Rhodium(II) acetate-catalyzed stereoselective synthesis, SAR and anti-HIV activity of novel oxindoles bearing cyclopropane ring

Article abstract of DOI:10.1016/j.ejmech.2011.01.037

Novel oxindole derivatives bearing substituted cyclopropane ring have been designed on the basis of docking studies with HIV-1 RT using the software DS 2.5 and synthesized as probable NNRTIs against HIV-1 using rhodium(II) acetate-catalyzed stereoselectiv

Full text of DOI:10.1016/j.ejmech.2011.01.037

European Journal of Medicinal Chemistry 46 (2011) 1181e1188
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Rhodium(II) acetate-catalyzed stereoselective synthesis, SAR and anti-HIV activity
of novel oxindoles bearing cyclopropane ringq
,
Garima Kumari a, Nutan b, Manoj Modi b, Satish K. Gupta b, Ramendra K. Singh a
*
a Nucleic Acids Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad 211002, India
b National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel oxindole derivatives bearing substituted cyclopropane ring have been designed on the basis of
docking studies with HIV-1 RT using the software DS 2.5 and synthesized as probable NNRTIs against
HIV-1 using rhodium(II) acetate-catalyzed stereoselective cyclopropanation reaction. The cyclopropane
isomer, having trans relationship with respect to carbonyl of lactam moiety and functional group on the
cyclopropane ring, was the major product in all cases along with a small amount of cis and methylene
products. The trans isomers interacted well with HIV-1 RT through H-bonding with amino acids, like
Lys101, Lys103, His235, Tyr318, constituting the non-nucleoside inhibitor binding pocket (NNIBP) during
docking experiments. However, the compounds showed very little activity when subjected to in vitro
Received 31 July 2010
Received in revised form
25 October 2010
Accepted 24 January 2011
Available online 31 January 2011
Keywords:
Oxindoles
Stereoselective synthesis
SAR
NNRTIs
Anti-HIV
anti-HIV-1 screening using b-galactosidase assay (TZM-bl cells) and GFP quantification (CEM-GFP cells).
The very low level of in vitro HIV inhibition, in comparison to predicted EC50 values on the basis of
computational studies, during CEM-GFP screening using AZT as positive control indicated that probably
the HIV RT is not the viral target and the molecules work through some different mechanism.
Ó 2011 Elsevier Masson SAS. All rights reserved.
MTT assay
1. Introduction
approved for clinical use [9,10] (Fig. 1). A combination of NRTIs and
NNRTIs with protease inhibitors, integrase inhibitors or fusion
HIV, the causative agent of AIDS, has been one of the major
challenges for almost three decades before the scientific commu-
nity all over the world. A large number of molecules are currently
being developed as antiviral agents targeting various viral enzymes
[1e7]. The enzyme HIV reverse transcriptase (HIV RT) is a vital
enzyme in the life cycle of HIV and is a prime target for developing
anti-HIV drug molecules.
The HIV RT inhibitors used currently are of two types: nucleos(t)
ide reverse transcriptase inhibitors (N(t)RTIs) and non-nucleoside
reverse transcriptase inhibitors (NNRTIs). The N(t)RTIs are
competitive inhibitors, interact at the dNTP binding site on HIV RT
and act as chain terminators, whereas the NNRTIs are non-
competitive inhibitors and bind at the allosteric site of HIV RT
located about 10 Å away from the dNTP binding site [8], change the
conformation of HIV RT and make it unsuitable for accepting
incoming dNTPs required for cDNA synthesis. Till date, four NNRTIs -
nevirapine, delavirdine, efavirenz and etravirine, have been
inhibitors has led to the development of Highly Active Antiretroviral
Therapy (HAART), the current way of treating AIDS patients.
Heterocyclic molecules consisting of cyclopropane functionality
have been reported to exhibit interesting biological properties
[11e19]. The approved NNRTIs against HIV, viz. nevirapine and
efavirenz, too possess cyclopropane moiety as structural unit.
Oxindoles have also been studied, in past, for their anti-HIV activity
[20,21]. On the basis of molecular modeling studies, we have
designed and synthesized some novel oxindole derivatives using
Rh2(OAc)4 as catalyst for stereocontrolled products. The oxindole
derivatives have been screened for their anti-HIV activity.
2. Chemistry
The cyclopropane derivatives of oxindoles were synthesized
starting from substituted isatin. Isatin was first reacted with tosyl
hydrazine to yield tosyl hydrazone, which was then treated with
NaOH to get the diazolactams [21]. The diazolactam was treated
with olefin(s) in the presence of Rh2(OAc)4 under N2 atmosphere to
yield cis and trans cyclopropane products along with a small
amount of olefin product when allyl alcohol was used, whereas
with allyl chloride no olefin side product was formed (Scheme 1).
q
Work presented in Nucleic Acids Symposium (Series No. 52) held in Japan.
* Corresponding author. Present address: INSA Exchange Fellow, Institute of
Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106,
Warszawa, Poland. Tel.: þ91 532 2461005; fax: þ91 532 2461005.
E-mail address: rksinghsrk@gmail.com (R.K. Singh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.037

1182
G. Kumari et al. / European Journal of Medicinal Chemistry 46 (2011) 1181e1188
Table 1
Yield of cis-trans isomers and their HPLC analysesa
Compound
R
R0
Yield (%)a
Retention time (min)
on RP-HPLC
3a e Trans
3b e Cis
4a e Trans
4b e Cis
5a e Trans
5b e Cis
6a e Trans
6b e Cis
7a e Trans
7b e Cis
8a e Trans
8b e Cis
9a e Trans
9b e Cis
eNO2
e
eCH2OH
e
45
15.5
16.1
16.0
16.5
14.8
15.5
14.2
14.8
13.6
14.3
13.9
14.3
14.8
15.7
13.7
14.4
9
66
8
72
11
49
6
53
4
51
7
eNO2
e
eCH2Cl
e
eOCF3
e
eCH2Cl
e
eOCF3
e
eCH2OH
e
eCH3
e
eCH2Cl
e
eCH3
e
eCH2OH
e
eH
e
eCH2Cl
e
60
9
57
5
10a e Trans
10b e Cis
eH
e
eCH2OH
e
a
Isolated by silica gel column chromatography using petroleum ether: ethyl
acetate (9:1) as eluent and analyzed by RP-HPLC (acetonitrile: H2O (70:30); flow
rate 1 mL/min).
Fig. 1. Approved NNRTIs as drug molecules against HIV-1.
nucleophilicity of carbonyl oxygen is also enhanced because of the
presence of nearby N in the lactam moiety. This stabilization can
only occur in the case of Tt leading to trans isomer and not in the
case of Tc leading to cis isomer. The present results thus derive
support from Doyle’s interpretation (Scheme 2).
Cyclopropanation reactions of diazolactams using rhodium(II)
acetate as catalyst are not so common like that of diazoacetates and
diazoketones. The products 3e10 were separated by silica gel
column chromatography using petroleum ether and ethyl acetate
(9:1) as eluent. The products 3ae10a were separated and further
analyzed using RP-HPLC (acetonitrile-H2O (70:30, v/v) as eluent)
and characterized using proton NMR spectroscopy.
3. Results and discussion
The trans isomer was formed as the major product in all cases
along with a small amount of the cis and alkene products (Table 1).
The formation of trans isomer as the major product was confirmed
by proton NMR analysis on the basis of higher coupling constant (J)
values. The formation of trans stereoisomer as a major product can
be explained using mechanistic model as proposed by Doyle
[22e24], which suggests that stereoisomeric pi-complexes are
reversibly formed. The R0 group of the alkene favoring an orienta-
tion away from the bulky metal, leads to transition states Tt and Tc.
Only the transition state leading to trans isomer is stabilized
because of interaction between developing electrophilic character
on carbon of alkene and nucleophilic carbonyl oxygen. The
3.1. Molecular modeling and biological evaluation
All molecules designed obeyed the Lipinski’s Rule of Five (Table 2).
The molecules were docked into the allosteric site of HIV-1 RT to
study molecular interactions. Docking studies revealed that when
eCH2OH was present as a substituent on the cyclopropane ring, one
H-bond was formed between eOH of the compound and Lys103 as
in the case of compound 3a, whereas in the case of compound 6a,
the H-bond was formed with Lys101. However, in the case of
compounds 8a and 10a, no H-bond was formed. In the case of 9a,
one H-bond was formed with the NH of the lactam moiety and
Lys101 of HIV RT. Both the amino acids, viz. Lys101 and Lys103, lie in
N2
NNHSO2C7H7
R
O
R
R
O
NaOH
(0.1 N)
TsNHNH2,
MeOH,rt
O
N
H
O
N
H
N
H
1a R= NO2
1b R= OCF3
1c R= CH3
1d R= H
2a R=NO2
2b R= OCF3
2c R= CH3
2d R= H
R= NO2, OCF3
CH3, H
CH2=CH-R', Rh2(OAc)4,
CH2Cl2
H
R'
H
H
H
H
H
R'
R
R
R
R'
O
+
+
O
O
N
H
NH
N
H
3a-10a
trans
3b-10b
cis
Scheme 1. Synthesis of oxindole derivatives bearing cyclopropane ring using Rh2(OAc)4 as stereoselective catalyst.

G. Kumari et al. / European Journal of Medicinal Chemistry 46 (2011) 1181e1188
1183
Scheme 2. Mechanism of cyclopropanation showing trans isomer as major product.
the non-nucleoside inhibitor binding pocket (NNIBP) of HIV-1 RT
and hence interactions with these amino acids are very crucial.
However, when eCH2Cl, a more hydrophobic group, was present as
a substituent on the cyclopropane ring, no H-bond was formed
between the eCl and amino acid residues of the NNIBP (Fig. 2).
In the case of compounds 3a and 7a, H-bonds were formed
between C]O of the oxindole moiety and His235 and Tyr318 of the
NNIBP. No such bonds were formed with 4a, 5a and 6a.
To further explore the SAR, the role of substituents at 5-position
of benzene ring of oxindole nucleus was analyzed. Four types of
derivatives, bearing a nitro group (3a and 4a), a trifluoromethoxy
group (5a and 6a), a methyl group (7a and 8a) and hydrogen (9a
and 10a) were studied. The nitro group in compound 3a formed
a H-bond with Lys101 in the NNIBP. In the case of compound 4a, the
nitro substituent alone formed seven H-bonds with Lys101 and
Tyr181 in the NNIBP. When the nitro group was replaced by tri-
fluoromethoxy group, methyl or hydrogen, no H-bond was formed
between these groups and amino acid residues in the NNIBP, as in
the case of compounds 5ae10a. But the predicted EC50 values of the
substituents having trifluoromethoxy group (5a and 6a) were much
higher than their counterparts. This may be because of better
orientation of these derivatives in the NNIBP. The predicted EC50
a computational assessment of level of interaction between the
molecules and HIV RT, are presented in Table 3, where the EC50
value for compound 6a was the lowest one.
Further, during docking studies, the lowering of docking energy
reflected the stability of HIV RT-ligand complexes. The results
indicated that most of the ligands moved into a stable position
comparable to nevirapine and efavirenz. However, compound 6a
formed most stable complex with lowest docking energy
(ꢀ50.81 kcal/mol) as shown in Table 4.
Analysis of the anti-HIV screening results (Table 5) confirmed
the computational data. All compounds have shown only slight
inhibition of HIV growth under in vitro condition using TZM-bl and
CEM-GFP cell lines. The low level of anti-HIV activity shown by the
molecules specially during CEM-GFP screening using AZT as posi-
tive control, in contrast to the predicted EC50 values on the basis of
docking studies with HIV RT, indicated towards the possibility of
HIV RT being not the viral target and the molecules adopt some
other mechanism to show anti-HIV activity. This observation is also
supported by the difference shown in the predicted and experi-
mental EC50 values in Table 3 and Table 5, respectively. However,
the compound 6a showed some better result. Because of very low
HIV-1 inhibition during preliminary investigations at two different
values (
mg/mL) of all derivatives on the basis of their Ludi_3 score,
concentrations, i.e., 2 and 20 mg/mL, no further experiments were
performed for calculation of EC50 and CC50 values.
In silico studies revealed that none of the molecules had orien-
tation similar to efavirenz. In the case of efavirenz, three H-bonds
were formed, two between NH and CO moieties with Lys101 and
one with CO moiety and Lys103 (Fig. 3). Thus, the lactam moiety is
crucial for interactions with HIV RT. However, in the present case,
only 3a, 8a and 9a formed H-bonds through their CO moiety with
His235 and Tyr318. Further, at C-5 position, in the case of efavirenz,
Cl is present, whereas in the case of 3ae10a, NO2, OCF3, CH3 and H
are present. Thus, it may be assumed that at 5-position on benzene
ring smaller electronegative groups are preferred for better orien-
tation of the molecules in the hydrophobic pocket.
Table 2
Oxindole derivatives 3ae10a in the eyes of Lipinski’s Rule.
Compound Formula Mol. Wt. H-acceptors H-donors logP TPSA
3
4
5
6
7
8
9
10
C11H10N2O4
C11H9N2O3
C12H9ClF3NO2 234.21
C12H10F3NO3
C12H12ClNO
C12H13NO2
234.21
252.65
6
5
3
4
2
3
2
3
2
1
1
2
1
2
1
2
0.54 95.15
1.84 74.92
3.48 38.33
1.89 58.56
2.44 29.10
1.21 40.33
2.19 29.10
1.95 49.35
273.21
203.24
221.68
189.08
207.66
C11H11NO2
C11H10ClNO

1184
G. Kumari et al. / European Journal of Medicinal Chemistry 46 (2011) 1181e1188
Table 3
Ludi_3 score and predicted EC50 of 3ae10a along with number of H-bonds formed
with NNIBP.
Compound R, R0
Ludi_3 Scorea Predicted EC50 No. of H-bonds
value (
mg/mL)
formed
3a
4a
5a
6a
7a
8a
9a
10a
NO2, CH2OH
NO2, CH2Cl
OCF3, CH2Cl
488
420
515
3.0
15.9
1.6
1.4
3.1
14.4
5.3
13.7
4
7
0
1
1
0
1
0
OCF3, CH2OH 530
CH3, CH2Cl
CH3, CH2OH
H, CH2OH
H, CH2Cl
483
418
453
416
a
Higher score predicts better interaction and thus better EC50 value.
5. Experimental
5.1. Chemistry
Silica gel G for TLC was obtained from E. Merck India Ltd. All
reactions were performed in oven-dried flasks and solvents used
were anhydrous. Melting points determined using an electro-
thermal apparatus are uncorrected. HPLC analysis was carried out
on 6 AD Binary Gradient Shimadzu HPLC systems. Mass spectra
were recorded on Micromass Quattro II. Fluorescence detection was
done using FLUOstar Optima, BMG Labtech, Germany. 1H NMR
spectra were recorded on a Bruker AVANCE 400 MHz Fourier
transform spectrometer and using an internal deuterium lock.
Chemical shifts are quoted in parts per million downfield from
tetramethylsilane.
5.1.1. General procedure for synthesis of compounds 3ae10a
Synthesis of desired oxindole derivatives through cyclo-
propanation of diazolactams started from the corresponding isatin,
which was reacted with tosyl hydrazine (1 equiv) in dry methanol.
The reaction mixture was allowed to stand overnight, 1(a, b)
obtained as a precipitate was filtered and washed with cold meth-
anol and dried. 1(a, b) was then treated with 0.1 N NaOH (1 equiv)
and the mixture was allowed to stand at room temperature for 24 h.
The diazoketone 2(a, b) obtained as a precipitate was then filtered
and air-dried. For cyclopropanation reaction, olefin (allyl chloride or
allyl alcohol) (10 equiv) in dry dichloromethane and Rh2(OAc)4
(0.001 equiv) were taken in a nitrogen-flushed round bottomed
flask, and to this, 2(a, b) dissolved in dry dichloromethane was
added slowly over 1 h with continuous stirring under N2 atmo-
sphere. Completion of reaction was confirmed by TLC, which
showed disappearance of 2. The reaction mixture was then filtered,
concentrated and the products 3e10 were isolated from the reac-
tion mixture by silica gel column chromatography using petroleum
ether and ethyl acetate (9:1, v/v) as eluent. The separation of the
Fig. 2. Binding of 3ae10a into allosteric site of wild-type HIV-1 RT (only main
surrounding residues are shown in green). (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
Table 4
4. Conclusion
Discovery Studio 2.5 docking results showing the stability of HIV-1 RT e ligand
complexes.
In summary, we have carried out Rh2(OAc)4 catalyzed stereo-
selective synthesis of novel oxindoles with cyclopropane moiety as
probable NNRTIs against HIV-1 on the basis of molecular modeling
studies using the software Discovery Studio 2.5. The compounds
showed very low anti-HIV activity under in vitro conditions using
TZM-bl and CEM-GFP cell lines. The compound 6a expressed a little
better result. It was assumed that although the compounds showed
some anti-HIV activity, HIV RT was not the target of the molecules
under study on the basis of in vitro experimental and computa-
tional results. All molecules, however, showed almost nil or very
little cytotoxicity, which indicated towards the possibility of
exploring the potential of oxindoles as anti-HIV molecules.
Ligand
Docked energy (kcal/mol)
Van der Waals
Electrostatic
Total
3a
4a
5a
6a
7a
8a
9a
10a
ꢀ33.37
ꢀ34.34
ꢀ36.71
ꢀ35.18
ꢀ37.67
ꢀ31.92
ꢀ29.21
ꢀ30.67
ꢀ38.53
ꢀ39.84
ꢀ14.05
ꢀ14.76
ꢀ5.98
ꢀ15.63
ꢀ4.49
ꢀ7.01
ꢀ4.68
ꢀ2.41
ꢀ7.46
ꢀ4.14
ꢀ47.42
ꢀ49.10
ꢀ42.69
ꢀ50.81
ꢀ42.16
ꢀ38.93
ꢀ33.89
ꢀ33.08
ꢀ45.99
ꢀ43.98
Nevirapine
Efavirenz

G. Kumari et al. / European Journal of Medicinal Chemistry 46 (2011) 1181e1188
1185
Table 5
Cytotoxicity and anti-HIV Activity of compounds 3ae10a.
Compound
Cell viability (%) by MTT assay
Using TZM-bl cells
Anti-HIV activity
Using CEM-GFP cells
10 20
Using TZM-bl cells
Using CEM-GFP cells
% Inhibition at 20
10
m
g
20
m
g
50
m
g
100
m
g
mg
m
g
50
mg
100
m
g
% Inhibition at 2
m
g
mg
(b
- galactosidase assay)
(GFP quantification)
3a
4a
5a
6a
7a
8a
9a
10a
100
102
91
90
93
102
99
93
107
102
95
93
91
100
98
92
104
101
66
62
88
95
92
87
109
92
60
60
87
93
89
86
119
101
94
91
92
105
96
91
127
118
74
72
89
101
95
87
122
103
37
33
85
100
92
83
116
76
02
03
81
100
90
88
5
6
9
10
8
6
03
04
03
07
05
03
06
02
4
7
isomers 3ae10a was done using C18 column (Phenomenex Luna
C18(2), 250 ꢁ 4.6 mm2, flow rate of 1 mL/min) on RP-HPLC
(acetonitrile - H2O (70:30, v/v) as eluent) and the isomers were
characterized using proton NMR spectroscopy.
CONH), 7.45e6.62 (m, 3H, Ar-H), 3.32 (d, 2H, CH2),1.52e1.32 (m,1H,
CH), 1.23 (d of d, J ¼ 6.8, 5.7 Hz, 1H), 1.03 (d of d, J ¼ 9.8, 5.8 Hz, 1H);
MS (EIþ, ): m/z (%) 291.03 (100); Anal. Calcd for C12H9ClF3NO2: C,
49.42; H, 3.11; Cl, 12.16; F, 19.54; N, 4.80 Found: C, 49.31; H, 3.21; Cl,
12.09; F, 19.63; N, 4.84.
5.1.1.1. (1R, 2R)-2-(Hydroxymethyl)-50-nitrospiro[cyclopropane-1,30-
indol]-20(10H)-one (3a). Mp 140e142 ꢂC; Rf ¼ 0.43 (DCM-MeOH,
5.1.1.7. (1R,
2R)-2-(Hydroxymethyl)-50-(trifluoromethoxy)spiro
9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
d
¼ 8.34e7.78 (m, 3H, Ar-
[cyclopropane-1,30-indol]-20(10H)-one (6a). Mp 121e122 ꢂC;
Rf ¼ 0.41 (DCM-MeOH, 9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
H), 8.02 (s, 1H, CONH), 3.53 (d, 2H, CH2), 1.76e1.52 (m, 1H, CH), 1.27
(d of d, J ¼ 10.1, 5.7 Hz, 1H), 0.9 (d of d, J ¼ 7.1, 5.7 Hz, 1H); MS (EIþ,
70 eV): m/z (%) 234.06 (100); Anal. Calcd for C11H10N2O4: C, 56.41;
H, 4.30; N, 11.96. Found: C, 56.32; H, 4.39; N, 11.84.
d
¼ 8.2 (s, 1H, CONH), 7.75e6.96 (m, 3H, Ar-H), 3.53 (d, 2H, CH2),
1.78e1.54 (m, 1H, CH), 1.28 (d of d, J ¼ 10.1, 5.7 Hz, 1H), 0.93 (d of d,
J ¼ 7.3, 5.7 Hz, 1H); MS (EIþ, 70 eV): m/z (%) 273.06 (100); Anal.
Calcd for C12H10F3NO3: C, 52.75; H, 3.69; F, 20.86; N, 5.13 Found: C,
52.24; H, 3.69; F, 20.98; N, 5.28.
5.1.1.2. (1R, 2S)-2-(Hydroxymethyl)-50-nitrospiro [cyclopropane-1,30-
indol]-20(10H)-one (3b). Mp 141e143 ꢂC; Rf ¼ 0.41 (DCM-MeOH,
9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
d
¼ 8.04e7.82 (m, 3H, Ar-
5.1.1.8. (1R,
2S)-2-(Hydroxymethyl)-50-(trifluoromethoxy)spiro
H), 7.92 (s, 1H, CONH), 3.49 (d, 2H, CH2), 1.55e1.35 (m, 1H, CH), 1.25
(d of d, J ¼ 6.7, 5.8 Hz, 1H), 1.00 (d of d, J ¼ 9.5, 5.8 Hz, 1H); MS (EIþ,
70 eV): m/z (%) 234.06 (100); Anal. Calcd for C11H10N2O4: C, 56.41;
H, 4.30; N, 11.96 Found: C, 56.32; H, 4.39; N, 11.84.
[cyclopropane-1,30-indol]-20(10H)-one (6b). Mp 121e123 ꢂC;
Rf ¼ 0.40 (DCM-MeOH, 9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
d
¼ 8.0 (s, 1H, -CONH), 7.47e6.67 (m, 3H, Ar-H), 3.47 (d, 2H, CH2),
1.55e1.35 (m, 1H, CH), 1.25 (d of d, J ¼ 6.7, 5.8 Hz, 1H), 1.00 (d of d,
J ¼ 9.5, 5.8 Hz, 1H); MS (EIþ, 70 eV): m/z (%) ¼ 273.06 (100); Anal.
Calcd for C12H10F3NO3: C, 52.75; H, 3.69; F, 20.86; N, 5.13 Found: C,
52.24; H, 3.69; F, 20.98; N, 5.28.
5.1.1.3. (1R, 2R)-2-(Chloromethyl)-50-nitrospiro[cyclopropane-1,30-
indol]-20(10H)-one (4a). Mp 149 ꢂC; Rf ¼ 0.47 (DCM-MeOH, 9.8:0.2);
1H NMR (400 MHz, DMSO-d6):
d
¼ 8.32e7.89 (m, 3H, Ar-H), 8.02 (s,
1H, CONH), 3.39 (d, 2H, CH2), 1.67e1.42 (m, 1H, CH), 1.26 (d of d,
J ¼ 10.2, 5.9 Hz,1H), 0.92 (d of d, J ¼ 7.3, 5.9 Hz, 1H); MS (EIþ, 70 eV):
m/z (%) m/z 252.03 (100); Anal. Calcd for C11H9ClN2O3: C, 52.29; H,
3.59; Cl, 14.03; N, 11.09. Found: C, 52.21; H, 3.65; Cl, 14.05; N, 11.02.
5.1.1.9. (1R, 2R)-2-(Chloromethyl)-50-methylspiro[cyclopropane-1,30-
indol]-20(10H)-one (7a). Mp 210e212 ꢂC; Rf ¼ 0.39 (DCM-MeOH,
5.1.1.4. (1R, 2S)-2-(Chloromethyl)-50-nitrospiro[cyclopropane-1,30-
indol]-20(10H)-one (4b). Mp 148e150 ꢂC; Rf ¼ 0.45 (DCM-MeOH,
9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
d
¼ 8.12e7.94 (m, 3H, Ar-H),
7.87 (s,1H, CONH), 3.34 (d, 2H, CH2),1.53e1.31 (m,1H, CH),1.24 (d of
d, J ¼ 6.8, 5.7 Hz,1H),1.02 (d of d, J ¼ 9.8, 5.8 Hz,1H); MS (EIþ, 70 eV):
m/z (%) 252.03 (100); Anal. Calcd for C11H9ClN2O3: C, 52.29; H, 3.59;
Cl, 14.03; N, 11.09. Found: C, 52.21; H, 3.65; Cl, 14.05; N, 11.02.
5.1.1.5. (1R, 2R)-2-(Chloromethyl)-50-(trifluoromethoxy)spiro[cyclo-
propane-1,30-indol]-20(10H)-one (5a). Mp 150e153 ꢂC; Rf ¼ 0.41
(DCM-MeOH, 9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
d
¼ 8.1(s, 1H,
CONH), 7.65e6.82 (m, 3H, Ar-H), 3.36 (d, 2H, CH2),1.76e1.52 (m,1H,
CH), 1.27 (d of d, J ¼ 10.1, 5.7 Hz,1H), 0.94 (d of d, J ¼ 7.2, 5.6 Hz,1H);
MS (EIþ, 70 eV): m/z (%) 291.03 (100); Anal. Calcd for C12H9ClF3NO2:
C, 49.42; H, 3.11; Cl, 12.16; F, 19.54; N, 4.80 Found: C, 49.31; H, 3.21;
Cl, 12.09; F, 19.63; N, 4.84.
5.1.1.6. (1R, 2S)-2-(Chloromethyl)-50-(trifluoromethoxy)spiro[cyclo-
propane-1,30-indol]-20(10H)-one (5b). Mp 152e153 ꢂC; Rf ¼ 0.38
(DCM-MeOH, 9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
d
¼ 7.9 (s, 1H,
Fig. 3. Binding of efavirenz into allosteric site of wild-type HIV-1 RT.

1186
G. Kumari et al. / European Journal of Medicinal Chemistry 46 (2011) 1181e1188
9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
d
¼ 8.12 (s, 1H, eCONH),
5.2. Biological evaluation
7.64e6.8 (m, 3H, Ar-H), 3.36 (d, 2H, eCH2), 2.33(s, 3H, CH3),
1.74e1.54 (m, 1H, eCH), 1.27 (d of d, J ¼ 10.1, 5.7 Hz,1H), 0.96 (d of d,
J ¼ 7.2, 5.6 Hz, 1H); MS (EIþ, 70 eV): m/z (%) ¼ 221.06 (100); Anal.
Calcd for C12H12ClNO: C, 65.02; H, 5.46; Cl, 15.99; N, 6.32 Found: C,
64.99; H, 5.43; Cl, 15.95; N, 6.30.
5.2.1. Anti-HIV screening using TZM-bl cell lines
The molecules were tested for their anti-HIV activity employing
TZM-bl cell lines. TZM-bl cells (2.0 ꢁ 105) were seeded in 12-well
plates and cultured overnight at 37 ꢂC in humidified atmosphere of
5% CO2. Infective HIV-1 virus isolate at a multiplicity of infection
(MOI) of 0.025 was treated at 37 ꢂC for 1 h with respective
5.1.1.10. (1R,
2S)-2-(Chloromethyl)-50-methylspiro[cyclopropane-
1,30-indol]-20(1’H)-one (7b). Mp 208e210 ꢂC; Rf ¼ 0.37(DCM-MeOH,
compounds in a total reaction volume of 100 mL. After incubation,
9.8:0.2); 1H NMR (44 MHz, DMSO-d6):
d
¼ 7.98 (s, 1H, -CONH),
the virus with various treatments was added to TZM-bl cells
growing in 12-well plates and was further incubated at 37 ꢂC for 4 h
in the humidified atmosphere of 5% CO2. After 4 h, medium was
removed, cells washed twice with serum free DMEM medium and
fresh growth medium (DMEM þ 10% FCS) was added. Various
compounds were added again in the respective wells and plates
were further incubated at 37 ꢂC for 48 h. After incubation, the cells
were washed twice with phosphate buffer saline and fixed in 1%
glutaraldehyde for 10 min. The cells were stained with 1 mL of
staining solution at 37 ꢂC for 24 h. The blue stained cells were
counted under the microscope. Inhibition was calculated by taking
the number of blue foci in experimental group divided by number
of blue foci in infected cells in absence of any test extract/AZT
multiplied by hundred. Percent inhibition was calculated by sub-
tracting above value from hundred.
7.44e6.68 (m, 3H, Ar-H), 3.32 (d, 2H, eCH2), 2.42(s, 3H, CH3),
1.54e1.34 (m, 1H, eCH), 1.24 (d of d, J ¼ 6.8, 5.7 Hz, 1H), 1.11 (d of d,
J ¼ 9.8, 5.8 Hz, 1H); MS (EIþ, 70 eV): m/z (%) ¼ 221.06 (100); Anal.
Calcd for C12H12ClNO: C, 65.02; H, 5.46; Cl, 15.99; N, 6.32 Found: C,
64.99; H, 5.43; Cl, 15.95; N, 6.30.
5.1.1.11. (1R, 2R)-2-(Hydroxymethyl)-50-methylspiro[cyclopropane-
1,30-indol]-20(10H)-one (8a). Mp 220e223 ꢂC; Rf ¼ 0.42(DCM-
MeOH, 9.8:0.2); 1H NMR (400 MHz, DMSO-d6):
d
¼ 8.16 (s, 1H,
eCONH), 7.82e6.94 (m, 3H, Ar-H), 3.56 (d, 2H, eCH2), 2.48 (s, 3H,
CH3),1.76e1.54 (m,1H, -CH),1.29 (d of d, J ¼ 10.1, 5.7 Hz,1H), 0.94 (d
of d, J ¼ 7.3, 5.7 Hz, 1H); MS (EIþ, 70 eV): m/z (%) ¼ 203.09 (100);
Anal. Calcd for C12H13NO2: C, 70.92; H, 6.45; N, 6.89 Found: C, 70.91;
H, 6.42; N, 6.86.
5.1.1.12. (1R, 2S)-2-(Hydroxymethyl)-50-methylspiro[cyclopropane-
5.2.2. Anti-HIV screening using CEM-GFP cell lines
1,30-indol]-20(10H)-one (8b). Mp 221e222 ꢂC; Rf ¼ 0.39(DCM-
CEM-GFP (5 ꢁ 106) cells were taken and washed in polybrene
MeOH, 9.8:0.2); 1H NMR (44 MHz, DMSO-d6):
d
¼ 8.0 (s, 1H,
(2 mg/mL) containing RPMI-1640 medium. Cells were resuspended
in 0.5 mL of RPMI-1640 medium containing 1 mg/mL polybrene and
eCONH), 7.49e6.68 (m, 3H, Ar-H), 3.48 (d, 2H, eCH2), 2.44 (s, 3H,
CH3), 1.54e1.36 (m, 1H, eCH), 1.26 (d of d, J ¼ 6.7, 5.8 Hz, 1H), 1.02 (d
of d, J ¼ 9.5, 5.8 Hz, 1H); MS (EIþ, 70 eV): m/z (%) ¼ 203.09 (100);
Anal. Calcd for C12H13NO2: C, 70.92; H, 6.45; N, 6.89 Found: C, 70.91;
H, 6.42; N, 6.86.
were infected with HIV-1 NL4.3 virus isolate at a multiplicity of
infection (MOI) of 0.05 for 4 h with intermittent mixing. After
infection, cells were washed twice with serum free RPMI-1640
media and were resuspended in 25 mL of complete medium.
Aliquots of 2.0 ꢁ 105 cells/mL were then seeded in 24 well culture
plates. Compounds were added to the respective wells in duplicate.
5.1.1.13. (1R, 2R)-2-(Hydroxymethyl)spiro[cyclopropane-1,30-indol]-
20(1’H)-one (9a). Mp 200e202 ꢂC; Rf ¼ 0.38(DCM-MeOH, 9.8:0.2);
Zidovudine (10 mM, AZT, a known antiretroviral drug and reverse
1H NMR (400 MHz, DMSO-d6):
d
¼ 8.14 (s, 1H, eCONH), 7.92e7.22
transcriptase inhibitor) was used as a positive reference control and
50% ethanol and distilled water were used as a vehicle negative
control. Plate was kept for incubation at 37 ꢂC in humidified
atmosphere of 5% CO2 for 5 days. On the 5th day 0.4 mL of cell
suspension was removed from each well and 1 mL of fresh
complete media was added. Test compounds and controls were
added in their respective wells and plate was further incubated at
37 ꢂC in humidified atmosphere of 5% CO2. On 8th day, cells were
(m, 4H, Ar-H), 3.39 (d, 2H, eCH2), 1.67e1.42 (m, 1H, eCH), 1.26 (d of
d, J ¼ 10.2, 5.9 Hz, 1H), 0.92 (d of d, J ¼ 7.3, 5.9 Hz, 1H); MS (EIþ,
70 eV): m/z (%) ¼ 189.08 (100); Anal. Calcd for C11H11NO2: C, 69.83;
H, 5.86; N, 7.40 Found: C, 69.81; H, 5.82; N, 7.34.
5.1.1.14. (1R, 2S)-2-(Hydroxymethyl)spiro[cyclopropane-1,30-indol]-
20(10H)-one (9b). Mp 199e201 ꢂC; Rf ¼ 0.37(DCM-MeOH, 9.8:0.2);
1H NMR (44 MHz, DMSO-d6):
d
¼ 7.98 (s, 1H, eCONH), 7.82e7.04
lysed with 150 mL of Promega cell culture lysis buffer and the lysate
(m, 4H, Ar-H), 3.34 (d, 2H, eCH2), 1.53e1.31 (m, 1H, eCH), 1.24 (d of
d, J ¼ 6.8, 5.7 Hz, 1H), 1.02 (d of d, 9.8, 5.8 Hz, 1H); MS (EIþ, 70 eV):
m/z (%) ¼ 189.08 (100); Anal. Calcd for C11H11NO2: C, 69.83; H, 5.86;
N, 7.40 Found: C, 69.81; H, 5.82; N, 7.34.
was centrifuged at 14,000 g for 10 min at 4 ꢂC. The supernatant was
transferred to black optiplate and absorbance read at an excitation
wavelength of 485 nm and emission at 520 nm using fluorimeter.
The results are expressed in percentage inhibition, calculated by
taking the GFP fluorescence in experimental group (i.e., in the
presence of test extract/AZT) divided by GFP fluorescence in
infected cells in the absence of test extract/AZT multiplied by
hundred. Percent inhibition was obtained by subtracting the above
value from hundred.
5.1.1.15. (1R, 2R)-2-(Chloromethyl)spiro[cyclopropane-1,30-indol]-20
(10H)-one (10a). Mp 189e190 ꢂC; Rf ¼ 0.43(DCM-MeOH, 9.8:0.2);
1H NMR (400 MHz, DMSO-d6):
d
¼ 8.12 (s, 1H, eCONH), 7.88e7.16
(m, 4H, Ar-H), 3.53 (d, 2H, eCH2), 1.76e1.52 (m, 1H, eCH), 1.27 (d of
d, J ¼ 10.1, 5.7 Hz, 1H), 0.9(d of d, J ¼ 7.1, 5.7 Hz,1H); MS (EIþ, 70 eV):
m/z (%) ¼ 207.05 (100); Anal. Calcd for C, 63.62; H, 4.85; Cl, 17.07; N,
6.75 Found: C, 63.56; H, 4.82; Cl, 17.06; N, 6.7.
5.2.3. MTT cell cytotoxicity assay
Cytotoxicity of compounds was assayed using MTT cell cyto-
toxicity assay employing TZM-bl cells. The MTT assay is based on
the reduction of the yellow colored 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehy-
drogenases of metabolically active cells (live cells) to a blue colored
formazan, which can be measured spectroscopically. Briefly, 3 ꢁ 103
TZM-bl cells were seeded in a 96 well plate in the absence or
presence of various concentrations of test compounds and incu-
bated at 37 ꢂC in a humidified atmosphere of 5% CO2 for 48 h. After
5.1.1.16. (1R, 2S)-2-(Chloromethyl)spiro[cyclopropane-1,30-indol]-20
(10H)-one (10b). Mp 188e190 ꢂC; Rf ¼ 0.42 (DCM-MeOH, 9.8:0.2);
1H NMR (44 MHz, DMSO-d6):
d
¼ 8.02 (s, 1H, eCONH), 8.78e7.02
(m, 4H, Ar-H), 3.49 (d, 2H, eCH2), 1.55e1.35 (m, 1H, eCH), 1.25 (d of
d, J ¼ 6.7, 5.8 Hz, 1H), 1.00 (d of d, J ¼ 9.5, 5.8 Hz, 1H); MS (EIþ,
70 eV): m/z (%) ¼ 207.05 (100); Anal. Calcd for C, 63.62; H, 4.85; Cl,
17.07; N, 6.75 Found: C, 63.56; H, 4.82; Cl, 17.06; N, 6.7.

G. Kumari et al. / European Journal of Medicinal Chemistry 46 (2011) 1181e1188
1187
incubation, 20
added to each well and incubated at 37 ꢂC for 3 h. After the incu-
bation, 100 L of MTT solvent (20% SDS and 50% dimethyl form-
m
L of MTT reagent at a concentration of 5 mg/mL was
the Input Site Sphere were free to move. A Smart Minimizer algo-
rithm with 1000 steps and r.m.s. gradient of 0.5 kcal/mol/Å was
used for ligand minimization.
m
amide in 1 ꢁ PBS) was added in each well. The absorbance (OD) was
read at 570 nm with reference filter at 690 nm (690 nm reading was
used as blank). The % cell viability was calculated from the equation,
The final step in docking was the scoring of the refined docked
poses. This was done using the Score Ligand Poses protocol of DS
2.5. The Ludi Energy Estimate [29e31] was used for the refined
poses. The ligand pose corresponding to the highest Ludi score [32]
was taken as the best-docked pose. Visualization was done using
PyMol software.
% Viability ¼ {[(OD drug treated cultures) ꢀ (OD untreated virus
control cultures)] / [(OD uninfected cultures) ꢀ (OD untreated virus
control cultures)]} ꢁ 100%
5.3.4. Validation of the docking methodology
5.3. Molecular modeling
The docking methodology was tested on nevirapine, a well-
known marketed NNRTI, which was docked into the allosteric site
of HIV-1 RT after extracting the ligand from the crystal structure.
The Ludi_3 score was found to be 718 and the corresponding Kd
The computational studies were carried out with the X-ray
crystal structure of HIV RT complexed with a ligand (PDB: ID 3E01).
All computational studies were done using Discovery Studio 2.5
(Accelrys Ltd., UK through Apsara Innovations Pvt Ltd, Bangalore,
India).
value was 0.06
value of 0.05
m
M.
M. This is in good agreement with the observed Kd
m
5.3.5. Calculation of H-acceptors, H-donors, TPSA and logP values
H-acceptors, H-donors and TPSA were calculated using Molins-
piration software and logP values using ChemDraw software.
5.3.1. Receptor setup
The target protein (PDB: ID 3E01) was taken, the ligand was
extracted, hydrogens were added and their positions were opti-
mized using the all-atom CHARMm forcefield [25,26] and the
Adopted Basis set Newton Raphson (ABNR) method available in DS
2.5 protocol until the root mean square (rms) gradient was less than
0.05 kcal/mol/Å. The minimized protein was defined as the receptor
using the binding site module of DS 2.5. The binding site was
defined from the volume of ligand method, which was modified to
accommodate all the important interacting residues in the allo-
steric site of HIV RT. The Input Site Sphere was defined over the
binding site, with a radius of 5 Å from the center of the binding site.
The protein, thus characterized, was taken as the target receptor for
the docking procedure.
Acknowledgments
The authors thank the Department of Biotechnology and Indian
Council of Medical Research, New Delhi, Government of India, for
financial support as a special drive for developing microbicides
against HIV. Garima Kumari thanks CSIR-UGC for providing
fellowship in the form of JRF and SRF. Special thanks are due to Prof.
K.V.R. Chary, TIFR, Mumbai, for 1H NMR spectra.
References
5.3.2. Ligand setup
[1] E. De Clercq, Int. J. Antimicrob. Agents 33 (2009) 307e320.
[2] K. Struble, J. Murray, B. Cheng, T. Gegeny, V. Miller, R. Gullick, AIDS 19 (2005)
747e756.
[3] A. Opar, Nat. Rev. Drug Discov. 6 (2007) 258e259.
[4] S. Sinha, R. Srivastava, E. De Clercq, R.K. Singh, Nucleosides Nucleotides
Nucleic Acids 23 (2004) 1815e1824.
[5] S. Sinha, R. Srivastava, B. Prusty, B.C. Das, R.K. Singh, Nucleosides Nucleotides
Nucleic Acids 26 (2007) 773e777.
[6] R.K. Singh, D. Rai, D. Yadav, A. Bhargava, J. Balzarini, E. De Clercq, Eur. J. Med.
Chem. 45 (2010) 1078e1086.
[7] R.K. Singh, D. Yadav, D. Rai, G. Kumari, C. Pannecoque, E. De Clercq, Eur. J. Med.
Chem. 45 (2010) 3787e3793.
[8] C. Shaw-Reid, B. Feuston, V. Munshi, K. Getty, J. Krueger, D.J. Hazuda,
M.A. Parniak, M.D. Miller, D. Lewis, Biochemistry 44 (2005) 1595e1606.
[9] E. De Clercq, Chem. Biodivers. 1 (2004) 44e64.
[10] J.J. Minuto, R. Haubrich, Futur. HIV Ther. 2 (2008) 525e537.
[11] H. Mitsuya, R. Yarchoan, S. Broder, Science 249 (1990) 1527e1533.
[12] J.S. Lambert, M. Seidlin, R.C. Reichman, C.S. Plank, M. Laverty, G.D. Morse,
C. Knupp, C. McLaren, C. Pettinelli, F.T. Valentine, R. Dolin, N. Engl. J. Med. 322
(1990) 1333e1340.
[13] J.R. Proudfoot, K.D. Hargrave, S.R. Kapadia, U.R. Patel, K.G. Grozinger, et al.,
J. Med. Chem. 38 (1995) 4830e4838.
[14] L.H. Li, D.H. Swenson, S.L.F. Schpok, S.L. Kuentzel, C.D. Dayton, W.C. Krueger,
Cancer Res. 42 (1982) 999e1004.
[15] L.H. Hurley, D.R. Needham-VanDevanter, C.-S. Lee, Proc. Natl. Acad. Sci. U.S.A.
84 (1987) 6412e6416.
[16] K. Weiland, T.P. Dooley, Biochemistry 30 (1991) 7559e7565.
[17] J.S. Wiseman, G. Tayrien, R.H. Abeles, Biochemistry 19 (1980) 4222e4231.
[18] J.S. Wiseman, J.S. Nichols, M. Kolpak, J. Biol. Chem. 257 (1982) 6328e6332.
[19] R.A. Pages, A. Burger, J. Med. Chem. 9 (1966) 766e768.
[20] T. Jiang, K.L. Kuhen, K. Wolff, H. Yin, K. Bieza, J. Caldwell, B. Bursulaya, T.Y.-
H. Wu, Y. He, Bioorg. Med. Chem. Lett. 16 (2006) 2105e2108.
[21] T. Jiang, K.L. Kuhen, K. Wolff, H. Yin, K. Bieza, J. Caldwell, B. Bursulaya,
T. Tuntland, K. Zhang, D. Karanewsky, Y. He, Bioorg. Med. Chem. Lett. 16
(2006) 2109e2112.
[22] G. Kumari, R.K. Singh, Nucleic Acids Res. Symp. Ser. (Oxf) 52 (2008) 265e266.
[23] W.H. Tamblyn, S.R. Hoffmann, M.P. Doyle, J. Organomet. Chem. 216 (1981)
C64eC68.
[24] M.P. Doyle, J.H. Griffin, V. Bagheri, R.L. Dorow, Organometallics 3 (1984)
53e61.
Using the built-and-edit module of DS 2.5, various ligands were
built, all-atom CHARMm forcefield parameterization was assigned
and then minimized using the ABNR method. A conformational
search of the ligand was carried out using a stimulated annealing
molecular dynamics (MD) approach. The ligand was heated to
a temperature of 700 K and then annealed to 200 K. Thirty such
cycles were carried out. The transformation obtained at the end of
each cycle was further subjected to local energy minimization, using
the ABNR method. The 30 energy-minimized structures were then
superimposed and the lowest energy conformation occurring in the
major cluster was taken to be the most probable conformation.
5.3.3. Docking and scoring
Docking of the HIV RT with different ligands was carried out
with CDOCKER protocol of DS 2.5. CDOCKER [27,28] is a grid-based
MD-simulated, annealing-based algorithm that uses CHARMm. The
receptor (protein) was held rigid, while the ligands were flexible
during the refinement. The basic strategy involves the generation of
several initial ligand orientations in the allosteric site of the target
protein followed by MD-based simulated annealing, and final
refinement by minimization.
Input Site Sphere parameters specify a sphere around the center
of the binding site, where CDOCKER experiment was performed.
The center of the Input Site Sphere was used for initial ligand
placement. Ten replicas for each ligand were generated and
randomly distributed around the center of the allosteric site. Each of
these was subjected to MD-based simulated annealing and final
refinement by minimization, leading to 10 minimized docked poses.
The docked complexes were further refined by the Ligand
Minimization protocol, during which the side-chain atoms within

1188
G. Kumari et al. / European Journal of Medicinal Chemistry 46 (2011) 1181e1188
[25] M.P. Doyle, R.L. Dorow, W.E. Buhro, J.H. Griffin, M.L. Tamblyn, Organometallics
3 (1984) 44e52.
[26] B.R. Brooks, R.E. Bruccoleri, B.D. Olafson, D.J. States, S. Swaminathan,
M. Karplus, J. Comput. Chem. 4 (1983) 187e217.
[27] F.A. Momany, R. Rone, J. Comput. Chem. 13 (1992) 888e900.
[28] G.Wu,D.H.Robertson,C.L.Brooks,M.Vieth,J.Comput.Chem.24(2003)1549e1562.
[29] C. Hamdouchi, B. Zhong, J. Mendoza, E. Collins, C. Jaramillo, J.E. Diego,
D. Robertson, C.D. Spencer, B.D. Anderson, S.A. Watkins, F. Zhang, H.B. Brooks,
Bioorg. Med. Chem. Lett. 15 (2005) 1943e1947.
[30] H.J. Böhm, J. Comput. Aided Mol. Des. 8 (1994) 243e256.
[31] H.J. Böhm, J. Comput. Aided Mol. Des. 8 (1994) 623e632.
[32] H.J. Böhm, J. Comput. Aided Mol. Des. 12 (1998) 309e323.