Bioorganic & Medicinal Chemistry Letters 24 (2014) 4460–4465

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

NaSH in the construction of thiophene ring fusedwith N-heterocycles: A rapid and inexpensive synthesisof novel small molecules as potential inducers of apoptosis

http://dx.doi.org/10.1016/j.bmcl.2014.07.0960960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.E-mail address: manojitpal@rediffmail.com (M. Pal).

N

N S

NHAr

R1

R2

R3N

N

SR

A C

N

N

SNHAr

R1R3

B R4

Figure 1. Reported apoptosis inducers thienopyrimidines5 A and B andthieno[2,3-b]pyrazine/quinoxaline based molecules C.

Sunder Kumar Kolli a, Ali Nakhi b, Raghavender Medishetti b,c, Swapna Yellanki b,c, Pushkar Kulkarni b,c,R. Ramesh Raju a,⇑, Manojit Pal b,⇑a Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, A.P., Indiab Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, Indiac Zephase Therapeutics (an incubated company at the DRILS), University of Hyderabad Campus, Gachibowli, Hyderabad 500046, India

a r t i c l e i n f o

Article history:Received 14 July 2014Revised 29 July 2014Accepted 31 July 2014Available online 8 August 2014

Keywords:ThiophenePyrazineQuinoxalineApoptosis

a b s t r a c t

A facile construction of a thiophene ring fused with N-heterocycles has been achieved via the reaction ofNaSH with 2-chloro-3-alkynyl quinoxalines/pyrazines leading to novel 2-substituted thieno[2,3-b]pyra-zine/quinoxaline derivatives as potential inducers of apoptosis. Some of them showed encouraging phar-macological properties when tested in zebrafish.

� 2014 Elsevier Ltd. All rights reserved.

designed

Thiophene fused N-heterocycles have gained considerableinterest in the area of medicinal chemistry and new drug discov-ery. For example, thienopyridines (ticlopidine, clopidogrel, prasu-grel etc.) have been explored as a class of well known drugtargeting the platelet adenosine diphosphate (ADP) 2 receptor.1

Thienopyrimidines have been investigated for their remarkableand versatile pharmacological properties2 including antimicrobial3

and anti-avian influenza virus (H5N1)4 activities. In spite of nota-ble and promising biological activities displayed by these classesof heterocycles the pharmacological studies of thiophene fusedwith N-heterocycles other than pyridine/pyrimidine are ratheruncommon in the literature. For example, pharmacological evalu-ation of compounds that belong to thienopyrazine or thienoqui-noxaline class is not known perhaps due to their limited orcumbersome accessibility. However, being drug like small organicmolecules these heterocycles seemed to have considerable prom-ise as potential bioactive agents and could be attractive for medic-inal/bioorganic chemistry efforts.

In the light of reported apoptotic properties of thienopyrimi-dines5 (Fig. 1, A and B) we anticipated that thieno[2,3-b]pyra-zine/thieno[2,3-b]quinoxaline framework (Fig. 1, C) could be a

new template for the design and synthesis of novel agents target-ing apoptosis. Apoptosis6 or regulated cell death or programmedcell death is a process by which unwanted cells are eliminatedunder physiological and pathological conditions. While its complexmechanism involves many pathways, defects in these pathwaysseemed to cause a number of human diseases including malig-nancy.6 Cytotoxic anticancer agents are known to induce apoptosisas cancer is associated with lower degree of apoptosis.

A number of methods have been reported for the synthesis ofbenzothiophenes7–9 including (i) electrophilic cyclization of o-alkynyl thioanisole8 and (ii) SNAr-type reaction, cyclization of o-bromo alkynylbenzenes with sodium sulfide.9 However, no generalmethod has been reported for the synthesis of thieno[2,3-b]pyra-zine/thieno[2,3-b]quinoxaline derivatives as these heterocyclesare rather uncommon in the literature. We decided to adopt a

N

N

Cl

R

NaSH.xH2O

N

N

SR

DMF, 90 oC1-2 h

3 4

Scheme 1. NaSH mediated synthesis of 2-substituted thieno[2,3-b]pyrazine/quin-oxaline derivatives 4.

Table 3Optimization of reaction conditionsa

NPh

NaSH.xH2ON

Ph

S. K. Kolli et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4460–4465 4461

strategy similar to the methodology (ii) for the synthesis of our tar-geted thiophene fused N-heterocycles. Herein we report our initialfindings on NaSH as a reagent for inexpensive and rapid synthesisof 2-substituted thieno[2,3-b]pyrazine/quinoxaline derivativesbased on SNAr-type reaction-cyclization strategy (Scheme 1). Tothe best of our knowledge only one example on the synthesis of2-phenylthieno[2,3-b]quinoxaline in low yield (34%) has beenreported earlier10 via the reaction of 2-chloro-3-phenylethynylqui-noxaline with relatively expensive sodium sulfide and no substratescope and generality of this reaction has been examined. Moreover,the use of NaSH is not common for the construction of thiophenering fused with N-heterocycles.

Table 1Synthesis of 3-alkynyl-2-chloroquinoxalines 3a–ka

N

N Cl

Cl N

N

Cl

R

R (2)

10% Pd/C, PPh3, CuI,Et3N, EtOH, 60 oC 3a-k1a

Entry Alkyne (2); R = T (h) Product (3) Yieldb (%)

1 2a; Ph 3 3a 802 2b; C6H3F2-m,m 3 3b 783 2c; (CH2)2Me 4 3c 754 2d; (CH2)3Me 3 3d 705 2e; (CH2)4Me 4 3e 736 2f; (CH2)5Me 3 3f 727 2g; (CH2)7Me 4 3g 788 2h; (CH2)9Me 4 3h 759 2i; CMe3 3 3i 70

10 2j; (CH2)3OH 3 3j 6811 2k; (CH2)3Cl 4 3k 77

a All reactions were performed by using 1a (1.256 mmol), terminal alkyne 2(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.

Table 2Synthesis of 3-alkynyl-2-chloropyrazines 3l–ra

N

N Cl

Cl N

N

Cl

R

R (2)

10% Pd/C, PPh3, CuI,Et3N, EtOH, 60 oC 3l-r1b

Entry Alkyne (2); R = T (h) Product (3) Yieldb (%)

1 2b 3 3l 802 2c 4 3m 753 2d 4 3n 764 2e 3 3o 705 2f 4 3p 756 2i 4 3q 707 2j 4 3r 72

a See footnote ‘a’ of Table 1 (1b was used in place of 1a).b Isolated yield.

Initially, we prepared the required starting material 3 via a Pd/C-Cu catalyzed mono alkynylation of 2,3-dichloroquinoxaline/pyr-azine (1) (Tables 1 and 2).

To establish the optimum reaction conditions for the synthesisof 4 we examined the reaction of 2-chloro-3-(phenylethynyl)quin-oxaline 3a with commercially available NaSH.xH2O (Sigma-Aldrich, Cat: 16,152-7) in various solvents. All these reactions wereperformed at refluxing temperature. While the reaction did notproceed in a nonpolar solvent like 1,4-dioxane, acetonitrile orTHF (Table 3, entries 1, 3 and 4) and chlorinated solvent e.g.DCM (dichloromethane), DCE (1,2-dichloroethane) or chloroform(Table 3, entries 6–8) the desired product 4a was obtained in medi-ocre or low yield after 18 h when EtOH (Table 3, entry 2) or MeOH(Table 3, entry 5) was used. To improve the product yield furtherwe examined the use of DMF. To our satisfaction, the reaction pro-

N Cl N S3a 4a

Solvent

Entry Solvent T (h) Yieldb (%)

1 1,4-Dioxane 24 ND2 Ethanol 18 603 Acetonitrile 24 ND4 THF 24 ND5 Methanol 18 406 DCM 24 ND7 DCE 24 ND8 Chloroform 24 ND9 DMF 1 85c

(ND = not detected)a All the reactions were performed by using 3a (0.377 mmol), NaSH.xH2O

(1.50 mmol) in a solvent (2 mL) at its refluxing temperature.b Isolated yield.c Reaction was performed at 90 �C.

Table 4Synthesis of 2-substituted thieno[2,3-b]pyrazine/quinoxaline derivatives 4a

N

N

Cl

R

NaSH.xH2O

N

N

SR

DMF, 90 oC

3 4

Entry Alkyne (3) Product (4); R = T (h) Yieldb (%)

1 3a 4a; Ph 1 852 3b 4b; C6H3F2-m,m 1 833 3c 4c; (CH2)2Me 2 724 3d 4d; (CH2)3Me 2 755 3e 4e; (CH2)4Me 2 776 3f 4f; (CH2)5Me 2 737 3g 4g; (CH2)7Me 2 768 3h 4h; (CH2)9Me 2 729 3i 4i; CMe3 1 80

10 3j 4j; (CH2)3OH 1 7111 3k 4k; (CH2)3Cl 1 6912 3l 4l; C6H3F2-m,m 1 7813 3m 4m; (CH2)2Me 2 7314 3n 4n; (CH2)3Me 1 7015 3o 4o; (CH2)4Me 1 7516 3p 4p; (CH2)5Me 1 6817 3q 4q; CMe3 1 8018 3r 4r; (CH2)3OH 2 62

a All the reactions were performed by using 3a (0.377 mmol), NaSH.xH2O(1.50 mmol) in DMF (2 mL) at 90 �C.

b Isolated yield.

Figure 2. X-ray crystal structure of 4a (ORTEP diagram). Thermal ellipsoidal diagram is drawn at 30% probability (hydrogen atoms are omitted for clarity).

Figure 3. Results of apoptosis assay: The percentage induction of apoptosis caused by compounds 4a–c, 4i and 4l at different concentrations along with Methotrexate. All thestatistical analysis was performed using GraphPad Prism� software. Compound 4c and 4i showed dose dependent increase in apoptotic activity. Compound 4a showedincreased apoptotic activity at 1 and 3 lM.

Control Methotrexate 30µM 4c at 30µM

4b at 30µM 4i at 30µM 4l at 30µM

4a at 30µM

Figure 4. Representative images of the embryos treated with compounds assayed for apoptosis.

4462 S. K. Kolli et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4460–4465

Figure 5. Results of teratogenicity assay: Each embryo was scored based on their level of toxicity from 5 being non toxic and 0.5 being highly toxic. Statistical analysis forscoring was done using GraphPad Prism� software using One-way ANOVA. The graph represents the teratogenic scoring given compared to the positive controlPhenobarbital. Compounds 4c, 4b and 4l, were found to be safe at all concentrations, whereas 4a showed mild toxicity at 30 lM. The compound 4i appeared to be toxic at30 lM but relatively less toxic at 10 lM.

4b 4i 4l 4a

4c

Figure 6. Representative zebrafish images of teratogenicity assay of compounds tested at 30 lM.

Figure 7. Results of hepatotoxicity assay: All the statistical analysis was done using GraphPad Prism� software. The following graph represents the qualitative data of % liversize, % liver degeneration & % yolk sac retention of three compounds at different concentrations when compared to positive control Amiodarone.

S. K. Kolli et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4460–4465 4463

4c

4b 4i 4l 4a

Figure 8. Representative zebrafish images of hepatotoxicity assay of compounds tested at 30 lM.

Figure 9. The EC50 (apoptosis) and NOAEL (overall NOAEL = lowest NOAEL) of testcompound 4c (EC50 = 4.46 lM & NOAEL = 30 lM), 4b (EC50 = 10.32 lM &NOAEL = 30 lM), 4i (EC50 = 4.86 lM & NOAEL = 1 lM), 4l (EC50 = 9.82 lM &NOAEL = 30 lM) and 4a (EC50 = 8.5 lM & NOAEL = 10 lM), The overall therapeuticindex (ratio of NOAEL/EC50) of 4c is 6.72, 4b is 2.90, 4i is 0.20, 4l is 3.05 and 4a is1.17.

4464 S. K. Kolli et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4460–4465

ceeded well in this solvent at 90 �C affording 4a in 85% yield within1 h (Table 3, entry 9).11 We were delighted with this observationand decided to use this condition for our further studies.

In order to expand the generality and scope of this methodologywe examined the reaction of various alkynes (Table 4). The hetero-aryl alkyne 3 containing groups like aryl (3a–b and 3l), alkyl (3c–iand 3m–q), hydroxyalkyl (3j and 3r), chloroalkyl (4k) substituentson the triple bond participated well in the reaction to give thedesired products in acceptable yields. Similar reactivities of 3 wereobserved irrespective of the presence of quinoxaline (3a–k) andpyrazine (3l–r) ring under the conditions employed. Altogethereighteen compounds were synthesized by using this method(Table 4) and characterized by spectral (NMR, IR and MS) data.Additionally, the molecular structure of 4a was further confirmedunambiguously by single crystal X-ray diffraction (Fig. 2).12

Table 5Summary of pharmacological evaluations of compounds 4c, 4b, 4i, 4l and 4a

Pharmacological evaluations

Tests Endpoint

Apoptosis Acridine Orange staining of apoptotic cellsTeratogenecity Morphological assessment of phenotypic changesHepatotoxicity Morphological assessment of phenotypic changesOverall Therapeutic Index Ratio of NOAEL/EC50 (Overall NOAEL = lowest NOAEL)

Having synthesized a number of target molecules we then eval-uated their potential to induce apoptosis in Zebrafish embryos13

along with a known drug methotrexate14 at 30 lM. Based on theirconsiderable effects in the present assay of apoptosis compounds4a–c, 4i and 4l were further tested at 1, 3, 10 and 30 lM along withmethotrexate (Figs. 3 and 4). It is evident that compound 4c and 4ishowed dose dependent increase in apoptotic activity, whereas 4ashowed increased apoptotic activity at 1 and 3 lM.

These compounds were also evaluated for their potential toxic-ities like teratogenicity and hepatotoxicity in Zebrafish embryo at arange of 1.0–30 lM. The toxicological evaluation was carried out ina blinded fashion. All the embryos in control group were foundnormal. Phenobarbital (3 mM) and Amiodarone (30 lM) was usedas positive controls for teratogenicity and hepatotoxicty assay,respectively. In the teratogenicity assay (Figs. 5 and 6) 4c, 4b and4l, were found to be safe at all concentrations, whereas 4a showedmild toxicity at 30 lM. Compound 4i was found to be toxic at30 lM but relatively less toxic at 10 lM. In hepatotoxicity assay(Figs. 7 and 8) all the embryos in control group were found normal.Amiodarone (30 lM) was used as a positive control that showedtoxic effects in this assay. The compounds 4c, 4b, 4i, 4l, 4a wereanalyzed for their hepatotoxicity at 1, 3, 10 and 30 lM. Amongthe compounds 4c, 4b and 4a was found to be safe at all concentra-tions whereas 4i was found to be toxic at 10 lM and lethal at30 lM. The compound 4l was found to be safe at 1, 3, 10 &30 lM. Based on the summary of EC50 values (apoptosis), NOAEL(No Observed Adverse Effect Level) and the overall therapeuticindex (Fig. 9 and Table 5) the safety order of the tested compoundsis as follows: 4c > 4l > 4b > 4a > 4i. Overall, the present class ofcompounds appeared to be novel apoptotic agents of furtherinterest.

In conclusion, a general, rapid and inexpensive synthesis of 2-substituted thieno[2,3-b]pyrazine/quinoxaline derivatives havebeen achieved via the reaction of NaSH with 2-chloro-3-alkynylquinoxalines/pyrazines under mild conditions. This is the firstexample of using NaSH to construct the thiophene ring fused withN-heterocycles via a SNAr-type reaction-cyclization strategy. These

Test compounds data

Positive Control Parameters 4c 4b 4i 4l 4a

Methotrexate EC50 4.46 10.32 4.89 9.82 8.5Phenobarbital NOAEL 30 30 3 30 10Amiodarone NOEAL 30 30 1 30 10— Therapeutic Index 6.72 2.90 0.20 3.05 1.17

S. K. Kolli et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4460–4465 4465

heterocycles were designed as potential inducers of apoptosis andwere tested for apoptosis, teratogenicity and hepatotoxicity in zeb-rafish embryos. Several of them showed encouraging pharmaco-logical properties and therefore seemed to have potentialmedicinal value. The newly developed synthetic methodology pre-sented here might be useful in accessing a library of small mole-cules based on thiophene fused with N-heterocycles.

Acknowledgments

The authors thank management of DRILS for support.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2014.07.096.

References and notes

1. Alexopoulos, D. Cardiology 2014, 127, 211. http://dx.doi.org/10.1159/000357399.

2. De Clercq, E. J. Med. Chem. 1986, 29, 1561.3. Hassan, N. A.; Hegab, M. I.; Rashad, A. E.; Fahmy, A. A.; Abdel-Megeid, F. M. E.

Nucleos. Nucleot. Nucleic Acids 2007, 26, 379.4. Rashad, A. E.; Shamroukh, A. H.; Randa, E. A. M.; Mostafa, A.; Elshesheny, R.;

Kandil, A.; Ali, M. A.; Banert, K. Eur. J. Med. Chem. 2010, 45, 5251.5. Cai, S. S.; Kemnitzer, W. E.; Sirisoma, N. S.; Zhang, H. -Z. US Patent Application

number US 2007/0099877 A1, May 3, 2007.6. Thompson, C. B. Science 1995, 267, 1456.7. Zhang, X.; Zeng, W.; Yang, Y.; Huang, H.; Liang, Y. Synlett 2013, 1687.

8. (a) Mehta, S.; Larock, R. C. J. Org. Chem. 2010, 75, 1652; (b) Cho, C.-H.;Neuenswander, B.; Larock, R. C. J. Comb. Chem. 2010, 12, 278; (c) Sanz, R.;Guilarte, V.; Hernando, E.; Sanjuán, A. M. J. Org. Chem. 2010, 75, 7443; (d) Cho,C.-H.; Neuenswander, B.; Lushington, G. H.; Larock, R. C. J. Comb. Chem. 2009,11, 900; (e) Mehta, S.; Waldo, J. P.; Larock, R. C. J. Org. Chem. 2009, 74, 1141; (f)Ebata, H.; Miyazaki, E.; Yamamoto, T.; Takimiya, K. Org. Lett. 2007, 9, 4499; (g)Zhou, Y.; Liu, W.-J.; Ma, Y.-G.; Wang, H.-L.; Qi, L.-M.; Cao, Y.; Wang, J.; Pei, J. J.Am. Chem. Soc. 2007, 129, 12386.

9. (a) Kashiki, T.; Shinamura, S.; Kohara, M.; Miyazaki, E.; Takimiya, K.; Ikeda, M.;Kuwabara, H. Org. Lett. 2009, 11, 2473; (b) Patel, M. V.; Rohde, J. J.; Gracias, V.;Kolasa, T. Tetrahedron Lett. 2003, 44, 6665.

10. Ames, D. E.; Ismail, M. J. Chem. Soc., Perkin Trans. 1 1980, 1384.11. (a) The use of NMP as a solvent was also found to be equally effective. However

we preferred DMF over NMP as the solubility of starting alkynes was found tobe better in DMF.(b) Though the reaction proceeded well when Na2S was used in place of NaSHin EtOH (cf Shartsberg, M. S.; Ivanchikova, I. D. ARKIVOC 2003, xiii, 87) but amixture of products i.e. 4a and the corresponding furo derivative (2-phenylfuro[2,3-b]quinoxaline) was obtained perhaps due to the participationof moisture contaminated with EtOH.

12. Crystal data of 4a: Molecular formula = C16H10N2S1, Formula weight = 262.33,Monoclinic, C2/c, a = 7.0273(12) Å, b = 4.8082(8) Å, c = 18.213(3) Å, V = 4500.00(2) Å3, T = 293 K, Z = 9, Dc = 1.416 Mg m�3, l(Mo-Ka) = 0.247 mm�1, 20983reflections were measured with 3216 unique reflections (Rint = 0.0298), ofwhich 3216 (I > 2r(I)) were used for the structure solution. Final R1 (wR2) = 0.0836 (0.2257), 295 parameters. Goodness of fit = 1.028. Crystallographicdata (excluding structure factors) for 4a have been deposited with theCambridge Crystallographic Data Center as supplementary publicationnumber CCDC No. 964922.

13. (a) Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish(Danio rerio), 4th ed.; University of Oregon Press: Eugene, OR, 2000; (b) Nakhi,A.; Archana, S.; Seerapu, G. P.; Chennubhotla, K. S.; Kumar, K. L.; Kulkarni, P.;Haldar, D.; Pal, M. Chem. Commun. 2013, 6268; (c) Panzica-Kelly, J. M.; Zhang,C. X.; Danberry, T. L.; Flood, A.; DeLan, J. W.; Brannen, K. C.; Augustine-Rauch,K. A. Birth Defects Res. B Dev. Reprod. Toxicol. 2010, 89, 382. http://dx.doi.org/10.1002/bdrb.20260.

14. Chen, Y.-X.; Lv, W.-G.; Chen, H.-Z.; Ye, F.; Xie, X. Eur. J. Obstet. Gynecol. Repord.Biol. 2009, 142, 107.