Syntheses and in vitro biological screening of 1-aryl-10H-[1,2,4] triazolo[3′,4′:3,4][1,2,4]triazino[5,6-b]indoles

Article abstract of DOI:10.1007/s00044-012-0342-1

Structurally diverse 1-aryl-10H-[1,2,4]triazolo[3′,4′:3,4][1,2, 4]triazino[5,6-b]indoles 4a-v were synthesized by regiospecific heterocyclizations. The designed molecular diversity was evaluated in vitro in parallel cell-based assays for cytotoxicity of viruses multiplication supporting cell lines and antiviral activity against viruses representative of two of three genera of the Flaviviridae family. The compound library was also tested against Retrovirus (HIV-1), two Picornaviruses (CVB-2 and Sb-1), and Paramyxoviridae (VSV) representative. Among double-stranded RNA (dsRNA) viruses, Reoviridae representative (Reo-1) was tested. Two representatives of DNA virus families were also included - HSV-1 (Herpesviridae) and VV (Poxviridae). The compounds 4m and 4o were found cytotoxic, having CC₅₀ values ranging from 4 to 30 μM. Moreover, compound 4v has exhibited significant activity (EC₅₀ = 3 μM) against BVDV.

Full text of DOI:10.1007/s00044-012-0342-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3675–3686
DOI 10.1007/s00044-012-0342-1
ORIGINAL RESEARCH
Syntheses and in vitro biological screening
of 1-aryl-10H-[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indoles
•
Kuldip Upadhyay Atul Manvar Roberta Loddo
•
•
•
Paolo La Colla Vijay Virsodiya Jalpa Trivedi
•
•
•
Ravi Chaniyara Anamik Shah
Received: 26 July 2012 / Accepted: 7 November 2012 / Published online: 30 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Structurally diverse 1-aryl-10H-[1,2,4]triazol-
o[30,40:3,4][1,2,4]triazino[5,6-b]indoles 4a–v were synthe-
sized by regiospecific heterocyclizations. The designed
molecular diversity was evaluated in vitro in parallel cell-
based assays for cytotoxicity of viruses multiplication
supporting cell lines and antiviral activity against viruses
representative of two of three genera of the Flaviviridae
family. The compound library was also tested against
Retrovirus (HIV-1), two Picornaviruses (CVB-2 and Sb-1),
and Paramyxoviridae (VSV) representative. Among dou-
ble-stranded RNA (dsRNA) viruses, Reoviridae represen-
tative (Reo-1) was tested. Two representatives of DNA
virus families were also included—HSV-1 (Herpesviridae)
and VV (Poxviridae). The compounds 4m and 4o were
found cytotoxic, having CC50 values ranging from 4 to
30 lM. Moreover, compound 4v has exhibited significant
activity (EC50 = 3 lM) against BVDV.
Introduction
The inherent toxicity and appearance of drug-resistant
viruses restricted the use of most antiviral agents, hence the
identification of novel antivirals and ‘‘lead’’ compounds are
of great interest from which new antivirals could be syn-
thesized (Buckwold et al., 2004, Kossakowski et al., 2009,
Cesarini et al., 2010). A livestock infection by the category
of Pestivirus genus (Flaviviridae family) creates significant
economic losses, and hence the development of safer,
efficacious, and relatively cheap anti-pestiviruses vaccine
is an unmet medical need (Paeshuyse et al., 2006). These
viruses cause a variety of clinical indications, such as ter-
atogenesis, abortion, respiratory problems, chronic wasting
disease, immune system dysfunction, and predisposition to
secondary viral and bacterial infections (Tonelli et al.,
2010). The Flaviviridae family contains viruses with sin-
gle-stranded positive sense RNA genomes (ssRNA?), and
encompasses three genera and several viruses those are
presently not classified to specific genera (Tonelli et al.,
Keywords 1,2,4-Triazines ꢀ Indoles ꢀ Triazoles ꢀ
In vitro ꢀ Cytotoxicity ꢀ Antiviral
V. Virsodiya
Division of Intellectual Property Right, R & D Centre,
Dishman Pharmaceuticals, Ahmedabad, India
K. Upadhyay
Department of Chemistry, Regional Forensic Science
Laboratory, Rajkot 360005, India
J. Trivedi
Department of Medicinal Chemistry, ISF College of Pharmacy,
Moga, Punjab 142001, India
Present Address:
A. Manvar
Centre for Synthesis & Chemical Biology, School of Chemistry
& Chemical Biology, University College Dublin, Belfield,
Dublin 4, Ireland
R. Chaniyara
New Drug Discovery Division, Wockhardt Research Centre,
Aurangabad, Maharashtra 431136, India
R. Loddo ꢀ P. L. Colla (&)
Dipartimento di Scienze e Tecnologie Biomediche, Sezione di
Microbiologia e Virologia Generale e Biotecnologie Microbiche,
A. Shah (&)
National Facility for Drug Discovery, Department of Chemistry,
Saurashtra University, Rajkot 360005, India
e-mail: anamik_shah@hotmail.com
`
Universita degli Studi di Cagliari, Cittadella Universitaria,
S.S. 554, Km 4500, 09042 Cagliari, Monserrato, Italy
e-mail: placolla@unica.it
123

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Med Chem Res (2013) 22:3675–3686
2010). The Hepacivirus genus includes the hepatitis C
virus (HCV) (Tonelli et al., 2010). Viruses such as GB
virus-A and GB virus-A-like agents, GB virus-D and GBV-
C or hepatitis G virus, which are currently not classified
within the Hepacivirus genus, are closely related to HCV
and represents unassigned members of Flaviviridae (To-
nelli et al., 2010; Carta et al., 2007). Moreover, this family
is also consisting the Flavivirus genus, with viruses, for
instance, Dengue Fever, Yellow Fever (YFV), West Nile,
Japanese encephalitis, and tick-borne encephalitis (TBEV),
and the Pestivirus genus, which consists of Bovine Viral
Diarrhea (BVDV), Border Disease, and Classical Swine
Fever viruses (Carta et al., 2011). Other important ssRNA?
viruses are those belonging to the Picornaviridae family.
These viruses cause a variety of diseases, such as cold,
heart infection, conjunctivitis, meningitis, and hepatitis
(Carta et al., 2007; Carta et al., 2011). This family includes
nine genera, some of which comprises major human
pathogens, such as Enterovirus (including Poliovirus,
Echovirus, Coxsackievirus), Rhinovirus, and Hepatovirus
(Giampieri et al., 2009). Currently, no particular vaccine is
available for the treatment of Picornaviridae infections as
well as for Flaviviridae members, except YFV.
Azoles and azines are ubiquitous heterocycles, which
are well-known for their versatile biological activities (Li
et al., 2009). Importantly, the fusion of the heterocyclic
nuclei enhances the pharmacological activities than its
parent nucleus (Abdel-Rahman et al., 2010). In a search of
novel therapeutic agents, versatile heterocyclic moieties
were appended in the 1,2,4-triazine nucleus via the inter-
action between functionalized 1,2,4-triazines with various
nucleophilic and electrophilic reagents (Abdel-Rahman
et al., 2010). In particular, 1,2,4-triazine motif is exploited
for pharmacological activities, as herbicides and pesticides,
as well as in dyes. In drug discovery, for examples, pyr-
rolo-triazines were found as potent PI3K inhibitors (Wang
et al., 2012) and various kinase inhibitors (Abraham et al.,
2011; Dyckman et al., 2011; Mesaros et al., 2012).
Moreover, they were also found as potent adenosine A2A
antagonists (Congreve et al., 2012), antitumors (Sztanke
et al., 2011), anticonvulsants (Sun et al., 2009), antimala-
rials (Ban et al., 2010), etc. Significant biological activities
were also associated with indole-fused 1,2,4-triazines
(Sivendran et al., 2010; Shelke and Bhosale, 2010; Gupta
et al., 2010; Ashour et al., 2012; Maarouf et al., 2012).
Importantly, the indolo-triazine (Buckwold et al., 2004)
(VP32947) was discovered as viral RNA-dependent RNA-
polymerase (EC50 = 0.03 lM, SI [111). Though remark-
able therapeutic worth of 1,2,4-triazine motif, the screening
in antiviral therapeutic area is still in its infancy (Mod-
zelewska-Banachiewicza and Kaminska, 2001; Elghandour
et al., 2006; Sztanke et al., 2007) (Fig. 1). Inspired by
literature reports, we have envisioned for the syntheses and
screening of a molecular diversity based on 1-aryl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indoles 4a–v.
Consequently, the development of novel, efficacious,
and inexpensive antiviral ‘‘lead’’ molecules to combat this
human pathogen is a pressing need (Stachulski et al.,
2011). BVDV is the prototype of the genus because it is
also used as a surrogate for HCV to develop new anti-HCV
agents (Tonelli et al., 2011). We have recently evaluated
various 9-aminoacridines (Tonelli et al., 2011) for in vitro
antiviral activity against a panel of RNA and DNA viruses.
Moreover, we have also probed in vitro a series of new
angular and linear [4,7]phenanthroline N-tricyclic systems
against the ssRNA? viruses (Carta et al., 2007). The
compounds were also evaluated against viruses represen-
tative of an additional genus of ssRNA? genomes [Human
immunodeficiency virus (HIV-1)], of double-stranded
RNA genomes (dsRNA) [Reovirus (Reo-1)], and of single-
stranded, negative-sense RNA genomes (ssRNA-) [respi-
ratory syncytial virus (RSV) and vesicular stomatitis virus
(VSV)]. Notably, phenantrolines were found promising
against members of the Flaviviridae and Picornaviridae
families.
Results and discussion
Chemistry
At the outset, compound 2 was synthesized according to
literature described procedure (Joshi and Chand, 1980).
The compounds 3a–v was synthesized by acid-catalyzed
condensation, utilizing aromatic aldehydes bearing ver-
satile electron donor and acceptors. Finally, they were
CO2Me
O
N
n-Pr
N
N
Ph
N
N
N
N
n-Pr
N
N
N
N
N
O
N
N
R
NH
O
MeOC
N
S
N
N
R1
N
N
R
N
H
N
O
NH2
H
R
N
R
Me
N
VP 32947 (Antiviral) (1d)
EC50 = 0.03 µM (SI > 111)
Present study
Antiviral activity
Antivirals (1a)
Antivirals (1b)
Antivirals (1c)
Fig. 1 Antiviral activity of 1,2,4-triazines condensed with other heterocycles (1)
123

Med Chem Res (2013) 22:3675–3686
3677
cyclized using thionyl chloride as a cyclizing agent,
afforded the target molecules 4a–v in moderate to good
yields with a wide range of functional group tolerance
(Scheme 1). The title compounds thus obtained generated
contradictions regarding their structural assignment. In
literatures, regioselectivity of electrocyclization of func-
tionalized 1,2,4-triazines, such as N-[1,2,4-triazine-3-yl]
nitrilimines (Shawali and Gomha, 2002), 3-azido-1,2,4-
triazines (Mojzych et al., 2005; Goodman and Paudler,
1977), and 3-hydrazino-1,2,4-triazino[5,6-b]indole (Joshi
and Chand, 1980), revealed that the comparative nucleo-
philicities of N2 or N4 in the 1,2,4-triazine ring governs
the course of orientation. In our case, the cyclization of
compounds 3a–v with one carbon cyclizing precursors
were only be electronically controlled and led to angu-
larly annulated 1,2,4-triazolo[30,40:3,4]-1,2,4-triazino[5,6-
b]indoles (compounds 4a–v), and not linearly cyclized,
1,2,4-triazolo[40,30:2,3]-1,2,4-triazino[5,6-b]indoles. The
mechanism for the preparation of compounds 4a–v is
proposed in Fig. 2. In the absence of steric hindrance in
the compounds 3a–v, the basicity of N4 became pre-
dominant over N2 of the 1,2,4-triazine ring, and hence
forced the orientation to afford angularly cyclized prod-
ucts; however, regio specificity was unaffected by the
nature of the functional groups presents in the compounds
3a–v. The mass spectra of the synthesized compounds
were proved to be most important tool to elucidate the
structures of the molecules. The m/z values of the relevant
fragments suggested about the significant structural fea-
tures like the mode of cyclization and aromatic nature of
the molecules. The appearance of an intense molecular
ion peak revealed the aromatic nature of the triazolo-
triazinoindole fused system, the molecular ion, in this
case, underwent fragmentation with loss of corresponding
benzonitrile, which was due to the benzonitrile di-radical
obtained by initial fragmentation of the triazole ring and
nitrogen molecule. An ion at m/z 103 was obtained by the
loss of nitrogen molecule and HCN (Fig. 3).
O
R
H
N
N
N
N
N
N
cat. AcOH (Glacial)
EtOH, 80 oC, 5h
70-81%
SOCl2
NH
NH2
NH
N
N
N
N
N
N
N
H
N
N
50-77 %
H
H
R
R
3a-v
2
4a-v
4a
4l
: 3,4-di OMe Ph
: 4-OMe Ph
: 4-SMe Ph
: 3-Br Ph
: 4-F Ph
: 2-Cl Ph
: 3-OPh Ph
: 2,5-diOMe Ph
: 3- OMe Ph
: 2- OMe Ph
: 3-Cl Ph
: 2-OH Ph
: 4-OH Ph
: 3-OH Ph
: 4-OH, 3-OMe Ph
: 4-Me Ph
4b
4c
4d
4e
4f
4g
4h
4i
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
: 4-Cl Ph
: 3-NO2 Ph
: 4-NO2 Ph
: Ph
4j
4k
: 2-NO2 Ph
: R = Anthryl
Scheme 1 Synthesis of compounds 4a–v
N
N
N
N
N
N
N
H
N
SOCl2
NH
N
N
N
N
NH
N
N
Cl
N
N
N
N
Cl
N
H
N
H
N
N
-H
Cl
H
H
R
N
R
R
R
R
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Compounds
N
N
N
N
(4a-v)
N
N
N
H
N
H
N
-H
H
H
H
R
R
H
H
R
Fig. 2 Proposed mechanism for the synthesis of compounds 4a–v
123

3678
Med Chem Res (2013) 22:3675–3686
Fig. 3 Mass fragmentation of
compound 4a
N
N
N
N
N
N
N
N
N
N
N
H
H
m/z: 174.08
m/z: 103.03
OMe
MeO
-N2
Compound 4a
m/z: 346.12
N
N
-HCN
N
H
m/z: 101.03
m/z: 126.05
Biology
Incorporation of electron-donating group at para position
(4c, R = 4-SMe Ph) and moderately electronegative group
(4p, R = 3-Cl Ph) exerted moderate antiviral activity with
EC50 values 45 and 69 lM against Reo-1 and BVDV
strains without causing cytotoxicity to their parent BHK-21
and MDBK cell lines, respectively. The regioisomerism of
the aryl substituents sincerely affected cytotoxicity and
antiviral activities. As such, 3,4-disubstituted compound 4a
(R = 3,4-diOMe Ph) exerted EC50 value of 55 lM against
Reo-1 strain without being cytotoxic to its parent BHK-21
cell-line at CC50 value [100 lM, while 4t (R = 4-OH,
3-OMe Ph) exerted antiviral activity at EC50 value 90 lM
against BVDV and 25 lM against Reo-1 strains, but
cytotoxic against BHK-21 cell-line with CC50 value
45 lM, whereas 2,5-disubstituted compound 4m was
inactive to all viruses representatives; however, it exerted
cytotoxicity to MT-4, MDBK, BHK-21, and Vero-76 cell
lines with CC50 values 4, 12, 30, and 15 lM, respectively.
In addition, notable cytotoxicities were exerted by the
compounds 4n (R = 3-OMe Ph) with CC50 values 47, 64,
and 65 lM on MT-4, MDBK, and Vero-76, 4o (R = 2-
OMe Ph) to all cell lines with CC50 values 5, 17, 23, and
20 lM, 4p (R = 3-Cl Ph) with CC50 values 29, 55, and
60 lM on MT-4, BHK-21 and Vero-76 cell lines, respec-
tively. Whereas, rest of the compounds were neither active
against any of the virus representative nor cytotoxic to any
cell lines employed.
The cytotoxicity and antiviral activities of the compounds
4a–v are summarized in Table 1 against viruses represen-
tative of two of three genera of the Flaviviridae family, i.e.,
Flaviviruses (YFV) and Pestiviruses (BVDV). The title
compounds were also screened against representatives of
other virus families. Among ssRNA? was a Retrovirus
(HIV-1), two Picornaviruses [Coxsackie Virus type B2
(CVB-2), and Poliovirus type-1 Sabin strain (Sb-1)];
among ssRNA- were Paramyxoviridae [Rhabdoviridae,
VSV, RSV] representative. Among dsRNA viruses, was a
Reoviridae representative [Respiratory Enteric Orphan
Virus type-1(Reo-1)]. Two representatives of DNA virus
families were also included: Herpes Simplex type 1 (HSV-
1, Herpesviridae) and Vaccinia Virus (VV, Poxviridae).
The indolo-triazine derivatives were presented antiviral
activities varied from moderate (4a, 4c, and 4p) to signif-
icant (4t and 4v). The data may serve as a good subject for
discussion of the influence of molecular structure of a
compound on its biological activity. It seems that antiviral
activity depends on the factors, such as type of substituents,
regioisomerism, and steric interactions associated with the
parent scaffold. The most active compound 4v (R =
9-anthryl) bearing sterically hindered group exerted EC50
value 3 lM against BVDV strain without cytotoxic
to parent MDBK cell-line at CC50 value [100 lM.
Table 1 Cytotoxicity and antiviral activities of compounds 4a–v
Compds MT-4 HIV-1 MDBK BVDV SI1
BHK- YFV
21
Reo-1 Vero- HSV-1 VV
76
VSV
CVB-2 Sb-1
RSV
CCa50
ECb50
CCc50
ECd50
CCe50
ECf50
ECf50
CCg50
ECh50
ECh50
ECh50
ECh50
ECh50
ECh50
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
4a
4b
4c
4d
4e
4f
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
–
–
–
–
–
–
[100 [100 55
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
[100 [100 45 90 [90 [90 [90 [90 [90 [90
[100 [100 [100 [100 [100 [100 [100
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
123

Med Chem Res (2013) 22:3675–3686
3679
Table 1 continued
Compds MT-4 HIV-1 MDBK BVDV SI1
BHK- YFV
21
Reo-1 Vero- HSV-1 VV
76
VSV
CVB-2 Sb-1
RSV
CCa50
ECb50
CCc50
ECd50
CCe50
ECf50
ECf50
CCg50
ECh50
ECh50
ECh50
ECh50
ECh50
ECh50
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
4g
4h
4i
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
–
–
–
–
–
–
–
–
–
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
4j
4k
4 l
4m
4n
4o
4p
4q
4r
[100 [100 [100 [100 [100 [100 [100 100
[100 [100
100
30
[100 [100 80
[30 [30 15
[80
[15
[65
[20
[60
[70
[80
[15
[65
[20
[60
[70
[80
[15
[65
[20
[60
[70
[80
[15
[65
[20
[60
[70
[80
[15
[65
[20
[60
[70
[80
[15
[65
[20
[60
[70
4
[4
12
[12
[64
[17
69
47
5
[47
[5
64
[100 [100 [100 65
17
23
[23
[55
[23
[55
20
60
29
[29
[100
[1.45 55
[100 [100 [100 [100
[100 [100 [100 [100
[100 [100 [100 [100
–
–
–
[100 [100 [100 70
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
[100 [100 [100 [100 [100 [100 [100 [100 [100 [100
4s
4t
58
[58
[100
90
[1.11 45
[100 [100 [100 C100 [100 [100 [100 [100 [100 [100
[33.33 [100 [100 [100 C100 [100 [100 [100 [100 [100 [100
[45
25
80
[80
[80
[80
[80
[80
[80
4u
4v
AZTi
[100 [100 [100 [100
–
56
50
–
[56
0.01
–
[100
3
–
–
–
–
–
–
–
–
–
–
–
–
–
NM
108j
–
1.8
–
–
2.5
–
–
–
–
–
–
–
–
NM
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3
–
–
–
–
23
–
18
–
–
–
–
176k
M
5255l
ACGm
1.8
–
–
–
Data represent mean value for three independent determinations. Variation among duplicate samples was less than 15 %. Antiviral activity is
given as EC50 (median effective concentration-the concentration of a drug (lM) required to induce a 50 % effect), and cytotoxicity is given as
CC50 (cytotoxic concentration-the amount of a drug (lM) at which 50 % cell become dead). SI (selectivity index) was determined as the ratio
between CC50 and EC50 for MDBK and BVDV (SI1)
a
Compd. concentration (lL) required to reduce the viability of mock-infected MT-4 (CD4? human T-cells containing an integrated HTLV-1
genome) cells by 50 %, as determined by the colorimetric MTT method
b
Compd. concentration (lL) required to reduce the viability of mock-infected MDBK (bovine normal kidney) cells by 50 %, as determined by
the MTT method
c
Compd. concentration (lL) required to reduce the viability of mock-infected BHK (Hamster normal kidney fibroblast) monolayers by 50 %,
as determined by the MTT method
d
Compd. concentration (lM) required to reduce the viability of mock-infected VERO-76 (Monkey normal kidney) monolayers by 50 %, as
determined by the MTT
e
Compd. concentration (lL) required to achieve 50 % protection of MT-4 cells from the HIV-1-induced cytopathogenicity, as determined by
the MTT method
f
Compd. concentration (lL) required to achieve 50 % protection of MDBK cells from the BVDV (Bovine Viral Diarrhea Virus)-induced
cytopathogenicity, as determined by the MTT method
g
Compd. concentration (lL) required to achieve 50 % protection of BHK (Kidney fibroblast) cells from the YFV (Yellow Fever Virus) and
Reo (Reovirus-1)-induced cytopathogenicity, as determined by the MTT method
h
Compd. concentration (lM) required to reduce the plaque number of HSV-1 (Herpesvirus-1), VV (Vaccinia Virus), VSV (Vesicular Stomatitis
Virus), CVB-2 (Coxsackievirus B2), Sb-1 (Poliovirus 1) and RSV (Respiratory Syncytial Virus) by 50 % in VERO-76 monolayers
i
AZT (30-azido-thymidine)
j
NM108 (20-b-methyl-guanosine)
NM176 (20-ethynyl-D-citidine)
k
l
M5255 (mycophenolic acid)
m
ACG (acyclo-guanosine)
123

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Med Chem Res (2013) 22:3675–3686
Conclusions and future directions
and glacial acetic acid (10.0 mol%) were added in ethanol
and heated at 80 °C for 5 h. On cooling the reaction mass
at ambient temperature, the crude products were precipitate
out. Ethanol was removed by filtration, and the products
were washed with diethyl ether and n-pentane and dried
under vacuum. No further purification was attempted.
In summary,
a
versatile 1-aryl-10H-[1,2,4]triazol-
o[30,40:3,4][1,2,4]triazino[5,6-b]indoles were designed,
synthesized, and evaluated in vitro in parallel cell-based
assays for cytotoxicity and antiviral activity against rep-
resentative strains of emergent and re-emergent human and
cattle viral infectious diseases. The screening results
revealed that the compound 4v exerted the most promising
activity with 3 lM EC50 value against BVDV strain devoid
of causing cytotoxicity to its parent MDBK cell-line. While
four distinct candidates 4a, 4c, 4p, and 4t have presented
antiviral activities from moderate to significant range with
diminutive cytotoxicity. In addition to these two deriva-
tives, 4m and 4o exerted significant cytotoxicity against all
RNA virus multiplication supporting cell lines and will be
evaluated for antiproliferative activity against hematolog-
ical and solid tumor cell lines.
General procedure for the syntheses of compounds (4a–v)
To an appropriate hydrazones 3a–v (5.0 mmol), thionyl
chloride (10 ml) was added under nitrogen atmosphere.
The resulting reaction mixture was heated at 75 °C in oil
bath for 4h. The progress of reaction was checked by TLC
(dichloromethane/methanol, 95/5). The excess of thionyl
chloride was removed by distillation under reduced pres-
sure. The residue was quenched in ice to leave the pre-
cipitates. The crude products were filtered and washed with
water and isopropanol and dried under vacuum to afford
pure title compounds.
Methods and materials
1-(3,4-Dimethoxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4a)
Chemistry
Yields: 77 % as a white solid, m.p. (°C): 289–291 (dec.).
IR (KBr, cm-1): 3352, 3128, 1517, 1487, 1463, 1298, 744.
1H-NMR (300 MHz, DMSO-d6) d = 12.28 (brs, 1H), 8.23
(d, J = 7.5 Hz, 1H, ArH), 8.08 (d, J = 6.0 Hz, 1H, ArH),
7.99 (s, 1H, ArH), 7.74 (t, J = 7.7, 7.2 Hz, 1H, ArH), 7.70
(d, J = 8.1 Hz, 1H, ArH), 7.46 (t, J = 7.8, 7.2 Hz, 1H,
ArH), 7.20 (d, J = 8.4 Hz, 1H, ArH), 3.90 (s, 6H,
2 9 OMe). 13C-NMR (75 MHz, DMSO-d6) d = 158.2,
155.5, 150.3, 149.3, 148.8, 137.9, 125.4, 123.7, 122.5,
119.2, 118.2, 116.9, 114.8, 112.9, 111.9, 109.6, 56.9, 55.2.
ESI–MS: m/z 346, 331, 304, 174, 126, 103, 101. Anal.
calcd for C19H15N6O2: C, 62.42; H, 4.07; N, 24.27. Found:
C, 62.46; H, 4.11; N, 24.32.
Chemicals and solvents were purchased from commercial
sources and were used without further purification. Melting
points were determined in open capillary tubes using
Electrothermal-9200 melting point apparatus and are
uncorrected. Yields refer to isolated compounds, estimated
to be [95 % pure as determined by 1H-NMR. TLC:
Macherey–Nagel, TLC plates AlugramÒ Sil G/UV254.
Detection under UV light at 254 nm. 1H-NMR spectra
were recorded on Bruker Avance II 300 MHz spectrometer
in DMSO-d6. Chemical shifts (d) are given in ppm relative
to TMS, coupling constants (J) are in Hz. All IR spectra
were recorded on Shimadzu FTIR 8400 Spectrophotometer
by KBr pellet method. All ESI–MS spectra were recorded
on Shimadzu GCMS-QP2010. The microanalysis was
performed on EuroVector EA3000.
1-(4-Methoxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4b)
Synthesis of 3-hydrazino-5H-[1,2,4]triazino[5,6-b]indole
(2)
Yields: 72 % as a white solid, m.p. (°C): 282–284. IR
(KBr, cm-1): 3412, 3128, 1612, 1581, 1558, 1504, 1257,
840. 1H-NMR (300 MHz, DMSO-d6) d = 13.04 (brs, 1H),
8.38 (d, J = 9.0 Hz, 2H, ArH), 8.33 (d, J = 7.8 Hz, 1H,
ArH), 7.82 (t, J = 7.5, 4.1 Hz, 1H, ArH), 7.58 (d,
J = 8.1 Hz, 1H, ArH), 7.46 (t, J = 7.5, 3.5 Hz, 1H, ArH),
7.24 (d, J = 9.0 Hz, 2H, ArH), 3.90 (s, 3H, OMe). 13C-
NMR (75 MHz, DMSO-d6) d = 158.6, 158.3, 154.5,
151.1, 139.0, 127.1, 125.1, 123.2, 122.0, 119.1, 117.3,
115.7, 114.2, 112.8, 55.9. ESI–MS: m/z 316, 301, 273, 174,
158, 126, 103, 101. Anal. calcd for C18H13N6O: C, 64.55;
H, 3.82; N, 26.57. Found: C, 64.61; H, 3.79, N, 26.62.
It was prepared according to literature described procedure.
Yields: 60 %, m.p. 275–278 °C (lit. m.p. 276–277 °C)
(Joshi and Chand, 1980).
General procedure for the syntheses of compounds
(3a–v) (Ram et al., 1987; Monge et al., 1987)
The above compounds were synthesized according to lit-
erature described method. To a three-neck flask, substituted
aromatic aldehydes (0.01 mol), compound 2 (0.01 mol)
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Med Chem Res (2013) 22:3675–3686
3681
1H-NMR (300 MHz, DMSO-d6) d = 12.59 (brs, 1H), 8.45
(d, J = 7.9, 1H, ArH), 7.75–7.63 (m, 3H, ArH), 7.55–7.35
(m, 4H, ArH). 13C-NMR (75 MHz, DMSO-d6) d = 157.9,
152.8, 150.2, 139.5, 132.6, 131.5, 129.9, 128.9, 128.8,
127.5, 124.5, 123.0, 119.5, 118.3, 115.7, 113.4. ESI–MS:
m/z 321, 322, 174, 137, 126, 103. Anal. calcd for
C16H9ClN6: C, 59.92; H, 2.83; N, 26.20. Found: C, 59.98;
H, 2.87; N, 26.21.
1-(4-Methylsulfanyl)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4c)
Yields: 69 % as a yellow solid, m.p. (°C): 241–243. IR
(KBr, cm-1): 3396, 3124, 1618, 1589, 1548, 1367, 891.
1H-NMR (300 MHz, DMSO-d6) d = 12.67 (brs, 1H), 8.23
(d, J = 7.5 Hz, 1H, ArH), 7.80 (d, J = 8.8 Hz, 2H, ArH),
7.71 (t, J = 7.7, 4.0 Hz, 1H, ArH), 7.69 (d, J = 8.1 Hz,
1H, ArH), 7.49 (t, J = 7.8, 4.2 Hz, 1H, ArH), 7.19 (d,
J = 8.4hz, 1H, ArH), 2.45 (s, 3H, SMe). 13C-NMR
(75 MHz, DMSO-d6) d = 158.3, 153.1, 151.0, 139.6,
135.3, 133.1, 131.4, 128.2, 122.3, 121.9, 119.0, 118.1,
115.2, 113.2, 15.3. ESI–MS: m/z 332, 317, 174, 126, 103,
101. Anal. calcd for C18H13N6S: C, 61.43; H, 3.64; N,
25.58. Found: C, 61.49; H, 3.59; N, 25.65.
1-(4-Chloro)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4g)
Yield: 73 % as a white solid, m.p. (°C): [300. IR (KBr,
cm-1): 3410, 3118, 1618, 1537, 1470, 1238, 829. 1H-NMR
(300 MHz, DMSO-d6) d = 12.12 (brs, 1H), 8.23 (d,
J = 7.9 Hz, 2H, ArH), 7.65–7.57 (m, 2H, ArH), 7.55 (d,
J = 7.6 Hz, 2H, ArH), 7.51–7.29 (m, 2H, ArH). 13C-NMR
(75 MHz, DMSO-d6) d = 158.3, 153.1, 152.0, 139.0,
135.2, 133.8, 130.5, 128.1, 123.2, 122.3, 119.2, 118.0,
115.3, 113.2. ESI–MS: m/z 322, 321, 174, 137, 126, 103.
Anal. calcd for C16H9ClN6: C, 59.92; H, 2.83; N, 26.20.
Found: C, 59.94; H, 2.85; N, 26.24.
1-(3-Bromophenyl)-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4d)
Yields: 75 % as a yellow solid, m.p. (°C):[300. IR (KBr,
cm-1): 3409, 3101, 1616, 1535, 1465, 1299, 898. 1H-NMR
(300 MHz, DMSO-d6) d = 12.28 (brs, 1H), 8.34 (t,
J = 3.7, 4.1 Hz, 1H, ArH), 7.72 (d, J = 8.1 Hz, 1H, ArH),
7.54 (t, J = 7.7, 7.2 Hz 1H, ArH), 7.46 (d, J = 4.2 Hz, 1H,
ArH), 7.15 (t, J = 7.7, 3.5 Hz, 1H, ArH), 7.23–6.60 (m,
3H, ArH). 13C-NMR (75 MHz, DMSO-d6) d = 158.2,
154.1, 152.0, 139.2, 133.2, 131.1, 130.1, 129.9, 125.9,
123.7, 123.0, 122.0, 118.1, 117.3, 115.1, 113.4. ESI–MS:
m/z 366, 367, 181, 174, 126, 103. Anal. calcd for
C16H9BrN6: C, 52.62; H, 2.48; N, 23.01. Found: C, 52.69;
H, 2.54; N, 23.05.
1-(3-Nitro)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4h)
Yield: 63 % as a yellow solid, m.p. (°C): [300. IR (KBr,
cm-1): 3408, 3116, 1620, 1523, 1455, 1299, 875. 1H-NMR
(300 MHz, DMSO-d6) d = 12.23 (brs, 1H), 8.67 (t,
J = 3.2 Hz, 1H, ArH), 8.62–8.22 (m, 2H, ArH), 8.19–8.17
(m, 1H, ArH), 7.77–7.63 (m, 2H, ArH), 7.52–7.32 (m, 2H,
ArH). 13C-NMR (75 MHz, DMSO-d6) d = 158.3, 153.5,
152.0, 148.2, 139.4, 133.6, 132.0, 131.1, 124.1, 123.9,
122.8, 120.8, 119.3, 118.2, 113.9, 112.9. ESI–MS: m/z 332,
174, 126, 103. Anal. calcd for C16H9N7O2: C, 58.01; H,
2.74; N, 29.60. Found: C, 58.09; H, 2.81; N, 29.65.
1-(4-Fluoro)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4e)
Yield: 67 % as a white solid, m.p. (°C): 164–165. IR (KBr,
cm-1): 3410, 3132, 1612, 1588, 1461, 1238, 756. 1H-NMR
(300 MHz, DMSO-d6) d = 12.22 (brs, 1H), 8.08 (d,
J = 6.8 Hz, 2H, ArH), 7.89 (d, J = 6.9 Hz, 1H, ArH),
7.66 (t, J = 7.3, 3.6 Hz, 1H, ArH), 7.55 (d, J = 7.9 Hz,
1H, ArH), 7.39 (t, J = 6.7, 3.6 Hz, 1H, ArH), 7.33 (d,
J = 8.7 Hz, 2H, ArH). 13C-NMR (75 MHz, DMSO-d6)
d = 165.2, 161.2, 159.1, 154.3, 152.4, 139.5, 129.3, 127.9,
124.2, 123.1, 119.2, 118.1, 116.2, 115.1, 113.7. ESI–MS:
m/z 305, 174, 126, 121, 103. Anal. calcd for C16H9FN6: C,
63.16; H, 2.98; N, 27.62. Found: C, 63.22; H, 2.92; N,
27.64.
1-(4-Nitro)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4i)
Yield: 64 % as a yellow solid, m.p. (°C): 253–255. IR
(KBr, cm-1): 3494, 3116, 1620, 1523, 1461, 1299, 856.
1H-NMR (300 MHz, DMSO-d6) d = 12.19 (brs, 1H),
8.35 (d, J = 7.2 Hz, 2H, ArH), 8.12–8.09 (m, 1H, ArH),
8.02 (d, J = 6.9 Hz, 2H, ArH), 7.59–7.57 (m, 1H, ArH),
7.53-7.35 (m, 2H, ArH). 13C-NMR (75 MHz, DMSO-d6)
d = 158.3, 154.1, 152.1, 147.4, 139.3, 133.1, 125.9,
125.2, 123.1, 122.9, 117.9, 117.2, 115.3, 113.4. ESI–MS:
m/z 332, 174, 126, 103. Anal. calcd for C16H9N7O2: C,
58.01; H, 2.74; N, 29.60. Found: C, 58.05; H, 2.80; N,
29.59.
1-(2-Chloro)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4f)
Yield: 70 % as a yellow solid, m.p. (°C): 247–249. IR
(KBr, cm-1): 3410, 3132, 1612, 1588, 1461, 1238, 756.
123

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Med Chem Res (2013) 22:3675–3686
3.89 (s, 3H, OMe). 13C-NMR (75 MHz, DMSO-d6)
d = 160.1, 155.4, 152.4, 150.0, 139.3, 130.1, 123.1, 122.9,
118.3, 116.2, 115.2, 112.6, 110.1, 107.7, 56.6, 55.9. ESI–
MS: m/z 346, 331, 304, 174, 126, 103, 101. Anal. calcd for
C19H15N6O2: C, 62.42; H, 4.07; N, 24.27. Found: C, 62.45;
H, 4.11; N, 24.32.
1-Phenyl-10H-[1,2,4]triazolo[30,40:3,4][1,2,4]triazino
[5,6-b]indole (4j)
Yield: 62 % as a white solid, m.p. (°C): 221–223. IR (KBr,
cm-1): 3425, 3114, 1614, 1587, 1485, 1230, 750. 1H-NMR
(300 MHz, DMSO-d6) d = 13.03 (1H, brs), 8.43 (d,
J = 6.6 Hz, 2H, ArH), 8.34 (d, J = 7.8 Hz, 1H), 7.82 (t,
J = 7.5, 7.8 Hz, 1H, ArH), 7.67 (m, 3H, ArH), 7.58 (d,
J = 7.8 Hz, 1H, ArH), 7.45 (t, J = 7.5 Hz, 1H, ArH). 13C-
NMR (75 MHz, DMSO-d6) d = 158.6, 154.2, 152.4,
139.4, 131.4, 129.9, 129.0, 126.4, 123.8, 123.1, 119.2,
118.1, 115.2, 113.3. ESI–MS: m/z 285, 176, 126, 103, 101.
Anal. calcd for C16H10N6: C, 67.12; H, 3.52; N, 29.35.
Found: C, 67.15; H, 3.57; N, 29.41.
1-(3-Methoxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4n)
Yield: 69 % as a yellow solid, m.p. (°C): 205–207. IR
(KBr, cm-1): 3404, 3143, 1608, 1554, 1456, 1315, 885.
1H-NMR (300 MHz, DMSO-d6) d = 12.20 (brs, 1H, NH
exchangeable), 8.14–8.10 (m, 2H, ArH), 7.85–7.77 (m, 3H,
ArH), 7.41–7.25 (m, 3H, ArH), 3.91 (s, 3H, OMe). 13C-
NMR (75 MHz, DMSO-d6) d = 162.2, 158.3, 154.1,
152.3, 139.3, 131.2, 128.4, 123.2, 122.8, 118.9, 118.7,
117.8, 115.9, 113.9, 112.7, 112.0, 55.6. ESI–MS: m/z 316,
301, 273, 174, 158, 126, 103, 101. Anal. calcd for
C18H13N6O: C, 64.55; H, 3.82; N, 26.57. Found: C, 64.59;
H, 3.84; N, 26.62.
1-(2-Nitro)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4k)
Yield: 57 % as a yellow solid, m.p. (°C): 279–281. IR
(KBr, cm-1): 3404, 3124, 1610, 1583, 1465, 1220, 846.
1H-NMR (300 MHz, DMSO-d6) d = 12.21 (brs, 1H, NH
exchangeable), 8.15–7.99 (m, 3H, ArH), 7.91–7.67 (m, 3H,
ArH), 7.51–7.31 (m, 2H, ArH). 13C-NMR (75 MHz,
DMSO-d6) d = 156.3, 152.6, 151.0, 149.9, 139.2, 134.5,
130.4, 127.0, 126.4, 123.8, 123.0, 122.4, 119.5, 118.1,
115.2, 111.9. ESI–MS: m/z 332, 174, 126, 103. Anal. calcd
for C16H9N7O2: C, 58.01; H, 2.74; N, 29.60. Found: C,
58.07; H, 2.73; N, 29.63.
1-(2-Methoxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4o)
Yield: 73 % as a yellow solid, m.p. (°C): 225–227. IR
(KBr, cm-1): 3423, 3124, 1616, 1587, 1483, 1253, 735.
1H-NMR (300 MHz, DMSO-d6) d = 12.21 (brs, 1H, NH
exchangeable), 8.17–8.13 (m, 2H, ArH), 7.68–7.63 (m, 2H,
ArH), 7.50–7.22 (m, 2H, ArH), 7.35–7.05 (m, 2H, ArH),
3.94 (s, 3H, OMe). 13C-NMR (75 MHz, DMSO-d6)
d = 158.1, 153.3, 152.1, 151.9, 139.3, 128.8, 128.5, 124.4,
123.8, 123.3, 119.2, 118.8, 118.5, 114.4, 114.2, 113.1,
55.7. ESI–MS: m/z 316, 301, 273, 174, 158, 126, 103, 101.
Anal. calcd for C18H13N6O: C, 64.55; H, 3.82; N, 26.57.
Found: C, 64.60; H, 3.80; N, 26.61.
1-(3-Phenoxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4l)
Yield: 59 % as a white solid, m.p. (°C): 283–285. IR (KBr,
cm-1): 3434, 3126, 1610, 1563, 1400, 1225, 750. 1H-NMR
(300 MHz, DMSO-d6) d = 12.17 (brs, 1H, NH
exchangeable), 8.14–8.07 (m, 2H, ArH), 7.63–7.51 (m, 3H,
ArH), 7.47–7.29 (m, 4H, ArH), 7.25–7.12 (m, 3H, ArH).
13C-NMR (75 MHz, DMSO-d6) d = 158.5, 157.1, 156.0,
154.3, 152.7, 139.8, 133.4, 131.1, 130.5, 123.9, 123.3,
122.6, 121.9, 121.2, 119.7, 118.0, 117.8, 115.6, 115.0,
111.7. ESI–MS: m/z 379, 195, 126, 103. Anal. calcd for
C22H14N6O: C, 69.83; H, 3.73; N, 22.21. Found: C, 69.87;
H, 3.79; N, 22.24.
1-(3-Chloro)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4p)
Yield: 68 % as a white solid, m.p. (°C): 234–236. IR (KBr,
cm-1): 3419, 3132, 1608, 1585, 1477, 1232, 896. 1H-NMR
(300 MHz, DMSO-d6) d = 12.23 (brs, 1H, NH
exchangeable), 8.15–8.11 (m, 2H, ArH), 8.01–7.97 (m, 2H,
ArH), 7.76–7.62 (m, 1H, ArH), 7.45–7.37 (m, 1H, ArH),
7.34–7.21 (m, 2H, ArH). 13C-NMR (75 MHz, DMSO-d6)
d = 158.3, 155.1, 152.1, 139.4, 134.2, 131.4, 130.1, 128.3,
127.2, 125.2, 124.3, 123.7, 119.3, 118.1, 115.4, 111.9.
ESI–MS: m/z 322, 321, 174, 137, 126, 103. Anal. calcd for
C16H9ClN6: C, 59.92; H, 2.83; N, 26.20. Found: C, 59.97;
H, 2.87; N, 26.26.
1-(2,5-Dimethoxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4m)
Yield: 72 % as a white solid, m.p. (°C): 249–250. IR (KBr,
cm-1): 3434, 3128, 1620, 1552, 1400, 1271, 894. 1H-NMR
(300 MHz, DMSO-d6) d = 12.17 (brs, 1H, NH
exchangeable), 8.14–8.11 (m, 2H, ArH), 7.67–7.56 (m, 2H,
ArH), 7.34–7.31 (m, 1H, ArH), 6.98 (d, J = 6.9 Hz, 1H,
ArH), 6.81 (d, J = 6.2 Hz, 1H, ArH), 3.95 (s, 3H, OMe),
123

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3683
1-(2-Hydroxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4q)
ArH), 7.50–7.45 (m, 2H, ArH), 7.35–7.17 (m, 2H, ArH),
6.95–6.89 (m, 1H, ArH), 4.87 (brs, 1H, OH exchangeable),
3.96 (s, 3H, OMe). 13C-NMR (75 MHz, DMSO-d6)
d = 158.4, 154.5, 152.1, 149.1, 148.9, 139.0, 123.1, 122.9,
122.1, 120.1, 118.9, 118.2, 115.3, 113.1, 112.7, 110.1,
57.2. ESI–MS: m/z 332, 317, 174, 126, 103. Anal. calcd for
C17H12N6O2: C, 61.44; H, 3.64; N, 25.29. Found: C, 63.49;
H, 3.65; N, 25.32.
Yield: 56 % as a yellow solid, m.p. (°C): [300 (dec.). IR
(KBr, cm-1): 3404, 3120, 1614, 1589, 1539, 1253, 740.
1H-NMR (300 MHz, DMSO-d6) d = 12.18 (brs, 1H, NH
exchangeable), 8.13–8.10 (m, 2H, ArH), 7.67–7.61 (m, 1H,
ArH), 7.57–7.55 (m, 1H, ArH), 7.47–7.31 (m, 2H, ArH),
7.24–7.07 (m, 2H, ArH), 4.11 (brs, 1H, OH exchangeable).
13C-NMR (75 MHz, DMSO-d6) d = 157.4, 154.3, 153.1,
152.1, 139.2, 130.5, 129.7, 124.1, 122.7, 121.5, 119.7,
119.1, 118.1, 115.2, 113.1, 111.3. ESI–MS: m/z 303, 174,
126, 103. Anal. calcd for C16H10N6O: C, 63.57; H, 3.33; N,
27.80. Found: C, 63.63; H, 3.37; N, 27.78.
1-(4-Methyl)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4u)
Yield: 71 % as a white solid, m.p. (°C): 287–289. IR (KBr,
cm-1): 3419, 3126, 1610, 1554, 1498, 1220, 844. 1H-NMR
(300 MHz, DMSO-d6) d = 12.20 (brs, 1H, NH
exchangeable), 8.59 (d, J = 7.1 Hz, 2H, ArH), 8.08–8.05
(m, 1H, ArH), 7.65–7.54 (m, 2H, ArH), 7.37–7.33 (m, 1H,
ArH), 7.25 (d, J = 6.8 Hz, 2H, ArH), 2.21 (s, 3H, Me).
13C-NMR (75 MHz, DMSO-d6) d = 158.7, 153.1, 151.2,
139.2, 137.2, 131.7, 129.8, 125.7, 123.9, 122.1, 119.2,
117.8, 115.3, 113.2, 20.9. ESI–MS: m/z 301, 174, 126, 117,
103. Anal. calcd for C17H12N6: C, 67.99; H, 4.03; N, 27.98.
Found: C, 68.03; H, 4.09; N, 27.95.
1-(4-Hydroxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4r)
Yield: 53 % as a yellow solid, m.p. (°C): [300 (dec.). IR
(KBr, cm-1): 3406, 3136, 1608, 1554, 1458, 1272, 844.
1H-NMR (300 MHz, DMSO-d6) d = 12.20 (brs, 1H, NH
exchangeable), 8.09–8.05 (m, 2H, ArH), 7.95 (d,
J = 6.6 Hz, 2H, ArH), 7.67–7.62 (m, 1H, ArH), 7.54–7.49
(m, 1H, ArH), 7.27–7.21 (m, 2H, ArH), 6.83 (d,
J = 6.5 Hz, 2H, ArH), 5.21 (brs, 1H, OH exchangeable).
13C-NMR (75 MHz, DMSO-d6) d = 158.8, 157.3, 154.2,
152.1, 139.0, 128.3, 123.1, 122.3, 122.9, 119.4, 118.2,
115.3, 114.4, 113.2. ESI–MS: m/z 303, 174, 126, 103.
Anal. calcd for C16H10N6O: C, 63.57; H, 3.33; N, 27.80.
Found: C, 63.62; H, 3.35; N, 27.82.
1-(9-Anthryl)-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4v)
Yield: 76 % as a yellow solid, m.p. (°C): [300 (dec.). IR
(KBr, cm-1): 3404, 3128, 1614, 1554, 1480, 1271, 759. 1H-
NMR (300 MHz, DMSO-d6) d = 12.37 (brs, 1H, NH
exchangeable), 7.72 (d, J = 7.1 Hz, 1H, ArH), 7.68 (t,
J = 6.8, 3.4hz, 1H, ArH), 7.62 (d, J = 7.8 Hz, 1H, ArH),
7.51 (t, J = 7.2, 3.3 Hz, 1H, ArH), 7.40–6.56 (m, 11H,
ArH). 13C-NMR (75 MHz, DMSO-d6) d = 156.2, 152.2,
141.3, 139.3, 135.3, 133.4, 131.3, 130.1, 127.5, 126.9, 126.2,
125.2, 123.1, 122.2, 119.2, 118.3, 115.2, 113.1. ESI–MS: m/
z 387, 279, 174, 126, 103. Anal. calcd for C24H14N6: C,
74.60; H, 3.65; N, 21.75. Found: C, 74.67; H, 3.70; N, 21.81.
1-(3-Hydroxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b]indole (4s)
Yield: 50 % as a yellow solid, m.p. (°C): 254–256. IR
(KBr, cm-1): 3304, 3136, 1610, 1554, 1490, 1319, 877.
1H-NMR (300 MHz, DMSO-d6) d = 12.18 (brs, 1H, NH
exchangeable), 8.14–8.11 (d, J = 7.2 Hz, 2H, ArH),
7.84–7.67 (m, 2H, ArH), 7.50–7.38 (m, 2H, ArH),
7.29–6.93 (m, 2H, ArH), 4.48 (brs, 1H, OH exchangeable).
13C-NMR (75 MHz, DMSO-d6) d = 158.5, 157.1, 154.1,
151.9, 139.1, 131.3, 130.9, 122.8, 122.4, 119.9, 118.4,
117.5, 117.2, 115.2, 114.1, 112.2. ESI–MS: m/z 303, 174,
126, 103. Anal. calcd for C16H10N6O: C, 63.57; H, 3.33; N,
27.80. Found: C, 63.60; H, 3.36; N, 27.87.
Biology
Cell-based assays
Compounds were dissolved in DMSO at 100 mM and then
diluted in culture medium. Cell lines were purchased from
American Type Culture Collection (ATCC). The absence
of mycoplasma contamination was checked periodically by
the Hoechst staining method. Cell lines supporting the
multiplication of RNA viruses were the following: CD4?
human T-cells containing an integrated HTLV-1 genome
(MT-4); Madin Darby Bovine Kidney (MDBK); Baby
Hamster Kidney (BHK-21); and Monkey kidney (Vero 76)
cells.
1-(3-Methoxy-4-Hydroxy)phenyl-10H-
[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[5,6-b] indole (4t)
Yield: 70 % as a yellow solid, m.p. (°C): 258–260. IR
(KBr, cm-1): 3404, 3128, 1609, 1560, 1460, 1288, 788.
1H-NMR (300 MHz, DMSO-d6) d = 12.17 (brs, 1H, NH
exchangeable), 8.15–8.07 (m, 1H, ArH), 7.63–7.61 (m, 1H,
123

3684
Med Chem Res (2013) 22:3675–3686
Cytotoxicity assays
compounds, were added. After 3–4 days incubation at
37 °C, cell viability was determined by the MTT method
(Pauwels et al., 1998). Activity of compounds against
CVB-2 strain, Sb-1, RSV, VSV, VV, and HSV-1, in
infected Vero 76 cells, was determined by plaque reduction
assays in Vero 76-cell monolayers. To this end, Vero 76
cells were seeded in 24-well plates at a density of
2 9 105 cells/well and were allowed to form confluent
monolayers by incubating overnight in growth medium at
37 °C in a humidified CO2 (5 %) atmosphere. Then,
monolayers were infected with appropriate virus dilutions
(250 ll) to give 50–100 PFU/well. Following the removal
of unadsorbed virus, Dulbecco’s modified Eagle’s medium
(500 ll), supplemented with 1 % inactivated FCS and
0.75 % methyl cellulose, with or without serial dilutions of
test compounds, were added. Cultures were incubated at
37 °C for 2 (Sb-1 and VSV) or 3 (CVB-2, VV and HSV-1)
or 5 days (RSV) and then fixed with PBS containing 50 %
ethanol and 0.8 % crystal violet, washed, and air-dried.
Plaques were then counted. 50 % effective concentrations
(EC50) were calculated by linear regression technique. The
cytotoxicity was evaluated in parallel with the antiviral
activity. AZT (30-Azido-thymidine), NM 108 (20-b-Methyl-
guanosine), NM 176 (20-Ethynyl-D-citidine), M 5255
(Mycophenolic acid), and ACG (Acyclo-guanosine) were
used as reference inhibitors of ssRNA?, ssRNA2, and
DNA viruses, respectively.
The compounds were probed for their in vitro cytotoxicity
and antiviral activities against representatives of different
virus families by cell-based assays. Compounds were dis-
solved in DMSO at 100 mM and then diluted in culture
medium. The cytotoxicity tests were run in parallel with
antiviral assays. MDBK, BHK, and Vero 76 cells were
resuspended in 96-multi-well plates at an initial density of
6 9 105, 1 9 106, and 5 9 105 cells/ml, respectively, in
maintenance medium, with or without serial dilutions of
test compounds. Cell viability was determined after
48–120 h at 37 °C in a humidified CO2 (5 %) atmosphere
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-
lium bromide (MTT) method. The cell numbers of Vero 76
monolayers were determined by staining with the crystal
violet dye. For cytotoxicity evaluations, exponentially
growing cells derived from human hematological tumors
[CD4? human T-cells containing an integrated HTLV-1
genome (MT-4)] were seeded at an initial density of
1 9 105 cells/ml in 96-well plates in RPMI-1640 medium,
supplemented with 10 % fetal calf serum (FCS), 100 U/ml
penicillin G, and 100 lg/ml streptomycin. Cell cultures
were then incubated at 37 °C in a humidified, 5 % CO2
atmosphere in the absence or presence of serial dilutions of
test compounds. Cell viability was determined after 96 h at
37 °C by MTT method (Pauwels et al., 1998).
Acknowledgments The authors are thankful to the ‘‘National
Facility for Drug Discovery through New Chemical Entities (NCEs)
Development and Instrumentation Support to Small Manufacturing
Pharma Enterprise’’ under Drugs and Pharmaceutical Research Sup-
port (DPRS) project jointly funded by DST (India), Gujarat Industries
Commissionerate (Government of Gujarat), and Saurashtra Univer-
sity, Rajkot (India).
Antiviral assays
The activity of compounds against HIV-1 was based on the
inhibition of virus-induced cytopathogenicity in MT-4 cells
acutely infected with a multiplicity of infection (m.o.i.) of
0.01. Briefly, RPMI (50 ll) containing 1 9 104 MT-4 was
added to each well of flat-bottom microtitre trays con-
taining RPMI (50 ll), with or without serial dilutions of
test compounds. Then, HIV-1 suspension (20 ll) contain-
ing 100 CCID50 was added. After 4-days incubation, cell
viability was determined by MTT method. Activity of
compounds against YFV and Reo-1 was based on the
inhibition of virus-induced cytopathogenicity in acutely
infected BHK-21 cells. Activities against BVDV, in
infected MDBK cells, were also based on inhibition of
virus induced cytopathogenicity. BHK and MDBK cells
were seeded in 96-well plates at a density of 5 9 104 and
3 9 104 cells/well, respectively, and were allowed to form
confluent monolayers by incubating overnight in growth
medium at 37 °C in a humidified CO2 (5 %) atmosphere.
Cell monolayers were then infected with 50 lL of a proper
Conflict of interest The authors declare that they have no conflict
of interest.
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