InCl3 mediated heteroarylation of indoles and their derivatization via C[sbnd]H activation strategy: Discovery of 2-(1H-indol-3-yl)-quinoxaline derivatives as a new class of PDE4B selective inhibitors for arthritis and/or multiple sclerosis

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InCl mediated heteroarylation of indoles and their

derivatization via C-H activation strategy: Discovery of

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3

2

-(1H-indol-3-yl)-quinoxaline derivatives a new class of

PDE4B selective inhibitors for arthritis and / or multiple sclerosis

Rajnikanth Sunke, Ramudu Bankala, B. Thirupataiah, E.V.V. Shivaji Ramarao, Sandeep Jetta, Hari Maduri

Doss, Raghavender Medishetti, Pushkar Kulkarni, Ravi Kumar Kapavarapu, Mahaboobkhan Rasool, Jayesh

Mudgal, Jessy E. Mathew, Gautham G. Shenoy, Kishore V. L. Parsa,* and Manojit Pal*

_R1

IC50:0.395 + 0.13 µM

100

R³

X

N

Y

8

0

Q

N

60

Cl

R²

R¹

40

X

N

Y

20

0

R³

0.001 0.01

0.1

1

10

100 1000

N

R²

3b (µM)

1

ACCEPTED MANUSCRIPT

European Journal of Medicinal Chemistry

journal homepage: www.elsevier.com

InCl mediated heteroarylation of indoles and their derivatization via C-H activation

3

strategy: Discovery of 2-(1H-indol-3-yl)-quinoxaline derivatives as a new class of

PDE4B selective inhibitors for arthritis and / or multiple sclerosis

a,‡

a,b

a

Rajnikanth Sunke, Ramudu Bankala, B. Thirupataiah, E.V.V. Shivaji Ramarao, Sandeep Jetta, Hari

c

a

Maduri Doss, Raghavender Medishetti, Pushkar Kulkarni, Ravi Kumar Kapavarapu, Mahaboobkhan

Rasool, Jayesh Mudgal, Jessy E. Mathew, Gautham G. Shenoy, Kishore V. L. Parsa, * and Manojit

c

b

a,

c,

Pal *

a

Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India

Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Madhav Nagar, 576 104, Karnataka, India

b

c

Immunopathology Lab, School of BioSciences and Technology, VIT University, Vellore-632014, India

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

3

A new class of PDE4 inhibitors were designed and synthesized via the InCl mediated

heteroarylation of indoles and their further derivatization through the Pd(II)-catalyzed C-H

activation strategy. This effort allowed us to discover a series of 2-(1H-indol-3-yl)-quinoxaline

based inhibitors possessing PDE4B selectivity over PDE4D and PDE4C. One of these

compounds i.e. 3b (PDE4B IC50 = 0.39 ± 0.13 µM with ~ 27 and > 250 fold selectivity for

PDE4B over PDE4D and C, respectively) showed effects in Zebrafish experimental autoimmune

encephalomyelitis (EAE) model of multiple sclerosis when dosed at 3, 10 and 30 mg/kg

intraperitoneally. Indeed, it halted the progression of the disease across all these doses tested. At

an intraperitoneal dose of 30 mg/kg the compound 3b showed promising effects in adjuvant

induced arthritic rats. The compound reduced paw volume, inflammation and pannus formation

Available online

Keywords:

Indole

Quinoxaline

PDE4

Arthritis

Multiple sclerosis

(

in the knee joints) as well as pro-inflammatory gene expression / mRNA levels significantly in

arthritic rats. Moreover, this compound was found to be selective towards PDE4 over other

families of PDEs in vitro and safe when tested for its probable toxicity (e.g. teratogenicity,

hepatotoxicity and cardiotoxicity) in Zebrafish.

2

1

. Introduction

managed [5] by the uses of single or combination of drugs chosen

from NSAIDs, corticosteroids, DMARDs (such as methotrexate,

leflunomide, and sulfasalazine) as well as biological agents (e.g.

TNF inhibitors and an anti-IL12/23 inhibitor) some patients do

not appear to achieve an adequate clinical response with these

treatments. Consequently, there appears to be an unmet need for

additional medications in the rheumatologist’s armamentarium

towards the treatment of psoriatic arthritis. Recently, a PDE4

inhibitor i.e. apremilast has been launched for the treatment of

psoriatic arthritis (Fig. 1) [6]. While the first-generation and

experimental PDE4 inhibitor rolipram has shown encouraging

results in mice models an appropriate and better PDE4 inhibitor

is yet to be developed for the potential treatment of multiple

sclerosis (MS, a demyelinating autoimmune disease). Notably,

roflumilast [7a] (Fig 1) (Daxas®, Nycomed) has been in the

European market for the treatment of chronic bronchitis since

Phosphodiesterase 4 or PDE4, one of the 11 families of PDEs

PDE1-PDE11) [1] is known to be the major PDE enzyme

(

involved in the cAMP-mediated regulation of functional

parameters in majority of inflammatory cells (e.g. neutrophils,

eosinophils, mast cells, macrophages, and dendritic cells) and

structural airway cells (including airway epithelium, endothelium

and smooth muscle). Thus, PDE4 selective inhibitors have been

reported to be useful for the treatment of psoriasis, psoriatic

arthritis,[2] and multiple sclerosis [3] in addition to asthma and

chronic obstructive pulmonary disease (COPD) [4]. Notably,

psoriatic arthritis (a chronic inflammatory arthritis in the

presence of skin and/or nail psoriasis) though currently being

-

---------------

‡

R. S. and R. B. contributed equally and share first authorship of this

work.

2

009 and in US market (Daliresp, Forest Lab) for exacerbations

*

Corresponding author: Tel.: +91 40 6657; fax: +91 40 6657 1581;

E-mail address: KishoreP@DRILS.ORG (KVLP);

during COPD since 2012. The other PDE-4 inhibitors available

manojitpal@rediffmail.com (M. Pal)

2

European Journal of Medicinal Chemistry

OCHF

2

ACCEPTED MANUSCRIPT

O

N

Cl

SO

2

Me

CN

N

O

_R1

_R3

OMe

_R1

AcHN

OEt

O

NH

N

Cl

N

R³

N

R²

OMe

O

N

D

_R2

B

C

Apremilast

Roflumilast

IC₅₀= 1.3 nM (PDE4B)

and 0.2 nM (PDE4D)

Fig. 3. Reported PDE4 inhibitor B and newly designed potential inhibitors

IC₅₀= 27-43 nM (PDE4B)

and 33 nM (PDE4D)

C.

Fig. 1. Marketed PDE4 inhibitors

in the market include crisaborole [7b] and ibudilast [7c,d].

However, all these inhibitors have suffered from some degree of

side effects including nausea and emesis [4]. One of the reasons

for these observed side effects was thought to be due to the non-

selective inhibition of PDE4 isoforms (see the IC₅₀values in Fig.

1

). Study have suggested that among the four isoforms e.g.

PDE4A, B, C, and D, the inhibition of PDE4B (that plays a key

role in inflammatory cell regulation) suppresses TNF-α

production via elevation of cAMP levels [8] whereas the

inhibition of PDE4D triggers the emetic response [9]. Moreover,

PDE4C (not generally found in pro-inﬂammatory cells) is highly

expressed in the CNS where most of the PDE4 inhibitors are

believed to promote their above mentioned side effects. Thus,

PDE4B selectivity over PDE4D and at the same time minimizing

PDE4C inhibition is a promising approach for the development

of better PDE4 inhibitors. While, efforts has been devoted to

identify inhibitors [9,10,11] possessing selectivity towards

PDE4B over PDE4D it is not known if they inhibit PDE4C.

Moreover, being a new class of selective inhibitors their potential

for the treatment of psoriatic arthritis, and/or multiple sclerosis

has not been evaluated. In our earlier effort we identified a novel

N-indolylmethyl substituted olanzapine based interesting and

^s^e^l^e^c^tⁱ^v^e^P^D^E⁴^Bⁱⁿ^hⁱ^bⁱ^t^o^r⁽^A^,^Fⁱ^g^.²⁾⁽^I^C⁵0 ~ 1.1 µM) that

Fig. 4. Docking of D into PDE4B (PDB ID: 1XLZ). White dotted lines

are H-bond interactions (see also Fig S-1 and S-2 in Suppl. file).

hydrophobicity of the molecule. Our design was further

supported by the docking study of a representative molecule D.

The molecular docking simulations were carried out using GRIP

method of docking in Biopredicta module of Vlife MDS

(

Molecular Design Suite) 4.6. The molecule D showed good

Me

N

Br

interactions with PDE4B e.g. (i) H-bond interactions with

SER282, SER429 and GLN417 (Fig. 4), (ii) hydrophobic

interactions with GLN284, SER282, PHE285, GLU421 and

PHE285 and (iii) π-stacking interactions with PHE414 residue of

PDE4B (see Fig S-2 in Suppl. file). However it showed only

hydrophobic but no H-bond and / or π-interactions with PDE4D

and C. Moreover, the docking score indicated possible selectivity

of D towards PDE4B (-83.64 kcal/mol) over PDE4D (-70.10

kcal/mol) and PDE4C (-60.41 kcal/mol). Notably, docking of a

non-selective inhibitor rolipram showed nearly equal interactions

with PDE4B (e.g. His234 and Gln443 residues) and PDE4D (e.g.

His160 and Gln369 residues) confirming it’s lack of selectivity in

silico too. Herein, we describe the PDE4 inhibitory properties of

compounds based on C (including D, Fig. 3) that allowed us to

discover a new class of inhibitors possessing PDE4B selectivity

over PDE4D and PDE4C and evaluate their potential for the

treatment of psoriatic arthritis and/or multiple sclerosis.

Me

N

S

N

SO Me

2

Fig. 2. PDE4 inhibitor (A) previously reported by our group [12].

showed >10 fold selectivity towards PDE4B over D, significant

inhibition of TNF-α, no major toxicities in RAW 264.7 and HEK

93T cells and no hepatotoxicity and changes in heart rate in

2

zebrafish embryo [12]. While selectivity was achieved but the

potency of this inhibitor was not up to our expectation. Therefore

identification of a better inhibitor was necessary. With this

objective we looked into our previously identified other class of

PDE4 inhibitors especially on 2-(1H-indol-3-yl)quinoline

derivatives B (Fig. 3) one of which showed considerable PDE4

inhibitory activity (EC₅₀~0.89 µM) when tested in a cell based

cAMP reporter assay [13]. These findings prompted us to design

structurally similar quinoxaline derivatives C (Fig. 3) as potential

and selective inhibitors of PDE4B. While replacing the quinoline

ring of B by a quinoxaline moiety was a subtle and obvious

modification but fortunately the outcome was positive.

Moreover, the substituents on the indole ring too played an

important role. It was known that the PDE4B pocket was more

hydrophobic in nature and bigger in size than PDE4D [11a, c]

2

.

Results and discussion

2.1. Chemistry

While methods to introduce aryl or heteroaryl group at C-2

and/or C-3 position of a quinoxaline ring are known in the

literature [14] compounds related to C were synthesized using an

AlCl induced C–C bond forming reaction [15] between 2,3-

dichloroquinoxaline and indoles (Method 1, Scheme 1) due to the

effectiveness of this methodology and easy availability of starting

materials. However, the methodology involved the use of a

stoichiometric amount of AlCl₃that is prone to generate

3

2

hence we aimed to introduce appropriate R group (in C, Fig. 3)

that preferably contributes towards overall optimal volume and

considerable amount of Al(OH) waste during the workup

3

AlCl

3

DCE, 80 ^o

C

ACCEPTED MANUSCRIPT

X

N

Y

of acrylic acid) were employed to react with a variety of

Q

1

compound 3 in the presence of Pd(OAc) , Cu(OAc) and TFA in

R

2 2

Method 1

X

N

Y

Cl

1,4-dioxane to afford the corresponding alkenylated product 6 in

good to acceptable yield (Table 2, see also Table S-3 in SI) (for

optimization of reaction conditions, see Table S-4 in SI). The

compound 7a-b was obtained via hydrolysis of 3t and 4a (Chart

1).

Q = benzene

ring; X = N;

Y = Cl

R³

1

Q

+

_R1

Method 2

N

R²

N

_R3

3-4

InCl , DCE, 80 ^o

C

3

_R2

Method 3

C-H activation

2

Q = benzene ring; X = N; Y = Cl

Q = benzene ring; X = CH; Y = CN

Q = no ring; X = C

Table 1: Effect of conditions on InCl

forming reaction between 1a and 2a.

catalyzed C-C bond

3

Analogues

a

5

Scheme 1. Reported (Method 1) and current method (Method

) of heteroarylation of indoles and their derivatization

Method 3).

2

(

process (especially during a scale up preparation). Moreover,

b

AlCl is not recoverable as well as recyclable. Thus a more

Entry

1.

Solvent

Temp (°C)

Time (h)

%yield

3

environmentally friendly and sustainable method was necessary

for the synthesis of target compounds based on C. As a less toxic

MeCN

DCE

60

2.5

1.0

3.0

2.0

4.0

4.5

4.0

2.0

0.5

1.0

64

92

73

26

47

45

38

21

as well as air and water-compatible Lewis acid catalyst InCl has

3

2

3

.

80

been used to catalyze various chemical transformations [16,17]

with high regio- and chemoselectivity. We now describe the first

DCM

60

InCl mediated heteroaryaltion of indoles leading to compounds

4.

DMF

100

60

3

and 4 (Method 2, Scheme 1) and their further derivatization via

5

6

7

.

Toluene

MeOH

PEG-400

DMSO

DCE

the direct C-H activation method i.e. the Fujiwara−Moritani

reaction [18] (Method 3, Scheme 1).

70

The InCl mediated heteroaryaltion of 2,3-dichloroquinoxaline

3

(1a) with 1-allyl-1H-indole (2a) was examined initially in a

8.

100

80

variety of solvents to establish the optimized reaction conditions

c

9

1

.

91

(

Table 1). Among the solvents examined 1,2-dichloroethane

DCE) was found to be effective (entry 2, Table 1) when the

d

0.

DCE

80

0

a

desired product 3a was isolated in 92% yield. The reaction also

proceeded in other solvents such as acetonitrile (MeCN),

dichoromethane (DCM), DMF, toluene, MeOH, PEG-400 and

DMSO but afforded 3a in low or poor yields (Entries 1 and 3-8,

Table 1). Notably, the reaction was completed within 0.5 h when

performed under ultrasound irradiation (entry 9, Table 1). The

All the reactions are carried out using 2,3-

dichloroquinoxaline 1a (1 mmol) and 1-allyl-1H-indole 2a (1

mmol), in the presence of InCl (0.5 mmol) in a solvent (5 mL)

3

under nitrogen.

b

Isolated yield.

reaction however, did not proceed in the absence of InCl (entry

c

3

The reaction was carried out under ultrasound using a

6

, Table 1) indicating the key role played by the catalyst in the

laboratory ultrasonic bath producing irradiation of 35 kHz.

present reaction. Nevertheless, based on above study InCl was

3

d

^T^h^e^r^e^a^c^tⁱ^oⁿ^w^a^s^p^e^r^f^o^r^m^e^dⁱⁿ^t^h^e^a^b^s^eⁿ^c^e^o^f^Iⁿ^C^l3^.

identified as a new and potential catalyst for the heteroarylation

of indoles. It was therefore necessary not only to assess the

generality and scope of this C-C bond forming reaction but also

its applicability in the synthesis of other analogs of 3a that were

required for conducting pharmacological evaluation as part of the

project goal. Accordingly, a large variety of indoles were reacted

with 1a and 2-chloroquinoline-3-carbonitrile (1b) (Scheme 2).

The reaction proceeded well in all these cases affording the

corresponding products (3) in good yield (Chart 1, see also Table

S-1 in SI). The use of 2-chloronicotinonitrile (1c) also afforded

the desired products (4) in good yields (Scheme 3, Chart 1, see

also Table S-2 and Scheme S-1 in SI). Finally, the C-2

alkenylation of indole ring of some of the compounds

synthesized was carried out using the C-H activation method

R¹

R³

InCl₃

0.5 equiv)

R¹

X

N

Y

X

N

Y

_R3

(

+

o

Cl

N

DCE, 80 C

R²

N

1-1.5 h

R²

1

2

3

1

a; X = N, Y = Cl

b; X = CH, Y = CN

R = OMe, Me, NO , Cl. Br, H

R = H, Me, Et, i-propyl, allyl, 2-butyl, Bn,

2

-(CH ) CMe Cl, CH CO Et, CH CONHPh,

2 2 2 2 2 2

CH CO(morpholinyl)

2

3

R = Cl

(

Table 2). The direct C-H bond oleﬁnation (the

Scheme 2. Synthesis of compound 3.

Fujiwara−Moritani reaction) [18] has attracted particular

attention in the recent past and a range of directing groups has

been reported to aid C(Ar)-H activation including quinolone

R¹

^Iⁿ^C^l3

CN

[

19a], pyridine [19b-e] etc. Recently, we have reported

(0.5 equiv)

R³

quinoxaline as a new directing group for the Pd (or Ru)-catalyzed

ortho C-H alkenylation of aniline derivatives leading towards the

synthesis of alkenyl substituted benzo[4,5]imidazo[1,2-

a]quinoxalines as inhibitors of PDE4 [20]. This idea (i.e. the

usefulness of alkenyl moiety for PDE4 inhibition) and the

strategy (i.e. alkenylation via C-H activation) was extended in the

present case for the synthesis of new indole derivatives (6). Thus

a range of olefin coupling partners e.g. 5a-c (Me, Et or t-Bu ester

CN

Cl

+

N

o

DCE, 80 C

1-1.5 h

N

_R2

N

R²

1

1c

2

R = OMe, Cl

4

2

R = allyl, CH CO Et

2

3

R = H

Scheme 3. Synthesis of compound 4.

4

European Journal of Medicinal Chemistry

ACCEPTED MANUSCRIPT

a,b

Chart 1. List of compound (3, 4 and 7) synthesized.

3

a (87)

3

d (82)

3

b (93)

3e (86)

3

c (91)

N

Cl

N

Ph

3

f (78)

3

h (87)

3

g (89)

3

i (92)

3j (88)

3

l (83)

3m (86)

3n (71)

3

k (80)

3

o (80)

3

r (84)

3

s (80)

3

p (84)

3q (85)

3

t (70)

3

u (70)

3

v (65)

3w (38)

3x (45)

3

y (42)

O

CN

N

O

3

z (65)

4

b (70)

4c (65)

c

7

a (33)

4

a (65)

c

7

b (75)

a

Reaction conditions for the synthesis of compound 3 and 4: compound 1 (1 mmol), compound 2 (1 mmol) and InCl (0.5 mmol)

3

in DCE (5 mL) at 80 °C for 1-1.5h under nitrogen.

b

Figure in the bracket indicates isolated yield in percentage (%).

c

Obtained via hydrolysis of 3t and 4a respectively using LiOH in THF (see Scheme S-1 in SI).

5

ACCEPTED MANUSCRIPT

a,b

Table 2. Synthesis of compound 6 via C-2 alkenylation of indole ring of 3.

4

1

_R1

OR

R

N

Cl

1

N

Cl

R = OMe, Me, Cl. Br, H

R³

O

5

R³

2

R = H, Me, i-propyl, allyl

3

R = H

4

Pd(OAc)

Cu(OAc)₂

2

R

= Me, Et, t-Bu

N

_R2

N

_R2

4

H

R O

3

TFA, 1,4-dioxane

O

6

o

80

C, 7-8 h

6

c (80)

6

d (77)

6

a (79)

6

b (83)

6

e (72)

N

Cl

N

O

t

O Bu

6

h (69)

6i (64)

6

f (75)

6g (66)

6

j (70)

Br

Cl

N

Cl

N

Cl

N

NH

O

t

O Bu

t

O Bu

6

k (81)

6l (73)

6m (77)

6o (66)

6

n (80)

6

p (69)

6q (73)

a

All the reactions were carried out using compound 3 (1 mmol), compound 5 (1.5 mmol), Pd(OAc) (5 mol%),

2

Cu(OAc) (1 mmol) and TFA (1.2 mmol) in 1,4-dioxane (2.5 mL) at 80 °C for 7-8 h under open air.

2

b

Figure in the bracket indicates isolated yield in percentage (%).

6

European Journal of Medicinal Chemistry

ACCEPTED MANUSCRIPT

Fig 5. In vitro evaluation of compounds against PDE4B at 10 µM.

2

.2. Biology

Most of the synthesized compounds e.g. 3, 4, 6 and 7 were

evaluated for their PDE4B inhibitory properties in vitro at 10 µM

using an enzyme based assay (Fig. 5) [21]. Rolipram, a well-

known inhibitor of PDE4 was used as a reference compound.

Compounds that showed > 75% inhibition i.e. 3a-c, 3i, 3j, 3t, 4c,

Table 3. In vitro data of most active compounds.

a

IC50 (µM)

Selectivity

(fold)

Entry

Compound

PDE4B

PDE4D

22.04±4.60

10.69±7.23

4.93±3.78

5.02±4.18

ND

1

3a

4.67±1.07

0.39±0.13

1.04±0.38

0.44±0.33

9.74±3.33

0.21±0.06

1.98±0.55

5.58±2.26

5.97±1.29

3.88±0.57

0.94±0.2

~4.8

6

a, 6b and 6q were taken for concentration dependent studies

2

3b

~27

and their IC₅₀values are presented in Table 3. Some of the

remaining compounds either did not show significant inhibition

or provided erratic data because of their precipitation in the assay

medium during the assay. While drawing the clear SAR

3

3c

~4.75

~11.40

4

3i

5

3j

(Structure-Activity-Relationship) conclusions from the current

6

3t

15.25±2.26

ND

~72

series of compounds was somewhat tricky some broad

observations noted are summarized in Fig 6. In general the

7

4c

“

OMe” group at C-5 of indole ring was beneficial for PDE4B

8

6a

ND

inhibition as well as selectivity. No substituent or a “Br” group

at this position contributed to moderate activities. No substituent

at C-6 position was favorable. An alkyl chain preferably having

three or four-carbon length or a -CH CO Et group at indole

9

6b

ND

10

6q

ND

2

1

Rolipram

0.88±0.3

nitrogen was effective compared to no substituent / other

substituent at this position. This perhaps contributed towards the

effective volume of the molecule to fit better into the PDE4B

pocket. Notably, presence of an alkenyl moiety at C-2 of the

indole ring did not improve the activity further but maintained a

moderate to good activity in several cases. The activity was

a

Data is represented as mean ± SD of at least 2 independent

experiments.

Cl / CN - low to

high activity

OMe - generally high activity

somewhat influenced by the nature of ester group attached to the

t

2

H / Br - moderate to good activity

alkenyl moiety, e.g. the bulky –CO Bu group was not tolerated

and the Me-/Et-ester was generally favorable. Generally, the 3-

chloro/cyano quinoxaline or pyridine ring at C-3 of indole was

effective though the 3-cyano quinoline ring at the same position

was found to be ineffective. Based on their promising activity

against PDE4B the dose response study against PDE4D was also

performed with the compound 3b and 3t (Fig 7). Indeed, the

compound 3b and 3t showed ~ 27 and 72 fold selectivity towards

PDE4B over PDE4D, respectively whereas rolipram did not

show any selectivity (Table 3). Moreover, these compounds did

not inhibit PDE4C significantly (~20-30% inhibition at 100 µM)

R¹

5

X

Y

3

4

H - good activity

R³

Q

6

7

N

alkyl chain of 3 or

4 carbon length or

CH COOEt group

N

2

alkenyl moiety - low_R2

to moderate activity

-

2

good activity

Q = benzene ring; X = N - low to good activity

Q = benzene ring; X = CH - no activity

Q = no ring; X = CH - moderate to good activity

(

Fig. 8) when rolipram inhibited PDE4C by 70% at 10 µM. Thus

their selectivity for PDE4B over PDE4C appeared to be > 250

fold. In another study compound 3b inhibited LPS-induced iNOS

(required for the nitric oxide production) and TNF-α expression

in macrophages (Fig. 9) indicating its potential for anti-

inflammatory effects. The compound 3b was also tested for its

selectivity towards PDE4 over other families of PDEs (e.g.

Fig. 6. Summary of SAR for PDE4B inhibitory activities of

compound 3, 4 and 6.

PDE1-PDE11) using an in vitro enzymatic assay [22]

.

Accordingly, the % inhibition shown by 3b at 10 µM was as

follows: 12% (PDE1A1), 23% (PDE1B), 39% (PDE1C), 3%

To gain some preliminary information regarding metabolic

stability / PK (pharmacokinetic) properties of compound 3b the

in vitro microsomal stability study was performed using the rat

liver microsomes. At a concentration of 5 µM the compound 3b

(

3

(

1

PDE2A1), 1% (PDE3A), 1% (PDE3B), 40% (PDE4A1A),

6% (PDE4A4B), 2% (PDE5A1), 2% (PDE6C), 3%

PDE7A1), 2% (PDE7B), 3% (PDE8A1), 9% (PDE9A2),

3% (PDE10A1), 15% (PDE10A2), 7% (PDE11A4). This

clearly indicated PDE4 selectivity of 3b over other PDEs.

7

ACCEPTED MANUSCRIPT

IC :0.395 + 0.13 µM

50

100

80

60

40

20

0

0.001 0.01

0.1

1

10

100 1000

Concentration of

Concentration

3b (µM)

IC : 10.69+7.23 µM

50

100

80

60

40

20

0

0.001 0.01

0.1

1

10

100 1000

Concentration

Concentration of

b (µM)

Fig. 8. Concentration dependent study of compound 3b and 3t

against PDE4C. The reference compound rolipram was used

at a concentration of 10 µM.

3

IC :0.21 + 0.06 µM

5

0

100

showed moderate stability after 60 min [i.e. the mean % of 3b

80

remaining after 60 min compared to 0 min ~ 42.25; half-life (t ,

1

/2

60

min) ~ 52.50 and intrinsic clearance (CL , µL/min/mg) ~ 52.80].

int

Then a single dose intravenous PK study of compound 3b was

performed in male BALBc mice (dose: 10 mg/kg b.w.).

Accordingly, the mean plasma PK parameters were determined

4

0

20

as follows: C_m_a_x(ng/mL) ~ 2850.23; C (ng/mL) ~ 5056.31;

0

T_m_a_x(h) ~ 0.08; AUC (h*ng/mL) ~ 1091.43; AUC

last

inf

0.001 0.01

0.1

1

10

100 1000

(

h*ng/mL) ~ 1163.89; AUC_e_x_t_r_a_p(%) ~ 6.23; Vss(L/kg) ~ 4.46;

Concentration of

t (µM)

CL(mL/min/kg) ~ 143.20; T (h) ~ 0.64; MRT (h) ~ 0.36.

3

1/2

last

Having observed promising and interesting in vitro activities of

compound 3b, we then evaluated its impact in a Zebrafish model

of multiple sclerosis (EAE; experimental autoimmune

encephalomyelitis) [23]. Notably, one of us was involved in

developing this model as a quick in vivo screening model for

multiple sclerosis [24]. The EAE was induced by immunization

with myelin oligodendrocyte glycoprotein – 35–55 (MOG) where

MOG in CFA (complete Freund's adjuvant) was injected

subcutaneously (s.c.) in the mid spine regions of zebrafish (using

IC : 15.25+2.26 µM

5

0

1

00

8

0

6

0

4

0

1

0 µl bevel-tipped Hamilton syringe with a volume of 5 µl/fish).

20

0

mL) at0.

6

00

0

1

ºC0.

f

0

o

1

r 300.1min i

1 10 100 10

n the presence of a00ir

Concentration of

(

A)

3t (µM)

Fig. 7. Concentration dependent study of compound 3b and 3t

against PDE4B and PDE4D.

8

European Journal of Medicinal Chemistry

1

0

8

6

4

2

0

ACCEPTED MANUSCRVe

I

hicle Control

MOG Control

P

T

(

B)

Gilenya - 1mg/kg

3

b 3 mg/kg

b 10 mg/kg

b 30 mg/kg

*

3

4

3

2

1

0

#

Compound 3b

1

2

3

4

5

6

7

No. of days

(

C)

D)

Fig. 10. Effect of compound 3b in Zebrafish EAE model. Data are

represented as mean + SEM. Statistical analysis was performed by Bonferroni

multiple comparison post-hoc test. # (compound 3b treated groups with MOG

control), p<0.001 was considered as significant.

3

30

5

2

1

5

0

5

0

5

0

No disease

1

*

Vehicle control

*

0

.9

.8

.7

.6

.5

.4

.3

3b

*

Methotrexate

3b+Methotrexate

*

Compound 3t

*

Fig. 9. Compound 3b abolishes LPS-induced iNOS and TNF-α

expression in macrophages. RAW 264.7 cells were incubated with 1, 3 and

1

0 µM of compound 3b (A,B) or compound 3t before stimulation with

µg/ml of LPS for 10 h and iNOS levels (A,C) and TNF-α mRNA (B,D)

were determined by western blotting and qPCR, respectively. Rolipram

served as the positive control. Data are represented as mean ±SD. Statistical

analysis was performed by unpaired student's t test. *, p <0.05 and #, p<0.001

was considered as significant.

2

5

0

The compound 3b was administered intraperitoneally at the dose

of 3, 10 and 30 mg/kg whereas positive control gilenya (a

sphingosine 1 phosphate receptor antagonist) was administered

orally at a dose of 1 mg/kg. The effects were evaluated for a

period of seven days. While gilenya robustly reduced the lesion

score to that of control, 3b did not reduce the lesion score

significantly (Fig. 10). However, it halted the progression of the

disease across all the doses tested. We were delighted with this

observation as 3b was the first PDE4B selective inhibitor to show

such effects and therefore was of further interest. Next, we

evaluated compound 3b in a rat adjuvant induced arthritis model.

At an intraperitoneal dose of 30 mg/kg the compound 3b

significantly reduced paw volume of adjuvant induced arthritic

rats similar to that of positive control 2 mg/kg of methotrexate

2

@

15

10

5

0

(

Fig 11A and Fig 11B). Further, it also reduced pain in arthritic

rats as observed by improved reaction time in the hot plate test

Fig 11C). Histopathological analysis of synovial tissues of knee

Figure 11. Effect of compound 3b in adjuvant induced arthritic rats. (A)

Paw thickness of adjuvant induced arthritic rats measured by Vernier calipers

(

B) Images of paw of treated / untreated rats. (C) Reaction time of adjuvant

induced arthritic rats assessed in the hot plate test. Data are represented as

mean + SEM. Statistical analysis was performed by Bonferroni multiple

comparison post-hoc test. * (compound 3b treated groups with vehicle

control), p<0.05, @, p <0.01 was considered as significant.

9

ACCEPTED MANU3₃₀5 SCRIPT

2

1

5

0

5

0

5

0

#

No disease

Methotrexate

Vehicle control

3

*

b+Methotrexate

Fig. 13. Compound 3b reduces pro-inflammatory gene expression in

adjuvant induced arthritic rats: qPCR analysis of (A) TNFα, (B) iNOS.

Analysis of serum levels of (C) TNFα, (D) IL-1β and (E) IL-6 cytokines in

adjuvant induced arthritic rats. Data are represented as mean + SEM.

Statistical analysis was performed by Bonferroni multiple comparison post-

hoc test. *, p <0.05 and #, p<0.001 was considered as significant.

*

#

*

#

3

b

joints revealed reduced synovial inflammation (synovitis) and

pannus formation in the knee joints of rats treated with

compound 3b. The combination therapy with methotrexate

further improved the lesion score (Fig 12). Consistent with

reduced synovial inflammation, the gene expression of pro-

inflammatory mediators such as TNF-α, IL-1β, IL-6 and iNOS

was dampened in compound 3b treated rats. Further, the mRNA

levels of TNFα, IL-1β, IL-6 and iNOS were also robustly

decreased in compound 3b administered rats (Fig. 13).

Fig. 12. Compound 3b reduces inflammation and pannus in synovial

tissues of knee joints of adjuvant induced arthritic rats. Histopathological

analysis and quantification of lesion scores of inflammation and pannus

formation in the knee joints of adjuvant induced arthritic rats. Data are

represented as mean + SEM. Statistical analysis was performed by Bonferroni

multiple comparison post-hoc test. *, p <0.05 and #, p<0.001 was considered

as significant.

(

A)

(B)

9

8

7

6

5

4

3

2

1

0

3

5

0

5

0

5

0

5

0

5

*

3

2

1

4

3

2

***

#

1

0

#

Compound 3b

Treatment

(

C)

(D)

1

12

10

8

6

4

25

20

15

10

*

6

4

#

5

0

2

0

Fig. 14. Compound 3b does not induce teratogenicity in Zebrafish

embryos. Top panel: Teratogenicity scores of compound 3b and positive

control phenobarbitol. Bottom panel: Representative images of teratogenicity

assay. Data are represented as mean + SEM. Statistical analysis was

performed by Bonferroni multiple comparison post-hoc test. *, p<0.05 was

considered as significant.

(

E)

10

European Journal of Medicinal Chemistry

AC

%

C

Li

E

ver

P

Si

T

ze ED MAph

NUSC

enob

arbi

RI

PTamiodarone and terfenadine were used

tol

,

%

Liver Degeneration

Yolk Sac Retention

individually as a positive control in these assays respectively. It

was observed that at the concentration of 1, 3, 10 and 100 µM

compound 3b was safe in all the toxicity assays tested (Fig 14-

16; Table 6). Overall, compound 3b has been identified as a

promising candidate for further preclinical development. While

the progress of 3b has been planned it was necessary to identify a

backup candidate as part of the drug discovery strategy.

Therefore we are focusing on compound 3t that showed better

potency and selectivity compared to 3b (Table 3).

5

00

400

*

**

*

**

300

200

*

**

100

0

Table 6. Results of zebrafish embryo toxicity study with

toxicological indices and major organs/systems affected in

a

positive control.

Compound 3b

Phenobarbital

3000

Test Concentrations (µM)

1, 3, 10, 30, 100

Concentration (µM)

Statistically Significant Toxic

Concentration (µM)

1

00

Positive

Control

Fig. 15. Compound 3b does not induce hepatotoxicity in Zebrafish

No Observed Adverse Effect

Level (NOAEL) (µM)

embryos. The above graph represents the qualitative data of % liver size, %

liver degeneration & % yolk sac retention in compound 3b treated embryos at

different concentrations when compared to positive control amiodarone. Data

are represented as mean + SEM. Statistical analysis was performed by

Bonferroni multiple comparison post-hoc test. ***, p<0.001 was considered

as significant.

30

Toxicity at MTC (maximum tolerated concentration)

Body Shape

Somites

Notochord

Tail

-

xxx

xx

-

x

-

Atria - Beats/Minute

Ventricles - Beats/Minute

200

150

100

Fins

-

Brain

-

Upper jaw

Heart

x

-

*

**

Intestine

Lower jaw

Liver

-

xxx

xx

5

0

*

**

x

-

0

xxx

Swim Bladder

-

Compound 3b

Concentration (µM)

a

Significance of sign: - (no effect), x (slightly toxic), xx

moderately toxic), xxx (severely toxic).

(

4

3

2

1

0

*

**

3. Conclusion

In conclusion, a new class of PDE4B inhibitors was designed

based on the docking of a representative compound into the

PDE4B, C and D in silico. These 2-(1H-indol-3-yl)-quinoxaline

based compounds were predicted to be selective towards PDE4B.

Their chemical synthesis was carried out via the InCl mediated

3

heteroaryaltion of indoles that involved a new C-C bond forming

reaction. Further derivatization of these compounds were

performed using a Pd(II)-catalyzed C-H activation strategy. A

range of target compounds and their analogues were prepared in

good yields. Evaluation of PDE4 inhibitory properties of

synthesized compounds allowed us to discover inhibitors

possessing PDE4B selectivity over PDE4D and PDE4C. One of

these compounds i.e. 3b (PDE4B IC50 = 0.39 ± 0.13 µM with ~

27 and > 250 fold selectivity for PDE4B over PDE4D and C,

respectively) showed effects in Zebrafish EAE model of multiple

sclerosis when dosed at 3, 10 and 30 mg/kg intraperitoneally.

Indeed, it halted the progression of the disease across all these

doses tested. At an intraperitoneal dose of 30 mg/kg the

compound 3b showed promising effects in adjuvant induced

arthritic rats. Indeed, this compound reduced paw volume,

inflammation and pannus formation (in the knee joints) as well as

pro-inflammatory gene expression / mRNA levels significantly in

Compound 3b

Treatment

Figure 16. Compound 3b does not induce cardiac toxicity in Zebrafish

embryos. The above graph represents the heart rate (top panel) and atria-

ventricular ratio (bottom panel) of compound 3b treated embryos at different

concentrations when compared to positive control terfenadine. Data are

represented as mean + SEM. Statistical analysis was performed by Bonferroni

multiple comparison post-hoc test. ***, p<0.001 was considered as

significant.

Having found to be active in two in vivo models the

compound 3b was then evaluated for its probable toxicity

specifically its potential ability to induce teratogenicity,

hepatotoxicity and cardiotoxicity in Zebrafish. Methotrexate,

1

arthritic rats. Moreover, this compound was fou

A

nd

C

to

C

b

E

e

P

se

T

le

E

cti

D

ve MA12

N

/2

U

0;

S

f

C

lowRI

rate: 1.0 mL/min; Diluent: ACN: WATER

P

T

towards PDE4 over other families of PDEs (e.g. PDE1-PDE11)

and safe when tested for its probable toxicity (e.g. teratogenicity,

hepatotoxicity and cardiotoxicity) in Zebrafish. Overall, as a new,

safe and selective inhibitor of PDE4B and with demonstrated

effects in two in vivo diseased models the compound 3b is of

further medicinal interest. Moreover, with the demonstrated

application of modern chemistry methodologies in accessing

NCEs to achieve PDE4B selectivity this research would be of

further interest.

(90:10); UV 220.0 nm, retention time 4.5 min.

4

.1.4. 2-(1-Allyl-5-methoxy-1H-indol-3-yl)-3-

chloroquinoxaline (3b)

Yield: 93%; light yellow solid; mp: 133-135 °C; R = 0.24

(10% EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.59

(s, 1H, C-2 indolyl H), 8.25 (d, J = 2.4 Hz, 1H, ArH), 8.14 (d, J

= 8.4 Hz, 1H, ArH), 7.97 (d, J = 8.0 Hz, 1H, ArH), 7.88-7.82 (m,

1H, ArH), 7.80-7.74 (m, 1H, ArH), 7.47 (d, J = 8.4 Hz, 1H,

ArH), 6.93 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 6.10-6.0 (m, 1H,

f

1

6

=

CH), 5.20 (dd, J = 10.4, 1.6 Hz, 1H, =CH ), 5.12 (dd, J = 17.4,

2

4

.

Experimental section

1.6 Hz, 1H, =CH ), 4.97 (d, J = 5.6 Hz, 2H, CH ), 3.84 (s, 3H,

2 2

13

OCH₃); C NMR (100 MHz, CDCl ) δ: 155.7, 148.6, 145.0,

3

.1. Chemistry

1

40.9, 138.8, 132.8, 132.5, 131.6, 130.1, 129.1, 128.3, 127.9,

118.1, 116.8, 113.2, 110.7 (2C), 104.5, 55.7 (OMe), 49.5

NCH ); MS (ES mass): 350.2 (M+1); HPLC: 99.8%, Column:

.1.1. General methods

(

2

Unless stated otherwise, reactions were performed under

nitrogen atmosphere using oven dried glassware. Reactions were

monitored by thin layer chromatography (TLC) on silica gel

plates (60 F254), using EtOAc/ n-hexane as solvent system and

visualizing with ultraviolet light or iodine spray. Flash

chromatography was performed on silica gel (230-400 mesh)

Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 %

TFA in water, mobile phase B: CH CN (T/%B): 0/20, 0.5/20,

3

2

/95, 8/95, 10/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN:

WATER (90:10); UV 220.0 nm, retention time 4.3 min.

4.1.5. 2-(1-(sec-Butyl)-5-methoxy-1H-indol-3-yl)-3-

chloroquinoxaline (3c)

1

13

using distilled hexane, ethyl acetate. H NMR and C NMR

spectra were recorded in CDCl or DMSO-d solution by using

3

6

Yield: 91%; pale yellow solid; mp: 110-112 °C; R = 0.23

f

4

00 and 100 MHz spectrometers, respectively. Proton chemical

1

(

=

1

10% EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.53

6

shifts (δ) are relative to tetramethylsilane (TMS, δ = 0.00) as

internal standard and expressed in ppm. Spin multiplicities are

given as s (singlet), d (doublet), dd (doublet of doublet), td

s, 1H, C-2 indolyl H), 8.19 (d, J = 2.4 Hz, 1H, ArH), 8.13 (d, J

7.6 Hz, 1H, ArH), 7.97 (d, J = 7.2 Hz, 1H, ArH), 7.87-7.82 (m,

H, ArH), 7.79-7.74 (m, 1H, ArH), 7.59 (d, J = 8.8 Hz, 1H,

ArH), 6.93 (dd, J = 9.2, 2.8 Hz, 1H, ArH), 4.66-4.58 (m, 1H,

(triplet of doublet), t (triplet) and m (multiplet) as well as b

(broad). Coupling constants (J) are given in hertz. MS spectra

NCH), 3.83 (s, 3H, OCH ), 1.99-1.85 (m, 2H, CH-CH ), 1.54 (d,

3

2

were obtained on a Agilent 6430 series Triple Quard LC-MS /

MS spectrometer. Melting points (mp) were determined by using

Buchi B-540 melting point apparatus and are uncorrected.

Chromatographic purity by HPLC (Agilent 1200 series Chem

Station software) was determined by using area normalization

method and conditions specified in each case are as follows:

column, mobile phase (range used), flow rate, detection

wavelength, and retention time.

13

J = 6.4 Hz, 3H, CH-CH ), 0.78 (t, J = 7.2 Hz, 3H, CH -CH );

C

3

2

3

NMR (100 MHz, DMSO-d ) δ: 155.4, 148.7, 144.7, 140.7, 138.5,

6

1

(

31.6, 131.1, 130.8, 129.8, 128.4, 128.1, 127.9, 112.8, 111.7,

10.0, 104.7, 55.7 (OMe), 53.8 (NCH), 29.6 (CH-CH ), 20.9

2

CH-Me), 10.9 (CH -Me); MS (ES mass): 366.0 (M+1); HPLC:

2

9

9.6%, Column: Agilent Eclipse Plus C-18 250 * 4.6 mm, 5 µm,

mobile phase A: 0.1 % TFA in water, mobile phase B: CH CN

3

(

T/%B): 0/5, 3/5, 10/95, 20/95, 22/5, 25/5; flow rate: 1.0

4

.1.2. General Procedure for the preparation of compound 3

mL/min; Diluent: ACN: WATER (90:10); UV 225.0 nm,

retention time 15.6 min.

and 4

A mixture of compound 1 (1.0 equiv), indole 2 (1.0 equiv) and

InCl (0.5 equiv) in 1,2-dichloroethane (5 mL) was stirred at 80

4.1.6. 2-Chloro-3-(1-ethyl-1H-indol-3-yl)quinoxaline (3d)

3

Yield: 82%; pale yellow solid; mp: 90-92 °C; R = 0.20 (10%

f

°

C for 1-1.5 h under a nitrogen atmosphere. After completion of

1

EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.72-8.70 (m,

3

the reaction, the mixture was poured into ice-cold water (15 mL),

stirred for 10 min and then extracted with ethylacetate (3 x 15

mL). The organic layers were collected, combined, washed with

cold water (2 x 20 mL), dried over anhydrous Na SO , filtered

1

H, ArH), 8.32 (s, 1H, C-2 indolyl H), 8.15 (d, J = 8.0 Hz, 1H,

ArH), 7.98 (d, J = 8.4 Hz, 1H, ArH), 7.75 (t, J = 7.6 Hz, 1H,

ArH), 7.68 (t, J = 7.2 Hz, 1H, ArH), 7.44-7.42 (m, 1H, ArH),

2

4

7

3

1

.37-7.31 (m, 2H, ArH), 4.32-4.25 (m, 2H, CH ), 1.58-1.54 (m,

2

and concentrated under vacuum. The residue obtained was

purified by column chromatography using ethylacetate/hexene to

give the desired product.

13

H, CH -CH ); C NMR (100 MHz, CDCl ) δ: 148.6, 145.0,

2

3

41.0, 138.9, 136.1, 131.7, 130.1, 129.1, 128.4, 127.8 (2C),

22.9, 122.8, 121.5, 110.9, 109.6, 41.6 (CH ), 15.3 (Me) ; MS

2

4

.1.3. 2-(1-Allyl-1H-indol-3-yl)-3-chloroquinoxaline (3a)

(ES mass): 308.1 (M+1); HPLC: 99.8%, Column: Symmetry C-

8 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water,

mobile phase B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20,

1

Yield: 87%; pale yellow solid; mp: 108-110 °C; R = 0.21

10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.71-

.68 (m, 1H, ArH), 8.30 (s, 1H, C-2 indolyl H), 8.17 (dd, J = 8.4,

.8 Hz, 1H, ArH), 7.99-7.97 (m, 1H, ArH), 7.78-7.73 (m, 1H,

f

1

3

(

8

0

3

1

2/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER (90:10);

UV 275.0 nm, retention time 4.4 min.

ArH), 7.71-7.67 (m, 1H, ArH), 7.43-7.39 (m, 1H, ArH), 7.36-

4.1.7.

2-Chloro-3-(1-ethyl-5-methoxy-1H-indol-3-

7

1

4

1

.31 (m, 2H, ArH), 6.12-6.02 (m, 1H, =CH), 5.30 (dd, J = 10.0,

.2 Hz, 1H, =CH ), 5.21 (dd, J = 17.2, 1.2 Hz, 1H, =CH ), 4.88-

yl)quinoxaline (3e)

2

13

Yield: 86%; pale yellow solid; mp: 126-128 °C; R

f

= 0.21

.86 (m, 2H, CH₂); C NMR (100 MHz, CDCl ) δ: 148.4, 145.1,

1

3

(10% EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.60

6

41.0, 138.9, 136.5, 132.5, 132.4, 130.1, 129.2, 128.4, 127.8,

(s, 1H, C-2 indolyl H), 8.25 (d, J = 2.4 Hz, 1H, ArH), 8.13 (d, J

27.7, 123.1, 122.7, 121.7, 118.1, 111.2, 109.9, 49.3 (CH ); MS

2

=

8.0 Hz, 1H, ArH), 7.97 (d, J = 8.0 Hz, 1H, ArH), 7.85 (t, J =

.6 Hz, 1H, ArH), 7.76 (t, J = 7.6 Hz, 1H, ArH), 7.54 (d, J = 8.8

Hz, 1H, ArH), 6.95 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 4.34 (q, J =

.2 Hz, 2H, CH ), 3.84 (s, 3H, OCH ), 1.42 (t, J = 7.2 Hz, 3H,

(

ES mass): 320.0 (M+1); HPLC: 99.8%, Column: Symmetry C-

8 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water,

mobile phase B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20,

7

1

3

7

2

3

1

2

European Journal of Medicinal Chemistry

13

CH -CH ); C NMR (100 MHz, DMSO-d₆

AC

) δ

:

C

15

E

5.5,

PT

14

E

8

D

.6, MA

O

N

CH

U

),

S

C

1.64IP(s

R

T

, 3H,CH ), 1.62 (s, 3H, CH ); C NMR (100

2

3

1

44.5, 140.7, 138.4, 133.7, 131.3, 131.0, 129.7, 128.3, 127.9,

12.8, 111.5, 109.9, 109.7, 105.0, 55.7 (OMe), 41.6 (NCH ), 15.8

MHz, CDCl ) δ: 155.6, 148.7, 145.0, 140.9, 138.8, 131.2, 130.1,

3

129.0 (2C), 128.3, 128.2, 127.9, 112.9, 110.5, 110.4, 104.5, 55.8

(OMe), 47.9 (NCH), 22.7 (2C, 2Me); MS (ES mass): 352.0

(M+1); HPLC: 99.8%, Column: Symmetry C-18 75 * 4.6 mm,

3.5µm, mobile phase A: 0.1 % TFA in water, mobile phase B:

2

(Me); MS (ES mass): 338.0 (M+1); HPLC: 97.7%, Column:

Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 %

TFA in water, mobile phase B: CH CN (T/%B): 0/20, 0.5/20,

3

2

/95, 10/95, 10.5/20, 12/20; flow rate: 1.0 mL/min; Diluent:

CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow rate:

3

ACN: WATER (80:20); UV 230.0 nm, retention time 4.3 min.

1.0 mL/min; Diluent: ACN: WATER (90:10); UV 220.0 nm,

retention time 4.5 min.

4

.1.8. 2-(1-Benzyl-1H-indol-3-yl)-3-chloroquinoxaline (3f)

4

.1.12.

2-(5-Bromo-1-isopropyl-1H-indol-3-yl)-3-

Yield: 78%; pale yellow solid; mp: 128-130 °C; R = 0.28

f

1

chloroquinoxaline (3j)

(

10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.73 (d,

3

J = 8.0 Hz, 1H, ArH), 8.35 (s, 1H, C-2 indolyl H), 8.01 (d, J =

Yield: 88%; pale yellow solid; mp: 198-200 °C; R = 0.23

f

1

8

1

.0 Hz, 1H, ArH), 7.99 (t, J = 7.2 Hz, 1H, ArH), 7.78-7.74 (m,

(10% EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.72

6

H, ArH), 7.71-7.67 (m, 1H, ArH), 7.37-7.30 (m, 5H, ArH), 7.20

(d, J = 2.0 Hz, 1H, ArH), 8.62 (s, 1H, C-2 indolyl H), 8.13-8.11

(m, 1H, ArH), 7.99-7.97 (m, 1H, ArH), 7.88-7.84 (m, 1H, ArH),

7.82-7.78 (m, 1H, ArH), 7.69 (d, J = 8.8 Hz, 1H, ArH), 7.42 (dd,

J = 8.8, 2.0 Hz, 1H, ArH), 4.94-4.87 (m, 1H, NCH), 1.56 (s, 3H,

13

(d, J = 6.4 Hz, 3H, ArH), 5.44 (s, 2H, CH₂); C NMR (100

MHz, CDCl ) δ: 148.4, 145.1, 141.0, 139.0, 136.6, 136.4, 132.8,

3

1

3

30.1, 129.3 (2C), 128.9 (2C), 128.5, 127.9 (2C), 127.8, 126.8,

13

23.3, 122.7, 121.8, 111.4, 110.1, 50.6 (CH ); MS (ES mass):

CH ), 1.55 (s, 3H, CH ); C NMR (100 MHz, DMSO-d ) δ:

2

3

6

70.1 (M+1); HPLC: 91.5%, Column: Symmetry C-18 75 * 4.6

148.1, 144.8, 141.7, 140.6, 138.8, 134.8, 131.3, 131.2, 130.3,

128.5, 128.0, 125.6, 124.8, 114.4, 113.2, 110.1, 48.2 (NCH), 22.6

(2C, 2Me); MS (ES mass): 401.9 (M+3); HPLC: 99.8%, Column:

Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 %

mm, 3.5µm, mobile phase A: 0.1 % TFA in water, mobile phase

B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow

3

rate: 1.0 mL/min; Diluent: ACN: WATER (80:20); UV 245.0

nm, retention time 4.0 min.

TFA in water, mobile phase B: CH CN (T/%B): 0/20, 0.5/20,

3

2

/95, 8/95, 10/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN:

4

.1.9. 2-Chloro-3-(1-isopropyl-1H-indol-3-yl)quinoxaline (3g)

WATER (90:10); UV 220.0 nm, retention time 5.4 min.

Yield: 89%; pale yellow solid; mp: 104-106 °C; R = 0.20

f

1

4.1.13.

chloroquinoxaline (3k)

2-(5-Bromo-1-methyl-1H-indol-3-yl)-3-

(

10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.68-

3

8

1

7

2

3

1

.66 (m, 1H, ArH), 8.40 (s, 1H, C-2 indolyl H ), 8.16-8.14 (m,

H, ArH), 7.99-7.77 (m, 1H, ArH), 7.77-7.73 (m, 1H, ArH),

.70-7.66 (m, 1H, ArH), 7.49-7.46 (m, 1H, ArH), 7.36-7.30 (m,

Yield: 80%; light yellow solid; mp: 137-139 °C; R = 0.22

(10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.86 (s,

1H, ArH), 8.22 (s, 1H, C-2 indolyl H), 8.14 (d, J = 8.4 Hz, 1H,

ArH), 7.94 (d, J = 8.0 Hz, 1H, ArH), 7.75 (t, J = 8.4 Hz, 1H,

ArH), 7.68 (t, J = 8.0 Hz, 1H, ArH), 7.39 (d, J = 8.8 Hz, 1H,

f

1

3

H, ArH), 4.83-4.76 (m, 1H, NCH), 1.65 (s, 3H, CH ), 1.63 (s,

3

13

H, CH₃); C NMR (100 MHz, CDCl ) δ: 148.6, 145.1, 141.0,

3

38.9, 136.0, 130.1, 129.1, 128.5, 128.4, 127.8, 127.7, 122.8,

22.6, 121.6, 111.0, 109.8, 47.7 (NCH), 22.7 (2C, 2Me); MS (ES

1

3

ArH), 7.20 (d, J = 8.8 Hz, 1H, ArH), 3.86 (s, 3H, NCH₃);

C

mass): 322.1 (M+1); HPLC: 99.8%, Column: Symmetry C-18 75

4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water, mobile

NMR (100 MHz, CDCl ) δ: 147.7, 144.6, 140.8, 138.9, 135.7,

3

*

134.1, 130.2, 129.4, 129.0, 128.4, 127.8, 125.9, 125.4, 115.2,

110.9, 110.4, 33.6 (NMe); MS (ES mass): 374.0 (M+3); HPLC:

99.8%, Column: Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile

phase B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20, 12/20;

3

flow rate: 1.0 mL/min; Diluent: ACN: WATER (80:20); UV

2

20.0 nm, retention time 4.7 min.

phase A: 0.1 % TFA in water, mobile phase B: CH CN (T/%B):

3

0

/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow rate: 1.0 mL/min;

Diluent: ACN: WATER (90:10); UV 220.0 nm, retention time

.8 min.

4

.1.10. 2-Chloro-3-(6-chloro-1-isopropyl-1H-indol-3-

yl)quinoxaline (3h)

4

Yield: 87%; light yellow solid; mp: 132-134 °C; R = 0.22

f

1

4.1.14. 2-Chloro-3-(1-methyl-1H-indol-3-yl)quinoxaline (3l)

Yield: 83%; pale yellow solid; mp: 143-145 °C; R = 0.21

(

10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.60 (d,

3

J = 8.4 Hz, 1H, ArH), 8.38 (s, 1H, C-2 indolyl H), 8.14 (d, J =

.4 Hz, 1H, ArH), 7.98 (d, J = 8.0 Hz, 1H, ArH), 7.77-7.73 (m,

H, ArH), 7.71-7.61 (m, 1H, ArH), 7.46 (d, J = 1.2 Hz, 1H,

f

1

8

1

(10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.78-

3

8.65 (m, 1H, ArH), 8.25 (s, 1H, C-2 indolyl H), 8.16-8.14 (m,

1H, ArH), 7.98-7.96 (m, 1H, ArH), 7.76-7.72 (m, 1H, ArH),

7.69-7.65 (m, 1H, ArH), 7.41-7.32 (m, 3H, ArH), 3.91 (s, 3H,

ArH), 7.28 (d, J = 1.2 Hz, 1H, ArH), 4.74-4.67 (m, 1H, NCH),

13

1

.64 (s, 3H, CH ), 1.63 (s, 3H, CH ); C NMR (100 MHz,

3

13

CDCl ) δ: 148.1, 144.9, 140.8, 139.0, 136.5, 130.2, 129.4, 129.0,

NCH₃); C NMR (100 MHz, CDCl ) δ: 148.5, 145.0, 141.0,

3

1

(

9

28.8, 128.3, 127.9, 126.2, 123.7, 122.1, 111.2, 109.8, 48.0

NCH), 22.6 (2C, 2Me); MS (ES mass): 355.9 (M+1); HPLC:

9.9%, Column: Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile

phase A: 0.1 % TFA in water, mobile phase B: CH CN (T/%B):

138.9, 137.1, 133.3, 130.1, 129.1, 128.4, 127.8, 127.6, 123.1,

122.7, 121.6, 110.9, 109.5, 33.4 (NMe); MS (ES mass): 294.0

(M+1); HPLC: 99.5%, Column: Symmetry C-18 75 * 4.6 mm,

3.5µm, mobile phase A: 0.1 % TFA in water, mobile phase B:

3

0

/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow rate: 1.0 mL/min;

Diluent: ACN: WATER (90:10); UV 220.0 nm, retention time

.3 min.

CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow rate:

1.0 mL/min; Diluent: ACN: WATER (90:10); UV 220.0 nm,

retention time 4.2 min.

3

5

4

.1.11.

2-Chloro-3-(1-isopropyl-5-methoxy-1H-indol-3-

4.1.15.

2-Chloro-3-(5-methoxy-1-methyl-1H-indol-3-

yl)quinoxaline (3i)

yl)quinoxaline (3m)

Yield: 92%; pale yellow solid; mp: 116-118 °C; R = 0.25

Yield: 86%; light orange solid; mp: 244-246 °C; R = 0.23

f

1

(

10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.39 (s,

(10% EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.58

3

6

1

7

H, C-2 indolyl H), 8.26 (d, J = 2.8 Hz, 1H, ArH), 8.12-8.10 (m,

H, ArH), 7.98-7.96 (m, 1H, ArH), 7.76-7.72 (m, 1H, ArH),

.69-7.65 (m, 1H, ArH), 7.37 (d, J = 8.8 Hz, 1H, ArH), 7.00 (dd,

(s, 1H, C-2 indolyl H), 8.26 (d, J = 2.4 Hz, 1H, ArH), 8.14 (d, J

= 8.0 Hz, 1H, ArH), 7.96 (d, J = 8.0 Hz, 1H, ArH), 7.86-7.82 (m,

1H, ArH), 7.78-7.74 (m, 1H, ArH), 7.48 (d, J = 8.8 Hz, 1H,

ArH), 6.96 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 3.92 (s, 3H, OCH₃),

J = 8.8, 2.4 Hz, 1H, ArH), 4.76-4.69 (m, 1H, NCH), 3.92 (s, 3H,

1

3

1

3

1

.85 (s, 3H, NCH₃); C NMR (100 MHz, DM

A

S

C

O-

C

d

E

)

P

δ:

T

15

E

5

D

.5, MAN

Y

U

iel

S

d:

C

6

R

5%IP; p

T

ale yellow solid; mp: 119-121 °C, R =0.25

6

f

1

48.6, 144.4, 140.7, 138.4, 135.2, 132.4, 131.0, 129.7, 128.3,

28.1, 127.9, 112.8, 111.5, 109.5, 104.9, 55.7 (OMe), 33.7

NMe); MS (ES mass): 324.1.0 (M+1); HPLC: 99.0%, Column:

X Bridge C-18 150 * 4.6 mm, 5 µm, mobile phase A: 0.1 % TFA

in water, mobile phase B: CH CN (T/%B): 0/10, 3/10, 10/95,

(10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.44

3

(dd, J = 10.37, 2.54 Hz, 1H, ArH), 8.37 (s, 1H, C-2 indolyl H),

8.17 (dd, J = 8.3, 0.9 Hz, 1H, ArH), 7.98 (dd, J = 8.2, 1.0 Hz,

1H, ArH), 7.74 (m, 2H, ArH), 7.32 (dd, J = 8.9, 4.4 Hz, 1H,

ArH), 7.09 (m, 1H, ArH), 6.06 (m, 1H, =CH), 5.26 (m, 2H,

(

3

13

2

0/95, 22/10, 25/10; flow rate: 1.0 mL/min; Diluent: ACN:

=CH ), 4.85 (d, J =5.6 Hz, 2H, NCH ); C NMR (100 MHz,

2

WATER (80:20); UV 225.0 nm, retention time 12.0 min.

CDCl ) δ: 160.4, 158.01 (C-F J = 234.5 Hz), 148.0, 144.68,

3

1

40.9, 139.0 (C-F J = 192.5 Hz), 133.7, 133.0, 132.3, 130.2,

29.3, 128.4, 128.3, 127.8, 118.4, 111.6, 111.4, 111.2, 111.2 (C-

4

.1.16. 2-Chloro-3-(5-nitro-1H-indol-3-yl)quinoxaline (3n)

Yield: 71%; light brown solid; mp: 306-308 °C; R = 0.25

F J = 4.5 Hz), 110.7, 110.6 (C-F J = 9.8 Hz), 108.4, 108.1 (C-F J

= 26.2 Hz), 49.6 (NCH ); MS (ES mass): 338.10 (M+1); HPLC:

f

1

(

10% EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ:

2

6

1

2.53 (s, 1H, NH, D O exchangeable), 9.51 (d, J = 2.0 Hz, 1H,

99.88%, Column: X Bridge C-18 150 * 4.6 mm, 5µm, mobile

phase A: 0.1 % HCOOH in water, mobile phase B: ACN (T%B):

0/20, 3/20, 10/95, 23/95, 25/20, 30/20; Flow rate: 1.0 mL/min;

Diluent: ACN: WATER (80:20); retention time 12.5 min.

2

ArH), 8.77 (d, J = 2.8 Hz, 1H, ArH), 8.15-8.12 (m, 2H, ArH),

8

1

.03-8.00 (m, 1H, ArH), 7.93-7.89 (m, 1H, ArH), 7.86-7.82 (m,

13

H, ArH), 7.71 (d, J = 9.2 Hz, 1H, ArH); C NMR (100 MHz,

DMSO-d ) δ: 147.6, 144.4, 142.3, 140.3, 139.8, 138.8, 134.0,

6

4

.1.21. 2-Chloro-3-(1-(3-chloro-3-methylbutyl)-1H-indol-3-

1

31.2, 130.4, 128.3, 127.9, 126.2, 119.6, 118.2, 112.9, 112.8; MS

ES mass): 323.1.0 (M-1); HPLC: 98.0%, Column: Symmetry C-

8 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water,

mobile phase B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20,

yl)quinoxaline (3w)

(

1

Yield: 38%; light yellow solid; mp: 208-210 °C; R = 0.40

(20% EtOAc-n-Hexane); H NMR (400 MHz, CDCl ) δ: 8.69

f

1

3

1

2/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER (90:10);

(dd, J = 5.2, 2.0 Hz, 1H, ArH), 8.32 (s, 1H, C-2 indolyl H), 8.16

(dd, J = 7.2, 1.2 Hz, 1H, ArH), 7.99 (dd, J = 7.2, 1.2 Hz, 1H,

ArH), 7.78-7.73 (m, 1H, ArH), 7.71-7.67 (m, 1H, ArH), 7.48 (dd,

J = 5.6, 1.2 Hz, 1H, ArH), 7.39-7.31 (m, 2H, ArH), 4.55-4.51 (m,

UV 220.0 nm, retention time 3.8 min.

4

.1.17. Compound 3o-3s

These compounds are known and prepared following the

reported method [15]. Spectral data of these compounds were

compared with the reported data.

2H, NCH

2

), 2.36-2.32 (m, 2H, NCH

C NMR (100 MHz, CDCl ) δ: 148.4, 145.0, 141.0, 139.0,

136.1, 132.1, 130.1, 129.2, 128.4, 127.8, 127.8, 123.2, 122.8,

21.7, 111.3, 109.6, 68.4 (C-Cl), 45.2 (NCH ), 43.7 (CH ), 32.6

2 2 3 2

-CH ), 1.70 (s, 6H, (CH ) );

13

3

1

2

4

.1.18. Ethyl-2-(3-(3-chloroquinoxalin-2-yl)-5-methoxy-1H-

(

2C, 2Me); MS (ES mass): 384.1 (M+1); HPLC: 99.5%; Column:

X BRIDGE C-18 150*4.6 mm, 5µm, mobile phase A: 0.1 %

Formic Acid in water, mobile phase B: CH CN (gradient) T/B%:

indol-1-yl)acetate (3t)

Yield: 70%; pale yellow solid; mp: 160-162 °C, R =0.27

3

f

1

0

/10, 3/10, 12/95, 23/95, 25/10, 30/10; flow rate: 1.0 mL/min;

(

10% EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.63

6

UV 190-800 nm, retention time 13.4 min.

s, 1H, C-2 indolyl H), 8.25 (d, J = 2.4 Hz, 1H, ArH), 8.21-8.11

m, 1H, ArH), 7.98 (dd, J = 8.2, 0.94 Hz, 1H, ArH), 7.91-7.83

m, 1H, ArH), 7.82-7.70 (m, 1H, ArH), 7.43 (d, J = 9.2 Hz, 1H,

4

.1.22. 2-(3-(3-Chloroquinoxalin-2-yl)-5-methoxy-1H-indol-1-

yl)-N-phenylacetamide (3x)

ArH), 6.93 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 5.29 (s, 2H, NCH₂),

.17 (m, 2H, OCH ), 3.84 (s, 3H, OCH ), 1.22 (t, J = 7.2 Hz, 3H,

4

Yield: 45%; pale yellow solid; mp: 214-216 °C; R

(30% EtOAc-n-Hexane); H NMR (400 MHz, DMSO-d

f

= 0.48

) δ:

2

3

13

1

CH₃); C NMR (100 MHz, DMSO-d ) δ: 169.0 (C=O), 155.6,

6

1

48.5, 144.5, 140.7, 138.6, 135.4, 132.2, 131.1, 129.0, 128.7,

10.45 (s, 1H, NH, D O exchangeable), 8.68 (s, 1H, C-2 indolyl

2

28.1, 127.0, 112.1, 111.6, 110.5, 105.0, 61.6 (OCH ), 55.7

H), 8.28 (d, J = 2.0 Hz, 1H, ArH), 8.16 (d, J = 8.4 Hz, 1H, ArH),

7.98 (d, J = 8.4 Hz, 1H, ArH), 7.86 (t, J = 7.2 Hz, 1H, ArH),

7.78 (t, J = 7.2 Hz, 1H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH),

7.46 (d, J = 8.8 Hz, 1H, ArH), 7.31 (t, J = 7.6 Hz, 2H, ArH),

7.06 (t, J = 7.2 Hz, 1H, ArH), 6.94 (dd, J = 6.8, 2.0 Hz, 1H,

2

(OMe), 48.0 (NCH ), 14.5 (Me); MS (ES mass): 396.10 (M+1);

2

HPLC: 99.84%, Column: X Bridge C-18 150 * 4.6 mm, 5µm,

mobile phase A: 0.1 % HCOOH in water, mobile phase B: CAN

(

T%B): 0/20, 3/20, 10/95, 23/95, 25/20, 30/20; Flow rate: 1.0

13

mL/min; Diluent: ACN: WATER (80:20); retention time 12.9

min.

ArH), 5.22 (s, 2H, NCH

MHz, DMSO-d ) δ: 174.9 (C=O), 166.1, 155.6, 148.6, 144.5,

40.7, 139.5, 139.1, 138.5, 135.9, 132.4, 131.2, 129.9, 129.3,

2

), 3.85 (s, 3H, OCH ); C NMR (100

3

6

1

5

9

4

.1.19.

Ethyl-2-(3-(3-chloroquinoxalin-2-yl)-1H-indol-1-

28.4, 128.2, 128.0, 124.0, 119.6, 112.9, 111.5, 110.1, 105.1,

yl)acetate (3u)

5.7 (OMe), 50.1 (NCH ); MS (ES mass): 443.10 (M+1); HPLC:

2

9.6%; Column: X BRIDGE C-18 150*4.6 mm, 5µm, mobile

Yield: 70%; pale yellow solid; mp: 194-196 °C, R =0.25

f

1

phase A: 0.1 % Formic Acid in water, mobile phase B: CH CN

(10% EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.66

3

6

(gradient) T/B%: 0/20, 3/20, 10/95, 23/95, 25/20, 30/20; flow

(d, J = 3.6 Hz, 2H, ArH), 8.17 (d, J = 8.4 Hz, 1H, ArH), 7.99 (d,

rate: 1.0 mL/min; UV 190-800 nm, retention time 11.2 min.

J = 8.4 Hz, 1H, ArH), 7.84 (m, 2H, ArH), 7.53 (d, J = 7.2 Hz,

1

2

H, ArH), 7.35-7.18 (m, 2H, ArH), 5.34 (s, 2H, NCH ), 4.18 (m,

2

13

4

.1.23. 2-(3-(3-Chloroquinoxalin-2-yl)-5-methoxy-1H-indol-1-

H, OCH ), 1.23 (t, J = 7.2 Hz, 3H, CH ); C NMR (100 MHz,

2

3

yl)-1-morpholinoethanone (3y)

DMSO-d ) δ: 169.0 (C=O), 148.4, 144.7, 140.7, 138.7, 137.2,

1

6

35.1, 131.1, 130.1, 128.5, 128.0, 127.5, 123.4, 122.9, 121.9,

10.9, 110.9, 61.6 (OCH ), 47.9 (NCH ), 14.5 (CH ); MS (ES

Yield: 42%; pale yellow solid; mp: 232-234 °C; R

(30% EtOAc-n-Hexane); H NMR (400 MHz, CDCl

f

= 0.42

1

) δ: 8.30 (s,

2

3

mass): 366.10 (M+1); HPLC: 98.83%, Column: X Bridge C-18

50 * 4.6 mm, 5µm, mobile phase A: 0.1 % HCOOH in water,

mobile phase B: ACN (T%B): 0/20, 3/20, 10/95, 23/95, 25/20,

0/20; Flow rate: 1.0 mL/min; Diluent: ACN: WATER (80:20);

retention time 12.9 min.

2H, C-2 indolyl H), 8.16-8.11 (m, 1H, ArH), 7.99 (dd, J = 7.2,

0.8 Hz, 1H, ArH), 7.79-7.66 (m, 2H, ArH), 7.25 (d, J = 7.6 Hz,

1

1H, ArH), 7.02 (d, J = 2.4 Hz, 1H, ArH), 5.02 (s, 2H, NCH

3.94 (s, 3H, OCH ), 3.73-3.61 (m, 6H, morpholine-O, NCH

3.55-3.47 (m, 2H, morpholine-NCH

CDCl ) δ: 165.0 (C=O), 155.8, 148.3, 144.9, 140.9, 139.0, 133.1,

),

2

3

2

),

13

); C NMR (100 MHz,

2

3

4

.1.20.

2-(1-Allyl-5-fluoro-1H-indol-3-yl)-3-

1

4

31.9, 130.1, 129.3, 128.3, 128.3, 127.9, 113.6, 111.7, 110.0,

05.0, 66.7 (OCH ), 66.3 (OCH ), 55.8 (OMe), 48.5 (NCH ),

chloroquinoxaline (3v)

2

5.6 (NCH ), 42.6 (NCH C=O); MS (ES mass): 437.10 (M+1);

2

1

4

European Journal of Medicinal Chemistry

HPLC: 99.3%; Column: X BRIDGE C-18 1

AC

50

*4

C

.6

E

mm, 5

PT

E

µ

D

m, MA15

NU

5.3,

SC

15

3

R

.3,IP14

T

2.8, 134.2, 131.9, 131.5, 127.3, 119.8, 119.3,

mobile phase A: 0.1 % Formic Acid in water, mobile phase B:

117.8, 112.8, 112.2, 111.8, 104.6, 103.9, 55.8 (OMe), 49.0

CH CN (gradient) T/B%: 0/20, 3/20, 10/95, 23/95, 25/20, 30/20;

(NCH ); MS (ES mass): 290.10 (M+1); HPLC: 97.55%, Column:

3

2

flow rate: 1.0 mL/min; UV 190-800 nm, retention time 10.1 min.

X Bridge C-18 150 * 4.6 mm, 5µm, mobile phase A: 0.1 %

HCOOH in water, mobile phase B: ACN (T%B): 0/20, 3/20,

4

.1.24.

2-(1-Allyl-5-methoxy-1H-indol-3-yl)quinoline-3-

1

0/95, 23/95, 25/20, 30/20; Flow rate: 1.0 mL/min; UV 190-800

carbonitrile (3z)

nm, Diluent: ACN: WATER (80:20); retention time 10.6 min.

Yield: 65%; Off white solid; mp: 138-140 °C, R =0.23 (10%

f

1

4.1.28. Synthesis of compound 7

EtOAc/ n-hexane); H NMR (400 MHz, DMSO-d ) δ: 9.06 (s,

6

1

H, ArH), 8.39 (s, 1H, C-2 indolyl H), 8.24 (d, J = 2.0 Hz, 1H,

To a solution of 3t or 4a (1 equiv.) in THF (5 mL) was added

LiOH (5 equiv) at the 0 ºC (ice bath) and the mixture was stirred

for 15 min at the same temperature. The temperature of the

reaction mixture was then allowed to reach room temp and

stirring continued for 5-7 h. After completion of the reaction

(TLC), the mixture was diluted with cold water (25 mL), washed

with EtOAc (2 x 10 mL) and the aqueous layer was collected.

The aqueous part was acidified (pH ~ 5-6) using dil HCl solution

and the mixture was extracted with EtOAc (3 x 10 mL). The

organic layers were collected, combined, washed with water (2 x

15 mL), dried over anhydrous Na SO , filtered and concentrated

ArH), 8.09 (d, J = 8.4 Hz, 1H, ArH), 8.01 (d, J = 8.0 Hz, 1H,

ArH), 7.92 (t, J = 7.6 Hz, 1H, ArH), 7.63 (t, J = 7.6 Hz, 1H,

ArH), 7.47 (d, J = 8.8 Hz, 1H, ArH), 6.93 (dd, J = 8.8, 2.4 Hz,

1

=

NMR (100 MHz, DMSO-d ) δ: 155.4, 152.9, 148.6, 145.6, 134.2,

1

H, ArH), 6.05 (m, 1H, =CH), 5.17 (dd, J = 31.7, 13.6 Hz, 2H,

13

CH ), 4.95 (d, J = 5.2 Hz, 2H, NCH ), 3.84 (s, 3H, OCH ); C

2 2 3

6

33.5, 131.9, 131.7, 128.9, 128.7, 127.7, 127.2, 123.8, 119.4,

17.9, 113.0, 112.5, 111.8, 104.9, 103.2, 55.7 (OCH ), 49.1

3

(NCH ); MS (ES mass): 340.10 (M+1); HPLC: 99.40%, Column:

2

X Bridge C-18 150 * 4.6 mm, 5µm, mobile phase A: 0.1 %

2

4

HCOOH in water, mobile phase B: ACN (T%B): 0/20, 3/20,

under low vacuum. The residue obtained was purified by using

column chromatography over silica gel (hexane-EtOAc as eluent)

to give the desired product.

1

0/95, 23/95, 25/20, 30/20; Flow rate: 1.0 mL/min; UV 254.0

nm, Diluent: ACN: WATER (80:20); retention time 11.8 min.

4

.1.25. Ethyl-2-(3-(3-cyanopyridin-2-yl)-5-methoxy-1H-indol-

4.1.29. 2-(3-(3-Chloroquinoxalin-2-yl)-5-methoxy-1H-indol-1-

1

-yl)acetate (4a)

yl)acetic acid (7a)

Yield: 65%; Off white solid; mp: 112-114 °C, R =0.25 (20%

Yield: 33%; pale yellow solid; mp: 223-225 °C; R = 0.42

f

1

EtOAc/n-hexane); H NMR (400 MHz, CDCl ) δ: 8.87 (dd, J =

(80% EtOAc-n-Hexane); H NMR (400 MHz, DMSO-d ) δ: 8.58

3

6

4

2

.8, 1.84 Hz, 1H, ArH ), 8.22 (s, 1H, C-2 indolyl H), 8.09 (d, J =

.4 Hz, 1H, ArH), 8.05-7.84 (m, 1H, ArH), 7.23-7.08 (m, 2H,

(s, 1H, C-2 indolyl H), 8.28 (d, J = 4.0 Hz, 1H, ArH), 8.13 (d, J

= 8.4 Hz, 1H, ArH), 7.95 (d, J = 8.4 Hz, 1H, ArH), 7.86-7.81 (m,

1H, ArH), 7.77-7.72 (m, 1H, ArH), 7.34 (d, J = 8.8 Hz, 1H,

ArH), 6.98 (dd, J = 8.9, 2.4 Hz, 1H, ArH), 4.91 (s, 2H, NCH₂),

.23 (m, 2H, OCH ), 3.90 (s, 3H, OCH ), 1.23 (t, J = 7.2 Hz, 3H,

4

ArH), 6.90-6.87 (m, 1H, ArH), 4.81 (s, 2H, NCH ), 3.84 (s, 3H,

2

3

2

13

CH₃); C NMR (100 MHz, CDCl ) δ: 167.7 (C=O), 156.5,

OCH₃); C NMR (100 MHz, DMSO-d ) δ: 155.4 (C=O), 148.7,

3

6

1

55.7, 152.5, 141.7, 132.1, 131.5, 127.5, 119.0, 118.8, 113.5,

144.4, 143.0, 140.7, 139.2, 138.3, 135.7, 132.4, 131.1, 129.6,

128.3, 127.9, 112.5, 111.8, 109.5, 104.9, 55.7 (2C, OMe &

13.4, 110.0, 104.8, 104.2, 61.9 (OCH ), 55.9 (OMe), 48.4

2

(

NCH ), 14.1 (CH -Me); MS (ES mass): 336.10 (M+1); HPLC:

NCH ); MS (ES mass): 368.0 (M+1); HPLC: 98.5%; Column: X

2

9

9.90%, Column: X Bridge C-18 150 * 4.6 mm, 5µm, mobile

BRIDGE C-18 150*4.6 mm, 5µm, mobile phase A: 0.1 %

phase A: 0.1 % HCOOH in water, mobile phase B: ACN (T%B):

/20, 3/20, 10/95, 23/95, 25/20, 30/20; Flow rate: 1.0 mL/min;

UV 190-800 nm, Diluent: ACN: WATER (80:20); retention time

2.9 min.

Formic Acid in water, mobile phase B: CH CN (gradient) T/B%:

0/20, 3/20, 10/95, 23/95, 25/20, 30/20; flow rate: 1.0 mL/min;

UV 190-800 nm, retention time 10.1 min.

3

0

1

4

.1.30.

2-(3-(3-Cyanopyridin-2-yl)-5-methoxy-1H-indol-1-

4

.1.26. 2-(1-Allyl-5-chloro-1H-indol-3-yl)nicotinonitrile (4b)

yl)acetic acid (7b)

Yield: 70%; Off white solid; mp: 74-76 °C; R = 0.21 (10%

Yield: 75%; white solid; mp: 217-219 °C; R = 0.40 (80%

f

1

EtOAc/n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.92 (d, J

EtOAc-n-Hexane); H NMR (400 MHz, DMSO-d ) δ: 13.61-

6

=

7

2.8 Hz, 1H, ArH ), 8.38 (d, J = 10.0 Hz, 2H, ArH), 8.32 (d, J =

.6 Hz, 1H, ArH), 7.60 (d, J = 8.4 Hz, 1H, ArH), 7.39 (m, 1H,

ArH), 7.31-7.25 (m, 1H, ArH), 6.09-5.99 (m, 1H, =CH), 5.23-

12.72 (bs, 1H, OH, D O Exchangeable ), 8.89 (dd, J = 2.8, 1.6

2

Hz, 1H, ArH), 8.28 (dd, J = 6.0, 2.0 Hz, 1H, ArH), 8.24 (s, 1H,

C-2 indolyl H), 7.88 (d, J = 2.40 Hz, 1H, ArH), 7.39-7.33 (m,

2H, ArH), 6.89 (dd, J = 6.4, 2.8 Hz, 1H, ArH), 5.11 (s, 2H,

13

5

.08 (m, 2H, =CH ), 4.98 (d, J = 4.8 Hz, 2H, NCH ); C NMR

2

13

(100 MHz, DMSO-d ) δ: 155.5, 153.9, 153.3, 144.5, 142.9,

NCH ), 3.77 (s, 3H, OCH ); C NMR (100 MHz, DMSO-d ) δ:

6

2

3

6

1

33.9, 132.4, 127.7, 126.2, 123.8, 123.0, 121.8, 120.2, 118.1,

170.4 (C=O), 156.2, 155.2, 153.3, 142.9, 132.6, 132.5, 127.1,

119.8, 119.1, 112.7, 112.3, 111.6, 104.6, 103.8, 55.8 (OMe), 48.3

12.8, 104.1, 49.1 (NCH ); MS (ES mass): 294.00 (M+1);

2

HPLC: 97.54%, Column: X Bridge C-18 150 * 4.6 mm, 5µm,

(NCH ); MS (ES mass): 308.1 (M+1); HPLC: 99.0%; column: X

2

mobile phase A: 0.1 % TFA in water, mobile phase B: ACN

BRIDGE C-18 150*4.6 mm, 5µm, mobile phase A: 0.1 %

(

T%B): 0/10, 3/10, 12/95, 23/95, 25/10, 30/10; Flow rate: 1.0

Formic Acid in water, mobile phase B: CH CN (gradient) T/B%:

3

mL/min; UV 190-800 nm, Diluent: ACN: WATER (80:20);

retention time 10.9 min.

0/20, 3/20, 10/95, 23/95, 25/20, 30/20; flow rate: 1.0 mL/min;

UV190-800 nm, retention time 8.5 min.

4

.1.27. 2-(1-Allyl-5-methoxy-1H-indol-3-yl)nicotinonitrile (4c)

4.1.31. Typical procedure for the preparation of (E)-alkyl 3-

(

3-(3-chloroquinoxalin-2-yl)-1-alkyl-5-substitited-1H-indol-2-

Yield: 65%; Off white solid; mp: 96-98 °C; R =0.23 (10%

f

1

yl)acrylate (6k)

EtOAc/n-hexane); H NMR (400 MHz, DMSO-d ) δ: 8.89 (d, J

6

=

3.2 Hz, 1H, ArH), 8.28 (dd, J = 14.4, 7.7 Hz, 2H, ArH), 7.88

A mixture of 2-(5-bromo-1H-indol-3-yl)-3-chloroquinoxaline

(

d, J = 2.0 Hz, 1H, ArH), 7.45 (d, J = 9.2 Hz, 1H, ArH), 7.34

dd, J = 8.0, 5.2 Hz, 1H, ArH), 6.90 (dd, J = 8.8, 2.0 Hz, 1H,

3q (0.280 mmol), ethyl acrylate 5a (0.421 mmol), Pd(OAc) (5

2

mol%), Cu(OAc) (0.280 mmol), and TFA (0.336 mmol) in 1,4-

2

ArH), 6.03 (m, 1H, =CH), 5.20 (d, J = 10.0 Hz, 1H, =CH ), 5.12

dioxane (2.5 mL) was heated at 80 °C under open air for 7h. The

progress of the reaction was monitored by TLC. After completion

of the reaction, reaction mixture was cooled to RT, diluted with

2

(

d, J = 17.6 Hz, 1H, =CH ), 4.92 (d, J = 5.12 Hz, 2H, NCH ),

2

13

3

.77 (s, 3H, OCH₃); C NMR (100 MHz, DMSO-d ) δ: 156.3,

6

1

5

ethyl acetate (15 mL) and passed through ce

A

lite

C

.

C

Th

E

e

P

re

T

su

E

lti

D

ng MAN

4

U

.1.3

SC

RIPT(E)-Methyl-3-(3-(3-chloroquinoxalin-2-yl)-1-

5

.

solution was washed with water (3 x 15 mL) followed by brine

solution (25 mL), dried over anhydrous Na SO , filtered and

isopropyl-1H-indol-2-yl)acrylate (6d)

2

4

Compound 6d was synthesized from 3g following a procedure

similar to that of compound 6k; Yield: 77%; light yellow solid;

mp: 130-132 °C; R = 0.24 (10% EtOAc/ n-hexane); H NMR

400 MHz, CDCl ) δ: 8.08-8.05 (m, 2H, ArH), 7.87-7.76 (m, 4H,

concentrated under reduced pressure. The residue was purified by

column chromatography using ethyl acetate–hexane to give

desired compound 6k.

1

f

(

3

4

.1.32. (E)-Ethyl-3-(3-(3-chloroquinoxalin-2-yl)-1-isopropyl-

ArH), 7.52 (d, J = 8.4 Hz, 1H, ArH), 7.44 (d, J = 7.2 Hz, 1H,

ArH), 7.32 (t, J = 7.6 Hz, 1H, ArH), 6.22 (d, J = 16.0 Hz, 1H,

5

-methoxy-1H-indol-2-yl)acrylate (6a)

=

CH), 4.83-4.76 (m, 1H, NCH), 3.34 (s, 3H, OCH ), 1.63 (m,

3

13

Compound 6a was synthesized from 3i following a procedure

3

H, CH ), 1.62 (s, 3H, CH ); C NMR (100 MHz, CDCl ) δ:

3

similar to that of compound 6k; Yield: 79%; pale yellow solid;

1

166.9 (C=O), 149.5, 147.4, 144.1, 140.6, 140.3, 136.5, 130.1

2C), 128.8, 128.4, 128.0 (2C), 125.6, 122.4, 120.0, 117.7, 111.9,

mp: 99-101 °C; R = 0.27 (10% EtOAc/ n-hexane); H NMR (400

f

(

MHz, CDCl ) δ: 8.11-8.08 (m, 1H, ArH), 8.05-8.02 (m, 1H,

3

1

4

11.6, 51.0 (OMe), 47.7 (NCH), 22.7 (2C, 2Me); MS (ES mass):

06.2 (M+1); HPLC: 99.8%, Column: Symmetry C-18 75 * 4.6

ArH), 7.76-7.69 (m, 4H, ArH), 7.43 (d, J = 8.8 Hz, 1H, ArH),

.02 (d, J = 8.8 Hz, 1H, ArH), 6.17 (d, J = 16.0 Hz, 1H,=CH),

7

4

mm, 3.5µm, mobile phase A: 0.1 % TFA in water, mobile phase

B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 10/95, 10.5/20, 12/20;

flow rate: 1.0 mL/min; Diluent: ACN: WATER (90:10); UV

.76-4.69 (m, 1H, NCH), 3.93 (s, 3H, OCH ), 3.68 (q, J = 7.2

3

Hz, 2H, OCH ), 1.62 (s, 3H, CH ), 1.60 (s, 3H, CH3), 0.73 (t, J =

2

3

13

7

(

.2 Hz, 3H, CH -CH ); C NMR (100 MHz, CDCl ) δ: 166.9

2

3

2

10.0 nm, retention time 4.0 min.

C=O), 154.0, 150.4, 148.2, 140.7, 140.5, 139.2, 131.7, 130.0

2C), 128.9, 128.3, 128.0, 126.5, 121.3, 115.7, 112.1, 111.7,

4.1.36. (E)-Ethyl-3-(1-allyl-3-(3-chloroquinoxalin-2-yl)-5-

1

08.2, 59.5 (OCH ), 56.6 (OMe), 47.7 (NCH), 22.7 (2C, 2Me) ,

methoxy-1H-indol-2-yl)acrylate (6e)

2

3.7 (OCH Me); MS (ES mass): 450.0 (M+1); HPLC: 99.9%,

2

Compound 6e was synthesized from 3b following a procedure

similar to that of compound 6k; Yield: 72%; light yellow solid;

Column: Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile phase A:

0

.1 % TFA in water, mobile phase B: CH CN (T/%B): 0/20,

1

3

mp: 102-104 °C; R = 0.28 (10% EtOAc/ n-hexane); H NMR

f

.5/20, 2/95, 10/95, 10.5/20, 12/20; flow rate: 1.0 mL/min;

(

400 MHz, CDCl ) δ: 8.11-8.04 (m, 2H, ArH), 7.77-7.70 (m, 3H,

3

Diluent: ACN: WATER (80:20); UV 210.0 nm, retention time

.0 min.

ArH), 7.58 (s, 1H, ArH), 7.38 (d, J = 8.8 Hz, 1H, ArH), 7.02 (d,

J = 8.8 Hz, 1H, ArH), 6.14 (d, J = 16.4 Hz, 1H, =CH), 6.12-6.00

4

.1.33.

(E)-Methyl-3-(3-(3-chloroquinoxalin-2-yl)-1-

(m, 1H, =CH allyl), 5.30 (d, J = 10.0 Hz, 1H, =CH ), 5.24 (d, J =

2

isopropyl-5-methoxy-1H-indol-2-yl)acrylate (6b)

17.6 Hz, 1H, =CH ), 4.81 (s, 2H, NCH ), 3.93 (s, 3H, OCH ),

2

3

.69 (q, J = 7.2 Hz, 2H, OCH ), 0.73 (t, J = 7.2 Hz, 3H, CH -

2

Compound 6b was synthesized from 3i following a procedure

13

CH₃); C NMR (100 MHz, CDCl ) δ: 166.9 (C=O), 154.2,

150.2, 148.2, 140.7, 140.5, 139.0, 132.5, 132.1, 132.0, 130.2,

1

MS (ES mass): 448.2 (M+1); HPLC: 97.7%, Column: Symmetry

C-18 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water,

mobile phase B: CH

1

3

similar to that of compound 6k; Yield: 83%; pale yellow solid;

1

mp: 110-112 °C; R = 0.28 (10% EtOAc/ n-hexane); H NMR

f

30.1, 128.9, 128.0, 126.6, 121.4, 118.3, 115.7, 112.4, 111.9,

08.4, 59.6 (OCH ), 56.6 (OMe), 49.3 (NCH ), 13.7 (CH -Me);

(

400 MHz, CDCl ) δ: 8.10-8.04 (m, 2H, ArH), 7.78-7.67 (m, 4H,

3

2

ArH), 7.44 (d, J = 8.8 Hz, 1H, ArH), 7.02 (d, J = 8.8 Hz, 1H,

ArH), 6.19 (d, J = 16.0 Hz, 1H, =CH), 4.80-4.68 (m, 1H, NCH),

3

1

5

.92 (s, 3H, OCH ), 3.15 (s, 3H, Ester CH ), 1.62 (s, 3H, CH ),

3

13

3

CN (T/%B): 0/20, 0.5/20, 2/95, 10/95,

.60 (s, 3H, CH₃); C NMR (100 MHz, CDCl ) δ: 167.4 (C=O),

3

0.5/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER

54.2, 150.4, 148.2, 140.7, 140.4, 139.5, 131.6, 130.1, 128.9,

28.2, 127.9, 126.7, 120.7, 115.4, 112.0, 111.8, 110.5, 108.2,

6.6 (OMe), 50.8 (Ester OMe), 47.7 (NCH), 22.7 (2C, 2Me); MS

(90:10); UV 210.0 nm, retention time 3.9 min.

4.1.37. (E)-Methyl-3-(5-bromo-3-(3-chloroquinoxalin-2-yl)-1-

(

1

ES mass): 436.0 (M+1); HPLC: 98.8%, Column: Symmetry C-

8 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water,

mobile phase B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 10/95,

methyl-1H-indol-2-yl)acrylate (6f)

Compound 6f was synthesized from 3k following a procedure

similar to that of compound 6k; Yield: 75%; pale yellow solid;

3

1

0.5/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER

1

mp: 126-128 °C; R = 0.25 (10% EtOAc/ n-hexane); H NMR

f

(80:20); UV 210.0 nm, retention time 3.9 min.

(

400 MHz, CDCl ) δ: 8.18-8.16 ( m, 1H, ArH), 8.13-8.10 (m, 1H,

3

4

.1.34. (E)-Ethyl-3-(3-(3-chloroquinoxalin-2-yl)-1-isopropyl-

ArH), 7.88-7.80 (m, 3H, ArH), 7.60 (s, 1H, ArH), 7.44-7.43 (m,

1

H-indol-2-yl)acrylate (6c)

1H, ArH), 7.31 (d, J = 8.8 Hz, 1H, ArH), 6.02 (d, J = 16.0 Hz,

1

3

1

H, =CH), 3.94 (s, 3H, NCH ), 3.72 (s, 3H, OCH ); C NMR

3

Compound 6c was synthesized from 3g following a procedure

(

100 MHz, CDCl ) δ: 166.4 (C=O), 148.1, 147.6, 141.1, 141.0,

3

similar to that of compound 6k; Yield: 80%; light yellow solid;

mp: 142-144 °C; R = 0.25 (10% EtOAc/ n-hexane); H NMR

1

136.9, 134.4, 132.0, 131.2, 130.6, 129.2, 128.4, 128.2, 127.4,

22.8, 122.0, 114.8, 114.2, 111.4, 51.9 (OMe), 31.4 (NMe); MS

ES mass): 458.1 (M+3); HPLC: 97.1%, Column: Symmetry C-

8 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water,

mobile phase B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 10/95,

f

1

(

1

(400 MHz, CDCl ) δ: 8.09-8.04 (m, 2H, ArH), 7.83-7.75 (m, 4H,

3

ArH), 7.51 (d, J = 8.0 Hz, 1H, ArH), 7.45 (d, J = 7.2 Hz, 1H,

ArH), 7.32 (t, J = 7.6 Hz, 1H, ArH), 6.23 (d, J = 15.6 Hz, 1H,

=

3

CH), 4.83-4.76 (m, 1H, NCH), 3.85 (q, J = 7.2 Hz, 2H, OCH₂),

1

0.5/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER

1

.63 (s, 3H, CH ), 1.62 (s, 3H, CH ), 0.83 (t, J = 7.2 Hz, 3H,

3

13

(90:10); UV 254.0 nm, retention time 4.2 min.

OCH -CH ); C NMR (100 MHz, CDCl ) δ: 166.5 (C=O),

2

3

1

5

49.7, 147.6, 143.6, 140.7, 140.3, 136.5, 130.2 (2C), 128.9,

28.5, 128.1, 128.0, 127.9, 125.6, 122.4, 120.1, 118.3, 111.5,

9.9 (OCH ), 47.7 (NCH), 22.7 (2C, 2Me), 13.7 (OCH Me); MS

4.1.38. (E)-Ethyl-3-(5-bromo-3-(3-chloroquinoxalin-2-yl)-1-

methyl-1H-indol-2-yl)acrylate (6g)

2

Compound 6g was synthesized from 3k following a procedure

(

1

ES mass): 420.2 (M+1); HPLC: 99.4%, Column: Symmetry C-

8 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water,

mobile phase B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 10/95,

similar to that of compound 6k; Yield: 66%; pale yellow solid;

1

mp: 125-127 °C; R = 0.26 (10% EtOAc/ n-hexane); H NMR

f

3

(

400 MHz, CDCl ) δ: 8.18-8.16 (m, 1H, ArH), 8.13-8.10 (m, 1H,

3

1

0.5/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER

ArH), 7.87-7.79 (m, 3H, ArH), 7.60 (s, 1H, ArH), 7.44 (dd, J =

.8, 1.2 Hz, 1H, ArH), 7.31 (d, J = 8.8 Hz, 1H, ArH), 6.03 (d, J

16.0 Hz, 1H, =CH), 4.18 (q, J = 7.2 Hz, 2H, OCH ), 3.95 (s,

(90:10); UV 210.0 nm, retention time 4.2 min.

8

=

2

1

6

European Journal of Medicinal Chemistry

13

3

H, NCH ), 1.25 (t, J = 7.2 Hz, 3H, CH₂

AC

-C

H

3

C

);EPC

T

NMR

E

D

MAN

4

U

.1.4

S

2

C

.

RI

(E)-tert-Butyl-3-(5-chloro-3-(3-chloroquinoxalin-2-

P

T

3

(100 MHz, CDCl ) δ: 166.0 (C=O), 148.1, 147.6, 141.1 (2C),

yl)-1H-indol-2-yl)acrylate (6k)

3

1

36.9, 134.5, 131.8, 131.1, 130.6, 129.2, 128.2, 127.3, 123.1,

Yield: 81%; light yellow solid; mp: 129-131 °C; R = 0.24

f

22.8, 122.5, 114.7, 114.2, 111.4, 60.8 (OCH ), 29.6 (NMe), 14.1

1

2

(

1

(

=

10% EtOAc/ n-hexane); H NMR (400 MHz, CDCl ) δ: 8.75 (s,

3

(CH -Me); MS (ES mass): 469.8 (M-1); HPLC: 97.6%, Column:

2

H, NH, D O exchangeable), 8.19-8.16 (m, 1H, ArH), 8.13-8.11

2

Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 %

TFA in water, mobile phase B: CH CN (T/%B): 0/20, 0.5/20,

2

m, 1H, ArH), 7.87-7.83 (m, 2H, ArH), 7.59 (d, J = 16.4 Hz, 1H,

CH), 7.54 (s, 1H, ArH), 7.38 (d, J = 8.8 Hz, 1H, ArH), 7.30 (d,

J = 9.2 Hz, 1H, ArH), 6.29 (d, J = 16.4 Hz, 1H, =CH), 1.48 (s,

3

/95, 10/95, 10.5/20, 12/20; flow rate: 1.0 mL/min; Diluent:

ACN: WATER (90:10); UV 210.0 nm, retention time 4.4 min.

13

9

H, (CH ) ); C NMR (100 MHz, DMSO-d ) δ: 165.5 (C=O),

3

6

4

.1.39. (E)-Methyl-3-(3-(3-chloroquinoxalin-2-yl)-1-methyl-

147.9, 147.6, 140.9, 140.8, 135.6, 134.9, 132.0, 131.7, 131.3,

129.3, 128.3, 128.1, 125.5, 125.0, 121.0, 120.6, 116.1, 113.9,

1

H-indol-2-yl)acrylate (6h)

8

0.6 (OC(Me) ), 28.1 (3C, (Me) ); MS (ES mass): 438.2 (M-1);

3

Compound 6h was synthesized from 3l following a procedure

HPLC: 95.5%, Column: Symmetry C-18 75 * 4.6 mm, 3.5µm,

mobile phase A: 0.1 % TFA in water, mobile phase B: CH CN

T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow rate: 1.0

similar to that of compound 6k; Yield: 69%; pale yellow solid;

1

3

mp: 189-191 °C; R = 0.24 (10% EtOAc/ n-hexane); H NMR

f

(

400 MHz, CDCl ) δ: 8.17-8.15 (m, 1H, ArH), 8.13-8.10 (m, 1H,

3

mL/min; Diluent: ACN: WATER (90:10); UV 240.0 nm,

retention time 4.4 min.

ArH), 7.87-7.82 (m, 3H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH),

.37 (d, J = 8.4 Hz, 1H, ArH), 7.38-7.34 (m, 1H, ArH),7.18 (t, J

8.0 Hz, 1H, ArH), 6.12 (d, J = 16.2 Hz, 1H, =CH), 3.97 (s,

7

=

3

1

4.1.43. (E)-Ethyl-3-(5-bromo-3-(3-chloroquinoxalin-2-yl)-1H-

13

H, NCH ), 3.72 (s, 3H, OCH ); C NMR (100 MHz, CDCl ) δ:

indol-2-yl)acrylate (6l)

3

66.7 (C=O), 148.9, 147.9, 141.1, 141.0, 138.4, 133.4, 132.5,

31.0, 130.5, 129.2, 128.2, 126.9, 124.6, 121.4, 120.9, 120.5,

15.2, 109.9, 51.8 (OMe), 30.9 (NMe); MS (ES mass): 378.0

Compound 6l was synthesized from 3q following a procedure

similar to that of compound 6k; Yield: 73%; pale yellow solid;

mp: 136-138 °C; R = 0.25 (10% EtOAc/ n-hexane); H NMR

1

f

(

3

M+1); HPLC: 97.7%, Column: Symmetry C-18 75 * 4.6 mm,

.5µm, mobile phase A: 0.1 % TFA in water, mobile phase B:

CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow rate:

(400 MHz, DMSO-d ) δ: 12.35 (s, 1H, NH, D O exchangeable),

6

2

8

1

=

.17-8.11 (m, 2H, ArH), 7.94 (d, J = 3.6 Hz, 2H, ArH), 7.76 (s,

H, ArH), 7.54-7.39 (m, 3H, ArH), 6.70 (d, J = 16.4 Hz, 1H,

3

1

.0 mL/min; Diluent: ACN: WATER (90:10); UV 245.0 nm,

retention time 3.9 min.

CH), 4.13 (q, J = 6.8 Hz, 2H, OCH2), 1.20-1.17 (m, 3H, CH -

2

13

CH₃); C NMR (100 MHz, DMSO-d ) δ: 166.2 (C=O), 147.8,

6

4

.1.40. (E)-tert-Butyl-3-(3-(3-chloroquinoxalin-2-yl)-1-

147.6, 140.5 (2C), 135.9, 134.5, 132.7, 131.8, 131.3, 129.3,

128.9, 128.3, 127.6, 123.5, 119.3, 116.1, 114.3, 113.5, 60.7

methyl-1H-indol-2-yl)acrylate (6i)

(

OCH2), 14.5 (CH -Me); MS (ES mass): 458.1 (M+2); HPLC:

2

Compound 6i was synthesized from 3l following a procedure

9

7.9%, Column: Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile

phase A: 0.1 % TFA in water, mobile phase B: CH CN (T/%B):

/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow rate: 1.0 mL/min;

Diluent: ACN: WATER (90:10); UV 245.0 nm, retention time

.0 min.

similar to that of compound 6k; Yield: 64%; light yellow solid;

1

3

mp: 123-125 °C; R = 0.26 (10% EtOAc/ n-hexane); H NMR

f

0

(

400 MHz, CDCl ) δ: 8.17-8.10 (m, 2H, ArH), 7.83-7.81 (m, 2H,

3

ArH), 7.75 (d, J = 16.0 Hz, 1H, =CH), 7.48 (d, J = 7.6 Hz, 1H,

ArH), 7.42 (s, 1H, ArH), 7.39-7.33 (m, 1H, ArH), 7.18 (s, 1H,

5

ArH), 6.07 (d, J = 16.0 Hz, 1H, =CH), 3.97 (s, 3H, NCH ), 1.44

4.1.44. (E)-Methyl-3-(5-bromo-3-(3-chloroquinoxalin-2-yl)-

1H-indol-2-yl)acrylate (6m)

3

13

(

s, 9H, (CH ) ); C NMR (100 MHz, CDCl ) δ: 165.6 (C=O),

3

1

8

4

48.9, 147.9, 141.1, 140.9, 138.4, 133.8, 131.3, 130.8, 130.4,

29.1, 128.2, 127.9, 126.9, 124.4, 123.2, 121.3, 120.5, 109.9,

Compound 6m was synthesized from 3q following a

procedure similar to that of compound 6k; Yield: 77%; light

0.9 (OCMe ), 31.5 (NMe), 28.0 (3C, 3Me); MS (ES mass):

3

yellow solid; mp: 291-293 °C; R = 0.25 (10% EtOAc/ n-hexane);

f

20.2 (M+1); HPLC: 98.8%, Column: Symmetry C-18 75 * 4.6

1

H NMR (400 MHz, CDCl ) δ: 8.72 (s, 1H, NH, D O

3

2

mm, 3.5µm, mobile phase A: 0.1 % TFA in water, mobile phase

B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow

rate: 1.0 mL/min; Diluent: ACN: WATER (90:10); UV 270.0

nm, retention time 3.4 min.

exchangeable), 8.20-8.17 (m, 1H, ArH), 8.14-8.12 (m, 1H, ArH),

3

7

1

.89-7.82 (m, 2H, ArH), 7.70 (s, 1H, ArH), 7.65 (d, J = 16.0 Hz,

H, =CH), 7.43 (dd, J = 8.8, 2.0 Hz, 1H, ArH), 7.35 (d, J = 8.8

Hz, 1H, ArH), 6.31 (d, J = 16.0 Hz, 1H, =CH), 3.77 (s, 3H,

13

4

.1.41. (E)-Methyl-3-(5-chloro-3-(3-chloroquinoxalin-2-yl)-

OCH₃); C NMR (100 MHz, DMSO-d ) δ: 166.7 (C=O), 147.8,

6

1

H-indol-2-yl)acrylate (6j)

147.7, 141.0, 140.9, 135.9, 134.4, 132.8, 131.8, 131.3, 129.3,

1

28.9, 128.3, 127.6, 123.6, 118.9, 116.3, 114.3, 113.5, 52.1

Compound 6j was synthesized from 3p following a procedure

(OMe); MS (ES mass): 444.1 (M+3); HPLC: 95.2%, Column:

similar to that of compound 6k; Yield: 70%; light yellow solid;

1

Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 %

TFA in water, mobile phase B: CH CN (T/%B): 0/20, 0.5/20,

2

mp: 156-158 °C; R = 0.28 (10% EtOAc/ n-hexane); H NMR

f

3

(

8

400 MHz, CDCl ) δ: 8.69 (s, 1H, NH, D O exchangeable), 8.24-

3

2

/95, 8/95, 10/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN:

.18 (m, 1H, ArH), 8.16-8.14 (m, 1H, ArH), 7.90-7.86 (m, 2H,

WATER (90:10); UV 245.0 nm, retention time 3.7 min.

ArH), 7.67 (d, J = 16.4 Hz, 1H, =CH), 7.56 (s, 1H, ArH), 7.41

d, J = 8.8 Hz, 1H, ArH), 7.32 (d, J = 9.2 Hz, 1H, ArH), 6.35 (d,

(

4.1.45. (E)-tert-Butyl-3-(5-bromo-3-(3-chloroquinoxalin-2-

13

J = 16.4 Hz, 1H, =CH), 3.79 (s, 3H, OCH₃); C NMR (100

yl)-1H-indol-2-yl)acrylate (6n)

MHz, DMSO-d ) δ: 166.7 (C=O), 147.8, 147.9, 140.9, 140.8,

6

Compound 6n was synthesized from 3q following a procedure

1

3

35.7, 134.6, 132.8, 131.8, 131.4, 129.3, 128.3, 128.2, 125.6,

25.2, 120.5, 118.8, 116.4, 113.9, 52.1 (OMe); MS (ES mass):

98.1 (M+1); HPLC: 98.0%, Column: Symmetry C-18 75 * 4.6

similar to that of compound 6k; Yield: 80%; light yellow solid;

1

mp: 122-124 °C; R = 0.26 (10% EtOAc/ n-hexane); H NMR

f

(

8

400 MHz, CDCl ) δ: 8.89 (s, 1H, NH, D O exchangeable), 8.19-

3

2

mm, 3.5µm, mobile phase A: 0.1 % TFA in water, mobile phase

.17 (m, 1H, ArH), 8.13-8.10 (m, 1H, ArH), 7.87-7.81 (m, 2H,

B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow

3

ArH), 7.70 (s, 1H, ArH), 7.59 (d, J = 16.0 Hz, 1H, =CH), 7.42

dd, J = 8.8, 1.2 Hz, 1H, ArH), 7.33 (d, J = 8.8 Hz, 1H, ArH),

rate: 1.0 mL/min; Diluent: ACN: WATER (90:10); UV 270.0

nm, retention time 4.4 min.

(

6

13

.29 (d, J = 16.0 Hz, 1H, =CH), 1.48 (s, 9H, (CH ) ); C NMR

3 3

(

100 MHz, DMSO-d ) δ: 165.5 (C=O), 147.9, 147.6, 140.9,

6

1

7

1

40.8, 135.9, 134.7, 131.9, 131.7, 131.3, 129

27.4, 123.6, 121.0, 115.9, 114.2, 113.4, 80.6 (OCMe ), 28.1

A

.3

C

, 1

C

2

E

8.8

P

,

T

12

E

8

D

.3, MAN

5

U

.

S

P

C

ha

R

rmIPac

T

ology

3

5

.1. In vitro assay for PDE4B

(3C, Me ); MS (ES mass): 484.2 (M+1); HPLC: 98.0%, Column:

3

Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile phase A: 0.1 %

5.1.1. Cells and Reagents

TFA in water, mobile phase B: CH CN (T/%B): 0/20, 0.5/20,

3

The Sf9 cells were obtained from ATCC (Washington D.C.,

USA) and were routinely maintained in Grace’s supplemented

medium (Invitrogen) with 10% FBS. cAMP was purchased from

SISCO Research Laboratories (Mumbai, India). PDElight HTS

cAMP phosphodiesterase assay kit was procured from Lonza

2

/95, 8/95, 10/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN:

WATER (90:10); UV 240.0 nm, retention time 4.5 min.

4

.1.46. (E)-Ethyl-3-(3-(3-chloroquinoxalin-2-yl)-5-methyl-

1

H-indol-2-yl)acrylate (6o)

(

Basel, Switzerland). PDE4B1 clone was procured from OriGene

Compound 6o was synthesized from 3r following a procedure

Technologies (Rockville, MD, USA). PDE4D2 enzyme was

purchased from BPS Bioscience (San Diego, CA, USA).

similar to that of compound 6k; Yield: 66%; light yellow solid;

1

mp: 129-131 °C; R = 0.27 (10% EtOAc/ n-hexane); H NMR

f

(

400 MHz, CDCl ) δ: 8.55 (s, 1H, NH, D O exchangeable), 8.22-

5.1.2. PDE4B protein production and purification

3

2

8

.19 (m, 1H, ArH), 8.15-8.13 (m, 1H, ArH), 7.88-7.82 (m, 2H,

PDE4B cDNA was sub-cloned into pFAST Bac HTB vector

Invitrogen) and transformed into DH10Bac (Invitrogen)

ArH), 7.68 (d, J = 16.0 Hz, 1H, =CH), 7.36 (d, J = 8.8 Hz, 2H,

ArH), 7.19 (d, J = 8.8 Hz, 1H, ArH), 6.29 (d, J = 16.0 Hz, 1H,

(

competent cells. Recombinant bacmids were tested for

integration by PCR analysis. Sf9 cells were transfected with

bacmid using Lipofectamine 2000 (Invitrogen) according to

manufacturer’s instructions. Subsequently, P3 viral titer was

amplified, cells were infected and 48 h post infection cells were

lysed in lysis buffer (50 mM Tris-HCl pH 8.5, 10 mM 2-

Mercaptoethanol, 1 % protease inhibitor cocktail (Roche), 1 %

NP40). Recombinant His-tagged PDE4B protein was purified as

previously described in a literature [21]. Briefly, lysate was

centrifuged at 10,000 rpm for 10 min at 4 ºC and supernatant was

collected. Supernatant was mixed with Ni-NTA resin (GE Life

Sciences) in a ratio of 4:1 (v/v) and equilibrated with binding

buffer (20 mM Tris-HCl pH 8.0, 500 mM-KCl, 5 mM imidazole,

=

CH), 4.24 (q, J = 7.2 Hz, 2H, OCH ), 2.43 (s, 3H, CH ), 1.31 (t,

2 3

13

J = 7.2 Hz, 3H, CH -CH ); C NMR (100 MHz, CDCl ) δ: 166.3

2

3

(C=O), 148.1, 147.8, 141.0 (2C), 135.0, 132.5, 132.4, 130.9 (2C),

1

6

30.4, 129.2, 128.1, 127.7, 127.2, 120.5, 117.4, 117.1, 111.0,

0.6 (OCH ), 29.6 (Me), 14.2 (CH -Me); MS (ES mass): 392.1

2

(M+1); HPLC: 97.3%, Column: Symmetry C-18 75 * 4.6 mm,

3

.5µm, mobile phase A: 0.1 % TFA in water, mobile phase B:

CH CN (T/%B): 0/20, 0.5/20, 2/95, 10/95, 10.5/20, 12/20; flow

rate: 1.0 mL/min; Diluent: ACN: WATER (80:20); UV 210.0

nm, retention time 3.4 min.

3

4

.1.47. (E)-Ethyl-3-(3-(3-chloroquinoxalin-2-yl)-1H-indol-2-

yl)acrylate (6p)

1

0 mM 2-mercaptoethanol and 10 % glycerol) in a ratio of 2:1

Compound 6p was synthesized from 3s following a procedure

(v/v) and mixed gently on rotary shaker for 1 hour at 4˚C. After

similar to that of compound 6k; Yield: 69%; light yellow solid;

1

incubation, lysate-Ni-NTA mixture was centrifuged at 4,500 rpm

for 5 min at 4˚C and the supernatant was collected as the flow-

through fraction. Resin was washed twice with wash buffer (20

mM Tris-HCl pH 8.5, 1 M KCl, 10 mM 2-Mercaptoethanol and

mp: 127-129 °C; R = 0.28 (10% EtOAc/ n-hexane); H NMR

f

(

8

3

400 MHz, DMSO-d ) δ: 12.19 (s, 1H, NH, D O exchangeable),

6

2

.17-8.12 (m, 2H, ArH), 7.95-7.93 (m, 2H, ArH), 7.57-7.49 (m,

H, ArH), 7.30 (t, J = 7.2 Hz, 1H, ArH), 7.10 (d, J = 7.2 Hz, 1H,

1

0% glycerol). Protein was eluted sequentially twice using

elution buffers (Buffer I: 20 mM Tris-HCl pH 8.5, 100 mM KCl,

50 mM imidazole, 10 mM 2-mercaptoethanol, 10% glycerol,

ArH), 6.73 (d, J = 16.0 Hz, 1H, =CH), 4.13 (q, J = 7.2 Hz, 2H,

13

OCH ), 1.20 (t, J = 7.2 Hz, 3H, CH -CH ); C NMR (100 MHz,

2

3

2

DMSO-d ) δ: 166.4 (C=O), 148.4, 147.6, 140.9, 140.7, 137.2,

6

Buffer II: 20 mM Tris-HCl pH 8.5, 100 mM KCl, 500 mM

imidazole, 10 mM 2-mercaptoethanol, 10% glycerol). Eluates

were collected in four fractions and analyzed by SDS-PAGE.

Eluates containing PDE4B protein were pooled and stored at -

1

(

35.9, 134.5, 133.3, 129.3, 128.3, 127.1, 125.1, 121.3, 118.3,

16.9, 116.1, 113.5, 112.2, 60.5 (OCH ), 14.5 (CH -Me); MS

2

+

ES mass): 377.2 (M ); HPLC: 96.1%, Column: Symmetry C-18

5 * 4.6 mm, 3.5µm, mobile phase A: 0.1 % TFA in water,

mobile phase B: CH CN (T/%B): 0/20, 0.5/20, 2/95, 8/95, 10/20,

7

8

0˚C in 50% glycerol until further use.

3

1

2/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER (90:10);

5.1.3. PDE4B enzymatic assay

UV 245.0 nm, retention time 4.0 min.

The inhibition of PDE4B enzyme was measured using

4

.1.48. (E)-Methyl-3-(3-(3-chloroquinoxalin-2-yl)-1H-indol-2-

PDElight HTS cAMP phosphodiesterase assay kit (Lonza)

according to manufacturer’s recommendations. Briefly, 10 ng of

PDE4B enzyme was pre-incubated either with DMSO (vehicle

control) or compound for 15 min before incubation with the

substrate cAMP (5 µM) for 1 h. The reaction was halted with

stop solution followed by incubation with detection reagent for

yl)acrylate (6q)

Compound 6q was synthesized from 3s following a procedure

similar to that of compound 6k; Yield: 73%; light yellow solid;

1

mp: 172-174 °C; R = 0.26 (10% EtOAc/ n-hexane); H NMR

f

(

400 MHz, CDCl ) δ: 8.76 (s, 1H, NH, D O exchangeable), 8.20-

3

2

1

0 minutes in dark. Luminescence values (RLUs) were measured

8

.17 (m, 1H, ArH), 8.14-8.11 (m, 1H, ArH ), 7.87-7.80 (m, 2H,

by a Multilabel plate reader (Perkin Elmer 1420 Multilabel

counter). The percentage of inhibition was calculated using the

following formula and IC s were computed using GraphPad

ArH), 7.70 (d, J = 16.4 Hz, 1H, =CH), 7.55 (d, J = 8.4 Hz, 1H,

ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.35 (t, J = 8.0 Hz, 1H,

ArH), 7.19 (t, J = 8.0 Hz, 1H, ArH), 6.35 (d, J = 16.4 Hz, 1H,

50

13

Prism Version 5.04 software.

=

CH), 3.76 (s, 3H, OCH₃); C NMR (100 MHz, DMSO-d ) δ:

6

1

9

66.8 (C=O), 148.4, 147.7, 140.9, 140.7, 137.3, 133.3, 133.2,

31.7, 131.3, 129.3, 128.3, 127.2, 125.1, 121.3, 121.0, 117.9,

17.0, 112.3, 52.0 (OMe); MS (ES mass): 364.1 (M+1); HPLC:

9.8%, Column: Symmetry C-18 75 * 4.6 mm, 3.5µm, mobile

5

.1.4. PDE4D and C enzymatic assay

phase A: 0.1 % TFA in water, mobile phase B: CH CN (T/%B):

3

0

/20, 0.5/20, 2/95, 8/95, 10/20, 12/20; flow rate: 1.0 mL/min;

Diluent: ACN: WATER (90:10); UV 205.0 nm, retention time

.5 min.

This assay was performed following a similar method as

described above using 0.5 ng commercially procured PDE4D2 or

PDE4C enzyme instead of 10 ng of in house purified PDE4B

without changing any other factors or conditions.

3

18

European Journal of Medicinal Chemistry

[

8] a) S. -L. C. Jin, L. Lan, M. Zoudilova, M. Conti, Specific role of

ACCEPTED MANUSCRIPT

phosphodiesterase 4B in lipopolysaccharide-induced signaling in

mouse macrophages. J. Immun. 175 (2005) 1523-1531.

b) M. A. Azam, N. S. Tripuraneni, Selective phosphodiesterase 4B

inhibitors: A review. Sci Pharm. 82 (2014) 453-481

Acknowledgements

Authors acknowledge the financial support received from

[9] A. Robichaud, P. B. Stamatiou, S.-L. C. Jin, N. Lachance, D.

MacDonald, F. Laliberte, S. Liu, Z. Huang, M. Conti, C.-C. Chan,

Deletion of phosphodiesterase 4D in mice shortens a2-

adrenoceptor-mediated anesthesia, a behavioral correlate of

emesis. J. Clin. Invest., 110 (2002) 1045-1371.

DBT,

New

Delhi,

India

Authors

(Grant

thank

No.

the

BT/PR22126/BRB/10/1585/2017).

Management of DRILS, Hyderabad, India, VIT University,

Vellore, India and Manipal University, Manipal, India for

encouragement and support.

[

10] a) B. Purushothaman, P. Arumugam, G. Kulsi, J. M. Song,

Design, synthesis, and biological evaluation of novel

catecholopyrimidine based PDE4 inhibitor for the treatment of

atopic dermatitis. Eur. J. Med. Chem. 145 (2018) 673-690

b) A. F. Donnell, P. J. Dollings, J. A. Butera, A. J. Dietrich, K. K.

Lipinski, A. Ghavami, W. D. Hirst, Identification of

pyridazino[4,5-b]indolizines as selective PDE4B inhibitors.

Bioorg. Med. Chem. Lett., 20 (2010) 2163-2167

c) K. Naganuma, A. Omura, N. Maekawara, M. Saitoh, N.

Ohkawa, T. Kubota, H. Nagumo, T. Kodama, M. Takemura, Y.

Ohtsuka, J. Nakamura, R. Tsujita, K. Kawasaki, H. Yokoi, M.

Kawanishi, Discovery of selective PDE4B inhibitors. Bioorg.

Med. Chem. Lett., 19 (2009) 3174-3176

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at xxxxxxxxx

Detailed tables and schemes and details of experimental

procedures for (i) docking studies, and (ii) in vitro and in vivo

experiments (file type: word).

d) M. Kranz, M. Wall, B. Evans, A. Miah, S. Ballantine, C.

Delves, B. Dombroski, J. Gross, J. Schneck, J. P. Villa, M. Neu,

D. O. Somers, Identification of PDE4B over 4D subtype-selective

inhibitors revealing an unprecedented binding mode. Bioorg. Med.

Chem., 17 (2009) 5336-5341.

Copies of spectra of all target compounds synthesized (file

type: PDF).

References and notes

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11] a) P. Srivani, D. Usharani, E. D. Jemmis, G. N. Sastry, Subtype

selectivity in phosphodiesterase 4 (PDE 4): a bottleneck in rational

drug design. Curr. Pharm. Des. 14 (2008) 3854-3872

[

1] a) M. Conti, J. Beavo, Biochemistry and physiology of cyclic

nucleotide phosphodiesterases: essential components in cyclic

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b) V. Boswell-Smith, D. Spina, C. P. Page, Phosphodiesterase

inhibitors. Br. J. Pharmacol. 147 (2006) S252-S257.

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S. Reid, J. Prestegard, B. Wang, H. Ke, Design, Synthesis, and

Structure-Activity Relationship, Molecular Modeling, and NMR

Studies of a Series of Phenyl Alkyl Ketones as Highly Potent and

Selective Phosphodiesterase-4 Inhibitors. J. Med. Chem. 51

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ACCEPTED MANUSCRIPT

Highlights

•

2-(1H-Indol-3-yl)-quinoxalines were designed as a new class of PDE4 inhibitors.

3

Synthesis involved InCl mediated heteroarylation/Pd(II)-catalyzed C-H activation.

Compound 3b (PDE4B IC₅₀~ 0.39 µM) showed PDEs family/PDE4 subtype

selectivity.

•

3b halted progression of the disease in Zebrafish EAE model of multiple sclerosis

3b also showed promising and benefecial effects in adjuvant induced arthritic rats.

Article Doi

InCl3 mediated heteroarylation of indoles and their derivatization via C[sbnd]H activation strategy: Discovery of 2-(1H-indol-3-yl)-quinoxaline derivatives as a new class of PDE4B selective inhibitors for arthritis and/or multiple sclerosis

DOI: 10.1016/j.ejmech.2019.04.020

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Full text of DOI:10.1016/j.ejmech.2019.04.020

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