Design and synthesis of 2-phenyl benzimidazole derivatives as VEGFR-2 inhibitors with anti-breast cancer activity

Article abstract of DOI:10.1111/cbdd.13433

Three new series of 2-phenyl benzimidazole-based derivatives were designed, synthesized, and evaluated for their in vitro cytotoxic activity against breast cancer (MCF-7) cell lines. Three compounds 8, 9, and 15 showed high cytotoxic activities, with IC

Full text of DOI:10.1111/cbdd.13433

(1072-1909-0003-0000 :Orcid ID )PROFESSOR AMANY SALAH MOSTAFA
Design and Synthesis of 2-Phenyl Benzimidazole Derivatives as VEGFR-2 Inhibitors with Anti-Breast Cancer
Activity
Amany S. Mostafa 1,*, Rania M. Gomaa 1 and Mohammad A. Elmorsy 1,2
1 Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516,
Egypt
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Delta University for Science and Technology, Egypt
ABSTRACT
Three new series of 2-phenyl benzimidazole-based derivatives were designed, synthesized and evaluated
for their in vitro cytotoxic activity against breast cancer (MCF-7) cell lines. Three compounds 8, 9 and 15
showed high cytotoxic activities, with IC50 values of 3.37, 6.30 and 5.84 µM, respectively, while they showed
comparable cytotoxicity to the standard drug doxorubicin against human normal cells, including non
tumorigenic breast epithelial cell line (MCF-10F), skin fibroblast cell line (BJ) and lung fibroblast cell line
(MRC-5). Six of the synthesized compounds were screened against vascular endothelial growth factor
receptor 2 (VEGFR-2) where compounds 8, 9, 12 and 15 exhibited an outstanding potency in comparison to
sorafenib, with IC50 values of 6.7-8.9 nM. Molecular docking study assessed the good binding patterns of
the most potent compounds with the reported conserved amino acids of VEGFR-2 active site.
KEYWORDS
Benzimidazoles; MCF-7; VEGFR-2; Anticancer; Molecular docking.
*Correspondence
Amany S. Mostafa, Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt; Tel:/Fax: +2050-2200-242; Email: amanysalah2002@yahoo.com
1. INTRODUCTION
Cancer remains a major threat to human life, representing the second main leading cause of death worldwide.[1] It is
estimated that 13.1 million people will die from cancer in 2030.[2] One in ten of all new types of cancer diagnosed
around the world each year is breast cancer, representing the most widespread type in women in both developing and
developed countries. VEGFR-2 plays an important role in regulating tumor cell proliferation, differentiation,
angiogenesis and apoptosis.[3-6] VEGF and its receptors VEGFR-1 and VEGFR-2 are mostly overexpressed in the
malignant epithelial cells of breast carcinoma,[7] suggesting its crucial role in the angiogenic progression and the
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metastasis of breast cancer.[8] Literature reviews showed that many heterocyclic scaffolds have been investigated as
anti-breast cancer agents.[9]
Benzimidazole ring system has emerged to have a great importance as bioactive substructure; owing to structure
similarity to the naturally occurring purines.[10] It is inspected to be one of the highest biologically active class of
compounds having diverse activities[11,12] like antimicrobial,[13,14] anti-inflammatory,[15,16] antiviral,[17] anti-tumor,[18]
anticancer,[19] anthelmintic,[20] antihypertensive[21] and anti-hepatitis C virus activity.[22]
2-Aryl benzimidazoles in particular have been widely explored as anticancer agents, targeting different tyrosine
kinases.[23] New series of 2-phenyl benzimidazoles I[24] and II[25] were reported as potent anti-breast cancer agents, with
IC50 values of 0.18 µM and 1.2 µM respectively against MCF-7. The past few years have witnessed the discovery of
new tumor angiogenesis inhibitors targeting VEGFR-2,[26,27] among which 2-subsituted benzimidazoles were embraced
in an exclusive category.[23,28] Successful trials produced many compounds with improved VEGFR-2 inhibitory activity
like compounds III[28] and IV[29] (Dovitinib), the drug in phase-II clinical study with IC50 values of 3.7 µM and 65 nM
respectively (Fig. 1).
In the light of bioisosterism approach in drug design,[30] and supported by computer-aided drug design, the
previous findings lead us to exploit the 2-phenyl benzimidazole scaffold for the design and synthesis of new derivatives
(Series 1-3) aiming for potential antitumor and VEGFR-2 inhibiting activity (Fig. 2).
The target compounds were successfully isolated in good yields and in vitro screened against both human breast
adenocarcinoma cell line (MCF-7) and VEGFR-2 enzyme, where significant results were obtained. Docking study was
performed to examine the mode of binding with the receptor site.
2. RESULTS AND DISCUSSION
2.1. Chemistry
Benzimidazole nucleus is considered to be a distinctive synthon for building up versatile biologically active molecules.
In the last decade, many efforts were devoted for synthesis of various benzimidazole derivatives.[31,32] The most
commonly-used synthetic approach typically involves the condensation of o-arylenediamines with carbonyl
compounds; like aldehydes, carboxylic acids or their derivatives; which often requires strong acidic conditions, using
elevated temperature or microwave irradiation.[33-36]
In the present investigation, synthesis of the target compounds is depicted in three schemes, where the key starting
compound; 3-(1H-benzo[d]imidazol-2-yl)aniline (1); was accomplished through a cyclocondensation reaction between
o-phenylenediamine and 3-aminobenzoic acid in the presence of polyphosphoric acid (PPA) as a catalyst. Compound 1
was N-alkylated with various phenacyl bromide derivatives to yield compounds 2-4. It was confirmed through 1H-NMR
analysis that alkylation takes place on the N-atom of the primary amino group in the purified isolated products (Scheme
1).
For further investigation of substitution effect on biological activity, additional N-alkylated compounds 5-9 were
synthesized through the reaction of 1 with a series of different benzyl chloride derivatives (Scheme 2).
Besides, a series of urea and thiourea derivatives were prepared by reacting 1 with various isocyanates and
isothiocyanates to produce the urea and thiourea derivatives 10-15 (Scheme 3).
All the synthesized compounds were achieved in pure forms and in quite good yields. They were all characterized
by different spectral analyses; that proved to be in agreement with the proposed structures.
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2.2. Biological Evaluation
2.2.1. Preliminary In vitro antitumor screening
All the newly synthesized compounds were tested against mammary gland breast cancer (MCF-7) cell lines using MTT
assay. The relative cell viability (%) for all compounds was determined at different concentrations and represented
graphically by Figure 3a,b.
Median inhibitory concentration (IC50 µM) was verified for all compounds, and compared with the standard anticancer
drug Doxorubicin (Dox.). Compound 8 showed the highest activity against MCF-7 cell line with outstanding IC50 value
of 3.37 µM, a superior value over the standard Doxorubicin. Compounds 15, 9, 12 and 2 showed excellent potency with
very close values to that of the standard drug with a range of IC50 values of 5.84-9.74 µM. However, the rest of
compounds showed moderate to low activities.
Besides, % growth inhibition of MCF-7 cells produced by the tested compounds at 100 µM was calculated,
compared with the standard at the same concentration and listed with IC50 values in (Table 1).
2.2.2. In vitro cytotoxicity against human normal cells
To verify the cytotoxic selectivity of the newly synthesized derivatives, the promising candidates with the highest
cytotoxic activity were chosen to be evaluated against different normal human cell lines. The immortal non tumorigenic
breast epithelial cell line (MCF-10F), the skin fibroblast cell line (BJ) and the human lung fibroblast cell line (MRC-5)
were employed in MTT assay, using Dox as a standard to assess the required IC50 values (Table 2). The compounds
exhibited varied degrees of cytotoxicity, with a limited increase in IC50 values than that of the standard Dox. These
results specifically assume that these compounds have cytotoxic selectivity comparable to that of Dox towards
cancerous cells over normal cell lines.
2.2.3. VEGFR-2 enzyme assay
In order to investigate the potential mechanism of action by which the target compounds could exert their antitumor
activity, VEGFR-2 enzyme assay was performed using ELISA-based VEGFR-TK assay.[37] Six compounds with the
highest antitumor activities were evaluated for VEGFR-2 inhibiting activity, using sorafenib as a reference drug. IC50
values of the tested compounds were precisely determined, where compounds 8, 9, 12 and 15 showed the highest
inhibitory activity against VEGFR-2, compared to the reference drug with IC50 values range of 6.7-8.9 nM. Compounds
6 and 2 showed good IC50 values of 9.7 and 11.1 respectively (Table 3).
2.2.4. Docking study
A computational study was carried to figure out the mode of interaction between the target compounds and the intended
receptor, hence interpreting the outstanding inhibitory pattern of this new set of compounds. PDB entry 2OH4,
representing the VEGFR-2 crystal structure was selected for this modeling study. Docking of the newly synthesized
compounds showed good binding for all compounds with conservative amino acids inside the target pocket, mainly Glu
(883) and Cys (917). Compounds 8 and 9 showed the highest docking scores. In compound 8, the binding was observed
throughout both the imidazole ring and nitro group via hydrophobic interaction and electron acceptor interaction,
respectively. It seems that the electron withdrawing effect of the nitro group afforded better binding with the negatively
charged thiolate moiety of cys (917). Compound 9 showed binding through the Cl atom which is an electron acceptor,
in addition to an extra binding with secondary amino group to Asp (1044). To investigate the binding similarity between
both compounds 8 and 9 with the original ligand, flexible alignment was applied. Good alignment was obtained,
highlighting a well matched binding mode with the receptor site (Figures 4-6).
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It was noticed that all compounds that contain 4-nitro group (8, 12 and 15) in their structure exhibited the essential
binding through this moiety with Cys (917). The biological assay augments this result, since the aforementioned
compounds showed excellent VEFGR-2 inhibition in the nano molar scale. The binding pattern of the reference drug
Sorafenib with VEGFR-2 enzyme binding site is illustrated in Figure 7.
3. EXPERIMENTAL
3.1. Chemistry
Melting points (ºC) were recorded using a capillary tube in an Electrothermal C14500 apparatus and are uncorrected.
Column chromatography was performed using 60 μm silica gel. NMR spectra were recorded on a Bruker Avance
spectrometer (500 MHz) in DMSO-d6 or CDCl3 at Mansoura University. Chemical shifts in ppm are given as δ values
against TMS as the internal standard. MS analyses were performed on JOEL JMS-600H spectrometer at the
Microanalytical Centre, Cairo University. Reactions were monitored by TLC performed on Silica gel plates (Merk 60
F250), using a mixture of petroleum ether: ethyl acetate (1:1) as eluting system. The spots were visualized by UV (366,
245 nm).
3-(1H-benzo[d]imidazol-2-yl)aniline (1)
A mixture of o-phenylenediamine (0.1mol) and m-amino benzoic acid (0.1mol) in PPA (15 ml) was refluxed for 4 h.
The reaction mixture was allowed to cool then poured over cold water, followed by slow addition of 10% ammonium
hydroxide solution with constant stirring till ph 8. The crude product was filtered off, washed with ice-cold water; the
resulting product was recrystallized from ethanol as buff powder, yield 65%, m.p. 248-250°C (Reported m.p. 247-
249°C).[38]
General procedure for preparing benzimidazoles 2-4:
To a solution of compound 1 (0.21 g, 1 mmol) in methanol (7 mL), substituted phenacyl bromide (1 mmol) was added,
and the reaction mixture was refluxed for 2 h. The mixture was concentrated under reduced pressure; the precipitated
product was filtered and purified using column chromatography on silica gel, using (petroleum ether/EtOAc, 3:1)
system.
2-(3-(1H-benzo[d]imidazol-2-yl)phenylamino)-1-phenylethanone (2)
Pale yellow solid, 62 % yield, m.p. 275-276 °C. 1H-NMR (400 MHz, DMSO-d6): 4.86 (2H, s, CH2), 7.13 (1H, d, J = 6.0
Hz, Ar-H), 7.42-7.45 (3H, m, Ar-H), 7.56-7.62 (4H, m, Ar-H), 7.72 (1H, dd, J = 7.2, 2.9 Hz, Ar-H), 7.84 (2H, dd, J =
6.0, 2.9 Hz, Ar-H), 8.13 (2H, d, J = 7.2 Hz, Ar-H). 13C-NMR (100 MHz, DMSO-d6):49.7, 110.3, 114.5, 115.0, 117.2,
118.8, 126.1, 128.8, 129.4, 130.3, 133.2, 133.4, 137.0, 139.3, 149.0, 150.3, 195.8. MS (m/z, %): 327 (26.3, M+),
328(12.4, M++1). Elemental analysis: Calcd. (Found) (%) for C21H17N3O: C 77.04 (77.09), H 5.23 (5.17), N 12.84
(12.77).
2-(3-(1H-benzo[d]imidazol-2-yl)phenylamino)-1-(4-chlorophenyl)ethanone (3)
Off-white solid, 58 % yield, m.p. 225-226 °C. 1H-NMR (500 MHz, DMSO-d6): 4.82, (2H, s, CH2), 5.61 (1H, s, NH),
7.06 (1H, d, J = 7.7 Hz, Ar-H), 7.38 (3H, dd, J = 6.3, 3.9 Hz, Ar-H), 7.52 (2H, dd, J = 6.0, 3.2 Hz, Ar-H), 7.68 (2H, d, J
= 8.5 Hz, Ar-H), 7.79 (2H, dd, J = 6.0, 3.2 Hz, Ar-H), 8.10 (2H, d, J = 8.5 Hz, Ar-H), 11.27 (1H, s, benzimidazole-NH).
13C-NMR (125 MHz, DMSO-d6):49.5, 110.0, 114.1, 115.4, 117.6, 118.3, 125.5, 129.0, 129.9, 130.8, 133.7, 138.6,
139.2, 149.1, 150.1, 195.3. MS (m/z, %): 361 (34.1, M+), 362 (2.5, M++1). Elemental analysis: Calcd. (Found) (%) for
C21H16ClN3O: C 69.71 (69.68), H 4.46 (4.48), N 11.61 (11.57).
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2-(3-(1H-benzo[d]imidazol-2-yl)phenylamino)-1-(4-bromophenyl)ethanone (4)
Dark yellow solid, 61 % yield, m.p. 255-256 °C. 1H-NMR (500 MHz, DMSO-d6): 4.81 (2H, s, CH2), 5.61 (1H, s, NH),
7.06 (1H, d, J = 7.8 Hz, Ar-H), 7.36– 7.40 (3H, m, Ar-H), 7.52 (2H, dd, J = 6.0, 3.1 Hz, Ar-H), 7.79 (2H, dd, J = 6.0,
3.1 Hz, Ar-H), 7.82 (2H, d, J = 8.5 Hz, Ar-H), 8.02 (2H, d, J = 8.5 Hz, Ar-H). 13C-NMR (125 MHz, DMSO-d6):50.1,
110.6, 114.6, 115.9, 117.3, 118.2, 126.0, 127.2, 128.3, 130.5, 130.6, 132.5, 134.6, 149.5, 150.6, 196.0. MS (m/z, %):
405 (15.7, M+), 406 (2.8, M++1). Elemental analysis: Calcd. (Found) (%) for C21H16BrN3O: C 62.08 (62.12), H 3.97
(3.90), N 10.34 (10.37).
General procedure for preparing benzimidazoles 5-9:
To a mixture of compound 1 (0.21 g, 1 mmol) and K2CO3 (0.14 g, 1 mmol) in acetone, the appropriate substituted
benzylamine (1 mmol) was added. The reaction mixture was refluxed for 12h. The solvent was removed under vacuum
and the crude solid was washed with water, filtered and crystallized from methanol.
3-(1H-benzo[d]imidazol-2-yl)-N-benzylaniline (5)
Light brown solid, 73 % yield, m.p. 140-141 °C. 1H-NMR (500 MHz, CDCl3): 4.16 (1H, s, CH2), 4.54 (1H, s, CH2),
6.49 (1H, d, J = 7.8 Hz, Ar-H), 6.98 (1H, t, J = 7.8 Hz, Ar-H), 7.03 (1H, d, J = 7.1 Hz, Ar-H), 7.07 – 7.22 (7H, m, Ar-
H), 7.39 (1H, d, J = 7.5 Hz, Ar-H), 7.48 (1H, s, Ar-H), 7.59 (1H, dd, J = 5.7, 3.2 Hz, Ar-H). 13C-NMR (125 MHz,
CDCl3): 46.7, 110.1, 113.0, 114.7, 115.0, 117.3, 118.7, 126.3, 128.7, 129.4, 130.2, 133.6, 137.4, 139.2, 148.9, 150.0.
MS (m/z, %): 299 (33.6, M+), 300 (5.4, M++1). Elemental analysis: Calcd. (Found) (%) for C20H17N3: C 80.24 (80.26),
H 5.72 (5.77), N 14.04 (14.08).
3-(1H-benzo[d]imidazol-2-yl)-N-(4-chlorobenzyl)aniline (6)
Reddish brown solid, 65 % yield, m.p. 218-219 °C. 1H-NMR (500 MHz, DMSO-d6): 4.42 (1H, s, CH2), 4.83 (1H, s,
CH2), 6.93 (1H, dd, J = 8.2, 1.6 Hz, Ar-H), 7.34 (2H, dd, J = 15.4, 8.2 Hz, Ar-H), 7.37 (1H, d, J = 2.1 Hz, Ar-H), 7.39
(1H, t, J = 7.8 Hz, Ar-H), 7.44 – 7.48 (3H, m, Ar-H), 7.51 – 7.54 (2H, m, Ar-H), 7.81 (2H, dd, J = 6.1, 3.2 Hz, Ar-H).
13C-NMR (125 MHz, DMSO-d6):47.2, 110.0, 113.4, 114.5, 115.6, 117.3, 118.2, 126.1, 127.9, 130.4, 132.4, 133.1,
137.7, 139.5, 148.3, 149.9. MS (m/z, %): 333 (30.1, M+), 334 (2.3, M++1). Elemental analysis: Calcd. (Found) (%) for
C20H16ClN3: C 71.96 (71.93), H 4.83 (4.79), N 12.59 (12.57).
3-(1H-benzo[d]imidazol-2-yl)-N-(4-methylbenzyl)aniline (7)
Off-white solid, 55 % yield, m.p. 145-146 °C. 1H-NMR (500 MHz, DMSO-d6): 2.25 (3H, s, CH3), 4.36 (1H, s, CH2),
4.75 (1H, s, CH2), 6.93 (1H, d, J = 8.0 Hz, Ar-H), 7.12 (2H, d, J = 6.0 Hz, Ar-H), 7.18 (1H, d, J = 8.0 Hz, Ar-H), 7.31
(1H, d, J = 8.0 Hz, Ar-H), 7.36 (1H, dt, J = 15.4, 8.2 Hz, Ar-H), 7.44 (1H, d, J = 7.7 Hz, Ar-H), 7.53 (3H, dt, J = 15.4,
8.2 Hz, Ar-H), 7.77 – 7.84 (2H, m, Ar-H). 13C-NMR (125 MHz, DMSO-d6):21.2, 47.8, 110.3, 113.1, 114.0, 115.2,
117.7, 117.9, 126.1, 127.7, 130.1, 133.3, 136.5, 137.6, 140.0, 148.1, 150.1. MS (m/z, %): 313 (17.6, M+), 314 (3.1,
M++1). Elemental analysis: Calcd. (Found) (%) for C21H19N3: C 80.48 (80.50), H 6.11 (6.15), N 13.41 (13.38).
3-(1H-benzo[d]imidazol-2-yl)-N-(4-nitrobenzyl)aniline (8)
Dark yellow solid, 60 % yield, m.p. 150-151 °C. 1H-NMR (500 MHz, DMSO-d6): 4.55 (2H, s, CH2), 6.92 (1H, d, J =
8.6 Hz, Ar-H), 7.34 (2H, d, J = 7.9 Hz, Ar-H), 7.38 (1H, t, J = 7.9 Hz, Ar-H), 7.53 (2H, dd, J = 6.1, 3.2 Hz, Ar-H), 7.65
(2H, d, J = 8.6 Hz, Ar-H), 7.78 (2H, dd, J = 6.1, 3.2 Hz, Ar-H), 8.19 – 8.22 (2H, m, Ar-H). 13C-NMR (125 MHz,
DMSO-d6): 48.0, 110.2, 113.2, 114.1, 115.3, 117.6, 118.4, 123.2, 126.0, 127.4, 133.1, 137.6, 146.7, 147.3, 148.0, 149.9.
MS (m/z, %): 344 (24.7, M+), 345 (6.5, M++1). Elemental analysis: Calcd. (Found) (%) for C20H16N4O2: C 69.76
(69.74), H 4.68 (4.74), N 16.27 (16.25).
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3-(1H-benzo[d]imidazol-2-yl)-N-(2,6-dichlorobenzyl)aniline (9)
Light brown solid, 73 % yield, m.p. 260-261 °C. 1H-NMR (500 MHz, DMSO-d6): 4.51 (2H, s, CH2), 7.03 (1H, dd, J =
8.3, 1.6 Hz, Ar-H), 7.37 – 7.43 (2H, m, Ar-H), 7.52 - 7.54 (3H, m, Ar-H), 7.55 (2H, d, J = 3.1 Hz, Ar-H), 7.64 (1H, s,
Ar-H), 7.83 (2H, dd, J = 6.1, 3.1 Hz, Ar-H). 13C-NMR (125 MHz, DMSO-d6): 43.1, 110.6, 113.4, 114.3, 116.1, 117.0,
118.8, 126.9, 127.7, 128.1, 133.1, 133.5, 137.6, 143.4, 148.0, 149.9. MS (m/z, %): 367 (52.1, M+), 368 (13.4, M++1).
Elemental analysis: Calcd. (Found) (%) for C20H15Cl2N3: C 65.23 (65.19), H 4.11 (4.14), N 11.41 (11.39).
General procedure for preparing benzimidazoles 10-15:
To a solution of compound 1 (0.21 g, 1 mmol) in THF, the appropriate isocyanate (isothiocyanate) (1 mmol) was added.
The reaction mixture was stirred at room temperature for 10 h, the produced solid was filtered, washed with water and
crystallized from THF.
1-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-3-benzylurea (10)
Light brown solid, 77 % yield, m.p. > 300 °C. 1H-NMR (500 MHz, DMSO-d6): 4.33 (2H, d, J = 5.9 Hz, CH2), 6.71 (1H,
t, J = 5.9 Hz, Ar-H), 7.18 (2H, dd, J = 5.9, 3.0 Hz, Ar-H), 7.24 (1H, m, Ar-H), 7.31 – 7.36 (4H, m, Ar-H), 7.39 (1H, t, J
= 7.9 Hz, Ar-H), 7.47 (1H, dd, J = 8.0, 1.1 Hz, Ar-H), 7.54-7.57 (2H, m, Ar-H), 7.67 (1H, d, J = 7.9 Hz, Ar-H), 8.32
(1H, s, CONH), 8.76 (1H, s, CONH). 13C-NMR (125 MHz, DMSO-d6): 43.2, 111.3, 118.5, 123.4, 124.7, 126.8, 127.4,
128.3, 131.2, 137.3, 137.5, 139.5, 140.3, 141.7, 149.8, 155.0. MS (m/z, %): 342 (19.5, M+), 343 (3.6, M++1). Elemental
analysis: Calcd. (Found) (%) for C21H18N4O: C 73.67 (73.61), H 5.30 (5.25), N 16.36 (16.35).
1-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-3-p-tolylurea (11)
White solid, 72 % yield, m.p. > 300 °C. 1H-NMR (500 MHz, DMSO-d6): 2.24 (3H, s, CH3), 7.10 (2H, d, J = 8.4 Hz, Ar-
H), 7.20 (2H, dd, J = 5.9, 3.0 Hz, Ar-H), 7.37 (2H, d, J = 8.4 Hz, Ar-H), 7.44 (1H, t, J = 7.8 Hz, Ar-H), 7.48 (1H, d, J =
8.4 Hz, Ar-H), 7.54-7.58 (2H, m, Ar-H), 7.74 (1H, d, J = 7.8 Hz, Ar-H), 8.38 (1H, s, Ar-H), 8.63 (1H, s, CONH), 8.81
(1H, s, CONH). 13C-NMR (125 MHz, DMSO-d6): 21.2, 111.3, 118.5, 119.4, 123.7, 124.8, 130.0, 131.1, 132.2, 137.5,
137.8, 138.6, 140.3, 141.8, 149.6, 155.1. MS (m/z, %): 342 (45.7, M+), 343 (13.3, M++1). Elemental analysis: Calcd.
(Found) (%) for C21H18N4O: C 73.67 (73.66), H 5.30 (5.37), N 16.36 (16.38).
1-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-3-(4-nitrophenyl)urea (12)
Dark yellow solid, 69 % yield, m.p. 165-166 °C. 1H-NMR (500 MHz, DMSO-d6): 7.17 (2H, dd, J = 5.7, 2.7 Hz, Ar-H),
7.47 (2H, dt, J = 15.4, 4.8 Hz, Ar-H), 7.54-7.57 (2H, m, Ar-H), 7.71 (2H, d, J = 9.2 Hz, Ar-H), 7.77 (1H, d, J = 7.4 Hz,
Ar-H), 8.18 (2H, d, J = 9.2 Hz, Ar-H), 8.39 (1H, s, Ar-H), 9.10 (1H, s, CONH), 9.48 (1H, s, CONH). 13C-NMR (125
MHz, DMSO-d6):111.1, 118.3, 119.0, 123.5, 124.8, 125.3, 131.0, 137.7, 137.9, 140.4, 141.8, 142.7, 143.1, 150.1,
155.0. MS (m/z, %): 373 (36.7, M+), 374 (4.6, M++1). Elemental analysis: Calcd. (Found) (%) for C20H15N5O3: C 64.34
(64.36), H 4.05 (4.10), N 18.76 (18.79).
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1-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-3-(naphthalen-1-yl)urea (13)
Buff solid, 67 % yield, m.p. 255-256 °C. 1H-NMR (500 MHz, DMSO-d6): 7.20 (2H, d, J = 3.6 Hz, Ar-H), 7.45 – 7.50
(2H, m, Ar-H), 7.53 – 7.58 (3H, m, Ar-H), 7.60 – 7.68 (3H, m, Ar-H), 7.77 (1H, d, J = 7.9 Hz, Ar-H), 7.94 (1H, d, J =
7.9 Hz, Ar-H), 8.05 (1H, d, J = 7.3 Hz, Ar-H), 8.15 (1H, d, J = 8.4 Hz, Ar-H), 8.45 (1H, s, Ar-H), 8.84 (1H, s, CONH),
9.26 (1H, s, CONH). 13C-NMR (125 MHz, DMSO-d6):111.3, 115.2, 116.4, 118.5, 123.1, 123.6, 124.3, 124.7, 124.9,
125.3, 126.0, 129.4, 131.3, 135.1, 136.9, 137.4, 138.0, 140.2, 141.8, 150.0, 154.9. MS (m/z, %): 378 (61.1, M+), 379
(10.9, M++1). Elemental analysis: Calcd. (Found) (%) for C24H18N4O: C 76.17 (76.20), H 4.79 (4.74), N 14.81 (14.87).
1-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-3-phenylthiourea (14)
Light brown solid, 51 % yield, m.p. 135-136 °C. 1H-NMR (500 MHz, DMSO-d6): 7.14 (1H, dd, J = 7.3, 2.7 Hz, Ar-H),
7.18 - 7.22 (3H, m, Ar-H), 7.29 – 7.38 (3H, m, Ar-H), 7.45 – 7.52 (3H, m, Ar-H), 7.56 (1H, d, J = 7.9 Hz, Ar-H), 7.91
(1H, d, J = 7.9 Hz, Ar-H), 8.29 (1H, s, Ar-H), 9.93 (1H, s, CSNH), 9.95 (1H, s, CSNH). 13C-NMR (125 MHz, DMSO-
d6):111.2, 118.4, 121.5, 123.4, 123.7, 123.9, 124.2, 128.8, 131.1, 137.7, 139.3, 139.8, 140.7, 150.1, 180.2. MS (m/z, %):
344 (37.3, M+), 345 (4.7, M++1). Elemental analysis: Calcd. (Found) (%) for C20H16N4S: C 69.74 (69.77), H 4.68
(4.70), N 16.27 (16.23).
1-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-3-(4-nitrophenyl)thiourea (15)
Yellow solid, 53 % yield, m.p. 133-134 °C. 1H-NMR (500 MHz, DMSO-d6): 7.20 (2H, dd, J = 5.7, 3.0 Hz, Ar-H), 7.52
– 7.59 (4H, m, Ar-H), 7.86 (2H, d, J = 8.9 Hz, Ar-H), 7.96 (1H, d, J = 7.2 Hz, Ar-H), 8.22 (2H, d, J = 8.9 Hz, Ar-H),
8.32 (1H, s, Ar-H), 10.42 (1H, s, CSNH), 10.50 (1H, s, CSNH). 13C-NMR (125 MHz, DMSO-d6):111.3, 118.5, 121.0,
123.2, 123.7, 123.9, 125.1, 131.0, 137.6, 139.5, 140.9, 143.3, 143.8, 150.0, 180.1. MS (m/z, %): 389 (17.6, M+), 390
(5.2, M++1). Elemental analysis: Calcd. (Found) (%) for C20H15N5O2S: C 61.68 (61.71), H 3.88 (3.86), N 17.98 (17.91).
3.2. Biology
3.2.1. Preliminary in vitro cytotoxic screening
MTT assay [39]
Cell lines and reagents: The mammary gland breast cancer cell lines (MCF-7) selected for the antitumor screening were
obtained from ATCC via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. The non
tumorigenic breast epithelial cell line (MCF-10F), the skin fibroblast cell line (BJ) and the human lung fibroblast cell
line (MRC-5) were obtained from The American Type Culture Collection. Doxorubicin, RPMI-1640 medium, MTT and
DMSO (sigma co., St. Louis, USA), Fetal Bovine serum (GIBCO, UK).
This colorimetric assay is based on conversion of the yellow tetrazolium bromide (MTT) to a purple formazan
derivative in viable cells. Cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum. Penicillin (100
units/ml) and streptomycin (100µg/ml) were added at 37°C in a 5% CO2 incubator. The cell lines were seeded in a 96-
well plate at a density of 1.0 x104 cells/well at 37°C. After incubation for 48 h, cells were treated with different
concentration of compounds; incubated for 24 h; then 20 µl of MTT solution (5mg/ml) was added and incubated for
further 4 h. DMSO (100 µL) was added into each well to dissolve the purple formazan formed. The absorbance was
recorded at 570 nm using a plate reader (EXL 800, USA). The % relative cell viability was calculated as (A570 of
treated samples/A570 of untreated sample) X 100.
3.2.2. VEGFR-2 enzyme assay
VEGFR-2 inhibition assay was performed for compounds 2, 6, 8, 9, 12 and 15 using the RayBio Human VEGFR2
ELISA (Enzyme-Linked Immunosorbent Assay) kit protocol (User Manual Cat#: ELH-VEGFR2-001). Manufacturer’s
protocol was applied using Sorafenib as a reference drug to measure the residual concentration of VEGFR-2 secreted in
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the conditioned medium of MCF7 cancer cell line. VEGFR-2 residual concentration (pg/mL) was calculated based on
the standard curve, and IC50 was determined for each compound.
3.2.3. Docking study
The synthesized compounds were built and energy minimized using MOE[40] software. PDB entry 2OH4 representing
the VEGFR-2 crystal structure was obtained from the protein data bank (PDB). The docking simulation process was
performed after protein preparation, where the protein was kept rigid, 100 ligand conformations were generated and the
docking energy was scored using Triangle matcher method and affinity dG refinement algorithm (100 run for each
pose).
4. CONCLUSION
In this work, three series of 2-phenyl benzimidazole-based compounds were designed and synthesized through readily
affordable synthetic pathways. Structural optimization of 2-aryl benzimidazole scaffold led us to discover new
derivatives with potent cytotoxic activities against MCF-7 cell lines. Compound 8 was even more potent than the
standard drug doxorubicin with IC50 value of 3.37 µM. Compounds 15 and 9 showed good cytotoxic activities with IC50
values of 5.84 and 6.30 µM, respectively. The rest of compounds exhibited moderate to low activity. VEGFR-2 enzyme
assay revealed that compounds 8, 9, 12 and 15 showed IC50 values comparable to that of the reference drug sorafenib,
ranging from 6.7 to 8.9 nM. Compounds 2 and 6 gave IC50 values of 8.9 and 11.1 nM. Docking scores of the most
active compounds with VEGFR-2 binding site were consistent with the in vitro screening results. The varied potencies
displayed by the three series against VEGFR-2 could be attributed to how far each compound can bind to the active site
of VEGFR-2. Type of substituent on the second aryl moiety seems to have a deep impact on the binding mode, where
electronic effect contributes to the donor-acceptor interaction with the conserved amino acids. Both nitro and chloro
groups act as electron acceptor, supporting the binding of compounds containing them. It is well noticed that active
compounds 8, 12 and 15 have 4-nitroaryl moiety in their structures, while compounds 9 and 6 have either 4-chloro or
2,5-dichloroaryl moieties, emphasizing that the presence of such electron withdrawing groups contributes much to the
enzyme inhibiting activity. In conclusion, six of the target compounds proved to have antitumor activities against MCF-
7 cell lines, with comparable cytotoxicity to that of Dox towards human normal cells, including MCF-10F, BJ and
MRC-5 cell lines, besides having VEGFR-2 inhibitory activities.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
Amany S. Mostafa https://orcid.org/0000-0003-1909-1072
List of figure legends
FIGURE 1. 2-Aryl benzimidazoles with breast cancer and VEGFR-2 inhibiting activity
FIGURE 2. The newly designed 2-phenyl benzimidazoles
SCHEME 1. Synthesis of compounds 2-4. Reagents and conditions: (a) PPA, 90-95°C, 4h; (b) the appropriate 4-
(un)substituted phenacyl bromide, MeOH, Reflux, 2 h
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SCHEME 2. Synthesis of compounds 5-9. Reagents and conditions: (a) Acetone, K2CO3, reflux, 12 h
SCHEME 3. Synthesis of compounds 10-15. Reagents and conditions: (a) THF, stirring, 10 h
FIGURE 3a,b. Relative % viability of MCF-7 cells for compounds 2-15
FIGURE 4a,b. 2D binding of compounds 8 (4a) and 9 (4b) with VEGFR-2 (pdb code 2OH4)
FIGURE 5. Compound 8 (blue) aligned with the co-crystallized ligand (orange) inside VEGFR-2
FIGURE 6. Compound 9 (blue) aligned with the co-crystallized ligand (orange) inside VEGFR-2
FIGURE 7. Docking of the reference drug Sorafenib with VEGFR-2
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TABLE 1. % Growth Inhibition and IC50 of the target compounds against MCF-7 cell lines
% Growth
Comp.
IC50 (µM)
Inhibition
82.8
71.9
55.9
65.8
89.6
52.7
94.1
93.9
61.1
54.8
88.3
54.1
51.3
85.3
93.8
2
9.74 0.2
33.55 2.5
38.40 1.4
44.89 0.6
12.56 1.8
35.60 3.7
3.37 2.1
6.30 2.7
44.91 1.1
49.14 1.9
7.18 3.5
39.50 3.2
14.51 2.4
5.84 4.3
4.17 3.0
3
4
5
6
7
8
9
10
11
12
13
14
15
Dox.
Data are presented as the mean SDs of three independent experiments
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TABLE 2. Cytotoxic activity (IC50) of compounds 2, 6, 8, 9, 12 and 15 against human normal cell lines
In vitro Cytotoxicity IC50 (µM)
Comp.
MCF-10F BJ
MRC-5
43.9 3.0
34.8 1.5
17.3 0.4
59.8 3.3
15.9 1.2
72.3 1.8
15.2 1.1
2
25.8 1.3
29.4 2.7
33.1 1.8
65.7 2.4
23.3 1.7
47.9 0.5
22.6 2.7
52.5 0.4
6
20.0 1.9
40.6 2.5
79.3 0.8
19.8 2.4
32.5 3.1
17.2 0.7
8
9
12
15
Dox.
Data are presented as the mean SDs of
three independent experiments
TABLE 3. VEGFR-2 inhibitory activity of compounds 2, 6, 8, 9, 12 and 15
Comp.
2
6
8
9
12
15
Sorafinib
VEGFR-2 (Cell line: MCF7)
IC50 (nM)
11.1 0.7 9.7 2.2 6.7 1.3 6.9 0.5 6.8 1.7 8.9 1.4 7.6 2.8
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.