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European Journal of Medicinal Chemistry 86 (2014) 270e278

Contents lists avai

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Preliminary communication

Ligand-free Pd-catalyzed CeN cross-coupling/cyclization strategy: Anunprecedented access to 1-thienyl pyrroloquinoxalines for the newapproach towards apoptosis

Sunder Kumar Kolli a, 1, Ali Nakhi b, 1, Sivakumar Archana b, c, Maneesha Saridena b,Girdhar Singh Deora d, Swapna Yellanki b, e, Raghavender Medisetti b, e,Pushkar Kulkarni b, e, 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 500046, Indiac Manipal College of Pharmaceutical Science, Manipal University, Manipal 576104, Indiad School of Pharmacy, University of Queensland, Brisbane QLD4072, Australiae Zephase Therapeutics (an incubated company at DRILS), University of Hyderabad Campus, Gachibowli, Hyderabad 500046, India

a r t i c l e i n f o

Article history:Received 2 April 2014Received in revised form14 August 2014Accepted 16 August 2014Available online 17 August 2014

Keywords:PalladiumPyrroloquinoxalinePDE4ApoptosisZebrafish

* Corresponding authors.E-mail addresses: manojitpal@rediffmail.com, palm

1 Equal contributor.

http://dx.doi.org/10.1016/j.ejmech.2014.08.0570223-5234/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

The link between PDE4 and apoptosis prompted us to design and synthesize for the first time a series ofnovel 1-thienyl pyrroloquinoxalines as potential PDE4 inhibitors/apoptotic agents. A ligand-free Pd-catalyzed CeN cross-coupling/cyclization strategy has been developed for the rapid and milder access tothis class of compounds some of which showed interesting pharmacological properties when testedin vitro and in zebrafish embryos.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

The development of robust chemical methodologies leading tonovel class of compounds for the exploration of new mechanisticpathways in pharmacology is the central focus of chemical science/chemical biology.

The cyclic adenosine monophosphate (cAMP) specific phos-phodiesterase 4 (PDE4) is one of the super family of enzymes calledPDEs (precisely PDE1ePDE11) each member of which degradeeither cAMP or cGMP (cyclic guanosine monophosphate) or both[1]. While PDE4 has been the target of several inflammatory dis-eases including COPD and asthma recent studies have indicatedthat inhibitors of PDE4 could be effective and selective promoters ofapoptosis in malignant cells without affecting normal cells [2]. Forexample, PDE4 inhibitors have been reported to induce apoptosis in

anojit@yahoo.co.uk (M. Pal).

served.

model of three lymphoid malignancies e.g. acute lymphoblasticleukemia (ALL), B-cell chronic lymphocytic leukemia (B-CLL) anddiffuse large B-cell lymphoma (DLBCL) (Fig. 1) [3]. Thus targetingPDE4 can be beneficial for the development of potential anticanceragents. In our endeavor for the development of novel inhibitors ofPDE4 [4] we became interested in the design and synthesis of 1-thieno substituted pyrrolo[2,3-b]quinoxalines as a new class ofPDE4 inhibitors that can induce apoptosis. Herein we report ourpreliminary results of this study.

In our earlier effort we have reported a series of 1,3-disubstituted pyrrolo[2,3-b]quinoxalines [5] (A, Fig. 2) as in-hibitors of PDE4 without inhibiting the luciferase [6]. In furthercontinuation of this work we decided to replace the 1-aryl group ofA by a thienyl moiety to afford new and potential inhibitors B(Fig. 2). We anticipated that due to the well known bioisosterism ofthiophene ring with benzene the newly designed molecules (B)would retain the PDE4 inhibitory properties of A. Additionally, inview of the fact that the thiophene ring being integral part ofseveral anticancer agents/drugs [7,8] as well as cytotoxic/apoptotic

Pluripotentstem cell

Multipotentialprogenitor

LymphoidprecursorMyeloid

precursor

B-Cellprecursor

T-Cell

B-CLL

ALL

DLBCLPDE4inhibitors

PDE4inhibitors

Apoptosis

PDE4inhibitors

Fig. 1. Partial representation of apoptosis induced by PDE4 inhibitors.[3].

N

N

NAr

S

N

N

N

A B

Fig. 2. Design of new PDE4 inhibitors/apoptotic agents.

N

N

NAr

SCO2Et

R = H / MeAr = C6H5= C4H4Me-p

S

H2N

OEt

O

Pd(OAc)2, tBuOKDMF, 80 oC, 2-3 h

4

5

R

N

N Cl

Cl N

N

Cl

Ar

Ar

10% Pd/C, PPh3CuI, Et3N, EtOH2-4 h, 60 oC 2

R R

1

Scheme 1. Synthesis of 1-thienyl substituted pyrrolo[2,3-b]quinoxalines (5) via Pd-catalyzed CeN cross-coupling/cyclization strategy.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278 271

agents [9] we expected potential apoptosis inducing properties ofB.

A number of methods have been reported for the synthesis of 1-aryl substituted pyrrolo[2,3-b]quinoxalines by our group [5,10] andothers [11]. For example, this class of compounds has been syn-thesized by the action of primary aliphatic or aromatic amines on 2-chloro-3-alkynylquinoxalines prepared via Sonogashira coupling of2,3-dichloroquinoxaline and terminal alkynes [11d]. Notably, whilethe synthesis of pyrrolo[2,3-b]quinoxaline having thienyl moiety atC-2 has been reported [11b] the preparation of its isomeric 1-thienyl substituted analog is not known in the literature. Ourinitial attempt to prepare this class of compounds via the reactionof a 2-aminothiophene derivative (e.g. ethyl 2-aminothiophene-3-carboxylate) with 2-chloro-3-alkynylquinoxaline under the re-ported conditions [11d] failed. We then focused on a Pd-basedstrategy i.e. a tandem Buchwald type coupling followed by intra-molecular cyclization in the same pot [12]. While palladium-catalyzed CeN cross-coupling/cyclization [13,14] of o-alkynylha-lo(hetero)arenes with primary amines, affording indoles andrelated heterocyclic derivatives have been reported earlier thesemethodologies involved the use of an expensive ligand e.g. tri-tert-butylphosphine or (silanyloxyphenyl)phosphine and longer reac-tion time (>10 h). We have observed that the Pd-catalyzed couplingof 2-chloro-3-alkynylquinoxalines (2) with ethyl 2-aminothiophene-3-carboxylate derivatives (4) proceeds smoothlyin the absence of any ligand to afford the desired 1-thienylsubstituted pyrrolo[2,3-b]quinoxalines (5) within 2e3 h (Scheme1).

2. Results and discussion

The starting material i.e. 2 was prepared from 2,3-dichloroquinoxaline according to a known procedure [6] whereas

4was prepared from the corresponding ketone (or equivalent) 3 byusing a Gewald type reaction (Table 1) [15].

To establish a relatively mild and faster optimized reactionconditions the coupling of 2-chloro-3-(phenylethynyl)quinoxaline(2a) with ethyl-2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (4d) was carried out at 80 �C in the presence of anumber of transition metal catalysts (Table 2). The reaction did notproceed in the presence of 10% Pd/C or Pd(PPh3)2Cl2 or CuI as acatalyst and Et3N as a base (entries 1e3, Table 2). While the changeof base from Et3N to tBuOK was also found to be unproductive(entry 4, Table 2) the change of catalyst from CuI to Cu(OAc)2afforded the desired product 4a albeit in low yield (entry 5, Table 2).The use of Et3N and DBU did not improve the product yield (entry 6and 7, Table 2). Notably, Pd(OAc)2 in place of Cu(OAc)2 increased theyield of 4a to 75% (entry 8, Table 2) with the significant decrease inreaction time though K2CO3 in place of tBuOK was found to beineffective (entry 8, Table 2). All these reactions were performed inDMF. The use of other solvents e.g. DMSO and acetonitrile was alsoexamined but found to be less effective. Overall, the combination ofPd(OAc)2 and tBuOK in DMF was found to be optimum and used toprepare the library of our target compounds (Table 3). Apart fromsynthesizing a range of 1-thienyl substituted pyrrolo[2,3-b]qui-noxalines (entries 1e21, Table 3) we also prepared 1-thienylsubstituted pyrrolo[2,3-b]pyrazine (entry 22, Table 3) successfullyto demonstrate the utility and scope of this methodology. Exceptfor few cases all the compounds were generally obtained in good to

acceptable yields within 2e3 h.While mechanistically (Scheme 2) the reaction seems to pro-

ceed via Pd-catalyzed heteroaryl amination of 2 followed by basemediated cyclization of the resultant 3-alkynyl quinoxalin-2-amineE-3 the reason for faster (2e3 h) reaction under milder (80 �C)conditions was not clearly understood. The higher reactivity ofchloro group at the azomethine carbon (i.e. CleC]Ne) towards thePd(0) catalyst generated in situ could be the reason for suchobservation. Additionally, the participation of the nitrogen lonepair in the resultant Pd(II)-complex E-1 [formed after oxidativeaddition of Pd(0) to the chloro compound 2] perhaps aided thefaster displacement of the chloro group by the anion of reactantamine 4 to afford the intermediate E2. The reductive elimination ofPd(0) from E2 completed the catalytic cycle affording the alkyne E-3which on intramolecular cyclization yielded the desired product 5(Scheme 2).

Most of the compounds synthesized (5aev) were evaluated fortheir PDE4B inhibitory properties in vitro using an enzyme basedassay [16]. The PDE4B isolated from Sf9 cells was used to assessthese compounds along with a reference compound rolipram, a

Table 1Preparation of ethyl 2-aminothiophene-3-carboxylate derivatives (4) via Gewaldreaction.a

SNH2

EtO

43

S8, morpholineEtOH, 60°C6-12 h

NCCH2CO2EtO

O

Entry Ketone Time (h) Product Yieldb (%)

1

3a

8

4a

75c

2

3b12

4b

72

3 Ph

O

3c12

S NH2

EtOOPh

4c

64

4

3d

6

4d

60

5

3e

8

4e

78

6

3f

12

4f

65

7

3g

7

4g

67

a All the 2-aminothiophene derivatives (2) were prepared by using the corre-sponding ketone (3) with an ethyl cyanoacetate (1.0 equiv) in the presence ofelemental sulfur (1.0 equiv), morpholine (1.0 equiv) in EtOH at 60 �C.

b Isolated yields.c The compound 4a was prepared by using 1,4-dithiane-2,5-dithiol (1.0 equiv),

ethyl cyanoacetate (1.0 equiv), Et3N (1.0 equiv) in DMF under a Gewald reactioncondition.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278272

well known inhibitor of PDE4. The compounds that showed sig-nificant inhibition of PDE4B at 30 mM include 5a (85%), 5v (62%) and5l (78%) [rolipram (89%)]. To understand their nature of in-teractions with the PDE4B protein docking studies were performedusing 5a, 5v and 5l (see Table S-1 and Figs. S-1, 2, 3, 4 in ESI). Thestudy suggested that both 5a (G Score �7.62) (Fig. 3) and 5l (GScore �7.31) interacted through an H-bond as well as a pepstacking interaction with the His-234 residue of PDE4B. Themolecule 5v (G Score �7.20) formed an H-bond with the Gln-443residue. Some hydrophobic interactions were also observed in allthese cases with Phe-446, Leu-393 and Tyr-233.

These inhibitors of PDE4 were then tested for their apoptoticactivities in Zebrafish embryos [17] at 1, 3, 10 and 30 mM along witha known drug methotrexate [18] at 30 mM (Figs. 4 and 5). All thesecompounds showed considerable effects in the present apoptosisassay. While the extent of apoptosis was increased up to 3 mM incase of compound 5v the embryos were found dead at 10 and30 mM. The compound 5l showed increased apoptotic activities at 3,10 and 30 mM compared to that observed at lower concentration i.e.1 mM. The compound 5a showed consistent increase in apoptoticactivities with the increase in concentrations up to 10 mM thoughthe activity was not increased further at 30 mM. Overall, the com-pound 5l and 5a appeared to be encouraging in this assay.

These compounds were also evaluated for their potential tox-icities like teratogenicity and hepatotoxicity in Zebrafish embryo ata range of 1.0e30 mM. The toxicological evaluation was carried outin a blinded fashion. All the embryos in control group were foundnormal. Phenobarbital (3 mM) and Amiodarone (30 mM) was usedas positive controls for teratogenicity and hepatotoxicty assay,respectively. In the teratogenicity assay (Figs. 6 and 7), the com-pound 5l was found to be non-toxic with no severe adverse effectsat all concentrations tested. The compound 5v was found to belethal to embryos at 10 and 30 mM whereas 50% embryos werefound alive at 3 mM though these embryos have shown majormorphological deformities. All the embryos were found alive at1 mM (indicating that 1 mM of 5v might be safer than 3 mM ofPhenobarbital) but had minor morphological deformities at swimbladder and lower jawwhen compared to the control. In the case ofcompound 5a out of 6 embryos, three at 3 mM and four at 10 and30 mM were found dead. At 1 mM all the embryos were found to besafe with minimal morphological deformities at lower jaw. In

Table 2Effect of reaction conditions on the CeN coupling/cyclization of 2a with 4d.a

N

NPh

Cl

CatalystBase

DMF80oC

N

N

NPh

SCO2Et+

SH2N

EtO2C

2a 4d 5d

Entry Catalyst Base Time (h) Yieldb (%)

1 10% Pd/C Et3N 12 ND2 Pd(PPh3)2Cl2 Et3N 12 ND3 CuI Et3N 12 ND4 CuI tBuOK 12 ND5 Cu(OAc)2 tBuOK 12 306 Cu(OAc)2 Et3N 12 ND7 Cu(OAc)2 DBU 12 208 Pd(OAc)2 tBuOK 3 759 Pd(OAc)2 K2CO3 12 ND

a All reactions were carried out by using 2a (1.89 mmol), 4d (1.89 mmol), catalyst(0.019 mmol), base (1.89 mmol) in DMF (3 mL) at 80 �C.

b Isolated yield. ND ¼ Product not detected.

Table 3Synthesis of 1-thienyl substituted pyrrolo[2,3-b]quinoxalines/pyrrolo[2,3-b]pyr-azine (5) via Pd-catalyzed CeN cross-coupling/cyclization strategy.a

N

NAr

ClN

N

NAr

T

R+

Pd(OAc)2tBuOK

DMF, 80 oC2-3 h2 4 5

R

T-NH2

Entry Alkyne (2); R & Ar¼ Amine(4)

Time(h)

Product (5) T¼ Yieldb

(%)

1 2a; H & Ph 4a 2S

CO2Et

5a76

2 2a 4b 3 SCO2Et

Me 5b

67

3 2a 4c 3 SCO2Et

Ph 5c

60

4 2a 4d 2 SCO2Et

5d

75

5 2a 4f 2 SCO2Et

5e

56

6 2a 4g 2S

CO2Et

NBoc 5f

68

7 2b; Me & Ph 4b 3 SCO2Et

Me 5g

71

8 2b 4c 3 SCO2Et

Ph 5h

70

9 2b 4d 2 SCO2Et

5i

65

10 2b 4e 2 SCO2Et

5j

72

Table 3 (continued )

Entry Alkyne (2); R & Ar¼ Amine(4)

Time(h)

Product (5) T¼ Yieldb

(%)

11 2c; H & C6H4Me-p 4b 3 SCO2Et

Me 5k

70

12 2c 4c 2 SCO2Et

Ph 5l

68

13 2c 4d 2 SCO2Et

5m

67

14 2c 4e 2 SCO2Et

5n

70

15 2c 4f 2 SCO2Et

5o

62

16 2c 4g 3

SCO2Et

NBoc 5p

66

17 2d; Me& C6H4Me-p 4b 3 SCO2Et

Me 5q

64

18 2d 4c 3 SCO2Et

Ph 5r

68

19 2d 4d 2 SCO2Et

5s

58

20 2d 4e 2 SCO2Et

5t

60

(continued on next page)

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278 273

Table 3 (continued )

Entry Alkyne (2); R & Ar¼ Amine(4)

Time(h)

Product (5) T¼ Yieldb

(%)

21 2d 4b 3 SCO2Et

Me 5u

53

22 2ec 4d 3 SCO2Et

5v

51

a All reaction were carried out by using 2 (1.89 mmol), Pd(OAc)2 (0.019 mmol), 4(1.89 mmol), tBuOK (1.89 mmol) in DMF (3 mL) at 80 �C.

b Isolated yield.c 2-Chloro-3-(phenylethynyl)pyrazine (2e) was used as the reactant alkyne.

Fig. 3. The binding mode and interactions of molecule 5a at the inhibitor binding siteof PDE4B (PDB ID: 1XMY).

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278274

hepatotoxicity assay (Figs. 8 and 9) the compound 5l showedtoxicity at 30 mM but found to be non-toxic at 1, 3 and 10 mM. Thecompound 5v though did not show toxicity at 1 mM but was foundto be toxic at 3 mM. Indeed the embryos were found dead at 10 and30 mM. The compound 5a did not show any toxicity at 1 and 3 mM,though the toxicity was increased significantly at 10 and 30 mM.Based on the summery (see Table S-2 and S-3 in ESI) of EC50 values(apoptosis), NOAEL (No Observed Adverse Effect Level) for terato-genicity and the overall therapeutic index (Fig. 10) the PDE4 in-hibitor 5l appeared to be a novel apoptotic agent of further interest[19].

3. Conclusions

In conclusion, we have developed a ligand/additive-free Pd-catalyzed CeN cross-coupling/cyclization of 2-chloro-3-alkynylquinoxalines with ethyl 2-aminothiophene-3-carboxylatederivatives for the rapid and milder access to novel 1-thienyl pyr-roloquinoxalines as potential PDE4 inhibitors. These compoundswere designed for the first time as potential apoptotic agents basedon the reported link between PDE4 and apoptosis. Some of thesecompounds showed encouraging PDE4 inhibitory propertiesin vitro that was supported by docking studies in silico. Thesecompounds were also screened for apoptosis, teratogenicity and

Pd(OAc)2

Pdo

N

N

PdII

ArR

Cl

2

4

N

N

PdII

ArR

N

S

CO2Et

N

NAr

R

N

S CO2Et

tBuOH

5

E-1

E-2

E-3

tBuOK

HN

S

CO2Et

H

tBuOK

Scheme 2. Proposed reaction mechanism.

hepatotoxicity in zebrafish embryos. In view of their observedpharmacological properties and the fact that most cytotoxic anti-cancer agents are known to induce apoptosis the present class ofPDE4 inhibitors seemed to have potential medicinal value. Thesynthetic methodology presented here may find application in theconstruction of library of small molecules based on 1-thienyl pyr-roloquinoxaline framework.

4. Experimental

4.1. General method for the preparation of N-thiophenyl substitutedpyrrole[2,3-b]quinoxalines/pyrazines (5)

A mixture of 2-chloro-3-arylethynyl substituted quinoxalines/pyrazine (2) (1.8938 mmol), and ethyl 2-aminothiophene-3-carboxylate derivative (4) (1.8938 mmol) and KO-tBu(1.8938 mmol) in DMF solvent (3 mL) was stirred at 80 �C for 2e3 hunder nitrogen atmosphere. After completion of the reaction asindicated by TLC, the reaction mixture was diluted with water(15mL) and extractedwith ethyl acetate (3� 10mL). The combined

Fig. 4. The percentage induction of apoptosis caused by compounds 5v, 5l and 5a atdifferent concentrations along with Methotrexate. All the statistical analysis wasperformed using GraphPad Prism® software.

Fig. 5. Representative images of the embryos treated with compounds assayed for apoptosis.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278 275

organic layers were collected, dried over anhydrous Na2SO4, filteredand concentrated under low vacuum. The residue was purified bycolumn chromatography on silica gel (Merck,100e200mesh) usingn-hexane/ethyl acetate to afford the desired product.

4.2. PDE4 enzymatic assay

The inhibition of PDE4 enzyme was measured using PDE lightHTS cAMP phosphodiesterase assay kit (Lonza) according to man-ufacturer's recommendations. Briefly, 10 ng of in house purifiedPDE4B1 enzyme was pre-incubated either with DMSO (vehicle

Fig. 6. Each embryo was scored based on their level of toxicity from 5 being non-toxicand 0.5 being highly toxic. Statistical analysis for scoring was done using GraphPadPrism® software using two-way ANOVA. The graph represents the teratogenic scoringgiven compared to the positive control Phenobarbital.

Fig. 8. All the statistical analysis was done using GraphPad Prism® software. The graphrepresents the qualitative data of % liver size, % liver degeneration & % yolk sacretention of three compounds at different concentrations when compared to positivecontrol Amiodarone.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278276

control) or compound for 15 min before incubation with the sub-strate cAMP (5 mM) for 1 h. The reaction was halted with stop so-lution and reaction mix was incubated with detection reagent for10 min in dark. Luminescence values (RLUs) were measured by aMultilabel plate reader (Perklin Elmer 1420Multilabel counter).Thepercentage of inhibition was calculated using the following for-mula: % inhibition ¼ [(RLU of vehicle control � RLU of inhibitor)/(RLU of vehicle control)] � 100.

Fig. 7. Representative images

4.3. Teratogenicity assay

(a) Drug exposure: 1 day post fertilization (dpf) embryos werecollected and checked for its developmental stage and condition.Embryos were de-chorinated using 0.5 mg/ml pronaseE treatmentfollowed by washes with E3 medium. Working concentrations ofthe compound was prepared by serially diluting the compound infinal concentration of 0.1% DMSO in E3 medium. Embryos (n ¼ 6)were distributed in 24 well plate followed by addition of drug andincubated at 28.5 �C until 5 dpf. (b)Morphological scoring: Embryoswere removed from drug solution, washed and allowed to anes-thetize using tricaine (0.008%) and observed for morphologicaltoxicity parameters like Body Shape, Somites, Notochord, Tail,

of teratogenicity assay.

Fig. 9. Representative images of hepatotoxicity assay.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278 277

Intestine, Fins, Brain, Upper jaw, Heart, Lower jaw, Liver and SwimBladder. Morphological assessment and scoring was done accord-ing to the procedure described [17b,c].

4.4. Hepatotoxicity assay

In this assay 4 dpf embryos were exposed to various concen-tration of test compound prepared from stock solutions asdescribed above. The embryos were distributed in 24 well platesalong with 250 ml of 0.1% DMSO with 6 embryos in each well. Eachwell is added with the respective working stock solutions to obtainthe final concentration of 1, 3, 10 and 30 mM concentration of the

Fig. 10. The EC50 (apoptosis) and NOAEL (teratogenicity) of test compound 5v(EC50 ¼ 1.732 mM & NOAEL ¼ 1.0 mM), 5l (EC50 ¼ 1.832 mM & NOAEL ¼ 3.0 mM) and 5a(EC50 ¼ 3.219 mM & NOAEL ¼ 1.0 mM). The overall therapeutic index (ratio of NOAEL/EC50) of 5v is 0.577, 5l is 1.637 and 5a is 0.310.

drug. The plate was incubated at 28 �C until 7 dpf. Embryos werewashed with E3 medium on 7 dpf and anesthetized using tricaine.The images of embryos treated with different compounds ofvarious concentrations are analyzed using Image J software fortheir liver size, liver degeneration and yolk sac retention and per-centages were calculated.

4.5. Apoptosis assay

24 hpf embryos were de-chorinated manually. 6 embryos weredistributed as two sets in each well of 24 well plates with 250 ml of0.1% DMSO. The working stock solutions were prepared by serialdilution as described earlier. Each well was added with 250 ml ofrespective concentration to obtain final working concentration.Embryos were incubated at 28 �C for 24 h and 48 h. The apoptoticeffects were checked at 24 h and 48 h by washing drug exposedembryos thricewith E3medium. Acridine orange (2 mg/ml) solutionof dye in E3 medium was added and incubated for 30 min. Theembryos were rinsed thoroughly twice in fresh E3 medium to washthe acridine orange solution. Stained embryos were anesthetizedwith tricaine and photographed under UV illumination using ZeissAxio CamMR camera attached to a Zeiss florescence microscope(GFP filter set: excitation 473, emission 520) under 5� magnifica-tion. The Images were taken and analyzed using Image J software.

Acknowledgments

AN thanks CSIR, for a research fellowship. Authors thank CSIR[Grant 02(0127)/13/EMR-II] and management of DRILS for supportand Dr. K. V. L. Parsa and his team for PDE4 assay.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278278

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2014.08.057.

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