Synthesis, biological screening and ADME prediction of benzylindole derivatives as novel anti-HIV-1, anti-fungal and anti-bacterial agents

Article abstract of DOI:10.1007/s00044-012-0463-6

Present study is focused on design, synthesis, and biological evaluation of substituted benzylindole derivatives as anti-HIV, anti-fungal, and anti-bacterial agents. Out of the reported compounds, compound B1 and B2 showed potent Anti-HIV activity, wherea

Full text of DOI:10.1007/s00044-012-0463-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4633–4640
DOI 10.1007/s00044-012-0463-6
ORIGINAL RESEARCH
Synthesis, biological screening and ADME prediction
of benzylindole derivatives as novel anti-HIV-1,
anti-fungal and anti-bacterial agents
•
A. M. Kashid P. N. Dube P. G. Alkutkar
•
•
•
K. G. Bothara S. N. Mokale S. C. Dhawale
•
Received: 2 July 2012 / Accepted: 31 December 2012 / Published online: 17 January 2013
Ó Springer Science+Business Media New York 2013
Abstract Present study is focused on design, synthesis,
and biological evaluation of substituted benzylindole
derivatives as anti-HIV, anti-fungal, and anti-bacterial
agents. Out of the reported compounds, compound B1 and
B2 showed potent Anti-HIV activity, whereas compound
B1–B4 showed good anti-fungal and anti-bacterial activity.
ADME properties of benzylindol analogs were analyzed
using Qikprop 2.5 tool of Schrodinger.
active anti-retroviral therapy (HAART), which employs a
combinational use of drugs (Este and Cihlar, 2010).
Currently, FDA-approved anti-HIV drugs belong to
seven different groups: nucleoside reverse transcriptase
inhibitors (NRTIs), nucleotide reverse transcriptase inhib-
itors (NtRTIs), nonnucleoside reverse transcriptase inhibi-
tors (NNRTIs), protease inhibitors (PIs), fusion inhibitors
(FIs), co-receptor inhibitors (CRIs), and integrase inhibi-
tors (INIs) (Mehellou and De Clercq, 2010).
Keywords HIV-1 ꢀ Reverse transcriptase inhibitor ꢀ
Benzylindol ꢀ ADME prediction
Although the HAART has brought about a substantial
decrease in the death rate, changing AIDS from a rapidly
lethal disease into a chronic manageable condition, the
retroviral infection can be only temporarily controlled but
not eradicated since, HIV-1 becomes almost undetectable
in the plasma for more than 2 years, persisting in
reservoirs.
Introduction
It has been recently reported that more than 33 million of
people were infected worldwide with human immunodefi-
ciency virus (HIV-1). The overall number of people living
with HIV has increased as a result of new infections and
the beneficial effects of the more widely available highly
Furthermore, the HAART efficacy has been limited by
the emergence of drug-resistant viral strains, drug-toxicity,
and the poor ability of patients to adhere to the prescribed
therapy and costs, so a refining of the current therapies and
the developing of new therapeutic paradigms are still
warranted (Sechi et al., 2009).
NNRTIs are a structurally diverse group of compounds
which binds to the viral enzyme RT, where it interacts with
a specific allosteric non-substrate binding pocket site.
NNRTIs resistance mutations arise rapidly and most often
directly in contact with the NNRTI molecule. Therefore,
attempts have been made to develop NNRTIs that are
active against resistance mutations. These attempts have
led to the identification of a number of compounds that are
active against resistance mutations. Currently, drugs which
are used to treat AIDS under NNRTIs are Nevirapine (1),
Efavirenz (2), Etravirine (3), Tenofovir desoproxil (4),
Delaviridine (5), and Rilpivirine (6) (Fig. 1) (Balzarini
et al., 2004).
A. M. Kashid (&) ꢀ P. N. Dube ꢀ P. G. Alkutkar ꢀ K. G. Bothara
Department of Pharmaceutical Chemistry, Sinhgad Institute of
Pharmacy, Narhe, Pune 411041, Maharashtra, India
e-mail: arunkashid2006@gmail.com
S. N. Mokale
Department of Pharmaceutical Chemistry, Y. B. Chavan College
of Pharmacy, Aurangabad, Maharashtra, India
S. C. Dhawale
Department of Pharmaceutical Chemistry, School of Pharmacy,
SRTM University, Nanded, Maharashtra, India
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Med Chem Res (2013) 22:4633–4640
Fig. 1 Clinically used NNRTIs
O
H3C
H
N
F3C
Cl
O
N
N
N
N
H
O
NH2
1. Nevirapine
2. Efavirenz
CN
CN
N
N
N
O
C
N
O
O
H2
(H3C)2HC
(H3C)2HC
O
O
C
O
P
H3C
CH3
N
H2
C
O
C
O
O
O
NH
CH3
O
N
Br
4. Tenofovir desoproxil
NH2
CN
3. Etravirine
CH3
SO2
CN
CH(CH3)2
HN
HN
H3C
CH3
N
N
N
N
HN
N
NH
H
O
N
5. Delaviridine
6. Rilpivirine
From the literature survey, it was observed that the
butterfly-like shape is an important factor in the binding of
the first generation NNRTIs. Despite their chemical
diversity, they assume very similar butterfly-like shape.
The butterfly structure has a hydrophilic centre as a ‘body’
and two hydrophobic moieties representing the ‘wings’.
Based on the above observation, we thought it was
logical to design a new molecular scaffold containing
benzylindol. Due to the importance of their indole back
bone, the compound showed a variety of biological activ-
ities such as anti-bacterial (Daisley and Shah, 1984; Varma
and Nobles, 1967), anti-fungal (Varma and Nobles, 1967;
Piscapo et al., 1987), anti-HIV (Pandeya et al., 1999,
2000), and anti-convulsant activity (Bhattacharya and
Chakraborti, 1998). Owing to the broad spectrum of che-
motherapeutic properties, it appears as an ideal drug for
AIDS treatment which suppresses HIV replication there by
acting as an anti-HIV drug as well as possesses efficacy
against opportunistic infections associated with AIDS like
tuberculosis, hepatitis, and other bacterial diseases.
In this study, we have focused on a new series of ben-
zylindole derivatives with various substituents at the ben-
zene-fused ring, also to ensure drug-like pharmacokinetic
profile of the designed NCEs, the ADME properties were
calculated.
Result and discussion
Chemistry
The synthetic route employed to obtain the title com-
pounds is depicted in Scheme 1. Dimethoxyindoles E1–
E4 were 3-acetylated by a Vilsmeier Haack reaction and
N-alkylated by treatment with 2,4-dichlorobenzyl chlo-
ride, in the presence of a catalytic amount of sodium
hydride to give intermediates C1–C4. Successively,
diketo esters B1–B4, obtained by coupling with diethyl
oxalate, were converted in the final derivatives A1–A4 by
hydrolysis in basic medium. The introduction of a nitro
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Med Chem Res (2013) 22:4633–4640
4635
Cl
Cl
H
H
N
ii
N
N
i
R2R1
R2R1
R2R1
CH3
CH3
O
O
Dimethoxy-1H-indol (E)
3-Acetyl-dimethoxy-1-(2,4-
dichlorobenzyl)-1H-indol
(D)
3-Acetyl-dimethoxy-1H-indol
(C)
Cl
Cl
Cl
Cl
N
N
iii
iv
R2R1
R2R1
COOEt
OH
COOH
O
O
OH
4-(1-(2,4-Chlorobenzyl)-dimethoxy-1H-indol-3-yl)
-2-hydroxy-4-oxobut-2-enoates
4-(1-(2,4-Chlorobenzyl)-dimethoxy-1H-indol-
3-yl)-2-hydroxy-4-oxobut-2-enoic acid
(B)
(A)
Scheme 1 Synthetic scheme of designed compounds (A1–A4, B1–
B4) Reagents: (i) phosphoryl chloride, dimethylacetamide, 4 N
NaOH; RT 12 h; (ii) DMF, sodium hydride, 2,4-dichlorobenzyl
chloride; 100 W, 5 min, 50 °C; (iii) diethyl oxalate, NaOCH3, THF;
250 W, 2 min, 50 °C (iv) 2 N NaOH; RT 1.5 h
group on 2,3-Dimethoxybenzaldehyde afforded a mixture
of 5- and 6-nitro derivatives which were separated by
flash chromatography on silica gel eluting with 30 %
EtOAc/cyclohexane (Fukuyama et al., 1998). The 2,3-
dimethoxy-6-nitrobenzaldehyde was converted into
the corresponding 2,3-dimethoxy-6,b-dinitrostyrene by
refluxing with nitromethane in the presence of glacial
acetic acid and ammonium acetate. Finally, 4,5-dim-
ethoxyindole (E1) was obtained by a Henry’s reductive
cyclization (Henry, 1895).
Methylation of the hydroxyl group with dimethyl sulfate
followed by reaction with nitromethane and reductive
cyclization, resulted in 6,7-dimethoxyindole (Rege et al.,
2006). For the designed NCES, yield obtained for each
product and their physicochemical properties are mentioned
in Table 1.
Biological activity
The standard nevirapine and test compounds (A1–A4, B1–
B4) were powdered finely and suspended in DMSO. The
RT inhibition assay was performed by using an RT assay
kit (Roche). The procedure for performing RT inhibition
assay was carried out as described in the kit protocol
(Table 2).
5,7-Dimethoxyindole was synthesized according to
Condie’s procedure, obtaining high yields. A solution of
3,5-dimethoxybenzaldehyde in nitromethane was refluxed
to afford 3,5-dimethoxy-b-nitrostyrene which was con-
verted in the nitro derivative and then into the desired
product by reductive cyclization (Condie et al., 2005).
A different synthetic pathway, according to a modifica-
tion of Rege’s procedure, was followed to obtain the
dimethoxy substitution at the 6-C and 7-C positions of the
indole nucleus. The treatment of commercially available
3-methoxy-4-hydroxybenzaldehyde with acetic anhydride
provided the O-acyl protected intermediate which, after
nitration, was hydrolyzed under basic conditions.
It has been observed from in vitro screening that newly
synthesized compounds possess RT inhibitory activity.
Among the synthesized compounds, B1–B4 showed a more
significant RT inhibitory activity. The compound in which
acid group was substituted at C3 position on the benzylindol
ring showed lesser RT inhibitory activity in comparison with
ester at C3 position except B3. The hydrophobic groups are
important for RT inhibitory activity at fused benzene ring.
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Table 1 Characterization of
synthesized compounds
(A1–A4, B1–B4)
Compound
ID
Derivatives
(R1R2)
Molecular
weight
%
yield
M.P.
(°C)
Rf
value
Solvent system
A-1
A-2
A-3
A-4
B-1
B-2
B-3
B-4
4,5-Dimethoxy
4,6-Dimethoxy
4,7-Dimethoxy
5,6-Dimethoxy
4,5-Dimethoxy
4,6-Dimethoxy
4,7-Dimethoxy
5,6-Dimethoxy
450.25
45.76
56.98
28.66
60.12
21.34
20.00
17.88
27.12
120–122
163–165
193–195
175–177
210–212
133–135
171–173
240–242
0.69
0.71
0.59
0.61
0.65
0.76
0.53
0.59
n-Hexane: ethyl
acetate (9:1)
478.32
Significant descriptors required for predicting the drug-like
properties of molecules are reported here. These properties
are as follows,
Table 2 In vitro reverse transcriptase assay data of synthesized
derivatives (A1–A4, B1–B4)
Compound ID
Optical density
% RT inhibition
1. Molecular weight (mol_MW) (150–650)
2. Octanol/water partition coefficient (LogP (o/w)) (-2
to 6.5)
Control
A-1
0.879
0.192
0.183
0.151
0.188
0.108
0.109
0.119
0.168
0.008
0.11
78.18
79.20
82.84
78.63
87.72
87.61
86.47
80.90
99.92
A-2
3. Aqueous solubility (QPlogS) (-6.5 to 0.5)
4. Apparent MDCK cell permeability (QPPMDCK) (\25
poor, [500 great)
A-3
A-4
B-1
5. Brain/blood partition coefficient (QPlogBB) (-3.0 to
B-2
1.2)
B-3
6. Percent human oral absorption (C80 % is high, B25 %
is poor)
B-4
Nevirapine
All the structures showed significant values for the
properties analyzed (Table 3) and showed drug-like char-
acteristics based on Lipinski’s rule of 5. The ADME values
of design compound inhibitors are given in Table 3.
The first three properties are based on Lipinski rule of
five, molecular weight (mol_MW) less than 650, partition
coefficient between octanol and water (logP (o/w))
between -2 and 6.5, and solubility (QPlogS) greater than
-7.
However, while comparing more and less bulky substituents
at this position, the methoxy group showed increased
activity. This indicates that the ring substituents are required
for RT inhibiting activity at C4 and C7 position.
Compounds B1–B4 showed good anti-fungal and anti-
bacterial activity. Compound B1 and B3 has shown
excellent activity against the Aspergillus niger and Penic-
illum notatum. Compound B3 and B4 has shown compa-
rable activity with ketoconazole for Aspergillus fumigatus,
whereas against the Candida albicans activity was found to
excellent than ketoconazole. Compound B2 has shown
excellent activity against Candida albicans and Penicillum
notatum as compared to ketoconazole.
Brain/blood partition coefficient (QPlogBB) parameter
indicated the ability of the drug to pass through the blood–
brain barrier which is mandatory for inhibition of HIV
infection. Whereas QPPMDCK predicted apparent MDCK
cell permeability in nm/s. MDCK cells are considered to be
a good mimic for the blood–brain barrier.
Compound B1 and B4 has shown excellent activity
against Bacillus substilis and S.typhi as compared to cip-
rofloxacin. Compound B2 showed better anti-bacterial
activity against Bacillus substilis, whereas B3 showed good
activity against S.typhi as compared to ciprofloxacin (Figs.
2 and 3).
Higher the value of MDCK cell, higher the cell per-
meability. All designed compounds had shown ADME
properties in acceptable range.
Conclusions
ADME properties
We have designed, synthesized, and evaluated 4-[1-(2,4-
dichlorobenzyl)-dimethoxy-1H-indol-3-yl]-2-hydroxy-
4-oxobut-2-enoic acid for reverse transcriptase inhibition
Total 44 descriptors and pharmaceutically relevant prop-
erties of benzylindol analogs were analyzed using Qikprop.
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4637
Table 3 Prediction of ADMET
properties of synthesized
compounds (A1–A4, B1–B4)
Compound
ID
Molecular
weight
LogP
(o/w)
LogS
LogBB
PMDCK
Violation
of rule of 5
% Oral
absorption
A-1
A-2
A-3
A-4
B-1
B-2
B-3
B-4
450.20
450.20
450.20
450.20
478.32
478.32
478.32
478.32
4.881
4.826
4.839
4.727
5.554
5.552
5.579
5.415
-5.79
-1.355
-1.496
-1.475
-1.571
-1.042
-1.126
-1.132
-1.139
143.841
114.201
113.699
97.473
0
0
0
0
1
1
1
1
88.194
85.893
86.184
84.219
100.00
96.511
96.772
95.466
-5.956
-5.896
-5.941
-6.642
-6.805
-6.851
-6.634
1,410.7
1,221.2
1,209.1
1,140.1
3-Acetyl-4,5-dimethoxy-1H-indole (D1)
activity. All the title compounds possess RT inhibitory
activity. The –OCH3 group of benzylindol ring helps to
form the hydrogen bonding with in hydrophobic pocket of
enzyme and thus increases the affinity of compounds
toward RT enzyme inhibition. It is further concluded that
compounds with benzylindol ring having ester group sub-
stitution on C3 position and combining hydrophobic group
like dichloro benzyl ring substitution on 1H-indol position
showed higher activity. This may form the possible but-
terfly-like conformation of 1,3 substitutions on benzylindol
ring required for RT inhibition. The presence of methoxy
group at fused benzene position may lead to change in the
conformation of butterfly structure.
White solid (166 mg, 76 %); IR (KBr) (m, cm-1) 3,260,
1,720, 1,593, 1,497, 1,315, 1,217, 1,153, 1,042, 820, 805.
3-Acetyl-4,6-dimethoxy-1H-indole (D2)
White solid (101 mg, 46 %); IR (KBr)) (m, cm-1) 3,265,
1,728, 1,595, 1,522, 1,347, 1,214, 1,178.
3-Acetyl-4,7-dimethoxy-1H-indole (D3)
IR (KBr) (m, cm-1) 3,250, 1,727, 1,591, 1,470, 1,150, 768.
3-Acetyl-5,6-dimethoxy-1H-indole (D4)
Materials and methods
White solid (176 mg, 81 %); IR (KBr) (m, cm-1) 3,300,
1,730, 1,630, 1,590, 1,110, 980.
Melting points were determined on Veego VMP-D digital
1
melting point apparatus and were uncorrected. H NMR
General procedure for the synthesis of 3-acetyl-
spectra were recorded on a FTNMR VARIAN MERCURY
YH-300 spectrometer (300 MHz) with TMS as internal
standard and DMSO as a solvent. Mass spectra were recor-
ded on time of flight mass spectrometer. FT-IR spectra were
recorded on JASCO FT-IR 4100 using KBr powder. The lab
system multiscan microplate reader was used to determine
absorbance of the sample to calculate the % inhibition.
dimethoxy-1-(2,4-dichlorobenzyl)-1H-indole
The appropriate 3-acetyl-dimethoxy-1H-indole derivative
(219 mg, 1 mmol) was dissolved in DMF (1 mL) at 0 °C and
dry sodium hydride (120 mg, 5 mmol) was added. The mixture
was stirred for 2 min. 2,4-Dichlorobenzyl chloride (293 mg,
1.5 mmol) was added drop wise, and the resulting solution was
placed in a cylindrical quartz tube (diam. 2 cm). The reaction
mixture was then stirred and irradiated in a microwave oven at
100 W and at continuous temperature (50 °C) for 5 min. A
saturated NaHCO3 solution was added. The reaction mixture
was extracted with ethyl acetate and dried over Na2SO4. After
removal of the solvent under reduced pressure, the residue was
powdered by treatment with diethyl ether and crystallized from
a mixture of diethyl ether and dichloromethane.
General procedure for the synthesis of 3-acetyl-
dimethoxy-1H-indole
Phosphoryl chloride (0.92 mL, 10 mmol) was added to ice
cold dimethylacetamide (2.79 mL, 30 mmol) with stirring
and cooling in ice. The suitable dimethoxyindole derivative
(177 mg, 1 mmol) was added and reaction mixture was
stirred at room temperature for 12 h, then poured over ice
and basified with a 4 N aqueous sodium hydroxide solu-
tion. The mixture was extracted with ethyl acetate and
dried over Na2SO4. After removal of the solvent under
reduced pressure, the residue was powdered by treatment
with diethyl ether and recrystallized from dichloromethane.
3-Acetyl-4,5-dimethoxy-1-(2,4-dichlorobenzyl)-1H-indole
(C1)
Oil (255 mg, 78 %); IR (KBr) (m, cm-1) 3,250, 1,760,
1,583, 1,494, 1,316, 1,267, 1,205, 1,148, 1,051, 825, 726.
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3-Acetyl-4,6-dimethoxy-1-(2,4-dichlorobenzyl)-1H-indole (C2)
Ethyl 4-[1-(2,4-dichlorobenzyl)-4,7-dimethoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (B3)
White solid (242 mg, 74 %): IR (KBr) (m, cm-1) 3,310,
1,790, 1,487, 1,320, 760, 614.
Yellow solid (408 mg, 94 %): IR (KBr) (m, cm-1) 3,240,
2,720, 1,760, 1,140, 930, 705; 1H NMR (CDCl3): d  1.22
(t, 3H, CH3), 3.73 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.09
(q, 2H, CH2), 5.32 (s, 2H, CH2), 6.23 (s, 1H, CH),
6.53–6.61 (m, 1H, ArH), 7.11–7.29 (m, 4H, ArH), 7.51 (d,
1H, ArH), 8.50 ppm (s, 1H, ArH); 13C NMR (DMSO-d6) d
ppm: 188 (C=O), 185 (C–OH), 170 (COOH), 140–128
(aromatic), 112 (ethylene), 56–54 (aliphatic, CH).
3-Acetyl-4,7-dimethoxy-1-(2,4-dichlorobenzyl)-1H-indole
(C3)
IR (KBr) (m, cm-1) 3,410, 3,318, 1,780, 1,294, 925, 710.
3-Acetyl-5,6-dimethoxy-1-(2,4-dichlorobenzyl)-1H-indole (C4)
White solid (219 mg, 67 %): IR (KBr) (m, cm-1) 3,380,
3,260, 1,690, 1,160, 839, 695.
Ethyl 4-[1-(2,4-dichlorobenzyl)-5,6-dimethoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (B4)
Yellow solid (384 mg, 90 %): IR (KBr) (m, cm-1) 3,120,
2,640, 1,760, 1,453, 920, 716; 1H NMR (CDCl3): d  1.32
(t, 3H, CH3), 3.82 (s, 6H, OCH3), 4.19 (q, 2H, CH2), 5.47
(s, 2H, CH2), 6.29 (s, 1H, CH), 7.15 (s, 1H, ArH),
7.20–7.26 (m, 2H, ArH), 7.39–7.43 (m, 2H, ArH),
7.95–7.98 ppm (m, 2H, ArH); 13C NMR (DMSO-d6) d
ppm: 189 (C=O), 185 (C–OH), 171 (COOH), 140–131
(aromatic), 109 (ethylene), 56–53 (aliphatic, CH).
General procedure for the synthesis of 4-[1-
(2,4-dichlorobenzyl)-dimethoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate
A mixture of suitable 3-acetyl-dimethoxy-1-(2,4-dichlorob-
enzyl)-1H-indole (378 mg, 1 mmol) diethyl oxalate (219 mg,
1.5 mmol) and a catalytic amount of NaOCH3 in anhydrous
THF (2 mL) was placed in a cylindrical quartz tube (diam.
2 cm), then stirred and irradiatedat continuoustemperature, in
a microwave oven for two subsequent periods in the same
conditions (250 W, 2 min, 50 °C). The solvent was evapo-
rated under reduced pressure; the collected solid was washed
with ether and crystallized from ethanol/diethyl ether.
General procedure for the synthesis of 4-[1-(2,4-
dichlorobenzyl)-dimethoxy-1H-indol-3-yl]-2-hydroxy-
4-oxobut-2-enoic acid
A methanol solution (5 mL) of derivatives (478 mg,
1 mmol) was treated with 2 N NaOH (5 mL, 50 mmol)
and stirred at room temperature for 1.5 h. Then the reaction
mixture was acidified with conc. HCl to afford a solid that
was collected and recrystallized from ethanol/diethyl ether.
Ethyl 4-[1-(2,4-dichlorobenzyl)-4,5-dimethoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (B1)
Yellow solid (410 mg, 96 %): IR (KBr) (m, cm-1) 3,410,
320, 1,698, 1,335, 735; 1H NMR (CDCl3): d  1.20 (t, 3H,
CH3), 3.75 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 4.09 (q, 2H,
CH2), 5.35 (s, 2H, CH2), 6.47 (s, 1H, CH), 6.94 (d, 1H,
ArH), 7.11e7.17 (m, 3H, ArH), 7.25–7.30 (m, 2H, ArH),
7.80 ppm (s, 1H, ArH); 13C NMR (DMSO-d6) d ppm: 190
(C=O), 187 (C–OH), 169 (COOH), 138–128 (Aromatic),
114 (ethylene), 56–54 (aliphatic, CH).
4-[1-(2,4-Dichlorobenzyl)-4,5-dimethoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (A1)
Yellow solid (199 mg, 50 %): IR (KBr) (m, cm-1) 3,360,
2,930, 1,760, 1,715, 920, 739, 695; 1H NMR (CDCl3): d 
3.76 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 5.46 (s, 2H, CH2),
7.08–7.19 (m, 3H, ArH–CH), 7.29–7.39 (m, 4H, ArH),
8.67 (s, 1H, ArH), 13.74 (bs, 1H, OH), 15.83 ppm (bs, 1H,
OH); 13C NMR (DMSO-d6) d ppm: 189 (C=O), 187
(C–OH), 165 (COOH), 148–138 (aromatic), 111 (ethyl-
ene), 60–53 (aliphatic, CH). MS: m/z 450.05 (M, 100),
249.05 (55).
Ethyl 4-[1-(2,4-dichlorobenzyl)-4,6-dimethoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (B2)
Yellow solid (419 mg, 98 %): IR (KBr) (m, cm-1) 3,230,
1,725, 1,495, 130, 710, 630; 1H NMR (CDCl3): d  1.22 (t,
3H, CH3), 3.73 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 4.09 (q,
2H, CH2), 5.32 (s, 2H, CH2), 6.23 (s, 1H, CH), 6.53-6.61
(m, 1H, ArH), 7.11–7.29 (m, 4H, ArH), 7.51 (d, 1H, ArH),
8.50 ppm (s, 1H, ArH); 13C NMR (DMSO-d6) d ppm: 187
(C=O), 184 (C–OH), 171 (COOH), 140–128 (aromatic),
110 (ethylene), 56–54 (aliphatic, CH).
4-[1-(2,4-Dichlorobenzyl)-4,6-dimethoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (A2)
Yellow solid (199 mg, 50 %): IR (KBr) (m, cm-1) 3,250,
2,830, 1,720, 1,453, 1,160, 710, 670; H NMR (CDCl3): d
1
 3.76 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 5.45 (s, 2H,
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Med Chem Res (2013) 22:4633–4640
4639
CH2), 6.41 (s,1H, CH), 6.76 (s,1H, ArH), 7.17–7.37 (m,
5H, ArH), 8.39 ppm (s, 1H, ArH); 13C NMR (DMSO-d6) d
ppm: 190 (C=O), 188 (C–OH), 167 (COOH), 138–128
(aromatic), 113 (ethylene), 56–53 (aliphatic, CH). MS:
m/z 450.07 (M, 100), 249.07 (55).
assay kit (Roche). The procedure for RT inhibition assay
was performed as described in the kit protocol. Briefly, the
reaction mixture consists of template primer complex,
dNTPs, and RT enzyme in the lysis buffer with or without
inhibitors. After 1 h incubation at 37 °C, the reaction
mixture was transferred to streptavidine-coated microtiter
plate (MTP). The biotin-labeled dNTPs that are incorpo-
rated in the template due to activity of RT were bound to
streptavidine. The unbound dNTPs were washed using
wash buffer, and anti-DIG-POD was added to the MTP.
The DIG-labeled dNTPs incorporated in the template were
bound to anti-DIG-POD Antibody. The unbound anti-DIG-
POD was washed and the peroxide substrate (ABST) was
added to the MTP. A colored reaction product was pro-
duced during the cleavage of the substrate catalyzed by a
peroxide enzyme. The absorbance of the sample was
determined as optical density (OD) at 405 nm using micro
titer plate ELISA reader. The % inhibition was calculated
using the following formula.
4-[1-(2,4-Dichlorobenzyl)-4,7-dimethoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (A3)
Yellow solid (82 %): IR (KBr) (m, cm-1) 3,310, 2,830,
1,720, 1,320, 910, 710; 1H NMR (CDCl3): d  3.76 (s, 3H,
OCH3), 3.98 (s, 3H, OCH3), 5.45 (s, 2H, CH2), 6.41 (s,1H,
CH), 6.76 (s,1H, ArH), 7.17–7.37 (m, 5H, ArH), 8.39 ppm
(s, 1H, ArH); 13C NMR (DMSO-d6) d ppm: 188 (C=O),
186 (C–OH), 164 (COOH), 138–128 (aromatic), 113
(ethylene), 56–53 (aliphatic, CH). MS: m/z 450.09 (M,
100), 249.09 (55).
4-[1-(2,4-Dichlorobenzyl)-5,6-dimethoxy-1H-indol-3-yl]-
ꢀ
ꢁ
2-hydroxy-4-oxobut-2-enoic acid (A4)
OD at 405 nm with inhibitor
% Inhibition ¼ 100 ꢁ
ꢂ 100
OD at 405 nm without inhibitor
Yellow solid (183 mg, 56 %): IR (KBr) (m, cm-1) 3,360,
2,810, 1,720, 1,490, 1,130, 740, 610; H NMR (CDCl3): d
1
 3.78 (s, 6H, OCH3), 5.46 (s, 2H, CH2), 6.93 (s, 1H, CH),
7.14–7.20 (m, 2H, ArH), 7.25 (s, 1H, ArH), 7.42–7.46 (m,
2H, ArH), 7.69 (s, 1H, ArH), 8.77 ppm (s, 1H, ArH); 13C
NMR (DMSO-d6) d ppm: 191 (C=O), 188 (C–OH), 169
(COOH), 138–128 (aromatic), 112 (ethylene), 56–53 (ali-
phatic, CH); MS: m/z 450.01 (M, 100), 249.01 (55).
Anti-microbial assay
The present study has focused on the determination of
minimum inhibitory concentration (MIC) of benzylindol
derivative series. MIC is important in diagnostic labora-
tories to confirm resistance of microorganisms to an anti-
microbial agent and also to monitor the activity of new
anti-microbial agents. An MIC is generally regarded as the
most basic laboratory measurement of the activity of an
anti-microbial agent against an organism.
In vitro anti-HIV activity
The standard nevirapine (1) and test compounds (A1–A4
and B1–B4) were powdered finely and suspended in
DMSO. Finally, 20 lg sample was used for in vitro assay.
The RT inhibition assay was performed by using an RT
MIC is the lowest concentration of an anti-microbial that
will inhibit the visible growth of a microorganism after
Fig. 2 MIC of compounds
against Candida albicans,
Aspergillus niger, Penicillium
notatum, and Aspergillus
fumigates
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4640
Med Chem Res (2013) 22:4633–4640
Fig. 3 MIC of compounds
against (A1–A4, B1–B4)
Bacillus subtilis and S. typhi
Daisley RW, Shah VK (1984) Synthesis and antibacterial activity of
some 5-nitro-2(3H)-one and their NMannich bases. J Pharm Sci
73:407–409
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viral research and therapy. Antivir Res 85:25–33
Fukuyama YI, Kodama C, Ochi M, Kataoka M, Shibata K (1998)
Antimicrobial indolequinones from the mid-intestinal gland of
the muricid gastropod Drupella fragum. Tetrahedron
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Henry L (1895) formation synthetique d’alcools niters. C.R. Hebd
Acad Sci 120:1265–1268
overnight incubation. MIC values can be determined by a
number of standard test procedures. The most commonly
employed methods are the tube dilution method and agar
dilution methods. Serial dilutions are made of the products
in bacterial growth media. The test organisms are then
added to the dilutions of the products, incubated, and
scored for growth. This procedure is a standard assay for
anti-microbial.
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imental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv
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discovery: where do we stand and where do we go? J Med Chem
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ADME prediction
ADME properties were calculated using Qikprop 2.5 tool
of Schrodinger. It predicts both physicochemically signif-
icant descriptors and pharmacokinetically relevant prop-
erties. QikProp provides ranges for comparing a particular
molecule’s properties with those of 95 % of known drugs.
Qikprop evaluates the acceptability of analogs based on
Lipinski’s rule of five (Lipinski et al., 1997), which is
essential to ensure drug-like pharmacokinetic profile while
using rational drug design. All the analogs were neutralized
before being used by Qikprop.
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coupling methods for the synthesis of haplophytine. Org Lett
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