Ligand-free Pd-catalyzed C-N cross-coupling/cyclization strategy: An unprecedented access to 1-thienyl pyrroloquinoxalines for the new approach towards apoptosis

Article abstract of DOI:10.1016/j.ejmech.2014.08.057

The link between PDE4 and apoptosis prompted us to design and synthesize for the first time a series of novel 1-thienyl pyrroloquinoxalines as potential PDE4 inhibitors/apoptotic agents. A ligand-free Pd-catalyzed C-N cross-coupling/cyclization strategy h

Full text of DOI:10.1016/j.ejmech.2014.08.057

European Journal of Medicinal Chemistry 86 (2014) 270e278
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Ligand-free Pd-catalyzed CeN cross-coupling/cyclization strategy: An
unprecedented access to 1-thienyl pyrroloquinoxalines for the new
approach 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., India
b Dr. Reddy's Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500046, India
c Manipal College of Pharmaceutical Science, Manipal University, Manipal 576104, India
d School of Pharmacy, University of Queensland, Brisbane QLD4072, Australia
e 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
a b s t r a c t
Article history:
The link between PDE4 and apoptosis prompted us to design and synthesize for the first time a series of
novel 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 to
this class of compounds some of which showed interesting pharmacological properties when tested
in vitro and in zebrafish embryos.
Received 2 April 2014
Received in revised form
14 August 2014
Accepted 16 August 2014
Available online 17 August 2014
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Palladium
Pyrroloquinoxaline
PDE4
Apoptosis
Zebrafish
1. Introduction
model of three lymphoid malignancies e.g. acute lymphoblastic
leukemia (ALL), B-cell chronic lymphocytic leukemia (B-CLL) and
The development of robust chemical methodologies leading to
novel class of compounds for the exploration of new mechanistic
pathways 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 called
PDEs (precisely PDE1ePDE11) each member of which degrade
either 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 indicated
that inhibitors of PDE4 could be effective and selective promoters of
apoptosis in malignant cells without affecting normal cells [2]. For
example, PDE4 inhibitors have been reported to induce apoptosis in
diffuse large B-cell lymphoma (DLBCL) (Fig. 1) [3]. Thus targeting
PDE4 can be beneficial for the development of potential anticancer
agents. In our endeavor for the development of novel inhibitors of
PDE4 [4] we became interested in the design and synthesis of 1-
thieno substituted pyrrolo[2,3-b]quinoxalines as a new class of
PDE4 inhibitors that can induce apoptosis. Herein we report our
preliminary 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 further
continuation of this work we decided to replace the 1-aryl group of
A by a thienyl moiety to afford new and potential inhibitors B
(Fig. 2). We anticipated that due to the well known bioisosterism of
thiophene ring with benzene the newly designed molecules (B)
would retain the PDE4 inhibitory properties of A. Additionally, in
view of the fact that the thiophene ring being integral part of
several anticancer agents/drugs [7,8] as well as cytotoxic/apoptotic
* Corresponding authors.
E-mail addresses: manojitpal@rediffmail.com, palmanojit@yahoo.co.uk (M. Pal).
Equal contributor.
1
http://dx.doi.org/10.1016/j.ejmech.2014.08.057
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278
271
4 was prepared from the corresponding ketone (or equivalent) 3 by
using a Gewald type reaction (Table 1) [15].
Pluripotent
stem cell
Multipotential
progenitor
To establish a relatively mild and faster optimized reaction
conditions 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 a
number of transition metal catalysts (Table 2). The reaction did not
proceed in the presence of 10% Pd/C or Pd(PPh3)2Cl2 or CuI as a
catalyst and Et3N as a base (entries 1e3, Table 2). While the change
Lymphoid
precursor
ALL
Myeloid
precursor
PDE4
inhibitors
t
of base from Et3N to BuOK was also found to be unproductive
(entry 4, Table 2) the change of catalyst from CuI to Cu(OAc)2
afforded 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 6
and 7, Table 2). Notably, Pd(OAc)2 in place of Cu(OAc)2 increased the
yield of 4a to 75% (entry 8, Table 2) with the significant decrease in
reaction time though K2CO3 in place of tBuOK was found to be
ineffective (entry 8, Table 2). All these reactions were performed in
DMF. The use of other solvents e.g. DMSO and acetonitrile was also
examined but found to be less effective. Overall, the combination of
Pd(OAc)2 and tBuOK in DMF was found to be optimum and used to
prepare the library of our target compounds (Table 3). Apart from
synthesizing a range of 1-thienyl substituted pyrrolo[2,3-b]qui-
noxalines (entries 1e21, Table 3) we also prepared 1-thienyl
substituted pyrrolo[2,3-b]pyrazine (entry 22, Table 3) successfully
to demonstrate the utility and scope of this methodology. Except
for few cases all the compounds were generally obtained in good to
PDE4
inhibitors
B-CLL
Apoptosis
B-Cell
precursor
PDE4
inhibitors
T-Cell
DLBCL
Fig. 1. Partial representation of apoptosis induced by PDE4 inhibitors.[3].
N
N
N
N
Ar
N
N
S
A
B
Fig. 2. Design of new PDE4 inhibitors/apoptotic agents.
Ar
R
N
N
R
N
N
Cl
Cl
Ar
agents [9] we expected potential apoptosis inducing properties of
B.
10% Pd/C, PPh3
CuI, Et3N, EtOH
2-4 h, 60 oC
Cl
A number of methods have been reported for the synthesis of 1-
aryl substituted pyrrolo[2,3-b]quinoxalines by our group [5,10] and
others [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 of
2,3-dichloroquinoxaline and terminal alkynes [11d]. Notably, while
the synthesis of pyrrolo[2,3-b]quinoxaline having thienyl moiety at
C-2 has been reported [11b] the preparation of its isomeric 1-
thienyl substituted analog is not known in the literature. Our
initial attempt to prepare this class of compounds via the reaction
of 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-based
strategy 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 and
related heterocyclic derivatives have been reported earlier these
methodologies 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 coupling
1
2
R = H / Me
Ar = C6H5
= C4H4Me-p
H2N
O
R
N
N
S
Ar
CO2Et
OEt
N
4
S
Pd(OAc)2, tBuOK
DMF, 80 oC, 2-3 h
5
Scheme 1. Synthesis of 1-thienyl substituted pyrrolo[2,3-b]quinoxalines (5) via Pd-
catalyzed CeN cross-coupling/cyclization strategy.
acceptable yields within 2e3 h.
While mechanistically (Scheme 2) the reaction seems to pro-
ceed via Pd-catalyzed heteroaryl amination of 2 followed by base
mediated cyclization of the resultant 3-alkynyl quinoxalin-2-amine
E-3 the reason for faster (2e3 h) reaction under milder (80 ꢀC)
conditions was not clearly understood. The higher reactivity of
chloro group at the azomethine carbon (i.e. CleC]Ne) towards the
Pd(0) catalyst generated in situ could be the reason for such
observation. Additionally, the participation of the nitrogen lone
pair in the resultant Pd(II)-complex E-1 [formed after oxidative
addition of Pd(0) to the chloro compound 2] perhaps aided the
faster displacement of the chloro group by the anion of reactant
amine 4 to afford the intermediate E2. The reductive elimination of
Pd(0) from E2 completed the catalytic cycle affording the alkyne E-
3 which on intramolecular cyclization yielded the desired product 5
(Scheme 2).
of
2-chloro-3-alkynylquinoxalines
(2)
with
ethyl
2-
aminothiophene-3-carboxylate derivatives (4) proceeds smoothly
in the absence of any ligand to afford the desired 1-thienyl
substituted pyrrolo[2,3-b]quinoxalines (5) within 2e3 h (Scheme
1).
Most of the compounds synthesized (5aev) were evaluated for
their PDE4B inhibitory properties in vitro using an enzyme based
assay [16]. The PDE4B isolated from Sf9 cells was used to assess
these compounds along with a reference compound rolipram, a
2. Results and discussion
The starting material i.e.
dichloroquinoxaline according to a known procedure [6] whereas
2
was prepared from 2,3-

272
S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278
Table 1
well known inhibitor of PDE4. The compounds that showed sig-
nificant inhibition of PDE4B at 30 M include 5a (85%), 5v (62%) and
Preparation of ethyl 2-aminothiophene-3-carboxylate derivatives (4) via Gewald
m
reaction.a
5l (78%) [rolipram (89%)]. To understand their nature of in-
teractions with the PDE4B protein docking studies were performed
using 5a, 5v and 5l (see Table S-1 and Figs. S-1, 2, 3, 4 in ESI). The
study suggested that both 5a (G Score ꢁ7.62) (Fig. 3) and 5l (G
EtO
O
O
NCCH2CO2Et
Score ꢁ7.31) interacted through an H-bond as well as a
pep
NH2
S8, morpholine
EtOH, 60°C
6-12 h
S
stacking interaction with the His-234 residue of PDE4B. The
molecule 5v (G Score ꢁ7.20) formed an H-bond with the Gln-443
residue. Some hydrophobic interactions were also observed in all
these cases with Phe-446, Leu-393 and Tyr-233.
3
4
Entry
1
Ketone
Time (h)
Product
Yieldb (%)
These inhibitors of PDE4 were then tested for their apoptotic
activities in Zebrafish embryos [17] at 1, 3, 10 and 30
a known drug methotrexate [18] at 30 M (Figs. 4 and 5). All these
compounds showed considerable effects in the present apoptosis
assay. While the extent of apoptosis was increased up to 3 M in
case of compound 5v the embryos were found dead at 10 and
30 M. The compound 5l showed increased apoptotic activities at 3,
10 and 30 M compared to that observed at lower concentration i.e.
M. The compound 5a showed consistent increase in apoptotic
activities with the increase in concentrations up to 10 M though
the activity was not increased further at 30 M. Overall, the com-
mM along with
m
8
75c
m
3a
4a
m
m
1
m
m
2
3
12
12
72
64
m
3b
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 at
4b
EtO
a range of 1.0e30
in a blinded fashion. All the embryos in control group were found
normal. Phenobarbital (3 mM) and Amiodarone (30 M) was used
mM. The toxicological evaluation was carried out
O
O
Ph
Ph
m
NH2
3c
S
as 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 effects
at all concentrations tested. The compound 5v was found to be
4c
lethal to embryos at 10 and 30
found alive at 3 M though these embryos have shown major
morphological deformities. All the embryos were found alive at
M (indicating that 1 M of 5v might be safer than 3 mM of
Phenobarbital) but had minor morphological deformities at swim
bladder and lower jaw when compared to the control. In the case of
mM whereas 50% embryos were
4
5
6
8
60
78
m
3d
3e
4d
1
m
m
compound 5a out of 6 embryos, three at 3
mM and four at 10 and
30 M were found dead. At 1 M all the embryos were found to be
m
m
safe with minimal morphological deformities at lower jaw. In
4e
Table 2
6
12
65
67
Effect of reaction conditions on the CeN coupling/cyclization of 2a with 4d.a
N
4f
Ph
Ph
CO2Et
Catalyst
Base
EtO2C
H2N
4d
N
N
N
N
3f
+
S
Cl
DMF
80oC
S
2a
5d
7
7
4g
Entry
Catalyst
Base
Time (h)
Yieldb (%)
1
2
3
4
5
6
7
8
9
10% Pd/C
Pd(PPh3)2Cl2
CuI
Et3N
Et3N
Et3N
12
12
12
12
12
12
12
3
ND
ND
ND
ND
30
ND
20
75
3g
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 of
elemental sulfur (1.0 equiv), morpholine (1.0 equiv) in EtOH at 60 ꢀC.
CuI
tBuOK
tBuOK
Et3N
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Pd(OAc)2
Pd(OAc)2
b
Isolated yields.
DBU
c
The compound 4a was prepared by using 1,4-dithiane-2,5-dithiol (1.0 equiv),
tBuOK
K2CO3
ethyl cyanoacetate (1.0 equiv), Et3N (1.0 equiv) in DMF under a Gewald reaction
condition.
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.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278
Table 3 (continued )
273
Table 3
Synthesis of 1-thienyl substituted pyrrolo[2,3-b]quinoxalines/pyrrolo[2,3-b]pyr-
Entry Alkyne (2); R & Ar¼ Amine
(4)
Time
(h)
Product (5) T¼
Yieldb
(%)
azine (5) via Pd-catalyzed CeN cross-coupling/cyclization strategy.a
Ar
T-NH2
Pd(OAc)2
tBuOK
R
N
N
R
N
N
Ar
+
N
T
CO2Et
DMF, 80 oC
2-3 h
11
12
2c; H & C6H4Me-p 4b
3
2
70
68
Cl
S
S
2
4
5
Me
5k
5l
Entry Alkyne (2); R & Ar¼ Amine
(4)
Time
(h)
Product (5) T¼
Yieldb
(%)
CO2Et
Ph
2c
2c
4c
CO2Et
1
2
2a; H & Ph
4a
4b
2
3
76
67
S
S
5a
CO2Et
CO2Et
Me
S
S
13
14
15
4d
2
2
2
67
70
62
2a
5b
5c
5m
CO2Et
CO2Et
3
4
2a
2a
4c
3
2
60
75
S
S
2c
2c
4e
Ph
5n
CO2Et
4d
CO2Et
5d
S
4f
CO2Et
5o
S
S
5
6
2a
2a
4f
2
2
56
68
5e
CO2Et
CO2Et
S
16
2c
4g
3
66
4g
N
N
Boc
5p
Boc
5f
CO2Et
Me
17
18
2d; Me & C6H4Me-p 4b
3
3
64
68
S
CO2Et
7
8
2b; Me & Ph
4b
4c
3
3
71
70
S
S
5q
Me
5g
CO2Et
CO2Et
Ph
2d
2d
4c
S
S
2b
5r
Ph
5h
CO2Et
CO2Et
19
20
4d
2
2
58
S
S
9
2b
2b
4d
4e
2
2
65
72
5s
5i
CO2Et
CO2Et
10
S
2d
4e
60
(continued on next page)
5j
5t

274
S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278
Table 3 (continued )
Entry Alkyne (2); R & Ar¼ Amine
(4)
Time
(h)
Product (5) T¼
Yieldb
(%)
CO2Et
21
2d
4b
3
53
S
S
Me
5u
5v
CO2Et
22
2ec
4d
3
51
a
All reaction were carried out by using 2 (1.89 mmol), Pd(OAc)2 (0.019 mmol), 4
t
(1.89 mmol), BuOK (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 site
of PDE4B (PDB ID: 1XMY).
hepatotoxicity assay (Figs. 8 and 9) the compound 5l showed
toxicity at 30
compound 5v though did not show toxicity at 1
to be toxic at 3 M. Indeed the embryos were found dead at 10 and
30 M. The compound 5a did not show any toxicity at 1 and 3
though the toxicity was increased significantly at 10 and 30
mM but found to be non-toxic at 1, 3 and 10
mM. The
mM but was found
m
hepatotoxicity in zebrafish embryos. In view of their observed
pharmacological properties and the fact that most cytotoxic anti-
cancer agents are known to induce apoptosis the present class of
PDE4 inhibitors seemed to have potential medicinal value. The
synthetic methodology presented here may find application in the
construction of library of small molecules based on 1-thienyl pyr-
roloquinoxaline framework.
m
m
m
M,
M.
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].
4. Experimental
3. Conclusions
4.1. General method for the preparation of N-thiophenyl substituted
pyrrole[2,3-b]quinoxalines/pyrazines (5)
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-carboxylate
derivatives for the rapid and milder access to novel 1-thienyl pyr-
roloquinoxalines as potential PDE4 inhibitors. These compounds
were designed for the first time as potential apoptotic agents based
on the reported link between PDE4 and apoptosis. Some of these
compounds showed encouraging PDE4 inhibitory properties
in vitro that was supported by docking studies in silico. These
compounds were also screened for apoptosis, teratogenicity and
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 h
under nitrogen atmosphere. After completion of the reaction as
indicated by TLC, the reaction mixture was diluted with water
(15 mL) and extracted with ethyl acetate (3 ꢂ 10 mL). The combined
Pd(OAc)2
Ar
R
N
N
2
Pdo
N
Ar
E-3
CO2Et
R
N
N
S
PdII Cl
tBuOH
E-1
tBuOK
R
CO2Et
Ar
5
HN
N
N
S
PdII
CO2Et
N
H
tBuOK
E-2
S
4
Fig. 4. The percentage induction of apoptosis caused by compounds 5v, 5l and 5a at
different concentrations along with Methotrexate. All the statistical analysis was
performed using GraphPad Prism® software.
Scheme 2. Proposed reaction mechanism.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278
275
Fig. 5. Representative images of the embryos treated with compounds assayed for apoptosis.
organic layers were collected, dried over anhydrous Na2SO4, filtered
4.2. PDE4 enzymatic assay
and concentrated under low vacuum. The residue was purified by
column chromatography on silica gel (Merck, 100e200 mesh) using
n-hexane/ethyl acetate to afford the desired product.
The inhibition of PDE4 enzyme was measured using PDE light
HTS cAMP phosphodiesterase assay kit (Lonza) according to man-
ufacturer's recommendations. Briefly, 10 ng of in house purified
PDE4B1 enzyme was pre-incubated either with DMSO (vehicle

276
S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278
Fig. 8. All the statistical analysis was done using GraphPad Prism® software. The graph
represents the qualitative data of % liver size, % liver degeneration & % yolk sac
retention of three compounds at different concentrations when compared to positive
control Amiodarone.
Fig. 6. Each embryo was scored based on their level of toxicity from 5 being non-toxic
and 0.5 being highly toxic. Statistical analysis for scoring was done using GraphPad
Prism® software using two-way ANOVA. The graph represents the teratogenic scoring
given compared to the positive control Phenobarbital.
4.3. Teratogenicity assay
(a) Drug exposure: 1 day post fertilization (dpf) embryos were
collected and checked for its developmental stage and condition.
Embryos were de-chorinated using 0.5 mg/ml pronaseE treatment
followed by washes with E3 medium. Working concentrations of
the compound was prepared by serially diluting the compound in
final concentration of 0.1% DMSO in E3 medium. Embryos (n ¼ 6)
were distributed in 24 well plate followed by addition of drug and
incubated at 28.5 ꢀC until 5 dpf. (b) Morphological scoring: Embryos
were removed from drug solution, washed and allowed to anes-
thetize using tricaine (0.008%) and observed for morphological
toxicity parameters like Body Shape, Somites, Notochord, Tail,
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 for
10 min in dark. Luminescence values (RLUs) were measured by a
Multilabel plate reader (Perklin Elmer 1420 Multilabel counter).The
percentage of inhibition was calculated using the following for-
mula: % inhibition ¼ [(RLU of vehicle control ꢁ RLU of inhibitor)/
(RLU of vehicle control)] ꢂ 100.
Fig. 7. Representative images of teratogenicity assay.

S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278
277
Fig. 9. Representative images of hepatotoxicity assay.
Intestine, Fins, Brain, Upper jaw, Heart, Lower jaw, Liver and Swim
Bladder. Morphological assessment and scoring was done accord-
ing to the procedure described [17b,c].
drug. The plate was incubated at 28 ꢀC until 7 dpf. Embryos were
washed with E3 medium on 7 dpf and anesthetized using tricaine.
The images of embryos treated with different compounds of
various concentrations are analyzed using Image J software for
their liver size, liver degeneration and yolk sac retention and per-
centages were calculated.
4.4. Hepatotoxicity assay
In this assay 4 dpf embryos were exposed to various concen-
tration of test compound prepared from stock solutions as
described above. The embryos were distributed in 24 well plates
4.5. Apoptosis assay
along with 250
well is added with the respective working stock solutions to obtain
the final concentration of 1, 3, 10 and 30 M concentration of the
ml of 0.1% DMSO with 6 embryos in each well. Each
24 hpf embryos were de-chorinated manually. 6 embryos were
m
distributed as two sets in each well of 24 well plates with 250
0.1% DMSO. The working stock solutions were prepared by serial
dilution as described earlier. Each well was added with 250 l of
ml of
m
respective concentration to obtain final working concentration.
Embryos were incubated at 28 ꢀC for 24 h and 48 h. The apoptotic
effects were checked at 24 h and 48 h by washing drug exposed
embryos thrice with E3 medium. Acridine orange (2 mg/ml) solution
of dye in E3 medium was added and incubated for 30 min. The
embryos were rinsed thoroughly twice in fresh E3 medium to wash
the acridine orange solution. Stained embryos were anesthetized
with tricaine and photographed under UV illumination using Zeiss
Axio CamMR camera attached to a Zeiss florescence microscope
(GFP filter set: excitation 473, emission 520) under 5ꢂ magnifica-
tion. The Images were taken and analyzed using Image J software.
Acknowledgments
Fig. 10. The EC50 (apoptosis) and NOAEL (teratogenicity) of test compound 5v
AN thanks CSIR, for a research fellowship. Authors thank CSIR
[Grant 02(0127)/13/EMR-II] and management of DRILS for support
and Dr. K. V. L. Parsa and his team for PDE4 assay.
(EC50 ¼ 1.732
m
m
M & NOAEL ¼ 1.0
m
M), 5l (EC50 ¼ 1.832
m
M & NOAEL ¼ 3.0
mM) and 5a
(EC50 ¼ 3.219
M & 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.

278
S.K. Kolli et al. / European Journal of Medicinal Chemistry 86 (2014) 270e278
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