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: TheirCu-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, Indiab Chemical Synthesis & Process Technologies, Department of Chemistry, University of Delhi, New Delhi 110 007, Indiac School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad 500 046, India

a r t i c l e i n f o

Article history:Received 10 July 2012Revised 13 August 2012Accepted 15 August 2012Available online 22 August 2012

Keywords:Pyrrolo[2,3-b]quinoxalineSulfonamideCu(OAc)2

Luciferase

0960-894X/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.bmcl.2012.08.056

⇑ Corresponding author. Tel.: +91 40 6657 1500; faE-mail address: manojitpal@rediffmail.com (M. Pa

a b s t r a c t

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 exhibitedinhibitory 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 herecan alert users of luciferase reporter gene assays for possible false positive results due to the direct inhi-bition of luciferase.

� 2012 Elsevier Ltd. All rights reserved.

Firefly luciferase, an enzyme from the firefly Photinus pyralis wasfirst cloned and expressed in Escherichia coli and subsequently inmammalian cells.1,2 In the presence of adenosine 50-triphosphate(ATP) this enzyme catalyzes the conversion of its substrate luciferinto luciferyl adenylate which on oxidation by molecular oxygen pro-duce an electronically excited state of oxyluciferin (Scheme 1).3 Thesecond step emits a photon of visible light as oxyluciferin returns tothe ground state. The cloning of firefly luciferase cDNA createdenormous interest for possible applications of the gene as a toolin scientific research. For example, due to the structural similaritiesbetween the catalytic site of the enzyme and the opioid binding siteof the receptor luciferase has been proposed as a model for the l-opioid receptor.4 At present, because of its high sensitivity and easeof use luciferase as a reporter gene has found wide applications inhigh throughput screening techniques.5 It is used as a reporter toassess the transcriptional activity in cells that are transfected witha genetic construct containing the luciferase gene under the controlof 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 ofagents such as oxyluciferin, AMP,6,7 substrate-like compounds suchas luciferin8 and ATP analogues,7 and dissimilar compounds such aspifithrin-R,9 lipoic acid,10 and N-tosylphenylalanine chloromethylketone (TPCK).11 Recently, a library containing structural analogues

ll rights reserved.

x: +91 40 6657 1581.l).

of quinoline A has been described as luciferase inhibitors which in-clude a potent inhibitor B (Fig. 1).12,13 Subsequent screening of aseries 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 mayinterfere the early drug discovery process it is therefore vital andnecessary 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 attractedour particular attention especially in the design and identificationof 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 interestedin 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-trifluoroacetamidoquinoxalineswhich in turn were prepared via selective amination of 2,3-dihaloquinoxaline followed by Sonogashira coupling.17a Alternatively,1,2-disubstituted pyrrolo[2,3-b]quinoxalines were synthesized bythe 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

N

N

Cl

R

N

N

NH

R

Z = SO2CH3, SO2C6H4CH3-pR' = CH3 /H; R = alkyl / aryl

NH2-ZCu(OAc)2

Et3N, DMF80 °C, 4-8 h

2 3

R' R'

Scheme 2. Preparation of 2-substituted-1H-pyrrolo[2,3-b]quinoxalines 3.

NH2

NH2

(COOH)2

NH

HN

HCl / H2Oreflux, 6h

R R

R = H;R = CH

Scheme 3. Synthesis of 2,3-dich

N

SHO

S

N CO

O

LuciferinATP

PPi

N

SHO

S

N CO

O PO

OO

O

OH OH

N

N

N

N

NH2

Luciferyl-AMP

O2

CO2 + AMP + H+

N

SO

S

N O

Excited state of oxyluciferin ( )

*

*Light

Oxyluciferin

Mg2+Luciferase

Scheme 1. Luciferase catalyzed conversion of luciferin to luciferyl-AMP followedby oxidation to the excited state of oxyluciferin (⁄) which on return to the groundstate emits light.

N

N

N

N CH3H3C

H3C

H3C N

N

N

D E

Figure 2. Design of target compound F based on k

N NH

O

Ph N NH

O OMe

OMeA B

Ph

N NH

O

Ph

C

Figure 1. Examples of known luciferase inhibitors.

6434 A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441

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-butylsulfonamide as an ammonia surrogate has been reported in thePd-mediated coupling of 2-alkynyl bromobenzene leading to 2-arylindoles.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 whichare presented. To the best of our knowledge the present strategyhas not been explored earlier for the preparation of this class ofcompounds having free NH group.

The precursor of 3-alkynyl-2-chloro quinoxalines (2), that is,2,3-dichloro quinoxalines (1) were prepared by the condensationof phenylenediamines with diethyl oxalate to give the correspond-ing 1,4-dihydroquinoxaline-2,3-dione which on treatment withPOCl3 afforded the desired 2,3-dichloquinoxalines (1, Scheme3).19 On coupling with various terminal alkynes under a modified

Sonogashira conditions the compound 1 afforded the required3-alkynyl-2-chloro quinoxalines (2). To establish the optimizedreaction 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 in70% yield along with dialkynyl derivative 2aa when 10% Pd/C-PPh3-CuI was used as catalyst complex20a and Et3N as a base (Entry1, Table 1). The yield of 2a was decreased when piperidine wasused in place of Et3N (Entry 2, Table 1). The use of other Pd

O

O

POCl3

N

N Cl

Clreflux, 6h

R

72%3; 68%

R = H (1a) 68%R = CH3 (1b) 65%

loroquinoxaline derivatives.

N

N

N

alkylor aryl

F

nown bioactive fused N-heterocycles D via E.

Table 2Pd/C-CuI mediated synthesis of 3-alkynyl-2-chloroquinoxalines (2)a

N

N Cl

Cl N

N

Cl

R

R

10% Pd/C, PPh3CuI, Et3NEtOH, 60 °C 2

R' R'

1

Entry Halide (1) Alkyne Time (h) Product (2) Yieldb(%)

1 1a (R0 = H) 2

N

N

Cl

2a 70

2 1a

CH3

4

N

N

Cl

CH3

2b 62

3 1a 3

N

N

Cl

2c 65

4 1aOH

4

N

N

Cl

OH 2d 60

5 1aOH

N

N

Cl

OH

2e 68

6 1a

(CH2)3 CH3

4

N

N

Cl

(CH2)3 CH3

2f 65

7 1a

Si(CH3)3

4

N

N

Cl2g 40c

8 1b (R0 = CH3) 4

N

N

Cl

H3C 2h 63d

(continued on next page)

Table 1Pd-mediated coupling of 1 with phenyl acetylene under various conditionsa

N

N Cl

Cl N

N

Cl

Ph

N

NPh

Ph

+Ph

Pd cat-CuI

2a 2aa1aBase

Entry Pd-catalysts Base Time (h) %Yieldb

2a 2aa

1 10%Pd/Cc Et3N 2 70 182 10%Pd/Cc Piperidine 5 61 263 Pd(PPh3)4 Et3N 4 45 474 Pd(PPh3)2Cl2 Et3N 5 48 42

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.c PPh3 (0.0502 mmol) was used.

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441 6435

Table 2 (continued)

Entry Halide (1) Alkyne Time (h) Product (2) Yieldb(%)

9 1b

CH3

4

N

N

Cl

H3C

CH3

2i 65d

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.d The other product, for example, dialkynyl derivative formed was not isolated and characterized in this case.

Table 3Effect of catalysts/base on the coupling-cyclization of 2a to 3aa

N

N

Cl

Ph

Catalystbase, DMF

N

N

NH

PhCH3SO2NH2

2a 3a

Entry Catalyst Base Product (3a)b

1 10% Pd/C Et3N ND2 (PPh3)2PdCl2 Et3N ND3 CuI Et3N ND4 Cu(OAc)2 Et3N 53%5 Cu(OAc)2 DBU 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.b Isolated yield. ND = not detected.

6436 A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441

catalysts e.g. Pd(PPh3)4 or Pd(PPh3)2Cl2 resulted in a �1:1 mixtureof 2a and 2aa (Entries 3 and 4, Table 1). Previously, the use ofPd(PPh3)2Cl2, CuI, Et3N in DMSO provided 2a in good yield.17b

However, in compared to Pd(PPh3)2Cl2 or other Pd-catalysts thePd/C has several advantages, for example, it is less expensive, sta-ble, easily separable from the product by simple filtration and isrecyclable. Moreover, due to the well known uses of Pd/C forhydrogenation reaction in industry the Pd/C-based strategy has

Table 4Effect of solvent on Cu(OAc)2 mediated coupling-cyclization of 2a/2ha

Entry Alkyne 2/Solvent Z-SO2NH2 Time (h

1 2a/1,4-Dioxane CH3 7/80

2 2h/1,4-Dioxane CH3 7/80

3 2a/DMF CH3 4/110

4 2a/DMF C6H4CH3-p 6/110

5 2a/DMSO CH3 4/100

a All the reactions were carried out by using 2a (0.9469 mmol), sulfonamide (1.4204b Isolated yield.

potential for scale up synthesis. Thus, combination of 10% Pd/C-CuI-PPh3 and Et3N in EtOH was chosen as the preferred conditionsfor the present alkynylation reaction and was used to prepareother 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 of2-chloro-3-(phenylethynyl)quinoxaline (2a) with methane sulfon-amide (NH2SO2CH3) as an ammonia surrogate in presence of a

)/temp (�C) Product (3) Yieldb (%)

N

N

NPh

SO2CH33aa

40

N

N

NPh

SO2CH3

H3C

3ab

43

N

N

NH

Ph

3a

53

3a 42

N

N

OH

Ph

2ab

52

mmol), and Et3N (1.4204 mmol) in a solvent (3 mL).

Table 5Cu(OAc)2 mediated synthesis of 2-substituted-1H-pyrrolo[2,3-b]quinoxalinesa

N

N

Cl

R

Cu(OAc)2 Et3NDMF, 80 °C

N

N

NH

R

32

R' R'CH3SO2NH2

Entry Alkyne (2) Time (h) Product (3) Yieldb (%)

1 2a 4 N

N

NH3a

53

2 2b 6 N

N

NH

CH3

3b

52

3 2c 8 N

N

NH

3c

48

4 2d 8 N

N

NH

OH

3d

51

5 2f 8N

N

NH

3e

58

6 2g 4 N

N

NH

3f

51

7 2h 6 N

NH3C

NH

3g

53

8 2i 6 N

NH3C

NH

CH3

3h

50

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).b Isolated yield.

Figure 3. Thermal ellipsoidal diagram of the compound 3a (20% probability,hydrogen atoms are omitted for clarity).

N

NR

NHS OOCH3

N

N

NSO

O

CH3

Cu(II)

R

2CH3SO2NH2

E-1

BH

E-3

3

N

NR

NS OOCH3

Cu(II)

E-2

BHCu(OAc)2

N

N

NR

SOO

CH3

Cu(II)

B

B

(B = Et3N)

E-4

B

Scheme 4. Proposed mechanism for the formation of pyrrolo[2,3-b]quinoxalines(3).

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441 6437

catalyst and base (Table 3). We envisioned that the reaction mayproceed via formation of two C–N bonds, that is, (i) coupling of sul-fonamide with 2a followed by (ii) intramolecular cyclization. Theinitial use of catalysts like 10% Pd/C, (PPh3)2PdCl2 and CuI in thepresence 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

Table 6Inhibition of luciferase by compounds 3aa, 3a and 3c at 30 lM

Entry Compound (30 lM) % PDE4B inhibition % Luciferase inhibition

1 3aa 94.69 ± 1.1 92.37 ± 2.22 3a 98.27 ± 14.8 98.42 ± 4.13 3c 99.97 ± 5.3 99.16 ± 3.7

6438 A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441

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 solventused. Thus, the Cu(OAc)2 mediated coupling-cyclization of 2a and2h was examined in a number of solvents (Table 4). The reactionafforded 1-methylsulfonyl substituted pyrrolo[2,3-b]quinoxaline(3aa or 3ab) when performed in 1,4-dioxane (Entries 1 and 2, Table4) but deprotected pyrrolo[2,3-b]quinoxaline (3a) in DMF (Entries3 and 4, Table 4). The use of DMSO provided 3-(phenylethynyl)qui-noxalin-2-ol (2ab) as a result of displacement of the chloro groupof 2a by a hydroxyl group (Entry 5, Table 4) the source of whichseemed 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) preparedwere well characterized by spectral (NMR, IR and MS) data, themolecular structure of 3a was further confirmed unambiguouslyby single crystal X-ray diffraction (Fig 3).21a The data was collectedat ambient temperature (298 K) on a Bruker SMART APEX CCD sin-gle crystal diffractometer using graphite monochromated Mo-Karadiation (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 withinthe range of 2.720–2.750 Å, and 2.730–2.770 Å it gives two types ofinteractions, that is, N2� � �H15 = 2.721 Å, N1� � �H9 = 2.741 Å, and

Figure 4. 3D binding orientation and H-bond interaction

N1� � �H15 = 2.734 Å, N2� � �H9 = 2.764 Å for the compound 3a.Through these weak van der Waals interactions21c it gives a 1Dnetwork in its crystal packing (see Fig. in ESI).

A plausible mechanism for the formation pyrrole ring fusedwith quinoxaline moiety is shown in Scheme 4. The reaction seemsto 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 thecompound 3. In the presence of Cu catalyst the reaction proceedsvia activation of the triple bond of E-1 by coordination to the tran-sition metal salt to form the p-complex E-2. The nucleophilicity ofthe sulfonamide nitrogen is enhanced in the presence of Et3N viagenerating a corresponding anion which participates in the intra-molecular nucleophilic attack to the metal coordinated triple bondin an endo dig fashion. This provides the metal-vinyl species E-3which upon subsequent in situ protonation regenerates the cata-lyst producing the N-sulfonyl derivative E-4. In a polar solvent suchas DMF the E-4 undergoes desulfonation to give the desired 2-substituted-1H-pyrrolo[2,3-b]quinoxalines (3) which perhaps wasnot favored in a less polar solvent such as 1,4-dioxane.22

All the compounds synthesized were initially tested for theirphosphodiesterase 4 (PDE4) inhibitory properties at 30 lM usinga luciferase reporter gene assay. We observed that a number ofcompounds 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 hitsrather than their real inhibition of PDE 4B. The results of this studyfor 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 thesecompounds are due to their inhibition of luciferase enzyme.23

Since examples of luciferase inhibitors are not common in the

s of ligand 3aa with firefly luciferase binding pocket.

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.

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441 6439

Table 7Docking simulation parameters of compounds 3aa, 3a and 3b

Entry Compound Docking score Lipophilic EvdWa Sitemapb LowMWc Binding energy (Kcal/mol)d

1 3aa �5.70 �6.1 �0.4 �0.5 �55.322 3a �7.10 �5.7 �0.4 �0.5 �57.083 3c �6.73 �4.7 �0.7 �0.4 �59.74

a Chemscore lipophilic pair term and fraction of the total protein–ligand vdW energy.b Ligand/receptor non-H-bonding polar/hydrophobic and hydrophobic/hydrophilic complementarity terms.c Reward for ligands with low molecular weight.d BE = E complex (minimized) � [E ligand (minimized) + E receptor].

Figure 7. Docked alignment of molecules 3aa, 3a, and 3c at the binding site pocket of luciferase enzyme.

6440 A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441

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.

To understand the binding modes of compounds 3aa, 3a, and 3cat 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 modeof the compounds with the binding site of the target protein wherethe 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, interactionwas observed between (i) one of the oxygen of SO group of 3aa andSer 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 aromaticrings were also observed in all these cases. These include pi–pistacking 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 stabilityto ligand–receptor complex. The docking simulation parametersobtained after docking of these molecules into the Firefly luciferaseprotein are summarized in Table 7. For the validation, the co-crystalligand M24 or [50-O-[(R)-[({3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]phenyl}carbonyl)oxy](hydroxy) phosphoryl]24 was re-dockedat the active site of protein. Overall, the data shown, suggests thatthese molecules interacted well with luciferase (Fig. 7).

In conclusion, the present research disclosed for the first timeluciferase inhibitory properties of 2-substituted pyrrolo[2,3-b]quinoxalines in vitro. These compounds were synthesized inmoderate yield from 3-alkynyl-2-chloroquinoxalines (preparedvia Pd/C-Cu mediated coupling of 2,3-dichloroquinoxaline withterminal 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 alsorepresents the first synthesis of pyrrolo[2,3-b]quinoxalines havingfree NH group. The crystal structure analysis of a representativecompound and its supramolecular interactions are presented.The luciferase inhibitory properties of some of these compoundssynthesized and tested were supported by their docking studieswhich suggested H-bonding interactions including one betweenone of the nitrogen of fused quinoxaline moiety and the Ala348 residue of luciferase. Overall, the present research can alertusers of luciferase reporter gene assays for achieving possiblefalse positive results that may occur due to the direct luciferaseinhibition.

Acknowledgments

The authors thank Professor J. Iqbal for encouragement andsupport. A. Nakhi thanks CSIR, New Delhi, India for awarding a Se-nior Research Fellowship. M. P. thanks DST, New Delhi, India forfinancial support (Grant no. SR/S1/OC-53/2009).

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6433–6441 6441

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.056.

References and notes

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.

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, 5625.19. 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 a solution of 2-chloro-3-(alkynyl)quinoxaline (2,1.8938 mmol) in DMF (4 mL), Cu(OAc)2 (0.0189 mmol),methanesulfonamide (2.8409 mmol) and triethylamine (2.8409 mmol) wereadded. The mixture was heated for the time indicated in Table 5. Aftercompletion of the reaction, the mixture was extracted with ethyl acetate(3 � 50 mL), washed with water (3 � 25 mL), dried over anhydrous sodiumsulphate (Na2SO4) and filtrated. The organic layer was collected andconcentrated under vacuum. The residue was purified by columnchromatography on silica gel using EtOAc-hexane.

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 = 2373.067) Å3, T = 298 K, Z = 8, Dc = 1.367 Mg m�3, (Mo-Ka) = 0.71073 mm�1, 10583reflections were measured with 2099 unique reflections (Rint = 0.0330), ofwhich 2099 (I > 2r(I)) were used for the structure solution. Final R1 (wR2) = 0.0670 (0.1879), 172 parameters. The final Fourier difference synthesisshowed minimum and maximum peaks of �0.326 and +0.434 e.Å�3,respectively. Goodness of fit = 1.068. Crystallographic data (excludingstructure factors) for 3a have been deposited with the CambridgeCrystallographic Data Centre as supplementary publication numbers CCDC890934.; (b) Sheldrick, G. M. SHELX-97; Program for Crystal Structure Solutionand Analysis: University of Gottingen, Gottingen, Germany, 1997; (c) Bondi, A.J. Phys. Chem. 1964, 68, 441.

22. Perhaps the polar amidic carbonyl group of DMF (e.g., Me2N–CH@O M Me2N+@CH–O�) 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 ofthe reviewers for his comments on this aspect of the proposed reactionmechanism.

23. The inhibition of luciferase was measured by using detection reagentcomponent of PDElight HTS assay kit (Lonza) according to manufacturer’srecommendations. Briefly, detection reagent containing luciferase enzyme andits substrate was incubated with 10 lM ATP and DMSO (vehicle control) or30 lM compound for 15 min. Luminescence values (RLUs) were measured by aMultilabel plate reader (Perklin Elmer 1420 Multilabel counter). Thepercentage of inhibition was calculated using the following formula: %inhibition = [(RLU of vehicle control � RLU of inhibitor)/(RLU of vehiclecontrol)] � 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 tothe binding affinity of a ligand with receptor [N–H� � �:N (13 kJ/mol or 3.1 kcal/mol) and N–H� � �:O (8 kJ/mol or 1.9 kcal/mol)]. In our molecular modelingstudies we kept optimum parameters (maximum distance 3.0 Å, minimumdonor angle 120� and minimum acceptor angle 90�) for the hydrogen bondingbetween ligand and receptor. In docking studies, the distance between theacceptor and donor atoms were found optimum, the molecule 3aa is having 2.92(SO� � �HN) and 2.7 (N� � �NH), the molecule 3a is having 2.90 (NH� � �O) and 2.63(N� � �NH), and the molecule 3c is having 2.80 (NH� � �O) and 3.00 (N� � �NH) (ligandgroups are presented in bold face). Thus, these hydrogen bonding interactionscontribute to the ligand binding affinity and to stabilize the ligand at theluciferase binding pocket as well (see Figs. 4–6).