AlCl3 induced (hetero)arylation of 2,3-dichloroquinoxaline: A one-pot synthesis of mono/disubstituted quinoxalines as potential antitubercular agents

Article abstract of DOI:10.1016/j.bmc.2012.01.012

A direct and single-step method has been developed for the synthesis of mono and 2,3-disubstituted quinoxalines by using a AlCl₃ induced (hetero)arylation of 2,3-dichloroquinoxaline. Both symmetrical and unsymmetrical 2,3-disubstituted quinoxal

Full text of DOI:10.1016/j.bmc.2012.01.012

Bioorganic & Medicinal Chemistry 20 (2012) 1711–1722
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Bioorganic & Medicinal Chemistry
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AlCl3 induced (hetero)arylation of 2,3-dichloroquinoxaline: A one-pot
synthesis of mono/disubstituted quinoxalines as potential antitubercular agents
K. Shiva Kumar a, D. Rambabu a,b, Sandhya Sandra a, Ravikumar Kapavarapu a, G. Rama Krishna c,
M.V. Basaveswara Rao d, Kiranam Chatti a, C. Malla Reddy c, Parimal Misra a, Manojit Pal a,
⇑
a Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
b Department of Chemistry, K.L. University, Vaddeswaram, Guntur 522 502, AP, India
c Department of Chemical Sciences, Indian Institute of Science Education and Research, Kolkata, West Bengal 741252, India
d Department of Chemistry, Krishna University, Machilipatnam 521 001, AP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A direct and single-step method has been developed for the synthesis of mono and 2,3-disubstituted
quinoxalines by using a AlCl3 induced (hetero)arylation of 2,3-dichloroquinoxaline. Both symmetrical
and unsymmetrical 2,3-disubstituted quinoxalines can be prepared conveniently by using this method
under appropriate reaction conditions. The reaction proceeds via C–C bond formation and can be utilized
for the preparation of a variety of quinoxaline derivatives from readily available starting materials and
reagents. The molecular structure of a representative compound was confirmed by single crystal X-ray
diffraction study. Some of the compounds synthesized were tested for chorismate mutase inhibitory
properties in vitro and one compound showed promising activity representing one of the few examples
of chorismate mutase inhibition by a heteroarene based small molecule.
Received 14 November 2011
Revised 7 January 2012
Accepted 9 January 2012
Available online 17 January 2012
Keywords:
Quinoxalines
Indole
AlCl3
X-ray
Ó 2012 Elsevier Ltd. All rights reserved.
Chorismate mutase
1. Introduction
2,3-disubstituted quinoxaline derivatives synthesis of which was
carried out by an inexpensive and scalable C–C bond forming
The quinoxaline ring has been found to be integral part of a
variety of biologically active compounds and many pharmaceutical
agents1 including several antibiotics such as Echinomycin, Levo-
mycin and Actinoleutin. Both 2-substituted and 2,3-disubstituted
aryl or heteroaryl quinoxaline derivatives have shown remarkable
pharmacological activities. For example, compound A was found to
be an inhibitor of p38 alpha mitogen-activated protein kinase2
whereas compound B showed low nanomolar activity against Ja-
nus kinase 2 (JAK2) and potently suppressed proliferation of SET-
2 cells in vitro (Fig. 1).3 Compounds C and D were identified as
selective antagonists at human A(1) and A(3) adenosine receptors.4
A number of quinoxaline derivatives have exhibited antibacterial
activities.5,6 3-Aryl and heteroaryl indoles on the other hand have
shown promising antibacterial properties.7 Thus combining the
structural features of quinoxaline and indole moiety in a single
molecule represented by E (Fig. 2) was thought to be an attractive
template for the identification and development of promising anti-
bacterial agents. Due to our continuing interest in the identifica-
tion of novel anti-tuberculosis agents8,9 we now wish to report
the pharmacological evaluation of a series of 2-substituted and
reaction.
A number of methods are known to introduce aryl or heteroaryl
group at C-2 and/or C-3 position of a quinoxaline ring.10,11 While
all these approaches have their own merit and synthetic value
O
F
N
N
F
F
N
N
N
N
N
N
NH
HN
B
A
N
HN
HN
O
Cl
N
N
N
N
N
H
D
C
⇑
Corresponding author. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail address: manojitpal@rediffmail.com (M. Pal).
Figure 1. Quinoxaline-containing biologically active compounds.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.012

1712
K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
Table 1
N
Effect of solvents on AlCl3 mediated reaction of 1 with 2aa
N
N
N
N
H
N
N
Cl
OH
N
H
OH
E
AlCl3
N
N
Cl
Cl
+
solvent
heat
Figure 2. Design of new quinoxaline derivatives as potential antibacterial agents.
OH
OH
3a
1
2a
1. R1H
2. R2H
N
N
R2
R1
N
N
Cl
R
N
N
Cl
RH
Entry
Solvent
Temp (°C)
Time (h)
Yieldb (%)
Cl AlCl3, DCE
80 °C
1
2
3
4
5
6
ClCH2CH2Cl
ClCH2Cl
EtOAc
CH3CN
Toluene
CHCl3
80
60
80
80
80
60
1.0
1.5
1.5
2.0
3.0
2.0
90
85
80
83
65
80
AlCl3, DCE
80 °C
3
1
4
Scheme 1. AlCl3 mediated mono and bis (hetero)arylation of 2,3-dichloro
quinoxaline.
a
All the reactions were carried out using compound 1 (1.0 equiv), 2a (1.0 equiv)
their use for the preparation of mono aryl or heteroaryl substituted
quinoxalines however, remained inconvenient due to the lack of
control or selectivity.11 Only one method has been reported for
the selective mono arylation of a quinoxaline ring using Suzuki
coupling involving the use of palladium catalysts and boronic acids
at 75–80 °C for 24 h.12 In several cases the required boronic acids
are either not available or often involved cumbersome preparation.
We therefore were in need of a more convenient, practical and
straightforward method for the preparation of 2-substituted and
2,3-disubstituted quinoxalines. In recent years we have been work-
ing on AlCl3-induced direct heteroarylation of various aromatic and
heterocyclic compounds via C–C bond forming reaction13 and the
methodology has found applications in the preparation of com-
pounds of potential pharmacological interest.14 In view of cost
effectiveness and easy availability of starting materials we chose
to adopt this AlCl3 induced C–C bond forming reaction for our pur-
pose (Scheme 1). To the best of our knowledge no successful mono
or di (hetero)arylation of the quinoxaline ring system using a sim-
ilar methodology has hitherto been described in the literature.
and AlCl3 (1.1 equiv) in a solvent (5 mL).
b
Isolated yield.
proceeded well in all these cases providing the corresponding
2-aryl (Table 2, entries 1–3) or indolyl substituted quinoxalines
(Table 2, entries 4–10) in good to excellent yields. As mentioned
earlier the use of 2.0 equiv of AlCl3 and indole afforded 2,3-
diindolyl substituted quinoxaline exclusively without any mono
substituted product (Table 1, entries 11 and 12). All the com-
pounds synthesized were well characterized by spectral (NMR,
MS and IR) data. Additionally, the molecular structure of a repre-
sentative compound 3g was established unambiguously by single
crystal X-ray diffraction (Fig. 3).15
Prompted by the results of selective mono (hetero)arylation of
2,3-dichloroquinoxaline (1) we then decided to explore the succes-
sive substitution of both the chloro group of 1 by two different
(hetero)arenes in a single pot. Accordingly, 1.0 equiv of dichloro
compound 1 was reacted with two different arenes/heteroarenes
(1.0 equiv each) in a sequential manner when unsymmetrically
substituted quinoxaline 4 containing two different arenes/hetero-
arenes was isolated (Table 3).
To demonstrate the utility of the present (hetero)arylation of
quinoxaline ring further structure elaboration of a representative
compound 3e was carried out under Suzuki12 and Sonogashira16
conditions. The chloro derivative 3e was reacted smoothly with
phenylboronic acid and 3,3-dimethylbut-1-yne separately to give
the corresponding aryl (6) and alkyne coupled product (5) respec-
tively (Scheme 2).
Mechanistically the heteroarylation reaction proceeds through
the complexation of AlCl3 with one of the nitrogens of 2,3-dichlo-
roquinoxaline followed by nucleophilic attack by a suitable arene
or heteroarene at the adjacent carbon and finally release of AlCl3
affording the desired product 3 (Scheme 3). Similarly, complexa-
tion of AlCl3 with the second nitrogen facilitated the formation of
product 4. It is evident that the nucleophilicity of the reacting are-
nes or heteroarenes is crucial in these cases, and therefore, the
reaction proceeded smoothly with electron-rich arenes or
heteroarenes.
2. Results and discussion
2.1. Chemistry
For the generation of quinoxaline based small molecule library
2,3-dichloroquinoxaline12 (1) was chosen as the starting com-
pound and resorcinol (2a) was used as an arylating agent for our
initial study. Due to the presence of two replaceable and chemi-
cally identical chloro groups in the quinoxaline 1 it was necessary
to establish an optimized reaction condition initially to obtain
mono substituted product 3a. Accordingly, to avoid the second
substitution the reaction of 1 and 2a (1.0 equiv of each compound)
was carried out in the presence of 1.1 equiv AlCl3 in various sol-
vents and results are summarized in Table 1. Among the solvents
examined 1,2-dichloroethane (DCE), CH2Cl2, EtOAc, CH3CN and
CHCl3 (Table 1, entries 1–4 and 6) were found to be effective
whereas the use of toluene provided lower yield of the desired
product 3a (Table 1, entry 5). The best result however was ob-
tained both in terms of reaction time and product yield using
1,2-dichloroethane (Table 1, entry 1). No formation of disubsti-
tuted product was observed in this case. It is worthy to mention
that the use of 2.0 equiv of reactant, that is, (hetero)arene as well
as AlCl3 led to the formation of 2,3-diaryl substituted quinoxaline
(see later).
2.2. Pharmacology
Due to our interest in the identification of novel heterocycle
based small molecules as inhibitors of chorismate mutase8,9 or
CM (EC 5.4.99.5) some of the compounds synthesized were tested
for CM inhibiting properties in vitro. The shikimate pathway for
the biosynthesis of aromatic amino acids such as phenylalanine
and tyrosine involves the Claisen rearrangement of chorismate to
prephenate in the presence of CM.17 Due to the absence of this
With the optimized reaction condition in hand we then decided
to test the generality and scope of this AlCl3 mediated route to
2-substituted quinoxaline. Thus a number of activated arenes
and indoles (2) were reacted with 2,3-dichloroquinoxaline (1) in
the presence of AlCl3 and results are shown in Table 2. The reaction

K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
1713
Table 2
Synthesis of 2-(hetero)aryl substituted 3-chloroquinoxaline (3) via AlCl3-mediated C–C bond forming reaction between 1 and (hetero)arenes (2)a
N
N
Cl
R
AlCl3
N
N
Cl
Cl
RH
+
DCE
80 οC
2
1
3
Entry
RH; (2)
Products (3)
Time (h)
1.0
% Yieldb
OH
N
Cl
OH
N
1
90
90
OH
OH
OH
OH
2a
2b
3a
N
N
Cl
2
3
0.5
0.5
3b
OH
N
N
Cl
OH
2c
90
3c
N
Cl
Cl
N
H
N
4
5
0.5
0.5
85
85
NH
2d
3d
3e
N
N
N
N
2e
H3CO
OCH3
N
N
Cl
N
H
6
7
8
0.5
0.5
0.5
81
80
84
2f
NH
NH
3f
N
N
Cl
OCH3
N
H
H3CO
Cl
2g
3g
Cl
Br
N
N
Cl
N
H
2h
NH
NH
3h
Cl
Br
N
N
N
H
9
0.5
86
2i
3i
(continued on next page)

1714
K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
Table 2 (continued)
Entry
RH; (2)
Products (3)
Time (h)
0.5
% Yieldb
F
F
N
N
Cl
F
N
H
F
10
90
2j
NH
N
3j
N
N
11
2e
0.5
88c
N
3h
NH
N
N
12
2d
0.5
90c
NH
3i
a
All the reactions were carried out using compound 1 (1.0 equiv), an appropriate (hetero)arene 2 (1.0 equiv) and AlCl3 (1.1 equiv) in dichloroethane (5 mL) at 80 °C.
Isolated yields after column chromatography.
The reaction was carried out using 2.0 equiv of heteroarene (2e or 2d) and AlCl3.
b
c
Figure 3. ORTEP representation of the compound 3g (thermal ellipsoids are drawn at 50% probability level).
pathway in animals but not in bacteria CM is considered as a novel
2,3-disubstituted compounds (4) when tested at 50
the mono-substituted derivatives compound 3j showed significant
inhibition of CM at 50 M (Table 4). In a dose–response study com-
pound 3j showed dose dependent inhibition of CM with an IC50 va-
lue of 19.74 M (Fig. 4). To understand the nature of interactions of
compound 3j with the CM a docking study was performed using
the energy minimization and conformational search with the MAC-
ROMODEL application in the Schrodinger package. The protein CM
(PDB ID-2F6L) crystal structure was retrieved from the protein data
bank and was used for our study after performing necessary steps.
The docking results of compound 3j with CM protein showed
arene–cation interaction with Arg 72 and Lys 60 and Hydrogen
bonding interaction with Arg134 residue of CM (Fig. 5). The binding
energy ꢀ12.77 Kcal molꢀ1 suggested good interactions between 3j
and CM.
lM. Among
target for the identification of effective antibacterial agents.18
Notably, only a few small molecules have been reported to possess
inhibitory activity against CM. The assay19,20 used to test our com-
pounds involved determination of activity of enzyme CM which
catalyzes the conversion of chorismate to prephenate. Thus deter-
mination of activity of CM is based on the direct observation of
conversion of chorismic acid to prephenate spectrophotometrically
at OD274. This reaction was performed in the presence of test
compounds to determine their CM inhibiting activities. A known
inhibitor of CM, that is, 4-(3,5-dimethoxyphenethylamino)-3-
nitro-5-sulfamoylbenzoic acid21 was prepared and used as a refer-
ence compound the IC50 value of which was fund to be less than
l
l
10
lM. Our results are summarized in Table 4. In general the mono
substituted compounds (3) were found to be superior than the

K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
1715
Table 3
Table 3 (continued)
Synthesis of unsymmetrical 2,3-disubstituted quinoxaline (4) via AlCl3-induced
(hetero)arylation of compound 1a
Entry
Reactant (2)
Product (4)
Time (h) Yieldb (%)
R1H
R2H
1. R1H
2. R2H
N
N
R2
R1
N
N
Cl
Cl AlCl3, DCE
80 o
C
N
1
4
N
N
Entry
Reactant (2)
Product (4)
Time (h) Yieldb (%)
R1H
R2H
7
2i
2e
1.0
80
NH
Br
N
N
4g
F
OH
NH
F
1
2i
2c
1.0
1.0
1.0
85
83
78
Br
NH
N
N
8
2i
2j
1.0
80
Br
4a
HO
NH
N
4h
OH
N
a
All the reactions were carried out using compound 1 (1.0 equiv), two different
appropriate arene or heteroarene (2) each 1.0 equiv and AlCl3 (2.2 equiv) in DCE
2
2i
2a
NH
(5 mL) at 80 °C.
b
Isolated yields after column chromatography.
Br
4b
3. Conclusions
In conclusion, we have reported a new and efficient method for
the synthesis of mono and 2,3-disubstituted quinoxalines from 2,3-
dichloroquinoxaline in the presence of AlCl3. Both symmetrical and
unsymmetrical 2,3-disubstituted quinoxalines can be prepared by
using this method under appropriate reaction conditions. The pres-
ent operationally simple and single-step AlCl3 induced (hetero)ary-
lation of 2,3-dichloroquinoxaline proceeds via C–C bond forming
reactions and can be utilized for the preparation of a variety of quin-
oxaline derivatives from readily available starting materials and re-
agents. This methodology does not require the use of expensive
transition metal catalysts or organometallic reagents, and therefore
has the potential to become useful alternative towards the direct
one pot synthesis of library of small molecules based on quinoxa-
line. To the best of our knowledge this is the first example of (het-
ero)arylation of quinoxaline ring using AlCl3 mediated method.
Some of the compounds synthesized were tested for chorismate
mutase inhibiting properties in vitro and one compound showed
promising activity representing one of the few examples of choris-
mate mutase inhibition by a heteroarene based small molecule.
Docking studies of this compound at the active site of chorismate
mutase suggest it interacts well. The quinoxaline scaffold presented
here therefore has potential for the identification and development
of novel small molecule based inhibitors of chorismate mutase
thereby new anti tubercular agents for further evaluation.
N
OH
NH
3
2h
2c
N
Cl
4c
OCH3
NH
N
4
5
6
2d
2g
2d
2i
1.0
1.0
1.0
78
80
78
N
NH
4d
Br
NH
N
N
2i
NH
4e
Br
4. Experimental section
4.1. Chemistry
NH
N
N
2a
4.1.1. General methods
Unless stated otherwise, reactions were performed under a
nitrogen atmosphere. Reactions were monitored by thin layer chro-
matography (TLC) on silica gel plates (60 F254), visualizing with
ultraviolet light or iodine spray. Flash chromatography was per-
formed on silica gel (100–200 and 230–400 mesh) using hexene,
HO
OH
4f

1716
K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
PhB(OH)2
N
N
Cl
N
N
N
N
10% Pd/C, PPh3, CuI
Pd(PPh3)4
2M K2CO3
toluene – ethanol
reflux, 24 h
Ethanol, Et3N
60-65 °C, 8 h
N
N
N
5
3e
6
Scheme 2. Structural elaboration of compound 3e.
N
R2
R1
N
N
Cl
AlCl3
R2H
N
N
Cl
Cl
N
Cl
R1
Cl
R1H
R1
N
N
AlCl3
AlCl3
3
4
Scheme 3. Probable reaction mechanism leading to product 3 and subsequently 4.
Table 4
Inhibition of chorismate mutase by compounds 3 and 4 in vitro
Entry
Compounds (3 and 4)
% Inhibitiona @ 50
lM
1
2
3
4
5
6
7
8
9
10
3a
3b
3f
3g
3j
4a
4d
4e
4f
31
35
36
42
64
35
45
31
21
27
4h
a
Average of three experiments.
Figure 5. Docking of compound 3j at the active site of CM.
ethyl acetate, dichloromethane. 1H NMR and 13C NMR spectra were
determined in CDCl3, DMSO-d6, Acetone-d6 solution by using 400
and 100 MHz spectrometers, respectively. Proton chemical shifts
(d) are relative to tetramethylsilane (TMS, d = 0.00) as internal stan-
dard and expressed in ppm. Spin multiplicities are given as s (sin-
glet), d (doublet), t (triplet) and m (multiplet) as well as b
(broad). Coupling constants (J) are given in hertz. Infrared spectra
were recorded on a FT-IR spectrometer. Melting points were deter-
mined using melting point apparatus and are uncorrected. MS spec-
tra were obtained on a mass spectrometer.
Figure 4. Dose dependent chorismate mutase inhibition (S = substrate, E = enzyme)
and IC50 value of 3j.

K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
1717
4.1.2. General procedure for the preparation of compound 3
A mixture of 2,3-dichloroquinoxaline (1, 1.0 equiv), an appro-
priate (hetero)arene (2, 1.0 equiv) and AlCl3 (1.1 equiv) in dichloro-
ethane (5 mL) was stirred at 80 °C for time indicated in Table 2
under a nitrogen atmosphere. After completion of the reaction,
the mixture was poured into ice-cold water (15 mL), stirred for
10 min and then extracted with ethylacetate (3 ꢁ 20 mL). The or-
ganic layers were collected, combined, washed with cold water
(2 ꢁ 20 mL), dried over anhydrous Na2SO4 and concentrated under
vacuum. The residue obtained was purified by column chromatog-
raphy using ethylacetate/hexene to give the desired product.
(2C), 124.9, 124.3, 122.5, 107.4; HPLC: 97.3%, column: Zorbax
, mobile phase A: 0.05% Formic Acid
XDB C-18 150 ꢁ 4.6 mm 5
l
in water mobile phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/
95, 15/50, 12/95, 15/50, 18/50; flow rate: 1.0 mL/min; UV
225 nm, retention time 6.55 min; IR (KBr) vmax 3158, 3065, 1621,
1508, 1276 cmꢀ1; m/z (CI) 307 (M+1, 100%); HRMS (ESI) calcd for
C
18H12N2OCl (M+H)+ 307.0638, found 307.0635.
4.1.6. 2-Chloro-3-(1H-indol-3-yl)quinoxaline (3d)
N
N
Cl
4.1.3. 4-(3-Chloroquinoxalin-2-yl)benzene-1,3-diol (3a)
N
N
Cl
OH
NH
Yellow solid; mp 261–263 °C; 1H NMR (400 MHz, DMSO-d6) d
11.90 (bs s, 1H), 8.61 (d, J = 2.4 Hz, 1H), 8.55 (d, J = 3.2 Hz, 1H),
7.98 (d, J = 1.2 Hz, 1H), 7.96 (d, J = 1.2 Hz, 1H), 7.87–7.82 (m, 1H),
7.79–7.75 (m, 1H), 7.53–7.51 (m, 1H), 7.26–7.19 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) d 149.0, 144.9, 140.8, 138.6, 136.7,
131.1 (2C), 130.0, 128.5, 128.0, 127.0, 123.1, 122.6, 121.4, 112.5,
OH
Yellow solid; mp 176–178 °C; 1H NMR (400 MHz, DMSO-d6) d
9.75 (bs s, 1H), 9.64 (bs s, 1H), 8.10–8.08 (m, 1H), 8.05–8.02 (m,
1H), 7.89–7.85 (m, 2H), 7.15 (d, J = 8.4 Hz, 1H), 6.40–6.34 (m,
2H); 13C NMR (100 MHz, DMSO-d6) d 160.3, 156.9, 153.3, 148.5,
140.7, 140.5, 131.7, 131.0, 130.9, 129.1, 128.1, 116.3, 107.0,
111.0; HPLC: 99.6%, column: Zorbax XDB C-18 150 ꢁ 4.6 mm 5
l,
mobile phase A: 0.05% Formic Acid in water mobile phase B:
CH3CN (gradient) T/%B: 0/70, 2/70, 9/95, 12/95, 15/70, 18/70; flow
rate: 1.0 mL/min; UV 219 nm, retention time 4.59 min; IR (KBr)
vmax 3174, 1542, 1437, 1130 cmꢀ1; m/z (CI) 280 (M+1, 100%);
102.9; HPLC: 97.8%, column: Zorbax XDB C-18 150 ꢁ 4.6 mm 5
l,
mobile phase A: 0.05% Formic Acid in water mobile phase B:
CH3CN (gradient) T/%B: 0/20, 2/20, 9/95, 12/95, 15/20, 18/20; flow
rate: 1.0 mL/min; UV 240 nm, retention time 7.75 min; IR (KBr)
vmax 3289, 3167, 1623, 1598, 1339, 1209 cmꢀ1; m/z (CI) 273
HRMS (ESI) calcd for
280.0642.
C
16H11N3Cl (M+H)+ 280.0642, found
(M+1, 100%); HRMS (ESI) calcd for
273.0431, found 273.0421.
C
14H10N2O2Cl (M+H)+
4.1.7. 2-Chloro-3-(1-methyl-1H-indol-3-yl)quinoxaline (3e)
4.1.4. 2-(3-Chloroquinoxalin-2-yl)naphthalen-1-ol (3b)
N
N
Cl
N
N
Cl
N
OH
Yellow solid; mp 150–152 °C; 1H NMR (400 MHz, DMSO-d6) d
8.65 (d, J = 8.0 Hz, 1H), 8.58 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.95
(d, J = 8.0 Hz, 1H), 7.85–7.81 (m, 1H), 7.79–7.74 (m, 1H), 7.56 (d,
J = 8.0 Hz, 1H), 7.32–7.28 (m, 2H), 3.93 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 148.2, 144.3, 140.4, 138.2, 136.9, 134.5,
130.7, 129.6, 128.1, 127.6, 127.1, 122.8, 122.5, 121.3, 110.4, 109.6,
Yellow solid; mp 219–220 °C; 1H NMR (400 MHz, CDCl3) d 8.18–
8.13 (m, 2H), 7.91–7.82 (m, 4H), 7.41–7.39 (m, 2H), 7.39–7.38 (m,
1H), 7.23–7.22 (m, 1H), 6.75 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
d 153.7, 152.0, 149.0, 141.2, 141.0, 133.2, 131.9, 131.3, 131.2,
129.4, 128.6, 128.4, 128.0, 127.5, 123.6, 123.5, 118.6, 116.7; HPLC:
99.4%, column: Zorbax XDB C-18 150 ꢁ 4.6 mm 5
l, mobile phase
33.1; HPLC: 99.8%, column: Zorbax XDB C-18 150 ꢁ 4.6 mm 5
l,
A: 0.05% Formic Acid in water mobile phase B: CH3CN (gradient)
T/%B: 0/70, 2/70, 9/95, 12/95, 15/70, 18/70; flow rate: 0.8
mL/min; UV 235 nm, retention time 3.77; IR (KBr) vmax 3274,
3071, 1587, 1343, 1053 cmꢀ1; m/z (CI) 305 (M-1, 100%); HRMS
(ESI) calcd for C18H12N2OCl (M+H)+ 307.0638, found 307.0633.
mobile phase A: 0.05% Formic Acid in water mobile phase B: CH3CN
(gradient) T/%B: 0/70, 2/70, 9/95, 12/95, 15/70, 18/70; flow rate:
1.0 mL/min; UV 219 nm, retention time 7.15 min; IR (KBr) vmax
3051, 2925, 1607, 1533, 1373 cmꢀ1; m/z (CI) 294 (M+1, 100%);
HRMS (ESI) calcd for C17H13N3Cl (M+H)+ 294.0798, found 294.0703.
4.1.5. 1-(3-Chloroquinoxalin-2-yl)naphthalen-2-ol (3c)
4.1.8. 2-Chloro-3-(5-methoxy-1H-indol-3-yl)quinoxaline (3f)
N
N
Cl
OH
OCH3
N
N
Cl
NH
Yellow solid; mp 217–218 °C; 1H NMR (400 MHz, DMSO-d6) d
10.63 (bs s, 1H), 8.24 (d, J = 8.2 Hz, 1H), 8.15–8.12 (m, 2H), 7.95–
7.92 (m, 2H), 7.51–7.46 (m, 2H), 7. 47–7.41 (m, 2H), 7.00–6.98
(m, 1H); 13C NMR (100 MHz, DMSO-d6) d 154.7, 153.4, 147.6,
140.7, 140.5, 132.2, 131.4, 131.0, 129.0, 128.8, 128.0, 127.1, 125.0
Brown solid; mp 252–253 °C; 1H NMR (400 MHz, DMSO-d6) d
11.79 (bs s, 1H), 8.53 (d, J = 2.8 Hz, 1H), 8.20 (d, J = 2.4 Hz, 1H),
8.13 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.86–7.82 (m, 1H),
7.78–7.74 (m, 1H), 7.42 (d, J = 9.2 Hz, 1H), 6.90–6.87 (m, 1H),

1718
K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
3.83 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 155.3, 149.0, 144.7,
140.8, 138.5, 131.7, 131.5, 131.2, 129.8, 128.4, 128.0, 127.7,
113.1, 113.0, 110.7, 104.6, 55.7; HPLC: 97.6%, column: X DB C-18
column: Zorbax XDB C-18 150 ꢁ 4.6 mm 5
l, mobile phase A:
0.05% Formic Acid in water mobile phase B: CH3CN (gradient) T/
%B: 0/70, 2/70, 9/95, 12/95, 15/70, 18/70; flow rate: 1.0 mL/min;
UV 220 nm, retention time 6.91 min; IR (KBr) vmax 3211, 1536,
1436, 1130 cmꢀ1; m/z (CI) 357 (M+1, 100%); HRMS (ESI) calcd for
150 ꢁ 4.6 mm 5
l, mobile phase A: 0.05% Formic Acid in water,
mobile phase B: CH3CN (gradient) T/%B: 0/70, 2/70, 9/95, 12/95,
15/70, 18/70; flow rate: 1.0 mL/min; UV 225 nm, retention time
4.04 min; IR (KBr) vmax 3225, 2945, 1533, 1429, 1225 cmꢀ1; m/z
(CI) 310 (M+1, 100%); HRMS (ESI) calcd for C17H13N3OCl (M+H)+
310.0747, found 310.0760.
C
16H10N3ClBr (M+H)+ 357.9747, found 357.9763.
4.1.12. 2-Chloro-3-(5,6-difluoro-1H-indol-3-yl)quinoxaline (3j)
F
N
N
Cl
4.1.9. 2-Chloro-3-(6-methoxy-1H-indol-3-yl)quinoxaline (3g)
F
N
N
Cl
NH
OCH3
Yellow solid; mp 237–238 °C; 1H NMR (400 MHz, DMSO-d6) d
12.04 (bs s, 1H), 8.63 (s, 1H), 8.56–8.51 (m, 1H), 8.19 (d,
J = 8.4 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.87–7.76 (m, 2H), 7.56–
7.52 (m, 1H); 13C NMR (100 MHz, DMSO-d6) d 148.2, 146.5,
144.5, 140.6, 138.7, 132.7, 132.0, 131.9, 131.1, 130.2, 128.6,
127.9, 122.6, 111.5, 109.6, 109.4; HPLC: 98.5%, column: Zorbax
NH
Brown solid; mp 236–238 °C; 1H NMR (400 MHz, DMSO-d6) d
11.70 (bs s, 1H), 8.52 (d, J = 8.8 Hz, 1H), 8.44 (d, J = 3.2 Hz, 1H),
8.12 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 9.6 Hz, 1H), 7.85–7.81 (m, 1H),
7.77–7.73 (m, 1H), 7.01 (d, J = 2.4 Hz, 1H), 6.87–6.84 (m, 1H),
3.80 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 156.8, 148.9, 144.7,
140.8, 138.5, 137.7, 131.1, 130.2, 129.8, 128.4, 128.0, 123.6,
121.1, 111.4, 111.1, 95.3, 55.6; HPLC: 98.2%, column: Zorbax XDB
XDB C-18 150 ꢁ 4.6 mm 5
l, mobile phase A: 0.05% Formic Acid
in water mobile phase B: CH3CN (gradient) T/%B: 0/80, 2/80, 9/
98, 12/98, 15/80, 18/80; flow rate: 1.0 mL/min; UV 225 nm, reten-
tion time 98.50 min; IR (KBr) vmax 3224, 1537, 1479, 1158 cmꢀ1
;
C-18 150 ꢁ 4.6 mm 5
l, mobile phase A: 0.05% Formic Acid in
water mobile phase B: CH3CN (gradient) T/%B: 0/70, 2/70, 9/95,
m/z (CI) 316 (M+1, 100%); HRMS (ESI) calcd for C16H9N3ClF2
(M+H)+ 316.0448, found 316.0453.
12/95, 15/70, 18/70; flow rate: 1.0 mL/min; UV 220 nm, retention
time 4.40 min; IR (KBr) vmax 3460, 3260, 2925, 1622, 1447 cmꢀ1
;
4.1.13. 2,3-Di(1H-indol-3-yl)quinoxaline (3h)
m/z (CI) 310 (M+1, 100%); HRMS (ESI) calcd for C17H13N3OCl
(M+H)+ 310.0747, found 310.0749.
4.1.10. 2-Chloro-3-(5-chloro-1H-indol-3-yl)quinoxaline (3h)
NH
N
Cl
N
N
Cl
N
NH
NH
Yellow solid; mp 236–238 °C; 1H NMR (400 MHz, DMSO-d6) d
11.38 (bs s, 2H), 8.02–8.00 (m, 2H), 7.99–7.93 (m, 2H), 7.71–7.69
(m, 2H), 7.42–7.37 (m, 4H), 7.14–7.11 (m, 2H), 7.03–7.00 (m,
2H); 13C NMR (100 MHz, DMSO-d6) d 150.1 (2C), 140.0 (2C),
136.5 (2C), 129.3 (2C), 128.5 (2C), 128.2 (2C), 126.4 (2C), 122.4
(2C), 121.5 (2C), 120.6 (2C), 114.6 (2C), 112.3 (2C); IR (KBr) vmax
3396, 3244, 2927, 1535, 1431, 1123 cmꢀ1; m/z (CI) 361 (M+1,
100%); HRMS (ESI) calcd for C24H17N4 (M+H)+ 361.1453, found
361.1446.
Brown solid; mp 280–282 °C; 1H NMR (400 MHz, DMSO-d6) d
12.08 (bs s, 1H), 8.61–8.59 (m, 2H), 8.14 (d, J = 8.8 Hz, 1H), 7.99–
7.96 (m, 1H), 7.87–7.84 (m, 1H), 7.80–7.77 (m, 1H), 7.54 (d,
J = 8.8 Hz, 1H), 7.27–7.24 (m, 1H); 13C NMR (100 MHz, DMSO-d6)
d 148.4, 144.7, 140.6, 138.8, 135.3, 132.5, 131.3, 130.2, 128.5,
128.1, 128.0, 126.1, 123.1, 121.7, 114.1, 110.7; HPLC: 97.3%, col-
umn: Zorbax XDB C-18 150 ꢁ 4.6 mm 5
l, mobile phase A: 0.05%
Formic Acid in water mobile phase B: CH3CN (gradient) T/%B: 0/
80, 2/80, 9/98, 12/98, 15/80, 18/80; flow rate: 1.0 mL/min; UV
225 nm, retention time 97.28 min; m/z (CI) 314 (M+1, 100%); IR
(KBr) vmax 3211, 2599, 1536, 1440 cmꢀ1; HRMS (ESI) calcd for
4.1.14. 2,3-Bis(1-methyl-1H-indol-3-yl)quinoxaline (3i)
C
16H10N3Cl2 (M+H)+ 314.0252, found 314.0260.
N
4.1.11. 2-(5-Bromo-1H-indol-3-yl)-3-chloroquinoxaline (3i)
N
N
Br
N
N
Cl
N
Yellow solid; mp 208–210 °C; 1H NMR (400 MHz, DMSO-d6) d
8.18–8.16 (m, 2H), 8.04–8.02 (m, 2H), 7.66–7.64 (m, 2H), 7.56–
7.45 (m, 4H), 7.25–7.22 (m, 2H), 7.17–7.14 (m, 2H), 3.78 (s, 6H);
13C NMR (100 MHz, DMSO-d6) d 149.2 (2C), 139.5 (2C), 136.8
(2C), 132.1 (2C), 128.7 (2C), 128.0 (2C), 126.5 (2C), 121.9 (2C),
121.4 (2C), 120.2 (2C), 113.0 (2C), 110.2 (2C), 32.7 (2C); HPLC:
NH
Yellow solid; mp 285–287 °C; 1H NMR (400 MHz, DMSO-d6) d
12.08 (bs s, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.59–8.57 (m, 1H), 8.12–
8.10 (m, 1H), 7.98–7.96 (m, 1H), 7.87–7.76 (m, 2H), 7.51–7.48
(m, 1H), 7.38–7.34 (m, 1H); 13C NMR (100 MHz, DMSO-d6) d
148.4, 144.8, 140.6, 138.8, 135.5, 132.4, 131.3, 130.3, 128.7,
128.5, 128.0, 125.7, 124.7, 114.5, 114.2, 110.6; HPLC: 98.9%,
99.7%, column: Zorbax XDB C-18 150 ꢁ 4.6 mm 5
l, mobile phase
A: 0.05% Formic Acid in water mobile phase B: CH3CN (gradient)

K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
1719
T/%B: 0/70, 2/70, 9/95, 12/95, 15/70, 18/70; flow rate: 1.0 mL/min;
UV 225 nm, retention time 20.0 min; IR (KBr) vmax 3470, 1534,
1373, 1091 cmꢀ1; m/z (CI) 389 (M+1, 100%).
151.0, 140.8, 139.2, 135.2, 131.2, 130.1 (2C), 129.3 (2C), 128.8,
128.5, 125.1, 125.1, 119.3, 114.1, 113.6 (2C), 107.5, 103.2; HPLC:
98.8%, column: X Bridge C-18 150 ꢁ 4.6 mm 5
l, mobile phase A:
0.05% Formic Acid in water, mobile phase B: CH3CN (gradient) T/
%B: 0/50, 2/50, 9/95, 12/95, 15/50, 18/50; flow rate: 1.0 mL/min;
UV 225 nm, retention time 6.52 min; IR (KBr) vmax 3416, 2958,
2923, 1733, 1249 cmꢀ1; m/z (CI) 432 (M+1, 100%).
4.1.15. General procedure for the preparation of compound 4
A mixture of 2,3-dichloroquinoxaline (1, 1.0 equiv), an appropri-
ate (hetero)arene (2, 1.0 equiv) and AlCl3 (1.0 equiv) in dichloroeth-
ane (5 mL) was stirred at 85 °C for 0.5 h under a nitrogen
atmosphere. The reaction mixture was cooled to room temperature
to which was add the second (hetero)arene (2, 1.0 equiv) and an
additional amount of AlCl3 (1.0 equiv). The reaction mixture was
stirred for 0.5 h at 85 °C under a nitrogen atmosphere. After comple-
tion of the reaction (indicated by TLC), the mixture was poured into
ice-cold water (15 mL), stirred for 10 min and then extracted with
ethylacetate (3 ꢁ 20 mL). The organic layers were collected, com-
bined, washed with cold water (2 ꢁ 20 mL), dried over anhydrous
Na2SO4 and concentrated under vacuum. The residue obtained
was purified by column chromatography using ethylacetate/hexene
to give the desired product.
4.1.18. 1-(3-(5-Chloro-1H-indol-3-yl)quinoxalin-2-yl)
naphthalen-2-ol (4c)
N
OH
N
NH
4.1.16. 1-(3-(5-Bromo-1H-indol-3-yl)quinoxalin-2-yl)naph
thalen-2-ol (4a)
Cl
Yellow solid; mp 192–194 °C; 1H NMR (400 MHz, DMSO-d6) d
11.32 (bs s, 1H), 9.89 (bs s, 1H), 8.82 (s, 1H), 8.19 (d, J = 8.4 Hz,
1H), 8.01–7.98 (m, 2H), 7.92–7.84 (m, 2H), 7.77–7.73 (m, 1H),
7.34–7.23 (m, 4H), 7.17–7.15 (m, 1H), 7.07–7.05 (m, 1H), 6.38 (d,
J = 2.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 153.1, 151.3,
151.2, 141.1 (2C), 139.6, 134.9, 133.4, 130.9, 130.7, 129.1, 129.0,
128.7 (2C), 128.3, 128.1, 127.6, 125.8, 123.7, 123.6, 122.9, 122.2,
119.9, 118.9, 113.7, 112.8; HPLC: 98.0%, column: Zorbax XDB C-
N
OH
N
NH
Br
18 150 ꢁ 4.6 mm 5
l, mobile phase A: 0.05% Formic Acid in water
mobile phase B: CH3CN (gradient) T/%B: 0/70, 2/70, 9/98, 12/98,
Yellow solid; mp 171–173 °C; 1H NMR (400 MHz, Acetone-d6) d
10.42 (bs s, 1H), 9.19 (bs s, 1H), 8.70 (bs s, 1H), 8.25 (d, J = 8.0 Hz,
1H), 8.04–8.00 (m, 2H), 7.93–7.87 (m, 2H), 7.79–7.75 (m, 1H),
7.35–7.28 (m, 6H), 6.61 (s, 1H); 13C NMR (100 MHz, Acetone-d6)
d 152.7, 151.3, 150.9, 141.3, 139.8, 135.3, 135.1, 133.5, 128.9
(2C), 128.8, 128.7, 128.6, 128.4, 128.4, 128.1, 126.9, 125.5, 125.0,
123.8, 123.3, 120.2, 118.5, 113.7, 113.3, 113.2; HPLC: 98.9%, col-
15/70, 18/70; flow rate: 0.8 mL/min; UV 225 nm, retention time
5.84 min; IR (KBr)
(M+1, 100%).
v
max 3745, 3213, 3181, 1533 cmꢀ1; m/z (CI) 422
4.1.19. 2-(1H-indol-3-yl)-3-(6-methoxy-1H-indol-3-yl)quinox-
aline (4d)
umn: Symmetry (R) C-18 74 ꢁ 4.6 mm 3.5
l, mobile phase A:
0.05% Formic Acid in water, mobile phase B: CH3CN (gradient) T/
%B: 0/50, 2/50, 9/95, 12/95, 15/50, 18/50; flow rate: 1.0 mL/min;
UV 225 nm, retention time 7.12 min; IR (KBr) vmax 3511, 3412,
3060, 2582, 1534, 1428 cmꢀ1; m/z (CI) 467 (M+2, 100%).
OCH3
NH
N
4.1.17. 4-(3-(5-Bromo-1H-indol-3-yl)quinoxalin-2-yl)benzene-
1,3-diol (4b)
N
NH
HO
N
OH
N
Brown solid; mp 160–162 °C; 1H NMR (400 MHz, CDCl3) d 8.27
(s, 2H), 8.12–8.10 (m, 2H), 8.05 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 8.8 Hz,
1H), 7.69 (d, J = 6.8 Hz, 2H), 7.38–7.33 (m, 2H), 7.23–7.13 (m, 3H),
6.83–6.80 (m, 2H), 3.84 (s, 3H); 13C NMR (100 MHz, CDCl3) d 156.7,
149.9, 149.7, 140.4, 140.4, 136.7, 135.9, 128.8 (2C), 128.6 (2C),
126.6, 126.4, 125.7, 122.7, 122.5, 121.6, 120.9, 120.6, 115.5,
115.4, 111.2, 110.7, 94.5, 55.5; HPLC: 98.4%, column: X Bridge
NH
Br
Yellow solid; mp 169–171 °C; 1H NMR (400 MHz, DMSO-d6) d
11.52 (bs s, 1H), 9.61 (bs s, 1H), 9.35 (bs s, 1H), 8.86 (s, 1H), 8.07
(d, J = 7.6 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.77–7.70 (m, 2H),
7.37–7.30 (m, 2H), 7.16 (d, J = 7.6 Hz, 1H), 6.78 (s, 1H), 6.43–6.33
(m, 2H); 13C NMR (100 MHz, DMSO-d6) d 159.7, 156.3, 152.9,
C-18 150 ꢁ 4.6 mm 5
l, mobile phase A: 0.05% Formic Acid in
water mobile phase B: CH3CN (gradient) T/%B: 0/50, 9/95, 12/95,
15/50, 18/50; flow rate: 0.8 mL/min; UV 225 nm, retention time
6.79 min; IR (KBr) vmax 3372, 2960, 2924, 1735, 1246 cmꢀ1; m/z
(CI) 389 (M-1, 100%).

1720
K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
4.1.20. 2-(5-Bromo-1H-indol-3-yl)-3-(1H-indol-3-yl)quinoxaline
4.1.23. 2-(5-Bromo-1H-indol-3-yl)-3-(5, 6-difluoro-1H-indol-3-
(4e)
yl)quinoxaline (4h)
F
F
Br
NH
N
N
Br
NH
N
N
NH
NH
Brown solid; mp 213–215 °C; 1H NMR (400 MHz, CDCl3) d 8.44
(bs s, 1H), 8.33 (bs s, 1H), 8.24 (s, 1H), 8.15 (d, J = 6.4 Hz, 1H), 8.11
(d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.71 -7.70, (d, J = 6.8 Hz,
2H), 7.38–7.32 (m, 3H), 7.23–7.16 (m, 3H), 7.10–7.07 (m, 1H);
13C NMR (100 MHz, DMSO-d6) d 149.9, 149.6, 140.0 (2C), 136.4,
135.0, 129.6 (2C), 129.5, 128.5 (2C), 128.2, 127.8, 126.1, 125.0,
123.7, 122.4, 121.1, 120.6, 114.5, 114.3, 113.9, 113.4, 112.3; IR
(KBr) vmax 3411, 2962, 2923, 2875, 1732, 1248 cmꢀ1; m/z (CI)
439 (M+1, 100%).
Brown solid; mp 172–174 °C; 1H NMR (400 MHz, CDCl3) d 8.38
(bs s, 1H), 8.31 (bs s, 1H), 8.13–8.10 (m, 3H), 7.90–7.85 (m, 1H),
7.73–7.71 (m, 2H), 7.33–7.31 (m, 2H), 7.27–7.25 (m, 2H), 7.17–
7.13 (m, 1H); 13C NMR (100 MHz, CDCl3) d 148.9, 148.7, 140.5,
140.3, 134.5, 130.9, 130.8, 129.5, 129.4, 128.6, 127.9, 127.7,
127.2, 125.9, 123.9, 122.7, 121.7, 121.7, 115.5, 115.0, 114.5,
112.7, 108.5, 108.3; IR (KBr) vmax 3453, 3411, 3239, 2927, 2358,
1713, 1474 cmꢀ1; m/z (CI) 475 (M+1, 100%).
4.1.21. 4-(3-(5-Bromo-1H-indol-3-yl)quinoxalin-2-yl)benzene-
1,3-diol (4f)
4.1.24. Preparation of 2-(3,3-Dimethylbut-1-ynyl)-3-(1-methyl-
1H-indol-3-yl)quinoxaline (5)
Br
N
NH
N
N
N
N
HO
OH
A mixture of the 2-chloro-3-(1-methyl-1H-indol-3-yl)quinoxa-
line (3e) (0.68 mmol), 10% Pd/C (0.013 mmol), PPh3 (0.12 mmol),
CuI (0.03 mmol), and triethylamine (2.04 mmol) in ethanol
(5 mL) was stirred at 25–30 °C for 30 min under a nitrogen atmo-
sphere. To this was added 3,3-dimethylbut-1-yne compound (2)
(1.06 mmol). The mixture was initially stirred at room temperature
for 1 h and then at 60–65 °C for 8 h. After completion of the reac-
tion, the mixture was cooled to room temperature, diluted with
EtOAc (50 mL), and filtered through celite. The filtrated was col-
lected, washed with water (3 ꢁ 30 mL), dried over anhydrous
Na2SO4, and concentrated. The crude residue was purified by col-
umn chromatography on silica gel using hexane/ethyl acetate to
give the desired product as a yellow solid; mp 184–185 °C; 1H
Brown solid; mp 242–244 °C; 1H NMR (400 MHz, CDCl3) d 12.20
(bs s, 1H), 8.39 (bs s, 1H), 8.29 (s, 1H), 8.12–8.10 (m, 1H), 7.96 (d,
J = 8.4 Hz, 1H), 7.73–7.68 (m, 2H), 7.37–7.7.28 (m, 4H), 6.57 (d,
J = 2.0 Hz, 1H), 6.07–6.05 (m, 1H), 5.18 (bs s, 1H); 13C NMR
(100 MHz, DMSO-d6) d 159.7, 156.3, 152.9, 151.0, 140.8, 139.2,
135.2, 131.2, 130.2, 129.2 (2C), 128.9, 128.8, 128.4, 125.2, 125.0,
119.3, 114.1, 113.7, 113.6, 107.5, 103.3; IR (KBr) vmax 3854, 3295,
1620, 1532 cmꢀ1; m/z (CI) 432 (M+1, 100%).
4.1.22. 2-(5-Bromo-1H-indol-3-yl)-3-(1-methyl-1H-indol-3-
yl)quinoxaline (4g)
NMR (400 MHz, DMSO-d6)
d 8.68–8.67 (m, 1H), 8.08 (d,
J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.82–7.79 (m, 1H), 7.75–
7.66 (m, 2H), 7.59 (d, J = 8.4 Hz, 1H), 7.34–7.25 (m, 2H), 3.94 (s,
3H), 1.43 (s, 9H); 13C NMR (100 MHz, DMSO-d6) d 150.3, 149.5,
140.5, 140.3, 136.9, 130.5, 129.4, 129.3, 129.0, 128.9 (2C), 122.4,
121.5, 121.1, 111.2, 110.6, 103.9, 80.1, 33.3, 30.3 (3C), 28.5; IR
(KBr) vmax 3464, 2951, 2220, 1534, 1372 cmꢀ1; m/z (CI) 340
(M+1, 100%); HRMS (ESI) calcd for C23H22N3 (M+H)+ 340.1820,
found 340.1814.
N
Br
N
N
NH
Yellow solid; mp 164–166 °C; 1H NMR (400 MHz, CDCl3) d 8.52
(s, 1H), 8.21–8.16 (m, 3H), 7.69–7.63 (m, 3H), 7.36–7.33 (m, 3H),
7.25–7.20 (m, 3H), 7.05–7.01 (m, 1H), 3.78 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 149.4, 149.2, 140.5, 140.4, 137.1, 134.5,
130.8, 129.0, 129.0, 128.7, 128.6, 128.3, 127.9, 126.6, 125.7,
124.4, 122.3, 121.5, 120.6, 114.7, 114.6, 114.6, 112.6, 109.5, 33.1;
4.1.25. Preparation of 2-(1-methyl-1H-indol-3-yl)-3-
phenylquinoxaline (6)
HPLC: 98.0%, column: Zorbax XDB C-18 150 ꢁ 4.6 mm 5
l, mobile
N
phase A: 0.05% Formic Acid in water mobile phase B: CH3CN (gra-
dient) T/%B: 0/80, 2/80, 9/95, 12/98, 15/80, 18/80; flow rate:
1.0 mL/min; UV 248 nm, retention time 5.82 min; IR (KBr) vmax
3423, 2951, 2933, 2855, 1632 cmꢀ1; m/z (CI) 454 (M+2, 100%).
N
N

K. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 1711–1722
1721
The percentage of enzyme inhibition caused by the test com-
pound is calculated by the following formula
A mixture of the 2-chloro-3-(1-methyl-1H-indol-3-yl)quinoxa-
line (3e) (0.68 mmol), phenyl boronic acid (1.49 mmol), and
Pd(PPh3)4 (6 mol%) under a nitrogen atmosphere. To this was added
toluene (5 mL), EtOH (0.7 mL), and K2CO3 (2.0 M in water, 2.2 mL)
and the mixture was stirred and heated under reflux for 24 h. after
completion of the reaction (indicated by TLC) the mixture was
cooled to room temperature and diluted with CH2Cl2 (15 mL). The
organic layer was separated and collected and the aqueous phase
was extracted with CH2Cl2 (2 ꢁ 15 mL). The combined organic ex-
tracts were washed with water (20 mL), dried over anhydrous
Na2SO4, filtered, and concentrated. The crude residue was purified
by column chromatography on silica gel using hexane/ethyl acetate
to afford the desired product as a yellow solid; mp 158–160 °C; 1H
NMR (400 MHz, DMSO-d6) d 8.37 (d, J = 8.4 Hz, 1H), 8.12 (d,
J = 7.6 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.84–7.81 (m, 1H), 7.77–
7.74 (m, 1H), 7.62–7.60 (m, 2H) 7.52–7.46 (m, 4H), 7.24 (t,
J = 7.4 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.77 (s, 1H), 3.65 (s, 3H);
13C NMR (100 MHz, DMSO-d6) d 153.7, 149.5, 141.2, 140.3, 137.3,
137.1, 129.4 (4C), 128.9, 128.6, 127.3, 127.0, 123.1, 122.9, 122.8,
122.4, 121.5, 121.1, 110.8, 110.6, 33.3; IR (KBr) vmax 2924, 2859,
1533, 1371 cmꢀ1; m/z (CI) 336 (M+1, 100%); HRMS (ESI) calcd for
% Inhibition = 100ꢀresidual activity of CM
ꢀ
ꢁ
ðS þ E þ CÞ ꢀ ðS þ CÞ
Residual activity of CM ¼
ꢁ 100
ðS þ EÞ ꢀ ðSÞ
S = Substrate absorbance at 274 nm; E = Enzyme absorbance at
274 nm; C = Compound absorbance at 274 nm.
Dose–response study of the compound 3j against chorismate
mutase activity was carried out using the concentration from
1 lM to 100 lM.
4.4. Docking study
The molecular docking was carried out with XP Glide applica-
tion of schrodinger software with MASTERO interface 9.2 The mol-
ecule 3j was docked in chorismate mutase protein of
Mycobacterium tuberculosis.
4.5. Procedure
C
23H18N3 (M+H)+ 336.1500, found 336.1501.
In the present study we have performed the energy minimiza-
tion and conformational search with the MACROMODEL applica-
tion in the Schrodinger package. The study molecule was energy
minimized for flexibility and then the conformational search was
performed. We used OPLS_2005 force field and water as implicit
solvent. We have followed the PRCG (Polak-Ribier conjugate gradi-
ent) method of minimization with 500 iterations with a threshold
gradient on 0.05 kJ/mol. The conformational search was based on
Montecarlo multiple minimum torsional sampling. The ligands
were then finally prepared with LIGPREP application.
4.2. Single crystal X-ray data for compound 3g15
Single crystals suitable for X-ray diffraction of 3g were grown
from methanol. The crystals were carefully chosen using a stereo
zoom microscope supported by a rotatable polarizing stage. The
data was collected at room temperature on Bruker’s KAPPA APEX
II CCD Duo with graphite monochromated Mo-K
a radiation
(0.71073 Å). The crystals were glued to a thin glass fiber using
FOMBLIN immersion oil and mounted on the diffractometer. The
intensity data were processed using Bruker’s suite of data process-
ing programs (SAINT), and absorption corrections were applied
using SADABS.22 The crystal structure was solved by direct meth-
ods using SHELXS-97 and the data was refined by full matrix
least-squares refinement on F2 with anisotropic displacement
parameters for non-H atoms, using SHELXL-97.23
The protein chorismate mutase (PDB ID-2F6L) crystal structure
was retrieved from the protein data bank and it was then refined
with the PROTEIN PREPERATION WIZARD application in which
the hydrogen’s were added and missing side chains and loops were
0
filled with PRIME application. Waters were observed within the 5ÅA
0
distance and waters were deleted beyond 5ÅA from het (hetero-
atom) groups. Finally the protein is then optimized and minimized
with impref using OPLS_2005 force filed. GRID based Flexible
Docking was done in the present study.
Crystal data of 3g: Molecular formula = C17H11ClN3O, Formula
weight = 308.74, Crystal system = Monoclinic, space group = P2(1)/
n, a = 9.0594 (12) Å, b = 9.1332(11) Å, c = 17.852(2) Å, V= 1445.3(3)
Å3, T = 298 K, Z = 4, Dc = 1.419 Mg m–3
,
l
(Mo K
a ,
) = 0.71073 mm–1
Acknowledgements
19170 reflections measured, 2480 independent reflections, 2156
The authors thank Professor Seyed E. Hasnain and Professor J.
Iqbal for encouragement and DBT, New Delhi, India for financial
support (Grant No. BT/01/COE/07/02). K.S. thanks UGC, New Delhi,
India for a Dr. D.S. Kothari Post doctoral fellowship [No. F.4–2/
2006(BSR)/13–24/2010(BSR)].
observed reflections [I > 2.0r(I)], R1_obs = 0.035, Goodness of fit =
1.040.
4.3. Pharmacology20,21
4.3.1. Chorismate mutase activity assay
Mycobacterium tuberculosis chorismate mutase (MtCM) gene
was PCR amplified and cloned into expression vector pET22b.
MtCM was purified from over expressed culture of BL21 (DE3) har-
boring pET22b/MtCM by Ni-NTA affinity chromatography.
Activity of chorismate mutase enzyme is based on the direct
observation of conversion of Chorismate to prephenate Spectro-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.012.
photometrically at OD274. The reaction volume of 100 ll contained
References and notes
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