Novel antiviral compounds against gastroenteric viral infections

Article abstract of DOI:10.1002/ardp.201400387

Viral gastroenteritis is a serious viral infection which affects a large number of individuals around the world, most of them being children. The infection may occur due to different viruses, for example, coxsackievirus, adenovirus, and rotavirus. There i

Full text of DOI:10.1002/ardp.201400387

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Full Paper
Novel Antiviral Compounds against Gastroenteric Viral Infections
Mosaad S. Mohamed1, Rania H. Abd El-Hameed1, Amira I. Sayed1, and Sameh H. Soror2,3
1
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Ain Helwan,
Helwan, Egypt
2
Biochemistry and Molecular Biology Department, Faculty of Pharmacy, Helwan University, Ain Helwan,
Cairo, Egypt
3
Center for Scientific Excellence “Helwan Structural Biology Research (HSBR)”, Cairo, Egypt
Viral gastroenteritis is a serious viral infection which affects a large number of individuals around the
world, most of them being children. The infection may occur due to different viruses, for example,
coxsackievirus, adenovirus, and rotavirus. There is no available cure for such infections, and the treatment
mainly depends on hospitalization and administration of nutritional supports. A new antiviral agent
against gastroenteritis viral infection will be a breakthrough in healthcare. Pyrrole and pyrrolopyrimidine
derivatives are well known for their biological activity as antibacterial, antifungal, and anticancer agents.
These compounds also proved to possess antiviral activity. Here, we synthesized novel pyrrole and
pyrrolopyrimidine compounds and examined their antiviral activity. We synthesized several new pyrrole,
pyrrolo[2,3-d]pyrimidine, and pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. The characteri-
zation of all synthesized compounds was based on microanalysis and spectral data. Moreover, we
determined the non-toxic doses of these compounds on BGM, Hep-2, and MA-104 cells. We tested all the
synthesized compounds for their antiviral activities against coxsackievirus B4, adenovirus type 7, and
rotavirus Wa strain. Several compounds exhibited significant activities as antiviral agents.
Keywords: Adenovirus / Coxsackievirus B4 / Direct antiviral agents / Pyrrole / Pyrrolo[2,3-d]pyrimidine /
Pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine / Rotavirus / Viral gastroenteritis
Received: October 18, 2014; Revised: December 24, 2014; Accepted: January 9, 2015
DOI 10.1002/ardp.201400387
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Middle East and North African countries [2] and approaches
US$ 1 billion per year in USA [3].
Introduction
Children are frequently affected by gastroenteritis due to
the mode of transmission (oral–fecal route). This condition is
exacerbated by the close contact between children and poor
hygiene [4]. Annually, about two billion cases of diarrheal
diseases occur among children under the age of five globally.
About 1.5 million of the affected children die from diarrheal
diseases, mostly in developing countries. This makes diarrheal
diseases the second most common cause of death among
children under the age of five following pneumonia [5].
Several viruses can cause gastroenteritis; the most common
are coxsackievirus, adenovirus, and rotavirus. Rotavirus is the
leading cause of severe gastroenteritis in the pediatric
population worldwide [6], adenovirus is the second most
common virus causing gastroenteritis in young children [7],
and coxsackievirus is considered the least dangerous cause of
gastroenteritis among the three viruses [8].
Viral gastroenteritis is one of the most common infections in
the world. Due to its significant morbidity, it constitutes a
major threat especially to vulnerable individuals such as
children, elderly, and immunocompromised individuals. The
viral gastroenteritis constitutes around 21–40% of infectious
diarrhea cases in developed countries [1]. Viral gastroenteritis
represents not only a health risk but also an economical and
financial burden through direct and indirect costs. Hospitali-
zation costs ranged from US$ 1.8 to 4.6 million annually in the
Correspondence: Prof. Sameh H. Soror, Biochemistry and Mo-
lecular Biology Department, Faculty of Pharmacy, Helwan
University, Ain Helwan, University Campus, Cairo 11795, Egypt.
E-mail: sameh_soror@pharm.helwan.edu.eg
Fax: þ20-2-2554-1601
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
M. S. Mohamed et al.
the difference in their melting points, difference in finger print
regioninIRspectraofbothandalsochemicalshiftofNH-proton
in 1H NMR as it is for 4-hydrazinopyrrolo[2,3-d]pyrimidines
appears at the aromatic region (6.7–7.9 ppm) but in 3-amino-4-
imino-pyrrolo[2,3-d]pyrimidines it appears in range >9.2 ppm.
We used pyrrolo[2,3-d]pyrimidines 9a,b as starting materi-
als for synthesis of several pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]-
pyrimidines 10a,b–14a,b via refluxing with triethylorthofor-
mate, acetic anhydride, carbon disulfide, ethyl cyanoacetate,
and chloroacetylchloride, respectively [35–39, 44–46], as
revealed in Scheme 2. The structure of these compounds
was proven by microanalysis and spectral data. We ruled out
the isomeric analogs of pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]-
pyrimidine on the ground of chemical shift magnitude of the
triazole and pyrimidine ring protons. The triazolo[4,3-c]-
pyrimidine protons (C3-H and C5-H) appeared at d 8–8.9 ppm,
but that of triazolo[1,5-c]pyrimidines (C2-H and C5-H)
appeared at 9.5–9.8 ppm [40].
Acute viral gastroenteritis infections require hospitalization
but treatment is mainly palliative. To the best of our
knowledge, there is no available specific antiviral treatment
for these infections. Few recent reports described synthesis of
antiviral agents against norovirus [9, 10]. There is a need to
develop antiviral drugs that can be used for treatment of
different viral gastroenteritis infections.
Several synthetic chemical compounds showed antiviral
activity. It is noted that the pyrrole nucleus plays a vital role in
many biological activities [11–15]. Also, pyrrolo[2,3-d]pyrimi-
dines as 7-deaza analogs of biogenic purines are of
considerable interest both from chemical and biological
points of view. This heterocyclic system is widely distributed
in nature, being a part of the antibiotics [16] tubercidin,
toyocamycin, sangivamycin, and cadeguomycin. Many syn-
thetic pyrrolo[2,3-d]pyrimidine derivatives show antibacterial
[17–19], antifungal [20], anti-inflammatory [21–23], antican-
cer [24, 25], antiviral [26–29], analgesic [30], antihyperglyce-
mic [31], and anticonvulsant [32] activities.
Biological results
Cytotoxicity
In this study, we report synthesis and biological activity of
novel pyrrole and pyrrolopyrimidine derivatives as antiviral
drugs for gastroenteritis viral infections.
The assessment of the cytotoxicity of any chemotherapeutic
agent (including antiviral compounds) is an essential part of
the biological evaluation of any new therapeutic compounds,
as it should determine that neither acute nor long-term
toxicity should occur to the host. Cytotoxicity of all synthe-
sized compounds was examined on three different cell lines:
BGM, Hep-2, and MA-104. There were variations in the
susceptibilities of the cell lines to the toxicity of the tested
compounds, where the BGM showed the highest resistance,
followed by Hep-2 and the least resistant was the MA-104 cell
line. This may be due to the differences between the origins of
these cell lines. Table 1 shows the CC50 for all the tested
compounds. The CC50 was determined using two different
techniques: by counting the number of viable cells and by
examining the effect on cell morphology. The CC50 for the
compounds which exhibited antiviral activity: 5b, 7f, 10a, and
12a, were 144, 144, 125, and 115 mM, respectively.
Results and discussion
Chemistry
Condensation of benzoin and primary amines in refluxing
toluene resulted in the formation of a-aminoketone inter-
mediates, which were condensed, without isolation, with
malononitrile to yield 2-amino-pyrrole-3-carbonitriles 1a–d
[17, 18]. Synthesisofpyrroles1c,dwaspreviouslyreported[19].
Refluxing of pyrroles 1a,b and acetic anhydride gave
acetylated products 2a,b, while refluxing with a mixture of
HCl/acetic acid (1:3) made 2-methyl-pyrrolo[2,3-d]pyrimidines
3a,b [33]. We obtained pyrrolo[2,3-d]pyrimidin-4-ones 4a,b
through the reaction of 1a,b with formic acid [12, 21, 28], which
were then refluxed with phosphorus oxychloride [17, 18] to
produce the respective 4-chloro derivatives 5a,b. Compounds
5a,b were refluxed with thiourea, which resulted in the
corresponding 4-thiones 6a,b [34]. 4-Aryl amino derivatives
7a–h were prepared via a reaction of 4-chloropyrrolopyrimi-
dines 5a,b with some aryl amines in the presence of catalytic
amount of triethylamine [18] as revealed in Scheme 1.
Antiviral assay
The non-toxic doses of the compounds were tested against
coxsackievirus B4, adenovirus type 7, and rotavirus Wa strain.
Considerable antiviral activity was observed with some of the
tested compounds. Four compounds among the tested ones
showed activity against coxsackievirus B4, adenovirus type 7,
and rotavirus Wa strain with different degrees of activity and
specificity. Figure 1 showed the activity of compound 5b,
which was highly specific against coxsackievirus B4, the viral
inhibition reached 76.6%. Compound 7f achieved consider-
able activity against all tested viruses ranged from a maximum
inhibition of coxsackievirus B4 (83.3%) followed by rotavirus
Wa strain (60%) and the least activity toward adenovirus type
7 (50%) as demonstrated in Fig. 2. Moreover, compound 7f
showed significantly higher activity against coxsackievirus B4
compared to its activity against rotavirus Wa strain or
adenovirus type 7. Compound 10a showed antiviral activity
We refluxed pyrroles 1c,d, individually, with triethylortho-
formate to produce 2-(ethoxymethylene)amino-pyrroles 8a,b,
which upon stirring with hydrazine hydrate gave 3-amino-4-
imino-pyrrolo[2,3-d]pyrimidines 9a,b [35–39], not 4-hydrazi-
nylpyrrolopyrimidines as reported before [40–43]. The struc-
tures of these compounds were proven chemically in our
previous publication [19] by unambiguous synthesis of 4-
hydrazinylpyrrolopyrimidine derivatives by condensation of 4-
chloro analogs with hydrazine hydrate and second by
microanalysis and spectral data. The formation of 3-amino-4-
imino-pyrrolo[2,3-d]pyrimidines, rather than the formation of
4-hydrazinylpyrrolo[2,3-d]pyrimidines, was confirmed through
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Antiviral Compounds against Gastroenteric Viral Infections
Ph
CN
Ph
Ph
O
+
RNH2
Ph
NHCOCH3
2a,b
N
R
OH
iii
i
O
Ph
O
H
Ph
CN
Ph
N
ii
iv
v
CH3
Ph
NH
R
N
Ph
NH2
Ph
N
R
N
R
1a d
3a,b
R = cyclohexyl, 3-Cl-C6H4,
4-CH3-C6H4, 4-CH3O-C6H4
O
Cl
H
Ph
Ph
N
N
vi
N
N
Ph
Ph
N
N
R
R
5a,b
4a,b
vi
viii
R'
N
H
S
H
Ph
Ph
N
N
N
N
Ph
Ph
N
N
R
R
7a h
6a,b
R' = C6H5, 2-CH3-C6H4, 3-CH3-C6H4,
4-CH3-C6H4
Scheme 1. Synthesis of compounds 1a–d to 7a–h. Reaction conditions: (i) HCl, toluene; (ii) CH2(CN)2; (iii) Ac2O; (iv) HCl/AcOH (1:3); (v)
HCOOH; (vi) POCl3; (vii) CS(NH2)2; (viii) R0NH2, TEA.
against both coxsackievirus B4 (63.3%) and rotavirus Wa strain
(53.3%) as shown in Fig. 3. Statistical analysis did not show any
significant difference in activity against the two viruses.
Finally, compound 12a showed considerable antiviral activity
against all tested viruses ranged, as it appears in Fig. 4, from
maximum inhibition of coxsackievirus B4 (80%) followed by
rotavirus Wa strain (66.7%) and adenovirus type 7 (53.3%).
Statistical analysis showed significant difference in activity
against coxsackievirus B4 compared to the activity against
rotavirus Wa strain and adenovirus type 7. The other tested
compounds did not show antiviral effect against the tested
viruses.
Structure-activity relationship
In the present study, we described a straight forward and
efficient synthesis of novel pyrrole, pyrrolo[2,3-d]pyrimidine
and pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives
as antiviral agents.
In order to analyze structure-activity relationship, two
structural components were considered: the nature of the
heterocyclic nucleus and the nature of the substituent.
First, regarding the influence of the heterocyclic nucleus
nature, we observed that pyrrolo[2,3-d]pyrimidine and
pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives ac-
quired significant antiviral activity over all the synthesized
pyrrole derivatives 1a–d, 2a,b, and 8a,b, which did not exhibit
significant activity.
If we compare the activity of the different compounds
against coxsackievirus B4, as shown in Fig. 5, there was no
significant difference in potency between compounds 5b, 7f,
10a, and 12a, which showed the highest potency.
As to the nature of the substituent: the pyrrolo[2,3-d]-
pyrimidine nucleus, 2-methyl-pyrrolo[2,3-d]pyrimidin-4-ones
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
M. S. Mohamed et al.
Ph
CN
Ph
CN
i
Ph
NH2
Ph
N
N
R
CHOEt
N
R
R
a
b
CH3
1c,d
8a,b
OCH3
ii
CH2Cl
N
N
N
H
N
Ph
NH2
N
N
N
Ph
N
Ph
iii
vii
N
Ph
N
R
N
Ph
N
N
Ph
N
R
10a,b
9a,b
14a,b
R
vi
iv
v
CH3
CH2CN
S
N
N
N
N
N
N
H
N
Ph
Ph
N
Ph
N
N
Ph
N
Ph
N
R
N
N
R
Ph
N
11a,b
13a,b
12a,b
R
Scheme 2. Synthesis of compounds 8a,b–14a,b. Reaction conditions: (i) CH(OEt)3; (ii) NH2NH2; (iii) CH(OEt)3; (iv) Ac2O; (v) C2S; (vi)
CNCH2CO2C2H5; (vii) ClCH2COCl.
3a,b, pyrrolo[2,3-d]pyrimidin-4-ones 4a,b, pyrrolo[2,3-d]py-
rimidine-4-thiones 6a,b and 3-amino-4-imino-pyrrolo[2,3-d]-
pyrimidines 9a,b were inactive. Substitution by 4-chloro group
of pyrrolo[2,3-d]pyrimidine induced the selective and potent
antiviral (against coxsackievirus) compound 5b, its IC50 was
108 mM (Table 2), while substitution with N-aryl group
resulted in an active compound against all viral strains,
compound 7f, its IC50 for coxsackievirus, adenovirus, and
rotavirus were 82, 144, and 123 mM respectively. While
comparing the activity of compounds 5b and 7f, it can be
inferred that replacement of 4-chloro group of compound 5b
with 4-(o-tolyl)-amino group (compound 7f) leads to
increasing the antiviral activity against coxsackievirus B4
from (IC50: 108 mM) to (IC50: 82 mM) in addition to its activity
against rotavirus Wa strain and adenovirus type 7.
On the other hand for substituents on position 2 of pyrrolo-
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus, 2-methyl-, 2-
cyanomethyl-, and 2-chloromethyl-pyrrolo[3,2-e][1,2,4]tria-
zolo[1,5-c]pyrimidines 11a,b, 13a,b and 14a,b were inactive.
The activity of these derivatives appeared only for pyrrolo[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine 10a against coxsackievirus
B4 and rotavirus Wa strain (IC50 were 137 and 162 mM
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Antiviral Compounds against Gastroenteric Viral Infections
Table 1. CC50 values of the tested compounds on BGM, Hep-2 and MA-104 cell lines.
Compounds
CC50 for BGM cell line (mM)
CC50 for Hep-2 cell line (mM)
CC50 for MA-104 cell line (mM)
1a
1b
1c
1d
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
175.716
189.261
200.321
191.550
182.529
145.673
130.378
121.395
189.466
150.807
180.459
168.148
155.634
169.115
157.456
152.638
130.833
152.638
148.001
143.740
123.206
123.206
98.644
118.624
153.319
171.847
124.542
167.677
144.404
162.225
138.396
155.715
136.206
153.337
111.123
128.769
175.716
189.261
200.321
191.550
182.529
145.673
130.378
97.116
146.430
162.224
171.703
191.550
182.529
145.673
104.302
97.116
189.466
150.807
128.899
144.127
129.695
96.637
112.468
130.833
130.833
130.833
148.001
143.740
102.672
123.206
98.644
118.624
153.319
147.297
124.542
143.723
144.404
162.225
115.329
155.715
113.505
153.337
88.899
189.466
150.807
154.679
168.148
129.695
144.956
157.456
152.638
130.833
152.638
148.001
143.740
102.672
123.206
123.305
142.349
153.319
171.847
149.451
167.677
144.404
162.225
138.396
155.715
136.206
153.337
133.348
150.231
107.308
respectively as shown in Table 2) and pyrrolo[3,2-e][1,2,4]-
triazolo[1,5-c]pyrimidine-2-thione 12a against all the tested
viruses.
These compounds may inhibit the rotavirus, adenovirus,
and coxsackievirus through inhibition of their viral polymer-
ases based on what has been reported regarding pyrrolopyr-
imidine derivatives [47–48]. Further investigation is required
to study the mechanism of action of these new compounds.
Among these compounds, compound 5b exhibited very high
specificity toward coxsackievirus B4, which renders this
molecule as a lead compound for drug discovery of antiviral
agents against coxsackievirus B4.
Experimental
Chemistry
All chemicals used as starting materials and reagents in this
study were reagent grade and were purchased from Merck
(Darmstadt, Germany). All melting points were uncorrected
and measured using Electro-thermal IA 9100 apparatus
(Shimadzu, Japan); IR spectra were recorded as potassium
bromide pellets on a Perkin-Elmer 1650 spectrophotometer
(USA), Faculty of Science, Cairo University, Cairo, Egypt. 1H
NMR spectra were determined on a Varian Mercury (300 MHz)
Conclusion
In this study, we synthesized novel pyrrole, pyrrolo[2,3-d]-
pyrimidine and pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine
derivatives. Some of the synthesized compounds were potent
inhibitors for gastroenteric viruses such as adenovirus,
coxsackievirus, and rotavirus.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
M. S. Mohamed et al.
Figure 1. Antiviral activity of non-toxic doses of tested
compound 5b against coxsackievirus B4, adenovirus type 7,
and rotavirus Wa strain. Compound 5b showed high specificity
toward coxsackievirus B4 and it exhibited no activity against
adenovirus type 7 and rotavirus Wa strain.
Figure 3. Antiviral activity of non-toxic doses of tested
compound 10a against coxsackievirus B4, adenovirus type 7,
and rotavirus Wa strain. Compound 10a was active against
coxsackievirus B4, and rotavirus Wa strain, it did not exhibit any
activity against adenovirus type 7. There was no significant
difference in the activity of compound 10a toward coxsack-
ievirus compared to its activity against rotavirus.
spectrometer (Varian UK) and chemical shifts were expressed
as ppm against TMS as internal reference (National Research
Center, Dokki, Cairo, Egypt). Mass spectrometric analysis
is carried out using a TSQ Quantum Access MAXtriplequadru-
pole system. Data acquisition and processing is 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. Column chromatography was per-
formed on (Merck) silica gel 60 (particle size 0.06–0.20 mm).
All new compounds yielded spectral data consistent with the
proposed structure and microanalysis within Æ0.4% of the
theoretical values. Compounds 1c,d were prepared before
[19], the other synthesized compounds are new and were
confirmed with spectral data.
General procedure for the synthesis of 2-amino-4,5-
diphenyl-1-substituted-1H-pyrrole-3-carbonitrile (1a,b)
A mixture of benzoin (2 g, 0.01 mol), the appropriate amine
(0.01 mol) and conc. HCl (6–8 drops) in toluene (50 mL) was
heated under reflux for 36 h, then cooled. Malononitrile
(0.66 mg, 0.01 mol) was added, followed by a catalytic amount
of pyridine (1.5 mL) portion-wise and left to reflux until a solid
Figure 4. Antiviral activity of non-toxic doses of tested
compound 12a against coxsackievirus B4, adenovirus type 7,
and rotavirus Wa strain. Compound 12a was active against
coxsackievirus B4, adenovirus type 7, and rotavirus Wa strain.
Compound 7f showed higher selectivity toward coxsackievirus,
there was significant difference in activity toward coxsack-
ievirus compared to its activity against adenovirus and rotavirus
with p < 0.001 and p < 0.01, respectively. Compound 12a was
more active against rotavirus than adenovirus, there was
significant difference in activity with p < 0.05.
Figure 2. Antiviral activity of non-toxic doses of tested
compound 7f against coxsackievirus B4, adenovirus type 7,
and rotavirus Wa strain. Compound 7f was active against
coxsackievirus B4, adenovirus type 7, and rotavirus Wa strain.
Compound 7f showed higher selectivity toward coxsackievirus,
there was significant difference in activity toward coxsack-
ievirus compared to its activity against adenovirus and rotavirus
with p < 0.001 and p < 0.05, respectively.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Antiviral Compounds against Gastroenteric Viral Infections
N-(3-Cyano-1-cyclohexyl-4,5-diphenyl-1H-pyrrol-2-yl)-
acetamide (2a)
Brown solid in 62%; m.p.: 203–205°C; IR (KBr) y (cmÀ 1): 3427
1
ÀÀ
–
(NH), 2217 (CÀÀ N), 1672 (C O); H NMR (DMSO-d , 300 MHz) d
–
6
ÀÀ
(ppm): 1.19–2.00 (m, 10H, cyclohexyl), 2.3 (s, 3H, CH3-CO), 3.88
(m, 1H, CH-N cyclohexyl), 7.01–8.6 (m, 10H, Ar-H), 11.6 (s, 1H,
NH, D2O exchangeable); ESI-MS m/z: 383.23 (Mþ, 3.25%);
Anal. calcd. for C25H25N3O (383.50): C, 78.30; H, 6.57; N, 10.96;
O, 4.17%. Found: C, 78.53; H, 6.29; N, 10.73; O, 4.35%.
N-(1-(3-Chlorophenyl)-3-cyano-4,5-diphenyl-1H-pyrrol-2-
yl)acetamide (2b)
Figure 5. Comparison of the activity of the different com-
pounds against coxsackievirus B4. There was no significant
difference in the activity of the different compounds (5b, 7f,
10a, and 12a) toward the coxsackievirus B4.
Light brown solid in 72%; m.p.: 190–192°C; IR (KBr) y (cmÀ 1):
3350 (NH), 2219 (CÀÀ N), 1699 (C O); 1H NMR (DMSO-d6,
ÀÀ
–
–
ÀÀ
300 MHz) d (ppm): 2.1 (s, 3H, CH3-CO), 7.27–8.36 (m, 14H, Ar-
H), 11.73 (s, 1H, NH, D2O exchangeable); ESI-MS m/z: 411.26
(Mþ, 35Cl, 7.6%), 413.32 (Mþþ2, 37Cl, 2.5%); Anal. calcd. for
was formed. The solvent was evaporated under reduced
pressure and the residue was recrystallized from methanol to
give compounds 1a,b.
C25H18ClN3O (411.88): C, 72.90; H, 4.40; Cl, 8.61; N, 10.20; O,
3.88%. Found: C, 72.68; H,4.21; Cl, 8.40; N, 9.95; O, 4.05%.
General procedure for the synthesis of 5,6-diphenyl-2-
methyl-7-substituted-3,7-dihydro-pyrrolo[2,3-d]-
pyrimidin-4-one (3a,b)
The appropriate aminopyrrole (1a,b) (0.01 mol) was refluxed
in a mixture of hydrochloric acid (5 mL) and acetic acid (15 mL)
for 18 h. The reaction mixture was cooled, poured onto ice,
and the formed solid was filtered off, dried, and recrystallized
from methanol to afford 3a,b.
2-Amino-1-cyclohexyl-4,5-diphenyl-1H-pyrrole-3-
carbonitrile (1a)
Brown solid in 65%; m.p.: 283–285°C; IR (KBr) y (cmÀ 1): 3439,
1
ÀÀ
3315 (NH2), 2217 (CÀÀ N); H NMR (DMSO-d6, 300 MHz) d (ppm):
ÀÀ
1.4–2.1 (m, 10H, cyclohexyl), 3.5 (m, 1H, CH-N cyclohexyl), 5.04
(s, 2H, NH2, D2O exchangeable), 7.05–7.75 (m, 10H, Ar-H); ESI-
MS m/z: 341.25 (Mþ, 6%); Anal. calcd. for C23H23N3 (341.46): C,
80.90; H, 6.79; N, 12.31%. Found: C, 81.11; H, 6.91; N, 12.12%.
7-Cyclohexyl-5,6-diphenyl-2-methyl-3H-pyrrolo[2,3-d]-
2-Amino-1-(3-chlorophenyl)-4,5-diphenyl-1H-pyrrole-3-
pyrimidin-4(7H)-one (3a)
carbonitrile (1b)
Grey solid in 62%; m.p.: 250–252°C; IR (KBr) y (cmÀ 1): 3425
Light brown solid in 78%; m.p.: 275–277°C; IR (KBr) y (cmÀ 1):
(NH), 1685 (C O), 1582 (C N); H NMR (DMSO-d , 300 MHz) d
1
–
–
–
–
6
1
ÀÀ
3441, 3340 (NH2), 2209 (CÀÀ N); H NMR (DMSO-d6, 300 MHz) d
(ppm): 1.41–2.00 (m, 10H, cyclohexyl), 2.22 (s, 3H, CH3), 3.88
(m, 1H, CH-N cyclohexyl), 7.11–8.20 (m, 10H, Ar-H), 11.85 (s,
1H, NH, D2O exchangeable); ESI-MS m/z: 383.33 (Mþ, 3%);
Anal. calcd. for C25H25N3O (383.50): C, 78.30; H, 6.57; N, 10.96;
O, 4.17%. Found: C, 78.11; H, 6.71; N, 11.19; O, 3.99%.
ÀÀ
(ppm): 6.07 (s, 2H, NH2, D2O exchangeable), 7.04–7.9 (m, 14H,
Ar-H); ESI-MS m/z: 369.10 (Mþ, 35Cl, 2%), 371.23 (Mþþ2, 37Cl,
0.66%); Anal. calcd. for C23H16ClN3 (369.86): C, 74.69; H, 4.36;
N, 11.36%. Found: C, 74.50; H, 4.13; N, 11.52%.
General procedure for the synthesis of N-(3-cyano-4,5-
diphenyl-1-substituted-1H-pyrrol-2-yl)acetamide (2a,b)
The appropriate aminopyrrole (1a,b) (0.01 mol) in acetic
anhydride (40 mL) was heated under reflux for 36 h, then
cooled, poured onto ice water, and neutralized with
ammonia to give precipitate, which was filtered off, dried,
and recrystallized from methanol, to give compounds 2a,b.
7-(3-Chlorophenyl)-5,6-diphenyl-2-methyl-3H-pyrrolo[2,3-
d]pyrimidin-4(7H)-one (3b)
Greyish brown solid in 72%; m.p.: 245–247°C; IR (KBr) y (cmÀ 1):
3395 (NH), 1707 (C O), 1589 (C N); 1H NMR (DMSO-d6,
–
–
–
–
300 MHz) d (ppm): 2.1 (s, 3H, CH3), 7.1–8.2 (m, 14H, Ar-H),
11.42 (s, 1H, NH, D2O exchangeable); ESI-MS m/z: 411.32 (Mþ,
35Cl, 2%), 413 (Mþþ2, 37Cl, 0.67); Anal. calcd. for C25H18ClN3O
Table 2. IC50 (mM) of compounds 5b, 7f, 10a, and 12a against coxsackievirus B4, adenovirus type 7, and rotavirus Wa
strain.
Compounds
IC50 for coxsackievirus B4 (mM)
IC50 for adenovirus type 7 (mM)
IC50 for rotavirus Wa strain (mM)
5b
7f
10a
12a
108
82
137
—
144
—
—
123
162
115
97
138
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
7

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
M. S. Mohamed et al.
(411.88): C, 72.90; H, 4.40; Cl, 8.61; N, 10.20; O, 3.88%. Found:
C, 72.73; H, 4.11; Cl, 8.89; N, 10.52; O, 3.62%.
General procedure for the synthesis of 5,6-diphenyl-7-
substituted-3,7-dihydro-pyrrolo[2,3-d]pyrimidine-4-
thione (6a,b)
General procedure for the synthesis of 5,6-diphenyl-7-
substituted-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one (4a,b)
The appropriate aminopyrrole (1a,b) (0.01 mol) in formic acid
(20 mL, 85%) was heated under reflux for 36 h, cooled, poured
onto ice water to give a precipitate 4a,b, which was filtered
off, dried, and recrystallized from ethanol.
A mixture of 4-chloropyrrolopyrimidine (5a,b) (0.01 mol) and
thiourea (1.5 g, 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 6a,b.
7-Cyclohexyl-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidine-
7-Cyclohexyl-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidin-
4(7H)-one (4a)
4(7H)-thione (6a)
Brown solid in 64%; m.p.: 205–207°C; IR (KBr) y (cmÀ 1): 3445
1
Yellowish brown solid in 65%; m.p.: >300°C; IR (KBr) y (cmÀ 1):
(NH), 1605 (C N), 1260 (C S); H NMR (DMSO-d , 300 MHz) d
–
–
–
–
6
3365 (NH), 1698 (C O), 1595 (C N); 1H NMR (DMSO-d6,
(ppm): 1.4–2.0 (m, 10H, cyclohexyl), 3.61 (m, 1H, CH-N
cyclohexyl), 6.9–7.8 (m, 10H, Ar-H), 9.2 (s, 1H, C2-H), 11.7 (s,
1H, NH, D2O exchangeable); ESI-MS m/z: 385.11 (Mþ, 2.5%);
Anal. calcd. for C24H23N3S (385.52): C, 74.77; H, 6.01; N, 10.90;
S, 8.32%. Found: C, 74.89; H, 6.27; N, 10.63; S, 8.15%.
–
–
–
–
300 MHz) d (ppm): 1.1–2.3 (m, 10H, cyclohexyl), 3.3 (m, 1H, CH-
N cyclohexyl), 7.1–8.0 (m, 10H, Ar-H), 8.3 (s, 1H, C2-H), 11.9 (s,
1H, NH, D2O exchangeable); ESI-MS m/z: 369.30 (Mþ, 11%);
Anal. calcd. for C24H23N3O (369.46): C, 78.02; H, 6.27; N, 11.37;
O, 4.33%. Found: C, 78.24; H, 6.03; N, 11.52; O, 4.18%.
7-(3-Chlorophenyl)-5,6-diphenyl-3H-pyrrolo[2,3-d]-
pyrimidine-4(7H)-thione (6b)
7-(3-Chlorophenyl)-5,6-diphenyl-3H-pyrrolo[2,3-d]-
pyrimidin-4(7H)-one (4b)
Dark brown solid in 69%; m.p.: 215–217°C; IR (KBr) y (cmÀ 1):
Yellowish brown solid 69%; m.p.: >300°C; IR (KBr) y (cmÀ 1):
3375 (NH), 1585 (C N), 1255 (C S); 1H NMR (DMSO-d6,
–
–
–
–
3382 (NH), 1680 (C O), 1587 (C N); 1H NMR (DMSO-d6,
300 MHz) d (ppm): 7.2–8.0 (m, 14H, Ar-H), 8.8 (s, 1H, C2-H), 11.8
(s, 1H, NH, D2O exchangeable); ESI-MS m/z: 413.23 (Mþ, 35Cl,
3%); 415.31 (Mþþ2, 37Cl, 1%); Anal. calcd. for C24H16ClN3S
(413.92): C, 69.64; H, 3.90; Cl, 8.57; N, 10.15%. Found: C, 69.41;
H, 3.70; Cl, 8.73; N, 10.36%.
–
–
–
–
300 MHz) d (ppm): 7.0–8.0 (m, 14H, Ar-H), 9.1 (s, 1H, C2-H), 11.6
(s, 1H, NH, D2O exchangeable); ESI-MS m/z: 397.23 (Mþ, 35Cl,
3%), 399 (Mþþ2, 37Cl, 1.10%); Anal. calcd. for C24H16ClN3O
(397.86): C, 72.45; H, 4.05; Cl, 8.91; N, 10.56; O, 4.02%. Found:
C, 72.24; H, 4.28; Cl, 8.70; N, 10.31; O, 4.29%.
General procedure for the synthesis of 5,6-diphenyl-7-
substituted-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-arylamine
(7a–h)
A mixture of 4-chloro pyrrolopyrimidine (5a,b) (0.01 mol), the
appropriate amine (0.01 mol) 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 7a–h.
General procedure for the synthesis of 4-chloro-5,6-
diphenyl-7-substituted-7H-pyrrolo[2,3-d]pyrimidine (5a,b)
The appropriate pyrrolopyrimidinone (4a,b) (0.01 mol) 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 5a,b.
4-Chloro-7-cyclohexyl-5,6-diphenyl-7H-pyrrolo[2,3-d]-
pyrimidine (5a)
7-Cyclohexyl-N,5,6-triphenyl-7H-pyrrolo[2,3-d]pyrimidin-
Dark grey solid in 73%; m.p.: 267–269°C; IR (KBr) y (cmÀ 1):
4-amine (7a)
1
Brown solid in 62%; m.p.: 200–202°C; IR (KBr) y (cmÀ 1): 3337
–
–
–
3059, 2929 (CH), 1620 (C C), 1585 (C N); H NMR (DMSO-d ,
–
6
1
–
300 MHz) d (ppm): 1.3–2.1 (m, 10H, cyclohexyl), 3.6 (m, 1H, CH-
N cyclohexyl), 7.0–8.0 (m, 10H, Ar-H), 8.4 (s, 1H, C2-H); ESI-MS
m/z: 387.26 (Mþ, 35Cl, 2%), 389.14 (Mþþ2, 37Cl, 0.65%); Anal.
calcd. for C24H22ClN3 (387.90): C, 74.31; H, 5.72; Cl, 9.14; N,
10.83%. Found: C, 74.49; H,5.46; Cl, 9.30; N, 10.61%.
(NH), 1612 (C N); H NMR (DMSO-d , 300 MHz) d (ppm): 1.2–
–
6
1.7 (m, 10H, cyclohexyl), 3.9 (m, 1H, CH-N cyclohexyl), 7.0–7.9
(m, 15H, Ar-H), 8.9 (s, 1H, C2-H), 10.9 (s, 1H, NH, D2O
exchangeable); ESI-MS m/z: 444.23 (Mþ, 2%); Anal. calcd. for
C
30H28N4 (444.57): C, 81.05; H, 6.35; N, 12.60%. Found: C,
81.31; H,6.10; N, 12.85%.
4-Chloro-7-(3-chlorophenyl)-5,6-diphenyl-7H-pyrrolo[2,3-
d]pyrimidine (5b)
7-Cyclohexyl-5,6-diphenyl-N-o-tolyl-7H-pyrrolo[2,3-d]-
Dark grey solid in 79%; m.p.: 278–280°C; IR (KBr) y (cmÀ 1): 3045,
pyrimidin-4-amine (7b)
2960(CH), 1615(C C), 1571 (C N); HNMR(DMSO-d , 300 MHz)
Light brown solid in 67%; m.p.: 190–192°C; IR (KBr) y (cmÀ 1):
1
–
–
–
–
6
1
–
d (ppm): 6.8–7.9 (m, 14H, Ar-H), 8.3 (s, 1H, C2-H); ESI-MS m/z:
3359 (NH), 1585 (C N); H NMR (DMSO-d , 300 MHz) d (ppm):
–
6
416.20(Mþ, 35Cl, 2%), 418.22(Mþþ2, 37Cl,1.3%);Anal. calcd. for
1.1–1.9 (m, 10H, cyclohexyl), 2.2 (s, 3H, CH3), 3.7 (m, 1H, CH-N
cyclohexyl), 6.8–7.8 (m, 14H, Ar-H), 9.0 (s, 1H, C2-H), 11.5 (s, 1H,
NH, D2O exchangeable); ESI-MS m/z: 458.25 (Mþ, 2.5%); Anal.
C24H15Cl2N3 (416.30): C, 69.24; H, 3.63; Cl, 17.03; N, 10.09%.
Found: C, 69.67; H, 3.41; Cl, 16.85; N, 10.41%.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
8

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Antiviral Compounds against Gastroenteric Viral Infections
calcd. forC31H30N4 (458.60): C, 81.19; H, 6.59; N, 12.22%.
Found: C, 80.90; H, 6.30; N, 12.41%.
under reduced pressure to give precipitates which were
recrystallized from methanol to yield compounds 8a,b.
7-Cyclohexyl-5,6-diphenyl-N-m-tolyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine (7c)
2-(Ethoxymethylene)amino-1-(4-methylphenyl)-4,5-
diphenyl-1H-pyrrole-3-carbonitrile (8a)
Brown solid in 69%; m.p.: 217–219°C; IþR (KBr) y (cmÀ 1): 3359
Brown solid in 61%; m.p.: 172–174°C; IR (KBr) y (cmÀ 1): 2207
1
À
–
–
(NH), 1585 (C N); ESI-MS m/z: 458.38 (M , 4%); Anal. calcd. for
(CÀ N), 1566 (C N), 1226 (C–O); H NMR (DMSO-d , 300 MHz) d
–
–
6
À
C
31H30N4 (458.60): C, 81.19; H, 6.59; N, 12.22%. Found: C,
(ppm): 2.12 (t, 3H, CH3*-CH2), 2.4 (s, 3H, CH3), 3.5 (q, 2H, CH3-
CH2*), 6.8–7.9 (m, 15H, Ar-H, CH); ESI-MS m/z: 405 (Mþ, 22%);
Anal. calcd. for C27H23N3O (405.50): C, 79.97; H, 5.72; N, 10.36;
O, 3.95%. Found: C, 79.68; H, 5.52; N, 10.48; O, 4.23%.
81.42; H, 6.42; N, 12.39%.
7-Cyclohexyl-5,6-diphenyl-N-p-tolyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine (7d)
Brown solid in 71%; m.p.: 221–223°C; IR (KBr) y (cmÀ 1): 3359
2-(Ethoxymethylene)amino-1-(4-methoxyphenyl)-4,5-
diphenyl-1H-pyrrole-3-carbonitrile (8b)
þ
–
(NH), 1585 (C N); ESI-MS m/z: 458.34 (M , 2.4%); Anal. calcd.
–
for C31H30N4 (458.60): C, 81.19; H, 6.59; N, 12.22%. Found: C,
81.01; H, 6.77; N, 12.00%.
Dark brown solid in 68%; m.p.: 189–191°C; IR (KBr) y (cmÀ 1):
2205 (CÀÀ N), 1554 (C N), 1246 (C–O); 1H NMR (DMSO-d6,
ÀÀ
–
–
ÀÀ
300 MHz) d (ppm): 2.2 (t, 3H, CH3*-CH2), 3.42 (q, 2H, CH3-
CH2*), 3.5 (s, 3H, OCH3), 6.7–8.0 (m, 15H, Ar-H, CH); ESI-MS
m/z: 421 (Mþ, 17.4%); Anal. calcd. for C27H23N3O2 (421.50): C,
76.94; H, 5.50; N, 9.97; O, 7.59%. Found: C, 77.21; H, 5.83; N,
10.14; O, 7.28%.
7-(3-Chlorophenyl)-N,5,6-triphenyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine (7e)
Brownsolidin61%;m.p.:211–213°C;IR(KBr)y(cmÀ 1):3435(NH),
1
–
1607 (C N); H NMR (DMSO-d , 300 MHz) d (ppm): 6.9–8.0 (m,
–
6
19H, Ar-H), 9.2 (s, 1H, C2-H), 11.9 (s, 1H, NH, D2O exchangeable);
ESI-MSm/z:472.27(Mþ, 35Cl, 6%),474.30(Mþþ2,37Cl,2%);Anal.
calcd. for C30H21ClN4 (472.97): C, 76.18; H, 4.48; Cl, 7.50; N,
11.85%. Found: C, 76.34; H, 4.21; Cl, 7.32; N, 11.57%.
General procedure for the synthesis of 3-amino-4-imino-
5,6-diphenyl-7-substituted-7H-pyrrolo[2,3-d]pyrimidine
(9a,b)
A mixture of the appropriate 2-(ethoxymethylene)amino-
pyrrole (8a,b) (0.01 mol) and hydrazine hydrate (5 mL, 80%) in
ethanol (20 mL) was stirred at room temperature for 6 h. The
solid formed was recrystallized from methanol to yield
compounds 9a,b.
7-(3-Chlorophenyl)-5,6-diphenyl-N-o-tolyl-7H-pyrrolo[2,3-
d]pyrimidin-4-amine (7f)
Brown solid in 60%; m.p.: 223–225°C; IR (KBr) y (cmÀ 1): 3448
þ
(NH), 1598 (C N); ESI-MS m/z: 486.90 (M , 35Cl, 2.6%), 489
–
–
(Mþþ2, 37Cl, 0.87%); Anal. calcd. for C31H23ClN4 (486.99): C,
76.46; H, 4.76; Cl, 7.28; N, 11.50%. Found: C, 76.62; H, 4.91; Cl,
7.41; N, 11.23%.
3-Amino-4-imino-7-(4-methylphenyl)-5,6-diphenyl-7H-
pyrrolo[2,3-d]pyrimidine (9a)
Grey solid in 89%; m.p.: 188–190°C; IR (KBr) y (cmÀ 1): 3353, 3312
1
–
7-(3-Chlorophenyl)-5,6-diphenyl-N-m-tolyl-7H-pyrrolo-
(NH ), 3166 (NH), 1576 (C N); H NMR (DMSO-d , 300 MHz) d
–
2
6
[2,3-d]pyrimidin-4-amine (7g)
(ppm):2.25(s,3H,CH3),5.36(s,2H,NH2,D2Oexchangeable),6.7–
7.9 (m, 14H, Ar-H), 8.27 (s, 1H, C2-H), 9.21 (s, 1H, NH); ESI-MS m/z:
391 (Mþ, 18%); Anal. calcd. for C25H21N5 (391.34): C, 76.73; H,
5.37; N, 17.90%. Found: C, 76.37; H, 5.59; N, 17.64%.
Brownsolidin61%;m.p.:229–231°C;IR(KBr)y(cmÀ 1):3448(NH),
1598 (C N); ESI-MS m/z: 487.30 (M , 35Cl, 2%), 489.42 (Mþþ2,
þ
–
–
37Cl,0.86%);Anal.calcd.forC31H23ClN4 (486.99):C,76.46;H,4.76;
Cl, 7.28; N, 11.50%. Found: C, 76.20; H, 4.85; Cl, 7.55; N, 11.72%.
3-Amino-4-imino-7-(4-methoxyphenyl)-5,6-diphenyl-7H-
7-(3-Chlorophenyl)-5,6-diphenyl-N-p-tolyl-7H-pyrrolo[2,3-
pyrrolo[2,3-d]pyrimidine (9b)
d]pyrimidin-4-amine (7h)
Dark grey solid in 93%; m.p.: 196–198°C; IR (KBr) y (cmÀ 1):
Brown solid in 63%; m.p.: 213–215°C; IR (KBr) y (cmÀ 1): 3448
3402, 3326 (NH ), 3158 (NH), 1616 (C N), 1234 (C–O); H NMR
1
–
–
2
1
–
(NH), 1598 (C N); H NMR (DMSO-d , 300 MHz) d (ppm): 3.0 (s,
(DMSO-d6, 300 MHz) d (ppm): 3.54 (s, 3H, OCH3), 5.4 (s, 2H,
NH2, D2O exchangeable), 6.7–7.6 (m, 14H, Ar-H), 8.3 (s, 1H, C2-
H), 9.3 (s, 1H, NH); ESI-MS m/z: 407 (Mþ, 29.5%); Anal. calcd.
for C25H21N5O (407.34): C, 73.71; H, 5.16; N, 17.19; O, 3.63%.
Found: C, 73.38; H, 4.82; N, 17.34; O, 3.87%.
–
6
3H, CH3), 7.1–7.9 (m, 18H, Ar-H), 9.6 (s, 1H, C2-H), 11.7 (s, 1H,
NH, D2O exchangeable); ESI-MS m/z: 486.27 (Mþ, 35Cl, 3%),
488.68 (Mþþ2, 37Cl, 1%); Anal. calcd. for C31H23ClN4 (486.99):
C, 76.46; H, 4.76; Cl, 7.28; N, 11.50%. Found: C, 76.71; H, 4.53;
Cl, 7.01; N, 11.35%.
General procedure for the synthesis of 8,9-diphenyl-7-
substituted-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]-
pyrimidine (10a,b)
The appropriate 4-imino-pyrrolopyrimidine (9a,b) (0.01 mol)
was heated under reflux for 8 h in triethylorthoformate
(15 mL), the solvent was removed under reduced pressure to
General procedure for the synthesis of 2-
(ethoxymethylene)amino-4,5-diphenyl-1-substituted-1H-
pyrrole-3-carbonitrile (8a,b)
Pyrroles (1c,d) (0.01 mol) were heated under reflux in
triethylorthoformate (30 mL) for 8 h. The solvent was removed
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
9

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
M. S. Mohamed et al.
give precipitates which were recrystallized from methanol to
7-(4-Methylphenyl)-8,9-diphenyl-7H-pyrrolo[3,2-e][1,2,4]-
yield compounds 10a,b.
triazolo[1,5-c]pyrimidine-2-thione (12a)
Light brown solid in 44%; m.p.: 223–225°C; IR (KBr) y (cmÀ 1):
1
–
–
7-(4-Methylphenyl)-8,9-diphenyl-7H-pyrrolo[3,2-e][1,2,4]-
3228 (NH), 1607 (C N); H NMR (DMSO-d , 300 MHz) d (ppm):
6
triazolo[1,5-c]pyrimidine (10a)
2.32 (s, 3H, CH3), 6.8–8.0 (m, 14H, Ar-H), 9.2 (s, 1H, C5-H), 9.33
Light brown solid in 59%; m.p.: 214–216°C; IR (KBr) y (cmÀ 1):
(s, 1H, NH); ESI-MS m/z: 433 (Mþ, 10.7%); Anal. calcd. for
1
–
–
1588 (C N); H NMR (DMSO-d , 300 MHz) d (ppm): 2.4 (s, 3H,
C26H19N5S (433.54): C, 72.03; H, 4.42; N, 16.15; S, 7.40%.
6
CH3), 7.0–7.6 (m, 14H, Ar-H), 9.71 (s,1H, C3-H), 9.73 (s, 1H,C5-H);
ESI-MSm/z:401(Mþ,24%);Anal.calcd.forC26H19N5 (401.47):C,
77.79; H, 4.77; N, 17.44%. Found: C, 77.55; H, 4.98; N, 17.13%.
Found: C, 72.32; H, 4.15; N, 15.98; S, 7.67%.
7-(4-Methoxyphenyl)-8,9-diphenyl-7H-pyrrolo[3,2-e]-
[1,2,4]triazolo[1,5-c]pyrimidine-2-thione (12b)
7-(4-Methoxyphenyl)-8,9-diphenyl-7H-pyrrolo[3,2-e]-
Dark brown solid in 54%; m.p.: 229–231°C; IR (KBr) y (cmÀ 1):
3123 (NH), 1576 (C N), 1234 (C–O); 1H NMR (DMSO-d6,
–
[1,2,4]triazolo[1,5-c]pyrimidine (10b)
–
Brown solid in 63%; m.p.: 221–223°C; IR (KBr) y (cmÀ 1): 1609
300 MHz) d (ppm): 3.4 (s, 3H, OCH3), 6.7–7.7 (m, 14H, Ar-H), 9.3
(s,1H, C5-H), 9.38 (s, 1H, NH); ESI-MS m/z: 449 (Mþ, 26%); Anal.
calcd. for C26H19N5OS (449.54): C, 69.47; H, 4.26; N, 15.58; S,
7.13 O, 3.56%. Found: C, 69.76; H, 4.11; N, 15.33; S, 7.43 O,
3.88%.
1
–
(C N), 1235 (C–O); H NMR (DMSO-d , 300 MHz) d (ppm): 3.42
–
6
(s, 3H, OCH3), 6.8–7.6 (m, 14H, Ar-H), 9.35 (s,1H, C3-H), 9.63 (s,
1H, C5-H); ESI-MS m/z: 417 (Mþ, 2.5%); Anal. calcd. for
C
26H19N5O (417.47): C, 74.80; H, 4.59; N, 16.78; O, 3.83%.
Found: C, 74.55; H, 4.32; N, 16.99; O, 4.12%.
General procedure for the synthesis of 2-cyanomethyl-8,9-
diphenyl-7-substituted-7H-pyrrolo[3,2-e][1,2,4]triazolo-
[1,5-c]pyrimidine (13a,b)
A mixture of the appropriate 4-imino-pyrrolopyrimidine
(9a,b) (0.01 mol) and ethyl cyanoacetate (0.02 mol) was
refluxed in absolute ethanol (15 mL) for 8 h. After removal
of ethanol precipitates were formed which were filtered off,
dried, and recrystallized from methanol to yield compounds
13a,b.
General procedure for the synthesis of 2-methyl-8,9-
diphenyl-7-substituted-7H-pyrrolo[3,2-e][1,2,4]triazolo-
[1,5-c]pyrimidine (11a,b)
The appropriate 4-imino-pyrrolopyrimidine (9a,b) (0.01 mol)
was heated under reflux for 8 h in acetic anhydride (30 mL),
cooled, poured onto ice water and neutralized with ammonia
to give precipitates which were filtered off, dried, and
recrystallized from ethanol to yield compounds 11a,b.
2-Methyl-7-(4-methylphenyl)-8,9-diphenyl-7H-pyrrolo-
2-Cyanomethyl-7-(4-methylphenyl)-8,9-diphenyl-7H-
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (11a)
pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (13a)
Brown solid in 68%; m.p.: 205–207°C; IR (KBr) y (cmÀ 1): 1612
Brown solid in 37%; m.p.: 212–214°C; IR (KBr) y (cmÀ 1): 2206
1
1
À
–
–
–
(C N); H NMR (DMSO-d , 300 MHz) d (ppm): 2.31 (s, 3H, CH -
(CÀ N), 1583 (C N); H NMR (DMSO-d , 300 MHz) d (ppm): 2.3
–
6
3
6
À
Ph),2.4(s,3H,CH3-triazole),7.0–7.9(m,14H,Ar-H),9.5(s,1H,C5-
H); ESI-MS m/z: 415 (Mþ, 30.9%); Anal. calcd. for C27H21N5
(415.50):C,78.05;H,5.09;N,16.86%.Found:C,77.84;H,5.33;N,
17.09%.
(s, 3H, CH3), 3.6 (s, 2H, CH2), 6.9–8.1 (m, 14H, Ar-H), 9.42 (s,1H,
C5-H); ESI-MS m/z: 440 (Mþ, 8%); Anal. calcd. for C28H20N6
(440.51): C, 76.35; H, 4.58; N, 19.08%. Found: C, 76.64; H, 4.91;
N, 18.75%.
2-Methyl-7-(4-methoxyphenyl)-8,9-diphenyl-7H-pyrrolo-
2-Cyanomethyl-7-(4-methoxyphenyl)-8,9-diphenyl-7H-
pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (13b)
Brown solid in 44%; m.p.: 218–220°C; IR (KBr) y (cmÀ 1): 2206
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (11b)
Brown solid in 58%; m.p.: 216–218°C; IR (KBr) y (cmÀ 1): 1615
1
1
À
–
(C N), 1212 (C–O); H NMR (DMSO-d , 300 MHz) d (ppm): 2.42
(CÀ N), 1604 (C–N), 1261 (C–O); H NMR (DMSO-d6, 300 MHz) d
–
6
À
(s, 3H, CH3-triazole), 3.5 (s, 3H, OCH3-Ph), 6.8–7.8 (m, 14H, Ar-
H), 9.45 (s,1H, C5-H); ESI-MS m/z: 431 (Mþ, 28%); Anal. calcd.
for C27H21N5O (431.50): C, 75.16; H, 4.91; N, 16.23; O, 3.71%.
Found: C, 75.41; H, 5.17; N, 16.55; O, 3.63%.
(ppm): 3.43 (s, 2H, CH2), 3.56 (s, 3H, OCH3), 6.7–7.9 (m, 14H, Ar-
H), 9.5 (s, 1H, C5-H); ESI-MS m/z: 456 (Mþ, 26.3%); Anal. calcd.
for C28H20N6O (456.51): C, 73.67; H, 4.42; N, 18.41; O, 3.50%.
Found: C, 73.93; H, 4.66; N, 18.15; O, 3.81%.
General procedure for the synthesis of 8,9-diphenyl-7-
substituted-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]-
pyrimidine-2-thione (12a,b)
General procedure for the synthesis of 2-chloromethyl-8,9-
diphenyl-7-substituted-7H-pyrrolo[3,2-e][1,2,4]triazolo-
[1,5-c]pyrimidine (14a,b)
The appropriate 4-imino-pyrrolopyrimidine (9a,b) (0.01 mol),
CS2 (0.76 g, 0.01 mol) and KOH (0.01 mol) in absolute
ethanol (15 mL) were refluxed for 11 h. After removal of
ethanol precipitates were formed which were filtered off,
dried, and recrystallized from ethanol to yield compounds
12a,b.
A mixture of the appropriate 4-imino-pyrrolopyrimidine (9a,
b) (0.01 mol) and chloroacetylchloride (0.02 mol) was
refluxed in absolute ethanol (15 mL) for 8 h. After removal
of ethanol precipitates were formed which were filtered off,
dried, and recrystallized from methanol to yield compounds
14a,b.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
10

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Antiviral Compounds against Gastroenteric Viral Infections
2-Chloromethyl-7-(4-methylphenyl)-8,9-diphenyl-7H-
Cell viability assay
pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (14a)
It was done by Trypan blue dye exclusion method [50]. BGM,
Hep-2 and MA-104 cell lines were grown in 12-well tissue
culture plates (2 Â 105 cells/mL). After 24 h incubation, the same
assay described above for tested samples cytotoxicity was
followed by applying 100 mL of tested samples dilutions (serial
dilutions) per well. After 72 h, the medium was removed, cells
were trypsinized and an equal volume of 0.4% (w/v) Trypan
blue dye aqueous solution was added to cell suspension. Viable
cell were counted under the phase contrast microscope.
Dark brown solid in 65%; m.p.: 198–200°C; IR (KBr) y (cmÀ 1):
1
–
1616 (C N); H NMR (DMSO-d , 300 MHz) d (ppm): 2.31 (s, 3H,
–
6
CH3), 4.2 (s, 2H, CH2), 6.8–8.0 (m, 14H, Ar-H), 9.3 (s,1H, C5-H);
ESI-MS m/z: 449 (Mþ, 35Cl, 20.5%), 451 (Mþþ2, 37Cl, 6.8%);
Anal. calcd. for C27H20ClN5 (449.95): C, 72.08; H, 4.48; Cl, 7.88;
N, 15.56%. Found: C, 71.94; H, 4.77; Cl, 8.16; N, 15.67%.
2-Chloromethyl-7-(4-methoxyphenyl)-8,9-diphenyl-7H-
pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (14b)
Light brown solid in 70%; m.p.: 207–209°C; IR (KBr) y (cmÀ 1):
Determination of coxsackievirus B4, adenovirus type 7,
and rotavirus Wa strain titers using plaque assay
1
–
1567 (C N), 1237 (C–O); H NMR (DMSO-d , 300 MHz) d (ppm):
–
6
3.4 (s, 3H, OCH3), 4.4 (s, 2H, CH2), 6.7–8.0 (m, 14H, Ar-H), 9.44
(s, 1H, C5-H); ESI-MS m/z: 465 (Mþ, 35Cl, 44.3%), 467 (Mþþ2,
37Cl, 14.8%); Anal. calcd. for C27H20ClN5O (465.95): C, 69.60; H,
4.33; Cl, 7.61; N, 15.03; O, 3.43%. Found: C, 69.89; H, 4.56; Cl,
7.92; N, 14.84; O, 3.14%.
Non-toxic dilutions were mixed (100 mL) with 100 mL of
different doses of coxsackievirus B4 (1 Â 105, 1 Â 106,
1 Â 107) and the same doses of adenovirus type 7 and
rotavirus Wa strain. The infectivity of rotavirus stocks were
activated with 10 mg/mL trypsin for 30 min at 37°C. The
mixture was incubated for 0.5 h at 37°C. The inoculation of
(100 mL) 10 fold dilutions of treated and untreated coxsack-
ievirus B4, adenovirus type 7, rotavirus Wa strain was carried
out separately into BGM, Hep-2, MA-104 cell lines respectively
in 12-well plates. After 1 h of incubation for adsorption at 37°
C in a 5% CO2–water vapor atmosphere without constant
rocking, the plates were rocked intermittently to keep the
cells from drying. After adsorption, 1 mL of 2Â media
(Dulbecco’s Modified Eagle Medium, Gibco-BRL (DMEM) plus
1 mL 1% agarose) was added to each well, 0.5 mg/mL was
added to the media–agarose mixture in the case of rotavirus
Wa strain and the plates were incubated at 37°C in a 5% CO2–
water vapor atmosphere. After the appropriate incubation
period, the cells were stained with 0.4% crystal violet after
formalin fixation, and the number of plaques counted. The
viral titers were then calculated, and expressed as plaque-
forming units per milliliter (pfu/mL) [51].
Antiviral screening
Cell culture
The cell lines BGM, Hep-2, and MA-104 were obtained from
the Holding Company for Biological Products and Vaccines,
VACSERA, Egypt. Cell lines were routinely cultured in 96-well
plates (Greiner-Bio One, Germany) in DMEM (GIBCO BRL), at
37°C in a humidified incubator of 5% (v/v) CO2.
Cytotoxicity test
The cytotoxicity test was performed according to methods
reported [47, 48]. Briefly, all samples (50 mg) were dissolved
in 1 mL DMSO containing 24 mL of 100Â of antibiotic/
antimycotic mixture for decontamination of the samples.
One hundred milliliters of the dissolved samples were diluted
by serial dilution. One hundred milliliters of each dilution
were inoculated in BGM, Hep-2, and MA-104 cell lines
previously cultured in 96-well plates to estimate the non-
toxic dose of the tested samples. Cytotoxicity assay was done
using cell morphology evaluation by inverted light micro-
scope and cell viability test applying Trypan blue dye
exclusion method.
Data analysis
IC50 for each compound were obtained from dose–effect
curves. The IC50 is the concentration of the compound that
causes 50% inhibition of the virus. The dose–effect curve was
plotted from the average of four assays with five concen-
trations within the inhibitory range of the compound.
Cell morphology evaluation by inverted light microscopy
BGM, Hep-2, and MA-104 cell cultures (2 Â 105 cells/mL) were
prepared separately in 96-well tissue culture plates (Greiner-
Bio One, Germany) and incubated for 24 h at 37°C in
humidified 5% (v/v) CO2 atmosphere. Confluent cell mono-
layers were formed, the medium was removed from each well
and replenished with 100 mL of serial dilution of different
samples tested prepared in DMEM (GIBCO BRL). For cell
controls 100 mL of DMEM without samples was added. All
cultures were incubated at 37°C in a humidified 5% (v/v) CO2
atmosphere for 72 h. Cell morphology was observed daily for
microscopically detectable morphological alterations, such as
loss of confluence, cell rounding and shrinking, cytoplasm
granulation and vacuolization. Morphological changes were
scored [49].
A.I.S. is supported by a grant from the Academy of Scientific
Research and Technology (ASRT) in Egypt.
The authors have declared no conflict of interest.
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