Aurora B, PLK1, FAK, and VEGFR2) [20]. Also, compound 4 (Pubchem ID 47037197), a 2-phenyl substituted benzimidazole, resulting from the virtual screening of the Pubchem database [21, 22], has shown multi-targeting activity against EGFR, VEGFR2, and PDFGR in addition to the inhibition of cancer cell lines (HepG-2) and kinase enzyme inhibition [21, 22]. Furthermore, it was reported that albendazole, methyl[6(propylthio)-1H-benzimidazol-2-yl]carbamate 5, the widely used drug for the treatment of hydatid disease, suppresses VEGF levels, HIF-1a and has anti-angiogenic effect in noncancerous models of angiogenesis (Fig. 1) [23] both in vitro (via treatment of human umbilical vein endothelial cells (HUVECs) with albendazole leading to down-regulation of the VEGFR2) and in vivo (via inhibition of hyperoxia-induced retinal angiogenesis using albendazole). These results were reported and provided new insights into the effective antiangiogenic role of albendazole [24]. Furthermore, the novel benzimidazole analog 6 (AC1-004) was discovered to downregulate VEGF and EPO target genes of HIF-1 and to inhibit
Figure 1. Chemical structures of 2-subsituted benzimidazoles reported in the literature with potent anti-angiogenic activity.
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the in vitro tube formation of HUVEC leading to its potential inhibitory activity on angiogenesis [25, 26]. The nucleus of 2-furylbenzimidazole has shown outstanding activity against VEGF-induced angiogenesis in tumor tissues both in vitro and in vivo. 2-(5-Methyl-2-furyl)-1Hbenzimidazole (NP-184) 7 inhibited the angiogenic functions of HUVEC in vitro and reduced the angiogenesis rate in vivo in material plug assay model (Fig. 1) [27, 28]. The structural modification of compound NP-184 resulted in compound (PJ-8) 8, which extremely inhibited VEGF–VEGFR2 and suppressed the tumor-induced angiogenesis in vivo [29]. These remarkable results were the key elements in our research with the aim of discovering new analogs for the lead compound NP-184 exhibiting effective inhibition for VEGFinduced angiogenesis. This work is a continuation of our previous research in designing new anti-angiogenic 2-furylbenzimidazoles (submitted for publication) [30]. We have carried out the synthesis, biological activity, and molecular docking studies of a series of new analogs through the functionalization and modification on position 1 of the benzimidazole nucleus forming alkyl chains, substituted hydrazides, semicarbazides, thiosemicarbazides, and heterocyclic rings and/or position 5 of the 2-furyl ring (Fig. 2).
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The synthesized compounds 14–18 (Schemes 1 and 2) were tested for their antitumor activity against MCF-7 breast cancer cell lines. We have previously studied the cytotoxic activity of the 2-substituted benzimidazoles against MCF-7 cancer cell line in our lab and we have observed remarkable selectivity against this cancer cell line [31]. We have synthesized several compounds in our lab that have shown remarkable cytotoxicity against MCF-7 cancer cell line and studied their inhibitory action against VEGF kinase enzyme using double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to measure the level of human VEGF in samples. These bioactivity studies were done in comparison to the reference drug tamoxifen. In order to understand their biological effect, a molecular modeling study was carried out with these new inhibitors using a pharmacophore-based approach. With LigandScout [32], the key features for the inhibition of VEGFR2 were compiled into a 3D-pharmacophore model from the ligand (K11) co-crystallized in the binding pocket of the receptor (PDB entry: 3EWH) [33]. Docking studies using GOLD [34] were conducted to generate the docking poses of the synthesized compounds, which were ranked based on their ability to fulfill the key interactions compiled in the 3D-pharmacophore model. Based on this study, the structure– activity relationship of the synthesized 2-(5-methyl-2-furyl)-1Hbenzimidazoles were rationalized and discussed.
Results and discussion Chemistry
Figure 2. Schematic representation showing the designing strategy of 2-(2-furyl)-1H-benzimidazole derivatives.
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The target compounds 14–18 were synthesized according to the reaction sequences outlined in Schemes 1 and 2. Compounds 11a,b, 12a,b, and 13a,b were previously prepared in our lab (submitted for publication) [30]. 2-[2-(Furan-2-yl)1H-benzo[d]imidazol-1-yl]acetohydrazide and the 5-methyl derivatives 13a,b were converted to the Schiff bases 14a–l (Scheme 1) via condensation with various selected aldehydes and with various functionalized methyl aryl ketones using ethanol/acetic acid (24:1) mixture [35]. Nucleophilic attack of 13a to phenylisocyanate and methylisothiocyanate was successfully performed in refluxing ethanol to give 1-[2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl) acetyl]-4-phenylsemicarbazide 15 and 1-[2-(2-(furan-2-yl)-1Hbenzo[d]imidazol-1-yl)acetyl]-4-methylthiosemicarbazide 18, respectively [36]. The cyclization of the acid hydrazide compound 13b with acetyl acetone in glacial acetic acid at 150°C resulted in the formation of 2-[2-(furan-2-yl)-1H-benzo[d] imidazol-1-yl]-1-(3,5-dimethyl-1H-pyrazol-1-yl)ethanone 16 [37]. Compound 13a was condensed with various alkyl and aryl chlorides in K2CO3/acetone at room temperature to obtain 17a–c (Scheme 2). www.archpharm.com
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Scheme 1. Synthetic pathway of compounds 14a–l. Reagents and conditions: (i) p-toluenesulfonic acid, DMF, 100°C; (ii) ethyl 2-bromoacetate, K2CO3, acetone, RT; (iii) hydrazine hydrate, ethanol, 90°C; (iv) aryl aldehydes or ketones, ethanol/acetic acid (24:1), reflux.
Bioactivity Cytotoxicity against human breast cancer cell line MCF-7 Cytotoxicity of the synthesized compounds 14–18 was tested using Skehan et al. method [38] in human breast cancer cell line MCF-7. The cytotoxicity results were compared to that of ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tamoxifen. Compound 14k exhibited higher potency against MCF-7cell line (IC50 ¼ 7.80 mg/mL) than that of tamoxifen (IC50 ¼ 8.00 mg/mL), while compounds 14e and 14g are equipotent to tamoxifen with IC50 ¼ 8.63 and 9.23 mg/mL, respectively. The rest of the compounds exhibited promising www.archpharm.com
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Scheme 2. Synthetic pathway of compounds 15–18. Reagents and conditions: (i) phenylisocyanate, ethanol, reflux; (ii) acetylacetone, g. AcOH, 150°C; (iii) chloro compounds, K2CO3, acetone, RT; (iv) methylisothiocyanate, ethanol, reflux.
activity against MCF-7 cell line with IC50 ¼ 11.20–16.00 mg/mL and their order of reactivity was 6f, 6h, 6b, 8, 3b, 4a, 6c, 7, and 6i in a descending order. The rest of the compounds exhibited moderate activity against MCF-7 cell line as shown in Table 1.
In vitro VEGF inhibition in human breast cancer cell line MCF-7 VEGF stimulates angiogenesis in tumor tissues thus promoting their nourishment and progression [1–5]. The discovery of ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
new chemotherapeutic agents by blocking the VEGF-induced angiogenesis has attracted much attention in the last few years [11, 12]. Since the 2-furylbenzimidazole nucleus showed promising inhibition against VEGF-induced angiogenesis in previous studies [27–29], we have synthesized several analogs of the 2-furylbenzimidazole nucleuses 14–18 and measured their inhibition potency in vitro against human VEGF in human breast cancer cell line MCF-7. This biological in vitro study was done using doubleantibody sandwich ELISA to assay the level of VEGF in human www.archpharm.com
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Table 1. Effect of the synthesized compounds on the breast cancer cell line MCF-7 and the VEGF level (pg/mL) in the breast cancer cell line MCF-7.
R1
R2
IC50 (mg/mL)a)
11a 11b
H CH3
H H
21.50 14.00
4460.90 (15%) 280.24 (95%)
12a
H
15.20
2000.82 (62%)
12b
CH3
17.80
3760.70 (28%)
13a
H
20.20
4010.45 (24%)
13b
CH3
22.40
760.40 (86%)
14a
H
37.90
5000.40 (5%)
14b
H
11.80
196.80 (96%)
14c
H
15.40
2190.66 (58%)
14d
H
16.60
3700.00 (30%)
14e
H
8.63
196.76 (96%)
14f
H
11.20
184.30 (97%)
14g
CH3
9.23
146.66 (97%)
14h
CH3
11.30
180.90 (97%)
Compound
VEGF (pg/mL)b)
(Continued) ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 1. (Continued)
Compound
R1
14i
H
16.00
3600.90 (31%)
14j
H
21.80
4230.50 (19%)
14k
H
7.80
104.30 (98%)
14l
H
22.10
4770.63 (9%)
15
H
16.00
2300.14 (56%)
16
CH3
13.90
240.80 (95%)
17a
H
22.10
4800.00 (9%)
17b
H
21.80
4670.23 (11%)
17c
H
18.70
4050.45 (23%)
18
H
17.50
2600.40 (51%)
DMSO Tamoxifen
– –
– 8.00
5250.00 (–) 110.75 (98%)
a) b)
R2
– –
IC50 (mg/mL)a)
VEGF (pg/mL)b)
Data were expressed as mean of six independent experiments. Values between brackets indicate percentage changes as compared with control of untreated cells.
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breast cancer cell line MCF-7 as compared to the inhibition for the untreated cells. The screening results (Table 1) showed that eight compounds (14b, 14e, 14f, 14g, 14h, 14k, 11b, and 16) were found to be potent inhibitors of VEGF in the MCF-7 cancer cell line, their percentage of inhibition ranges from 95 to 98% as compared to the positive drug, tamoxifen (98%). These results were consistent with cell cytotoxicity activity against MCF-7 cell line, where these eight compounds exhibited excellent activity with IC50 ranging from 7.80 to 13.90 mg/mL.
Molecular modeling Recent studies showed that the molecular mechanism of 2-subsituted benzimidazoles inhibiting angiogenesis can be due to inhibition of VEGFR [15–20]. A computational study was carried out to comprehend how these compounds could interact with this receptor, and to explain their structure– activity relationships. PDB entry 3EWH [33] representing the VEGFR2 crystal structure was selected for this modeling study because of the co-crystallized benzimidazole and because of its high resolution. Using LigandScout [32], a 3D-pharmacophore from the benzimidazole inhibitor (K11) co-crystallized in the protein-binding site was generated (Fig. 3). The generated structure-based pharmacophore represents important binding features for the inhibition of VEGFR2 and was used to direct the selection of the most plausible binding poses of the novel compounds. The pharmacophore includes
Figure 3. 3D-pharmacophore for ligand interaction with VEGFR2 developed from PDB entry 3EWH. Hydrophobic contacts are shown as yellow spheres and hydrogen bonds are shown as green and red arrows for donors and acceptors, respectively.
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three main hydrophobic sub-sites. Relevant residues are LEU889, ILE892, and ILE888 for sub-site I, PHE1047, LEU840, ALA866, and VAL848 for sub-site II, and finally GLU885, VAL899, and THR916 in the last sub-site (Fig. 3). Additionally, four key hydrogen bonds were included into the pharmacophore model: on the one hand, a donor to GLU885 and acceptor to ASP1046 stabilize the inhibitor in the center of the cavity and on the other hand, a hydrogen bond donor and acceptor toward CYS919 stabilize the terminal pyrimidine ring. Lastly, a complete exclusion volume sphere coat was added to define the steric extent of the binding site. The crystal structure of VEGFR was prepared as described in the Experimental Section and used for the docking experiment. The docking program GOLD was used to sample plausible docking poses for all compounds. The generated docking conformations were ranked based on their ability to fulfill the interaction features reported in the above-mentioned 3Dpharmacophore. The VEGFR2 inhibitory potency of most of the newly synthesized 2-furylbenzimidazoles may be attributed to the presence of the acetohydrazide moiety, which is common in all the compounds and can act as a hydrophilic anchor within a rather hydrophobic binding cleft. The acid hydrazide group is able to stabilize the compounds in the binding site by bringing the two key hydrogen acceptor and donor groups toward ASP1046 and GLU885, respectively. This docking study showed that novel compounds stabilized by this central interaction were deeply embedded into the hydrophobic binding cleft of VEGFR. Due to the much smaller size than the co-crystallized inhibitor (K11), the docked compounds could not fill the whole binding cleft, but bridge two hydrophobic sub-sites (I and III) and are stabilized by the central hydrogen bonds. Furthermore, the studied compounds have an extra furyl group that is accommodated by the hydrophobic residues VAL848, PHE845, and PHE1047 (Fig. 4). For instance, compound 14k (Fig. 4), which displayed one of the highest inhibitory potencies, shows an excellent complementarity with the reference 3D-pharmacophore. The crucial hydrogen bonds were identified between GLU885 and ASP1046 and the acid hydrazide group of the ligand, indicating a good overall orientation of the ligand in the cavity. In the first sub-site, the phenyl ring is stabilized by the residues LEU889, ILE892, and ILE888, while the benzimidazole ring interacts with the residues VAL899 and THR916 of the third enzyme hydrophobic sub-site. The second hydrophobic region in the binding pocket was not filled. Instead, a new hydrophobic area was covered by the furyl group of the inhibitor. Interestingly, the double bond, and in particular its cis conformation, is of high importance for the orientation of the inhibitor in the enzyme pocket. Consequently, series 14 www.archpharm.com
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Surprisingly, the methyl group substitution on the acid hydrazide group (in compounds 14g and 14h) has no significant effect on the activity, as it shows no hydrophobic contact with the surrounding residues. Moreover, cyclization of the acid hydrazide (in compound 16) shows good binding in the pocket, although it does not keep the hydrogen donor group toward GLU885. This could be explained by the increased rigidity of the compound, the ring keeping all other binding features in the right orientation within the binding cleft.
Conclusion
Figure 4. Plausible binding mode for compound 14k in 2D (up) and 3D (down) showing favorable hydrophobic contacts (yellow spheres) and two hydrogen bonds with GLU885 (green) and ASP1046 (red). For clarity, not all interacting amino acids are shown in the 3D view.
and compound 16 have superior activity over other compounds (compound 15, series 17, and compound 18), which cannot fulfill these key interactions due to absence of the cis double bond. As the pocket is highly hydrophobic, lipophilic features either on the phenyl (sub-site I) or furyl rings are crucial for good binding and therefore for the activity. This is clearly the case for compounds 14b and 14k, which have an ortho methyl substitution on the phenyl ring, and with 14f and 14e, which are ortho substituted with chlorine and methoxy, respectively, 14g with ortho methyl on the furyl ring, and 14h with two methyl groups on both phenyl and furyl rings. In contrast, removing the hydrophobic moiety (compound 14a), or replacing it with hydrophilic moieties like amino or hydroxyl group (as in compounds 14l or 14j, respectively), or changing the phenyl ring for a less lipophilic ring like pyridine or furyl (in compound 14c and 14d, respectively) dramatically decreases inhibitory potency. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The scope of this research work was the synthesis of new (2furyl)-1H-benzimidazoles as expected inhibitors of VEGFinduced angiogenesis. In this study, as a completion to our previous study made in part I (submitted for publication) [30], we have synthesized compounds 14–18 through the functionalization at position 1 of the benzimidazole nucleus forming various hydrazides, semicarbazides, thiosemicarbazide, alkyl chains, and heterocyclic rings using improved synthetic pathways to reach our target compounds. In vitro studies of the VEGF inhibition of the synthesized compounds in human breast cancer cell line MCF-7 showed that compounds 14b, 14e, 14f, 14g, 14h, 14k, and 16 are quite potent to the reference drug, tamoxifen with high percentage of inhibition in addition to excellent cell cytotoxicity activity against MCF-7 cell line with IC50 ranging from 7.80 to 13.90 mg/mL in comparison to that of tamoxifen (IC50 ¼ 8.00 mg/mL). Molecular modeling study revealed the importance of the acid hydrazide group – common in all synthesized compounds – in stabilizing the docked inhibitors in the binding site of VEGFR2 with two hydrogen bonds toward ASP1046 and GLU885. Moreover, the cis double bond seems to be of high importance for good orientation in the binding pocket of compounds such as 14a–i. Additionally, this study revealed that the lipophilic features on the phenyl group of inhibitors 14b, 14k, 14f, 14e, 14g, and 14h can create hydrophobic contacts with the residues found in sub-site I, which consequently affects their inhibition potency.
Experimental Physical measurements Microanalyses and spectral data of the compounds were performed in the Micro analytical labs, National Research Centre and Micro analytical Laboratory Center, Faculty of Science, Cairo University, Cairo, Egypt. The IR spectra (4000–400 cm1) were recorded using KBr pellets in a Jasco FT/IR 300E Fourier transform infrared spectrophotometer on a Perkin Elmer FT-IR 1650 (spectrophotometer). The melting points were determined on a Stuart SMP30 Melting Point Apparatus and are uncorrected. The www.archpharm.com
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1 H and 13C NMR spectra were recorded using Jeol EX-270 MHz and 500 MHz NMR spectrophotometers using DMSO-d6 as a solvent. Chemical shifts are reported in parts per million (ppm) from the tetramethylsilane resonance in the indicated solvent. Coupling constants are reported in Hertz (Hz); spectral splitting partners are designed as follows: singlet (s); doublet (d); triplet (t); and multiplet (m). The mass spectra were carried out using Finnigan mat SSQ 7000 (Thermo. Inst. Sys., Inc., USA) spectroscopy at 70 eV. Reaction progress was monitored using thin layer chromatography (TLC) analysis on Silica Gel 60 F254 plates (Merck).
General procedure for the preparation of compounds 14a–i To a solution of the acid hyrazide 13a or 13b (0.01 mol) in ethanol/ acetic acid (24:1, 25 mL), 0.02 mol of aryl aldehydes namely benzaldehyde, 4-methylbenzaldehyde, 3-pyridinecarboxaldehyde, furfural, 4-methoxybenzaldehyde, and 4-chlorobenzaldehyde and arylacetophenones namely acetophenone, 4-hydroxyacetophenones, 4-methylacetophenones, and 4-aminoacetophenones was added. The reaction mixture was heated under reflux for 5 h, and then cooled. The separated solids were filtered, dried, and crystallized from acetic acid.
N 0 -Benzylidene-2-(2-(furan-2-yl)-1H-benzo[d]imidazol1-yl)acetohydrazide (14a) Yield ¼ 90% (3.10 g). m.p.: 250–253°C. TLC Rf ¼ 0.70 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.74 (s, 2H, CH2), 6.70 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.13 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.20–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.40–7.42 (m, 2H, H4 þ H7 benzimidazole), 7.60–7.62 (m, 3H, H3 0 þ H4 0 þ H5 0 phenyl), 7.75–7.76 (m, 2H, H2 0 þ H6 0 phenyl), 7.80 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.07 (s, 1H, –– CH), 11.79 (s, 1H, NH, D2O exchangeable). IR (cm1): 3427 and 3185 (NH and OH), 1681 (C –– O), 1614 (C –– N). MS: m/z 345, 16% (Mþþ1); m/z 344, 64% (Mþ); m/z 343, 14% (Mþ1); m/z 197, 100%. Anal. calcd. for C20H16N4O2 (344.13): C, 69.86; H, 4.66; N, 16.23. Found C, 69.77; H, 4.70; N, 16.29.
2-[2-(Furan-2-yl)-1H-1,3-benzodiazol-1-yl]-N 0 -[(1Z)(4-methylphenyl)methylidene]acetohydrazide (14b) Yield ¼ 85% (3.06 g). m.p.: 249–252°C. TLC Rf ¼ 0.57 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 2.30 (s, 3H, CH3), 5.70 (s, 2H, CH2), 6.67 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.12 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.21–7.22 (m, 2H, H5 þ H6 benzimidazole), 7.24–7.25 (d, J ¼ 7.6 Hz, 2H, H3 0 þ H5 0 phenyl), 7.55–7.56 (m, 2H, H4 þ H7 benzimidazole), 7.63–7.64 (m, 2H, J ¼ 7.6Hz, H2 0 þ H6 0 phenyl), 7.83 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.02 (s, 1H, HC –– N), 11.73 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 21.59, 46.57, 111.01, 127.74, 119.39, 122.82, 123.19, 127.59, 128.05, 129.97, 131.82, 136.90, 140.44, 142.99, 144.85, 144.94, 145.41, 145.93, 148.25, 154.72, 168.82. IR (cm1): 3390 and 3220 (NH and OH), 1678 (C –– O), 1611 (C –– N). MS: m/z 359, 16% (Mþþ1); m/z 358, 54% (Mþ); m/z 357, 12% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H18N4O2 (358.14): C, 70.38; H, 5.06; N, 15.63. Found C, 70.43; H, 5.09; N, 15.67.
2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 0 ((pyridin-3-yl)methylene)acetohydrazide (14c) Yield ¼ 92% (3.17 g). m.p.: 235–237°C. TLC Rf ¼ 0.62 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.77 (s, 2H, CH2), 6.67 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.14 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.22–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.45–7.47 (m, 2H, H5 0 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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pyridine), 7.59–7.65 (m, 2H, H4 þ H7 benzimidazole), 7.88 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.02 (s, 1H, HC –– N), 8.19–8.21 (m, 2H, H4 0 pyridine), 8.58–8.60 (m, 2H, H6 0 pyridine), 8.90–8.92 (s, 2H, H2 0 pyridine), 11.97 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSOd6) d: 46.52, 110.96, 112.69, 112.86, 119.41, 122.92, 123.28, 124.47, 130.48, 134.23, 136.84, 142.12, 142.94, 144.89, 145.44, 145.86, 149.17, 151.14, 162.22. IR (cm1): 3429 and 3200 (NH and OH), 1695 (C –– O), 1607 (C –– N). MS: m/z 346, 13% (Mþþ1); m/z 345, 51% (Mþ); m/z 344, 9% (Mþ1); m/z 197, 100%. Anal. calcd. for C19H15N5O2 (345.35): C, 66.08; H, 4.38; N, 20.28. Found C, 66.15; H, 4.45; N, 20.36.
2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 0 ((furan-2-yl)methylene)acetohydrazide (14d) Yield ¼ 85% (2.94 g). m.p.: 240–241°C. TLC Rf ¼ 0.60 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.70 (s, 2H, CH2), 6.62 (dd, J ¼ 3.05 Hz, 1H, H4 0 2-furyl), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2furyl), 6.94 (dd, J ¼ 3.05 Hz, 1H, H3 0 2-furyl), 7.12 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.22–7.25 (m, 2H, H5 þ H6 benzimidazole), 7.59–7.63 (m, 2H, H4 þ H7 benzimidazole), 7.80 (dd, J ¼ 3.05 Hz, 1H, H5 0 2furyl), 7.88 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 7.95 (s, 1H, HC –– N), 11.70 (s, 1H, NH, D2O exchangeable). IR (cm1): 3423 and 3200 (NH and OH), 1688 (C –– O), 1620 (C –– N). MS: m/z 335, 17% (Mþþ1); m/z 334, 84% (Mþ); m/z 333, 40% (Mþ1); m/z 197, 100%. Anal. calcd. for C18H14N4O3 (345.35): C, 64.66; H, 4.22; N, 16.76. Found C, 64.69; H, 4.27; N, 16.70.
N 0 -(4-Methoxybenzylidene)-2-(2-(furan-2-yl)-1Hbenzo[d]imidazol-1-yl)acetohydrazide (14e) Yield ¼ 82% (3.08 g). m.p.: 275–277°C. TLC Rf ¼ 0.77 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 3.77 (s, 3H, OCH3), 5.71 (s, 2H, CH2), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 6.98–7.00 (d, J ¼ 9 Hz, 2H, H2 0 þ H6 0 phenyl), 7.12 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.20–7.22 (m, 2H, H5 þ H6 benzimidazole), 7.60–7.62 (m, 2H, H4 þ H7 benzimidazole), 7.69–7.70 (dd, J ¼ 9 Hz, 2H, H3 0 þ H5 0 phenyl), 7.80 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.05 (s, 1H, –– CH), 11.70 (s, 1H, NH, D2O exchangeable). IR (cm1): 3423 and 3200 (NH and OH), 1688 (C –– O), 1620 (C –– N). MS: m/z 375, 16.8% (Mþþ1); m/z 374, 84% (Mþ); m/z 373, 40% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H18N4O3 (374.39): C, 67.37; H, 4.85; N, 14.96. Found C, 67.43; H, 4.89; N, 15.07.
N 0 -(4-Chlorobenzylidene)-2-(2-(furan-2-yl)-1Hbenzo[d]imidazol-1-yl)acetohydrazide (14f) Yield ¼ 86% (3.26 g), m.p.: 230–232°C, TLC Rf ¼ 0.77 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.77 (s, 2H, CH2), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.13 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.20–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.47–7.50 (d, J ¼ 8.4 Hz, 2H, H2 0 þ H6 0 phenyl), 7.60–7.64 (m, 2H, H4 þ H7 benzimidazole), 7.78–7.79 (d, J ¼ 8.4 Hz, 2H, H3 0 þ H5 0 phenyl), 7.86 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.10 (s, 1H, –– CH), 11.80 (s, 1H, NH, D2O exchangeable). IR (cm1): 3440 and 3215 (NH and OH), 1691 (C –– O), 1608 (C –– N). MS: m/z 380, 17% (Mþþ1); m/z 379, 24% (Mþ); m/z 378, 52% (Mþ1); m/z 197, 100%. Anal. calcd. for C20H15ClN4O2 (378.81): C, 63.41; H, 3.99; Cl, 9.36; N, 14.79. Found C, 63.48; H, 4.04; Cl, 9.30; N, 14.85.
N 0 -Benzylidene-2-(2-(5-methylfuran-2-yl)-1Hbenzo[d]imidazol-1-yl)acetohydrazide (14g) Yield ¼ 94% (3.36 g). m.p.: 232–235°C. TLC Rf ¼ 0.70 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 2.21 (s, 3H, CH3), www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2014, 347, 291–304
5.74 (s, 2H, CH2), 6.31 (d, J ¼ 3.05 Hz, 1H, H4 2-furyl), 7.05 (d, J ¼ 3.05 Hz, 1H, H3 2-furyl), 7.19–7.21 (m, 2H, H5 þ H6 benzimidazole), 7.42–7.43 (m, 3H, H3 0 þ H4 0 þ H5 0 phenyl protons), 7.60– 7.61 (m, 2H, H4 þ H7 benzimidazole), 7.75–7.76 (m, 2H, H2 0 þ H6 0 phenyl protons), 8.10 (s, 1H, CH –– ) 11.79 (s, 1H, NH, D2O exchangeable). IR (cm1): 3428 (NH), 1679 (C –– O), 1610 (C –– N). MS: m/z 359, 19% (Mþþ1); m/z 358, 72% (Mþ); m/z 357, 26% (Mþ1); m/z 260, 100%; m/z 211, 66.0%. Anal. calcd. for C21H18N4O2 (358.14): C, 70.38; H, 5.06; N, 15.63. Found C, 70.46; H, 5.02; N, 15.68.
2-[2-(5-Methylfuran-2-yl)-1H-1,3-benzodiazol-1-yl]-N 0 [(1Z)-(4-methylphenyl)methylidene]acetohydrazide (14h) Yield ¼ 88% (3.29 g), m.p.: 240–243°C, TLC Rf ¼ 0.67 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 2.20 (s, 3H, CH3 phenyl), 2.29 (s, 3H, CH3 2-furyl), 5.72 (s, 2H, CH2), 6.26 (d, J ¼ 3.05 Hz, 1H, H4 2-furyl), 6.99 (d, J ¼ 3.05 Hz, 1H, H3 2-furyl), 7.19–7.21 (m, 2H, H5 þ H6 benzimidazole), 7.23–7.25 (d, J ¼ 7.6 Hz, 2H, H3 0 þ H5 0 phenyl protons), 7.57–7.61 (m, 2H, H4 þ H7 benzimidazole), 7.63–7.65 (d, J ¼ 7.6 Hz, 2H, H2 0 þ H6 0 phenyl protons), 8.10 (s, 1H, CH), 11.7 (s, 1H, NH, D2O exchangeable). 13 C NMR (DMSO-d6) d: 13.76, 21.57, 46.52, 101.23, 108.93, 110.92, 113.63, 119.17, 122.65, 122.92, 127.52, 127.65, 129.99, 131.84, 137.05, 137.85, 140.42, 143.01, 144.21, 144.73, 154.15, 169.19. IR (cm1): 3430 and 3189 (NH and OH), 1676 (C –– O), 1612 (C –– N). MS: m/z 373, 26% (Mþþ1); m/z 372, 88% (Mþ); m/z 371, 33% (Mþ1); m/z 255, 18%. Anal. calcd. for C22H20N4O2 (372.42): C, 70.95; H, 5.41; N, 15.04. Found C, 71.04; H, 5.49; N, 15.09.
2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 0 (1-phenylethylidene)acetohydrazide (14i) Yield ¼ 85% (3.04 g), m.p.: 245–248°C, TLC Rf ¼ 0.65 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 2.30 (s, 3H, CH3), 5.77 (s, 2H, CH2), 6.69 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.10 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.21–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.39–7.41 (m, 3H, H3 0 þ H4 0 þ H5 0 phenyl), 7.60–7.63 (m, 2H, H4 þ H7 benzimidazole), 7.86–7.89 (m, 1H, H2 0 þ H6 0 phenyl), 7.90 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 11.06 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 14.29, 46.85, 111.01, 112.72, 119.39, 122.82, 123.19, 126.88, 128.69, 128.95, 129.22, 129.82, 133.73, 136.89, 138.52, 142.99, 144.91, 145.44, 145.94, 149.43, 169.82. IR (cm1): 3427 and 3179 (NH and OH), 1683 (C –– O), 1610 (C –– N). MS: m/z 359, 24% (Mþþ1); m/z 358, 85% (Mþ); m/z 357, 11% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H18N4O2 (358.14): C, 70.38; H, 5.06; N, 15.63. Found C, 70.30; H, 5.01; N, 15.69.
2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 0 (1-(4-hydroxyphenyl)ethylidene)acetohydrazide (14j) Yield ¼ 76% (2.84 g). m.p.: 220–223°C. TLC Rf ¼ 0.75 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 2.30 (s, 3H, CH3), 5.73 (s, 2H, CH2), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 6.73–6.82 (d, J ¼ 8.4 Hz, 2H, H2 0 þ H6 0 phenyl), 7.11 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.21–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.58–7.65 (m, 2H, H4 þ H7 benzimidazole), 7.78–7.80 (d, J ¼ 8.4 Hz, 2H, H3 0 þ H5 0 phenyl), 7.87 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 10.00 (s, 1H, OH, D2O exchangeable), 11.00 (s, 1H, NH, D2O exchangeable). IR (cm1): 3421 and 3250 (NH and OH), 1675 (C –– O), 1603 (C –– N). MS: m/z 375, 24.4% (Mþþ1); m/z 374, 100.0% (Mþ); m/z 373, 13% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H18N4O3 (374.14): C, 67.37; H, 4.85; N, 14.96. Found C, 67.32; H, 4.94; N, 15.07. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2-Furylbenzimidazoles as Anti-Angiogenic Agents
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2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 00 (1-p-tolylethylidene)acetohydrazide (14k) Yield ¼ 72% (2.68 g). m.p.: 230–233°C. TLC Rf ¼ 0.65 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 2.27 (s, 3H, CH3phenyl), 2.30 (s, 3H, CH3), 5.72 (s, 2H, CH2), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.11 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.18–7.21 (m, 2H, H5 þ H6 benzimidazole), 7.22–7.24 (d, J ¼ 6 Hz, 2H, H3 0 þ H5 0 phenyl), 7.60–7.64 (m, 2H, H4 þ H7 benzimidazole), 7.76–7.77 (d, J ¼ 6 Hz, 2H, H2 0 þ H6 0 phenyl), 7.87 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 11.00 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 14.22, 21.38, 46.95, 110.98, 112.64, 119.40, 122.79, 122.95, 123.16, 123.28, 126.79, 129.52, 129.74, 135.77, 136.92, 139.42, 143.04, 144.90, 145.41, 145.97, 149.47, 169.69. IR (cm1): 3423 and 3186 (NH and OH), 1677 (C –– O), 1615 (C –– N). MS: m/z 373, 27% (Mþþ1); m/z 372, 100% (Mþ); m/z 371, 17% (Mþ1); m/z 197, 96%. Anal. calcd. for C22H20N4O2 (372.42): C, 70.95; H, 5.41; N, 15.04. Found C, 71.05; H, 5.37; N, 15.08.
N 0 -(1-(4-Aminophenyl)ethylidene)-2-(2-(furan-2-yl)-1Hbenzo[d]imidazol-1-yl)acetohydrazide (14l) Yield ¼ 80% (2.98 g). m.p.: 238–240°C. TLC Rf ¼ 0.80 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 2.18 (s, 3H, CH3), 5.45 (NH2, D2O exchangeable), 5.7 (s, 2H, CH2), 6.53–6.56 (d, J ¼ 8.4 Hz, 2H, H3 0 þ H5 0 phenyl), 6.67 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.10 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.20–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.57–7.62 (m, 4H, (H2 0 þ H6 0 ) phenyl þ (H4 þ H7) benzimidazole), 7.89 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 10.7 (s, 1H, NH, D2O exchangeable). IR (cm1): 3421 and 3286 (NH, NH2, and OH), 1681 (C –– O), 1608 (C –– N). MS: m/z 374, 30% (Mþþ1); m/z 373, 76.2% (Mþ); m/z 372, 47% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H19N5O2 (373.41): C, 67.55; H, 5.13; N, 18.76. Found C, 67.50; H, 5.19; N, 18.84.
1-(2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetyl)-4phenylsemicarbazide (15) To a solution of the acid hyrazide 13a (0.01 mol) in 30 mL ethanol, phenyl isocyanate (0.01 mol) was added, The reaction mixture was heated under reflux for 12 h. The final product was monitored using TLC. Excess solvent was evaporated under reduced pressure. The separated solids were filtered, dried, and crystallized from ethanol. Yield ¼ 65% (2.44 g). m.p.: 225–229°C. TLC Rf ¼ 0.60 (petroleum ether/ethyl acetate/ethanol, 1:1:1). 1H NMR (DMSOd6) d: 5.30 (s, 2H, CH2), 6.70 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 6.91–6.93 (m, 1H, H4 0 phenyl), 7.21 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.22–7.24 (m, 2H, H5 þ H6 benzimidazole), 7.27–7.29 (m, 2H, H3 0 þ H5 0 phenyl) 7.39–7.41 (m, 2H, H2 0 þ H6 0 phenyl), 7.59–7.63 (m, 2H, H4 þ H7 benzimidazole), 7.90 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.10 (s, 1H, NH, D2O exchangeable), 9.00 (s, 1H, NH, D2O exchangeable), 10.20 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 46.53, 110.97, 112.62, 113.10, 118.69, 119.12, 119.51, 122.59, 122.82, 122.96, 123.32, 129.23, 136.65, 139.99, 142.96, 144.74, 145.28, 145.49, 155.71, 167.55. IR (cm1): 3450 (enolic OH), 3400, 3307, and 3204 (NH), 3000 (H-bonded OH), 1700 (C –– O), 1680 (C –– O), 1602 (C –– N). MS: m/z 283, 13% (Mþ NHC6H5); m/z 256, 44% (Mþ CONHC6H5); m/z 197, 100%; m/z 184, 53%. Anal. calcd. for C20H17N5O3 (375.38): C, 63.99; H, 4.56; N, 18.66. Found C, 64.07; H, 4.50; N, 18.69.
1-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-(2-(5-methylfuran-2yl)-1H-benzo[d]imidazol-1-yl)ethanone (16) A mixture of hydrazide 13b (0.01 mol) and acetyl acetone (0.02 mol) in glacial acetic acid (10 mL) and few drops of DMF www.archpharm.com
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was stirred at room temperature overnight. After dilution with water, the solid precipitated was filtered, dried, and crystallized from acetic acid. Yield ¼ 70% (2.34 g), m.p.: 176–179°C, TLC Rf ¼ 0.57 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 1.70 (s, 3H, CH3 pyrazole), 2.10 (s, 3H, CH3 pyrazole), 2.30 (s, 3H, CH3 2-furyl), 5.47 (s, 2H, CH2), 6.20 (d, J ¼ 3.05 Hz, 1H, H4 2-furyl), 6.52 (s, 1H, CH), 7.00 (d, J ¼ 3.05 Hz, 1H, H3 2-furyl), 7.10–7.20 (m, 2H, H5 þ H6 benzimidazole), 7.51–7.59 (m, 2H, H4 þ H7 benzimidazole). IR (cm1): 1672 (C –– O), 1610 (C –– N). MS: m/z 335, 5% (Mþþ1); m/z 334, 72% (Mþ); m/z 255, 13%; m/z 238, 38%; m/z 210, 74%; m/z 183, 37%. Anal. calcd. for C19H18N4O2 (334.37): C, 68.25; H, 5.43; N, 16.76. Found C, 68.31; H, 5.53; N, 16.84.
General procedure for the preparation of compounds 17a–c To a solution of the acid hyrazide 13a (0.01 mol) and anhydrous K2CO3 (10.6 g, 0.1 mol) in dry acetone, chloro compounds (0.01 mol) were added dropwise namely: benzoyl chloride, benzyl chloride, and acetyl chloride. The reaction mixture was stirred for 3 h at room temperature. The final product was monitored using TLC. Excess acetone was evaporated using reduced pressure and the reaction mixture was poured onto cold water and the precipitate was filtered, dried, and recrystallized from ethanol.
N 0 -{2-[2-(Furan-2-yl)-1H-1,3-benzodiazol-1-yl]acetyl}benzohydrazide (17a) Yield ¼ 83% (2.98 g). m.p.: 265–267°C. TLC Rf ¼ 0.55 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.30 (s, 2H, CH2), 6.71 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.22 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.25–7.27 (m, 2H, H5 þ H6 benzimidazole), 7.45–7.47 (m, 2H, H4 þ H7 benzimidazole), 7.48–7.50 (m, 1H, H4 0 phenyl), 7.53–7.64 (m, 2H, H3 0 þ H5 0 phenyl), 7.82–7.90 (m, 2H, H2 0 þ H6 0 phenyl), 7.93 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 10.50 (s, 1H, NH, D2O exchangeable), 11.50 (s, 1H, NH, D2O exchangeable). IR (cm1): 3433 and 3207 (NH and OH), 1697 (C –– O), 1604 (C –– N). MS: m/z 361, 7% (Mþþ1); m/z 360, 40% (Mþ); m/z 359, 8% (Mþ1); m/z 197, 70%. Anal. calcd. for C20H16N4O3 (360.37): C, 66.66; H, 4.48; N, 15.55. Found C, 66.74; H, 4.53; N, 15.62.
Arch. Pharm. Chem. Life Sci. 2014, 347, 291–304
7.63 (m, 2H, H4 þ H7 benzimidazole), 7.80 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 9.90 (s, 1H, NH, D2O exchangeable), 10.30 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 22.30, 46.39, 111.05, 112.57, 113.10, 119.16, 122.71, 122.94, 123.29, 136.76, 142.88, 145.19, 145.89, 168.69, 173.19. IR (cm1): 3413 and 3259 (NH and OH), 1678 (C –– O), 1610 (C –– N). MS: m/z 299, 7% (Mþþ1); m/z 298, 17% (Mþ); m/z 297, 7% (Mþ1); m/z 197, 100% (2-furylbenzimidazole þ CH2). Anal. calcd. for C15H14N4O3 (298.31): C, 60.40; H, 4.73; N, 18.78. Found C, 60.48; H, 4.81; N, 18.84.
1-(2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetyl)-4methylthiosemicarbazide (18) To a solution of the acid hyrazide 13a (0.01 mol) in 30 mL ethanol, methyl isothiocyanate (0.01 mol) was added, the reaction mixture was heated under reflux for 10 h. The final product was monitored using TLC. Excess solvent was evaporated under reduced pressure. The separated solids were filtered, dried, and crystallized from ethanol. Yield ¼ 75% (2.81 g). m.p.: 255–257°C. TLC Rf ¼ 0.70 (petroleum ether/ethyl acetate/ethanol, 1:1:1). 1H NMR (DMSO-d6) d: 2.88 (s, 3H, CH3), 5.20 (s, 2H, CH2), 6.72 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.16 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.21– 7.28 (m, 2H, H5 þ H6 benzimidazole), 7.55–7.57 (m, 1H, H4 benzimidazole), 7.60–7.62 (m, 1H, H7 benzimidazole), 7.88 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.10 (s, 1H, NH, D2O exchangeable), 9.00 (s, 1H, NH, D2O exchangeable), 10.20 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 31.54, 46.23, 110.97, 112.60, 119.49, 122.97, 123.14, 123.28, 136.67, 142.87, 144.63, 145.26, 145.56, 167.40, 171.15. IR (cm1): 3400 and 3235 (NH), 2800 (H-bonded OH), 1676 (C –– O), 1611 (C –– N), 1160 (C –– S). MS: m/z 331, 2% (Mþþ2); m/z 330, 3% (Mþþ1); m/z 329, 32% (Mþ); m/z 311, 21% (Mþ H2O); m/z 298, 28%; m/z 197, 95% (base peak). Anal. calcd. for C15H15N5O2S (375.38): C, 54.70; H, 4.59; N, 21.26. Found C, 54.76; H, 4.64; N, 21.33.
Bioactivity materials and methods Cytotoxicity against human breast cancer cell line MCF-7
Yield ¼ 80% (2.77 g). m.p.: 277–230°C. TLC Rf ¼ 0.67 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 4.72 (s, 2H, CH2 benzylic), 5.70 (s, 2H, CH2), 6.34 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 6.53 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 6.92–7.09 (m, 2H, H5 þ H6 benzimidazole), 7.25–7.33 (m, 2H, H4 þ H7 benzimidazole), 7.45–7.47 (m, 5H, H2 0 þ H3 0 3þ H4 0 þ H5 0 þ H6 0 phenyl), 7.63 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 9.00 (s, 1H, NH, D2O exchangeable), 9.30 (s, 1H, NH, D2O exchangeable). IR (cm1): 3403 and 3150 (NH and OH), 1683 (C –– O), 1615 (C –– N). MS: m/z 346, 31% (Mþ); m/z 345, 7% (Mþ1); m/z 241, 21% (2-furylbenzimidazole þ CH2CONH); m/z 197, 100% (2-furylbenzimidazole þ CH2), m/z 184, 60% (2-furylbenzimidazole). Anal. calcd. for C20H18N4O2 (346.38): C, 69.35; H, 5.24; N, 16.17. Found C, 69.40; H, 5.32; N, 16.25.
The antitumor activity against MCF-7 cells was assessed in the National Cancer Institute, Cancer Biology Department, Cairo, Egypt. The cytotoxicity of the synthesized compounds 11–18 was tested using the method of Skehan et al. [38]. Cells were plated in 96-multiwell plates (104 cells/well) for 24 h before treatment with the compounds to allow attachment of cells to wall of the plate. Different concentrations of the compound under test (0.0, 5.0, 12.5, 25.0, and 50.0 mg/mL) were added to the cell monolayer. Six wells were prepared for each individual dose. Monolayer cells were incubated with the compounds for 48 h at 37°C and in atmosphere of 5% CO2. After 48 h, cells were fixed, washed, and stained with sulforhodamine B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris–EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration was plotted to get the survival curve of each tumor cell line after the specified compound.
N 0 -Acetyl-2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (17c)
In vitro VEGF inhibition in human breast cancer cell line MCF-7
Yield ¼ 75% (2.23 g). m.p.: 248–250°C, TLC Rf ¼ 0.78 (petroleum ether/ethyl acetate, 1:2).1H NMR (DMSO-d6) d: 1.82 (s, 3H, CH3), 5.25 (s, 2H, CH2), 6.70 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.17 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.19–7.25 (m, 2H, H5 þ H6 benzimidazole), 7.52–
The effect of tested compounds on the level of human VEGF was determined using human tumor cell lines including breast cancer cell line MCF-7 obtained from the American Type Culture Collection (Rockville, MD, USA). The tumor cells were
N 0 -Benzyl-2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (17b)
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maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (Gibco), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37°C in humidified atmosphere containing 5% CO2. Cells at a concentration of 0.50 106 were grown in a 25 cm2 flask in 5 mL of complete culture medium. The cells in culture medium were treated with 20 mL of IC50 values of the compounds or the standard reference drug, tamoxifen dissolved in DMSO, then incubated for 24 h at 37°C, in a humidified 5% CO2 atmosphere. The cells were harvested and homogenates were prepared in saline using a tight pestle homogenizer until complete cell disruption. The kit uses a double-antibody sandwich ELISA to assay the level of human VEGF in samples. VEGF was added to monoclonal antibody enzyme well, which is pre-coated with human VEGF monoclonal antibody, incubation; then, VEGF antibodies labeled with biotin were added, and combined with streptavidin–HRP to form immune complex; then incubated and washed again to remove the uncombined enzyme. Then chromogen solution A and B was added, the color of the liquid changes to blue, and by the effect of acid, the color finally becomes yellow. The chroma of color and the concentration of the human TRK of sample were positively correlated and the optical density was determined at 450 nm. The level of human VEGF in samples was calculated (ng/L) as duplicate determinations from the standard curve.
Modeling study Structures of the studied inhibitors were built using MOE (Chemical Computing Group, Montreal, CA). The crystal structure of VEGFR in complex with ligand K11 was retrieved from the Protein Data Bank (PDB code: 3EWH) [33]. The protein was prepared (hydrogen atoms added, water removed, and ligand extracted) in GOLD 5.1 (CCDC, Cambridge, UK) [34] using default parameters unless otherwise stated. The docking program GOLD was used to perform the docking of compounds series 14, 15, 16, 17, and 18 into the crystal structure of VEGFR. The binding site was defined by specifying all residues around the original ligand, within a sphere of 7 Å radius. The top 100 docked solutions were kept for each ligand. The program LigandScout [32] was used to generate a 3D-pharmacophore from the ligand of the PDB structure 3EWH using default parameters. The developed pharmacophore was used to prioritize plausible docking poses of the studied compounds. Docking poses visualization was done using LigandScout. The most plausible conformation was selected for each ligand based on its ability to match the highest amount of chemical interactions described in the 3D-pharmacophore.
The authors are grateful to the National Cancer Institute, Cancer Biology Department, Cairo, Egypt, to perform the antitumor activity against the MCF-7 cancer cell line. Deep thanks are expressed to the “Science &Technology Development Fund” for financially supporting the manuscript through the fund of the STDF project no. 1517. The authors have declared no conflict of interest.
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Full Paper Synthesis, Biological Evaluation, and Docking Studies of New 2-Furylbenzimidazoles as Anti-Angiogenic Agents: Part II Ahmed Temirak1, Yasser M. Shaker1, Fatma A. F. Ragab2, Mamdouh M. Ali3, Salwa M. Soliman4, Jeremie Mortier4,5, Gerhard Wolber4, Hamed I. Ali6,7, and Hoda I. El Diwani1 1
2 3
4 5 6
7
Division of Pharmaceutical and Drug Industries, Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, Cairo, Egypt Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Cairo University, Giza, Egypt Division of Genetic Engineering and Biotechnology, Department of Biochemistry, National Research Center, Cairo, Egypt Department of Pharmaceutical Chemistry, Institute of Pharmacy, Free University of Berlin, Berlin, Germany Department of Organic Chemistry, Free University of Berlin, Berlin, Germany Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Round Rock, TX, USA Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Helwan University, Cairo, Egypt
The 2-(5-methyl-2-furyl)-1H-benzimidazole moiety has shown promising activity against vascular endothelial growth factor (VEGF)-induced angiogenesis. In part I of this study, we have synthesized new analogs and tested their anti-angiogenic potentials. Here, we continue our previous study with different new analogs. Some compounds show promising cytotoxic activity against the human breast cancer cell line MCF-7, with IC50 in the range of 7.80–13.90 mg/mL, and exhibited remarkable in vitro inhibition against VEGF in the MCF-7 cancer cell line, with 95–98% of inhibition in comparison to tamoxifen as reference (IC50: 8.00 mg/mL, % of inhibition ¼ 98%). Additionally, a molecular docking study was carried out to gain insight into plausible binding modes and to understand the structure– activity relationships of the synthesized compounds. Keywords: Angiogenesis / Cytotoxicity / 2-(2-Furyl)-1H-benzimidazoles / Molecular modeling / Vascular endothelial growth factor (VEGF) Received: September 29, 2013; Revised: November 12, 2013; Accepted: November 15, 2013 DOI 10.1002/ardp.201300356
:
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Introduction Angiogenesis, which is the formation of new capillary blood vessels from preexisting ones [1], is a crucial process that promotes tumor growth, survival, and metastasis through supplying these proliferating tissues with oxygen and nutrients [2–4]. Angiogenesis is stimulated in tumor tissues through the production and secretion of angiogenic factors like vascular endothelial growth factor (VEGF) [5], plateletCorrespondence: Dr. Yasser M. Shaker, Division of Pharmaceutical and Drug Industries, Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, 12622 Cairo, Egypt. E-mail: [email protected], [email protected] Fax: þ20 2 3337093
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derived growth factor (PDGF) [6], basic fibroblast growth factor (bFGF) [7], endothelial growth factor (EGF) [8], and angiopoietin [9]. The VEGF signaling pathway that acts through the VEGF receptor 2 (VEGFR2) plays a key role in the regulation of tumor angiogenesis, in which the binding of VEGF to VEGFR2 leads to receptor dimerization, followed by the autophosphorylation of tyrosine residues in the intracellular kinase domain, resulting in potent mitogenic and chemotactic effects on endothelial cells [10]. The discovery of new tumor angiogenesis inhibitors targeting VEGFR has been the scope of many researchers during the past few years [11, 12]. New inhibitors have passed the clinical trials and are marketed as novel drugs targeting
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angiogenesis such as multikinase inhibitors; sunitinib and sorafenib and the monoclonal antibody bevacizumab. These agents have shown proven efficacy against many cancers that encourage researchers to move forward in this area [13–16]. There are many examples in the literature of 2-subsituted benzimidazoles as potential inhibitors for angiogenesis through targeting the VEGFR (Fig. 1). 4-(2-(4-Bromophenylamino)-1-methyl-1H-benzo[d]imidazol-5-yloxy)-N-methylpyridine2-carboxamide 1 was reported as a novel 2-arylbenzimidazole compound showing potential RAF kinase inhibition. It inhibits four RTKs (CSF1R kinase, VEGFR kinase, c-Kit, and PDGFR) [17]. Moreover, dovitinib (TKI-258) 2, a modified scaffold of 3benzimidazol-2-ylhydroquinolin-2-one has shown selective inhibition of FGFR and VEGFR’s. It exhibited promising efficacy against metastatic renal cell carcinoma in clinical trials (Fig. 1) [18, 19]. A series of 2-anilino-4-(benzimidazol-2-yl)pyrimidines 3 showed multikinase inhibition with antiproliferative activity toward four cancer-related protein kinases
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(Aurora B, PLK1, FAK, and VEGFR2) [20]. Also, compound 4 (Pubchem ID 47037197), a 2-phenyl substituted benzimidazole, resulting from the virtual screening of the Pubchem database [21, 22], has shown multi-targeting activity against EGFR, VEGFR2, and PDFGR in addition to the inhibition of cancer cell lines (HepG-2) and kinase enzyme inhibition [21, 22]. Furthermore, it was reported that albendazole, methyl[6(propylthio)-1H-benzimidazol-2-yl]carbamate 5, the widely used drug for the treatment of hydatid disease, suppresses VEGF levels, HIF-1a and has anti-angiogenic effect in noncancerous models of angiogenesis (Fig. 1) [23] both in vitro (via treatment of human umbilical vein endothelial cells (HUVECs) with albendazole leading to down-regulation of the VEGFR2) and in vivo (via inhibition of hyperoxia-induced retinal angiogenesis using albendazole). These results were reported and provided new insights into the effective antiangiogenic role of albendazole [24]. Furthermore, the novel benzimidazole analog 6 (AC1-004) was discovered to downregulate VEGF and EPO target genes of HIF-1 and to inhibit
Figure 1. Chemical structures of 2-subsituted benzimidazoles reported in the literature with potent anti-angiogenic activity.
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the in vitro tube formation of HUVEC leading to its potential inhibitory activity on angiogenesis [25, 26]. The nucleus of 2-furylbenzimidazole has shown outstanding activity against VEGF-induced angiogenesis in tumor tissues both in vitro and in vivo. 2-(5-Methyl-2-furyl)-1Hbenzimidazole (NP-184) 7 inhibited the angiogenic functions of HUVEC in vitro and reduced the angiogenesis rate in vivo in material plug assay model (Fig. 1) [27, 28]. The structural modification of compound NP-184 resulted in compound (PJ-8) 8, which extremely inhibited VEGF–VEGFR2 and suppressed the tumor-induced angiogenesis in vivo [29]. These remarkable results were the key elements in our research with the aim of discovering new analogs for the lead compound NP-184 exhibiting effective inhibition for VEGFinduced angiogenesis. This work is a continuation of our previous research in designing new anti-angiogenic 2-furylbenzimidazoles (submitted for publication) [30]. We have carried out the synthesis, biological activity, and molecular docking studies of a series of new analogs through the functionalization and modification on position 1 of the benzimidazole nucleus forming alkyl chains, substituted hydrazides, semicarbazides, thiosemicarbazides, and heterocyclic rings and/or position 5 of the 2-furyl ring (Fig. 2).
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The synthesized compounds 14–18 (Schemes 1 and 2) were tested for their antitumor activity against MCF-7 breast cancer cell lines. We have previously studied the cytotoxic activity of the 2-substituted benzimidazoles against MCF-7 cancer cell line in our lab and we have observed remarkable selectivity against this cancer cell line [31]. We have synthesized several compounds in our lab that have shown remarkable cytotoxicity against MCF-7 cancer cell line and studied their inhibitory action against VEGF kinase enzyme using double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to measure the level of human VEGF in samples. These bioactivity studies were done in comparison to the reference drug tamoxifen. In order to understand their biological effect, a molecular modeling study was carried out with these new inhibitors using a pharmacophore-based approach. With LigandScout [32], the key features for the inhibition of VEGFR2 were compiled into a 3D-pharmacophore model from the ligand (K11) co-crystallized in the binding pocket of the receptor (PDB entry: 3EWH) [33]. Docking studies using GOLD [34] were conducted to generate the docking poses of the synthesized compounds, which were ranked based on their ability to fulfill the key interactions compiled in the 3D-pharmacophore model. Based on this study, the structure– activity relationship of the synthesized 2-(5-methyl-2-furyl)-1Hbenzimidazoles were rationalized and discussed.
Results and discussion Chemistry
Figure 2. Schematic representation showing the designing strategy of 2-(2-furyl)-1H-benzimidazole derivatives.
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The target compounds 14–18 were synthesized according to the reaction sequences outlined in Schemes 1 and 2. Compounds 11a,b, 12a,b, and 13a,b were previously prepared in our lab (submitted for publication) [30]. 2-[2-(Furan-2-yl)1H-benzo[d]imidazol-1-yl]acetohydrazide and the 5-methyl derivatives 13a,b were converted to the Schiff bases 14a–l (Scheme 1) via condensation with various selected aldehydes and with various functionalized methyl aryl ketones using ethanol/acetic acid (24:1) mixture [35]. Nucleophilic attack of 13a to phenylisocyanate and methylisothiocyanate was successfully performed in refluxing ethanol to give 1-[2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl) acetyl]-4-phenylsemicarbazide 15 and 1-[2-(2-(furan-2-yl)-1Hbenzo[d]imidazol-1-yl)acetyl]-4-methylthiosemicarbazide 18, respectively [36]. The cyclization of the acid hydrazide compound 13b with acetyl acetone in glacial acetic acid at 150°C resulted in the formation of 2-[2-(furan-2-yl)-1H-benzo[d] imidazol-1-yl]-1-(3,5-dimethyl-1H-pyrazol-1-yl)ethanone 16 [37]. Compound 13a was condensed with various alkyl and aryl chlorides in K2CO3/acetone at room temperature to obtain 17a–c (Scheme 2). www.archpharm.com
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Scheme 1. Synthetic pathway of compounds 14a–l. Reagents and conditions: (i) p-toluenesulfonic acid, DMF, 100°C; (ii) ethyl 2-bromoacetate, K2CO3, acetone, RT; (iii) hydrazine hydrate, ethanol, 90°C; (iv) aryl aldehydes or ketones, ethanol/acetic acid (24:1), reflux.
Bioactivity Cytotoxicity against human breast cancer cell line MCF-7 Cytotoxicity of the synthesized compounds 14–18 was tested using Skehan et al. method [38] in human breast cancer cell line MCF-7. The cytotoxicity results were compared to that of ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tamoxifen. Compound 14k exhibited higher potency against MCF-7cell line (IC50 ¼ 7.80 mg/mL) than that of tamoxifen (IC50 ¼ 8.00 mg/mL), while compounds 14e and 14g are equipotent to tamoxifen with IC50 ¼ 8.63 and 9.23 mg/mL, respectively. The rest of the compounds exhibited promising www.archpharm.com
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Scheme 2. Synthetic pathway of compounds 15–18. Reagents and conditions: (i) phenylisocyanate, ethanol, reflux; (ii) acetylacetone, g. AcOH, 150°C; (iii) chloro compounds, K2CO3, acetone, RT; (iv) methylisothiocyanate, ethanol, reflux.
activity against MCF-7 cell line with IC50 ¼ 11.20–16.00 mg/mL and their order of reactivity was 6f, 6h, 6b, 8, 3b, 4a, 6c, 7, and 6i in a descending order. The rest of the compounds exhibited moderate activity against MCF-7 cell line as shown in Table 1.
In vitro VEGF inhibition in human breast cancer cell line MCF-7 VEGF stimulates angiogenesis in tumor tissues thus promoting their nourishment and progression [1–5]. The discovery of ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
new chemotherapeutic agents by blocking the VEGF-induced angiogenesis has attracted much attention in the last few years [11, 12]. Since the 2-furylbenzimidazole nucleus showed promising inhibition against VEGF-induced angiogenesis in previous studies [27–29], we have synthesized several analogs of the 2-furylbenzimidazole nucleuses 14–18 and measured their inhibition potency in vitro against human VEGF in human breast cancer cell line MCF-7. This biological in vitro study was done using doubleantibody sandwich ELISA to assay the level of VEGF in human www.archpharm.com
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Table 1. Effect of the synthesized compounds on the breast cancer cell line MCF-7 and the VEGF level (pg/mL) in the breast cancer cell line MCF-7.
R1
R2
IC50 (mg/mL)a)
11a 11b
H CH3
H H
21.50 14.00
4460.90 (15%) 280.24 (95%)
12a
H
15.20
2000.82 (62%)
12b
CH3
17.80
3760.70 (28%)
13a
H
20.20
4010.45 (24%)
13b
CH3
22.40
760.40 (86%)
14a
H
37.90
5000.40 (5%)
14b
H
11.80
196.80 (96%)
14c
H
15.40
2190.66 (58%)
14d
H
16.60
3700.00 (30%)
14e
H
8.63
196.76 (96%)
14f
H
11.20
184.30 (97%)
14g
CH3
9.23
146.66 (97%)
14h
CH3
11.30
180.90 (97%)
Compound
VEGF (pg/mL)b)
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Table 1. (Continued)
Compound
R1
14i
H
16.00
3600.90 (31%)
14j
H
21.80
4230.50 (19%)
14k
H
7.80
104.30 (98%)
14l
H
22.10
4770.63 (9%)
15
H
16.00
2300.14 (56%)
16
CH3
13.90
240.80 (95%)
17a
H
22.10
4800.00 (9%)
17b
H
21.80
4670.23 (11%)
17c
H
18.70
4050.45 (23%)
18
H
17.50
2600.40 (51%)
DMSO Tamoxifen
– –
– 8.00
5250.00 (–) 110.75 (98%)
a) b)
R2
– –
IC50 (mg/mL)a)
VEGF (pg/mL)b)
Data were expressed as mean of six independent experiments. Values between brackets indicate percentage changes as compared with control of untreated cells.
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breast cancer cell line MCF-7 as compared to the inhibition for the untreated cells. The screening results (Table 1) showed that eight compounds (14b, 14e, 14f, 14g, 14h, 14k, 11b, and 16) were found to be potent inhibitors of VEGF in the MCF-7 cancer cell line, their percentage of inhibition ranges from 95 to 98% as compared to the positive drug, tamoxifen (98%). These results were consistent with cell cytotoxicity activity against MCF-7 cell line, where these eight compounds exhibited excellent activity with IC50 ranging from 7.80 to 13.90 mg/mL.
Molecular modeling Recent studies showed that the molecular mechanism of 2-subsituted benzimidazoles inhibiting angiogenesis can be due to inhibition of VEGFR [15–20]. A computational study was carried out to comprehend how these compounds could interact with this receptor, and to explain their structure– activity relationships. PDB entry 3EWH [33] representing the VEGFR2 crystal structure was selected for this modeling study because of the co-crystallized benzimidazole and because of its high resolution. Using LigandScout [32], a 3D-pharmacophore from the benzimidazole inhibitor (K11) co-crystallized in the protein-binding site was generated (Fig. 3). The generated structure-based pharmacophore represents important binding features for the inhibition of VEGFR2 and was used to direct the selection of the most plausible binding poses of the novel compounds. The pharmacophore includes
Figure 3. 3D-pharmacophore for ligand interaction with VEGFR2 developed from PDB entry 3EWH. Hydrophobic contacts are shown as yellow spheres and hydrogen bonds are shown as green and red arrows for donors and acceptors, respectively.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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three main hydrophobic sub-sites. Relevant residues are LEU889, ILE892, and ILE888 for sub-site I, PHE1047, LEU840, ALA866, and VAL848 for sub-site II, and finally GLU885, VAL899, and THR916 in the last sub-site (Fig. 3). Additionally, four key hydrogen bonds were included into the pharmacophore model: on the one hand, a donor to GLU885 and acceptor to ASP1046 stabilize the inhibitor in the center of the cavity and on the other hand, a hydrogen bond donor and acceptor toward CYS919 stabilize the terminal pyrimidine ring. Lastly, a complete exclusion volume sphere coat was added to define the steric extent of the binding site. The crystal structure of VEGFR was prepared as described in the Experimental Section and used for the docking experiment. The docking program GOLD was used to sample plausible docking poses for all compounds. The generated docking conformations were ranked based on their ability to fulfill the interaction features reported in the above-mentioned 3Dpharmacophore. The VEGFR2 inhibitory potency of most of the newly synthesized 2-furylbenzimidazoles may be attributed to the presence of the acetohydrazide moiety, which is common in all the compounds and can act as a hydrophilic anchor within a rather hydrophobic binding cleft. The acid hydrazide group is able to stabilize the compounds in the binding site by bringing the two key hydrogen acceptor and donor groups toward ASP1046 and GLU885, respectively. This docking study showed that novel compounds stabilized by this central interaction were deeply embedded into the hydrophobic binding cleft of VEGFR. Due to the much smaller size than the co-crystallized inhibitor (K11), the docked compounds could not fill the whole binding cleft, but bridge two hydrophobic sub-sites (I and III) and are stabilized by the central hydrogen bonds. Furthermore, the studied compounds have an extra furyl group that is accommodated by the hydrophobic residues VAL848, PHE845, and PHE1047 (Fig. 4). For instance, compound 14k (Fig. 4), which displayed one of the highest inhibitory potencies, shows an excellent complementarity with the reference 3D-pharmacophore. The crucial hydrogen bonds were identified between GLU885 and ASP1046 and the acid hydrazide group of the ligand, indicating a good overall orientation of the ligand in the cavity. In the first sub-site, the phenyl ring is stabilized by the residues LEU889, ILE892, and ILE888, while the benzimidazole ring interacts with the residues VAL899 and THR916 of the third enzyme hydrophobic sub-site. The second hydrophobic region in the binding pocket was not filled. Instead, a new hydrophobic area was covered by the furyl group of the inhibitor. Interestingly, the double bond, and in particular its cis conformation, is of high importance for the orientation of the inhibitor in the enzyme pocket. Consequently, series 14 www.archpharm.com
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Surprisingly, the methyl group substitution on the acid hydrazide group (in compounds 14g and 14h) has no significant effect on the activity, as it shows no hydrophobic contact with the surrounding residues. Moreover, cyclization of the acid hydrazide (in compound 16) shows good binding in the pocket, although it does not keep the hydrogen donor group toward GLU885. This could be explained by the increased rigidity of the compound, the ring keeping all other binding features in the right orientation within the binding cleft.
Conclusion
Figure 4. Plausible binding mode for compound 14k in 2D (up) and 3D (down) showing favorable hydrophobic contacts (yellow spheres) and two hydrogen bonds with GLU885 (green) and ASP1046 (red). For clarity, not all interacting amino acids are shown in the 3D view.
and compound 16 have superior activity over other compounds (compound 15, series 17, and compound 18), which cannot fulfill these key interactions due to absence of the cis double bond. As the pocket is highly hydrophobic, lipophilic features either on the phenyl (sub-site I) or furyl rings are crucial for good binding and therefore for the activity. This is clearly the case for compounds 14b and 14k, which have an ortho methyl substitution on the phenyl ring, and with 14f and 14e, which are ortho substituted with chlorine and methoxy, respectively, 14g with ortho methyl on the furyl ring, and 14h with two methyl groups on both phenyl and furyl rings. In contrast, removing the hydrophobic moiety (compound 14a), or replacing it with hydrophilic moieties like amino or hydroxyl group (as in compounds 14l or 14j, respectively), or changing the phenyl ring for a less lipophilic ring like pyridine or furyl (in compound 14c and 14d, respectively) dramatically decreases inhibitory potency. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The scope of this research work was the synthesis of new (2furyl)-1H-benzimidazoles as expected inhibitors of VEGFinduced angiogenesis. In this study, as a completion to our previous study made in part I (submitted for publication) [30], we have synthesized compounds 14–18 through the functionalization at position 1 of the benzimidazole nucleus forming various hydrazides, semicarbazides, thiosemicarbazide, alkyl chains, and heterocyclic rings using improved synthetic pathways to reach our target compounds. In vitro studies of the VEGF inhibition of the synthesized compounds in human breast cancer cell line MCF-7 showed that compounds 14b, 14e, 14f, 14g, 14h, 14k, and 16 are quite potent to the reference drug, tamoxifen with high percentage of inhibition in addition to excellent cell cytotoxicity activity against MCF-7 cell line with IC50 ranging from 7.80 to 13.90 mg/mL in comparison to that of tamoxifen (IC50 ¼ 8.00 mg/mL). Molecular modeling study revealed the importance of the acid hydrazide group – common in all synthesized compounds – in stabilizing the docked inhibitors in the binding site of VEGFR2 with two hydrogen bonds toward ASP1046 and GLU885. Moreover, the cis double bond seems to be of high importance for good orientation in the binding pocket of compounds such as 14a–i. Additionally, this study revealed that the lipophilic features on the phenyl group of inhibitors 14b, 14k, 14f, 14e, 14g, and 14h can create hydrophobic contacts with the residues found in sub-site I, which consequently affects their inhibition potency.
Experimental Physical measurements Microanalyses and spectral data of the compounds were performed in the Micro analytical labs, National Research Centre and Micro analytical Laboratory Center, Faculty of Science, Cairo University, Cairo, Egypt. The IR spectra (4000–400 cm1) were recorded using KBr pellets in a Jasco FT/IR 300E Fourier transform infrared spectrophotometer on a Perkin Elmer FT-IR 1650 (spectrophotometer). The melting points were determined on a Stuart SMP30 Melting Point Apparatus and are uncorrected. The www.archpharm.com
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1 H and 13C NMR spectra were recorded using Jeol EX-270 MHz and 500 MHz NMR spectrophotometers using DMSO-d6 as a solvent. Chemical shifts are reported in parts per million (ppm) from the tetramethylsilane resonance in the indicated solvent. Coupling constants are reported in Hertz (Hz); spectral splitting partners are designed as follows: singlet (s); doublet (d); triplet (t); and multiplet (m). The mass spectra were carried out using Finnigan mat SSQ 7000 (Thermo. Inst. Sys., Inc., USA) spectroscopy at 70 eV. Reaction progress was monitored using thin layer chromatography (TLC) analysis on Silica Gel 60 F254 plates (Merck).
General procedure for the preparation of compounds 14a–i To a solution of the acid hyrazide 13a or 13b (0.01 mol) in ethanol/ acetic acid (24:1, 25 mL), 0.02 mol of aryl aldehydes namely benzaldehyde, 4-methylbenzaldehyde, 3-pyridinecarboxaldehyde, furfural, 4-methoxybenzaldehyde, and 4-chlorobenzaldehyde and arylacetophenones namely acetophenone, 4-hydroxyacetophenones, 4-methylacetophenones, and 4-aminoacetophenones was added. The reaction mixture was heated under reflux for 5 h, and then cooled. The separated solids were filtered, dried, and crystallized from acetic acid.
N 0 -Benzylidene-2-(2-(furan-2-yl)-1H-benzo[d]imidazol1-yl)acetohydrazide (14a) Yield ¼ 90% (3.10 g). m.p.: 250–253°C. TLC Rf ¼ 0.70 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.74 (s, 2H, CH2), 6.70 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.13 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.20–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.40–7.42 (m, 2H, H4 þ H7 benzimidazole), 7.60–7.62 (m, 3H, H3 0 þ H4 0 þ H5 0 phenyl), 7.75–7.76 (m, 2H, H2 0 þ H6 0 phenyl), 7.80 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.07 (s, 1H, –– CH), 11.79 (s, 1H, NH, D2O exchangeable). IR (cm1): 3427 and 3185 (NH and OH), 1681 (C –– O), 1614 (C –– N). MS: m/z 345, 16% (Mþþ1); m/z 344, 64% (Mþ); m/z 343, 14% (Mþ1); m/z 197, 100%. Anal. calcd. for C20H16N4O2 (344.13): C, 69.86; H, 4.66; N, 16.23. Found C, 69.77; H, 4.70; N, 16.29.
2-[2-(Furan-2-yl)-1H-1,3-benzodiazol-1-yl]-N 0 -[(1Z)(4-methylphenyl)methylidene]acetohydrazide (14b) Yield ¼ 85% (3.06 g). m.p.: 249–252°C. TLC Rf ¼ 0.57 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 2.30 (s, 3H, CH3), 5.70 (s, 2H, CH2), 6.67 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.12 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.21–7.22 (m, 2H, H5 þ H6 benzimidazole), 7.24–7.25 (d, J ¼ 7.6 Hz, 2H, H3 0 þ H5 0 phenyl), 7.55–7.56 (m, 2H, H4 þ H7 benzimidazole), 7.63–7.64 (m, 2H, J ¼ 7.6Hz, H2 0 þ H6 0 phenyl), 7.83 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.02 (s, 1H, HC –– N), 11.73 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 21.59, 46.57, 111.01, 127.74, 119.39, 122.82, 123.19, 127.59, 128.05, 129.97, 131.82, 136.90, 140.44, 142.99, 144.85, 144.94, 145.41, 145.93, 148.25, 154.72, 168.82. IR (cm1): 3390 and 3220 (NH and OH), 1678 (C –– O), 1611 (C –– N). MS: m/z 359, 16% (Mþþ1); m/z 358, 54% (Mþ); m/z 357, 12% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H18N4O2 (358.14): C, 70.38; H, 5.06; N, 15.63. Found C, 70.43; H, 5.09; N, 15.67.
2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 0 ((pyridin-3-yl)methylene)acetohydrazide (14c) Yield ¼ 92% (3.17 g). m.p.: 235–237°C. TLC Rf ¼ 0.62 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.77 (s, 2H, CH2), 6.67 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.14 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.22–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.45–7.47 (m, 2H, H5 0 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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pyridine), 7.59–7.65 (m, 2H, H4 þ H7 benzimidazole), 7.88 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.02 (s, 1H, HC –– N), 8.19–8.21 (m, 2H, H4 0 pyridine), 8.58–8.60 (m, 2H, H6 0 pyridine), 8.90–8.92 (s, 2H, H2 0 pyridine), 11.97 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSOd6) d: 46.52, 110.96, 112.69, 112.86, 119.41, 122.92, 123.28, 124.47, 130.48, 134.23, 136.84, 142.12, 142.94, 144.89, 145.44, 145.86, 149.17, 151.14, 162.22. IR (cm1): 3429 and 3200 (NH and OH), 1695 (C –– O), 1607 (C –– N). MS: m/z 346, 13% (Mþþ1); m/z 345, 51% (Mþ); m/z 344, 9% (Mþ1); m/z 197, 100%. Anal. calcd. for C19H15N5O2 (345.35): C, 66.08; H, 4.38; N, 20.28. Found C, 66.15; H, 4.45; N, 20.36.
2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 0 ((furan-2-yl)methylene)acetohydrazide (14d) Yield ¼ 85% (2.94 g). m.p.: 240–241°C. TLC Rf ¼ 0.60 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.70 (s, 2H, CH2), 6.62 (dd, J ¼ 3.05 Hz, 1H, H4 0 2-furyl), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2furyl), 6.94 (dd, J ¼ 3.05 Hz, 1H, H3 0 2-furyl), 7.12 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.22–7.25 (m, 2H, H5 þ H6 benzimidazole), 7.59–7.63 (m, 2H, H4 þ H7 benzimidazole), 7.80 (dd, J ¼ 3.05 Hz, 1H, H5 0 2furyl), 7.88 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 7.95 (s, 1H, HC –– N), 11.70 (s, 1H, NH, D2O exchangeable). IR (cm1): 3423 and 3200 (NH and OH), 1688 (C –– O), 1620 (C –– N). MS: m/z 335, 17% (Mþþ1); m/z 334, 84% (Mþ); m/z 333, 40% (Mþ1); m/z 197, 100%. Anal. calcd. for C18H14N4O3 (345.35): C, 64.66; H, 4.22; N, 16.76. Found C, 64.69; H, 4.27; N, 16.70.
N 0 -(4-Methoxybenzylidene)-2-(2-(furan-2-yl)-1Hbenzo[d]imidazol-1-yl)acetohydrazide (14e) Yield ¼ 82% (3.08 g). m.p.: 275–277°C. TLC Rf ¼ 0.77 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 3.77 (s, 3H, OCH3), 5.71 (s, 2H, CH2), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 6.98–7.00 (d, J ¼ 9 Hz, 2H, H2 0 þ H6 0 phenyl), 7.12 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.20–7.22 (m, 2H, H5 þ H6 benzimidazole), 7.60–7.62 (m, 2H, H4 þ H7 benzimidazole), 7.69–7.70 (dd, J ¼ 9 Hz, 2H, H3 0 þ H5 0 phenyl), 7.80 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.05 (s, 1H, –– CH), 11.70 (s, 1H, NH, D2O exchangeable). IR (cm1): 3423 and 3200 (NH and OH), 1688 (C –– O), 1620 (C –– N). MS: m/z 375, 16.8% (Mþþ1); m/z 374, 84% (Mþ); m/z 373, 40% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H18N4O3 (374.39): C, 67.37; H, 4.85; N, 14.96. Found C, 67.43; H, 4.89; N, 15.07.
N 0 -(4-Chlorobenzylidene)-2-(2-(furan-2-yl)-1Hbenzo[d]imidazol-1-yl)acetohydrazide (14f) Yield ¼ 86% (3.26 g), m.p.: 230–232°C, TLC Rf ¼ 0.77 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.77 (s, 2H, CH2), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.13 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.20–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.47–7.50 (d, J ¼ 8.4 Hz, 2H, H2 0 þ H6 0 phenyl), 7.60–7.64 (m, 2H, H4 þ H7 benzimidazole), 7.78–7.79 (d, J ¼ 8.4 Hz, 2H, H3 0 þ H5 0 phenyl), 7.86 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.10 (s, 1H, –– CH), 11.80 (s, 1H, NH, D2O exchangeable). IR (cm1): 3440 and 3215 (NH and OH), 1691 (C –– O), 1608 (C –– N). MS: m/z 380, 17% (Mþþ1); m/z 379, 24% (Mþ); m/z 378, 52% (Mþ1); m/z 197, 100%. Anal. calcd. for C20H15ClN4O2 (378.81): C, 63.41; H, 3.99; Cl, 9.36; N, 14.79. Found C, 63.48; H, 4.04; Cl, 9.30; N, 14.85.
N 0 -Benzylidene-2-(2-(5-methylfuran-2-yl)-1Hbenzo[d]imidazol-1-yl)acetohydrazide (14g) Yield ¼ 94% (3.36 g). m.p.: 232–235°C. TLC Rf ¼ 0.70 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 2.21 (s, 3H, CH3), www.archpharm.com
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5.74 (s, 2H, CH2), 6.31 (d, J ¼ 3.05 Hz, 1H, H4 2-furyl), 7.05 (d, J ¼ 3.05 Hz, 1H, H3 2-furyl), 7.19–7.21 (m, 2H, H5 þ H6 benzimidazole), 7.42–7.43 (m, 3H, H3 0 þ H4 0 þ H5 0 phenyl protons), 7.60– 7.61 (m, 2H, H4 þ H7 benzimidazole), 7.75–7.76 (m, 2H, H2 0 þ H6 0 phenyl protons), 8.10 (s, 1H, CH –– ) 11.79 (s, 1H, NH, D2O exchangeable). IR (cm1): 3428 (NH), 1679 (C –– O), 1610 (C –– N). MS: m/z 359, 19% (Mþþ1); m/z 358, 72% (Mþ); m/z 357, 26% (Mþ1); m/z 260, 100%; m/z 211, 66.0%. Anal. calcd. for C21H18N4O2 (358.14): C, 70.38; H, 5.06; N, 15.63. Found C, 70.46; H, 5.02; N, 15.68.
2-[2-(5-Methylfuran-2-yl)-1H-1,3-benzodiazol-1-yl]-N 0 [(1Z)-(4-methylphenyl)methylidene]acetohydrazide (14h) Yield ¼ 88% (3.29 g), m.p.: 240–243°C, TLC Rf ¼ 0.67 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 2.20 (s, 3H, CH3 phenyl), 2.29 (s, 3H, CH3 2-furyl), 5.72 (s, 2H, CH2), 6.26 (d, J ¼ 3.05 Hz, 1H, H4 2-furyl), 6.99 (d, J ¼ 3.05 Hz, 1H, H3 2-furyl), 7.19–7.21 (m, 2H, H5 þ H6 benzimidazole), 7.23–7.25 (d, J ¼ 7.6 Hz, 2H, H3 0 þ H5 0 phenyl protons), 7.57–7.61 (m, 2H, H4 þ H7 benzimidazole), 7.63–7.65 (d, J ¼ 7.6 Hz, 2H, H2 0 þ H6 0 phenyl protons), 8.10 (s, 1H, CH), 11.7 (s, 1H, NH, D2O exchangeable). 13 C NMR (DMSO-d6) d: 13.76, 21.57, 46.52, 101.23, 108.93, 110.92, 113.63, 119.17, 122.65, 122.92, 127.52, 127.65, 129.99, 131.84, 137.05, 137.85, 140.42, 143.01, 144.21, 144.73, 154.15, 169.19. IR (cm1): 3430 and 3189 (NH and OH), 1676 (C –– O), 1612 (C –– N). MS: m/z 373, 26% (Mþþ1); m/z 372, 88% (Mþ); m/z 371, 33% (Mþ1); m/z 255, 18%. Anal. calcd. for C22H20N4O2 (372.42): C, 70.95; H, 5.41; N, 15.04. Found C, 71.04; H, 5.49; N, 15.09.
2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 0 (1-phenylethylidene)acetohydrazide (14i) Yield ¼ 85% (3.04 g), m.p.: 245–248°C, TLC Rf ¼ 0.65 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 2.30 (s, 3H, CH3), 5.77 (s, 2H, CH2), 6.69 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.10 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.21–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.39–7.41 (m, 3H, H3 0 þ H4 0 þ H5 0 phenyl), 7.60–7.63 (m, 2H, H4 þ H7 benzimidazole), 7.86–7.89 (m, 1H, H2 0 þ H6 0 phenyl), 7.90 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 11.06 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 14.29, 46.85, 111.01, 112.72, 119.39, 122.82, 123.19, 126.88, 128.69, 128.95, 129.22, 129.82, 133.73, 136.89, 138.52, 142.99, 144.91, 145.44, 145.94, 149.43, 169.82. IR (cm1): 3427 and 3179 (NH and OH), 1683 (C –– O), 1610 (C –– N). MS: m/z 359, 24% (Mþþ1); m/z 358, 85% (Mþ); m/z 357, 11% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H18N4O2 (358.14): C, 70.38; H, 5.06; N, 15.63. Found C, 70.30; H, 5.01; N, 15.69.
2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 0 (1-(4-hydroxyphenyl)ethylidene)acetohydrazide (14j) Yield ¼ 76% (2.84 g). m.p.: 220–223°C. TLC Rf ¼ 0.75 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 2.30 (s, 3H, CH3), 5.73 (s, 2H, CH2), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 6.73–6.82 (d, J ¼ 8.4 Hz, 2H, H2 0 þ H6 0 phenyl), 7.11 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.21–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.58–7.65 (m, 2H, H4 þ H7 benzimidazole), 7.78–7.80 (d, J ¼ 8.4 Hz, 2H, H3 0 þ H5 0 phenyl), 7.87 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 10.00 (s, 1H, OH, D2O exchangeable), 11.00 (s, 1H, NH, D2O exchangeable). IR (cm1): 3421 and 3250 (NH and OH), 1675 (C –– O), 1603 (C –– N). MS: m/z 375, 24.4% (Mþþ1); m/z 374, 100.0% (Mþ); m/z 373, 13% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H18N4O3 (374.14): C, 67.37; H, 4.85; N, 14.96. Found C, 67.32; H, 4.94; N, 15.07. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N 00 (1-p-tolylethylidene)acetohydrazide (14k) Yield ¼ 72% (2.68 g). m.p.: 230–233°C. TLC Rf ¼ 0.65 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 2.27 (s, 3H, CH3phenyl), 2.30 (s, 3H, CH3), 5.72 (s, 2H, CH2), 6.68 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.11 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.18–7.21 (m, 2H, H5 þ H6 benzimidazole), 7.22–7.24 (d, J ¼ 6 Hz, 2H, H3 0 þ H5 0 phenyl), 7.60–7.64 (m, 2H, H4 þ H7 benzimidazole), 7.76–7.77 (d, J ¼ 6 Hz, 2H, H2 0 þ H6 0 phenyl), 7.87 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 11.00 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 14.22, 21.38, 46.95, 110.98, 112.64, 119.40, 122.79, 122.95, 123.16, 123.28, 126.79, 129.52, 129.74, 135.77, 136.92, 139.42, 143.04, 144.90, 145.41, 145.97, 149.47, 169.69. IR (cm1): 3423 and 3186 (NH and OH), 1677 (C –– O), 1615 (C –– N). MS: m/z 373, 27% (Mþþ1); m/z 372, 100% (Mþ); m/z 371, 17% (Mþ1); m/z 197, 96%. Anal. calcd. for C22H20N4O2 (372.42): C, 70.95; H, 5.41; N, 15.04. Found C, 71.05; H, 5.37; N, 15.08.
N 0 -(1-(4-Aminophenyl)ethylidene)-2-(2-(furan-2-yl)-1Hbenzo[d]imidazol-1-yl)acetohydrazide (14l) Yield ¼ 80% (2.98 g). m.p.: 238–240°C. TLC Rf ¼ 0.80 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 2.18 (s, 3H, CH3), 5.45 (NH2, D2O exchangeable), 5.7 (s, 2H, CH2), 6.53–6.56 (d, J ¼ 8.4 Hz, 2H, H3 0 þ H5 0 phenyl), 6.67 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.10 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.20–7.23 (m, 2H, H5 þ H6 benzimidazole), 7.57–7.62 (m, 4H, (H2 0 þ H6 0 ) phenyl þ (H4 þ H7) benzimidazole), 7.89 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 10.7 (s, 1H, NH, D2O exchangeable). IR (cm1): 3421 and 3286 (NH, NH2, and OH), 1681 (C –– O), 1608 (C –– N). MS: m/z 374, 30% (Mþþ1); m/z 373, 76.2% (Mþ); m/z 372, 47% (Mþ1); m/z 197, 100%. Anal. calcd. for C21H19N5O2 (373.41): C, 67.55; H, 5.13; N, 18.76. Found C, 67.50; H, 5.19; N, 18.84.
1-(2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetyl)-4phenylsemicarbazide (15) To a solution of the acid hyrazide 13a (0.01 mol) in 30 mL ethanol, phenyl isocyanate (0.01 mol) was added, The reaction mixture was heated under reflux for 12 h. The final product was monitored using TLC. Excess solvent was evaporated under reduced pressure. The separated solids were filtered, dried, and crystallized from ethanol. Yield ¼ 65% (2.44 g). m.p.: 225–229°C. TLC Rf ¼ 0.60 (petroleum ether/ethyl acetate/ethanol, 1:1:1). 1H NMR (DMSOd6) d: 5.30 (s, 2H, CH2), 6.70 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 6.91–6.93 (m, 1H, H4 0 phenyl), 7.21 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.22–7.24 (m, 2H, H5 þ H6 benzimidazole), 7.27–7.29 (m, 2H, H3 0 þ H5 0 phenyl) 7.39–7.41 (m, 2H, H2 0 þ H6 0 phenyl), 7.59–7.63 (m, 2H, H4 þ H7 benzimidazole), 7.90 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.10 (s, 1H, NH, D2O exchangeable), 9.00 (s, 1H, NH, D2O exchangeable), 10.20 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 46.53, 110.97, 112.62, 113.10, 118.69, 119.12, 119.51, 122.59, 122.82, 122.96, 123.32, 129.23, 136.65, 139.99, 142.96, 144.74, 145.28, 145.49, 155.71, 167.55. IR (cm1): 3450 (enolic OH), 3400, 3307, and 3204 (NH), 3000 (H-bonded OH), 1700 (C –– O), 1680 (C –– O), 1602 (C –– N). MS: m/z 283, 13% (Mþ NHC6H5); m/z 256, 44% (Mþ CONHC6H5); m/z 197, 100%; m/z 184, 53%. Anal. calcd. for C20H17N5O3 (375.38): C, 63.99; H, 4.56; N, 18.66. Found C, 64.07; H, 4.50; N, 18.69.
1-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-(2-(5-methylfuran-2yl)-1H-benzo[d]imidazol-1-yl)ethanone (16) A mixture of hydrazide 13b (0.01 mol) and acetyl acetone (0.02 mol) in glacial acetic acid (10 mL) and few drops of DMF www.archpharm.com
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was stirred at room temperature overnight. After dilution with water, the solid precipitated was filtered, dried, and crystallized from acetic acid. Yield ¼ 70% (2.34 g), m.p.: 176–179°C, TLC Rf ¼ 0.57 (petroleum ether/ethyl acetate, 1:1). 1H NMR (DMSO-d6) d: 1.70 (s, 3H, CH3 pyrazole), 2.10 (s, 3H, CH3 pyrazole), 2.30 (s, 3H, CH3 2-furyl), 5.47 (s, 2H, CH2), 6.20 (d, J ¼ 3.05 Hz, 1H, H4 2-furyl), 6.52 (s, 1H, CH), 7.00 (d, J ¼ 3.05 Hz, 1H, H3 2-furyl), 7.10–7.20 (m, 2H, H5 þ H6 benzimidazole), 7.51–7.59 (m, 2H, H4 þ H7 benzimidazole). IR (cm1): 1672 (C –– O), 1610 (C –– N). MS: m/z 335, 5% (Mþþ1); m/z 334, 72% (Mþ); m/z 255, 13%; m/z 238, 38%; m/z 210, 74%; m/z 183, 37%. Anal. calcd. for C19H18N4O2 (334.37): C, 68.25; H, 5.43; N, 16.76. Found C, 68.31; H, 5.53; N, 16.84.
General procedure for the preparation of compounds 17a–c To a solution of the acid hyrazide 13a (0.01 mol) and anhydrous K2CO3 (10.6 g, 0.1 mol) in dry acetone, chloro compounds (0.01 mol) were added dropwise namely: benzoyl chloride, benzyl chloride, and acetyl chloride. The reaction mixture was stirred for 3 h at room temperature. The final product was monitored using TLC. Excess acetone was evaporated using reduced pressure and the reaction mixture was poured onto cold water and the precipitate was filtered, dried, and recrystallized from ethanol.
N 0 -{2-[2-(Furan-2-yl)-1H-1,3-benzodiazol-1-yl]acetyl}benzohydrazide (17a) Yield ¼ 83% (2.98 g). m.p.: 265–267°C. TLC Rf ¼ 0.55 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 5.30 (s, 2H, CH2), 6.71 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.22 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.25–7.27 (m, 2H, H5 þ H6 benzimidazole), 7.45–7.47 (m, 2H, H4 þ H7 benzimidazole), 7.48–7.50 (m, 1H, H4 0 phenyl), 7.53–7.64 (m, 2H, H3 0 þ H5 0 phenyl), 7.82–7.90 (m, 2H, H2 0 þ H6 0 phenyl), 7.93 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 10.50 (s, 1H, NH, D2O exchangeable), 11.50 (s, 1H, NH, D2O exchangeable). IR (cm1): 3433 and 3207 (NH and OH), 1697 (C –– O), 1604 (C –– N). MS: m/z 361, 7% (Mþþ1); m/z 360, 40% (Mþ); m/z 359, 8% (Mþ1); m/z 197, 70%. Anal. calcd. for C20H16N4O3 (360.37): C, 66.66; H, 4.48; N, 15.55. Found C, 66.74; H, 4.53; N, 15.62.
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7.63 (m, 2H, H4 þ H7 benzimidazole), 7.80 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 9.90 (s, 1H, NH, D2O exchangeable), 10.30 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 22.30, 46.39, 111.05, 112.57, 113.10, 119.16, 122.71, 122.94, 123.29, 136.76, 142.88, 145.19, 145.89, 168.69, 173.19. IR (cm1): 3413 and 3259 (NH and OH), 1678 (C –– O), 1610 (C –– N). MS: m/z 299, 7% (Mþþ1); m/z 298, 17% (Mþ); m/z 297, 7% (Mþ1); m/z 197, 100% (2-furylbenzimidazole þ CH2). Anal. calcd. for C15H14N4O3 (298.31): C, 60.40; H, 4.73; N, 18.78. Found C, 60.48; H, 4.81; N, 18.84.
1-(2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetyl)-4methylthiosemicarbazide (18) To a solution of the acid hyrazide 13a (0.01 mol) in 30 mL ethanol, methyl isothiocyanate (0.01 mol) was added, the reaction mixture was heated under reflux for 10 h. The final product was monitored using TLC. Excess solvent was evaporated under reduced pressure. The separated solids were filtered, dried, and crystallized from ethanol. Yield ¼ 75% (2.81 g). m.p.: 255–257°C. TLC Rf ¼ 0.70 (petroleum ether/ethyl acetate/ethanol, 1:1:1). 1H NMR (DMSO-d6) d: 2.88 (s, 3H, CH3), 5.20 (s, 2H, CH2), 6.72 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.16 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.21– 7.28 (m, 2H, H5 þ H6 benzimidazole), 7.55–7.57 (m, 1H, H4 benzimidazole), 7.60–7.62 (m, 1H, H7 benzimidazole), 7.88 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 8.10 (s, 1H, NH, D2O exchangeable), 9.00 (s, 1H, NH, D2O exchangeable), 10.20 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d: 31.54, 46.23, 110.97, 112.60, 119.49, 122.97, 123.14, 123.28, 136.67, 142.87, 144.63, 145.26, 145.56, 167.40, 171.15. IR (cm1): 3400 and 3235 (NH), 2800 (H-bonded OH), 1676 (C –– O), 1611 (C –– N), 1160 (C –– S). MS: m/z 331, 2% (Mþþ2); m/z 330, 3% (Mþþ1); m/z 329, 32% (Mþ); m/z 311, 21% (Mþ H2O); m/z 298, 28%; m/z 197, 95% (base peak). Anal. calcd. for C15H15N5O2S (375.38): C, 54.70; H, 4.59; N, 21.26. Found C, 54.76; H, 4.64; N, 21.33.
Bioactivity materials and methods Cytotoxicity against human breast cancer cell line MCF-7
Yield ¼ 80% (2.77 g). m.p.: 277–230°C. TLC Rf ¼ 0.67 (petroleum ether/ethyl acetate, 1:2). 1H NMR (DMSO-d6) d: 4.72 (s, 2H, CH2 benzylic), 5.70 (s, 2H, CH2), 6.34 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 6.53 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 6.92–7.09 (m, 2H, H5 þ H6 benzimidazole), 7.25–7.33 (m, 2H, H4 þ H7 benzimidazole), 7.45–7.47 (m, 5H, H2 0 þ H3 0 3þ H4 0 þ H5 0 þ H6 0 phenyl), 7.63 (dd, J ¼ 3 Hz, 1H, H5 2-furyl), 9.00 (s, 1H, NH, D2O exchangeable), 9.30 (s, 1H, NH, D2O exchangeable). IR (cm1): 3403 and 3150 (NH and OH), 1683 (C –– O), 1615 (C –– N). MS: m/z 346, 31% (Mþ); m/z 345, 7% (Mþ1); m/z 241, 21% (2-furylbenzimidazole þ CH2CONH); m/z 197, 100% (2-furylbenzimidazole þ CH2), m/z 184, 60% (2-furylbenzimidazole). Anal. calcd. for C20H18N4O2 (346.38): C, 69.35; H, 5.24; N, 16.17. Found C, 69.40; H, 5.32; N, 16.25.
The antitumor activity against MCF-7 cells was assessed in the National Cancer Institute, Cancer Biology Department, Cairo, Egypt. The cytotoxicity of the synthesized compounds 11–18 was tested using the method of Skehan et al. [38]. Cells were plated in 96-multiwell plates (104 cells/well) for 24 h before treatment with the compounds to allow attachment of cells to wall of the plate. Different concentrations of the compound under test (0.0, 5.0, 12.5, 25.0, and 50.0 mg/mL) were added to the cell monolayer. Six wells were prepared for each individual dose. Monolayer cells were incubated with the compounds for 48 h at 37°C and in atmosphere of 5% CO2. After 48 h, cells were fixed, washed, and stained with sulforhodamine B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris–EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration was plotted to get the survival curve of each tumor cell line after the specified compound.
N 0 -Acetyl-2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (17c)
In vitro VEGF inhibition in human breast cancer cell line MCF-7
Yield ¼ 75% (2.23 g). m.p.: 248–250°C, TLC Rf ¼ 0.78 (petroleum ether/ethyl acetate, 1:2).1H NMR (DMSO-d6) d: 1.82 (s, 3H, CH3), 5.25 (s, 2H, CH2), 6.70 (dd, J ¼ 3 Hz, 1H, H4 2-furyl), 7.17 (dd, J ¼ 3 Hz, 1H, H3 2-furyl), 7.19–7.25 (m, 2H, H5 þ H6 benzimidazole), 7.52–
The effect of tested compounds on the level of human VEGF was determined using human tumor cell lines including breast cancer cell line MCF-7 obtained from the American Type Culture Collection (Rockville, MD, USA). The tumor cells were
N 0 -Benzyl-2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (17b)
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maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (Gibco), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37°C in humidified atmosphere containing 5% CO2. Cells at a concentration of 0.50 106 were grown in a 25 cm2 flask in 5 mL of complete culture medium. The cells in culture medium were treated with 20 mL of IC50 values of the compounds or the standard reference drug, tamoxifen dissolved in DMSO, then incubated for 24 h at 37°C, in a humidified 5% CO2 atmosphere. The cells were harvested and homogenates were prepared in saline using a tight pestle homogenizer until complete cell disruption. The kit uses a double-antibody sandwich ELISA to assay the level of human VEGF in samples. VEGF was added to monoclonal antibody enzyme well, which is pre-coated with human VEGF monoclonal antibody, incubation; then, VEGF antibodies labeled with biotin were added, and combined with streptavidin–HRP to form immune complex; then incubated and washed again to remove the uncombined enzyme. Then chromogen solution A and B was added, the color of the liquid changes to blue, and by the effect of acid, the color finally becomes yellow. The chroma of color and the concentration of the human TRK of sample were positively correlated and the optical density was determined at 450 nm. The level of human VEGF in samples was calculated (ng/L) as duplicate determinations from the standard curve.
Modeling study Structures of the studied inhibitors were built using MOE (Chemical Computing Group, Montreal, CA). The crystal structure of VEGFR in complex with ligand K11 was retrieved from the Protein Data Bank (PDB code: 3EWH) [33]. The protein was prepared (hydrogen atoms added, water removed, and ligand extracted) in GOLD 5.1 (CCDC, Cambridge, UK) [34] using default parameters unless otherwise stated. The docking program GOLD was used to perform the docking of compounds series 14, 15, 16, 17, and 18 into the crystal structure of VEGFR. The binding site was defined by specifying all residues around the original ligand, within a sphere of 7 Å radius. The top 100 docked solutions were kept for each ligand. The program LigandScout [32] was used to generate a 3D-pharmacophore from the ligand of the PDB structure 3EWH using default parameters. The developed pharmacophore was used to prioritize plausible docking poses of the studied compounds. Docking poses visualization was done using LigandScout. The most plausible conformation was selected for each ligand based on its ability to match the highest amount of chemical interactions described in the 3D-pharmacophore.
The authors are grateful to the National Cancer Institute, Cancer Biology Department, Cairo, Egypt, to perform the antitumor activity against the MCF-7 cancer cell line. Deep thanks are expressed to the “Science &Technology Development Fund” for financially supporting the manuscript through the fund of the STDF project no. 1517. The authors have declared no conflict of interest.
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