Design, synthesis, assessment, and molecular docking of novel pyrrolopyrimidine (7-deazapurine) derivatives as non-nucleoside hepatitis C virus NS5B polymerase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.03.046

Hepatitis C virus (HCV) infection is highly persistent and presents an unmet medical need requiring more effective treatment options. This has spurred intensive efforts to discover novel anti-HCV agents. The RNA-dependent RNA polymerase (RdRp), NS5B of HC

Full text of DOI:10.1016/j.bmc.2016.03.046

Accepted Manuscript
Design, Synthesis, Assessment, and Molecular Docking of Novel Pyrrolopyri-
midine (7-deazapurine) Derivatives as Non-Nucleoside Hepatitis C Virus NS5B
Polymerase Inhibitors
Mosaad S. Mohamed, Amira I. Sayed, Mohammed A. Khedr, Sameh H. Soror
PII:
S0968-0896(16)30208-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.046
BMC 12895
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 January 2016
16 March 2016
27 March 2016
Please cite this article as: Mohamed, M.S., Sayed, A.I., Khedr, M.A., Soror, S.H., Design, Synthesis, Assessment,
and Molecular Docking of Novel Pyrrolopyrimidine (7-deazapurine) Derivatives as Non-Nucleoside Hepatitis C
Virus NS5B Polymerase Inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/
j.bmc.2016.03.046
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Design, Synthesis, Assessment, and Molecular Docking of Novel
Pyrrolopyrimidine (7-deazapurine) Derivatives as Non-Nucleoside Hepatitis C
Virus NS5B Polymerase Inhibitors

Mosaad S. Mohamed a, Amira I. Sayed a, Mohammed A. Khedrb, Sameh H. Soror c,d
a) Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Ain
Helwan, Cairo, Egypt.
b) Department of pharmaceutical chemistry, faculty of pharmacy, Helwan University, Ain Helwan,
Cairo, Egypt.
c) Biochemistry and Molecular Biology Department, Faculty of Pharmacy, Helwan University, Ain
Helwan, Cairo, Egypt.
d) Center for scientific excellence “Helwan Structural Biology Research (HSBR)”, Cairo, Egypt.
Corresponding author: sameh_soror@pharm.helwan.edu.eg, Fax;00202-2554-1601
Department of Biochemistry and molecular Biology,
Faculty of Pharmacy,
Helwan University Ain Helwan,
University campus, Postcode 11795
Cairo, Egypt
1

Abstract
Hepatitis C virus (HCV) infection is highly persistent and presents an unmet medical need requiring
more effective treatment options. This has spurred intensive efforts to discover novel anti-HCV
agents. The RNA-dependent RNA polymerase (RdRp), NS5B of HCV, constitutes a selective target
for drug discovery due to its absence in human cells; also, it is the centerpiece for viral replication.
Here, we synthesized novel pyrrole, pyrrolo[2,3-d]pyrimidine and pyrrolo[3,2-e][1,2,4]triazolo[4,3-
c]pyrimidine derivatives. The non-toxic doses of these compounds on Huh 7.5 cell line were
determined and their antiviral activity against HCVcc genotype 4a was examined. Compounds 7j, 7f,
5c, 12i and 12f showed significant anti HCV activity. The percent of reduction for the non-toxic
doses of 7j, 7f, 5c, 12i and 12f were 90 %, 76.7±5.8%, 73.3±5.8%, 70% and 63.3±5.8% respectively.
The activity of these compounds was interpreted by molecular docking against HCV NS5B
polymerase enzyme.
2

1. Introduction
Hepatitis C virus (HCV) is one of the major global health problems. HCV infection is
responsible for 350,000 death cases annually [1]. HCV infection is particularly a national problem in
Egypt. It is one of the top five death-causing diseases in the country. The incidence rate reaches
14.5 % among Egyptian population, which represents the highest prevalence of hepatitis C worldwide
[2]. According to the Egyptian ministry of health, 1,000,000 new cases are identified each year [3].
Furthermore, some independent studies have reported that 500,000 new patients are added to HCV
victims in Egypt each year, among them 70,000 are children [4].
There are six major genotypes of HCV and they differ in their geographical distribution.
Genotype 1 is the most common HCV genotype around the world: the subtype 1a and 1b are
prevalent in USA and Europe respectively, whereas, genotype 4 is the dominant HCV variant in the
Egyptian population [5].
The standard of care (SOC) for chronic HCV infection is using the combination of pegylated
interferon-α and ribavirin. This therapy is poorly tolerated, and there is variation in response among
HCV genotypes. The sustained virological response (SVR) obtained by the combination therapy was
markedly higher in patients infected with genotype 2 or genotype 3 than those infected with genotype
1 or genotype 4. Several studies have reported that interferon therapy is partially ineffective against
genotype 4 which represent an obstacle in the treatment of Egyptian patients [6,7]. There are
tremendous efforts to achieve interferon-free HCV-therapy through using direct-acting antivirals
(DAAs) [8].
Replication of the HCV viral RNA is controlled by host factors as well as viral factors, and
both can serve as drug targets. Possible direct targets in HCV are the viral NS3/4A protease, NS4B,
NS5A, and NS5B polymerase [8]. Recently, FDA has approved different DAAs Anti-HCV:
Telaprevir and Boceprevir, which are NS3/4A protease inhibitors, Daclatasvir and Ledispasvir as
3

NS5A inhibitors and Sofosbuvir, which is a nucleotide analog polymerase inhibitor. A new regime
for HCV has been developed recently depending on the NS5B inhibitor “Sofosbuivir” in combination
either with Ribavirin or Ribavirin and Interferon [1]. Other Interferon- and Ribavirin free-
combinations are in clinical trials [9,10].
The HCV polymerase (NS5B) is an essential enzyme in the life cycle of the hepatitis C virus.
The main role of NS5B is to assemble the replicase complex at the endoplasmic reticulum membrane
and amplify the genetic material through RNA-dependent RNA polymerase (RdRp) activity. Due to
its central role in the virus replication, and its absence in human body it became a preferable selective
target during the design of anti-HCV drugs [11,12]. The NS5B RdRp shares the structural
characteristics of nucleic acid polymerase. NS5B RdRp is composed of three domains: thumb, finger
and palm. The catalytic site is located at the interface of the thumb and finger domains (Figure 1),
which represents an interesting target for novel anti-HCV drugs [13].
Figure 1: 3D structure of RdRp of HCV
4

The HCV NS5B polymerase inhibitors are classified as nucleoside inhibitors, e.g.
toyocamycin, triciribine derivatives and Valopicitabine (Figure 2a) [14-23], and non-nucleoside
inhibitors (NNIs), e.g. Deleobuvir, Tegobuvir, Filibuvir and sterobuvir (Figure 2b). Purine
nucleoside analogs as pyrrolopyrimidine nucleosides are good examples of HCV NS5B polymerase
inhibitors [16-23]. To date there is no pyrrolopyrimidine non-nucleosides that are acting as HCV
NS5B polymerase inhibitors. The main aim of this work was to synthesize pyrrolopyrimidine
derivatives as non-nucleoside purine analogs and evaluate their antiviral activity against HCV
genotype 4. Finally, we will predict the binding mode of the synthesized compounds by using
molecular docking against HCV NS5B polymerase.
X
NH2
Et
Y
N
N
N
N
H2N
N
HO
HO
N
N
O
N
O
HO
O
N
N
Me
OH
Z
O
O
O
Me
Me
HO
OH
Y
CN
X
Z
H
H
H
Me
HO
OH
1, S-Me
2, S-Me
NH2
Valopicitabin (NM283) 6
C(=NH)-NHOH
3, NH-Me C(=NH)-NHOH
Triciribine derivative 5
4,
NH2
H
Toyocamycin derivatives 1-4
Figure 2a: NS5B nucleoside inhibitors as anti HCV
5

O
HO
F
F
F
F
F
F
F
N
N
N
N
N
H
N
Br
N
N
N
N
N
O
GS-9190
Tegobuvir
Bl-207127
Deleobuvir
F
H
N
O
H
O
O
N
N
N
N
N
O
N
O
H
OH
N
S
S
N
H
OH
O
O
PF-868554
Filibuvir
ANA-598
Setrobuvir
Figure 2b: Non-nucleoside NS5B inhibitors as anti HCV
6

2. Results and discussion
Chemical synthesis
The synthesis of 2-aminopyrrole-3-carbonitriles requires the availability of α-aminoketones
[24-30]. These are usually unstable compounds, obtained in situ, via either reaction of α-hydroxy
ketones with amines in acidic media, reaction of α-halo ketones with amines, or from α-amino acids
via Dakin-West reaction [31]. Our target pyrroles 1a-c were synthesized via condensation of benzoin
with aryl amines in refluxing toluene that resulted in the formation of α-aminoketone intermediate.
The formed intermediate was condensed, without isolation, with malononitrile to produce 2-amino-
pyrrole-3-carbonitriles derivatives 1a-c.
Based on the importance of the pyrrolopyrimidine nucleoside analogs as HCV inhibitors [19-
23], the chemical synthesis plan was to cyclize pyrroles 1a-c to pyrrolopyrimidines using different
reagents. Thus, Herein, pyrrolo[2,3-d]pyrimidines 2a-c were prepared by condensation of 2-
aminopyrrole-3-carbonitriles 1a-c with formic acid. Although a literature survey indicated that the
reaction of compounds analogous to 1a-c with acetic anhydride gave the corresponding pyrimidine
derivatives [32,33], in our hands, condensation of 1a-c, independently, with acetic anhydride gave the
acetylated analogous 3a-c. The structures of these compounds were proved by microanalysis and
13
spectral data, where the IR spectra showed bands for NH, CN and C=O groups, in addition to C
NMR spectra that revealed CN and C=O groups, also, 1H NMR and MS spectra were consistent with
the structures. On the other hand, refluxing compounds 1a-c with a mixture of acetic acid/HCl (3:1)
gave the corresponding pyrrolo[2,3-d]pyrimidines 4a-c. Unambiguously, 4a-c were also obtained by
refluxing of 3a-c with a mixture of acetic acid/HCl (3:1) (Scheme 1). The IR spectra of compounds
4a-c revealed bands for NH and C=O groups and the absence of CN group band, 1H NMR and MS
spectra were consistent with their structures.
7

O
Ph
NH
Ph
Ph
O
N
Ph
+ RNH2
N
R
2a-c
Ph
CN
OH
i
iii
Ph
NHCOCH3
N
R
3a-c
Ph
CN
O
Ph
Ph
ii
iv
v
Ph
NH2
NH
R
N
R
1a-c
=
O
R= Cyclohexyl, 3-Cl-C6H4,
4-Cl-C6H4
Ph
NH
v
CH3
N
Ph
N
R
4a-c
Scheme 1: Synthesis of compounds 1a-c – 4a-c. (i) toluene, HCl; (ii) CH2(CN)2, pyridine; (iii)
HCOOH; (iv)Ac2O; (v) AcOH/HCl(3:1).
Pyrrolopyrimidines 2a-c were converted to the corresponding 4-chloro derivatives 5a-c by
condensation with phosphorus oxychloride. 4-Chloropyrrolo[2,3-d]pyrimidines 5a-c were reacted,
independently, with thiourea, certain aryl amines or hydrazine hydrate to afford the respective
pyrrolopyrimidine-4-thiones 6a-c, 4-aryl amino 7a-l and 4-hydrazino derivatives 8a-c (Scheme 2).
8

S
Ph
NH
N
Ph
N
R
6a-c
ii
R'
N
HN
Cl
O
Ph
N
Ph
Ph
N
N
NH
iii
i
Ph
N
R
N
Ph
Ph
N
R
N
R
7a-l
2a-c
5a-c
R'= C6H5, 2-CH3-C6H4, 3-CH3C6H4
, 4-CH3-C6H4
R= Cyclohexyl, 3-Cl-C6H4,
4-Cl-C6H4
iv
7a, R= Cyclohexyl, R'= C6H5
7b, R= Cyclohexyl, R'= 2-CH3-C6H4
7c
, R= Cyclohexyl, R'= 3-CH3-C6H4
7d, R= Cyclohexyl, R'= 4-CH3-C6H4
7e, R= 3-Cl-C6H4, R'= C6H5
7f, R= 3-Cl-C6H4, R'= 2-CH3-C6H4
7g, R= 3-Cl-C6H4, R'= 3-CH3-C6H4
HN NH2
Ph
N
7h
, R= 3-Cl-C6H4, R'= 4-CH3-C6H4
7i, R= 4-Cl-C6H4, R'= C6H5
N
Ph
7j, R= 4-Cl-C6H4, R'= 2-CH3-C6H4
N
7k
,R= 4-Cl-C6H4, R'= 3-CH3-C6H4
7l, R= 4-Cl-C6H4, R'= 4-CH3-C6H4
R
8a-c
Scheme 2: Synthesis of compounds 5a-c – 8a-c. (i) POCl3; (ii) CS(NH2)2, EtOH; (iii) R'NH2,TEA,
EtOH; (iv) NH2NH2.H2O (99%), EtOH.
4-Hydrazinopyrimidine derivatives were extensively used in many reports [34-43] as starting
material for the synthesis of several fused tricyclic heterocycles. Analogously, in this work, we
synthesized certain pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives 9a-c, 10a-c, 11a-c and
14a-c by the reaction of 4-hydrazinopyrrolopyrimidines 8a-c with formic acid, acetic anhydride,
9

carbon disulfide or ethyl chloroformate, respectively. Also, 8a-c were reacted with aryl aldehydes in
presence of triethylamine to afford 4-(2-arylhydrazinyl)pyrrolo[2,3-d]pyrimidines 12a-i and finally
they reacted with acetylacetone to yield 4-(3,5-dimethyl-1H-pyrazol-1-yl)pyrrolo[2,3-d]pyrimidines
13a-c as revealed in Scheme 3. The structures of all synthesized compounds were substantiated by
microanalysis and spectral data.
N
N
N
CH3
N
Ph
N
Ph
N
N
Ph
N
N
R
Ph
N
R
H
10a-c
9a-c
N
ii
N
S
i
Ph
N
HN NH2
H
N
Ph
N
N
O
iii
N
vi
Ph
N
R
Ph
N
11a-c
N
Ph
N
R
N CH
R'
N
HN
Ph
8a-c
N
R
v
Ph
14a-c
iv
N
R= Cyclohexyl, 3-Cl-C6H4,
4-Cl-C6H4
CH3
N
N
Ph
N
12a-i
R'= -C6H5, 4-OCH3-C6H4,
4-Cl-C6H4
R
H3C
Ph
N
12a, R= Cyclohexyl, R'= C6H5
12b, R= Cyclohexyl, R'= 4-OCH3-C6H4
12c
N
, R= Cyclohexyl, R'= 4-Cl-C6H4
12d, R= 3-Cl-C6H4, R'= C6H5
N
12e, R= 3-Cl-C6H4, R'= 4-OCH3-C6H4
, R= 3-Cl-C6H4, R'= 4-Cl-C6H4
12g, R= 4-Cl-C6H4, R'= C6H5
Ph
N
12f
13a-c
R
12h, R= 4-Cl-C6H4, R'= 4-OCH3-C6H4
12i
, R= 4-Cl-C6H4, R'= 4-Cl-C6H4
Scheme 3: Synthesis of compounds 9a-c – 14a-c. (i) HCOOH; (ii) Ac2O; (iii) CS2, EtOH; (iv)
R’CHO, EtOH; (v) (CH3CO)2CH2, EtOH; (vi) ClCOOEt, pyridine.
10

Antiviral activity and SAR analysis
Before determining the antiviral activity, the cytotoxicity of the synthesized compounds was
assessed. For this purpose, we used the cell line Huh 7.5. The CC50 was determined using two
different methods: first by counting the viable cells and secondly by examining the cell morphology
(Table 1).
Table 1: CC50 and IC50 for the synthesized compounds
Compounds CC50 (µM)
IC50* for HCV genotype4 (µM)
176
189
189
190
151
151
183
146
146
130
97
>150
>150
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
>150
>150
>150
>150
>150
>150
>150
>150
>150
97
>150
155
168
120
130
145
121
158
153
>150
>150
108.1±12.01
>150
>150
>150
>150
>150
11

131
153
148
144
103
123
127
144
1023
123
157
146
146
178
190
190
172
161
138
188
184
138
148
140
158
140
151
131
140
113
131
>150
>150
7c
7d
>150
7e
92.4±10.3
>150
7f
7g
>150
7h
>150
7i
82.1±10.3
>150
7j
7k
>150
7l
>150
8a
>150
8b
>150
8c
>150
9a
>150
9b
>150
9c
>150
10a
10b
10c
11a
11b
11c
12a
12b
12c
12d
12e
12f
12g
12h
12i
>150
>150
>150
>150
>150
>150
>150
>150
>150
>150
102.9±9.4
>150
>150
93.6±9.4
12

156
147
147
171
183
160
>150
>150
>150
>150
>150
>150
13a
13b
13c
14a
14b
14c
* IC50 is represented as the mean of 3 measurements ±SD
The non-toxic doses of the synthesized compounds were measured to determine their activity
against HCV genotype 4a. Only five compounds showed considerable antiviral activity. Figure 3
showed the viral inhibition of compounds 5c, 7f, 7j, 12f and 12i. The CC50 for these compounds were
120, 144, 144, 131 and 131 µM, respectively. It was observed that compound 7j showed maximum
antiviral activity (90%) with less toxicity, compound 7f activity was (76.7±5.8%), compound 5c
activity was (73.3±5.8%), compound 12i activity was (70%) and compound 12f had the least activity
(63.3±5.8%).
Chart Title
3.5
3
2.5
2
1.5
1
0.5
73.3±5.8
0
1
Compounds
Figure 3: Antiviral activity of non-toxic doses of tested compounds 5c, 7f, 7j, 12f and 12i against
HCVcc genotype 4a (number of determinations= 3).
13

It was observed that pyrrolo[2,3-d]pyrimidine analogues possessed significant anti-HCV
activity when compared to the synthesized pyrrole derivatives 1a-c, 3a-c and pyrrolo[3,2-
e][1,2,4]triazolo[4,3-c]pyrimidine derivatives 9a-c, 10a-c, 11a-c and 14a-c which did not exhibit any
activity. Regarding SAR, the critical inspection of the biological activity results indicate that 5,6-
diphenyl-7-(3- or 4-chlorophenyl)pyrrolo[2,3-d]pyrimidines with appropriate substitution (chloro or
much better arylamino) at position 4 could be a useful scaffold as anti-HCV genotype 4a (compounds
5c, 7f and 7j). Also, substitution at 4-position by arylamino group is essential for antiviral activity
with comparative least toxicity as indicated by the effect of compounds 7j and 7f, Table 1. Extending
the substituent at position-4, even replacing the aryl amino by aryl hydrazone function results in
decreasing the antiviral activity with increasing cytotoxicity (compounds 12f and 12i), Table 1,
Figure 4. These results are in accordance with the results of molecular docking reported herein (next
section), in addition to their consistency with the previous study [20].
Cl
H3C
Cl
NH-NH2
N
Ph
Ph
N
HN
HN
Ph
Ph
N
Ph
Ph
Ph
Ph
N
N
N
N
N
N
N
N
N
N
5c (73.3±5.8%)
7j (90%)
Cl
12i (70%)
8c (0%)
Cl
Cl
Cl
Cl
H3C
HN
Cl
NH-NH2
N
Ph
Ph
N
N
N
N
Ph
Ph
HN
Ph
Ph
N
Ph
Ph
N
N
N
N
N
N
N
Cl
5b(0%)
12f (63.3±5.8%)
Cl
Cl
Cl
8b (0%)
7f (76.7±5.8%)
14

Figure 4: SAR of the newly synthesized Pyrrolopyrimidine derivatives.
Molecular docking
Many trials were done that revealed some potent inhibitors of HCV RdRp. Most of the
reported inhibitors were in complex with the enzyme at distances may reach 30 Å from the palm
domain within the groove between the thumb and palm (Figure 1) [45].
In this work a number of pyrrolo[2,3-d]pyrimidine derivatives were synthesized and screened
for their HCV inhibitory activity. The binding modes of these compounds within the HCV RdRp
catalytic site at the palm domain and the thumb region were computed. The docking results were
compared to a reported substituted 3,3-dimethylhexahydro-1H-dibenzo[b,e][1,4]diazepin-1-one.
According to the docking results, none of the docked compounds formed coordinate with Mg2+ found
in the catalytic site. The placement of all poses was found to locate at 10.69 Å from the Mg2+, which
indicates that they are still close to the palm domain but at much better location than the previously
reported inhibitors. The calculated entropy conformation score for all the ligands was neglected
(0.00) or very low (1.40) which confirmed the strong binding within the docking site.
Clash penalty score, which indicates the quality of binding, was also calculated. It ranged
from a low value for compound 2b (1.41), compound 6b (1.71), compound 7j (2.00) and compound
5c (2.27) and was found at its highest level for compound 4b (11.36).
From all compounds with less ligand entropy conformation score and clash penalty score only
compound 7j showed low lipophilic contribution score (-13.97). As mentioned previously as the
ligand entropy conformation score and clash penalty score are very low as the binding affinity is
improved and that is very clear for the values of calculated affinities of compounds 7j, 6b, 5c, and 2b
that were -24.92, -23.80, -23.4 and -23.27 kcal/mol respectively. This can interpret the activity of
15

compounds 7j and 5c. Compounds 12f and 12i were ranked just after 5c according to their docking
scores that were -22.43 and -22.31 kcal/mol respectively. They showed neglected entropy
conformation score and relatively good clash score when compared to other active compounds
(Table 2).
Table 2: Molecular model assessment scores
Compounds Docking score a
Kcal/mol
Lipo score b
Clash score c
Rot score d
-16.59
-21.67
-18.33
-19.39
-23.27
-21.21
-19.51
-21.65
-21.31
-18.60
-20.22
-21.84
-15.08
-19.33
-23.42
-16.11
-23.80
-20.49
-19.15
-17.65
-16.10
-15.48
-21.38
-20.92
-20.66
-20.82
-19.64
-24.92
-21.80
-20.38
-22.43
-22.31
-8.27
-10.89
-7.08
-12.01
-10.63
-12.47
-11.40
-13.21
-9.46
6.15
4.09
2.58
5.04
1.41
4.85
6.12
9.04
2.77
2.86
11.36
4.44
9.44
4.77
2.27
3.46
1.71
5.21
7.56
4.89
10.13
5.23
7.06
3.21
5.32
6.77
8.82
2.00
8.81
9.49
2.94
3.04
1.40
0.00
0.00
1.40
0.00
0.00
1.40
0.00
0.00
1.40
0.00
0.00
1.40
0.00
0.00
1.40
0.00
0.00
1.40
1.40
1.40
1.40
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
7d
7e
7f
-9.78
-13.59
-11.84
-13.91
-11.80
-7.55
-10.57
-11.44
-11.22
-13.89
-11.18
-13.41
-8.89
-11.96
-14.49
-11.87
-11.83
-12.37
-13.97
-12.47
-12.77
-13.36
-12.68
7g
7h
7i
7j
7k
7l
12f
12i
16

Regarding the ligand-receptor interactions, we can find that compound 7j showed a clear
hydrogen bond with Gln 446 and that was confirmed by both MOE 2014.09 docking (Figure 5A) and
Leadit 2.1.2 (Figure 6C).
17

Figure 5: Molecular model for binding of 7J
A) Binding mode of compound 7j forming a hydrogen bond with Gln 446. B) The placement of
compound 7j within the same site of the reported inhibitor.
By using Leadit 2.1.2 software, it was discovered that Gln 446 can have a unique binding with
its C=O and NH2 groups to form two different bonds with the aniline NH and the pyrimidine C=N
group respectively. On the other hand compound 7f showed a hydrogen bond with Gln 446 as well,
where compound 5c had a hydrogen bond with Tyr 448 within the same site (Figure 6A,B).
Compounds 12f showed two kinds of hydrogen bonds with Gly 449 (NH) and Gln 446 (C=O), while
compound 12i was involved in hydrogen bonds with Tyr 448 (NH) and Gly 449 (NH) as shown in
(Figure 6D, E).
18

Figure 6: Suggested-binding modes of compounds 5c, 7f, 7j, 12f and 12i.
19

3. Conclusion
In this study, we synthesized novel pyrrole, pyrrolo[2,3-d]pyrimidine and pyrrolo[3,2-
e][1,2,4]triazolo[4,3-c]pyrimidine derivatives and tested their activity towards HCV genotype 4a.
Pyrrole and pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives do not exhibit any activity
against HCV genotype 4a, however, some pyrrolo[2,3-d]pyrimidine derivatives showed reasonable
inhibition of the virus. The binding mode for the active compounds was predicted by a molecular
docking study. Compound 7j exhibited the highest potency as anti HCV which renders this molecule
as a lead compound for drug discovery of anti-HCV agents.
4. Experimental
Chemistry
All chemicals used in this study were purchased from Merck (Darmstadt, Germany) as
reagent grade. All melting points were uncorrected and determined using Electro-thermal IA 9100
apparatus (Shimadzu, Japan). We recorded the IR spectra as potassium bromide pellets on a Perkin-
Elmer 1650 spectrophotometer (USA), Faculty of Science, Cairo University, Cairo, Egypt. The 1H-
NMR (300 MHz) and 13C-NMR (75 MHz) spectra were determined by Varian Mercury spectrometer
(Varian UK) and chemical shifts were expressed as ppm against TMS as internal reference (The Main
Chemical warfare Laboratories, Almaza, Cairo, Egypt). A TSQ Quantum Access MAX triple
quadrupole system was used for mass spectrometric analysis. Data acquisition and processing was
performed using Thermo Scientific Xcalibur 2.1 software (Faculty of Pharmacy, Helwan University,
Cairo, Egypt). Microanalyses were operated using Vario, Elmentar apparatus (Shimadzu, Japan)
(Organic Microanalysis Unit, Faculty of Science, Cairo University, Cairo, Egypt). The progress of
20

reactions was monitored by TLC sheets pre coated with UV fluorescent silica gel (Merck 60 F254)
using chloroform/methanol (3:1) and spots were visualized using UV-light and iodine vapor. All new
compounds yielded spectral data consistent with the proposed structure and microanalysis within
±0.4% of the theoretical values. Compounds 1a,b-6a,b and 7a-h were prepared as we described
before [44].
Preparation of 2-Amino-1-(4-chlorophenyl)-4,5-diphenyl-1H-pyrrole-3-carbonitrile (1c)
A mixture of benzoin (2.12 g, 10 mmol), 4-chloroaniline (1.27 g, 10 mmol) and conc. HCl (6-
8 drops) in toluene (50 mL) was heated under reflux for 36 h then cooled. Malononitrile (0.66 g, 10
mmol) was added, followed by a catalytic amount of pyridine (1.5 mL) portion-wise and left to reflux
until a solid mass was formed. The solvent was evaporated under reduced pressure and the residue
was recrystallized from methanol to give compound 1c.
Yield: 75%; m.p.: 290-292 oC; IR (KBr) υ (cm-1): 3441, 3340 (NH2), 2209 (C≡N); MS (ESI) m/z:
369.19 (M+, 35Cl, 5%), 371.23 (M++2, 37Cl, 1.58%); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 5.39 (s,
2H, NH2, D2O exchangeable), 7.03-7.96 (m, 14H, Ar-H); Anal. Calcd for C23H16ClN3 (369.86): C,
74.69; H, 4.36; N, 11.36%. Found: C, 74.42; H, 4.49; N, 11.61%.
Preparation of 7-(4-chlorophenyl)-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2c)
The appropriate aminopyrrole(1c) (3.699 g, 10 mmol) in formic acid (20 mL, 85%) was
heated under reflux for 36 h, cooled, poured onto ice-water to give a precipitate 2c which was filtered
off, dried, and recrystallized from ethanol.
o
Yield: 71%; m.p.: > 300 C; IR (KBr) υ (cm-1): 3382 (NH), 1680 (C=O), 1587 (C=N); MS
35
37
1
(ESI) m/z: 397.11 (M+, Cl, 2%), 399 (M++2, Cl, 0.62% ); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 6.99-7.91 (m, 14H, Ar-H), 8.25 (s, 1H, C2-H), 11.89 (s, 1H, NH, D2O exchangeable); Anal.
21

Calcd for C24H16ClN3O (397.86): C, 72.45; H, 4.05; N, 10.56 %. Found: C, 72.19; H, 4.32; N,
10.72%.
Preparation of N-(1-(4-chlorophenyl)-3-cyano-4,5-diphenyl-1H-pyrrol-2-yl)acetamide (3c)
The appropriate aminopyrrole(1c) (3.699 g, 10 mmol) in acetic anhydride (40 mL) was heated
under reflux for 5 h, then cooled, poured onto ice-water and neutralized with ammonia to give a
precipitate, which was filtered off, dried, and recrystallized from methanol, to give compound 3c.
o
Yield: 65%; m.p.: 210-212 C; IR (KBr) υ (cm-1): 3350 (NH), 2219 (C≡N), 1699 (C=O); MS (ESI)
35
37
1
m/z: 411.52 (M+, Cl, 4%), 413.61(M++2, Cl, 1.36%); H NMR (DMSO-d6, 300 MHz) δ (ppm):
13
1.90 (s, 3H, CH3-CO), 7.07-7.91 (m, 14H, Ar-H), 11.40 (s, 1H, NH, D2O exchangeable); C NMR
(DMSO-d6) δ (ppm): 21.74 (CH3), 116.15 (C≡N), 124.04, 126.41,126.92, 127.81, 128.46, 128.65,
128.74, 128.89, 129.03, 129.39, 130.20, 131.37, 132.98, 134.48, 134.88, 135.89 (sp2 carbon atoms),
171.28 (C=O); Anal. Calcd for C25H18ClN3O (411.88): C, 72.90; H, 4.40; N, 10.20%. Found: C,
72.60; H, 4.59; N, 10.45%.
Preparation of 7-(4-chlorophenyl)-2-methyl-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one
(4c)
The appropriate aminopyrrole (1c) (3.699 g, 10 mmol) or its acetyl derivative (3c) (4.119 g,
10mmol) was refluxed, independently, in a mixture of acetic acid (15mL) and hydrochloric acid
(5mL) for 5 h. The reaction mixture was cooled, poured onto ice and the formed solid was filtered
off, dried and recrystallized from methanol to afford 4c.
o
Yield: 68%; m.p.: 257-259 C; IR (KBr) υ (cm-1): 3395 (NH), 1707 (C=O), 1589(C=N); MS (ESI)
35
37
1
m/z: 411.55(M+, Cl, 4%), 413.70(M++2, Cl, 1.32%); H NMR (DMSO-d6, 300 MHz) δ (ppm):
2.32 (s, 3H, CH3), 7.13-7.92 (m, 14H, Ar-H), 11.41 (s, 1H, NH, D2O exchangeable); Anal. Calcd for
C25H18ClN3O (411.88): C, 72.90; H, 4.40; N, 10.20 %. Found: C, 73.21; H, 4.65; N, 10.36%.
Preparation of 4-chloro-7-(4-chlorophenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidine (5c)
22

The appropriate pyrrolopyrimidinone (2c) (3.979 g, 10 mmol) was heated under reflux in
phosphorus oxychloride (30 mL) for 15 h, then cooled and poured onto ice-water to yield precipitate,
which was recrystallized from ethanol to give compounds 5c.
o
Yield: 75%; m.p.: 252-254 C; IR (KBr) υ (cm-1): 3045, 2960 (CH), 1615 (C=C), 1571 (C=N); MS
(ESI) m/z: 416.36 (M+, 35Cl, 3.2%), 418.31(M++2, 37Cl, 2.13%), 420.4(M++4, Cl37, 1.07%); 1H NMR
(DMSO-d6, 300 MHz) δ (ppm): 7.0-7.91 (m, 14H, Ar-H), 8.44 (s, 1H, C2-H); Anal. Calcd for
C24H15Cl2N3 (416.30): C, 69.24; H, 3.63; N, 10.09 %. Found: C, 68.95; H, 3.79; N, 10.25%.
Preparation of 7-(4-chlorophenyl)-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidine-4(7H)-thione (6c)
A mixture of 4-chloropyrrolopyrimidine (5c) (4.163 g, 10 mmol) and thiourea (1.5g, 0.02
mol) was heated under reflux in dry ethanol (30 mL) for 10 h, then cooled, poured onto ice-water, to
give precipitate, which were filtered off, dried, and recrystallized from methanol to give compounds
6c.
o
Yield: 70%; m.p.: 185-187 C; IR (KBr) υ (cm-1): 3375 (NH), 1585 (C=N), 1255(C=S); MS (ESI)
35
37
1
m/z: 413.37 (M+, Cl, 2%), 415.34 (M++2, Cl, 0.65%); H NMR (DMSO-d6, 300 MHz) δ (ppm):
7.14-7.91 (m, 14H, Ar-H), 8.86 (s, 1H, C2-H), 10.15 (s, 1H, NH, D2O exchangeable); Anal. Calcd
for C24H16ClN3S (413.92): C, 69.64; H, 3.90; N, 10.15 %. Found: C, 69.78; H, 4.11; N, 10.01%.
General procedure for the synthesis of 5,6-diphenyl-7-(4-chlorophenyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-yl)-arylamine (7i-l):
A mixture of 4-chloropyrrolopyrimidine (5c) (4.163 g, 10 mmol), the appropriate amine (10
mmol) and few drops of triethylamine was heated under reflux in absolute ethanol for 8 h, then
cooled and poured onto ice-water to give precipitate, which were filtered off, dried, and recrystallized
from methanol to give compounds 7i-l.
7-(4-Chlorophenyl)-N,5,6-triphenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (7i)
23

Yield: 61%; m.p.: 180-182 oC; IR (KBr) υ (cm-1): 3435 (NH), 1607 (C=N); MS (ESI) m/z: 472 (M+,
37
1
35Cl, 3%), 474.21(M++2, Cl, 0.9%); H NMR (DMSO-d6, 300 MHz) δ (ppm): 6.90-7.90 (m, 19H,
Ar-H), 8.44 (s, 1H, C2-H), 11.11 (s, 1H, NH, D2O exchangeable); Anal. Calcd for C30H21ClN4
(472.97): C, 76.18; H, 4.48; N, 11.85%. Found: C, 76.00; H, 4.71; N, 12.02%.
7-(4-Chlorophenyl)-5,6-diphenyl-N-o-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (7j)
o
Yield: 60%; m.p.: 210-212 C; IR (KBr) υ (cm-1): 3448 (NH), 1598 (C=N); MS (ESI) m/z: 487.22
35
37
(M+, Cl, 5%), 489.11(M++2, Cl, 1.71%);1H NMR (DMSO-d6, 300 MHz) δ (ppm): 1.99 (s, 3H,
CH3), 6.88-7.91 (m, 18H, Ar-H), 8.38 (s, 1H, C2-H), 11.11 (s, 1H, NH, D2O exchangeable); Anal.
Calcd for C31H23ClN4 (486.99): C, 76.46; H, 4.76; N, 11.50%. Found: C, 76.18; H, 4.54; N, 11.76%.
7-(4-Chlorophenyl)-5,6-diphenyl-N-m-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (7k)
o
Yield: 63%; m.p.: 202-204 C; IR (KBr) υ (cm-1): 3448(NH), 1598(C=N); MS (ESI) m/z: 485.22
35
1
(M+-2H, Cl, 2.8%) ; H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.10 (s, 3H, CH3), 6.84-7.91 (m,
18H, Ar-H), 8.40 (s, 1H, C2-H), 11.67 (s, 1H, NH, D2O exchangeable); Anal. Calcd for C31H23ClN4
(486.99): C, 76.46; H, 4.76; N, 11.50%. Found: C, 76.70; H, 4.44; N, 11.29%.
7-(4-Chlorophenyl)-5,6-diphenyl-N-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (7l)
o
Yield: 65%; m.p.: 195-197 C; IR (KBr) υ (cm-1): 3448 (NH), 1598 (C=N); MS (ESI) m/z: 487.21
35
37
1
(M+, Cl, 6%), 489.25(M++2, Cl, 1.9%); H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.25 (s, 3H,
CH3), 7.0-7.91 (m, 18H, Ar-H), 8.38(s, 1H, C2-H), 10.95 (s, 1H, NH, D2O exchangeable); Anal.
Calcd for C31H23ClN4 (486.99): C, 76.46; H, 4.76; N, 11.50%. Found: C, 76.21; H, 4.95; N, 11.81%.
General procedure for the synthesis of 5,6-Diphenyl-4-hydrazinyl-7-substituted-7H-pyrrolo[2,3-
d]pyrimidines (8a-c):
24

A mixture of 4-chloro pyrrolopyrimidine 5a-c (10 mmol) and hydrazine hydrate (0.5 mL, 10
mmol) was heated under reflux in absolute ethanol for 8 h, cooled, poured onto ice water to give
precipitates which were filtered off, dried, and recrystallized from methanol to give compounds 8a-c.
7-Cyclohexyl-5,6-diphenyl-4-hydrazinyl-7H-pyrrolo[2,3-d]pyrimidine (8a)
Yield: 70%; m.p.: 202-204 oC; IR (KBr) υ (cm-1): 3412-3322 (NH2), 3237(NH), 1533 (C=N); MS
1
(ESI) m/z: 383.35(M+, 2%); H NMR (DMSO-d6, 300 MHz) δ (ppm): 1.40-1.95 (m, 10H,
cyclohexyl), 3.92 (m, 1H, CH-N cyclohexyl), 5.34 (s, 2H, NH2 D2O exchangeable), 7.12-7.69 (m,
10H, Ar-H), 8.12 (s, 1H, C2-H), 8.58 (s, 1H, NH, D2O exchangeable); Anal. Calcd for C24H25N5
(383.49): C, 75.17; H, 6.57; N, 18.26%. Found: C, 75.35; H, 6.82; N, 18.01%.
7-(3-Chlorophenyl)-5,6-diphenyl-4-hydrazinyl-7H-pyrrolo[2,3-d]pyrimidine (8b)
Yield: 72%; m.p.: 212-214 oC; IR (KBr) υ (cm-1): 3427-3385 (NH2), 3217(NH), 1558 (C=N); MS
35
37
(ESI) m/z: 411.27 (M+, Cl, 4%), 413.50 (M++2, Cl, 1.4%);1H NMR (DMSO-d6, 300 MHz) δ
(ppm): 6.28 (s, 2H, NH2 D2O exchangeable), 7.0-7.85 (m, 14H, Ar-H), 8.20 (s, 1H, C2-H), 8.40 (s,
1H, NH, D2O exchangeable); Anal. Calcd for C24H18ClN5 (411.89): C, 69.98; H, 4.40; N, 17.00%.
Found: C, 69.71; H, 4.71; N, 16.83%.
7-(4-Chlorophenyl)-5,6-diphenyl-4-hydrazinyl-7H-pyrrolo[2,3-d]pyrimidine (8c)
Yield: 75%; m.p.: 220-222 oC; IR (KBr) υ (cm-1): 3427-3385 (NH2), 3217(NH), 1558 (C=N); MS
35
37
1
(ESI) m/z: 411.22 (M+, Cl, 8%), 413.23 (M++2, Cl, 2.6%); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 5.08(s, 2H, NH2 D2O exchangeable), 7.0-7.79 (m, 14H, Ar-H), 7.90 (s, 1H, C2-H), 8.10 (s,
1H, NH, D2O exchangeable); Anal. Calcd for C24H18ClN5 (411.89): C, 69.98; H, 4.40; N, 17.00%.
Found: C, 69.68; H, 4.19; N, 17.25%.
25

General procedure for the synthesis of
e][1,2,4]triazolo[4,3-c]pyrimidines (9a-c):
8,9-diphenyl-7-substituted-7H-pyrrolo[3,2-
The appropriate hydrazine 8a-c (10 mmol) was heated under reflux for 8 h in formic acid (20
mL, 85 %), cooled, poured onto ice water to give a precipitate which was filtered off, dried, and
recrystallized from ethanol to yield compounds 9a-c.
7-Cyclohexyl-8,9-diphenyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine (9a)
Yield: 68%; m.p.: 280-282 oC; IR (KBr) υ (cm-1): 1618 (C=N); MS (ESI) m/z: 394.49(M++1H, 8%);
1H NMR (DMSO-d6, 300 MHz) δ (ppm): 1.41-1.96 (m, 10H, cyclohexyl), 3.82 (m, 1H, CH-N
cyclohexyl), 7.17-7.70 (m, 10H, Ar-H), 8.16 (s, 1H, C3-H), 8.83 (s, 1H, C5-H); Anal. Calcd for
C25H23N5 (393.48): C, 76.31; H, 5.89; N, 17.80 %. Found: C, 76.49; H, 5.62; N, 18.07%.
7-(3-Chlorophenyl)-8,9-diphenyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine (9b)
Yield: 70%; m.p.: 266-268 oC; IR (KBr) υ (cm-1): 1566 (C=N); MS (ESI) m/z: 421.42 (M+, 35Cl, 2%),
423.39 (M++2, 37Cl, 0.7%); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 7.0-7.87 (m, 14H, Ar-H), 8.39
(s, 1H, C3-H), 8.90 (s, 1H, C5-H); Anal. Calcd for C25H16ClN5 (421.88): C, 71.17; H, 3.82; N,
16.60%. Found: C, 71.36; H, 4.05; N, 16.36%.
7-(4-Chlorophenyl)-8,9-diphenyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine (9c)
35
Yield: 72%; m.p.: 287-289 oC; IR (KBr) υ (cm-1): 1566 (C=N); MS (ESI) m/z: 421.77 (M+, Cl,
2.9%), 423.80 (M++2, 37Cl, 0.9%); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 7.07-7.94 (m, 14H, Ar-
H), 8.12 (s, 1H, C3-H), 8.84 (s, 1H, C5-H); Anal. Calcd for C25H16ClN5 (421.88): C, 71.17; H, 3.82;
N, 16.60%. Found: C, 71.01; H, 3.61; N, 16.89%.
General procedure for the synthesis of 8,9-Diphenyl-3-methyl-7-substituted-7H-pyrrolo[3,2-
e][1,2,4]triazolo[4,3-c]pyrimidines (10a-c):
26

The appropriate hydrazine 8a-c (10 mmol) was heated under reflux for 5 h in acetic anhydride
(30 mL), cooled, poured onto ice water and neutralized with ammonia to give a precipitate which was
filtered off, dried, and recrystallized from ethanol to yield compounds 10a-c.
7-Cyclohexyl-8,9-diphenyl-3-methyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine (10a)
1
Yield: 58%; m.p.: 260-262 oC; IR (KBr) υ (cm-1): 1554 (C=N); MS (ESI) m/z: 407(M+, 4%); H
NMR (DMSO-d6, 300 MHz) δ (ppm): 1.51-1.99 (m, 10H, cyclohexyl), 2.27 (s, 3H, CH3), 3.93 (m,
1H, CH-N cyclohexyl), 7.13-7.65 (m, 10H, Ar-H), 8.64 (s, 1H, C5-H); Anal. Calcd for C26H25N5
(407.51): C, 76.63; H, 6.18; N, 17.19%. Found: C, 76.89; H, 6.00; N, 17.35%.
7-(3-Chlorophenyl)-8,9-diphenyl-3-methyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine (10b)
Yield: 60%; m.p.: 280-282 oC; IR (KBr) υ (cm-1): 1612 (C=N); MS (ESI) m/z: 435.13 (M+, 35Cl, 4%),
437.21 (M++2, 37Cl, 1.25%); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.32 (s, 3H, CH3), 7.0-7.79
(m, 14H, Ar-H), 8.68 (s, 1H, C5-H); Anal. Calcd for C26H18ClN5 (435.91): C, 71.64; H, 4.16; N,
16.07%. Found: C, 71.39; H, 4.41; N, 16.31%.
7-(4-Chlorophenyl)-8,9-diphenyl-3-methyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine (10c)
Yield: 59%; m.p.: 275-277 oC; IR (KBr) υ (cm-1): 1612 (C=N); MS (ESI) m/z: 435.28 (M+, 35Cl, 7%),
437.30 (M++2, 37Cl, 2.34%); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.26 (s, 3H, CH3), 7.01-7.90
(m, 14H, Ar-H), 8.80 (s, 1H, C5-H); Anal. Calcd for C26H18ClN5 (435.91): C, 71.64; H, 4.16; N,
16.07%. Found: C, 71.89; H, 4.39; N, 15.85%.
General procedure for the synthesis of
8,9-Diphenyl-7-substituted-2H-pyrrolo[3,2-
e][1,2,4]triazolo[4,3-c]pyrimidine-3(7H)-thiones (11a-c):
A mixture of the appropriate hydrazine 8a-c (10 mmol) and carbon disulfide (0.8 mL, 10
mmol) was heated under reflux for 3 h in absolute ethanol (30 mL), cooled, poured onto ice water to
give a precipitate which was filtered off, dried, and recrystallized from ethanol to yield compounds
11a-c.
27

7-Cycloxexyl-8,9-diphenyl-2H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-3(7H)-thiones (11a)
Yield: 60 %; m.p.: 276-278 oC; IR (KBr) υ (cm-1): 3287(NH), 1649(C=S),1595 (C=N); MS (ESI)
1
m/z: 425(M+, 4%); H NMR (DMSO-d6, 300 MHz) δ (ppm): 1.29-1.88 (m, 10H, cyclohexyl), 3.93
(m, 1H, CH-N cyclohexyl), 7.11-7.83 (m, 10H, Ar-H), 8.12 (s, 1H, C5-H), 12.24 (s, 1H, NH, D2O
exchangeable); Anal. Calcd for C25H23N5S (425.55): C, 70.56; H, 5.45; N, 16.46%. Found: C, 70.81;
H, 5.73; N, 16.20%.
7-(3-Chlorophenyl)-8,9-diphenyl-2H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-3(7H)-thiones
(11b)
Yield: 61%; m.p.: 288-290 oC; IR (KBr) υ (cm-1): 3314(NH), 1652(C=S), 1567 (C=N); MS (ESI)
35
37
1
m/z: 453.44 (M+, Cl, 5%), 455.50 (M++2, Cl, 1.7%); H NMR (DMSO-d6, 300 MHz) δ (ppm):
7.0-7.90 (m, 14H, Ar-H), 8.25 (s, 1H, C5-H), 11.98 (s, 1H, NH, D2O exchangeable); Anal. Calcd for
C25H16ClN5S (453.95): C, 66.15; H, 3.55; N, 15.43%. Found: C, 66.32; H, 3.24; N, 15.56%.
7-(4-Chlorophenyl)-8,9-diphenyl-2H-pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-3(7H)-thiones
(11c)
o
Yield: 63%; m.p.: 280-282 C; IR (KBr) υ (cm-1): 3314(NH), 1652(C=S), 1567 (C=N); MS (ESI)
35
37
1
m/z: 453.61 (M+, Cl, 6%), 455.59 (M++2, Cl, 2.1%); H NMR (DMSO-d6, 300 MHz) δ (ppm):
6.99-7.83 (m, 14H, Ar-H), 8.03 (s, 1H, C5-H), 11.96 (s, 1H, NH, D2O exchangeable); Anal. Calcd for
C25H16ClN5S (453.95): C, 66.15; H, 3.55; N, 15.43%. Found: C, 66.41; H, 3.78; N, 15.64%.
General procedure for the synthesis of 4-(2-(Aryl)-hydrazinyl)-5,6-diphenyl-7-substituted-7H-
pyrrolo[2,3-d]pyrimidines (12a-i):
A mixture of the appropriate hydrazine 8a-c (10 mmol) and the appropriate aromatic aldehyde
(10 mmol) was heated under reflux in absolute ethanol for 8 h, cooled, poured onto ice water to give
precipitates which were filtered off, dried, and recrystallized from methanol to give compounds 12a-i.
4-(2-Benzylidenehydrazinyl)-7-cyclohexyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidines (12a)
28

o
Yield: 50%; m.p.: 253-255 C; IR (KBr) υ (cm-1): 3344(NH), 1586 (C=N); MS (ESI) m/z: 471 (M+,
1
6%); H NMR (DMSO-d6, 300 MHz) δ (ppm): 1.42-1.97 (m, 10H, cyclohexyl), 3.89 (m, 1H, CH-N
cyclohexyl), 7.07-7.87 (m, 15H, Ar-H), 8.07 (s, 1H, C2-H), 8.86 (s, 1H, N=CH), 9.75 (s, 1H, NH,
D2O exchangeable); Anal. Calcd for C31H29N5 (471.60): C, 78.95; H, 6.20; N, 14.85%. Found: C,
78.70; H, 6.47; N, 15.04%.
7-Cyclohexyl-5,6-diphenyl-4-(2-(4-methoxybenzylidene)hydrazinyl)-7H-pyrrolo[2,3-d]pyrimidines
(12b)
o
Yield: 54%; m.p.: 270-272 C; IR (KBr) υ (cm-1): 3411(NH), 1611 (C=N); MS (ESI) m/z: 502.62
1
(M++1H, 15%); H NMR (DMSO-d6, 300 MHz) δ (ppm): 1.41-1.99 (m, 10H, cyclohexyl), 3.8 (m,
1H, CH-N cyclohexyl), 4.04 (s, 3H, OCH3), 7.11-7.86 (m, 14H, Ar-H), 8.10 (s, 1H, C2-H), 8.57 (s,
1H, N=CH), 9.80 (s, 1H, NH, D2O exchangeable); Anal. Calcd for C32H31N5O (501.62): C, 76.62; H,
6.23; N, 13.96 %. Found: C, 76.84; H, 6.59; N, 13.75%.
4-(2-(4-Chlorobenzylidene)hydrazinyl)-7-Cyclohexyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidines
(12c)
o
Yield: 52%; m.p.: 279-281 C; IR (KBr) υ (cm-1): 3339(NH), 1594 (C=N); MS (ESI) m/z: 506.60
35
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(M+, Cl, 20%), (M++2, Cl, 6.63%); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 1.45-1.99 (m, 10H,
cyclohexyl), 3.91 (m, 1H, CH-N cyclohexyl), 7.10-7.90 (m, 14H, Ar-H), 8.15 (s, 1H, C2-H), 8.64 (s,
1H, N=CH), 9.95 (s, 1H, NH, D2O exchangeable); Anal. Calcd for C31H28ClN5 (506.04): C, 73.58; H,
5.58; N, 13.84 %. Found: C, 73.31; H, 5.41; N, 13.59 %.
4-(2-Benzylidenehydrazinyl)-7-(3-chlorophenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidines (12d)
o
Yield: 51%; m.p.: 276-278 C; IR (KBr) υ (cm-1): 3315(NH), 1619 (C=N); MS (ESI) m/z: 499.86
(M+, 35Cl, 10%), 501.99(M++2, 37Cl, 3.25%); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 6.99-7.91 (m,
19H, Ar-H), 8.15 (s, 1H, C2-H), 8.71 (s, H, N=CH), 10.03 (s, H, NH, D2O exchangeable); Anal.
Calcd for C31H22ClN5 (499.99): C, 74.47; H, 4.43; N, 14.01 %. Found: C, 74.76; H, 4.69; N, 13.80 %.
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