Synthesis, molecular modeling, and biological evaluation of novel benzimidazole derivatives as inhibitors of hepatitis C virus RNA replication

Article abstract of DOI:10.1248/cpb.c13-01009

In this study, synthesis and docking studies of a series of new benzimidazole derivatives linked to substituted pyrimidines either through the methylenethio linkage or its bioisosteric methylene amino bridge were carried out. All the synthesized compounds were evaluated for their hepatitis C virus (HCV) RNA replication-inhibitory activity. Compounds 4d, 4f, and 4h were found to be more potent than VX-950 (IC_50/90of 4d=0.123/0.321, 4f=0.145/0.345, 4h=0.129/0.432, VX-950=0.20/0.45μM, respectively) and 6d (IC_50/90=0.116/0.452μM) displayed activity very similar to that of the standard. Compounds 4d, 4f, 4h, and 6d were potent HCV RNA replication inhibitors and are good drug candidates for further investigations.

Full text of DOI:10.1248/cpb.c13-01009

8
56
Regular Article
Chem. Pharm. Bull. 62(9) 856–866 (2014)
Vol. 62, No. 9
Synthesis, Molecular Modeling, and Biological Evaluation of Novel
Benzimidazole Derivatives as Inhibitors of Hepatitis C Virus RNA
Replication
a
a
,b,†
Hoda Ibrahim El Diwani, Heba Tawfik Abdel-Mohsen, Ismail Salama,*
c
a
a
Fatma Abdel-Fattah Ragab, Mostafa Mahmoud Ramla, Shadia Ahmed Galal,
Mohamed Mostafa Abdalla, Abeer Abdel-Wahab, and Maha Adel El Demellawy
d
e
e
a
Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries
b
Research, National Research Center; Cairo, Egypt: Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Suez Canal University; Ismailia, Egypt: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo
University; Cairo, Egypt: Research Unit, Saco Pharm. Co.; 6th October City, Cairo, Egypt: and Medical
Biotechnology Department (MBD), Genetic Engineering & Biotechnology Research Institute (GEBRI), City of
Scientific Research & Technology Applications; Alexandria, Egypt.
c
d
e
Received December 31, 2013; accepted May 20, 2014
In this study, synthesis and docking studies of a series of new benzimidazole derivatives linked to sub-
stituted pyrimidines either through the methylenethio linkage or its bioisosteric methylene amino bridge
were carried out. All the synthesized compounds were evaluated for their hepatitis C virus (HCV) RNA
replication-inhibitory activity. Compounds 4d, 4f, and 4h were found to be more potent than VX-950
(
(
IC50/90 of 4dꢀ0.123/0.321, 4fꢀ0.145/0.345, 4hꢀ0.129/0.432, VX-950ꢀ0.20/0.45µM, respectively) and 6d
IC50/90ꢀ0.116/0.452µM) displayed activity very similar to that of the standard. Compounds 4d, 4f, 4h, and 6d
were potent HCV RNA replication inhibitors and are good drug candidates for further investigations.
Key words synthesis; benzimidazole; pyrimidine; hepatitis C virus; viral RNA replication inhibitor
As estimated by WHO, 170 million persons worldwide are contains a six-stranded-barrel that ends with a helix. The ac-
infected with hepatitis C virus (HCV) which causes chronic tive site (His: 57, Asp: 81, and Ser: 139) is located between
liver disease. HCV is a positive stranded RNA virus and is these two regions and is formed by a shallow solvent exposed
1,2)
a member of the Flaviviridae family of viruses. Combined pocket to which the inhibitors bind in an extended conforma-
18–21)
therapy of HCV infections with pegylated alpha interferon tion.
IFN-α) and ribavirin is only effective in 50–60% of infected Sofosbuvir (GS-7977), a uridine nucleotide analogue, and
(
individuals and is associated with serious side effects such as an inhibitor of HCV NS5B polymerase is effective against
depression, flu-like symptoms, fatigue and hemolytic anemia HCV genotypes 1, 2, 3, 4, and 6. Sofosbuvir, especially in
3
,4)
caused by ribavirin.
There is an unmet need for potent and selective inhibitors tion in the treatment of chronic hepatitis C.
of HCV replication. Significant research efforts are currently Currently, different anti-HCV treatments depend on
directed towards targeting viral enzymes especially NS3-4A the inhibition of HCV NS3-4A serine protease. Telaprevir
interferon-free combinations, represents a very promising op-
2
2)
5
–8)
serine protease
merase which are both required for virus propagation.
The NS3-4A serine protease has been an attractive target
in the development of new antivirals with activities against valent but reversible complex with the HCV NS3-4A serine
and NS5B RNA-dependent RNA poly- (VX-950), boceprevir and BILN 2061 (Fig. 1) are examples of
9
–11)
23,24)
well known HCV NS3-4A serine protease inhibtors.
Telaprevir, a small molecule peptidomimetic, forms a co-
9
–11)
HCV since it is essential for viral replication.
protease activity of NS3 in complex with the NS4A cofactor is excellent efficacy in the clinical setting.
responsible for the proteolytic cleavage at four junctions of the The benzimidazole scaffold is common in different anti-
HCV polyprotein precursor: NS3-NS4A (self cleavage), NS4A- HCV agents with different modes of actions.
The serine protease in a slow process (slow-on). It was discovered to have
2
3–27)
7,12–17)
NS4B, NS4B-NS5A, and NS5A-NS5B.
Derivatives of bis-benzimidazolemethane were discovered
Crystal structure of the serine protease domain of the NS3, to be highly potent, reversible and selective serine protease
1
8,19)
28,29)
with its essential cofactor NS4A,
complex adopts a chymotrypsin/trypsin-like fold with struc-
turally conserved regions typical of small chymotrypsin-like azole derivatives having a pyrimidothiomethylene group at
revealed that the NS3-4A inhibitors e.g., CRA-6336
(Fig. 2).
Recently, we have synthesized a novel series of benzimid-
2
0,21)
proteases.
–93 of NS3 and residues 21–34 of NS4A) contains an eight- activity against different cell lines. In this study we were
stranded β-barrel motif, with one of the strands contributed by interested in finding out whether replacing the 2-methylbenz-
The N-terminal region of NS3-4A (residues the 2-position and it was found that it has a potent antitumor
3
0)
1
2
0)
the NS4A cofactor. The C-terminal region (residues 94–175) imidzole moiety of bis-benzimidazolemethane CRA-6336
with 2-pyrimidomethylenethio moiety or its bioisosteric 2-
pyrimidomethyleneamino group would have a positive impact
The authors declare no conflict of interest.
†
on the inhibitory activity against HCV. Hence, introducing
Present address: Department Chemistry and Pharmacy, Emil Fischer Cen-
ter, Friedrich-Alexander University; Schuhstrasse 19, D-91052 Erlangen, new scaffolds for the development of wide range of antiviral
Germany.
agents.
*
To whom correspondence should be addressed. e-mail: ismail.salama.mohamed@fau.de
© 2014 The Pharmaceutical Society of Japan

September 2014
857
After the successful discovery of the new scaffold, we start- boxylic group.
ed to carry out different modifications and the correspond-
ing inhibitory activity was examined. These modifications Results and Discussion
included the introduction of different substituents on the 4-
Chemistry A convergent synthesis was designed for the
phenyl ring of the pyrimidine moiety, bioisosteric replacement preparation of series 4a–h and 5a–h. The first part of this
of the phenyl ring with heterocyclic rings, and hydrolysis of synthesis involved the preparation of 2-chloromethyl-1H-
3
0,31)
the 5-cyano substituent on the pyrimidine nucleus to car- benzo[d]imidazole 1 as previously reported
(Chart 1).
The second part of this synthesis involved the preparation of
different amino and mercapto pyrimidines 2a–h and 3a–h
by ternary condensation of the appropriate aldehyde with
thiourea or guanidine hydrochloride and ethylcyanoacetate
3
0,32–34)
according to reported procedures
(Chart 1). Aiming to
study the influence of substitution at different positions of the
phenyl ring linked to pyrimidine, different substituted benzal-
dehydes were used to obtain the two series 2a–f and 3a–f. In
order to investigate the effect of the replacement of the phenyl
ring by five membered heterocycles, 2-thiophenecarbaldehyde
or 2-furancarbaldehyde were chosen to produce the desired
pyrimidines 2g,h and 3g,h respectively. Dehydrohalogenation
between the two parts was then carried out in basic medium
to obtain the required target compounds 4a–h and 5a–h,
which were subsequently hydrolyzed by refluxing with 80%
H SO acid to obtain the carboxylic acid derivatives 6a–h and
2
4
7a–h (Chart 2).
Inhibition of HCV Viral RNA Replication As a first ex-
periment compounds 4a and 5a were tested for their HCV in-
hibitory activity (Table 1, entries 2 and 10). Both IC50 and IC90
were evaluated. VX-950 was used as a standard. It was found
Fig. 1. Structures of Known HCV NS3-4A Serine Protease Inhibitors
Fig. 2. Structure of CRA-6336
Reagents and conditions: a) i: chloroacetic acid, 4N-HCl, reflux, 6h, ii: aq. NH , 81%. b) K CO , ethanol, reflux, 8h, 20–34%. 2a, 3a: R=phenyl; 2b, 3b: R=4-chloro-
3
2
3
phenyl; 2c, 3c: R=4-fluorophenyl; 2d, 3d: R=4-hydroxyphenyl; 2e, 3e: R=2-chlorophenyl; 2f, 3f: R=2-hydroxyphenyl; 2g, 3g: R=2-thienyl; 2h, 3h: R=2-furyl.
Chart 1

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58
Vol. 62, No. 9
Reagents and conditions: a) triethylamine, tetrahydrofuran, 24h, 73–81%; b) K CO , acetone, rt, 10h then reflux for 6h, 73–81%; c) K CO , DMF, rt, 2h then reflux for
2
3
2
3
1
5
0h, 73–81%; d) i: 80% sulphuric acid, reflux, 4h, ii: aq. NH , 90–96%. 4a, 5a, 6a, 7a: R=phenyl; 4b, 5b, 6b, 7b: R=4-chlorophenyl; 4c, 5c, 6c, 7c: R=4-fluorophenyl; 4d,
3
d, 6d, 7d: R=4-hydroxyphenyl; 4e, 5e, 6e, 7e: R=2-chlorophenyl; 4f, 5f, 6f, 7f: R=2-hydroxyphenyl; 4g, 5g, 6g, 7g: R=2-thienyl; 4h, 5h, 6h, 7h: R=2-furyl.
Chart 2
Table 1. The Results of HCV RNA Replication Inhibitory Activity (IC50, IC ) (µM) and the Cytotoxic Activity CC (µM) of the Tested Compounds
90
50
and Standard
a)
b)
c)
Entry
Product
IC50 (± S.D.)µM after 48h
IC90 (± S.D.)µM after 48h
CC50 (± S.D.)µM
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1
0.25± 0.02
0.345± 0.04
0.312± 0.03
0.231± 0.01
0.123± 0.02
0.245± 0.05
0.145± 0.04
0.355± 0.03
0.129± 0.02
0.263± 0.02
0.235± 0.02
0.277± 0.02
0.465± 0.04
0.356± 0.03
0.277± 0.02
0.116± 0.01
0.298± 0.02
0.285± 0.02
0.255± 0.02
0.181± 0.01
0.546± 0.05
0.512± 0.05
0.546± 0.05
0.657± 0.06
0.87± 0.08
0.65± 0.05
0.744± 0.06
0.654± 0.05
0.563± 0.04
0.321± 0.05
0.564± 0.05
0.345± 0.04
0.765± 0.07
0.432± 0.07
0.678± 0.05
0.456± 0.05
0.654± 0.04
1.12± 0.11
0.879± 0.09
0.678± 0.07
0.452± 0.05
0.666± 0.04
0.987± 0.09
0.764± 0.07
0.535± 0.06
1.23± 0.1
137± 6
4a
4b
4c
4d
4e
4f
245± 13
453± 18
245± 8
265± 9
312± 11
311± 12
456± 15
657± 19
765± 19
986± 25
643± 22
564± 30
523± 34
545± 33
656± 32
978± 47
766± 43
587± 12
876± 16
647± 23
987± 32
953± 33
673± 17
964± 24
90± 1.2
4g
4h
5a
5b
5c
6a
6b
6c
6d
6e
6f
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
6g
6h
7c
7d
7e
7f
0.987± 0.1
1.43± 0.14
1.78± 0.16
2.12± 0.2
7h
d)
VX-950
0.20± 0.0001
0.45± 0.0003
a) Compound concentration required to achieve 50% inhibition of HCV RNA replication. b) Compound concentration required to achieve 90% inhibition of HCV RNA
replication. c) Compound concentration required to reduce the cell viability by 50% as determined by MTT method. d) The standard used in comparison. *Data expressed as
means± S.D. for three independent experiments.
that both compounds 4a and 5a have excellent inhibitory activ- out and some of the synthesized compounds were further test-
ities (IC50/90=0.345/0.744µM, IC50/90=0.263/0.678µM, respec- ed for their anti-HCV activity and for their cytotoxic activity.
tively) when compared with VX-950 (IC50/90=0.20/0.45µM) It was found that all of the tested compounds have significant
(
Table 1, entry 26). The cytotoxic activity of both compounds activity. Comparing the inhibitory activity values revealed that
was also tested against the viability hepatocyte. It was found compounds 4d, f, and h were more potent than the standard.
that 4a and 5a (50% cytotoxic concentration CC =245 µM It was also observed that bioisosteric replacement of CH –S
5
0
2
and CC =765 µM, respectively) (Table 1, entries 2 and 10) linkage in series 4a–h and 6a–h by CH –NH bridge in series
5
0
2
have lower cytotoxic activity when compared with VX-950 5a–h and 7a–h, resulted in a decrease or total loss of activ-
which has a value of CC =90µM (Table 1, entry 26). This ity except in case of 5b where high potency was observed.
5
0
result revealed the perfect selectivity of the synthesized scaf- The IC50/90 values of 4c, 6c (Table 1, entries 4 and 15) were
fold against the virus in comparison to the host cell. After the 0.231/0.563 and 0.277/0.678µM, respectively while the IC50/90
successful finding of the HCV inhibitory activity of these two values of 5c, 7c (Table 1, entries 12 and 21) were 0.277/0.654
compounds 4a and 5a, different modifications were carried and 0.546/1.23µM, respectively. The effect of hydrolyzing the

September 2014
859
Fig. 3. Docking Pose of 4d in the Binding Site of NS3-4A Serine Protease (PDB Code 2FM2)
(a) Solid surface representation of NS3 active site with 4d (stick model, colored by atom type). On the protein surface, acidic residues are colored in red, basic residues
are in blue, hydrophobic residues are in white, polar residues are in green. (b) The NS3 residues are shown as stick models and colored by residue type, the structure of 4d
is presented as CPK model.
Fig. 4. Docking Pose of 6d in the Binding Site of NS3-4A Serine Protease (PDB Code 2FM2)
(a) Solid surface representation of NS3 active site with 6d (stick model, colored by atom type). On the protein surface, acidic residues are colored in red, basic residues
are in blue, hydrophobic residues are in white, polar residues are in green. (b) The NS3 residues are shown as stick models and colored by residue type, the structure of 6d
is presented as CPK model.
cyano group in series 4a–h and 5a–h to give series 6a–h and 4d, 6d, 7d exhibited higher activities when compared with
7
a–h, respectively on the inhibitory activity was also studied. the corresponding substitutions in the same series (Table 1,
Comparing the IC50/90 of 4a–h and 5c (Table 1, entries 2–9, entries 5, 16, and 22). This may be due to hydrogen bond-
2) with the IC50/90 of 6a–h and 7c (Table 1, entries 13–21) ing of the OH group with the enzyme. The higher potency of
revealed the higher activity of the cyano containing series the 4-hydroxy derivatives 4d, 6d, and 7d than the 2-hydroxy
4a–h and 5c). This can be attributed to the steric demanding derivatives 4f, 6f, and 7f, respectively, may be due to more
1
(
effect of the carboxylic group in 6a–h and 7c which hinders favorable steric factors and best fitting with the enzyme. By
the perfect fitting to the enzyme and also the cyano group is comparing all of the tested compounds with VX-950 as a
positioned in a hydrophobic area in the binding site. The only standard, we found that compounds 4d, 4f, 4h, 6d, 6h showed
exceptions were 4d and 4g (Table 1, entries 5 and 8) in which higher inhibitory activity than the standard (Table 1, entries 5,
compounds 6d and 6g (Table 1, entries 16 and 19) having a 7, 9, 16, and 20).
carboxylic substituent showed higher activity. Subsequently,
We have also evaluated the CC50 of all the tested com-
the effect of different substituents in the 6-position of the pounds against hepatocytes. It was found that all of the tested
pyrimidine moiety was examined in each series. It was found compounds have a lower cytotoxic activity when compared
that the compounds having p-hydroxyphenyl group namely, with VX-950 (Table 1).

8
60
Vol. 62, No. 9
Molecular Modeling Calculations In order to rationalize Experimental
the described biological results, docking studies of compounds
Chemistry Melting points are uncorrected and were
recorded on a Gallenkamp thermometer melting point ap-
35–37)
4
d and 6d were undertaken. The program GOLD 5.0.1
with the GoldScore fitness function, which uses bond paratus. IR spectra (KBr) were recorded on Bruker Vector
strengths, was used for the docking calculations. 22 and JASCO FT/IR 300E Fourier transformer spectropho-
Analysis of key protein–ligand contacts and binding affinity tometer. NMR spectra were recorded on Varian GEMINI 200
of the poses provides insight into binding of 4d and 6d to the (200MHz) and JEOL EX-270 and 500MHz spectrometers
active sites of NS3-4A serine protease.
using DMSO-d6 as a solvent. Mass spectra were recorded
For the lowest energy poses of 4d, hydrogen bond interac- on FINNGAN MAT SSQ 7000. Elemental analysis was per-
tions were observed between the p-OH and Gln41 (3.08Å). formed in the microanalytical laboratory of the National Re-
This was consistent with the observed SAR (Figs. 3a,b).
search Center. The synthesized compounds were named using
For the lowest energy poses of 6d, both oxygens of the ChemDraw Ultra software (v 10.0).
carboxylate forms H bond with the NH group of backbone of
Preparation of 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-
Ser139 (ca. 1.8, 2.3Å) and with the NH group of the backbone 1,6-dihdyro-4-(aryl or heteroaryl)-6-oxopyrimidine-5-car-
of Gly137 (ca. 3Å). The p-OH has a distance of 3.38Å from bonitriles (4a–f)). General Method A solution of the ap-
3
2,33)
Gln41 (Figs. 4a,b). The binding to Ala156 residue is vital for propriate thiouracil 2a–f
all the NS3-4A inhibitors.
(42.17mmol) in dry tetrahydro-
38)
furan (60mL) containing triethylamine (0.5mL) was stirred
The IC50/90 of 4d (0.123/0.321µM) compared to that of for 1h. A solution of 1 (7g, 42.17mmol) in dry tetrahydro-
VX-950 (0.20/0.45 µM) may be attributed to the novel feature furan was then added portionwise and the reaction mixture
of 4d, it extends substantially toward the P′ side of the active was allowed to stir at room temperature for additional 24h,
site. Compound 4d derives a significant part of its binding poured onto crushed ice with stirring. The precipitated prod-
energy from contacts on both P and P′ sides. Interaction with uct was filtered off and recrystallized from the appropriate
Lys136 of the protein may be a reason for the improved po- solvent.
tency of this inhibitor.
These additional interactions of compound 4d versus oxo-4-phenylpyrimidine-5-carbonitrile (4a) was prepared ac-
2-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-6-
3
0)
VX-950 justify its higher activity. The active site pockets that cording to the reported method.
compound 4d occupy are depicted in Figs. 3a and b.
2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(4-chloro-
The residues that induce common interactions with both 4d phenyl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (4b):
1
and 6d are Lys136, Ser139, Phe154, Ala156, Ala157, Arg155, Yield 75%; mp 183–185°C (from MeOH); H-NMR (500MHz,
and Cys159 (Figs. 3, 4). Those residues are common in DMSO-d ) δ: 4.67 (s, 2H, CH –S), 7.18–7.22 (m, 2H, H5 and
6
2
NS3-4A inhibitor binding as it has been widely discussed in H6 benzimidazole), 7.28–7.33 (m, 2H, H3′ and H5′ phenyl),
21,39)
previous literature.
As previously reported, hydrophobic 7.50–7.55 (m, 2H, H4 and H7 benzimidazole), 7.87–7.91 (m,
interactions highly contribute in the binding affinity of the 2H, H2′ and H6′ phenyl), 8.21 (br, 1H, NH pyrimidine, D O
2
majority of NS3-4A serine protease inhibitors. The residues exchangeable), 10.17 (br, 1H, NH benzimidazole, D O ex-
2
−1
Ile132, Leu135, Lys136, Phe154, Ala156, Ala157, and Cys159 changeable); IR (KBr, cm ) 3394 (NH benzimidazole), 3125
play important roles in hydrophobic binding. (NH pyrimidine), 3056 (CH arom.), 2924 (CH aliph.), 2209
The higher activity of cyanopyrimidines compared to their (CN), 1647 (C=O), 1629 (C=N benzimidazole), 1552, 1518
carboxy analogues may be attributed to that they are directed (C=N pyrimidine, C=C). MS (electron ionization (EI)) m/z
+
·
towards a hydrophobic area in the binding site (Phe43).
393/395 (M , 19%/6% Cl pattern), 180 (100%). Anal. Calcd
As shown in Figs. 3a and 4a, it was observed that methy- for C H ClN OS: C, 57.94; H, 3.08; N, 17.78; S, 8.14. Found:
19
12
5
lenethio bridge fitted into the hydrophobic cavity formed by C, 57.75; H, 3.00; N, 17.57; S, 8.36.
Ala156, Ile132, Leu135, and Phe154. This may explain why
2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(4-fluoro-
compounds which have methylenethio bridge have higher in- phenyl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (4c): Yield
1
hibitory activity than compounds which have methyleneamino 76%; mp 250–252°C (from MeOH); H-NMR (500MHz,
bridge.
DMSO-d ) δ: 4.79 (s, 2H, CH –S), 7.22–7.26 (m, 2H, H5 and
6
2
H6 benzimidazole), 7.30–7.39 (m, 2H, H3′ and H5′ phenyl),
7.55–7.60 (m, 2H, H4 and H7 benzimidazole), 7.91–7.98 (m,
Conclusion
Successfully, through the present study, a new series of 2H, H2′ and H6′ phenyl), 10.04 (br, 2H, OH enolic and NH
−1
potent HCV inhibitors having the benzimidazole-2-yl-methyl- benzimidazole, D O exchangeable); IR (KBr, cm ) 3365
2
thiopyrimidine scaffold with high potency was discovered. (NH benzimidazole), 3200–2500 (br, enolic OH), 3051 (CH
The docking results showed that the 2-(4-hydroxyphenyl)- arom.), 2963 (CH aliph.), 2212 (CN), 1628 (C=N benzimid-
pyrimidine is a good scaffold to be explored, and that 4-po- azole), 1530, 1505 (C=N pyrimidine, C=C); MS (EI) m/z 377
+
·
+·
sitioned substituents that can perform H-bonding interactions (M , 3%), 379 (M +2, 0.71%), 57 (100%). Anal. Calcd for
with the enzyme are important. C H FN OS: C, 60.47; H, 3.20; N, 18.56; S, 8.50. Found: C,
The most active compound 4d gave better results than Tela- 60.25; H, 3.45; N, 18.32; S, 8.24.
19
12
5
previr (VX-950). The presence of the 4-OH on the phenyl ring
2-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-4-(4-
linked to pyrimidine in compound 4d was found to have great hydroxyphenyl)-6-oxopyrimidine-5-carbonitrile (4d): Yield
1
impact on activity. The new potent compounds need further 74%; mp 200–202°C (from MeOH); H-NMR (500MHz,
pharmacological investigation to be used as HCV inhibitors in DMSO-d ) δ: 4.50 (s, 2H, CH –S), 7.01–7.20 (m, 2H, H5 and
6
2
the near future.
H6 benzimidazole), 7.31–7.67 (m, 4H, 4 phenyl H), 7.70–7.89
m, 2H, H4 and H7 benzimidazole), 8.20 (br, 1H, NH py-
(

September 2014
861
rimidine, D O exchangeable), 11.45 (s, 1H, OH phenolic,
2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(furan-2-
2
D O exchangeable), 12.40 (br, 1H, NH benzimidazole, D O yl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (4h): Yield
2
2
−1
1
exchangeable); IR (KBr, cm ) 3351 (NH benzimidazole), 80%; mp 223–225°C (from EtOH); H-NMR (200MHz,
3
259 (br, NH pyrimidine, H bonded OH phenolic), 3063 (CH DMSO-d ) δ: 7.12–7.20 (m, 2H, H3′ and H4′ furyl), 7.25–7.29
6
arom.), 2925 (CH aliph.), 2217 (CN), 1654 (C=O), 1608 (C=N (m, 1H, H5′ furyl), 7.48–7.52 (m, 2H, H5 and H6 benzimid-
benzimidazole), 1565, 1515 (C=N , C =C pyrimidine); MS (EI) azole), 7.88–8.02 (m, 2H, H4 and H7 benzimidazole), 8.42 (br,
+
·
+·
m/z 375 (M , 2%), 377 (M +2, 1%), 217 (100%). Anal. Calcd 1H, NH pyrimidine, D O exchangeable), 12.14 (br, 1H, NH
2
−1
for C H N O S: C, 60.78; H, 3.50; N, 18.66; S, 8.54. Found: benzimidazole, D O exchangeable); IR (KBr, cm ) 3446 (NH
19
13
5
2
2
C, 60.54; H, 3.42; N, 18.34; S, 8.28.
benzimidazole), 3139 (NH pyrimidine), 3034 (CH arom.), 2913
2
-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(2-chloro- (CH aliph.), 2216 (CN), 1655 (C=O), 1620 (C=N benzimid-
phenyl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (4e): azole), 1599, 1521 (C=N pyrimidine, C=C); MS (EI) m/z 349
1
+·
+·
Yield 76%; mp 210–212°C (from MeOH); H-NMR (500MHz, (M , 1%), m/z 351 (M +2, 1%), m/z 55 (100%). Anal. Calcd
DMSO-d ) δ: 4.51 (s, 2H, CH –S), 7.45–7.58 (m, 6H, H5 and for C H N O S: C, 58.44; H, 3.18; N, 20.05; S, 9.18. Found:
6
2
17 11
5
2
H6 benzimidazole+4 phenyl H), 7.70–7.88 (m, 2H, H4 and H7 C, 58.30; H, 3.47; N, 20.25; S, 9.23.
benzimidazole), 8.01 (br, 1H, NH pyrimidine, D O exchange-
Preparation of 2-((1H-Benzo[d]imidazol-2-yl)methyl-
2
able), 11.35 (br, 1H, NH benzimidazole, D O exchangeable); amino)-1,6-dihydro-4-(Aryl or Heteroaryl)-6-oxopyrimi-
2
−1
IR (KBr, cm ) 3387 (NH benzimidazole), 3136 (br, NH dine-5-carbonitriles (5a–h). General Method A solution
pyrimidine), 3022 (CH arom.), 2922 (CH aliph.), 2204 (CN), of compound 1 (2g, 12.05mmol) in dry dimethylformamide
1
652 (C=O), 1609 (C=N benzimidazole), 1561, 1514 (C=N (7mL), was added to a mixture of the appropriate aminopy-
+
·
pyrimidine, C=C); MS (EI) m/z 393/ 395 (M , 10%/4% Cl rimidine 3a–h (12.05mmol) and anhydrous potassium car-
pattern); 164 (100%). Anal. Calcd for C H ClN OS: C, 57.94; bonate (1.66g, 12.05mmol) in dimethylformamide (10mL).
19
12
5
H, 3.08; N, 17.78; S, 8.14. Found: C, 57.90; H, 2.95; N, 17.67; The reaction mixture was allowed to stir for 2h at room
S, 7.78.
temperature then heated under reflux for additional 10h. After
-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-4-(2- cooling, it was poured into ice-water with continuous stirring;
hydroxyphenyl)-6-oxopyrimidine-5-carbonitrile (4f): Yield the formed precipitate was filtered and recrystallized from the
2
1
7
3%; mp 193–195°C (from MeOH); H-NMR (500MHz, appropriate solvent.
DMSO-d ) δ: 4.44 (s, 2H, CH –S), 7.12–7.24 (m, 2H, H5 and
2-((1H-Benzo[d]imidazol-2-yl)methylamino)-1,6-dihydro-6-
H6 benzimidazole), 7.35–7.67 (m, 4H, 4 phenyl H), 7.75–7.90 oxo-4-phenyl pyrimidine-5-carbonitrile (5a): Yield 78%; mp
6
2
1
(
m, 2H, H4 and H7 benzimidazole), 8.25 (br, 1H, NH py- 116–118°C (from EtOH); H-NMR (200MHz, DMSO-d ) δ:
6
rimidine, D O exchangeable), 11.22 (s, 1H, OH phenolic, D O 5.25 (s, 2H, CH –NH), 6.47 (br, 1H, CH –NH, D O exchange-
2
2
2
2
2
exchangeable), 12.52 (br, 1H, NH benzimidazole, D O ex- able), 7.38–8.49 (m, 9H, Ar-H), 9.81 (br, 1H, NH pyrimidine,
2
−1
changeable); IR (KBr, cm ) 3354 (NH benzimidazole), 3206 D O exchangeable), 11.74 (br, 1H, NH benzimidazole, D O
NH pyrimidine, H bonded OH phenolic), 3030 (CH arom.), exchangeable); IR (KBr, cm ) 3388 (NH benzimidazole),
924 (CH aliph.), 2216 (CN), 1664 (C=O), 1627 (C=N b e n z - 3306 (NH–CH ), 3137 (NH pyrimidine), 3051 (CH arom.),
2
2
−1
(
2
2
imidazole), 1588, 1558 (C=N pyrimidine, C=C ) . Anal. Calcd 2928 (CH aliph.), 2210 (CN), 1688 (C=O), 1625 (C=N b e n z -
for C H N O S: C, 60.79; H, 3.49; N, 18.66; S, 8.54. Found: imidazole), 1593, 1517 (C=N pyrimidine, C=C); MS (EI) m/z
1
9
13
5
2
+
·
C, 60.57; H, 3.35; N, 18.42; S, 8.34
342 (M , 1%), m/z 91 (100%). Anal. Calcd for C H N O: C,
19 14 6
General Method for Preparation of 4g,h To a well 66.69; H, 4.09; N, 24.55. Found: C, 66.85; H, 4.34; N, 24.88.
3
2,33)
stirred mixture of the appropriate thiouracil 2g,h
(
1
1
2-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(4-chloro-
(5b):
12.05mmol) and anhydrous potassium carbonate (1.66g, phenyl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile
2.05mmol) in dry acetone (25mL), a solution of 1 (2g, Yield 75%; mp 195–197°C (from EtOH); H-NMR (200MHz,
2.05mmol) in dry acetone was added portion wise. The re- DMSO-d ) δ: 5.76 (s, 2H, CH –NH), 5.88 (br, 1H, CH –NH,
1
6
2
2
action mixture was stirred for 10h at room temperature then D O exchangeable), 6.60 (br, 1H, NH pyrimidine, D O ex-
2
2
heated under reflux for additional 6h, cooled, filtered and changeable), 7.24 (d, J=7.4Hz, 2H, H3′ and H5′ 4-Cl-phenyl),
the solid was washed with acetone. The organic solvent was 7.35–7.43 (m, 2H, H5 and H6 benzimidazole), 7.63–7.75 (m,
evaporated under reduced pressure and the residue was recrys- 2H, H4 and H7 benzimidazole), 7.89 (d, J=7.4Hz, 2H, H2′
tallized from the appropriate solvent.
and H6′ 4-Cl-phenyl), 11.80 (br, 1H, NH benzimidazole, D O
2
1
−
2
-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-6- exchangeable); IR (KBr, cm ) 3375 (NH benzimidazole,
oxo-4-(thiophen-2-yl)pyrimidine-5-carbonitrile (4g): Yield 81%; NH–CH ), 3102 (NH pyrimidine), 3051 (CH arom.), 2980 (CH
2
1
mp 200–202°C (from EtOH); H-NMR (200MHz, DMSO- aliph.), 2212 (CN), 1671 (C=O), 1619 (C=N benzimidazole),
d ) δ: 4.80 (s, 2H, CH –S), 7.16–7.24 (m, 2H, H3′ and H4′ 1597, 1517 (C=N pyrimidine, C=C); MS (EI) m/z 375 (M ,
+
·
6
2
thienyl), 7.28–7.33 (m, 1H, H5′ thienyl), 7.51–7.58 (m, 2H, H5 2%), 164 (100%). Anal. Calcd for C H ClN O: C, 60.56; H,
19
13
6
and H6 benzimidazole), 7.98–8.12 (m, 2H, H4 and H7 benz- 3.48; N, 22.31. Found: C, 60.86; H, 3.68; N, 22.64.
imidazole), 8.50 (br, 1H, NH pyrimidine, D O exchangeable), 2-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(4-fluoro-
2.20 (br, 1H, NH benzimidazole, D O exchangeable); IR phenyl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (5c):
KBr, cm ) 3380 (NH benzimidazole), 3156 (NH pyrimidine), Yield 80%; mp 170–172°C (from EtOH); H-NMR (200MHz,
059 (CH arom.), 2970 (CH aliph.), 2209 (CN), 1680 (C=O ) , DMSO-d ) δ: 5.74 (s, 2H, CH –NH), 6.15 (br, 1H, CH –NH,
2
1
(
2
−1
1
3
6
2
2
1
621 (C=N benzimidazole), 1550, 1525 (C=N pyrimidine, C= D O exchangeable), 7.22–7.30 (m, 2H, H5 and H6 benzimid-
2
·
+
C); MS (EI) m/z 365 (M , 2%), m/z 84 (100%). Anal. Calcd azole), 7.60–7.72 (m, 4H, phenyl H), 7.93–8.10 (m, 2H, H4
for C H N OS : C, 55.87; H, 3.04; N, 19.17; S, 17.55. Found: and H7 benzimidazole), 8.98 (br, 1H, NH pyrimidine, D O
1
7
11
5
2
2
C, 55.47; H, 3.43; N, 19.28; S, 17.60.
exchangeable), 12.82 (br, 1H, NH benzimidazole, D O ex-
2

8
62
Vol. 62, No. 9
−1
changeable); IR (KBr, cm ) 3468 (NH benzimidazole), 3323 (CN), 1657 (C=O), 1611 (C=N benzimidazole), 1566.0, 1506
+
·
(
(
NH–CH ), 3201 (NH pyrimidine), 3056 (CH arom.), 2926 (C=N pyrimidine, C=C); MS (EI) m/z 348 (M , 4%), 132
2
CH aliph.), 2201 (CN), 1678 (C=O), 1624 (C=N benzimid- (100%). Anal. Calcd for C H N OS: C, 58.60; H, 3.48; N,
17 12 6
azole), 1598, 1555 (C=N pyrimidine, C=C); MS (EI) m/z 361 24.13; S, 9.20. Found: C, 58.76; H, 3.75; N, 24.46; S, 9.26.
+
·
(
M +1, 22%), m/z 260 (100%). Anal. Calcd for C H FN O:
2-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(furan-2-
19
13
6
C, 63.32; H, 3.64; N, 23.33. Found: C, 63.14; H, 3.30; N, 23.48. yl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (5h): Yield
1
2
-((1H-Benzo[d]imidazol-2-yl)methylamino)-1,6-dihydro-4- 78%; mp 150–152°C (from MeOH); H-NMR (200MHz,
(
7
4-hydroxyphenyl)-6-oxopyrimidine-5-carbonitrile (5d): Yield DMSO-d ) δ: 4.90 (br, 1H, CH –NH, D O exchangeable),
6
2
2
1
8%; mp 230–232°C (from MeOH); H-NMR (200MHz, 5.88 (s, 2H, CH –NH), 7.12–7.28 (m, 3H, H5 and H6 benz-
2
DMSO-d ) δ: 4.88 (br, 1H, CH –NH, D O exchangeable), imidazole, H4′ furyl), 7.56–7.64 (m, 3H, H4 and H7 benz-
6
2
2
5
.50 (s, 2H, CH –NH), 7.12–7.18 (m, 2H, H3′ and H5′ phe- imidazole, H3′ furyl), 7.80–7.87 (m, 1H, H5′ furyl), 8.04 (br,
2
nyl), 7.29–7.46 (m, 2H, H5 and H6 benzimidazole), 7.62–7.78 1H, NH pyrimidine, D O exchangeable), 12.49 (br, 1H, NH
2
−1
(
m, 2H, H2′ and H6′ phenyl), 8.09–8.29 (m, 2H, H4 and H7 benzimidazole, D O exchangeable); IR (KBr, cm ) 3448
2
benzimidazole), 8.33 (br, 1H, NH pyrimidine, D O exchange- (NH benzimidazole), 3278 (NH–CH ), 3183 (NH pyrimidine),
2
2
able), 11.50 (br, 2H, OH phenolic and NH benzimidazole, 3030 (CH arom.), 2950 (CH aliph.), 2203 (CN), 1684 (C=O ) ,
−
1
D O exchangeable); IR (KBr, cm ) 3347 (NH benzimidazole, 1625 (C=N benzimidazole), 1591, 1513 (C=N pyrimidine, C=
2
+
·
NH–CH ), 3183 (br, NH pyrimidine, H bonded OH phenolic), C); MS (EI) m/z 332 (M , 2%), 262 (100%). Anal. Calcd for
2
3
056.6 (CH arom.), 2926 (CH aliph.), 2208 (CN), 1661 (C=O ) , C H N O : C, 61.44; H, 3.65; N, 25.29. Found: C, 61.23; H,
17 12 6 2
1610 (C=N benzimidazole), 1549, 1511 (C=N pyrimidine, C= 3.42; N, 25.53.
+
·
C); MS (EI) m/z 358 (M , 2%), 259 (100%). Anal. Calcd for
Preparation of 2-((1H-Benzo[d]imidazol-2-yl)methyl-
C H N O : C, 63.67; H, 3.95; N, 23.45. Found: C, 63.95; H, thio or Methylamino)-1,6-dihydro-4-(aryl or heteroaryl)-
19
14
6
2
4.00; N, 23.53.
6-oxopyrimidine-5-carboxylic Acids (6a–h, 7a–h). Gen-
2
-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(2-chloro- eral Method A solution of compounds 4a–h or 5a–h
phenyl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile
Yield 79%; mp 213–215°C (from EtOH); H-NMR (200MHz, 4h. The reaction mixture was cooled, filtered and the filtrate
(5e): (6.02mmol) in 80% sulphuric acid (5mL) was refluxed for
1
DMSO-d ) δ: 5.22 (s, 2H, CH –NH), 5.42 (br, 1H, CH –NH, was diluted with cold water and neutralized with aqueous am-
6
2
2
D O exchangeable), 7.01–7.22 (m, 2H, H5 and H6 benzimid- monium hydroxide. The formed precipitate was filtered, dried
2
azole), 7.29–7.68 (m, 6H, H4 and H7 benzimidazole, phenyl and recrystallized from dimethylformamide/ethanol.
H), 8.33 (br, 1H, NH pyrimidine, D O exchangeable), 13.02
2-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-6-
2
(
br, 1H, NH benzimidazole, D O exchangeable); IR (KBr, oxo-4-phenylpyrimidine-5-carboxylic Acid (6a): Yield 95%;
2
−1
1
cm ) 3401 (NH benzimidazole, NH–CH ), 3151 (NH py- mp >300°C (from MeOH); H-NMR (200MHz, DMSO-d )
rimidine), 3058 (CH arom.), 2922 (CH aliph.), 2219 (CN), 1652 δ: 5.82 (s, 2H, CH –S), 6.85 (br, 1H, NH pyrimidine, D O
2
6
2
2
(
C=O), 1623 (C=N benzimidazole), 1549, 1511 (C=N pyrimi- exchangeable), 7.12–7.30 (m, 2H, H5 and H6 benzimidazole),
+
·
dine, C=C); MS (EI) m/z 376/378 (M , 20%/7% Cl pattern), 7.34–7.53 (m, 3H, H3′, H4′, H5′ phenyl), 7.73–7.85 (m, 2H,
32 (100%). Anal. Calcd for C H ClN O: C, 60.56; H, 3.48; H2′ and H6′ phenyl), 8.03–8.15 (m, 2H, H4 and H7 benzimid-
1
19
13
6
N, 22.31. Found: C, 60.85; H, 3.64; N, 22.67.
azole), 11.15 (br, 1H, COOH, D O exchangeable), 11.71 (br,
2
−1
2
-((1H-Benzo[d]imidazol-2-yl)methylamino)-1,6-dihydro-4- 1H, NH benzimidazole, D O exchangeable); IR (KBr, cm )
2
(
7
2-hydroxyphenyl)-6-oxopyrimidine-5-carbonitrile (5f): Yield 3417 (NH benzimidazole), 3127 (br, H bonded OH carboxylic,
4%; mp 256–258°C (from MeOH); H-NMR (200MHz, NH pyrimidine), 3050 (CH arom.), 2937 (CH aliph.), 1705
1
DMSO-d ) δ: 5.33 (s, 2H, CH –NH), 5.38 (br, 1H, CH –NH, (C=O carboxylic), 1661 (C=O amide), 1620 (C=N benzimid-
6
2
2
D O exchangeable), 7.05–7.28 (m, 2H, H5 and H6 benzimid- azole), 1509, 1560 (C=N pyrimidine, C=C ) . Anal. Calcd for
2
azole), 7.30–7.65 (m, 6H, H4 and H7 benzimidazole, phenyl C H N O S: C, 60.31; H, 3.74; N, 14.81; S, 8.47. Found: C,
19
14
4
3
H), 8.33 (br, 1H, NH pyrimidine, D O exchangeable), 13.14 60.65; H, 4.00; N, 15.02; S, 8.78.
2
(
br, 2H, OH phenolic and NH benzimidazole, D O exchange-
2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(4-chloro-
2
−1
able); IR (KBr, cm ) 3428 (NH benzimidazole), 3321 (br, phenyl)-1,6-dihydro-6-oxopyrimidine-5-carboxylic Acid (6b):
NH–CH , H bonded OH phenolic), 3108 (NH pyrimidine), Yield 90%; mp >300°C (from MeOH); H-NMR (200MHz,
3
1
2
055 (CH arom.), 2933 (CH aliph.), 2213 (CN), 1664 (C=O ) , DMSO-d ) δ: 5.83 (s, 2H, CH –S), 7.22 (br, 1H, NH pyrimi-
6
2
1
612 (C=N benzimidazole), 1581.0, 1521 (C=N pyrimidine, dine, D O exchangeable), 7.54–7.57 (m, 4H, H5 and H6 benz-
2
·
+
C=C); MS (EI) m/z 357 (M , 4%), 57.27 (100%). Anal. Calcd imidazole, H3′ and H5′ phenyl), 7.73–7.76 (m, 4H, H4 and H7
for C H N O : C, 63.67; H, 3.95; N, 23.45. Found: C, 63.93; benzimidazole, H2′ and H6′ phenyl),11.15 (br, 2H, COOH and
H, 3.61; N, 23.59.
19
14
6
2
13
NH benzimidazole, D O exchangeable); C-NMR (70MHz,
2
2
-((1H-Benzo[d]imidazol-2-yl)methylamino)-1,6-dihydro-6- DMSO-d ) δ: 27.0 (CH ), 119.0–139.0 (Ar-C), 149.0–154.0 (C=
6
2
oxo-4-(thiophen-2-yl)pyrimidine-5-carbonitrile (5g): Yield N groups), 162.5 (C=O amide), 168.0 (C=O carboxylic); IR
1
−1
8
1%; mp 185–187°C (from MeOH); H-NMR (200MHz, (KBr, cm ) 3419 (NH benzimidazole), 3281 (br, H bonded
DMSO-d ) δ: 4.84 (br, 1H, CH –NH, D O exchangeable), 5.40 OH carboxylic), 3179 (NH pyrimidine), 3055 (CH arom.), 2960
6
2
2
(
2
s, 2H, CH –NH), 7.05–7.40 (m, 3H, thienyl H), 7.45–7.82 (m, (CH aliph.), 1702 (C=O carboxylic), 1654 (C=O amide), 1624
H, H5 and H6 benzimidazole), 7.96–8.10 (m, 2H, H4 and H7 (C=N benzimidazole), 1541, 1521 (C=N pyrimidine, C=C ) .
2
benzimidazole), 8.88 (br, 1H, NH pyrimidine, D O exchange- Anal. Calcd for C H ClN O S: C, 55.27; H, 3.18; N, 13.57; S,
2
19 13
4
3
able), 12.96 (br, 1H, NH benzimidazole, D O exchangeable); 7.77. Found: C, 55.56; H, 3.52; N, 13.86; S, 7.90.
2
−1
IR (KBr, cm ) 3347 (NH benzimidazole, NH–CH ), 3152
2-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(4-fluoro-
2
(
NH pyrimidine), 3055 (CH arom.), 2928 (CH aliph.), 2215 phenyl)-1,6-dihydro-6-oxopyrimidine-5-carboxylic Acid (6c):

September 2014
863
1
−1
Yield 91%; mp >300°C (from MeOH); H-NMR (200MHz, benzimidazole, D O exchangeable); IR (KBr, cm ) 3406 (NH
2
DMSO-d ) δ: 5.53 (s, 2H, CH –S), 7.28 (br, 1H, NH py- benzimidazole), 3216 (br, H bonded OH carboxylic), 3109
6
2
rimidine, D O exchangeable), 7.42–7.53 (m, 4H, H5 and H6 (NH pyrimidine), 3055 (CH arom.), 2923 (CH aliph.), 1701
2
benzimidazole, H3′ and H5′ phenyl), 7.66–7.73 (m, 4H, H4 (C=O carboxylic), 1654 (C=O amide), 1626 (C=N benzimid-
and H7 benzimidazole, H2′ and H6′ phenyl), 11.22 (br, 2H, azole), 1562, 1530 (C=N pyrimidine, C=C ) . Anal. Calcd for
COOH and NH benzimidazole, D O exchangeable); IR (KBr, C H N O S : C, 53.11; H, 3.15; N, 14.58; S, 16.68. Found: C,
2
17 12
4
3 2
−1
cm ) 3406 (NH benzimidazole), 3216 (br, H bonded OH car- 53.42; H, 3.35; N, 14.75; S, 16.23.
boxylic), 3109 (NH pyrimidine), 3055 (CH arom.), 2923 (CH
2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(furan-2-
aliph.), 1701 (C=O carboxylic), 1654 (C=O amide), 1626 (C= yl)-1,6-dihydro-6-oxopyrimidine-5-carboxylic Acid (6h): Yield
1
N benzimidazole), 1562, 1530 (C=N pyrimidine, C=C ) . Anal. 94%; mp >300°C (from MeOH); H-NMR (200MHz, DMSO-
Calcd for C H FN O S: C, 57.57; H, 3.31; N, 14.14; S, 8.09. d ) δ: 5.23 (s, 2H, CH –S), 5.79–5.88 (m, 1H, H4′ furyl),
19
13
4
3
6
2
Found: C, 57.96; H, 3.56; N, 14.47; S, 8.32.
-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-4- 7.54–7.63 (m, 3H, H4 and H7 benzimidazole, H5′ furyl), 8.00
4-hydroxyphenyl)-6-oxopyrimidine-5-carboxylic Acid (6d): (br, 1H, NH pyrimidine, D O exchangeable), 12.40 (br, 2H,
7.13–7.31 (m, 3H, H5 and H6 benzimidazole, H3′ furyl),
2
(
2
1
Yield 96%; mp >300°C (from MeOH); H-NMR (200MHz, COOH and NH benzimidazole, D O exchangeable); IR (KBr,
DMSO-d ) δ: 5.50 (s, 2H, CH –S), 7.21–7.29 (m, 2H, H3′ and cm ) 3388 (NH benzimidazole), 3176 (br, H bonded OH car-
2
−1
6
2
H5′ phenyl), 7.34–7.45 (m, 2H, H5 and H6 benzimidazole), boxylic, NH pyrimidine), 3056 (CH arom.), 2920 (CH aliph.),
.52–7.58 (m, 2H, H2′ and H6′ phenyl), 8.12–8.22 (m, 2H, H4 1700 (C=O carboxylic), 1654 (C=O amide), 1617 (C=N b e n z -
7
and H7 benzimidazole), 8.35 (br, 1H, NH pyrimidine, D O imidazole), 1580, 1532 (C=N pyrimidine, C=C ) . Anal. Calcd
2
exchangeable), 10.50 (s, 1H, OH phenolic D O exchangeable); for C H N O S: C, 55.43; H, 3.29; N, 15.21; S, 8.70. Found:
2
17 12
4
4
1
1.15 (br, 2H, COOH and NH benzimidazole, D O exchange- C, 55.80; H, 3.19; N, 15.52; S, 8.98.
2
−1
able); IR (KBr, cm ) 3400 (NH benzimidazole), 3184 (br,
2-((1H-Benzo[d]imidazol-2-yl)methylamino)-1,6-dihydro-6-
H bonded OH carboxylic), 3109 (NH pyrimidine), 3055 (CH oxo-4-phenylpyrimidine-5-carboxylic Acid (7a): Yield 94%;
1
arom.), 2936 (CH aliph.), 1701 (C=O carboxylic), 1654 (C=O mp >300°C (from MeOH); H-NMR (200MHz, DMSO-d ) δ:
6
amide), 1627 (C=N benzimidazole), 1560, 1540 (C=N pyrimi- 5.07 (s, 2H, CH –NH), 6.31 (br, 1H, CH –NH, D O exchange-
2
2
2
dine, C=C ) . Anal. Calcd for C H N O S: C, 57.86; H, 3.58; able), 7.40–8.35 (m, 9H, Ar-H), 9.72 (br, 1H, NH pyrimidine,
19
14
4
4
N, 14.21; S, 8.13. Found: C, 58.00; H, 3.62; N, 14.52; S, 8.45.
D O exchangeable), 10.83 (br, 1H, COOH, D O exchangeable),
2
2
2
-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(2-chloro- 11.85 (br, 1H, NH benzimidazole, D O exchangeable); IR
2
−1
phenyl)-1,6-dihydro-6-oxopyrimidine-5-carboxylic Acid (6e): (KBr, cm ) 3406 (NH benzimidazole), 3219 (br, H bonded
Yield 93%; mp >300°C (from MeOH); H-NMR (200MHz, OH carboxylic, NH–CH ), 3117 (NH pyrimidine), 3053 (CH
DMSO-d ) δ: 5.36 (s, 2H, CH –S), 7.29–7.38 (m, 2H, H5 arom.), 2990 (CH aliph.), 1700 (C=O carboxylic), 1651 (C=O
1
2
6
2
and H6 benzimidazole), 7.83–7.90 (m, 6H, H4 and H7 benz- amide), 1619 (C=N benzimidazole), 1559, 1521 (C=N pyrimi-
imidazole, 4 phenyl H), 8.10 (br, 1H, NH pyrimidine, D O dine, C=C ) . Anal. Calcd for C H N O : C, 63.15; H, 4.19; N,
2
19 15
5
3
exchangeable), 9.90 (br, 1H, COOH, D O exchangeable), 12.50 19.38. Found: C, 63.45; H, 4.32; N, 19.55.
2
(
br, 1H, NH benzimidazole, D O exchangeable); IR (KBr,
2-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(4-chloro-
2
−1
cm ) 3379 (NH benzimidazole), 3230 (br, H bonded OH car- phenyl)-1,6-dihydro-6-oxopyrimidine-5-carboxylic Acid (7b):
boxylic), 3174 (NH pyrimidine), 3054 (CH arom.), 2930 (CH Yield 93%; mp >300°C (from MeOH); H-NMR (200MHz,
aliph.), 1707 (C=O carboxylic), 1664 (C=O amide), 1624 (C= DMSO-d ) δ: 5.74 (s, 2H, CH –NH), 6.13 (br, 1H, CH –NH,
1
6
2
2
N benzimidazole), 1560, 1542 (C=N pyrimidine, C=C ) . Anal. D O exchangeable), 7.09–7.25 (m, 2H, H5 and H6 benzimid-
2
Calcd for C H ClN O S: C, 55.27; H, 3.18; N, 13.57; S, 7.77. azole), 7.37 (d, J=8Hz, 2H, H3′ and H5′ phenyl), 7.51–7.62
19
13
4
3
Found: C, 55.55; H, 3.42; N, 13.77; S, 7.80.
(m, 2H, H4 and H7 benzimidazole), 7.77 (d, J=8Hz, 2H, H2′
2
-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-4- and H6′ phenyl), 8.00 (br, 1H, NH pyrimidine, D O exchange-
2
(
2-hydroxyphenyl)-6-oxopyrimidine-5-carboxylic Acid (6f): able), 10.13 (br, 1H, COOH, D O exchangeable), 12.60 (br,
Yield 91%; mp >300°C (from MeOH); H-NMR (200MHz, 1H, NH benzimidazole, D O exchangeable); IR (KBr, cm )
DMSO-d ) δ: 5.38 (s, 2H, CH –S), 7.08–7.62 (m, 8H, Ar-H), 3406 (NH benzimidazole), 3222 (br, H bonded OH carboxylic,
2
1
−1
2
6
2
8
.81 (br, 2H, OH phenolic and NH pyrimidine, D O exchange- NH–CH ), 3109 (NH pyrimidine), 3056 (CH arom.), 2920 (CH
2
2
able), 9.04 (br, 2H, COOH and NH benzimidazole, D O ex- aliph.), 1715 (C=O carboxylic), 1650 (C=O amide), 1609 (C=
2
−1
changeable); IR (KBr, cm ) 3390 (NH benzimidazole), 3212 N benzimidazole), 1559, 1539 (C=N pyrimidine, C=C ) . Anal.
br, H bonded OH carboxylic and OH phenolic, NH pyrimi- Calcd for C H ClN O : C, 57.65; H, 3.57; N, 17.70. Found: C,
(
19
14
5
3
dine), 3056 (CH arom.), 2920 (CH aliph.), 1702 (C=O carbox- 57.43; H, 3.67; N, 17.86.
ylic), 1650 (C=O amide), 1609 (C=N benzimidazole), 1562, 2-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(4-fluoro-
540 (C=N pyrimidine, C=C ) . Anal. Calcd for C H N O S: phenyl)-1,6-dihydro-6-oxopyrimidine-5-carboxylic Acid (7c):
1
19
14
4
4
1
C, 57.86; H, 3.58; N, 14.21; S, 8.13. Found: C, 57.66; H, 3.74; Yield 93%; mp >300°C (from MeOH); H-NMR (200MHz,
N, 14.52; S, 8.32. DMSO-d ) δ: 5.81 (s, 2H, CH –NH), 6.85 (br, 1H, CH –NH,
-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-6- D O exchangeable), 7.10 (br, 1H, NH pyrimidine, D O ex-
6
2
2
2
2
2
oxo-4-(thiophen-2-yl)pyrimidine-5-carboxylic Acid (6g): Yield changeable), 7.30–7.39 (m, 4H, H5 and H6 benzimidazole, H3′
1
9
3%; mp >300°C (from MeOH); H-NMR (200MHz, and H5′ phenyl), 7.77–7.84 (m, 4H, H4 and H7 benzimidazole,
DMSO-d ) δ: 5.84 (s, 2H, CH –S), 7.15–7.42 (m, 3H, thienyl H2′ and H6′ phenyl), 11.12 (br, 2H, COOH and NH benz-
6
2
13
H), 7.46–7.83 (m, 2H, H5 and H6 benzimidazole), 7.89–8.12 imidazole, D O exchangeable); C-NMR (70MHz, DMSO-
2
(
m, 2H, H4 and H7 benzimidazole), 8.58 (br, 1H, NH py- d ) δ: 27.0 (CH ), 114.0–137.0 (Ar-C), 151.0–165.0 (C–F, C=
6 2
rimidine, D O exchangeable), 12.94 (br, 2H, COOH and NH N groups), 166.0 (C=O amide), 167.5 (C=O carboxylic); IR
2

8
64
Vol. 62, No. 9
−1
(
KBr, cm ) 3420 (NH benzimidazole), 3166 (br, H bonded O carboxylic), 1659 (C=O amide), 1624 (C=N benzimid-
OH carboxylic, NH–CH , NH pyrimidine), 3033 (CH arom.), azole) 1560, 1542 (C=N pyrimidine, C=C ) . Anal. Calcd for
2
2
982 (CH aliph.), 1713 (C=O carboxylic), 1668 (C=O amide), C H N O S: C, 55.58; H, 3.57; N, 19.07; S, 8.73. Found: C,
17 13 5 3
1620 (C=N benzimidazole), 1543, 1512 (C=N pyrimidine, C= 55.86; H, 3.76; N, 19.35; S, 8.84.
C). Anal. Calcd for C H FN O : C, 60.16; H, 3.73; N, 18.47.
2-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(furan-2-
19
14
5
3
Found: C, 60.45; H, 3.86; N, 18.67.
yl)-1,6-dihydro-6-oxopyrimidine-5-carboxylic Acid (7h): Yield
1
2
-((1H-Benzo[d]imidazol-2-yl)methyamino)-1,6-dihydro-4- 92%; mp >300°C (from MeOH); H-NMR (200MHz, DMSO-
(
4-hydroxyphenyl)-6-oxopyrimidine-5-carboxylic Acid (7d): d ) δ: 4.88 (br, D O exchangeable, 1H, CH –NH), 5.85 (s,
6
2
2
1
Yield 96%; mp >300°C (from MeOH); H-NMR (200MHz, 2H, CH –NH), 7.04–7.25 (m, 3H, H5 and H6 benzimidazole,
2
DMSO-d ) δ: 5.23 (br, 1H, CH –NH, D O exchangeable), 5.50 H4′ furyl), 7.52–7.61 (m, 3H, H4 and H7 benzimidazole, H3′
6
2
2
(
7
s, 2H, CH –NH), 7.21–7.29 (m, 2H, H3′ and H5′ phenyl), furyl), 7.79–7.83 (m, 1H, H5′ furyl), 8.22 (br, 1H, NH py-
2
.34–7.45 (m, 2H, H5 and H6 benzimidazole), 7.52–7.58 (m, rimidine, D O exchangeable), 12.54 (br, 2H, COOH and NH
2
−1
2
H, H2′ and H6′ phenyl), 8.12–8.22 (m, 2H, H4 and H7 benz- benzimidazole, D O exchangeable); IR (KBr, cm ) 3300 (NH
2
imidazole), 8.35 (br, 1H, NH pyrimidine, D O exchangeable), benzimidazole, NH–CH ), 3159 (br, H bonded OH carboxylic,
2
2
1
0.50 (s, 1H, OH phenolic D O exchangeable); 11.15 (br, 2H, NH pyrimidine), 3050 (CH arom.), 2929 (CH aliph.), 1698
2
COOH and NH benzimidazole, D O exchangeable); IR (KBr, (C=O carboxylic, amide), 1620 (C=N benzimidazole), 1558,
2
−1
cm ) 3425 (NH benzimidazole), 3166 (br, H bonded OH car- 1538 (C=N pyrimidine, C=C ) . Anal. Calcd for C H N O : C,
boxylic and OH phenolic, NH–CH , NH pyrimidine), 3056 58.12; H, 3.74, N ; 19.94. Found: C, 58.32; H, 3.52; N, 19.75.
17
13
5
4
2
(
CH arom.), 2923 (CH aliph.), 1702 (C=O carboxylic), 1650
Hepatitis C Virus (HCV) Viral RNA Replication Inhibi-
(C=O amide), 1611 (C=N benzimidazole), 1546, 1520 (C=N tory Activity. Generation of HCV Replicon Cells The
8)
pyrimidine, C=C ) . Anal. Calcd for C H N O : C, 60.47; H, applied method was previously described by Lin et al. Paren-
4
19
15
5
4
.01; N, 18.56. Found: C, 60.64; H, 3.95; N, 18.75.
tal Huh-7 cells were cultured in Dulbecco’s modified Eagle’s
2
-((1H-Benzo[d]imidazol-2-yl)methylamino)-4-(2-chloro- medium (DMEM; JRH Biosciences, Lenexa, KS, U.S.A.)
phenyl)-1,6-dihydro-6-oxopyrimidine-5-carboxylic Acid (7e): containing 10% heat-inactivated fetal bovine serum (ΔFBS;
1
Yield 96%; mp >300°C (from MeOH); H-NMR (200MHz, JRH Biosciences), 2mM L-glutamine, and nonessential amino
DMSO-d ) δ: 4.90 (br, 1H, CH –NH, D O exchangeable), 5.50 acids (JRH Biosciences). The cells were transfected with an
6
2
2
(
s, 2H, CH –NH), 7.19–7.41 (m, 2H, H5 and H6 benzimid- in vitro-transcribed subgenomic HCV replicon RNA whose
2
azole), 7.54–7.79 (m, 6H, H4 and H7 benzimidazole and 4 sequence was identical to that of the I377neo/NS3-3′/wt repli-
4
0)
phenyl H), 9.40 (br, 1H, NH pyrimidine, D O exchangeable), con described by Lohmann et al. Stable cells containing the
2
1
0.50 (br, 2H, COOH and NH benzimidazole, D O exchange- self-replicating HCV replicon were selected and maintained
2
−1
able); IR (KBr, cm ) 3390 (NH benzimidazole), 3216 (br, in the presence of 250µg of G418 (Invitrogen, Carlsbad, CA,
H bonded OH carboxylic, NH–CH ), 3125 (NH pyrimidine), U.S.A.) per mL and were used for HCV replicon assays.
2
3
053 (CH arom.), 2930 (CH aliph.), 1700 (C=O carboxylic),
HCV Replicon Assay HCV replicon cells were plated in
4
1
651 (C=O amide), 1619 (C=N benzimidazole), 1560, 1538 a 96-well plate at a density of 10 cells per well in DMEM
(C=N pyrimidine, C=C ) . Anal. Calcd for C H ClN O : C, with 10% ΔFBS. On the following day (ca. 16h later), the
19 14 5 3
57.65; H, 3.57; N, 17.70. Found: C, 57.78; H, 3.77; N, 17.57.
culture medium was replaced with DMEM containing either
2
-((1H-Benzo[d]imidazol-2-yl)methylamino)-1,6-dihydro-4- no compound as a control or compounds serially diluted
(
2-hydroxyphenyl)-6-oxopyrimidine-5-carboxylic Acid (7f): in the presence of 2% ΔFBS and 0.5% dimethyl sulfoxide
1
Yield 90%; mp >300°C (from MeOH); H-NMR (200MHz, (DMSO). After the cells were incubated with the compounds
DMSO-d ) δ: 5.01 (br, 1H, CH –NH, D O exchangeable), 5.30 for 48h, the intracellular RNA was extracted with an RNeasy
6
2
2
(
s, 2H, CH –NH), 7.60–7.87 (m, 8H, Ar-H), 8.01 (br, 1H, NH 96 kit (Qiagen, Valencia, CA, U.S.A.). The level of HCV
2
pyrimidine, D O exchangeable), 10.22 (s., 1H, OH phenolic, RNA, both the positive strand and the negative strand, was
2
D O exchangeable), 11.82 (br, 1H, COOH, D O exchangeable), determined by a real-time multiplex quantitative reverse
2
2
1
3.32 (br, 1H, NH benzimidazole, D O exchangeable); IR transcription-polymerase chain reaction (RT-PCR) assay (the
2
−1
(
KBr, cm ) 3406 (NH benzimidazole), 3225 (br, H bonded Taqman assay) with a pair of HCV-specific primers (5′-CCA
OH carboxylic and OH phenolic, NH–CH ), 3056 (CH arom.), TGA ATC ACT CCC CTG TG-3′ and 5′-CCG GTC GTC
2
2
957 (CH aliph.), 1719 (C=O carboxylic), 1651 (C=O amide), CTG GCA ATT C-3′), an HCV-specific probe (5′,6-FAM-CCT
617 (C=N benzimidazole), 1559, 1533 (C=N pyrimidine, C= GGA GGC TGC ACG ACA CTC A-TAMRA-3′, where FAM
C). Anal. Calcd for C H N O : C, 60.47; H, 4.01; N, 18.56. is 6-carboxyfluorescein and TAMRA is 6-carboxytetrameth-
1
19
15
5
4
Found: C, 60.27; H, 3.75; N, 18.65.
-((1H-Benzo[d]imidazol-2-yl)methylamino)-1,6-dihydro-6- system (Applied Biosystems, Foster City, CA, U.S.A.). In
oxo-4-(thiophen-2-yl)pyrimidine-5-carboxylic Acid (7g): Yield each experiment, each datum point represents the average
ylrhodamine), and an ABI Prism 7700 sequence detection
2
1
9
1%; mp >300°C (from MeOH); H-NMR (200MHz, DMSO- of five replicates in cell culture. The IC50 was defined as the
d ) δ: 5.19 (br, 1H, CH –NH, D O exchangeable), 5.79 (s, 2H, concentration of compound at which the HCV RNA level in
6
2
2
CH –NH), 7.18–7.37 (m, 3H, thienyl H), 7.55–7.65 (m, 2H, H5 the replicon cells was reduced by 50%. To monitor any cy-
2
and H6 benzimidazole), 7.95–8.12 (m, 2H, H4 and H7 benz- totoxic effect, the viabilities of the replicon cells following
imidazole), 8.85 (br, 1H, NH pyrimidine, D O exchangeable), 48h of treatment with compound were determined by using
2
1
2.45 (br, 1H, COOH, D O exchangeable), 12.90 (br, 1H, NH a tetrazolium compound (3-[4,5-dimethylthiazol-2-yl]-5-[3-
2
−1
benzimidazole, D O exchangeable); IR (KBr, cm ) 3375 (NH carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium,
2
benzimidazole), 3170 (br, H bonded OH carboxylic, NH–CH2, inner salt (MTS))-based assay (CellTiter 96 AQueous One
NH pyrimidine), 3052 (Ar-H), 2930 (CH aliph.), 1705 (C= Solution Cell Proliferation Assay; Promega, Madison, WI,

September 2014
865
U.S.A.). The CC50 was defined as the concentration of the ChemScore function estimates the free energy of binding of
compound at which cell viability was reduced by 50%. The the ligand to a protein.
data obtained are presented in Table 1.
Docking. Generation of Structures and Exploration of
Acknowledgment This study was supported by the “US–
the Conformational Space Structure building and refine- Egypt Joint Science and Technology Board Fund” adminis-
ment for 4d and 6d was accomplished using SYBYLX 1.1 tered through the USDA (BIO9-002-015).
41)
molecular modeling software. The obtained structures were
used in this study and will be used in another 3D-QSAR References
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