Pyrrolo[2,3-b]quinoxalines as inhibitors of firefly luciferase: Their Cu-mediated synthesis and evaluation as false positives in a reporter gene assay

Article abstract of DOI:10.1016/j.bmcl.2012.08.056

2-Substituted pyrrolo[2,3-b]quinoxalines having free NH were prepared directly from 3-alkynyl-2-chloroquinoxalines in a single pot by using readily available and inexpensive methane sulfonamide (or p-toluene sulfonamide) as an ammonia surrogate. The reaction proceeded in the presence of Cu(OAc)₂ affording the desired product in moderate yield. The crystal structure analysis of a representative compound and its supramolecular interactions are presented. Some of the compounds synthesized exhibited inhibitory activities against luciferase that was supported by the predictive binding mode of these compounds with luciferase enzyme through molecular docking studies. The key observations disclosed here can alert users of luciferase reporter gene assays for possible false positive results due to the direct inhibition of luciferase.

Full text of DOI:10.1016/j.bmcl.2012.08.056

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6433–6441
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrrolo[2,3-b]quinoxalines as inhibitors of firefly luciferase: Their
Cu-mediated synthesis and evaluation as false positives in a reporter gene assay
Ali Nakhi a, Md. Shafiqur Rahman a,b, Ravada Kishore c, Chandana Lakshmi T. Meda a, Girdhar Singh Deora a,
Kishore V. L. Parsa a, Manojit Pal a,
⇑
a Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
b Chemical Synthesis & Process Technologies, Department of Chemistry, University of Delhi, New Delhi 110 007, India
c School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Substituted pyrrolo[2,3-b]quinoxalines having free NH were prepared directly from 3-alkynyl-
2-chloroquinoxalines in a single pot by using readily available and inexpensive methane sulfonamide
(or p-toluene sulfonamide) as an ammonia surrogate. The reaction proceeded in the presence of Cu(OAc)2
affording the desired product in moderate yield. The crystal structure analysis of a representative com-
pound and its supramolecular interactions are presented. Some of the compounds synthesized exhibited
inhibitory activities against luciferase that was supported by the predictive binding mode of these com-
pounds with luciferase enzyme through molecular docking studies. The key observations disclosed here
can alert users of luciferase reporter gene assays for possible false positive results due to the direct inhi-
bition of luciferase.
Received 10 July 2012
Revised 13 August 2012
Accepted 15 August 2012
Available online 22 August 2012
Keywords:
Pyrrolo[2,3-b]quinoxaline
Sulfonamide
Cu(OAc)2
Ó 2012 Elsevier Ltd. All rights reserved.
Luciferase
Firefly luciferase, an enzyme from the firefly Photinus pyralis was
first cloned and expressed in Escherichia coli and subsequently in
mammalian cells.1,2 In the presence of adenosine 50-triphosphate
(ATP) this enzyme catalyzes the conversion of its substrate luciferin
to luciferyl adenylate which on oxidation by molecular oxygen pro-
duce an electronically excited state of oxyluciferin (Scheme 1).3 The
second step emits a photon of visible light as oxyluciferin returns to
the ground state. The cloning of firefly luciferase cDNA created
enormous interest for possible applications of the gene as a tool
in scientific research. For example, due to the structural similarities
between the catalytic site of the enzyme and the opioid binding site
of quinoline A has been described as luciferase inhibitors which in-
clude a potent inhibitor B (Fig. 1).12,13 Subsequent screening of a
series of related N-pyridin-2-ylbenzamide analogues afforded an-
other potent inhibitor C (Fig. 1).14 Since emergence of ‘false posi-
tives’ due to the luciferase inhibition by small molecules may
interfere the early drug discovery process it is therefore vital and
necessary to identify those compounds that are active against lucif-
erase. Herein we report the first identification of drug-like pyrrolo-
quinoxalines as new and potential inhibitors of luciferase.
Due to their various pharmacological properties nitrogen het-
erocycles15 containing fused pyrrole and furan ring have attracted
our particular attention especially in the design and identification
of new chemical entities under new drug discovery program.
Accordingly, in view of antiviral activities of compound D (B-220)
against herpes simplex virus type 1 (HSV-1), cytomegalovirus
(CMV), and varicella-zoster virus (VZV)15a we became interested
in the synthesis of compound library based on F designed via E
(Fig. 2).
Only few methods have been reported for the synthesis of pyr-
rolo[2,3-b]quinoxalines.16,17 These include Pd-mediated intramo-
lecular ring closure of 2-alkynyl-3-trifluoroacetamidoquinoxalines
which in turn were prepared via selective amination of 2,3-dihalo
quinoxaline followed by Sonogashira coupling.17a Alternatively,
1,2-disubstituted pyrrolo[2,3-b]quinoxalines were synthesized by
the action of primary aliphatic or aromatic amines on 2-chloro-3-
alkynylquinoxalines prepared via Sonogashira coupling of 2,3-
dichloroquinoxaline and terminal alkynes.17b This method though
of the receptor luciferase has been proposed as a model for the l-
opioid receptor.4 At present, because of its high sensitivity and ease
of use luciferase as a reporter gene has found wide applications in
high throughput screening techniques.5 It is used as a reporter to
assess the transcriptional activity in cells that are transfected with
a genetic construct containing the luciferase gene under the control
of a promoter (i.e., the region of DNA that facilitates the transcrip-
tion of a particular gene) of interest. Notably, the luciferin–lucifer-
ase reaction has been reported to be inhibited by a number of
agents such as oxyluciferin, AMP,6,7 substrate-like compounds such
as luciferin8 and ATP analogues,7 and dissimilar compounds such as
pifithrin-R,9 lipoic acid,10 and N-tosylphenylalanine chloromethyl
ketone (TPCK).11 Recently, a library containing structural analogues
⇑
Corresponding author. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail address: manojitpal@rediffmail.com (M. Pal).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.056

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A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441
O
C
R
NH2-Z
R'
N
R'
N
HO
Cu(OAc)2
S
N
N
S
O
R
Et3N, DMF
80 °C, 4-8 h
N
H
N
N
Cl
3
Luciferin
2
Z = SO2CH3, SO2C6H4CH3-p
R' = CH3 /H; R = alkyl / aryl
ATP
Mg2+
PPi
Luciferase
Scheme 2. Preparation of 2-substituted-1H-pyrrolo[2,3-b]quinoxalines 3.
NH2
N
N
N
O
C
appeared to be effective for the preparation of 1,2-substituted ana-
logues was particularly not suitable for direct access to pyrrolo[2,3-
b]quinoxalines having free NH group. Recently, the use of t-butyl
sulfonamide as an ammonia surrogate has been reported in the
Pd-mediated coupling of 2-alkynyl bromobenzene leading to 2-aryl
indoles.18 This prompted us to examine the use of common sulfon-
amides as ammonia equivalent in the preparation of pyrrolo[2,3-
b]quinoxalines. We have observed that 3-alkynyl-2-chloro quinox-
alines (2) prepared from 2,3-dichloro quinoxaline (1) undergo cou-
pling-cyclization reaction with methane (or p-toluene)
sulfonamide in the presence of Cu(OAc)2 to give 2-substituted-
1H-pyrrolo[2,3-b]quinoxalines (3, Scheme 2) the results of which
are presented. To the best of our knowledge the present strategy
has not been explored earlier for the preparation of this class of
compounds having free NH group.
The precursor of 3-alkynyl-2-chloro quinoxalines (2), that is,
2,3-dichloro quinoxalines (1) were prepared by the condensation
of phenylenediamines with diethyl oxalate to give the correspond-
ing 1,4-dihydroquinoxaline-2,3-dione which on treatment with
POCl3 afforded the desired 2,3-dichloquinoxalines (1, Scheme
3).19 On coupling with various terminal alkynes under a modified
Sonogashira conditions the compound 1 afforded the required
3-alkynyl-2-chloro quinoxalines (2). To establish the optimized
reaction conditions for selective mono alkynylation of 1 the dichlo-
roquinoxaline (1a) was reacted with phenyl acetylene in the pres-
ence of various Pd catalysts and results are summarized in Table 1.
The coupling reaction afforded the mono alkynylated product 2a in
70% yield along with dialkynyl derivative 2aa when 10% Pd/C-
PPh3-CuI was used as catalyst complex20a and Et3N as a base (Entry
1, Table 1). The yield of 2a was decreased when piperidine was
used in place of Et3N (Entry 2, Table 1). The use of other Pd
O
P
O
HO
S
N
N
S
O
O
N
O
OH OH
Luciferyl-AMP
O2
CO2 + AMP + H+
*
O
O
S
N
N
S
Excited state of oxyluciferin ( )
*
Light
Oxyluciferin
Scheme 1. Luciferase catalyzed conversion of luciferin to luciferyl-AMP followed
by oxidation to the excited state of oxyluciferin (⁄) which on return to the ground
state emits light.
Ph
O
O
OMe
O
N
N
H
Ph
N
N
H
N
N
H
Ph
OMe
A
B
C
Figure 1. Examples of known luciferase inhibitors.
N
H3C
H3C
N
N
N
alkyl
or aryl
N
N
N
N
N
D
E
F
N
CH3
H3C
Figure 2. Design of target compound F based on known bioactive fused N-heterocycles D via E.
H
R
NH2
NH2
R
N
O
O
R
N
N
Cl
Cl
(COOH)2
POCl3
reflux, 6h
HCl / H2O
reflux, 6h
N
H
1a
R = H ( ) 68%
R = H; 72%
R = CH3; 68%
R = CH3 (1b) 65%
Scheme 3. Synthesis of 2,3-dichloroquinoxaline derivatives.

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441
6435
Table 1
Pd-mediated coupling of 1 with phenyl acetylene under various conditionsa
Ph
+
Ph
Ph
N
N
N
N
N
Cl
Cl
Ph
Pd cat-CuI
Base
Cl
N
1a
2aa
2a
Entry
Pd-catalysts
Base
Time (h)
%Yieldb
2a
2aa
1
2
3
4
10%Pd/Cc
10%Pd/Cc
Pd(PPh3)4
Et3N
Piperidine
Et3N
Et3N
2
5
4
5
70
61
45
48
18
26
47
42
Pd(PPh3)2Cl2
a
All of the reactions were carried out using 1a (1.256 mmol), phenyl acetylene (1.256 mmol), Pd catalyst (0.0125 mmol), CuI (0.0125 mmol)
and base (1.8844 mmol) in EtOH (4 mL) at 60 °C.
b
Isolated yield.
PPh3 (0.0502 mmol) was used.
c
Table 2
Pd/C-CuI mediated synthesis of 3-alkynyl-2-chloroquinoxalines (2)a
R
R'
N
N
R'
N
Cl
Cl
R
10% Pd/C, PPh3
CuI, Et3N
EtOH, 60 °C
Cl
N
1
2
Entry
1
Halide (1)
1a (R0 = H)
Alkyne
Time (h)
Product (2)
Yieldb(%)
2
2a
70
N
N
Cl
CH3
CH3
2
3
1a
1a
4
2b
2c
62
65
N
N
Cl
Cl
N
N
3
4
OH
OH
OH
N
4
5
1a
1a
2d
2e
60
68
N
N
Cl
Cl
OH
N
N
(CH2)3 CH3
Si(CH3)3
(CH2)3 CH3
6
7
1a
1a
4
4
2f
65
N
N
Cl
Cl
2g
40c
N
8
1b (R0 = CH3)
4
H3C
N
N
2h
63d
Cl
(continued on next page)

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A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441
Table 2 (continued)
Entry
Halide (1)
Alkyne
Time (h)
Product (2)
Yieldb(%)
CH3
CH3
9
1b
4
2i
65d
H3C
N
N
Cl
a
All the reactions were carried out by using 1 (1.256 mmol), terminal alkyne (1.256 mmol), 10% Pd/C (0.0125 mmol), PPh3 (0.0502 mmol), CuI (0.0125 mmol), and Et3N
(1.8844 mmol) in EtOH (4 mL).
b
Isolated yield.
c
After coupling followed by removal of –Si(CH3)3 group in the presence of KOH/MeOH.
The other product, for example, dialkynyl derivative formed was not isolated and characterized in this case.
d
Table 3
Effect of catalysts/base on the coupling-cyclization of 2a to 3aa
Ph
N
CH3SO2NH2
N
N
Ph
N
H
Catalyst
N
Cl
base, DMF
3a
2a
Entry
Catalyst
Base
Product (3a)b
1
2
3
4
5
10% Pd/C
(PPh3)2PdCl2
CuI
Cu(OAc)2
Cu(OAc)2
Et3N
Et3N
Et3N
Et3N
DBU
ND
ND
ND
53%
20%
a
All the reactions were carried out by using 2a (2.5125 mmol), catalyst (0.0251), base (3.7688 mmol), in DMF (3 mL) at 110 °C for 4 h.
Isolated yield. ND = not detected.
b
catalysts e.g. Pd(PPh3)4 or Pd(PPh3)2Cl2 resulted in a ꢀ1:1 mixture
of 2a and 2aa (Entries 3 and 4, Table 1). Previously, the use of
Pd(PPh3)2Cl2, CuI, Et3N in DMSO provided 2a in good yield.17b
However, in compared to Pd(PPh3)2Cl2 or other Pd-catalysts the
Pd/C has several advantages, for example, it is less expensive, sta-
ble, easily separable from the product by simple filtration and is
recyclable. Moreover, due to the well known uses of Pd/C for
hydrogenation reaction in industry the Pd/C-based strategy has
potential for scale up synthesis. Thus, combination of 10% Pd/C-
CuI-PPh3 and Et3N in EtOH was chosen as the preferred conditions
for the present alkynylation reaction and was used to prepare
other 3-alkynyl-2-chloro quinoxalines represented by 2 (Table 2).
Having prepared a range of 3-alkynyl-2-chloroquinoxalines (2)
via selective alkynylation of 1 we then examined the reaction of
2-chloro-3-(phenylethynyl)quinoxaline (2a) with methane sulfon-
amide (NH2SO2CH3) as an ammonia surrogate in presence of a
Table 4
Effect of solvent on Cu(OAc)2 mediated coupling-cyclization of 2a/2ha
Entry
Alkyne 2/Solvent
Z-SO2NH2
Time (h)/temp (°C)
Product (3)
Yieldb (%)
40
N
Ph
SO2CH3
1
2a/1,4-Dioxane
CH3
7/80
N
N
3aa
N
H3C
Ph
SO2CH3
2
2h/1,4-Dioxane
CH3
7/80
43
N
N
3ab
N
Ph
3
4
2a/DMF
2a/DMF
CH3
4/110
6/110
53
42
N
H
N
3a
C6H4CH3-p
3a
Ph
N
5
2a/DMSO
CH3
4/100
52
N
OH
2ab
a
All the reactions were carried out by using 2a (0.9469 mmol), sulfonamide (1.4204 mmol), and Et3N (1.4204 mmol) in a solvent (3 mL).
Isolated yield.
b

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441
6437
Table 5
Cu(OAc)2 mediated synthesis of 2-substituted-1H-pyrrolo[2,3-b]quinoxalinesa
R
R'
N
N
R'
N
N
CH3SO2NH2
R
N
H
Cl
Cu(OAc)2 Et3N
DMF, 80 °C
3
2
Entry
1
Alkyne (2)
Time (h)
4
Product (3)
Yieldb (%)
N
2a
53
N
N
H
3a
N
N
CH3
2
3
4
2b
2c
2d
6
8
8
N
H
3b
52
48
51
N
N
H
N
3c
N
N
H
OH
N
3d
N
N
5
2f
8
58
N
H
3e
N
6
7
8
2g
2h
2i
4
6
6
51
53
50
N
H
N
3f
H3C
H3C
N
N
N
3g
N
H
CH3
N
H
N
3h
a
All the reactions are carried out by using 2 (1.8938 mmol), Cu(OAc)2 (0.0189 mmol) CH3SO2NH2 (2.8409 mmol) and Et3N (2.8409 mmol) in DMF (4 mL).
Isolated yield.
b
Cu(II)
N
R
R
N
N
N
N
CH3SO2NH2
B
B
2
Cu(OAc)2
NH
S
BH
O
O
O
S
O
CH3
CH3
E-1
E-2
(B = Et3N)
Figure 3. Thermal ellipsoidal diagram of the compound 3a (20% probability,
hydrogen atoms are omitted for clarity).
Cu(II)
BH
Cu(II)
R
N
N
N
B
R
catalyst and base (Table 3). We envisioned that the reaction may
proceed via formation of two C–N bonds, that is, (i) coupling of sul-
fonamide with 2a followed by (ii) intramolecular cyclization. The
initial use of catalysts like 10% Pd/C, (PPh3)2PdCl2 and CuI in the
presence of Et3N was unsuccessful (Entries 1–3, Table 3). However,
the desired 2-phenyl-1H-pyrrolo[2,3-b]quinoxaline (3a) was iso-
lated when Cu(OAc)2/Et3N was used (Entry 4, Table 3). The use of
3
N
N
S
N
O
O
S
O
O
CH3
CH3
E-3
E-4
Scheme 4. Proposed mechanism for the formation of pyrrolo[2,3-b]quinoxalines
(3).

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A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441
Table 6
N1ꢁ ꢁ ꢁH15 = 2.734 Å, N2ꢁ ꢁ ꢁH9 = 2.764 Å for the compound 3a.
Through these weak van der Waals interactions21c it gives a 1D
network in its crystal packing (see Fig. in ESI).
Inhibition of luciferase by compounds 3aa, 3a and 3c at 30 lM
Entry
Compound (30
lM)
% PDE4B inhibition
% Luciferase inhibition
A plausible mechanism for the formation pyrrole ring fused
with quinoxaline moiety is shown in Scheme 4. The reaction seems
to proceed via in situ generation of 3-alkynyl substituted N-qui-
noxalin-2-yl methanesulfonamide E-1 which subsequently under-
goes Cu-mediated intramolecular cyclization to afford the
compound 3. In the presence of Cu catalyst the reaction proceeds
via activation of the triple bond of E-1 by coordination to the tran-
1
2
3
3aa
3a
3c
94.69 1.1
98.27 14.8
99.97 5.3
92.37 2.2
98.42 4.1
99.16 3.7
DBU in place of Et3N decreased the product yield (Entry 5, Table 3).
The reaction was also found to be sensitive to the nature of solvent
used. Thus, the Cu(OAc)2 mediated coupling-cyclization of 2a and
2h was examined in a number of solvents (Table 4). The reaction
afforded 1-methylsulfonyl substituted pyrrolo[2,3-b]quinoxaline
(3aa or 3ab) when performed in 1,4-dioxane (Entries 1 and 2, Table
4) but deprotected pyrrolo[2,3-b]quinoxaline (3a) in DMF (Entries
3 and 4, Table 4). The use of DMSO provided 3-(phenylethynyl)qui-
noxalin-2-ol (2ab) as a result of displacement of the chloro group
of 2a by a hydroxyl group (Entry 5, Table 4) the source of which
seemed to be the traces of water present in DMSO used. Thus, com-
bination of Cu(OAc)2/Et3N in DMF was used to prepare 2-substi-
tuted pyrrolo[2,3-b]quinoxalines represented by 3 (Table 5).20b
All the 2-substituted pyrrolo[2,3-b]quinoxalines (3) prepared
were well characterized by spectral (NMR, IR and MS) data, the
molecular structure of 3a was further confirmed unambiguously
by single crystal X-ray diffraction (Fig 3).21a The data was collected
at ambient temperature (298 K) on a Bruker SMART APEX CCD sin-
sition metal salt to form the p-complex E-2. The nucleophilicity of
the sulfonamide nitrogen is enhanced in the presence of Et3N via
generating a corresponding anion which participates in the intra-
molecular nucleophilic attack to the metal coordinated triple bond
in an endo dig fashion. This provides the metal-vinyl species E-3
which upon subsequent in situ protonation regenerates the cata-
lyst producing the N-sulfonyl derivative E-4. In a polar solvent such
as DMF the E-4 undergoes desulfonation to give the desired 2-
substituted-1H-pyrrolo[2,3-b]quinoxalines (3) which perhaps was
not favored in a less polar solvent such as 1,4-dioxane.22
All the compounds synthesized were initially tested for their
phosphodiesterase 4 (PDE4) inhibitory properties at 30 lM using
a luciferase reporter gene assay. We observed that a number of
compounds appeared as initial hits from this assay. However, fur-
ther testing of these compounds against luciferase inhibitions indi-
cated the emergence of these compounds as false positive hits
rather than their real inhibition of PDE 4B. The results of this study
for most active false positives are summarized in Table 6. It is evi-
dent from Table 6 that compounds 3aa, 3a and 3c are potent inhib-
itors of luciferase and the PDE4 inhibitions shown by these
compounds are due to their inhibition of luciferase enzyme.23
Since examples of luciferase inhibitors are not common in the
gle crystal diffractometer using graphite monochromated Mo-K
a
radiation (0.71073 Å).21b The compound 3a (50% ethylacetate/n-
hexane) crystallizes in the monoclinic C2/c space group. The supra-
molecular interactions between nitrogen and hydrogen are within
the range of 2.720–2.750 Å, and 2.730–2.770 Å it gives two types of
interactions, that is, N2ꢁ ꢁ ꢁH15 = 2.721 Å, N1ꢁ ꢁ ꢁH9 = 2.741 Å, and
Figure 4. 3D binding orientation and H-bond interactions of ligand 3aa with firefly luciferase binding pocket.

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441
6439
Figure 5. 3D binding orientation and H-bond interactions of ligand 3a with firefly luciferase binding pocket.
Figure 6. 3D binding orientation and H-bond interactions of ligand 3c with firefly luciferase binding pocket.

6440
A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441
Table 7
Docking simulation parameters of compounds 3aa, 3a and 3b
Entry
Compound
Docking score
Lipophilic EvdWa
Sitemapb
LowMWc
Binding energy (Kcal/mol)d
1
2
3
3aa
3a
3c
ꢂ5.70
ꢂ7.10
ꢂ6.73
ꢂ6.1
ꢂ5.7
ꢂ4.7
ꢂ0.4
ꢂ0.4
ꢂ0.7
ꢂ0.5
ꢂ0.5
ꢂ0.4
ꢂ55.32
ꢂ57.08
ꢂ59.74
a
b
c
Chemscore lipophilic pair term and fraction of the total protein–ligand vdW energy.
Ligand/receptor non-H-bonding polar/hydrophobic and hydrophobic/hydrophilic complementarity terms.
Reward for ligands with low molecular weight.
BE = E complex (minimized) ꢂ [E ligand (minimized) + E receptor].
d
Figure 7. Docked alignment of molecules 3aa, 3a, and 3c at the binding site pocket of luciferase enzyme.
literature hence the present class of compounds is of further inter-
est particularly to avoid interferences in the early stage of drug dis-
covery process.
In conclusion, the present research disclosed for the first time
luciferase inhibitory properties of 2-substituted pyrrolo[2,3-
b]quinoxalines in vitro. These compounds were synthesized in
moderate yield from 3-alkynyl-2-chloroquinoxalines (prepared
via Pd/C-Cu mediated coupling of 2,3-dichloroquinoxaline with
terminal alkynes) using readily available and inexpensive meth-
ane sulfonamide (or p-toluene sulfonamide) as an ammonia sur-
rogate in a single pot. The present Cu-mediated process also
represents the first synthesis of pyrrolo[2,3-b]quinoxalines having
free NH group. The crystal structure analysis of a representative
compound and its supramolecular interactions are presented.
The luciferase inhibitory properties of some of these compounds
synthesized and tested were supported by their docking studies
which suggested H-bonding interactions including one between
one of the nitrogen of fused quinoxaline moiety and the Ala
348 residue of luciferase. Overall, the present research can alert
users of luciferase reporter gene assays for achieving possible
false positive results that may occur due to the direct luciferase
inhibition.
To understand the binding modes of compounds 3aa, 3a, and 3c
at the molecular level, we carried out molecular docking simula-
tion studies of these molecules at the Firefly luciferase inhibitor-
binding site.24,25 The docking studies predicted good binding mode
of the compounds with the binding site of the target protein where
the pyrrolo quinoxaline moiety appeared to play an important role.
One of the nitrogen of quinoxaline moiety of 3aa, 3a and 3c inter-
acted with Ala 348 through an H-bond.26 Additionally, interaction
was observed between (i) one of the oxygen of SO group of 3aa and
Ser 314 (Fig. 4), (ii) NH of pyrrole ring of 3a and Ser 347 (Fig. 5) and
(iii) NH of pyrrole ring of 3c and Ser 314 as well as Gly 339 residue
(Fig. 6). Good hydrophobic interactions involving ligands aromatic
rings were also observed in all these cases. These include pi–pi
stacking between aromatic rings of 2-substituted-1H-pyrrolo[2,3-
b]quinoxalins and binding site residue (His 245, Phe 247) of the en-
zyme. This pi–pi stacking provides additional hydrophobic stability
to ligand–receptor complex. The docking simulation parameters
obtained after docking of these molecules into the Firefly luciferase
protein are summarized in Table 7. For the validation, the co-crystal
ligand M24 or [50-O-[(R)-[({3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-
3-yl]phenyl}carbonyl)oxy](hydroxy) phosphoryl]24 was re-docked
at the active site of protein. Overall, the data shown, suggests that
these molecules interacted well with luciferase (Fig. 7).
Acknowledgments
The authors thank Professor J. Iqbal for encouragement and
support. A. Nakhi thanks CSIR, New Delhi, India for awarding a Se-
nior Research Fellowship. M. P. thanks DST, New Delhi, India for
financial support (Grant no. SR/S1/OC-53/2009).

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441
6441
21. (a) Crystal data of 3a: Molecular formula = C16H10N3, Formula weight = 244.27,
Monoclinic, C2/c, a = 25.527 (4) Å, b = 4.5133 (7) Å, c = 23.957 (4) Å, V = 2373.0
Supplementary data
67) Å3, T = 298 K, Z = 8, Dc = 1.367 Mg mꢂ3, (Mo-K ) = 0.71073 mmꢂ1, 10583
a
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.056.
reflections were measured with 2099 unique reflections (Rint = 0.0330), of
which 2099 (I > 2 (I)) were used for the structure solution. Final R1 (w
r
R2) = 0.0670 (0.1879), 172 parameters. The final Fourier difference synthesis
showed minimum and maximum peaks of ꢂ0.326 and +0.434 e.Åꢂ3
,
References and notes
respectively. Goodness of fit = 1.068. Crystallographic data (excluding
structure factors) for 3a have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication numbers CCDC
890934.; (b) Sheldrick, G. M. SHELX-97; Program for Crystal Structure Solution
and Analysis: University of Gottingen, Gottingen, Germany, 1997; (c) Bondi, A.
J. Phys. Chem. 1964, 68, 441.
1. de Wet, J. R.; Wood, K. V.; Helinski, D. R.; DeLuca, M. Proc. Natl. Acad. Sci. U.S.A.
1985, 82, 7870.
2. de Wet, J. R.; Wood, K. V.; DeLuca, M.; Helinski, D. R.; Subramani, S. Mol. Cell.
Biol. 1987, 7, 725.
22. Perhaps the polar amidic carbonyl group of DMF (e.g., Me2N–
CH@O M Me2N+@CH–Oꢂ) was responsible for a nucleophilic attack on the N-
sulfonyl group (e.g., S@O) thereby cleavage of the S–N bond. We thank one of
the reviewers for his comments on this aspect of the proposed reaction
mechanism.
23. The inhibition of luciferase was measured by using detection reagent
component of PDElight HTS assay kit (Lonza) according to manufacturer’s
recommendations. Briefly, detection reagent containing luciferase enzyme and
3. McElroy, W. D.; Seliger, H. H.; White, E. H. Photochem. Photobiol. 1969, 10, 153.
4. Sudhaharan, T.; Reddy, A. R. Biochemistry 1998, 37, 4451.
5. Fan, F.; Wood, K. V. Assay Drug Dev. Technol. 2007, 5, 127.
6. Gates, B. J.; DeLuca, M. Arch. Biochem. Biophys. 1975, 169, 616.
7. Lee, R. T.; Denburg, J. L.; McElroy, W. D. Arch. Biochem. Biophys. 1970, 141, 38.
8. Denburg, J. L.; Lee, R. T.; McElroy, W. D. Arch. Biochem. Biophys. 1969, 134, 381.
9. Rocha, S.; Campbell, K. J.; Roche, K. C.; Perkins, N. D. BMC Mol. Biol. 2003, 4, 9.
10. Niwa, K.; Ohmiya, Y. Biochem. Biophys. Res. Commun. 2004, 323, 625.
11. Lee, R.; McElroy, W. D. Biochemistry 1969, 8, 130.
12. Auld, D. S.; Southall, N. T.; Jadhav, A.; Johnson, R. L.; Diller, D. J.; Simeonov, A.;
Austin, C. P.; Inglese, J. J. Med. Chem. 2008, 51, 2372.
13. http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid)411.
14. Heitman, L. H.; van Veldhoven, J. P. D.; Zweemer, A. M.; Ye, K.; Brussee, J.;
IJzerman, A. P. J. Med. Chem. 2008, 51, 4724.
15. Wilhelmsson, L. M.; Kingi, N.; Bergman, J. J. Med. Chem. 2008, 51, 7744.
16. Iijima, C.; Hayashi, E. Yakugaku Zasshi. 1977, 97, 712.
17. (a) Acardi, A.; Cacchi, S.; Fabrizi, G.; Paris, L. M. Tetrahedron Lett. 2004, 45, 2431;
(b) Ames, D. E.; Brohi, M. I. J. C. S. Perkin Trans. 1 1980, 1384.
18. Prakash, A.; Dibakar, M.; Selvakumar, K.; Ruckmani, K.; Sivakumar, M.
Tetrahedron Lett. 2011, 52, 5625.
its substrate was incubated with 10
30 M compound for 15 min. Luminescence values (RLUs) were measured by a
Multilabel plate reader (Perklin Elmer 1420 Multilabel counter). The
percentage of inhibition was calculated using the following formula:
lM ATP and DMSO (vehicle control) or
l
%
inhibition = [(RLU of vehicle control ꢂ RLU of inhibitor)/(RLU of vehicle
control)] ꢃ 100.
24. Auld, D. S.; Lovell, S.; Thorne, N.; Lea, W. A.; Maloney, D. J.; Shen, M.; Rai, G.;
Battaile, K. P.; Thomas, C. J.; Simeonov, A.; Hanzlik, R. P.; Inglese, J. Proc. Natl.
Acad. Sci. U.S.A. 2010, 107, 4878.
25. Glide, version 5.7; Schrodinger, LLC: New York, NY, 2011.
26. H-Bond parameters of compounds 3aa, 3a, and 3c with the receptor binding site of
luciferace: Hydrogen bonding is one of the major parameters that contributes to
the binding affinity of a ligand with receptor [N–Hꢁ ꢁ ꢁ:N (13 kJ/mol or 3.1 kcal/
mol) and N–Hꢁ ꢁ ꢁ:O (8 kJ/mol or 1.9 kcal/mol)]. In our molecular modeling
studies we kept optimum parameters (maximum distance 3.0 Å, minimum
donor angle 120° and minimum acceptor angle 90°) for the hydrogen bonding
between ligand and receptor. In docking studies, the distance between the
acceptor and donor atoms were found optimum, the molecule 3aa is having 2.92
(SOꢁ ꢁ ꢁHN) and 2.7 (Nꢁ ꢁ ꢁNH), the molecule 3a is having 2.90 (NHꢁ ꢁ ꢁO) and 2.63
(Nꢁ ꢁ ꢁNH), and the molecule 3c is having 2.80 (NHꢁ ꢁ ꢁO) and 3.00 (Nꢁ ꢁ ꢁNH) (ligand
groups are presented in bold face). Thus, these hydrogen bonding interactions
contribute to the ligand binding affinity and to stabilize the ligand at the
luciferase binding pocket as well (see Figs. 4–6).
19. Mao, L.; Sakurai, H.; Hirao, T. Synthesis 2004, 2535.
20. For a review, see: (a) Pal, M. Synlett 2009, 2896; (b) General procedure for the
preparation of 3: To
1.8938 mmol) in
a
solution of 2-chloro-3-(alkynyl)quinoxaline (2,
DMF (4 mL), Cu(OAc)2 (0.0189 mmol),
methanesulfonamide (2.8409 mmol) and triethylamine (2.8409 mmol) were
added. The mixture was heated for the time indicated in Table 5. After
completion of the reaction, the mixture was extracted with ethyl acetate
(3 ꢃ 50 mL), washed with water (3 ꢃ 25 mL), dried over anhydrous sodium
sulphate (Na2SO4) and filtrated. The organic layer was collected and
concentrated under vacuum. The residue was purified by column
chromatography on silica gel using EtOAc-hexane.