Synthesis and antiviral activity of β-carboline derivatives bearing a substituted carbohydrazide at C-3 against poliovirus and herpes simplex virus (HSV-1)

Article abstract of DOI:10.1016/j.ejmech.2009.07.005

Several novel 1,3-disubstituted β-carboline derivatives bearing a substituted carbohydrazide group at C-3 were synthesized and evaluated for their antiviral activity against vaccinal poliovirus (VP) and herpes simplex virus type 1 (HSV-1). The cytotoxicity and selectivity index of the active compounds were also evaluated. Among the synthesized derivatives, compounds 10 and 11 displayed potent activity against both vaccinal poliovirus and HSV-1 virus. Compound 10 presented the highest selectivity index (SI = 2446.8) against HSV-1 virus and low cytotoxicity (CC₅₀ = 1150.0 ± 67.3 μM). The virus yield inhibition assay showed that compound 10 was able to inhibit HSV-1 plaque formation before and during the virus adsorption. The characteristic small plaque pattern observed in compound-treated cells suggested that compound 10 inhibited viral dissemination to neighboring cells. A computational study for prediction of ADME properties of the novel synthesized β-carbolines derivatives was performed by determination of lipophilicity, topological polar surface area (TPSA), absorption (% ABS) and simple molecular descriptors, using Lipinski′s rule.

Full text of DOI:10.1016/j.ejmech.2009.07.005

European Journal of Medicinal Chemistry 44 (2009) 4695–4701
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiviral activity of b-carboline derivatives bearing a substituted
carbohydrazide at C-3 against poliovirus and herpes simplex virus (HSV-1)
Anelise S. Nazari Formagio a, Patricia R. Santos b, Karine Zanoli b, Tania Ueda-Nakamura c,
,
Lilian T. Du¨sman Tonin a, Celso V. Nakamura c, Maria Helena Sarragiotto a
*
a
´
´
Departamento de Qu´ımica, UEM, Universidade Estadual de Maringa, Avenida Colombo 5790, 87020-900 Maringa-PR, Brazil
b
c
´
´
Programa de Po´s-graduaç˜ao em Cieˆncias Farmaceˆuticas, UEM, Universidade Estadual de Maringa, Avenida Colombo 5790, 87020-900 Maringa-PR, Brazil
Departamento de Analises Clınicas, UEM, Universidade Estadual de Maringa, Avenida Colombo 5790, 87020-900 Maringa-PR, Brazil
´
´
´
´
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel 1,3-disubstituted b-carboline derivatives bearing a substituted carbohydrazide group at C-3
Received 29 October 2008
Received in revised form
20 May 2009
Accepted 9 July 2009
Available online 16 July 2009
were synthesized and evaluated for their antiviral activity against vaccinal poliovirus (VP) and herpes
simplex virus type 1 (HSV-1). The cytotoxicity and selectivity index of the active compounds were also
evaluated. Among the synthesized derivatives, compounds 10 and 11 displayed potent activity against
both vaccinal poliovirus and HSV-1 virus. Compound 10 presented the highest selectivity index
(SI ¼ 2446.8) against HSV-1 virus and low cytotoxicity (CC50 ¼ 1150.0 ꢀ 67.3
mM). The virus yield inhi-
bition assay showed that compound 10 was able to inhibit HSV-1 plaque formation before and during the
virus adsorption. The characteristic small plaque pattern observed in compound-treated cells suggested
that compound 10 inhibited viral dissemination to neighboring cells. A computational study for
Keywords:
1,3-Disubstituted b-carboline derivatives
Carbohydrazide derivatives
Antiviral activity
prediction of ADME properties of the novel synthesized
b-carbolines derivatives was performed by
determination of lipophilicity, topological polar surface area (TPSA), absorption (% ABS) and simple
molecular descriptors, using Lipinski0s rule.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
a disease under control in most countries [5]. However, the
members of this genus cause a wide array of illnesses, such as,
Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are
human herpes viruses belonging to family Herpesviridae [1]. These
types of HSV are responsible for mucocutaneous infections, mainly
in immunocompromised patients. HSV-1 provokes orofacial
lesions, while HSV-2 causes mucocutaneous genital infections [2].
Antiviral research on HSV preliminarily focuses on compounds
capable of targeting the viral polymerase. Acyclovir, a nucleoside
inhibitor of DNA polymerase, the first selective antiviral agent
introduced, still is the drug commonly employed in the treatment
of HSV infection [3]. However, the widespread use of Acyclovir has
led to the development of viral resistance against this drug [4].
Therefore, the search for new drugs against Acyclovir-resistant HSV
viruses is highly necessary.
meningitis, myocarditis, encephalitis, and respiratory diseases.
Although well studied, poliovirus remains one of most appropriate
model for the study of viral replication.
Recent studies have pointed to b-carboline alkaloids as a new
class of antiviral agents. Harman and some of its derivatives and
matairesinol, a compound isolated from Symplocos setchuenensis,
were found to inhibit the replication of HIV in H9 lymphocyte cells
[6]. Flazin, a b-carboline alkaloid isolated of Suillus granulatus was
found to possess anti-HIV activity [7]. A structure–activity study on
46 flazin synthesized analogues showed a potent anti-HIV activity
for flazinamide, which was considered a promising anti-HIV agent
[8]. Synthetic b-carboline derivatives bearing a guanidinium group-
terminated side chain at C-3 exhibited anti-HIV activity in MT4
cells, inhibiting HIV replication by interfering with TAT–TAR
interaction [9].
Poliovirus (PV) is a member of the genus Enterovirus belonging
to Picornaviridae family. This non-enveloped virus with a single
stranded RNA genome is the etiological agent of poliomyelitis,
The potentialities of b-carboline alkaloids as antiviral agents and
the importance of the search for new anti-HSV-1 drugs have led us
to study this class of compounds. Studies on structure–activity
relationship have demonstrated the influence of the substituents in
* Corresponding author. Tel.: þ55 44 3261 3657.
positions-1, -3, and -9 of the b-carboline skeleton for a variety of
E-mail address: mhsarragiotto@uem.br (M.H. Sarragiotto).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.005

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A.S. Nazari Formagio et al. / European Journal of Medicinal Chemistry 44 (2009) 4695–4701
synthetic
b-carboline derivatives [10–19]. Furthermore, antiviral
2.2. Antiviral activity
activity has been reported for several compounds possessing the
carbohydrazide moiety [20–23].
The novel
evaluated for their activity towards HSV-1 and vaccinal poliovirus
(VP). Compounds with EC50 > 100 M were considered inactive.
b-carboline-3-carbohydrazides derivatives 5–23 were
Based on these considerations, in this work, we synthesized
a series of novel
b-carboline derivatives bearing a substituted car-
m
bohydrazide group at C-3 and a phenyl-substituted group at C-1. All
derivatives were evaluated for their in vitro activity against herpes
simplex virus type 1 (HSV-1) and vaccinal poliovirus (VP). The
cytotoxicity and selectivity index of the active compounds were
determined. Studies to establish the step of the viral cycle affected
were also performed. A computational study for prediction of
The cytotoxicity to Vero cells and the selectivity index for the active
derivatives were also determined.
The antiviral assay results (Table 1) showed that five (6, 7,10–12)
of all tested compounds were active towards poliovirus, whereas
six (5, 8–11 and 20) displayed potent-to-weak activity against
HSV-1. The b-carboline-3-carbohydrazides 10 and 11, which have
ADME properties of the novel synthesized
b
-carbolines derivatives
a phenyl group at C-1, and a 4-methoxybenzylidene-carbohy-
drazide and 4-N,N-dimethylaminobenzylidene-carbohydrazide
groups at C-3, respectively, displayed potent activity against both
poliovirus and HSV-1 with low cytotoxicity and high selectivity
index.
was carried out by determination of lipophilicity, topological polar
surface area (TPSA), absorption (% ABS) and simple molecular
descriptors, using Lipinski’s rule.
Comparison of the EC50 data of all tested compounds showed
that the electronic nature of the R1 and R2 substituents on the phenyl
portions affects the antiviral activity to a significant extent. The
presence of an electron-withdrawing substituent, such as nitro or
2. Results and discussion
2.1. Chemistry
chlorine in the phenyl group attached to C-1 of the
b-carboline
The synthetic route for the preparation of
hydrazides 5–23 is presented in Scheme 1. Methyl tetrahydro-
boline-3-carboxylates 2a–e were prepared through Pictet–Spengler
condensation of -tryptophan 1 with 4-hydroxybenzaldehyde (a),
benzaldehyde (b), 4-methoxybenzaldehyde (c), 3-nitrobenzaldehyde
(d) and 4-nitrobenzaldehyde (e), in acid media, and subsequent
esterification of the corresponding carboxylic acids with methanol
b
-carboline-3-carbo-
skeleton or to the carbohydrazide moiety, resulted in inactive (13,14,
18, 19 and 21–23) or weakly active (8, 9, and 20) compounds.
Specifically for the most active compound 10, the substitution of the
4-methoxybenzylidene-carbohydrazide group for 3-nitrobenzy-
lidene-carbohydrazide or 2-chlorobenzylidene-carbohydrazide
groups led, respectively, to the inactive compounds 13 and 14.
Concerning the influence of the electron-donating groups on the
phenyl substituent, it was observed that the activity depends on
b-car-
L
and sulfuric acid [13,24]. Oxidation of methyl 1,2,3,4-tetrahydro
carboline-3-carboxilates 2a–e with sulfur under xylene reflux [15,25]
furnished methyl -carboline-3-carboxylates 3a–e. Conversion of 3a–
e to 1-(substituted-phenyl)- -carboline-3-carbohydrazides 4a–e was
b-
both nature of the substituent and its position, if at C-1 of the b-
carboline or on the carbohydrazide moiety. The substitution of the
1-(4-hydroxyphenyl) groups of the anti-PV active compounds 6
b
b
carried out by reactionwith hydrazine hydrate in ethanol under reflux
according to the procedures described in literature for similar
compounds [15]. Condensation of 4a–e with aromatic aldehydes
4-methoxybenzaldehyde, 4-dimethylaminobenzaldehyde, benzalde-
hyde, 4-nitrobenzaldehyde, and 2-chlorobenzaldehyde under reflux
in ethanol yielded the carbohydrazides 5–23.
All novel compounds were characterized by their spectral data
(IR, EIMS, 1H and 13C NMR). The 1H NMR spectra of carbohydrazides
5–23 showed signals at dH 8.40 integrating for one proton, and at dH
6.70–9.00, corresponding to the imine and aromatic hydrogens,
respectively, of the (substituted-benzylidene)carbohydrazide
group. The presence of this group was confirmed by the signals at dC
and
7 for 1-(4-methoxyphenyl) group resulted in inactive
compounds 16 and 17. Comparison of EC50 values of compounds 12
and 10 showed that the presence of a 4-methoxy substituent (R2) at
the phenyl of carbohydrazide moiety increase the activity of 10.
However, the change of the 4-methoxyphenyl group from the
carbohydrazide moiety (in compound 10) to the C-1 of
nucleus resulted in the inactive compound 17.
To establish the step in which compounds 6 and 10 affects the
VP and HSV-1 viral cycle, respectively, the virus yield inhibition
assay was performed before, during, and after virus infection. The
experiments were performed on Vero cell monolayers in 24-well
b-carboline
plates. The results showed that
b-carboline derivative 6 have no
147–150 (C]N), 158–162 (C]0), and
d
110–135 (aromatic carbons
of R2 group) in the 13C NMR spectra.
effect on poliovirus neither on adsorption (before) nor during virus
3
3
COOCH3
COOCH3
COOH
a, b
NH2
c
d
N
NH
1
N
H
N
H
N
H
1
R1
R1
N
1
2
3
"
4
"
2
O
CONHNH2
R2
3
e
N
"
1
N
"
6
N
H
H
N
H
N
H
1
'
'
2
6
R1
R1
5 - 23
'
4
4
Scheme 1. Reagents and conditions: (a) acetic acid, R1CHO, reflux, 2 h, and adjusted to pH ¼ 5 with NH4OH; 80–90%. (b) CH3OH, H2SO4, reflux, 48 h; 82–87%. (c) S, xylene, reflux,
48 h to 4ꢁ C, 3 h; 70–73%. (d) NH2NH2.H2O, EtOH, reflux, 48 h; 72–76%. (e) R2CHO, EtOH, H2SO4 (cat), reflux, 36 h; 60–78%.

A.S. Nazari Formagio et al. / European Journal of Medicinal Chemistry 44 (2009) 4695–4701
4697
Table 1
HSV-1 and poliovirus antiviral activity, cytotoxicity and selectivity index (SI) data for
b-carboline-3-carbohydrazide derivatives 5–23.
R1
R2
CC50
(
m
M)
PV
HSV-1
a
Comp
SIc
EC50
(
m
M)
SIc
b
b
EC50
(
m
M)
5
6
7
8
4-OH
4-OH
4-OH
4-OH
4-OH
H
H
H
H
H
4-OCH3
4-OCH3
4-OCH3
4-OCH3
3-NO2
3-NO2
4-NO2
4-NO2
4-NO2
4-OCH3
4-N(CH3)2
H
4-NO2
2-Cl
4-OCH3
4-N(CH3)2
H
4-NO2
2-Cl
4-OCH3
4-N(CH3)2
H
4-NO2
4-OCH3
4-N(CH3)2
4-OCH3
4-N(CH3)2
H
802.7 ꢀ 100.5
>100
nd
22.9 ꢀ 7.3
>100
>100
86.5 ꢀ 5.4
41.2 ꢀ 23.7
0.47 ꢀ 0.3
1.85 ꢀ 0.63
>100
>100
>100
>100
>100
35.0
nd
nd
2.3
0.7
2446.8
629.5
nd
nd
nd
nd
nd
nd
nd
nd
14.3
nd
900.2 ꢀ 94.5
2.67 ꢀ 1.24
1.03 ꢀ 0.17
>100
337.2
1202.8
nd
1238.9 ꢀ 487.6
199.5 ꢀ 80.2
9
29.8 ꢀ 10.1
>100
nd
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1150.0 ꢀ 67.3
0.87 ꢀ 0.5
0.87 ꢀ 0.04
1.95 ꢀ 1.7
>100
>100
>100
>100
>100
>100
>100
1321.8
1338.6
398.4
nd
nd
nd
nd
nd
nd
nd
1164.6 ꢀ 208.1
776.9 ꢀ 1.7
nd
nd
nd
nd
nd
nd
nd
>100
>100
>100
1049.6 ꢀ 100.5
>100
>100
>100
>100
nd
nd
nd
nd
73.2 ꢀ 7.6
nd
nd
nd
>100
>100
>100
nd
nd
nd ¼ not determined.
a
Concentration at which 50% cytotoxicity is observed.
Concentration at which 50% efficacy in antiviral assay is observed.
Selectivity index (CC50/EC50).
b
c
infection. Virucidal activity was also not detected (Table 2). The
interference was observed at the replication viral phase (after
infection) and was dose-dependent (Fig. 1A). In this phase, the viral
RNA is translated in a single highly autocatalytic polyprotein,
whose proteolytic cleavage products serve as capsid precursors and
replication proteins. These proteins are used to viral RNA replica-
tion [26].
calculations following Lipinski’s rule of five [28], which establishes
that the absorption or permeation of an orally administered
compound is more likely to be good if the drug satisfies the
following criteria: a) hydrogen bond donors ꢄ5 (OH and NH
groups); b) hydrogen bond acceptors ꢄ10 (N and O atoms); c)
molecular weight <500; d) calculated log P < 5. Compounds
violating more than one of these rules may present bioavailability
To determine whether compound 10 blocks HSV replication or
not, we investigated its action after virus infection. The effect of
compound 10 on HSV-1 was also assayed by incubating the virus
with the compound for 1 h before its addition to the Vero cells.
Interestingly the compound did not inhibit virus replication after
infection, but it was able to interfere in the early stage of the virus
problems. Our results revealed that the lipophilicity of all b-car-
boline-3-carbohydrazides is larger than 5.0; however, the molec-
ular weight (390.4 > MW < 480.4), hydrogen bond acceptors
(n-ON ¼ 5–9) and donors (n-OHNH ¼ 2–3) fulfill Lipinski’s rule.
These data may suggest the potential of these derivatives as new
antiviral agents.
infection at EC50 ¼ 113
m
M (Fig. 1B).
Accordingly, the calculated percent absorption of compounds
5–23 ranged between 53.2 and 84.8%. The compounds with the 4-
nitrophenyl groupatC-1 of theb-carbolineskeletonorattachedtothe
2.3. Computational study
carbohydrazide moiety showed high TPSA values, suggesting their
poor oral bioavailability and absorption. However, the most potent
compounds 6, 7, and 10–12 demonstrated better TPSA than ACV
(Acyclovir) (%ABS ¼ 67.9%), presenting % ABS between 76.7 and 84.8%.
In order to identify the compounds with potential drug-score,
A computational study of the synthesized
b-carboline-3-car-
bohydrazides 5–23 was performed by determination of Lipinski’s
rule and topological polar surface area (TPSA). Calculations were
performed using Molinspiration online property calculation toolkit
the most active b-carboline-3-carbohydrazide derivatives 6, 7, and
(http://www.molinspiration.com). Table
3 shows the Lipinski
10–12 were submitted to an in silico ADMET screening (available at
http://www.organic-chemistry.org). There are several approaches
that assess druglikeness of compounds based on topological
descriptors, fingerprints of molecular drug-like structures keys, or
other properties, such as clog P and molecular weight [29]. The
Osiris program (www.organic-chemistry.org/prog/peo) determines
the frequency of occurrence of each fragment within the collection
of treated drugs and within the supposedly non-drug-like collec-
tion of Fluka compounds. Positive values (0.1–10) indicate that the
molecule predominantly contains the better fragments, which are
frequently present in commercial drugs. The drug-score combines
druglikeness, clog P, log S, molecular weight, and toxicity risk in one
handy value than may be used to judge the compound overall
potential to qualify for a drug.
parameters, the calculated percent absorption (% ABS), and the
topological polar surface area (TPSA) of synthesized compounds 5–
23. The percent absorption was estimated using equation: %
ABS ¼ 109 ꢂ 0.345 ꢃ TPSA, according to Zhao et al. [27].
In vivo absorption of the synthesized
b-carboline-3-carbohy-
drazides was tentatively assessed by means of theoretical
Table 2
Effect of time treatment on antipoliovirus activity of compound 6 (n ¼ 1).
% Reduction of plaque formation
0.825
mM
1.65
m
M
3.3
m
M
6.6 mM
Before
During
After
0
0
0
0
0
0
32.0
0
0
0
66.3
0
0
0
88.3
0
In this work, we used the Osiris program to calculate the frag-
ment-based druglikeness and drug-score of the most active
compounds 6, 7, and 10–12 (Fig. 2). The data were compared with
Virucidal

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A.S. Nazari Formagio et al. / European Journal of Medicinal Chemistry 44 (2009) 4695–4701
poliovirus at non-cytotoxic concentrations. The high selectivity
100
75
50
25
0
A
indexes (SI) obtained for the active compounds suggest that the
derivatives are promising antiviral drugs. The most active
compounds 6, 7, 10, 11 and 12 presented better druglikeness values
than that of Acyclovir; however their drug-score values were
smaller than ACV, probably due to their low solubility and high
lipophilicity.
Compound 10, with a phenyl group at C-1 and a 4-methox-
ybenzylidene-carbohydrazide group at C-3, was the most active of
the synthesized derivatives, with EC50 of 0.47 mM and 0.87 mM
against HSV-1 and poliovirus, respectively. Our results confirmed
that compound 10 exerts a dose-dependent effect during the early
phases of HSV-1 infection in Vero cells. The anti-herpetic activity of
this compound could be due to other mechanisms than the
competition with the virus to bind to heparan sulfate (HS), the
major glycosaminoglycan on the cell surface, and glycoproteins on
the viral envelope [30], on the cell surface, as suggested by Mar-
chetti et al. [31]. Further studies are required to explore the antiviral
mechanism of this compound in detail.
0
1
2
3
4
5
6
7
Concentration (µM)
100
75
50
25
0
B
4. Experimental protocols
4.1. General
1H and 13C spectra were recorded in a Varian spectrometer
model Mercury plus BB at 300 MHz and 75 MHz, respectively. Mass
spectra (MS) were recorded in a Thermoelectron Corporation
Focus-DSQ II spectrometer. IR spectra were recorded on a BOMEM
spectrometer model MB-100. For TLC, Merck precoated plates
(silica gel 60 G254) were used. Silica gel 60 Merck (230–400
mesh) was used in column chromatography purification of
some compounds. All reagents were purchased from commercial
suppliers.
0
50
100
150
200
250
Concentration (µM)
Fig. 1. Effect of the compound 6 on poliovirus (A) and 10 on HSV-1 infection (B).
Compound was added before (A), during (C), and after virus infection (:) on Vero
cell monolayers. Virucidal activity (-) was also investigated. Antiviral activity of
compound 6 on poliovirus was only due its effect after virus infection, while the
compound 10 showed to be active on HSV-1 by inhibiting the adsorption/penetration
step or inactivating the virus particle. Data are means ꢀ standard deviation of triplicate
samples of a representative experiment. Each experiment was repeated at least three
times.
4.2. Synthesis
those calculated for ACV. Interestingly, derivatives 6, 7,10,11, and 12
presented druglikeness values (from 3.307 to 4.517) better than
ACV (ꢂ2.418). The drug-score values of the most active compounds
were smaller than ACV, probably due to their low solubility and
high lipophilicity. Compounds 6 and 11 presented high tumorigenic
risk, which is a direct indication of its non-potential drug-score.
4.2.1. General procedure for preparation of 1-(substituted-phenyl)-
N0-(substituted-benzylidene)-
b-carboline-3-carbohydrazides (5–24)
A solution of derivatives 4 (1 mmol) in water (10 mL) containing
two drops of concentrated sulfuric acid was refluxed for 30 min,
until complete dissolution. Then, a solution of aromatic aldehyde
(1 mmol) in ethanol (3 mL) was added. The resulting solution was
refluxed for 48 h. The mixture was poured into cold water and
neutralized with 10% aqueous sodium bicarbonate solution and the
precipitate formed was filtered and dried, furnishing the title
compounds 5–23 with 60–78% yield.
3. Conclusion
In conclusion, some
b-carboline derivatives containing
(substituted-benzylidene)carbohydrazide group at C-3 were
prepared and identified as novel antiviral agents against HSV-1 and
4.2.1.1. 1-(4-Hydroxyphenyl)-N0-(4-methoxybenzylidene)-9H-pyrido
[3,4-b]indole-3-carbohydrazide (5). Yield: 75%; mp: 172–174 ꢁC; IR
(KBr) nmax (cmꢂ1): 3442 (N–H), 1666 (C]O), 1509–1305 (C]C); 1H
6
NMR (300 MHz, DMSO-d6):
d 3.86 (3H, s, OCH3), 6.94 (2H, d,
0.3
J ¼ 8.7 Hz, H-300/H-500), 7.34 (1H, t, J ¼ 7.5 Hz, H-6), 7.58 (1H, t,
J ¼ 7.5 Hz, H-7), 7.65 (2H, d, J ¼ 7.5 Hz, H-30/H-50), 7.68 (1H, d,
J ¼ 7.5 Hz, H-8), 7.77 (2H, d, J ¼ 8.7 Hz, H-200/H-600), 8.13 (2H, d,
J ¼ 8.0 Hz, H-20/H-60), 8.23 (1H, d, J ¼ 7.5 Hz, H-5), 8.41 (1H, s,
N]CH), 8.94 (1H, s, H-4), 11.45–11.60 (s, 2H, NH); 13C NMR (DMSO-
4
0.2
2
0
0.1
d6):
d 54.5, 112.0, 113.0, 113.2, 119.6, 120.7, 120.8, 126.0, 127.7, 128.0,
0.0
128.1,128.3,128.4,129.4,134.3,137.0,137.8,140.4,141.0,147.2,160.3,
160.6; MS (m/z, relative intensity %): 436.06 (Mþ$, 0.1), 287 (73),
260. (79), 259 (100).
-0.1
-0.2
-2
-4
druglikeness
drug-score
4.2.1.2. 1-(4-Hydroxyphenyl)-N0-(4-N,N-dimethylaminobenzylidene)-
9H-pyrido[3,4-b]indole-3-carbohydrazide (6). Yield: 78%; mp: 245–
247 ꢁC; IR (KBr) nmax (cmꢂ1): 3427 (N–H), 1670 (C]O), 1520–1310
Fig. 2. Druglikeness and drug-score values of the most active compounds (6, 7, 10, 11,
12) and Acyclovir (ACV) using Osiris program.
(C]C); 1H NMR (300 MHz, CDCl3/CD3OD):
d 3.10 (6H, s, N(CH3)2),

A.S. Nazari Formagio et al. / European Journal of Medicinal Chemistry 44 (2009) 4695–4701
4699
Table 3
Predicted ADME, Lipinski parameters and molecular properties of the synthesized compounds 5–23 and of Acyclovir (ACV).
Comp
% ABS
TPSAa
Lipinski rulea
Log Sb
n-ON acceptors
n-OHNH donors
mi log P
MW
n violations
5
6
7
8
74.6
76.7
77.8
62.0
77.8
81.6
83.7
84.8
69.0
84.8
78.4
80.5
81.6
65.8
65.8
67.9
65.8
67.9
69.0
67.9
99.61
93.61
90.37
136.2
90.37
79.38
73.38
70.15
116.0
70.15
88.61
82.62
79.38
125.2
125.2
119.2
125.2
119.2
116.0
119.1
7
7
6
9
6
6
6
5
8
5
7
7
6
9
9
9
9
9
8
8
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
5.24
5.29
5.18
5.14
5.81
5.72
5.77
5.66
5.62
6.29
5.78
5.82
5.72
5.68
5.65
5.70
5.68
5.72
5.62
ꢂ1.61
436.5
449.5
406.4
451.4
440.9
420.5
433.5
390.4
435.4
424.9
450.5
463.5
420.5
465.5
465.5
478.5
465.5
478.5
435.4
225.2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
ꢂ6.48
ꢂ6.49
ꢂ6.46
ꢂ6.92
ꢂ7.19
ꢂ6.77
ꢂ6.79
ꢂ6.75
ꢂ7.21
ꢂ7.49
ꢂ6.79
ꢂ6.81
ꢂ6.77
ꢂ7.23
ꢂ7.23
ꢂ7.25
ꢂ7.23
ꢂ7.25
ꢂ7.21
ꢂ1.62
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
ACV
%ABS ¼ 109 ꢂ 0.345 ꢃ TPSA.
a
www.molinspiration.com/cgi-bin/properties.
www.organic-chemistry.org/prog/peo.
b
6.71 (2H, d, J ¼ 7.5 Hz, H-30/H-50), 7.71–7.58–7.66 (5H, m, Ar–H),
7.80 (2H, d, J ¼ 8.0 Hz, H-200/H-600), 8.07 (2H, d, J ¼ 7.5 Hz, H-20/H-60),
8.26 (1H, d, J ¼ 7.8 Hz, H-5), 8.39 (1H, s, N]CH), 9.13 (1H, s, H-4);
8.00 (2H, d, J ¼ 7.8, H-20/H-60), 8.45 (1H, d, J ¼ 8.1 Hz, H-5), 8.88 (1H,
s, N]CH), 9.86 (1H, s, H-4), 11.83–12.15 (2H, s, NH); 13C NMR
(DMSO-d6):
d 112.7, 113.5, 115.5, 120.2, 121.2, 122.1, 127.0, 127.5,
13C NMR (CDCl3/CD3OD):
d
56.6, 112.4, 112.6, 113.8, 120.2, 121.4,
128.2, 128.5, 129.4, 129.9, 130.4, 131.3, 132.0, 133.2, 134.2, 138.8,
141.4, 143.9, 158.9, 161.8; MS (m/z, relative intensity %): 440.01
(Mþ$, 1.4), 303 (90), 286 (40), 260 (97), 259 (100), 258 (33).
121.5, 128.5, 128.7, 128.8, 128.9, 130.1, 130.2, 134.8, 137.3, 138.2,
140.8, 141.6, 148.4, 150.5, 150.7, 160.7; MS (m/z, relative intensity %):
449.05 (Mþ$, 4.3), 287 (43), 260 (97), 259 (100).
4.2.1.6. 1-(Phenyl)-N0-(4-methoxybenzylidene)-9H-pyrido[3,4-b]
indole-3-carbohydrazide (10). Yield: 70%; mp: 218–220 ꢁC; IR (KBr)
nmax (cmꢂ1): 3365 (NH), 1683 (C]O), 1509–1303 (C]C); 1H NMR
4.2.1.3. 1-(4-Hydroxyphenyl)-N0-(benzylidene)-9H-pyrido[3,4-b]indole-
3-carbohydrazide (7). Yield: 83%; mp: 310–312 ꢁC; IR (KBr) nmax
(cmꢂ1): 3304 (N–H), 1657 (C]O), 1534–1314 (C]C); 1H NMR
(300 MHz, CDCl3/CD3OD):
d 3.86 (3H, s, OCH3), 6.97 (2H, d,
(300 MHz, CDCl3/CD3OD):
d
7.15 (2H, d, J ¼ 7.8 Hz, H-30/H-50), 7.37
J ¼ 8.7 Hz, H-300/H-500), 7.37 (1H, td, J ¼ 7.8; 2.5 Hz, H-6), 7.55–7.61
(3H, m, Ar–H), 7.64 (1H, d, J ¼ 7.8 Hz, H-8), 7.69 (1H, td, J ¼ 7.8 and
2.5 Hz, H-7), 7.81 (2H, d, J ¼ 8.7 Hz, H-200/H-600), 8.04 (2H, d,
J ¼ 7.0 Hz, H-20/H-60), N]CH: 8.23 (1H, s, N]CH), 8.27 (1H, d,
(1H, t, J ¼ 7.8 Hz; H-6), 7.55–7.40 (3H, m, Ar–H), 7.66 (1H, d,
J ¼ 7.8 Hz, H-8), 7.84 (2H, d, J ¼ 7.8 Hz, H-20/H-60), 7.95 (2H, d,
J ¼ 8.0 Hz; H-200/H-600), 8.27 (1H, d, J ¼ 8.0 Hz, H-5), 8.33 (1H, s,
N]CH), 8.93 (1H, s, H-4); 13C NMR (CDCl3/CD3OD):
d
112.1, 114.0,
J ¼ 8.1 Hz, H-5), 8.97 (1H, s, H-4); 13C NMR (CDCl3/CD3OD):
d 55.5,
116.0, 120.7, 121.9, 122.0, 127.8, 128.5, 128.7, 129.4, 129.8, 130.2,
130.5, 133.8, 135.2, 136.8, 138.2, 141.3, 148.5, 150.9, 158.1; MS (m/z,
relative intensity %): 406.04 (Mþ$, 5.8), 303 (76), 286 (37), 260 (91),
259 (100).
112.1, 114.3, 114.5, 121.1, 122.1, 122.3, 126.9, 128.5, 129.1, 129.3, 129.4,
129.6, 130.3, 130.6, 135.2, 137.9, 139.1, 141.0, 148.2, 161.4, 161.6; MS
(m/z, relative intensity %): 420.08 (Mþ$, 6.1), 287 (78), 270 (33), 244
(95), 243 (100), 242 (47).
4.2.1.4. 1-(4-Hydroxyphenyl)-N0-(4-nitrobenzylidene)-9H-pyrido[3,4-
b]indole-3-carbohydrazide (8). Yield: 80%; mp: 342–344 ꢁC; IR (KBr)
nmax (cmꢂ1): 3380 (N–H), 1675 (C]O), 1524–1310 (C]C); 1H NMR
4.2.1.7. 1-(Phenyl)-N0-(4-N,N-dimethylaminobenzylidene)-9H-pyr-
ido[3,4-b]indole-3-carbohydrazide (11). Yield: 75%; mp: 230–
232 ꢁC; IR (KBr) nmax (cmꢂ1): 3376 (NH), 1664 (C]O), 1519–1303
(300 MHz, DMSO-d6):
d
7.08 (2H, d, J ¼ 7.5 Hz, H-30/H-50), 7.35 (1H,
(C]C); 1H NMR (300 MHz, CDCl3/CD3OD):
d 3.04 (6H, s, N(CH3)2),
t, J ¼ 7.5 Hz, H-6), 7.60 (1H, t, J ¼ 7.5 Hz, H-7), 7.73 (1H, d, J ¼ 7.5 Hz,
H-8), 8.03 (2H, d, J ¼ 8.0 Hz, H-300/H-500), 8.13 (2H, d, J ¼ 7.5 Hz, H-20/
H-60), 8.34 (2H, d, J ¼ 8.0 Hz, H-200/H-600), 8.47 (1H, d, J ¼ 7.5 Hz, H-
5), 8.80 (1H, s, N]CH), 8.91 (1H, s, H-4),12.06–11.86 (2H, s, NH); 13C
6.35 (2H, d, J ¼ 6.0 Hz, H-300/H-500), 7.34 (1H, td, J ¼ 7.0 and 2.0 Hz, H-
6), 7.55 (1H, td, J ¼ 7.0 and 2.0 Hz, H-7), 7.57–7.64 (3H, m, Ar–H),
7.59 (2H, d, J ¼ 6.0 Hz, H-200/H-600), 7.66 (1H, d, J ¼ 7.0 Hz, H-8), 8.03
(2H, d, J ¼ 7.2 Hz, H-20/H-60), 8.13 (1H, s, N]CH), 8.24 (1H, d,
NMR (DMSO-d6):
d
112.7, 113.5,115.6,120.2, 122.1, 121.2,124.1,127.9,
J ¼ 7.8 Hz, H-5), 8.93 (1H, s, H-4); 13C NMR (CDCl3/CD3OD):
d 40.1,
128.5,129.5, 130.1,130.3,134.3, 138.4, 140.9, 141.2, 141.4,145.5,147.7,
158.6, 161.6; MS (m/z, relative intensity %): 451.03 (Mþ$, 2.5), 303
(75), 286 (29), 260 (91), 259 (100), 258 (32).
111.8,112.2,114.2,120.8, 121.3,122.0,128.5,128.8,129.2,129.3, 130.1,
130.5, 135.1, 137.9, 138.7, 141.2, 141.4, 149.9, 152.0, 161.1; MS (m/z,
relative intensity %): 433.09 (Mþ$, 25.5), 287 (42), 270 (30), 244
(100), 243 (95), 242 (46).
4.2.1.5. 1-(4-Hydroxyphenyl)-N0-(2-chlorobenzylidene)-9H-pyrido[3,4-
b]indole-3-carbohydrazide (9). Yield: 70%; mp: 360–362 ꢁC; IR (KBr)
nmax (cmꢂ1): 3354 (N–H), 1652 (C]O), 1515–1319 (C]C); 1H NMR
4.2.1.8. 1-(Phenyl)-N0-(benzylidene)-9H-pyrido[3,4-b]indole-3-carbo-
hydrazide (12). Yield: 74%; mp: 290–292 ꢁC; IR (KBr) nmax (cmꢂ1):
3327 (NH), 1665 (C]O), 1518–1309 (C]C); 1H NMR (300 MHz,
(300 MHz, DMSO-d6):
d
7.05 (2H, d, J ¼ 7.8 Hz, H-30/H-50), 7.31 (1H,
t, J ¼ 7.8 Hz, H-6), 7.56–7.46 (4H, m, Ar–H), 7.60 (1H, t, J ¼ 7.8 Hz, H-
CDCl3/CD3OD):
d
7.38 (1H, td, J ¼ 7.0 and 2.5 Hz, H-7), 7.40–7.70
7), 7.70 (1H, d, J ¼ 7.8 Hz, H-8), 7.95 (2H, d, J ¼ 8.0 Hz, H-200/H-600),
(7H, m, Ar–H), 7.61 (1H, d, J ¼ 7.0 Hz, H-8), 7.86 (2H, d, J ¼ 7.0 Hz,

4700
A.S. Nazari Formagio et al. / European Journal of Medicinal Chemistry 44 (2009) 4695–4701
H-200/H-600), 8.04 (2H, d, J ¼ 7.0 Hz, H-20/H-60), 8.26 (1H, d, J ¼ 7.8 Hz,
H-5), 8.28 (1H, s, N]CH), 8.97 (1H, s, H-4); 13C NMR (CDCl3/
J ¼ 8.5 Hz, H-3’/H-5’), 7.34 (1H, dd, J ¼ 8.0 and 2.0 Hz, H-6), 7.41 (3H,
m, Ar–H), 7.56 (1H, dd, J ¼ 8.0 and 2.0 Hz, H-7), 7.60 (1H, d,
J ¼ 8.0 Hz, H-8), 7.80 (2H, d, J ¼ 7.0 Hz, H-200/H-600), 7.97 (2H, d,
J ¼ 8.5 Hz, H-20/H-60), 8.17 (1H, d, J ¼ 8.0 Hz, H-5), 8.23 (1H, s,
CD3OD):
d 112.3, 114.5, 120.8, 121.8, 121.9, 127.7, 128.4, 128.7, 128.9,
129.1, 129.2, 130.4, 130.2, 130.5, 133.8, 135.3, 138.2, 141.4, 141.7,
148.5, 162.0; MS (m/z, relative intensity %): 390.08 (Mþ$, 4.2), 287
(72), 270 (32), 244 (88), 243 (100), 242 (46).
N]CH), 8.85 (1H, s, H-4); 13C NMR (CDCl3/CD3OD):
d 55.6, 112.2,
114.7, 114.2, 120.9, 122.1, 122.2, 127.9, 128.8, 128.9, 129.8, 130.4,
130.5, 130.6, 133.9, 135.1, 138.5, 141.2, 141.4, 148.4, 160.5, 160.6; MS
(m/z, relative intensity %): 420.08 (Mþ$, 6.4), 318 (21), 317 (100),
300 (47), 274 (96), 273 (66).
4.2.1.9. 1-(Phenyl)-N0-[1-(4-nitrobenzylidene)-9H-pyrido[3,4-b]indole-
3-carbohydrazide (13). Yield: 78%; mp: 178–180 ꢁC; IR (KBr) nmax
(cmꢂ1): 3422 (NH), 1665 (C]O), 1517–1339 (C]C); 1H NMR
(300 MHz, CDCl3/CD3OD):
d
7.33 (1H, t, J ¼ 7.8 Hz, H-6), 7.40–7.60
4.2.1.14. 1-(4-Methoxyphenyl)-N0-(4-nitrobenzylidene)-9H-pyrido[3,4-
b]indole-3-carbohydrazide (18). Yield: 68%; mp: 255–257 ꢁC; IR
(KBr) nmax (cmꢂ1): 3326 (NH), 1675 (C]O), 1523–1309 (C]C); 1H
(3H, m, Ar–H), 7.62 (1H, t, J ¼ 7.8 Hz, H-7), 7.68 (1H, d, J ¼ 7.8 Hz, H-
8), 8.00 (2H, d, J ¼ 8.7 Hz, H-200/H-600), 8.20 (2H, d, J ¼ 7.5 Hz, H-20/H-
60), H-300/H-500, 8.31 (2H, d, J ¼ 8.7 Hz, H-300/H-500), 8.48 (1H, d,
J ¼ 7.8 Hz, H-5), 8.78 (1H, s, N]CH), 8.98 (1H, s, H-4); 13C NMR
NMR (300 MHz, CDCl3/CD3OD):
d 3.96 (3H, s, OCH3), 7.20 (2H, d,
J ¼ 8.7 Hz, H-3’/H-5’), 7.35 (1H, dd, J ¼ 7.5; 2.5 Hz, H-6), 7.54 (1H,
dd, 7.5 and 2.0 Hz, H-7), 7.62 (1H, d, J ¼ 7.5 Hz, H-8), 8.00 (2H, d,
J ¼ 8.7 Hz, H-200/H-600), 8.03 (2H, d, J ¼ 8.7 Hz, H-20/H-60), 8.23 (2H,
d, J ¼ 8.7 Hz, H-300/H-500), 8.29 (1H, d, J ¼ 8.7 Hz, H-5), 8.42 (1H, s,
(CDCl3/CD3OD): d 112.7, 114.4, 120.4, 121.2, 122.2, 124.2, 127.9, 129.0,
129.2, 129.3, 129.4, 134.6, 137.3, 138.6, 140.9, 141.1, 141.6, 145.0, 147.7,
161.5; MS (m/z, relative intensity %): 435.05 (Mþ$, 3.7), 287 (63),
244 (85), 243 (100), 242 (43).
N]CH), 8.98 (1H, s, H-4); 13C NMR (CDCl3/CD3OD):
d 55.2, 112.3,
114.0, 114.3, 120.6, 120.9, 121.6, 123.8, 128.2, 128.7, 129.7, 130.0,
130.1, 130.3, 135.7, 137.4, 140.2, 141.5, 145.5, 148.7, 150.3, 160.4; MS
(m/z, relative intensity %): 465.06 (Mþ$, 3.9), 317 (100), 300 (42),
274 (95).
4.2.1.10. 1-(Phenyl)-N0-(2-chlorobenzylidene)-9H-pyrido[3,4-b]indole-
3-carbohydrazide (14). Yield: 72%; mp: 204–206 ꢁC; IR (KBr) nmax
(cmꢂ1): 3446 (NH), 1671 (C]O), 1519–1322 (C]C); 1H NMR
(300 MHz, CDCl3/CD3OD):
d 7.30–7.55 (6H, m, Ar–H), 7.62 (1H, d,
J ¼ 7.8 Hz, H-8), 7.66 (2H, d, J ¼ 7.5 Hz, H-30/H-50), 7.70 (1H, m, H-7),
8.03 (2H, d, J ¼ 8.1 Hz, H-20/H-60), 8.23 (1H, d, J ¼ 7.8 Hz, H-5), 8.29
(1H, dd, J ¼ 7.0 and 3.0 Hz, H-300), 8.96 (1H, s, H-4), 8.78 (1H, s,
4.2.1.15. 1-(3-Nitrophenyl)-N0-(4-methoxybenzylidene)-9H-pyrido[3,4-
b]indole-3-carbohydrazide (19). Yield: 80%; mp: 184–186 ꢁC; IR (KBr)
nmax (cmꢂ1): 3297 (NH), 1664 (C]O), 1528–1305 (C]C); 1H NMR
N]CH); 13C NMR (CDCl3/CD3OD):
d
112.3, 114.6, 120.8, 121.8, 121.9,
(300 MHz, CDCl3/CD3OD): d 3.84 (3H, s, OCH3), 6.97 (2H, d,
127.2, 127.9, 128.5, 128.9, 129.1, 129.2, 129.3, 129.4, 129.7, 130.4,
131.3, 134.1, 135.4, 137.7, 138.1, 141.8, 144.7, 162.4; MS (m/z, relative
intensity %): 424.14 (Mþ$, 0.01), 287 (75), 270 (33), 244 (91), 243
(100), 242 (45).
J ¼ 8.7 Hz, H-300/H-500), 7.35 (1H, t, J ¼ 7.8 Hz, H-6), 7.60 (1H, dd,
J ¼ 7.8 and 2.0 Hz, H-60), 7.69 (1H, d, J ¼ 8.1 Hz, H-8), 7.73 (2H, d,
J ¼ 8.7 Hz, H-200/H-600), 7.93 (1H, t, J ¼ 7.8 Hz, H-7), 8.34 (1H, d,
J ¼ 8.1 Hz, H-5), 8.39 (1H, dd, J ¼ 7.5; 3.0 Hz, H-40), 8.57 (1H, s,
N]CH), 8.63 (1H, d, J ¼ 8.7 Hz, H-50), 8.94 (1H, t, J ¼ 3.0 Hz, H-20),
4.2.1.11. 1-(4-Methoxyphenyl)-N0-(4-methoxybenzylidene)-9H-pyr-
ido[3,4-b]indole-3-carbohydrazide (15). Yield: 78%; mp: 145–
147 ꢁC; IR (KBr) nmax (cmꢂ1): 3283 (NH), 1672 (C]O), 1509–1304
8.97 (1H, s, H-4); 13C NMR (CDCl3/CD3OD):
d 54.9, 112.4, 113.9, 114.4,
120.3, 121.0, 121.6, 123.1, 123.5, 126.7, 128.6, 128.8, 129.8, 130.4,
134.6, 135.2, 138.2, 138.8, 138.9, 141.6, 148.1, 148.2, 160.7, 160.8; MS
(m/z, relative intensity %): 467.10 (Mþ$, 0.5), 332 (75), 289 (100),
288 (36), 261 (41), 242 (68).
(C]C); 1H NMR (300 MHz, CDCl3/CD3OD):
d 3.86 (3H, s, OCH3), 3.94
(3H, s, OCH3), 6.98 (2H, d, J ¼ 7.0 Hz, H-200/H-600), 7.18 (2H, d,
J ¼ 8.7 Hz, H-30/H-50), 7.35 (1H, d, J ¼ 7.5 Hz, H-6), 7.57 (1H, d,
J ¼ 7.5 Hz, H-8), 7.60 (1H, td, J ¼ 7.5 and 2.5 Hz, H-7), 7.79 (2H, d,
J ¼ 8.7 Hz, H-20/H-60),: 7.98 (2H, d, J ¼ 7.0 Hz, H-300/H-500), 8.20 (1H,
s, N]CH), 8.22 (1H, d, J ¼ 7.8 Hz, H-5), 8.96 (1H, s, H-4); 13C NMR
4.2.1.16. 1-(3-Nitrophenyl)-N0-(4-N,N-dimethylaminobenzylidene)-9H-
pyrido[3,4-b]indole-3-carbohydrazide (20). Yield: 85%; mp: 260–
262 ꢁC; IR (KBr) nmax (cmꢂ1): 3492 (NH), 1655 (C]O), 1522–1321
(CDCl3/CD3OD):
d
55.5, 55.4, 112.2, 114.2, 114.5, 114.7, 120.4, 120.8,
(C]C); 1H NMR (300 MHz, DMSO-d6):
d 3.04 (6H, s, N(CH3)2), 6.72
121.9, 126.6, 128.8, 129.4, 129.8, 130.3, 130.4, 135.1, 138.4, 141.2,
141.4,148.3,160.4,161.5,161.6; MS (m/z, relative intensity %): 450.10
(Mþ$, 8.1), 318 (22), 317 (100), 301 (15), 300 (45).
(2H, d, J ¼ 8.7 Hz, H-300/H-500), 7.35 (1H, t, J ¼ 7.8 Hz, H-6), 7.60 (1H, t,
J ¼ 7.8 Hz, H-7), 7.66 (1H, d, J ¼ 7.8 Hz, H-8), 7.68 (2H, d, J ¼ 8.7 Hz,
H-200/H-600), 7.88 (1H, dd, J ¼ 7.8 and 2.0 Hz, H-60), 7.90 (1H, d,
J ¼ 8.1 Hz, H-40), 8.29 (1H, d, J ¼ 7.8 Hz, H-5), N]CH: 8.38 (1H, s,
N]CH), 8.58 (1H, d, J ¼ 8.1 Hz, H-50), 8.97 (1H, s, H-4), 8.94 (1H, t,
J ¼ 2.0 Hz, H-20), 11.42–11.86 (2H, s, NH); 13C NMR (DMSO-d6):
4.2.1.12. 1-(4-Methoxyphenyl)-N0-(4-N,N-dimethylaminobenzylidene)-
9H-pyrido[3,4-b]indole-3-carbohydrazide (16). Yield: 83%; mp: 310–
312 ꢁC; IR (KBr) nmax (cmꢂ1): 3417 (NH), 1599 (C]O), 1514–1300
d
56.8, 110.7,111.8,113.7,119.8,120.5,120.6,120.9,122.5,123.0,128.1,
(C]C); 1H NMR (300 MHz, CDCl3/CD3OD):
d
3.03 (6H, s, N(CH3)2),
128.2, 129.1, 130.0, 134.1, 134.4, 137.6, 138.3, 138.5, 141.1, 147.6, 148.4,
150.8, 160.0; MS (m/z, relative intensity %): 480.06 (Mþ$, 1.3), 289
(76), 243 (46), 242 (70), 241 (38), 146 (100).
3.93 (3H, s, OCH3), 7.18 (2H, d, J ¼ 8.7 Hz, H-30/H-50), 7.36 (1H, t,
J ¼ 7.8 Hz, H-6), 7.58 (1H, d, J ¼ 7.8 Hz, H-8), 7.60 (1H, td, J ¼ 7.8 and
2.0 Hz, H-7), 7.67 (2H, d, J ¼ 7.5 Hz, H-300/H-500), 7.72 (2H, d,
J ¼ 8.7 Hz, H-20/H-60), 7.97 (2H, d, J ¼ 8.7 Hz, H-200/H-600), 8.14 (1H, s,
N]CH), 8.22 (1H, d, J ¼ 7.8 Hz, H-5), 8.90 (1H, s, H-4); 13C NMR
4.2.1.17. 1-(4-Nitrophenyl)-N0-(4-methoxybenzylidene)-9H-pyrido[3,4-
b]indole-3-carbohydrazide (21). Yield: 76%; mp: 166–168 ꢁC; IR (KBr)
nmax (cmꢂ1): 3288 (NH), 1668 (C]O), 1512–1309 (C]C); 1H NMR
(CDCl3/CD3OD):
d 40.3, 55.7, 111.9, 112.2, 113.9, 114.6, 120.9, 121.5,
122.2, 126.8, 128.9, 129.4, 129.6, 129.8, 130.5, 138.0, 138.9, 141.3,
144.9, 149.4, 160.6, 161.2, 161.4; MS (m/z, relative intensity %):
463.12 (Mþ$, 29.0), 317 (79), 300 (41), 274 (100), 273 (50).
(300 MHz, CDCl3/CD3OD): d 3.81 (3H, s, OCH3), 7.05 (2H, d,
J ¼ 8.7 Hz, H-300/H-500), 7.35 (1H, t, J ¼ 7.0 Hz, H-6), 7.64 (1H, t,
J ¼ 7.0 Hz, H-7), 7.70 (1H, d, J ¼ 7.0 Hz, H-8), 7.75 (2H, d, J ¼ 8.7 Hz,
H-200/H-600), 8.45 (2H, d, J ¼ 7.0 Hz, H-30/H-50), 8.50 (1H, d, J ¼ 7.0 Hz,
H-5), 8.55 (2H, d, J ¼ 7.0 Hz, H-20/H-60), 8.61 (1H, s, N]CH), 9.02
4.2.1.13. 1-(4-Methoxyphenyl)-N0-(benzylidene)-9H-pyrido[3,4-
b]indole-3-carbohydrazide (17). Yield: 72%; mp: 160–162 ꢁC; IR
(KBr) nmax (cmꢂ1): 3283 (NH), 1671 (C]O), 1513–1320 (C]C); 1H
(1H, s, H-4); 13C NMR (CDCl3/CD3OD):
d 55.9, 112.7, 114.4, 115.0,
120.9,121.1,122.4,123.7,127.0,128.7,130.3,130.6,134.7,138.2,139.4,
NMR (300 MHz, CDCl3/CD3OD):
d
3.91 (3H, s, OCH3), 7.16 (2H, d,
139.9, 141.7, 142.5, 143.6, 147.5, 148.4, 160.8; MS (m/z, relative

A.S. Nazari Formagio et al. / European Journal of Medicinal Chemistry 44 (2009) 4695–4701
4701
intensity %): 467.03 (Mþ$, 0.5), 332 (82), 288 (100), 287 (40), 243
(43), 242 (80).
4.3.2.4. Virucidal activity. The viral suspensions were pre-incu-
bated with different concentrations at 37 ꢁC for 1 h. The mixtures
were used to infect Vero cells for 1 h at 37 ꢁC in 5% CO2. Next, the
cell monolayer was washed with PBS and further incubated in
DMEM containing 0% carboxymethylcellulose.
In all of the experiments above, after 48 or 72 h incubation at
37 ꢁC, cell monolayers were fixed with 10% formaldehyde, stained
with solution of 0.5% crystal violet and the number of plaques was
counted. Controls consisted of either untreated Vero cells or
infected Vero cells.
4.2.1.18. 1-(4-Nitrophenyl)-N0-(4-N,N-dimethylaminobenzylidene)-
9H-pyrido[3,4-b]indole-3-carbohydrazide (22). Yield: 80%; IR (KBr)
nmax (cmꢂ1): 3420 (NH), 1660 (C]O), 1519–1322 (C]C); 1H NMR
(300 MHz, DMSO-d6):
d
2.98 (3H, s, CH3), 6.76 (2H, d, J ¼ 8.7 Hz, H-
300/H-500), 7.35 (1H, t, J ¼ 7.0 Hz, H-6), 7.59 (2H, d, J ¼ 8.7 Hz, H-200/H-
600), 7.63 (1H, t, J ¼ 7.0 Hz, H-7), 8.23 (2H, d, J ¼ 7.5 Hz, H-30/H-50),
8.47 (2H, d, J ¼ 7.5 Hz, H-20/H-60), 8.51 (1H, s, N]CH), 8.48 (1H, d,
J ¼ 7.0 Hz, H-5), 9.01 (1H, s, H-4), 12.12–1159 (2H, s, N–H). 13C NMR
(DMSO-d6):
d 60.3, 111.8, 112.7, 114.8, 120.6, 121.0, 122.4, 123.8,
Acknowledgments
128.5, 129.6, 130.3, 130.7, 134.7, 137.2, 138.1, 138.2, 139.7, 141.7, 147.5,
149.3, 159.4, 160.5. MS (m/z, relative intensity %): 478.03 (Mþ$, 4.5),
332 (79), 288 (100), 287 (40), 242.00 (78).
This work was supported by Conselho Nacional de Desenvolvi-
mento Cientı´fico e Tecnolo´gico (CNPq, Brazil). We thank CAPES and
CNPq for fellowships (A.S.N.F.; K.Z.; P.R.S. and L.T.D.T.)
4.2.1.19. 1-(4-Nitrophenyl)-N0-(benzylidene)-9H-pyrido[3,4-b]indole-
3-carbohydrazide (23). Yield: 73%; mp: 220–222 ꢁC; IR (KBr) nmax
(cmꢂ1): 3279 (NH), 1664 (C]O), 1521–1324 (C]C); 1H NMR
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4.3.1. Screening tests
Vero cells monolayers, infected or not with poliovirus (PV) or
herpesvirus (HSV-1), were treated with different concentrations of
each synthesized compounds and the antiviral activity was detec-
ted by Sulforhodamine B assay (SRB) [32]. The cytotoxicity was
evaluated against Vero cells after 72 h of drug exposure using the
SRB assay. EC50, CC50 and SI were determined and the results of all
tested compounds are listed in Table 1.
4.3.2. Virus yield inhibition assay
4.3.2.1. Effect on viral adsorption. Compounds were dissolved in
DMSO, diluted in serum free DMEM at different concentrations and
incubated with Vero cell monolayer pre-washed with phosphate-
buffered saline (PBS) in 24-well tissue culture plates for 1 h at 37 ꢁC
in 5% CO2. After removal of the unbound compound, the cells were
washed with PBS and then infected with 40–50 UFP/well of HSV-1
virus. After incubating for 1 h, the unabsorbed virus was removed;
the cell monolayer was washed with PBS and then incubated in
DMEM containing 0.5% carboxymethylcellulose.
4.3.2.2. Treatment during infection. The assay was performed as
described above, with the exception that the compound was added
together with the virus. After 1 h incubation, the solution con-
taining unabsorbed virus was removed, the cell monolayer was
washed with PBS and further incubated in DMEM containing 0.5%
carboxymethylcellulose.
4.3.2.3. Treatment after infection. To evaluate the effect of the
compounds on infected cells, monolayer Vero cells were washed
with PBS and infected. After 1 h incubation, the unabsorbed virus
was removed and the cell monolayer was washed with PBS and
then incubated with different concentrations of the compounds in
DMEM containing 0.5% carboxymethylcellulose.