uses of β-diketones for the synthesis of novel heterocyclic compounds and their antitumor evaluations

Article abstract of DOI:10.4314/bcse.v34i2.15

The reaction of the 3-oxo-N,3-diphenylpropan-amide (3) with either malononitrile or ethyl cyanoacetate in ammonium acetate gave the 1,2-dihydropyridine derivatives 6a or 6b, respectively. On the other hand, carrying the same reaction in the presence of triethylamine gave the 1,6-dihydropyridine derivatives 7a and 7b, respectively. Moreover, compound 3 reacted with 2-aminoprop-1-ene-1,1,3-tricarbonitrile to give the pyridine derivative 9. Compound 7b reacted with the active methylene derivatives 10a,b and 4a,b to give the naphthyridine derivatives 11a,b and 12a,b; respectively. Compound 3 was also used for the synthesis of thiophene derivatives 13a,b and 16a,b. In addition, the reaction of ethyl benzoylacetate (1) with o-phenylene diamine gave the benzimidazole derivative 18. The reactivity of the latter product towards different reagents was studied to give different products. The cytotoxicity of the newly synthesized products was studied towards some cancer and normal cell lines, in addition toxicity of compounds was measured and docking of the most active compounds was done. Compounds 6b, 7b, 9, 13a, 13b, 16a, 20b, 20c, 24b, 25 and 26b exhibited optimal cytotoxic effect against cancer tested cell lines. These active compounds were evaluated against c-Met kinase using foretinib as the reference drug where all compounds expressed higher activity than the reference drug.

Full text of DOI:10.4314/bcse.v34i2.15

Bull. Chem. Soc. Ethiop. 2020, 34(2), 385-405.
 2020 Chemical Society of Ethiopia and The Authors
DOI: https://dx.doi.org/10.4314/bcse.v34i2.15
ISSN 1011-3924
Printed in Ethiopia
USES OF -DIKETONES FOR THE SYNTHESIS OF NOVEL HETEROCYCLIC
COMPOUNDS AND THEIR ANTITUMOR EVALUATIONS
Rafat M. Mohareb1*, Mona M. Kamel2 and Yara R. Milad2,3
1Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt
2Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University,
P.O. BOX 11562, Cairo, Egypt
3Jean Coutu, 531 Jarry Est, Montreal Quebec, Canada
(Received May 28, 2020; Revised October 10, 2020; Accepted October 20, 2020)
ABSTRACT. The reaction of the 3-oxo-N,3-diphenylpropan-amide (3) with either malononitrile or ethyl
cyanoacetate in ammonium acetate gave the 1,2-dihydropyridine derivatives 6a or 6b, respectively. On the other
hand, carrying the same reaction in the presence of triethylamine gave the 1,6-dihydropyridine derivatives 7a and
7b, respectively. Moreover, compound 3 reacted with 2-aminoprop-1-ene-1,1,3-tricarbonitrile to give the pyridine
derivative 9. Compound 7b reacted with the active methylene derivatives 10a,b and 4a,b to give the naphthyridine
derivatives 11a,b and 12a,b; respectively. Compound 3 was also used for the synthesis of thiophene derivatives
13a,b and 16a,b. In addition, the reaction of ethyl benzoylacetate (1) with o-phenylene diamine gave the
benzimidazole derivative 18. The reactivity of the latter product towards different reagents was studied to give
different products. The cytotoxicity of the newly synthesized products was studied towards some cancer and
normal cell lines, in addition toxicity of compounds was measured and docking of the most active compounds was
done. Compounds 6b, 7b, 9, 13a, 13b, 16a, 20b, 20c, 24b, 25 and 26b exhibited optimal cytotoxic effect against
cancer tested cell lines. These active compounds were evaluated against c-Met kinase using foretinib as the
reference drug where all compounds expressed higher activity than the reference drug.
KEY WORDS: Ethyl benzoylacetate, Pyridine, Benzimidazole, Cytotoxicity
INTRODUCTION
It is of great interest to note that pyridine and pyrimidine derivatives were one of the most
classes of compounds due to their high pharmaceutical and biological values. Especially fused
pyridine derivatives have varieties of biological uses. Among such activities, they are known to
exhibit high pharmacological, CNS depressant [1, 2], neuroleptic [3] and tuberculostatic [4]
activities. In addition, a largenumber of pyridines were used as antimicrobial agents [5],
inhibitors of glycogen syntheses kinase-3 (GSK-3) [6] and potent antitumor agents [7].
Moreover, benzimidazoles represent a group of compounds with a large number of
pharmaceutical applications and many of them were basic nucleus of drugs structures. Some of
them were naturally occurring nucleotides that capable to interact with biopolymers to enhance
better biological properties. It was noticed that some 2-aminobenzimidazoles showed interesting
antimicrobial effect, especially, their corresponding carbamate derivatives were obtained in
good yields and exhibited significant in vivo antifilarial activity [8]. Within the field of drug
designing such group of compounds have a great affinity towards different enzymes and protein
receptors [9]. Optimization of benzimidazole-based structures has resulted in marketed drugs,
e.g. within the field of chemotherapy it was found that omeprazole [10] and pimobendan [11]
they were found not only as good therapeutically active drugs but also for treatment of heart
feeler problems. Moreover, a large number of benzimidazoles derivatives are well known for
their antimicrobial [12-15], anthelmintic [16], antiviral [17-18] and antifungal [19-21] activities.
Many benzimidazole containing compounds are known as anticancer agents [22-28]. Within the
field of topoisomerase inhibitors it was found that some benzimidazole was used as
__________
*Corresponding author. E-mail: raafat_mohareb@yahoo.com
This work is licensed under the Creative Commons Attribution 4.0 International License

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topoisomerase inhibitory drugs, e.g. Hoechst 33258 and Hoechst 33342 (Figure 1) [29, 30].
Within the field of DNA binders, it was also found that bis-benzimidazole derivatiives are used
as head to head binders [31]. Within the field of cancer chemotherapy, many drugs containing
benzimidazole nucleus are known through the market like RAF265 (CHIR-265; Novartis
Pharmaceuticals, Basel, Switzerland) and AZD6244 (ARRY-142886; AstraZeneca, London,
England). A very active known drug is RAF265 resulted in a reduction in tumor cell growth and
in tumor cell apoptosis [32].
Figure 1. Examples of topoisomerase inhibitors containing benzimidazole nucleus.
Due to such high importance of pyridine and benzimidazole derivatives was focused on the
efficient synthesis of new pyridine and benzimidazole derivatives starting from ethyl
benzoylacetate followed by their cytotoxic evaluations against human cancer and normal cell
lines.
EXPERIMENTAL
1
13C NMR and H NMR spectra were recorded on Bruker DPX200 instrument in CDCl3 and
DMSO with TMS as internal standard for protons and solvent signals as internal standard for
carbon spectra. Chemical shift values are mentioned in δ (ppm). Mass spectra were recorded on
EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument. Elemental analyses were
carried out by the Microanalytical Data Unit at Ludwig-Maximilians-Universität-München,
Germany. The progress of all reactions was monitored by TLC on 2 x 5 cm pre-coated silica gel
60 F254 plates of thickness of 0.25 mm (Merck).
General procedure for the synthesis of the 1,2-dihydropyridine derivatives 6a,b
Either of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) together with
ammonium acetate (0.50 g) were added to a dry solid of compound 3 (2.16 g, 0.01 mol). The
reaction mixture, in each case, was heated in an oil bath at 120 oC for 15 min. The solid product
formed after boiling with ethanol was collected by filtration.
6-Hydroxy-2-imino-1,4-diphenyl-1,2-dihydropyridine-3-carbonitrile (6a). Yellow crystals
o
(EtOH), yield 74% (2.12 g), mp 119-121 C. IR max cm-1: 3544-3260, 3056, 2957, 2207, 1688,
1
1662, 1604 (C=C). H NMR (DMSO-d6 400 MHz):  = 4.15 (s, 2H, CH2), 7.30-7.99 (m, 10H,
2C6H5), 10.21 (s, 1H, NH). 13C NMR (DMSO-d6 75 MHz):  = 49.5 (pyridine C-3), 103.6,
104.5 (C=C), 119.5 (CN), 120.19, 121.3, 123.9, 124.1, 124.8, 125.2, 125.5, 126.0, 134.5, 136.8
(two benzene, pyridine C), 165.9 (C=O), 175.2 (C=N). EIMS m/z 287 [M]+ (20); anal. calcd. for
C18H13N3O (287.11): C, 75.25; H, 4.56; N, 14.63. Found: C, 72.06; H, 4.72; N, 14.88.
Ethyl 6-hydroxy-2-imino-1,4-diphenyl-1,2-dihydropyridine-3-carboxylate (6b). Yellow crystals
o
(EtOH), yield 70% (2.33 g), mp 277-279 C. IR max cm-1: 3443-3259, 3092, 2959, 2888, 1695,
1689, 1636. 1H NMR (DMSO-d6 400 MHz):  = 1.14 (t, 3H, J = 7.03 Hz, OCH2CH3), 4,18 (q,
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2H, J = 7.03 Hz, OCH2CH3), 5.95 (s, 2H, CH2), 7.30-7.99 (m, 10H, 2C6H5), 10.21 (s, 1H, D2O
exchangeable, NH). 13C NMR (DMSO-d6 75 MHz) : 13.6 (OCH2CH3), 50.3 (OCH2CH3), 50.6
(pyridine C-3), 103.8, 104.9 (C=C), 116.5 (CN), 119.0, 120.5, 122.5, 123.6, 124.4, 124.6, 125.1,
125.7 (two benzene C), 163.8 (C=N), 165.1, 166.4 (2C=O), EIMS m/z 334 [M]+ (28). Anal.
calcd. for C20H18N2O3 (334.38): C, 71.84; H, 5.43; N, 8.38 %. Found: C, 71.69; H, 5.51; N, 8.49.
General procedure for the synthesis of the 1,6-dihydopyridine-2-carboxylate 7a,b
Either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added to a
solution of compound 3 (2.16 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine
(0.50 mL). The reaction mixture, in each case was heated under reflux for 3 h then evaporated
under vacuum. The solid product produced after triturating the remaining product with diethyl
ether was collected by filtration.
4-Amino-5-benzoyl-6-oxo-1-phenyl-1,6-dihydropyridine-2-carbonitrile (7a). Pale yellow
o
crystals (1,4-dioxane), yield 80% (2.12 g), mp 160-168 C. IR max cm-1: 3544-3260, 3058,
2957, 1705, 1689, 1632. 1H NMR (DMSO-d6 400 MHz):  = 4.28, 5.31 (2s, 4H, D2O
exchangeable, 2NH2), 6.15 (s, 1H, pyridine H-4), 7.31-7.68 (m, 10H, 2C6H5). 13C NMR
(DMSO-d6 75 MHz):  = 119.5, 120.1, 120.7, 122.0, 123.6, 124.7, 125.6, 127.3, 129.8, 130.2,
131.6, 139.4 (two benzene, pyridine C), 164.3, 166.4 (2 C=O). EIMS m/z 305 [M]+ (26). Anal.
calcd. for C18H15N3O2 (305.54): C, 70.81; H, 4.95; N, 13.76.
Ethyl 4-amino-5-benzoyl-6-oxo-1-phenyl-1,6-dihydropyridine-2-carboxylate (7b). Yellow
o
crystals (1,4-dioxane), yield 81% (2.47 g), mp 177-179 C. IR max cm-1: 3540-3320, 3065,
2955, 2889, 1718, 1688, 1633. 1H NMR (DMSO-d6 400 MHz): 4.25 (s, 2H, D2O
exchangeable, NH2), 6.11 (s, 1H, pyridine H-4), 7.28-7.67 (m, 10H, 2C6H5), 11.42 (s, 1H, D2O
exchangeable, OH). 13C NMR (DMSO-d6 75 MHz):  = 119.6, 120.2, 120.9, 122.5, 123.8,
124.3, 124.6, 125.7, 129.8, 130.2, 133.2, 139.4 (two benzene, pyridine C), 164.2, 166.8 (2
C=O). EIMS m/z 306.32 [M]+ (28). Anal. calcd. for C18H14N2O3 (306.32): C, 70.58; H, 4.61; N,
9.15. Found: C, 70.80; H, 4.88; N, 9.05.
2,4-Diamino-7-oxo-5,8-diphenyl-7,8-dihydro-1,8-naphthyridine-3-carbonitrile (9)
2-Aminoprop-1-ene-1,1,3-tricarbonitrile (8) (1.32 g, 0.01 mol) was added to a solution of
compound 3 (2.16 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL). The
reaction mixture was heated under reflux for 6 h then evaporated under vacuum. The solid
product produced after triturating the remaining product with diethyl ether was collected by
o
filtration. Pale yellow crystals (1,4-dioxane), yield 86% (3.03 g), mp 260-263 C. IR max cm-1:
1
3473, 3327, 3056, 2220, 1688, 1623. H NMR (DMSO-d6, 400 MHz)  = 4.92, 5.68 (2s, 4H,
D2O exchangeable, 2NH2), 6.80 (s, 1H pyridine H-6), 7.31-7.58 (m, 10H, 2C6H5). 13C NMR
(DMSO-d6, 75 MHz):  = 116.8 (CN), 120.1, 121.3, 122.3, 123.5, 124.0, 124.8, 125.1, 126.4,
128.1, 130.2, 131.2, 131.8, 133.2, 134.2 (two C6H5, two pyridine C), 162.4 (C=O), 169.5 (C=N).
EIMS m/z 353 [M]+ (22). Anal. calcd. for C21H15N3O (353.39): C, 71.38; H, 4.28; N, 19.82.
Found: C, 71.42; H, 4.31; N, 19.72.
General procedure for the synthesis of the 1,6-naphthridine derivatives 11a,b
Either acetylacetone (1.00 g, 0.01 mol) or ethyl acetoacetate (1.30 g, 0.01 mol) was added to a
solution of compound 7b (3.06 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine
(0.50 mL). The reaction mixture was heated under reflux for 4 h then poured onto ice/water and
the formed solid product was collected by filtration.
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8-Benzoyl-2,4-dimethyl-6-phenyl-1,6-naphthridine-5,7(1H,6H)-dione (11a). Orange crystals
o
(1,4-dioxane) yield 76% (2.81 g), mp 116-118 C. IR max cm-1: 3430-3261, 3065, 2919, 1688-
1662. 1H NMR (DMSO-d6 400 MHz)  = 2.51, 4.1 (2s, 6H, 2CH3), 5.95 (s, 1H, pyridine H-3),
7.25-7.54 (m, 10H, 2C6H5), 10.16 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO-d6 75
MHz) : 39.6, 49.8 (2 CH3), 119.9, 120.0, 123.9, 124.2, 124.8, 125.8, 126.0, 126.3, 129.3.2,
129.4, 130.6, 132.6, 134.1, 134.2 (two benzene C, two pyridine C), 165.8, 170.2, 175.2 (3
C=O). EIMS m/z 370 [M]+ (20). Anal. calcd. for C23H18N2O3 (370.41): C, 74.58; H, 4.90; N,
7.56. Found: C, 74.66; H, 4.69; N, 7.38.
8-Benzoyl-4-hydroxy-2-methyl-6-phenyl-1,6-naphthridine-5,7(1H,6H)-dione (11b). Yellow
o
crystals (1,4-dioxane), yield 80% (2.97 g), mp 97-99 C. IR max cm-1: 3550-3320, 3055, 2955,
1
2889, 1720-1688, 1633. H NMR (DMSO-d6 400 MHz)  = 2.51 (s, 3H, CH3), 5.45 (s, 1H,
pyridine H-3), 7.01-7.31 (m, 10H, 2C6H5), 8.20 (s, 1H, D2O exchangeable, NH), 9.01 (s, 1H,
D2O exchangeable, OH). 13C NMR (DMSO-d6 75 MHz) : 25.1, (CH3), 118.9, 119.8, 120.3,
123.6, 123.9, 124.2, 125.3, 125.6, 128.6, 129.4, 132.1, 133.6, 134.2, 134.6 (two benzene, two
pyridine C), 165.6, 172.0, 180.3 (3 C=O). EIMS m/z 372 [M]+ (18). Anal. calcd. for C22H16N2O4
(372.38): C, 70.96; H, 4.33; N, 7.52. Found: C, 70.82; H, 4.48; N, 7.63.
General procedure for the synthesis of the 5,6,7,8-tetrahydro-1,6-naphthyridine derivatives
12a,b
Either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added to a
solution of compound 7b (3.06 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine
(0.50 mL). The reaction mixture was heated under reflux for 8 h then poured onto ice/water and
the formed solid product was collected by filtration.
2-Amino-5,7-dioxo-4,6-diphenyl-5,6,7,8-tetrahydro-1,6-naphthyridine-3-carbonitrile
(12a).
o
Yellow crystals (1,4-dioxane), yield 88% (3.11 g), mp 140-143 C. IR max cm-1: 3409-3334,
1
3055, 2923, 2210, 1680-1662. H NMR (DMSO-d6 400 MHz): = 3.58, 4.20 (2s, 4H, D2O
exchangeable, 2NH2), 6.20 (s, 1H, pyridine H-3), 7.20 (s, 1H, pyridine H-3), 7.27-7.54 (m, 10H,
2C6H5). 13C NMR (DMSO-d6 75 MHz) : 48.9 (pyridine C-3), 119.9, 120.3, 120.6, 123.9, 125.8,
128.6, 129.3.2, 129.4, 132.6, 134.1, 140.4 (two benzene C, two pyridine C), 165.8, 166.3, 170.1
(3 C=O), 195.3 (C=N). EIMS m/z 372[M]+ (20); anal. calcd. for C21H16N4O3 (372.38): C, 67.73;
H, 4.33; N, 15.05. Found: C, 67.58; H, 4.04; N, 15.25.
2-Hydroxy-5,7-dioxo-4,6-diphenyl-5,6,7,8-tetrahydro-1,6-naphthyridine-3-carbonitrile (12b).
o
Yellow crystals (1,4-dioxane), yield 70% (2.48 g), mp 100-103 C. IR max cm-1: 3542-3260,
3093, 2956, 1688-1662, 1605. 1H NMR (DMSO-d6 400 MHz)  = s, 2H, D2O
exchangeable, NH2), 4.14 (s, 1H, pyridine H-3), 7.27-7.67 (m, 11H, 2C6H5, pyridine H-3), 10.19
(s, 1H, D2O exchangeable, OH). 13C NMR (DMSO-d6 75 MHz)  = 49.5 (pyridine C-3), 119.8,
120.2, 122.5, 123.6, 124.2, 124.6, 125.3, 125.8, 130.2, 132.5, 133.2, 134.1, 134.6. 135.2 (two
benzene C, two pyridine C), 165.6, 170.1 (2C=O), 171.1 (C=N). EIMS m/z 373 [M]+ (20). Anal.
calcd. for C21H15N3O4 (373.37): C, 67.56; H, 4.05; N, 11.25. Found: C, 67.33; H, 3.87; N, 11.41.
General procedure for the synthesis of the thiophene derivatives 13a,b
Each of elemental sulfur (0.32 g, 0.01 mol) and either malononitrile (0.66 g, 0.01 mol) or ethyl
cyanoacetate (1.13 g, 0.01 mol) was added to a solution of compound 3 (2.16 g, 0.01 mol) in
1,4-dioxane (40 mL) containing triethylamine (0.50 mL). The reaction mixture, in each case,
was heated under reflux for 3 h then poured onto ice/water containing few drops of hydrochloric
acid and the formed solid product was collected by filtration.
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5-Amino-4-cyano-N,3-diphenylthiophene-2-carboxamide (13a). Orange crystals (1,4-dioxane),
o
1
yield 90% (2.87 g), mp 120-122 C. IR max cm-1:3439-33259, 3056, 2923, 2211, 1687. H
NMR (DMSO-d6 400 MHz):  = 4.15 (s, 2H, D2O exchangeable, NH2), 7.31-7.58 (m, 10H,
2C6H5), 10.21 (s, 1H, D2O exchangeable, NH), .13C NMR (DMSO-d6 75 MHz):  = 117.5 (CN),
120.3, 121.6, 122.8, 123.6, 124.4, 124.8, 125.1, 125.8, 136.7, 139.5. 140.4, 142.2 (two benzene
C, thiophene C), 165.8 (C=O). EIMS m/z 319 [M]+ (26). Anal. calcd. for C18H13N3OS (319.38):
C, 67.69; H, 4.10; N, 13.16; S, 10.04. Found: C, 67.56; H, 3.89; N, 12.88; S, 10.38.
Ethyl 2-amino-4-phenyl-5-(phenylcarbamoyl)thiophene-3-carboxylate (13b). Orange crystals
o
(1,4-dioxane), yield 73% (2.67 g), mp 90-93 C. IR max cm-1: 3426-3261, 3056, 2955, 2890,
1687-1661, 1604 (C=C). 1H NMR (DMSO-d6 400 MHz)  = tJHz,
OCH2CH3, 4.18 (q, 2H, J = 6.09 Hz, OCH2CH3),(s, 2H, D2O exchangeable, NH2), 7.31-
7.99 (m, 10H, 2C6H5), 10.01 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO-d6 75 MHz) :
16.5 (OCH2CH3), 50.2 (OCH2CH3), 119.4, 119.9, 121.9, 123.8, 124.6, 125.1, 125.5, 126.2,
132.3, 133.1, 134.6, 135.3 (two C6H5, thiophene C), 165.8, 167.1 (2C=O). EIMS m/z 366 [M]+
(28). Anal. calcd. for C20H18N2O3S (366.43): C, 65.55; H, 4.95; N, 7.64; S, 8.75. Found: C,
65.72; H, 4.73; N, 7.73; S, 8.93.
General procedure for the synthesis of the 4-(phenylcarbamoyl)thiophene derivatives 16a,b
To a solution of compound 2 (2.39 g, 0.01 mol) in dimethylformamide (30 mL) containing
potassium hydroxide (0.56 g, 0.01 mol) phenylisothiocyanate was added. The reaction mixture
was stirred at room temperature for 24 h then to the reaction mixture either ethyl chloroacetate
(1.22 g, 0.01 mol) or (0.92 g, 0.01 mol) was added. The reaction mixture was stirred at room
temperature for an additional 24 h then poured onto ice/water containing few drops of
hydrochloric acid (till pH 6) and the formed solid product was collected by filtration.
Ethyl 3-phenyl-5-(phenylamino)-4-(phenylcarbamoyl)thiophene-2-carboxylate (16a). Yellow
o
crystals (1,4-dioxane), yield 86% (3.80 g), mp 160-163 C. IR max cm-1: 3397-3229, 3048,
1
2986, 2890, 1688-1668, 1624. H NMR (DMSO-d6 400 MHz)  = tJHz,
OCH2CH33.87(q, 2H, J = 7.21 Hz, OCH2CH3)7.43-7.49 (m, 15H, 3C6H5), 8.20, 10.39 (2s,
2H, D2O exchangeable, 2NH). 13C NMR (DMSO-d6 75 MHz)  = 16.6 (OCH2CH3), 50.6
(OCH2CH3), 120.0, 121.8, 122.6, 123.8, 123.9, 124.2, 124.5, 125.2, 125.3, 125.8, 126.3, 126.9,
130.4, 133.1, 138.0, 139.2 (three C6H5 and thiophene C), 165.8, 167.1 (2C=O). EIMS m/z 366
[M]+ (28). Anal. calcd. for C26H22N2O3S (442.53): C, 70.57; H, 5.01; N, 6.33; S, 7.24. Found: C,
70.29; H, 4.87; N, 6.58; S, 7.42.
5-Methyl-N,4-diphenyl-2-(phenylamino)thiophene-3-carboxamide (16b). Orange crystals (1,4-
dioxane), yield 79% (3.25 g), mp 175-177 oC. IR max cm-1: 3453-3422, 3054, 2932, 1720-1668.
1H NMR (DMSO-d6 400 MHz):  = 3.56 (s, 3H, CH3), 7.24-7.56 (m, 15H, 3C6H5), 10.51, 11.30
(2s, 2H, D2O exchangeable, 2NH). 13C NMR (DMSO-d6 75 MHz) : 16.6 (CH3), 119.2, 120.5,
120.8, 121.2, 121.7, 122.5, 123.6, 123.8, 124.8, 125.3, 125.6, 126.2, 130.4, 132.5, 133.8, 134.1
(three benzene C, thiophene C), 167.8, 172.5 (2C=O). EIMS m/z 412 [M]+ (20). Anal. calcd. for
C25H20N2O2S (412.51): C, 72.79; H, 4.89; N, 6.79; S, 7.77. Found: C, 72.95; H, 4.63; N, 6.56; S,
8.04.
2-(1H-Benzo[d]imidazol-2-yl)-1-phenylethanone (18)
Equimolar amounts of ethyl benzoylacetate (1.92 g, 0.01 mol) and o-phenylenediamine (1.09 g,
0.01 mol) were heated at 120 oC in the dry conditions in an oil bath for 10 min then the reaction
mixture was left to cool. The formed solid product was triturated with diethylether then
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Rafat M. Mohareb et al.
collected by filtration. Pale yellow crystals (1,4-dioxane), yield 72% (1.69 g), mp 180-183 oC.
IR max cm-1: 3437-3302, 3053, 2974, 1680, 1615. 1H NMR (DMSO-d6 400 MHz):  = 3.50 (s,
2H, CH2), 7.20-7.55 (m, 9H, C6H5, C6H4), 10.45 (s, 1H, D2O exchangeable, NH). 13C NMR
(DMSO-d6 75 MHz): = 49.8 (CH2), 118.2, 123.8, 124.9, 125.0, 125.3, 126.0, 126.4, 126.8,
128.3, 129.6 (benzene, indole C), 164.8 (C=O), 167.2 (C=N). EIMS m/z 236 [M]+ (90). Anal.
calcd. for C15H12N2O (236.27): C, 76.25; H, 5.12; N, 11.86. Found: C, 76.39; H, 4.82; N, 11.66.
General procedure for the synthesis of the benzo[d]imidazol derivatives 20a-c
Either benzaldehyde (1.06 g, 0.01 mol) 4-methoxybanzaldehyde (1.36 g, 0.01 mol) or 4-
chlorobenzaldehyde (1.40 g, 0.01 mol) was added to a solution of compound 18 (2.36 g, 0.01
mol) in 1,4-dioxane (40 mL) containing piperidine (0.50 mL). The reaction mixture, in each
case, was heated under reflux for 3 h then poured onto ice/water containing few drops of
hydrochloric acid. The formed oily product was triturated with ethanol and the precipitated
product was collected by filtration.
2-(1H-Benzo[d]imidazol-2-yl)-1,3-diphenylprop-2-en-1-one (20a). Pale yellow crystals (1,4-
o
1
dioxane), yield 68% (2.20 g), mp 183-187 C. IR max cm-1: 3537-3425, 3053, 1680, 1615. H
NMR (DMSO-d6 400 MHz):  = 6.06 (s, 1H, CH), 7.26-7.55 (m, 14H, 2C6H5, C6H4), 10.54 (s,
1H, D2O exchangeable NH). 13C NMR (DMSO-d675 MHz) : 85.1, 112.3 (C=C), 120.3, 122.2,
122.6, 123.1, 123.5, 124.0, 124.6, 125.0, 125.4, 125.7, 127.5, 127.3 (2C6H5, C6H4), 160.2 (CO),
173.4 (C=N). EIMS m/z 324[M]+ (48); anal. calcd. for C22H16N2O (324.38): C, 81.46; H, 4.97;
N, 8.64. Found: C, 81.66; H, 5.26; N, 8.47.
2-(1H-Benzo[d]imidazol-2-yl)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (20b). Orange
o
crystals (1,4-dioxane), yield 78% (2.76 g), mp 145-147 C. IR max cm-1: 3424-3244, 3058,
1710, 1666. 1H NMR (DMSO-d6 400 MHz)  = 3.50 (s, 3H, CH3), 6.08 (s, 1H, CH), 7.26-7.55
(m, 13H, C6H5, 2C6H4), 10.54 (s, 1H, D2O exchangeable NH). 13C NMR (DMSO-d6 75 MHz) :
38.7 (CH3), 89.2, 113.8 (C=C), 120.1, 122.4, 122.7, 123.0, 123.3, 124.1, 124.5, 125.2, 125.8,
125.3, 127.1, 127.2 (C6H5. 2C6H4), 161.2 (CO), 172.8 (C=N). EIMS m/z 354 [M]+ (25). Anal.
calcd. for C23H18N2O2 (354.41): C, 77.95; H, 5.12; N, 7.90. Found: C, 78.28; H, 5.08; N, 8.21.
2-(1H-Benzo[d]imidazol-2-yl)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (20c). Yellow
o
crystals (1,4-dioxane), yield 72% (2.57 g), mp 160-163 C. IR max cm-1: 3423-3294, 3055,
1
1720, 1660. H NMR (DMSO-d6 400 MHz)  = 6.08 (s, 1H, CH), 7.26-7.55 (m, 13H, C6H5,
2C6H4), 10.54 (s, 1H, D2O exchangeable NH). 13C NMR (DMSO-d6 75 MHz) = 87.3, 112.6
(C=C), 119.2, 122.4, 122.9, 123.0, 123.3, 124.2, 124.8, 125.3, 125.9, 126.1, 127.7, 128.6 (C6H5.
2C6H4), 161.4 (CO), 171.3 (C=N). EIMS m/z 358 [M]+ (38); anal. calcd. for C22H15ClN2O
(358.83): C, 73.64; H, 4.21; N, 7.81. Found: C, 73.75; H, 4.52; N, 7.95.
2-(3,5-Diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (21)
To a solution of compound 20a (3.24 g, 0.01 mol) in 1,4-dioxane (40 mL) hydrazine hydrate
(0.50 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured
onto ice/water containing few drops of hydrochloric acid and the formed solid product was
o
collected by filtration. Yellow crystals (EtOH), yield 68% (2.28 g), mp 221-223 C. IR max
1
cm-1: 3440-3302, 3054, 1662, 1608. H NMR (DMSO-d6 400 MHz)  = 7.28-7.74 (m, 14H,
2C6H5, C6H4), 10.12, 11.20 (2s, 2H, D2O exchangeable, 2NH), 13C NMR (DMSO-d6 75 MHz) :
118.3, 121.5, 121.9, 122.6, 123.8, 124.3, 124.5, 125.8, 126.0, 126.2, 127.6, 130.6, 139.2, 140.4
(2C6H5, C6H4, pyrazole C), 163.4, 170.0 (two C=N). EIMS m/z 336 [M]+ (66); anal. calcd. for
C22H16N4 (336.40): C, 78.55; H, 4.79; N, 16.66. Found: C, 78.39; H, 4.86; N, 16.41.
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1-Amino-4-benzoyl-3-(4-methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyridine-2-carbonitrile (22)
Malononitrile (0.66 g, 0.01 mol) was added to a solution of compound 20b (3.54 g, 0.01 mol) in
1,4-dioxane (40 mL) containing triethylamine (0.50 mL). The reaction mixture was heated
under reflux for 4 h then evaporated under vacuum. The remaining product was triturated with
ethanol and the formed solid product was collected by filtration. Yellow crystals (EtOH), yield
73% (3.05 g), mp 182-185 oC. IR max cm-1: 3438-3304, 3054, 2200, 1687, 1662, 1614. 1H NMR
(DMSO-d6 400 MHz)  = 3.50 (s, 3H, OCH3), 5.02 (s, 2H, D2O exchangeable, NH2), 7.21-7.55
(m, 13H, C6H5, 2C6H4), 13C NMR (DMSO-d6 75 MHz) : 46.8 (OCH3), 116.9 (CN), 120.3,
120.6, 121.5, 121.8, 122.3, 123.2, 123.7, 124.7, 124.8, 125.0, 125.5, 126.7, 130.4, 131.6, 137.7,
139.9 (C6H5, 2C6H4, pyridine C), 163.2 (C=O), 166.8 (C=N). EIMS m/z 418 [M]+ (48); anal.
calcd. for C26H18N4O2 (418.54): C, 74.63; H, 4.34; N, 13.39. Found: C, 74.49; H, 4.62; N, 13.61.
General procedure for the synthesis of the arylhydrazone derivatives 24a,b
o
To a cold solution (0-5 C) of compound 18 (2.36 g, 0.01 mol) in ethanol (50 mL) containing
sodium acetate (3.50 g, 0.50 mol) either benzenediazonium chloride (0.01 mol) or 4-chloro-
benzenediazonium chloride (0.01 mol [prepared by adding a cold solution of sodium nitrite
o
(0.70 g, in water (10 mL)) to a cold solution (0-5 C) of either aniline (0.93 g, 0.01 mol) or 4-
chloroaniline (1.27 g, 0.01 mol) in concentrated hydrochloric acid (12 mL) with continuous
stirring] was added with continuous stirring. The whole reaction mixture was left at room
temperature for 1 h then, the formed solid product was collected by filtration.
2-(1H-Benzo[d]imidazol-2-yl)-1-phenyl-2-(2-phenylhydrazono)ethanone (24a). Orange crystals
o
1
(EtOH), yield 88% (2.99 g), mp 167-169 C. IR max cm-1: 3440-3299, 3053, 1668, 1610. H
NMR (DMSO-d6 400 MHz)  = 7.26-7.55 (m, 14H, 2C6H5, C6H4), 8.05, 10.55 (2s, 2H, D2O
exchangeable, 2NH). 13C NMR (DMSO-d6 75 MHz) : 121.0, 121.4, 122.6, 123.1, 123.5, 124.2,
124.6, 125.0, 125.3, 126.0, 128,2, 128.4, 130.4, 130.9, 137.7, 139.9 (2C6H5, C6H4), 163.0
(C=O), 166.8, 168.4 (2C=N). EIMS m/z 340 [M]+ (28); anal. calcd. for C21H16N4O(340.13): C,
74.10; H, 4.74; N, 16.46. Found: C, 74.33; H, 4.59; N, 16.79.
2-(1H-Benzo[d]imidazol-2-yl)-1-phenyl-2-(2-(4-chlorophenylhydrazono)ethanone
(24b).
o
Orange crystals from (EtOH), yield 80% (2.99 g), mp 155-157 C. IR max cm-1: 3442-3302,
1
3055, 1666, 1610. H NMR (DMSO-d6 400 MHz)  = 7.26-7.58 (m, 13H, C6H5, 2C6H4), 8.08,
10.55 (2s, 2H, D2O exchangeable, 2NH). 13C NMR (DMSO-d6 75 MHz) : 120.4, 121.3, 122.7,
123.3, 123.8, 124.0, 124.4, 125.1, 125.5, 126.4, 128.1, 128.5, 130.7, 130.4, 138.2, 139.5 (C6H5,
2C6H4), 163.2 (C=O), 166.8, 168.6 (2C=N). EIMS m/z 374 [M]+ (56). Anal. calcd. for
C21H15N4O (374.83): C, 67.29; H, 4.03; N, 14.95. Found: C, 67.51; H, 3.86; N, 15.04.
(1,3-Diaminobenzo[4,5]imidazo[1,2-a]pyridin-4-yl)(phenyl)methanone (25)
Malononitrile (0.66 g, 0.01 mol) was added to a solution of compound 18 (2.36 g, 0.01 mol) or
3b (2.30 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL). The reaction
mixture was heated under reflux for 4 h then poured onto ice/water and the formed solid product
was collected by filtration. Yellow crystals (EtOH), yield 75% (2.26 g), mp 190-193 oC. IR max
cm-1: 3426-3201, 3055, 1670, 1570. 1H NMR (DMSO-d6 400 MHz)  = 4.10, 5.40 (2s, 4H, D2O
exchangeable, 2NH2), 7.26-7.55 (m, 10H, C6H5, C6H4, pyridine H-3). 13C NMR (DMSO-d6 75
MHz)  = 116.2, 120.3, 120.4, 121.1, 122.3, 122.7, 123.5, 123.9, 124.8, 128.1, 128.6, 130.4,
142,3, 142.6, 152.6 (C6H5, C6H4, pyridine C), 165.7 (C=O), 171.3 (C=N). EIMS: m/z = 302
[M]+ (24); anal. calcd. for C18H14N4O (302.34): C, 71.51; H, 4.67; N, 18.53. Found: C, 71.39; H,
4.48; N, 18.71.
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General procedure for the synthesis of the benzo[4,5]imidazo[1,2-a]pyridine derivatives 26a,b
Either acetylacetone (1.0 g, 0.01 mol) or ethyl acetoacetate (1.30 g, 0.01 mol) to a solution of
compound 18 (2.36 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL).
The reaction mixture was heated under reflux for 2 h then poured onto ice/water and the formed
solid product was collected by filtration.
(1,3-Dimethylbenzo[4,5]imidazo[1,2-a]pyridine-4-yl)(phenyl)methanone (26a). Yellow crystals
(EtOH), yield 68% (2.10 g), mp 130-133 oC. IR max cm-1: 3058, 1720, 1620. 1H NMR (DMSO-
d6 400 MHz)  = 2.42, 3.34 (2s, 6H, 2CH3), 6.61 (s, 1H, pyridine H-3), 7.27-7.36 (m, 9H, C6H5,
C6H4). 13C NMR (DMSO-d6 75 MHz)  = 24.3, 26.2 (2CH3), 118.6, 121.4, 121.8, 123.5, 124.5,
125.3, 125.8, 126.1, 127,3, 129.2, 130.4, 140.5, 143.0, 150.3 (C6H5, C6H4, pyridine C), 165.5
(C=O), 167.2 (C=N). EIMS m/z 300 [M]+ (68). Anal. calcd. for C21H16N4O (300.36): C, 79.98;
H, 5.37; N, 9.33. Found: C, 79.69; H, 5.48; N, 9.59.
1-Hydroxy-3-methylbenzo[4,5]imidazo[1,2-a]pyridine-4-yl)(phenyl)methanone (26b). Orange
o
crystals (EtOH), yield 77% (2.32 g), mp 145-147 C. IR max cm-1: 3435-3125, 3058, 1696,
1
1474. H NMR (DMSO-d6 400 MHz) = 3.34 (s, 3H, CH3), 6.63 (s, 1H, pyridine H-3), 7.27-
7.38 (m, 9H, C6H5, C6H4), 10.50 (s, 1H, D2O exchangeable, OH). 13C NMR (DMSO-d6 75 MHz)
 = 24.1 (CH3), 119.2, 122.0, 122.8, 123.1, 123.5, 124.7, 125.2, 126.3, 127.3, 129.2, 132.6,
142.1, 143.0, 144.6 (C6H5, C6H4, pyridine), 166.4 (C=O), 168.2 (C=N). EIMS m/z 302 [M]+
(28); anal. calcd. for C19H14N2O2 (302.33): C, 75.48; H, 4.67; N, 9.27. Found: C, 75.21; H, 4.59;
N, 9.41.
Chemicals
Chemical used through the anticancer evaluations of the synthesised compounds were obtained
from Sigma Chemical Co. (Saint Louis, USA).
Cell cultures
The cancer cell lines namely the human gastric cancer (NUGC and HR), human colon cancer
(DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF),
nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38) were kindly provided
by the National Cancer Institute (NCI, Cairo, Egypt). The cytotoxicity of the newly synthesised
compounds against the six cancer cell lines and the normal fibroblast were demonstrated
through Table 1.
c-Met enzymatic activity of the most active compounds
Using homogeneous time-resolved fluorescence (HTRF) assay as previously reported the c-Met
kinase activities of compounds 6b, 7b, 9, 13a, 13b, 16a, 20b, 20c, 24b, 25 and 26b were
evaluated. The IC50’s were expressed through Table 2 using foretinib as the positive control.
The data revealed that the three compounds expressed high enzymatic activity toward c-Met
with IC50’s much higher than that of the reference foretinib.
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Uses of -diketones for the synthesis of novel heterocyclic compounds
393
b
Table 1. Cytotoxicity of the newly synthesized compounds against a variety of cancer cell lines [IC50
(nM)].
Cytotoxocity (IC50 in nM)
Compound
NUGC
2140
66
DLDI
3228
80
HA22T
2314
238
HEPG2
2230
232
HONE1
3219
470
MCF
2240
128
WI38
na
6a
6b
na
na
na
na
na
na
na
na
na
380
na
na
na
na
na
na
na
665
na
na
na
na
na
na
7a
7b
9
2201
380
222
2210
1320
3270
1282
860
750
182
1135
2220
1240
480
1488
120
360
2395
1348
2870
1320
1028
539
2150
114
1128
1163
129
2244
1160
759
1220
560
1011
1128
1063
1158
98
1381
840
3261
33
2068
1340
2134
1446
330
1252
870
1025
299
2028
1210
2432
1260
1324
64
1060
324
2136
669
3188
666
2146
1504
1280
3430
2166
2271
1160
1220
1228
328
1240
1178
2177
1049
1140
1254
1186
1130
466
2143
1619
1460
1209
1313
360
11a
11b
12a
12b
13a
13b
16a
16b
18
20a
20b
20c
21
268
1062
2440
1562
682
268
2152
150
2160
122
350
3110
48
2315
210
2056
1655
1603
332
1145
1246
2868
890
330
2262
1080
1149
380
487
1445
32
22
24a
24b
25
26a
26b
CHS 828
2484
1240
82
670
71
3340
3013
260
229
3076
82
262
2742
559
25
2067
1245
15
18
aNUGC, gastric cancer, DLDI, colon cancer, HA22T, liver cancer, HEPG2, liver cancer; HONEI, nasopharyngeal
carcinoma; HR, gastric cancer; MCF, breast cancer; WI38, normal fibroblast cells.
Table 2. c-Met enzymatic activity of the most active compounds.
Compound No.
IC50 (nM) c-Met
0.24 ± 0.05
6b
7b
0.36 ± 0.28
9
0.42 ± 0.16
13a
13b
16a
20b
20c
24b
25
0.58 ± 0.28
0.61 ± 0.31
0.48 ± 0.19
0.53 ± 0.20
0.86 ± 0.45
0.50 ± 0.28
0.73 ± 0.25
26b
0.80 ± 0.32
Foretinib1.16 ± 0.17
RESULTS AN DISCUSSION
The present investigation emphasized mainly on two important things, of these one is to the
synthesis of molecules having nitrogen and or sulfur heterocyclic and the other is to determine
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Rafat M. Mohareb et al.
X
NC
Ph
C
NH4OAc
H2C
X
PhCOCH2CONHPh
+
CONHPh
120 oC
CN
3
5a, X = CN
b, X = COOEt
4a, X = CN
b, X = COOEt
PhNH2
2
PhCOCH2COOEt
1
NH
X
Ph
N
O
Ph
6a, X = CN
b, X = COOEt
O
O
Ph
N
Ph
NaOEt
+
4a,b
3
X
H2N
7a, X = NH2
b, X = OH
NH2
N
NC
CN
CN
H2N
NH4OAc
3
Ph
O
+
H2N
N
NC
120 oC
8
Ph
9
Scheme 1. Synthesis of compounds 6a,b; 7a,b and 9.
their cytotoxicity against cancer and normal cell lines. The synthetic strategies adopted for the
synthesis of the intermediates and target compounds are depicted in Schemes 1-4. Nitrogen
containing heterocyclic organic compounds having extra keto group show interesting chemical
properties as well as biological activity [33]. The reaction of ethyl benzoylacetate (1) with
aniline (2) gave the 3-oxo-N,3-diphenylpropanamide (3). Compound 3 reacted with either
malononitrile (4a) or ethyl cyanoacetate (4b) to give the 1,2-dihydropyridinederivatives 6a and
6b, respectively, through the acyclic intermediates 5a,b. The structure of the latter products was
based on analytical and spectral data. Thus, the 1H NMR spectrum of 6a showed the presence of
a singlet at  4.15 ppm indicating the presence of the CH2 group, a multiplet at 7.30-7.99 ppm
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Uses of -diketones for the synthesis of novel heterocyclic compounds
395
for the two phenyl groups and a singlet at  10.21 ppm (D2O exchangeable) corresponding to
the NH group. Moreover, the 13C NMR spectrum showed signal at 49.5 corresponding to the
pyridine C-3, two signals at 103.6, 104.5 for the C=C group, a signal at 119.5 for the CN
group, eight signals at 120.19, 132.9, 125.8, 129.4, 129.5, 134.5, 136,8, 139.5 equivalent to
two benzene and pyridine C and two signals at 165.9, 195.2 confirming the C=O and C=N
groups. On the other hand, the reaction of compound 3 with either malononitrile or ethyl
cyanoacetate in sodium ethoxide solution gave the 1,6-dihydropyridine derivatives 7a and 7b,
respectively. The reaction of compound 3 with the 2-aminoprop-1-ene-1,1,3-tricarbonitrile 8
gave the 7,8-dihydro-1,8-naphthyridine derivative 9 (Scheme 1) its structure was confirmed on
the basis of analytical and spectral data.
The high yield of compound 7b encouraged us to make further work in order to produce
pharmaceutically active fused pyridine derivatives. Thus, the reaction of compound 7b with
either acetylacetone (10a) or ethyl acetoacetate (10b) gave the pyrido[2,3-c]pyridine derivatives
11a and 11b, respectively. Furthermore, compound 7b reacted with either malononitrile or ethyl
cyanoacetate to give the pyrido[2,3-c]pyridine derivatives 12a and 12b, respectively. The
analytical and spectral data of 11a,b and 12a,b were in agreement with their proposed
structures.
O
O
O
O
Ph
Ph
O
Ph
O
N
O
Ph
N
1,4-dioxane
+
X
O
HN
Et3N
H2N
10a, X = CH3
b, X = OEt
Y
H3C
7b
11a, Y = CH3
b, Y = OH
O
O
Ph
O
Ph
N
1,4-dioxane
H2C
CN
X
7b
+
N
Et3N
4a, X = CN
b, X = COOEt
R
H2N
12a, R = NH2
b, R = OH
Ph
X
+
PhCOCH2CONHPh
S8
+
4a,b
NH2
PhHNOC
S
3
13a, X = CN
b, X = COOEt
Scheme 2. Synthesis of compounds 11a,b; 12a,b and 13a,b.
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Rafat M. Mohareb et al.
DMF/KOH
COPh
SK
PhHNOC
PhNCS
3
+
13
PhHN
14
Ph
PhHNOC
PhHN
H2C
Cl
COR
+
14
COR
S
15a, R = OC2H5
b, R = CH3
16a, R = OC2H5
b, R = CH3
H
N
NH2
NH2
120 oC
PhCOCH2COOEt
+
COPh
1
N
17
18
H
N
1,4-dioxane
18
CHO
+
X
COPh
N
19a, X = H
b, X = OCH3
c, X = Cl
HC
X
20a, X = H
b, X = OCH3
c, X = Cl
Scheme 3. Synthesis of compounds 16a,b; 19 and 20a-c.
The benzoylbenzanilide was studied to synthesis thiophene through the well known
Gewald’s thiophene synthesis [34, 35]. Thus, the reaction of compound 3 with elemental sulfur
and either malononitrile (4a) and ethyl cyanoacetate (4b) gave the thiophene derivatives 13a
and 13b, respectively (Scheme 2). On the other hand, compound 3 reacted with phenyl
isothiocyanate in basic dimethylformamide gave the intermediate potassium sulphide salt 14.
The latter intermediate reacted with either ethyl chloroacetate (15a) or chloroacetone (15b) to
give the thiophene derivatives16a and 16b, respectively. The structures of the latter products
were established on the basis of analytical and spectral data. Thus, the 1H NMR spectrum of 16a
showed a triplet at 0.71 ppm indicating the presence of the ester CH3 group, a quartet at 3.87
for the ester CH2 group, a mutiplet at  7.43-7.49 equivalent to the three phenyl protons and two
singlets at  8.20 and 10.39 for the two NH groups. The 13C NMR spectrum showed the
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Uses of -diketones for the synthesis of novel heterocyclic compounds
397
presence of a signal at 13.6 for the CH3 group, a signal at 39.6 equivalent to the CH2 group
and eleven signals at 110.0, 121.8, 125.8, 126.3, 128.4, 129.1, 130.4, 133.1, 138.0, 139.2,
140.2, 148.7 indicating the two C6H5 and thiophene C and two signals at 165.8, 195.1 for the
two C=O groups.
Next we moved towards the uses of ethyl benzoylacetate to synthesis of benzimidazole
o
derivatives. Thus, compound 1 reacted with orthophenylene diamine in an oil bath 120 C to
give the 2-(1H-benzo[d]imidazol-2-yl)-1-phenylethanone (18). The analytical and spectral data
of compound 18 were in agreement with its proposed structure. Compound 18 reacted with
either benzaldehyde (19a), 4-methoxybenzaldehyde (19b) or 4-chlorobenzaldehyde (19c) to
give the benzalidene derivatives 20a-c, respectively (Scheme 3).
H
N
Ph
20a
+
NH2NH2
N
N
Ph
N
H
21
N
N
COPh
1,4-dioxane
Et3N
20b
+
4a
H2N
OCH3
CN
-
+
N
22
NCl
H
N
N
EtOH/AcONa
COPh
18
18
+
N
X
NH
X
23a, X = H
b, X = 4-Cl
24a, X = H
b, X = 4-Cl
N
N
1,4-dioxane
COPh
+
3a
Et3N
NH2
H2N
25
O
O
N
piperidine
COPh
+
18
R
N
R
CH3
10a, R = CH3
b, R = OC2H5
26a, R = CH3
b, R = OH
Scheme 4. Synthesis of compounds 21; 24a,b; 25 and 26a,b.
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Rafat M. Mohareb et al.
Compound 20a reacted with hydrazine hydrate to give the pyrazole derivative 21. On the
other hand, 20b reacted with malononitrile (3a) to give the 1-amino-4-benzoyl-3-(4-
methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyridine-2-carbonitrile (22).
Compound 18 reacted with either benzenediazonium chloride (23a) or 4-
chlorobenzenediazonium chloride (23b) to give the phenylhydrazo derivative 24a and 24b,
respectively. On the other hand, it reacted with malononitrile (3a) in 1,4-dioxane containing
triethylamine to give the benzo[4,5]imidazo[1,2-a]pyridine derivative 25. Similarly compound
18 reacted with either either acetylacetone (10a) or ethyl acetoacetate (10b) to give the
benzo[4,5]imidazo[1,2-a]pyridine derivatives 26a and 26b, respectively (Scheme 4).
Structure activity relationship
From Table 1 the newly synthesized compounds were tested against the six cancer cell lines the
human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and
HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and a normal
fibroblast cells (WI38). Compounds 6b, 7b, 9, 13a, 13b, 16a, 20b, 20c, 24b, 25 and 26b
exhibited optimal cytotoxic effect against cancer cell lines, with IC50’s in the nM range.
Comparing the cytotoxicity of the 1,2-dihydropyridine derivatives 6a and 6b, it is obvious that
the ethyl ester group present in 6b is responsible for its high cytotoxicity. On the other hand
considering the 1,6-dihydropyridine 7a and 7b; the latter compound with the OH group showed
high cytotoxicity against the NUGC, DLDI, HA22T and HONE1 cell lines. The 2-(amino(6-
hydroxy-2-imino-1,4-diphenyl-1,2-dihydropyridin-3-yl)methylene)-malononitrile (9) showed
high cytotoxicity against NUGC, DLDI and HEPG2 cell lines. Such high potency of compound
9 is attributed to the presence of the dicyanethylidene moiety. Comparing the cytotoxicity of the
1,6-naphthridine derivatives 11a and 11b, it is obvious that the presence of the OH group in
compound 11b is responsible for its high cytotoxicity against HA22T and HEPG2 cell lines.
Such finding was confirmed through the comparison of the cytotoxicity of compounds 12a and
12b, where the last one showed higher cytotoxicity towards the six cancer cell lines. The
significant cytotoxicity of 12b appeared against the HEPG2 cell line with 870 nM.
For the thiophene derivatives 13a,b and 16a,b, it is clear from Table 1 that such compounds
showed significant cytotoxicity against the cancer cell lines. Compounds 13b and 16a with the
special optimal cytotoxicity which is attributed to the presence of the oxygen rich COOEt and
OCH3 moeties in both compound, respectively. It is of great interest to compare the cytotoxicity
of the un-substituted benzimidazole derivative 18 and the benzylidene derivatives 20a-c. It is
obvious that compound 18 showed lower cytotoxicity relative to 20a-c. In addition going
through the latter compounds, one can notice that the presence of the electronegative groups
OCH3 and Cl in 20b and 20c, respectively are responsible for the high cytotoxicity of such
compounds. Moreover, the Cl group showed more potency than the OCH3 group as cleared from
the greater cytotoxicity of 20c over 20b. The annulated compound 22 showed higher
cytotoxicity than the pyrazole derivative 21. The reaction of the benzimidazole derivative 18
with arylidenediazonium salts 23a and 23b gave the arylhydrazone derivatives 24a and 24b
leading to a remarkable increase of cytotoxicity of 24a,b. In addition compound 24b with Cl
group showed an optimal cytotoxicity against NUGC, DLDI, HA22T, HEPG2 and MCF cell
lines with IC50’s 380, 122, 33, 669 and 890 nM, respectively. It is of great value to note that
compound 24b with the Cl group showed more potency than 24a. Considering the imidazo[1,2-
a]pyridine derivatives 25 and 26a,b compound 26b showed the highest cytotoxticity among the
three compounds. Moreover, the nitrogen rich compound 25 showed higher cytotoxicity than
26a. It is of good worthy to notice that the ethyl 6-hydroxy-2-imino-1,4-diphenyl-1,2-
dihydropyridine-3-carboxylate (6b) and the (1-hydroxy-3-methylbenzo[4,5]imidazo[1,2-
a]pyridine-4-yl)(phenyl)methanone (26b) showed the maximum inhibitory effect towards the
six cancer cell lines among the tested compounds.
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Uses of -diketones for the synthesis of novel heterocyclic compounds
399
It is very clear from our present finding that the heterocyclic systems with halogen
substituted pattern OCH3, Cl or COOEt show greater cytotoxic property. In every case it was
observed that molecules with electronegative substitutions as compounds 6b, 7b, 13a,b; 16a,b;
20b, 20c, 25, and 26b showed higher cytotoxicity because they were bearing either oxygen or
chlorine substituted as well as comprised with similar structural features.
Toxicity
It is well known that bioactive compounds are often toxic to shrimp larvae. Thus, in order to
monitor these chemicals’ in vivo lethality to shrimp larvae (Artemia salina), results are given in
Table 3 for the compounds which exhibited optimal cytotoxic effect against cancer cell lines
which are the ten compounds 6b, 7b, 13a, 13b, 16a, 16b, 20b, 20c, 25 and 26b. The shrimp
lethality assay is considered as a useful tool for preliminary assessment of toxicity, and it has
been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria
toxins, pesticides, and cytotoxicity testing of dental materials, natural and synthetic organic
compounds [36].
Table 3. Toxicity of the most cytotoxic compounds.
Compound
No.
Cons.
(μg/mL)
Mortalitya
Toxicity
LC50
Upper
95% limit
Lower
95% limit
6b
10
100
1000
10
100
1000
10
100
1000
10
100
1000
10
100
1000
10
100
1000
10
100
1000
10
100
1000
10
0
0
8
2
6
8
0
5
10
1
4
10
1
6
10
0
1
10
0
6
10
4
8
10
2
5
8
Non toxic
890.22
-
-
7b
Harmful
Harmful
Harmful
Harmful
Harmful
Harmful
Very toxic
Very toxic
Non toxic
177.63
213.29
237.39
122.32
84.08
673.42
66.12
230.62
240.31
140.83
166.23
-
285.41
22.45
66.80
107.82
-
13a
13b
16a
16b
20b
20c
25
238.40
12.40
215.80
-
24.40
365.70
-
213.79
-
100
1000
10
26b
0
1000.2
100
1000
0
6
aTen organisms (A. salina) tested for each concentration.
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Rafat M. Mohareb et al.
In order to prevent the toxicity results from possible false effects originated from solubility
of compounds and DMSO’s possible toxicity effect, compounds were prepared by dissolving in
DMSO in the suggested DMSO volume ranges. It is clear from Table 3 that compounds 6b and
26b showed non toxicity against the tested organisms.
Molecular docking
The molecular studies were carried out using Molecular Operating Environment (MOE 2014).
All the minimizations were performed with MOE until a RMSD gradient of 0.01 kcal/mol Å
with MMFF94X force field and the partial charges were automatically calculated. Docking
simulations were performed using the crystal structure of NUGC (PDB ID: 4ASD) which
obtained from Protein Data Bank. Enzyme structure was checked for missing atoms, bonds and
contacts. Water molecules was removed. Protonate 3D application of MOE was used to add the
missing hydrogens and properly assign the ionization states. The ligand molecules were
constructed using the builder molecule and were energy minimized. The active site was
generated using the MOE-Alpha site finder. Ligands were docked within the active sites using
the MOE-Dock The generated poses were energy minimized using the MMFF94x force field.
Finally, the optimized poses were ranked using the GBVI/WSA DG free-energy estimates.
Docking poses were visually inspected and interactions with binding pocket residues were
analyzed.
Docking simulation was carried out to illustrate the binding mode and the interaction of the
active compounds 6b, 7b, 13a and 16b with the amino acids in the active site of the NUGC.
Docking study was performed using the crystal structure of NUGC (PDB ID: 4ASD) [20] which
has co-crystallized ligand (sorafenib, BAX) as inhibitor inside its active site. In the beginning,
docking study was validated by re-docking of co-crystallized ligand (BAX) inside the active site
of NUGC. The re-docking of the co-crystallized ligand, BAX, was carried out to indicate the
suitability of the used protocol for the planned docking study. The validation method was
achieved by removing the bound ligand from the complex followed by its docking back into the
binding site, which yielded root mean square deviation values RMSD of 0.88 Å with energy
score (S) -9.94 kcal/mol. The top pose obtained from the MOE docking simulation showed the
interactions of the co-crystallized ligand, BAX, with the key amino acids inside the active site.
Docking ligand interaction and electrostatic map of compounds 6b, 7b, 13a and 16b were
indicated through Figures 2-5, respectively.
(A)
(B)
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Uses of -diketones for the synthesis of novel heterocyclic compounds
401
(C)
Figure 2. (A) Docking of compound 6b, (B) Ligand interaction of compound 6b, and (C)
electrostatic map of compound 6b. Energy of compound 6b = -24.66.
(A)
(B)
(C)
Figure 3. (A) Docking of compound 7b, (B) ligand interaction of compound 7b, and (C)
electrostatic map of 7b. Energy of compound 7b = -20.26.
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Rafat M. Mohareb et al.
(A)
(B)
(C)
Figure 4. (A) Docking of compound 13a, (B) ligand interaction of compound 13a, and (C)
electrostatic map of compound 13a. Energy of compound 13b = -24.89.
(A)
(B)
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Uses of -diketones for the synthesis of novel heterocyclic compounds
403
(C)
Figure 5. (A) Energy of compound 16b, (B) ligand interaction of compound 16b, and (C)
electrostatic map of compound 16b. Energy of compound 16b = -26.98.
For Figures 2-5 the blue color indicating the structure of the target molecule and the green color
for the amino acid.
CONCLUSION
The present work, through simple synthetic approaches, led to the development of novel
pyridine, thiophene and imidazole derivatives that exhibited remarkable antitumor activities
against six tumor cell lines. As new class of heterocyclic compounds, eleven of the obtained
new compounds showed remarkable cytotoxicity against most of the six tumor cell lines. Most
of the active compounds were devoid of the typical nitrogen and or sulfur feature of heterocyclic
derivatives and so the activity could be attributed to some sort of electronegative substituents
through the synthesized compounds. In addition c-Met kinase inhibitions for the most active
compounds showed that all compounds exhibited inhibitions higher than the reference drug
foretinib. The results of the biological screening will encourage future work of heterocyclic
compounds derived from -diketones.
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