Design, synthesis and anxiolytic-like activity of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides

Accepted Manuscript

Design, synthesis and anxiolytic-like activity of 1-arylpyrrolo[1,2-a]pyra-

zine-3-carboxamides

G.V. Mokrov, O.A. Deeva, T.A. Gudasheva, S.A. Yarkov, M.A. Yarkova, S.B.

Seredenin

PII:

S0968-0896(15)00357-0

DOI:

http://dx.doi.org/10.1016/j.bmc.2015.04.049

BMC 12261

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

29 January 2015

13 April 2015

17 April 2015

Please cite this article as: Mokrov, G.V., Deeva, O.A., Gudasheva, T.A., Yarkov, S.A., Yarkova, M.A., Seredenin,

S.B., Design, synthesis and anxiolytic-like activity of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides, Bioorganic &

Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.04.049

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Design, synthesis and anxiolytic-like activity of

1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides.

G.V. Mokrov^*, O.A. Deeva, T.A. Gudasheva, S.A. Yarkov, M.A. Yarkova,

S.B. Seredenin

Federal State Budgetary Institution “Research Zakusov Institute of Pharmacology”;

Baltiyskaya str., 8, Moscow 125315, Russia.

*Corresponding author. Tel.: +7 926 1856846. Kosmonavta Volkova str., 29-1-310, Moscow 127299,

Russia. E-mail address: g.mokrov@gmail.com (G.V. Mokrov).

Abstract:

A series of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides were designed and

synthesized as 18 kDa translocator protein (TSPO) ligands. Anxiolytic-like activity of

compounds was evaluated in the open field test and elevated plus maze test.

Compounds 1a and 1b demonstrated high anxiolytic-like effect in the dose range of

0.1-1.0 mg/kg comparable with that of diazepam. The involvement of TSPO receptor

in the mechanism of anxiolytic-like activity of new compounds was proved by

antagonism of the most active compound 1a with TSPO selective inhibitor PK11195. In

vitro binding studies demonstrated high TSPO affinities for compounds 1a and 1b.

Keywords: anxiolytic activity, elevated plus maze test, open field test, PK11195,

pyrrolo[1,2-a]pyrazines, TSPO, vinyl azides

1

Graphical abstract:

2

1. Introduction

Diazepam and some other benzodiazepines are widely used for the treatment of anxiety

due to their fast action and potent efficacy. However most benzodiazepines produce negative

effects, such as development of tolerance, drug dependence, sedation, and withdrawal

symptoms, which limit their clinical use [1-3]. Some types of antidepressants, such as selective

serotonin reuptake inhibitors, are also used in the treatment of anxiety disorders, although

their anxiolytic effect is often delayed by several weeks [3]. Thus, considering the foregoing,

new antianxiety agents with fast onset and potent effect but minimum unfavorable side effects

are in high demand.

The 18 kDa translocator protein (TSPO) [4], which was first identified as a discrete

receptor for diazepam [5], is known to be structurally and functionally distinct from the

benzodiazepine site of the GABA_Areceptor which until recently was called central

benzodiazepine receptor [6, 7]. TSPO acts as a complex with the 32 kDa voltage-dependent

anion channel and the 30 kDa adenine nucleotide transporter [8]. TSPO is expressed in the

mitochondrial membrane of peripheral tissues, including heart, kidney, lung, and endocrine

glands, and in glial cells in the central nervous system (CNS) [4, 9]. Although the exact

physiological role of TSPO in the CNS is not yet completely defined, accumulated evidence

suggests that TSPO in the CNS promotes the synthesis of neurosteroids [10-12]. Indeed, TSPO

activates cholesterol transport from the outer to the inner mitochondrial membrane, which is

the rate-limiting step of neurosteroid biosynthesis [13].

Neurosteroids are potent positive allosteric modulators of GABA_Awhich plays an

important role in the pathophysiology of anxiety disorders; they have their own binding sites on

GABA_Areceptors, which are different from the binding site for benzodiazepines [14, 15]. Since

TSPO plays an important role in regulating the synthesis of neurosteroids, the ligands of this

receptor may be anxiolytics free from side-effects of benzodiazepines [16]. A wide variety of

TSPO ligands have already been reported; the classical ones are Ro5-4864 [17] and PK11195

[18] (Fig. 1).

3

O

CH₃

N

CH₃

Cl

Fig. 1. Structures of Ro5-4864 and PK11195

Anxiolytic-like activity was demonstrated in different animal models for the series of

TSPO-ligands created to date (Fig. 2). 2-Phenylimidazo[1,2-a]pyridine derivative CB-34 shows

marked anti-conflict effect in rats in the Vogel test [19]. In the elevated plus maze test, FGIN-1-

27 inhibits neophobia in a manner that is antagonized by PK11195 but not by flumazenil [20].

Indolylglyoxylamide A demonstrated anxiolytic-like effect in rats in the elevated plus maze test

[21]. Benzoxazolone derivative B showed anxiolytic-like effect in the rat model with improved

safety profile [22]. Compound YL-IPA08 caused significant suppression of enhanced anxiety and

contextual fear induced in the inescapable electric foot-shock-induced mouse model of post-

traumatic stress disorder and the time-dependent sensitization procedure [23].

The first TSPO ligand that showed anxiolytic effects in clinical trials was the benzoxazine

etifoxine [24]. This compound turned out to exert anxiolytic efficacy comparable with the

benzodiazepine lorazepam in patients suffering from adjustment disorders with anxiety [24].

However, etifoxine is also a weak, direct GABA_Areceptor modulator [25], and so the extent to

which the effects observed are related to TSPO binding is not yet clear. Another TSPO ligand

with potent antianxiety efficacy is XBD173 (AC-5216, emapunil). This compound demonstrated

high and rather selective affinity to TSPO. XBD173 enhances neurosteroidogenesis in the brain,

thereby exerting anxiolytic properties in animal models and in humans [26, 27]. Unfortunately a

Phase II study on the efficacy, safety and tolerability of XВD173 that has been performed by

Novartis in patients with generalized anxiety disorder showed the lack of efficacy

(ClinicalTrials.gov identifier: NCT00108836).

Therefore, the search of new highly effective anxiolytic drugs with quick action without

side effects among new TSPO ligands still has a big interest. The present paper describes

4

synthesis and evaluation of anxiolytic activity of new TSPO ligands belonging to 1-

arylpyrrolo[1,2-a]pyrazine-3-carboxamides.

H

N

F

O

N

CH₃

H₃C

N

H₃C

N

O

N

O

H₃C

Cl

H₃C

N

Cl

N

O

N

HCl

CH₃

N

Fig. 2.Structures of TSPO ligands exhibiting anxiolytic-like activity in animal models

5

2. Result and discussion

2.1. Design

Despite the large number of different TSPO ligands, they all comply to the specific

structural requirements [28-31]. These requirements are illustrated in Fig. 3 using PK11195 as

an example [28]. The core of a ligand must be a planar aromatic heterocycle (PAH). In any

position of the heterocycle should be an aromatic substituent (AS) linked with the core by C-C

single bond. In the position close to AS should be an amide group with lipophilic substituent

(LAS). One of the key roles in binding to TSPO plays carbonyl group (CG) of the amide

substituent. Amide group can be linked directly to the heterocyclic core, as well as can be

distant from it by one or two C-C bonds.

PAH

AS

N

Cl

CH₃

O

CG

CH₃

H₃C

Designed compounds

PK11195

LAS

Fig. 3. Structural requirements for TSPO ligands with PK11195 as an example [28] and

designed pyrrolo[1,2-a]pyrazines.

Based on these requirements we have suggested a new type of fused ring system -

pyrrolo[1,2-a]pyrazine derivatives. Structures of this type contain all the necessary

pharmacophore elements of TSPO ligands with amphiphilic pyrrolo[1,2-a]pyrazine heterocycle

as an aromatic core.

6

2.2. Chemistry

Vinyl azides domino approach was selected as a strategy for the synthesis of the target

compounds 1. This method was proposed for the synthesis of pyrrolo[1,2-a]pyrazines by [3+3]-

cycloaddition of vinylazides to 2-pyrrolealdehyde [32]. In our case retrosynthetic analysis

applying this approach leads to arylpyrrolketones 2 and 2-azidoacrylamides 3 (Scheme 1).

Scheme 1. Retrosynthetic analysis of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides.

Phenylpyrrolketone 2a was prepared according to Scheme 2 [33, 34]. Benzoyl chloride

4а was reacted with morpholine to give amide 5. Vilsmeier reaction of amide 5 with pyrrole in

the presence of phosphoryl chloride in DCE led to target ketone 2a.

Scheme 2. Reagents and conditions: (i) morpholine, CH₂Cl₂, Et₃N (ii) pyrrole, POCl₃, DCE,

then 10% aqueous Na₂CO₃.

All attempts to obtain pyrrole(2-halophenyl)ketones 2b-d [34, 35], from 2-halobenzoyl

chlorides using Scheme 2 failed. When using less bulky dimethylamine instead of morpholine

the desired compounds were obtained only in trace amounts. Presumably, the electron

withdrawing halogen atom in the ortho-position of the benzene ring prevents the successful

course of condensation of corresponding amides with pyrrole. The method described by Yadav,

which involve the reaction of pyrrole 6 with benzoyl chlorides (on the example of 2-

chlorobenzoylchloride 4c) in the presence of zinc in toluene solution [36], did not give the

7

declared ketone 2c in a yield more than 80% as described [35], but gave a mixture of ketones 2c

and 7 in the ratio of about 1:1 with a total yield of about 30% (Scheme 3). The best results were

obtained by Bailey method [34, 37] using the Grignard reaction (Scheme 3). Pyrrole 6 was

reacted with ethylmagnesium bromide in ether to give magnesium derivative of pyrrole 8,

which was consequently reacted with 2-halobenzoyl chlorides 4b-d to give the target pyrrole(2-

halophenyl)ketones 2b-d with the yields of 30-35% without side beta-addition products.

Scheme 3. Reagents and conditions: (i) Zn, toluene, RT, 2 h (ii) EtMgBr, Et₂O (iii) Et₂O,

then NH₄Cl, H₂O.

2-Azidoacrylamides 3 were synthesized according to Scheme 4 [38].

Compound

R¹

R²

3a

3b

3c

CH₃

CH₂Ph

n-C₄H₉

sec-C₄H₉

Scheme 4. Reagents and conditions: (i) Et₃N, CH₂Cl₂(ii) Br₂, CH₂Cl₂(iii) NaN₃, DMF, 60°C,

20 min.

8

Acryloylchloride 9 was reacted with amines 10 in the presence of triethylamine to give

the corresponding acrylamides 11, which has been reported before [39-41]. These amides 11

then were brominated by bromine into dibromoderivatives 12. The optimal way to convert

dibromides 12 into2-azidoakrylamides 3 appeared to be the reaction of compounds 12 with

sodium azide in DMF while heating the reaction mixture for 20 minutes at 60°C [42].

The [3+3]-cycloaddition reactions of arylpyrroleketones 2 with 2-azidoacrylamides 3

were carried out in dry DMF in the presence of Cs₂CO₃as a base [32] (Scheme 5).

Compound

R¹

R²

R³

H

Yield,%

61

57

1a

1b

1c

1d

1e

1f

CH₃

CH₂Ph

n-C₄H₉

sec-C₄H₉

H

F

54

50

Cl

Br

H

66

68

Scheme 5. Reagents and conditions: (i) Cs₂CO₃, DMF, RT, 24 h.

Pyrrolo[1,2-a]pyrazines 1 formation was characterized by appearance of the bright

yellow spots on TLC that fluoresce under ultraviolet. The reactions were completed after 24 h

of stirring the mixtures at the room temperature. The pure products were isolated by column

chromatography. The yields of the reactions were in the range between 50% and 68%.

Other synthetic rout was offered to obtain target 1-arylpyrrolo[1,2-a]pyrazine-3-

carboxamides 1 (Scheme 6) - the reaction of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxylic acids 16

with amines 10.

9

Compound

R¹

H

R²

CH₃

R³

H

Yield, %

45

53

1g

1h

1d

H

CH₃

CH₂Ph

n-C₄H₉

H

Cl

54

Scheme 6. Reagents and conditions: (i) NaN₃, DMF, 60°C, 20 min (ii) Cs₂CO₃, DMF, RT,

24 h (iii) NaOH, H₂O, then HCl (iv) POCl₃, Et₃N, DCM, kat. DMF, then HCl, H₂O.

The acids 16 were prepared starting from ethyl 2,3-dibromopropionate 13. This

compound was treated with sodium azide under conditions described for the amides 3 to

afford ethyl 2-azidoacrylate 14 [43]. Cyclocondensation reaction of vinylazide 14 with

arylpyrrolketones 2 in the presence of Cs₂CO₃yielded the esters 15. Saponification of the latter

led to the target acids 16. Reactions of the acids 16 with the amines 10 were carried out with

POCl₃and catalytic amount of DMF, and resulted in carboxamides 1 with not high yields ranging

from 45 to 54%.

Comparison of the two proposed synthetic routes with 1d as an example showed that

the first method using 2-azidoacrylamide 3b gave better yield (50% from ketone 2c) when the

method through the 1-arylpyrrolo[1,2-a]pyrazine-3-carboxylic acid 16b (24% from ketone 2c).

The structures of all synthesized 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides 1 were

confirmed by NMR and IR spectroscopy and elemental analysis. The characteristic signals in the

¹H NMR spectra of compounds 1 are the signal of H(4) in the pyrazine ring between δ 8.33-8.77

and three signals of the pyrrole ring protons between δ 6.50-7.60. It should be noted, that for

compounds containing a tertiary amide group the signal of H(4) was observed in the range of δ

10

8.33-8.53, however for compounds with a secondary amide group - in the interval of δ 8.73-

8.77. The difference in chemical shift of this proton for compounds with different types of

amide groups can be explained by the fact that the more bulky tertiary amide group goes out of

the pyrrolopyrazine cycle plane, resulting in smaller anti-shielding effect of the amide carbonyl

group on H(4), than in the case of the less bulky secondary amide group, which is almost in the

plane of heterocycle.

For compounds 1 containing two different substituents at amide nitrogen atom a

mixture of rotamers with the ratio from 1:1.4 to 1:1.8 (see Experimental) in the ¹H NMR spectra

were observed. This ratio was calculated from the integrals of the corresponding signals of

substituents in the amide group and primarily the signals of the methyl groups resonating at

δ 2.9-3.3. The highest value in the rotamers ratio (1:1.8) was observed for compound 1f. The

preferably rotamer is when the bulky sec-butyl substituent locates in trans-position relative to

the carbonyl group. This fact is confirmed by a large difference in chemical shifts between

CHCH₃protons of cis- (δ 4.72) and trans-rotamers (δ 4.38). As was shown for compound

PK11195 (see Fig. 1) with the same tertiary amide group, CHCH₃proton of cis-rotamer is less

shielded than in trans-rotamer (δ 4.80 and 3.89 respectively) [44].

11

2.3. Biological evaluation

2.3.1. Anxiolytic-like activity in the open field test

The anxiolytic-like activities of synthesized compounds were investigated in Balb/c mice

using the open-field test (OFT). This test is a common model for behavior evaluation in rodents

under emotional stress, forming as a response to a new environment and a threatening

situation [45-47]. In this paper we used the technique of illuminated «open field» where the

movement of the animal from the dark into the brightly illuminated arena creates additional

stressing factors, based on the natural desire of rodents to avoid brightly lit places [45]. The

activating effect on locomotor activity in animals with «freezing» reaction in OFT was taken as a

measure of the anxiolytic-like activity of compounds [47]. The total locomotor activity (TLA) was

calculated as sum of number of squares crossed in the periphery (SCP), in the central regions

(SCC), number of entries into the center (C) and number of rearing (R).

The results on the effect of compounds 1a-1h on the behavior of the animals in the OFT

are represented in the Table 1. These results revealed that compounds 1a, 1b, and 1c displayed

anxiolytic-like activities. Compound 1a in the doses of 0.1; 0.5 and 1.0 mg/kg, compound 1b in

the doses of 0.1 and 0.5 mg/kg, and compound 1c in the dose of 10.0 mg/kg significant

increased TLA of mice compared with control groups under the conditions of emotional stress

in OFT with P<0.05 (Table 1). Compounds 1e, 1f, 1g were inactive in the doses of 0.1-5.0 mg/kg.

Compound 1d in the dose of 0.5 mg/kg, and compound 1h in the dose of 1.0 mg/kg had a slight

negative effect, they significantly decreased TLA of mice compared with control groups.

Structure activity relationship analysis showed that the most potent activity is exhibited

by compounds that don’t contain substituents in the benzene ring (R³= H) and contain methyl

group (R¹= CH₃) and benzyl or n-butyl group (R²= CH₂Ph or n-Bu) as a substituents in the amide

group, i.e. compounds 1a and 1b. Varying the ortho substituent in benzene ring of compounds

with the same amide group (R¹= CH₃, R²= n-Bu) showed that the introduction of the fluorine

(1c) leads to decrease in activity, and chlorine (1d) or bromine (1e) - to its disappearance.

Removing each of the substituents in the amide group (R¹or R²) in compound 1a resulted in

disappearance of activity (1h: R¹= H, and 1g: R²= H). Replacement of n-Bu by the bulky sec-Bu

group (1f) also leads to the disappearance of activity.

The results of this study revealed that compounds 1a and 1b are active in OFT in a wide

range of doses.

12

Table 1. Anxiolytic-like activity of compounds 1 in the open field test.

Compound

Dose, mg/kg

Control

SCP

SCC

C

R

TLA

12,58 (6,56)

42,88 (10,58)*

48,25 (6,54)*

41,38 (8,21)*

18,13 (14,82)

0,0000

0,08 (0,29)

1,25 (2,76)

1,13 (1,73)

1,38 (2,07)

0,00 (0,00)

0,043

0,00 (0,00)

0,13 (0,35)

0,00 (0,00)

0,343

0,00 (0,00)

0,13 (0,35)

0,00 (0,00)

0,343

12,67 (6,67)

44,13 (12,48)*

49,50 (7,03)*

42,88 (8,29)*

18,13 (14,82)

0,0000

0,1

0,5

1a

1,0

5,0

p (ANOVA)

Control

0,1

7,38 (5,13)

19,63 (13,98)*

18,88 (13,59)*

10,50 (8,54)

13,00 (9,59)

0,141

0,63 (1,77)

2,38 (5,29)

11,63 (9,68)*

1,88 (2,95)

1,88 (4,12)

0,003

0,00 (0,00)

0,25 (0,46)

0,00 (0,00)

0,084

0,00 (0,00)

0,13 (0,35)

0,00 (0,00)

0,545

8,00 (5,76)

22,13 (18,49)*

30,88 (21,44)*

12,38 (8,96)

14,88 (12,97)

0,065

0,5

1b

1c

1d

1e

1f

1,0

5,0

p (ANOVA)

Control

0,5

8,00 (4,69)

10,25 (3,49)

6,13 (6,96)

13,38 (4,53)*

13,75 (8,14)

0,064

0,38 (0,74)

0,25 (0,71)

1,38 (1,92)

0,00 (0,00)

4,88 (4,39)*

0,0002

0,00 (0,00)

1,000

0,00 (0,00)

1,000

8,38 (4,96)

10,50 (4,07)

7,50 (6,97)

13,38 (4,53)

18,63 (8,45)*

0,025

1,0

5,0

10,0

p (ANOVA)

Control

0,1

7,38 (5,13)

3,75 (1,83)

14,13 (7,70)

1,88 (1,25)*

8,50 (5,81)

0,001

0,63 (1,77)

1,63 (4,60)

0,13 (0,35)

0,00 (0,00)

0,715

0,00 (0,00)

0,38 (1,06)

0,00 (0,00)

0,406

0,00 (0,00)

1,000

8,00 (5,76)

5,75 (5,23)

14,25 (7,74)

1,88 (1,25)*

8,50 (5,81)

0,003

1,0

0,5

5,0

p (ANOVA)

Control

0,1

8,00 (4,69)

6,63 (3,34)

7,25 (6,11)

7,25 (3,62)

7,38 (7,76)

0,929

0,38 (0,74)

0,25 (0,71)

0,38 (0,74)

0,38 (1,06)

0,50 (0,93)

0,957

0,00 (0,00)

1,000

0,00 (0,00)

1,000

8,38 (4,96)

6,88 (3,83)

7,63 (6,44)

7,63 (4,57)

7,88 (7,68)

0,975

0,5

1,0

5,0

p (ANOVA)

Control

0,1

5,88 (3,00)

5,50 (2,14)

8,75 (4,46)

3,38 (2,83)

3,75 (3,96)

0,054

0,75 (1,75)

0,00 (0,00)

0,084

0,00 (0,00)

1,000

0,00 (0,00)

1,000

6,63 (3,34)

5,50 (2,14)

8,75 (4,46)

3,38 (2,83)

3,75 (3,96)

0,054

0,5

1,0

5,0

p (ANOVA)

Control

0,1

3,50 (2,78)

5,38 (1,41)

4,63 (1,60)

9,75 (8,21)*

7,13 (5,33)

0,192

1,50 (2,07)

0,75 (1,49)

0,25 (0,71)

0,38 (0,74)

2,13 (2,36)

0,311

0,13 (0,35)

0,00 (0,00)

0,406

0,00 (0,00)

1,000

5,13 (3,94)

6,13 (1,13)

4,88 (1,96)

10,13 (8,24)

9,25 (6,41)

0,359

0,5

1g

1,0

5,0

p (ANOVA)

Control

0,1

5,88 (3,00)

4,00 (1,20)

7,63 (2,88)

3,00 (2,45)

5,75 (3,73)

0,022

0,75 (1,75)

0,00 (0,00)

0,88 (1,64)

0,00 (0,00)

0,75 (1,75)

0,337

0,00 (0,00)

1,000

0,00 (0,00)

1,000

6,63 (3,34)

4,00 (1,20)

8,50 (2,88)

3,00 (2,45)*

6,50 (3,89)

0,008

0,5

1h

1,0

5,0

p (ANOVA)

13

Notes:

The data are represented as M (SD), where M is the arithmetic mean; SD-standard deviation;

* – statistically significant differences (P < 0,05) compared with the control group under the Mann-

Whitney test.

SCP - number of squares crossed in the periphery

SCC - number of squares crossed in the central regions

C - number of entries into the center

R - number of rearing

TLA - total locomotor activity

2.3.2. Anxiolytic-like activity in the elevated plus-maze test

Compounds 1a and 1b that were found to be most active in the OFT were further

evaluated in the elevated plus-maze test (EPM) in CD-1 mice to effectively confirm their

anxiolytic-like activity. EPM has been widely validated to measure anxiety in rodents [48]. This

test is based on the natural aversion of rodents for open and elevated areas, as well as on their

natural spontaneous exploratory behavior in novel environments. The ability of compounds to

increase the percentage of open arms entries (% OAE) and the time spent in open arms (%

TSOA) was taken as a measure of their anxiolytic-like activity [37]. % OAE and % TSOA were

calculated using the following formulas:

% OAE = [number of open arms entries/(number of open + closed arms entries)] × 100

% TSOA = [time spent in open arms/(time spent in open + closed arms)] × 100

Diazepam was used as a positive control in a dose of 1.0 mg/kg [49].

The anxiolytic-like activities of compounds 1a, 1b in the doses of 0.1-5 mg/kg and

diazepam are shown in the Table 2. The results of EPM test proved anxiolytic-like activity of

selected compounds. Compound 1a in the dose range of 0.1-1.0 mg/kg and compound 1b in

the dose of 0.1 mg/kg significantly (P<0.05) increased % TSOA compared to control group, as

also observed with diazepam. Compound 1a in the dose of 1.0 mg/kg and compound 1b in the

dose of 0.1 mg/kg also increased % OAE (P<0.05) compared with the control.

Thus, compounds 1a and 1b demonstrated close and higher % TSOA and % OAE values

compared with that of diazepam, and appeared to have not a lower levels of anxiolytic-like

activity compared with diazepam.

14

Table 2. Anxiolytic-like activity of 1a and 1b in the elevated plus-maze test.

Time spent, sec

Number of entries

Compound Dose,mg/kg

% TSOA % OAE

Open

arms

Closed

arms

Open

arms

Closed

arms

36,38

(14,74)

145,75

(12,04)

3,75

(1,58)

7,75

(1,04)

19,36

(5,76)

31,51

(7,99)

Control

0,1

71,50*

(29,20)

105,75*

(8,36)

5,00

(2,39)

8,13

(2,36)

38,82*

(11,16)

37,32

(12,73)

63,88*

(19,03)

138,25

(17,63)

4,50

(1,31)

8,63

(2,13)

31,35*

(8,16)

34,50

(7,58)

0,5

1a

77,13*

(12,70)

117,75*

(10,90)

5,38

(1,41)

7,38

(1,92)

39,50*

(5,51)

42,17*

(5,65)

1,0

30,13

(7,10)

135,13

(28,12)

2,50

(0,76)

8,25

(1,75)

18,78

(5,60)

23,43

(6,18)

5,0

0,0002

0,001

0,008

0,779

<0,0001

0,0072

p (ANOVA)

Control

20,38

(11,75)

142,63

(16,62)

1,75

(0,71)

7,50

(0,93)

12,01

(5,59)

18,54

(6,08)

47,88*

(22,94)

91,25*

(16,57)

3,50*

(1,60)

8,38

(1,60)

33,49*

(14,47)

28,85*

(12,09)

0,1

31,00

(32,02)

109,38*

(16,80)

2,88

(2,47)

7,50

(1,69)

20,03

(18,68)

23,50

(16,37)

0,5

1b

17,75

(11,27)

110,88*

(25,75)

2,00

(1,07)

8,13

(2,64)

13,57

(7,26)

20,31

(9,06)

1,0

5,0

34,25

(40,34)

126,00

(16,80)

2,88

(3,04)

9,38

(2,39)

18,06

(19,86)

19,48

(17,18)

p (ANOVA)

Control

0,321

0,001

0,445

0,328

0,2067

0,4787

35,25

(23,84)

74,88*

(43,89)

168,50

(33,99)

142,38

(25,52)

2,50

(0,93)

4,13*

(1,13)

5,75

(1,39)

6,25

16,55

(7,97)

33,23*

(12,72)

30,16

(8,49)

41,67*

(13,55)

Diazepam

1,0

(2,76)

p (ANOVA)

0,0087

0,0742

0,0107

0,8731

0,0063

0,1141

Notes:

The data are represented as M (SD), where M is the arithmetic mean; SD-standard deviation;

* – statistically significant differences (P < 0,05) compared with the control group under the Mann-

Whitney test.

15

2.3.3. PK11195 influence on the anxiolytic-like effect of compounds in EPM test

To prove the involvement of TSPO receptor in the mechanism of the anxiolytic-like

activity of the new compounds we studied the behavior of animals when administered together

the most potent in the OFT and EPM compound 1a with PK11195.

PK11195 (1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline-carboxamide) is

a specific inhibitor of TSPO and it blocks the anxiolytic-like effect of TSPO ligands when co-

administered with them [50, 51].

We used 1a in the dose of 0.5 mg/kg that was chosen from the previous experiments,

and PK11195 in the dose of 10.0 mg/kg [52]. Mice behavior was evaluated in the EPM test. 1a

in the dose of 0.5 mg/kg caused pronounced anxiolytic-like effect in CD-1 mice by significantly

increasing the percentage of time spent in the open arms compared with control group

(P<0.05). Preliminary administration of PK11195 in the dose of 10.0 mg/kg completely blocked

the anxiolytic-like effect of 1a, significantly (P<0.05) reducing percentage of open arms entries

and time spent in the open arms (Table 3). PK11195 itself did not change % TSOA and % OAE

compared with control.

To show the specificity of the identified antagonism a study of co-administration of

TSPO inhibitor with diazepam was also performed. The data in Table 3 reveal that the

anxiolytic-like effect of GABA_Areceptor benzodiazepine site agonist diazepam is not eliminated

by PK11195.

The experimental results demonstrate the dependence of the anxiolytic-like effect of

compound 1a on interaction with TSPO receptor site.

16

Table 3. The influence of the specific TSPO (18 kDa) inhibitor PK11195 on the

anxiolytic-like effect of compound 1a.

Time spent, sec

Number of entries

Experimental

groups

Compound

% TSOA

% OAE

Closed

arms

Open

arms

Closed

arms

Open arms

Control

(CI+CI)

41,50

(15,99)

131,00

(18,02)

3,13

(2,23)

8,25

(1,91)

23,87

(7,58)

25,78

(13,16)

РК11195

(10,0

mg/kg)+ CI

30,13

(26,82)

167,25*

(13,92)

3,00

(2,98)

8,63

(2,39)

14,43

(11,82)

22,20

(17,77)

1a

CI+

1a (0,5

mg/kg)

78,50*

(18,59)

110,63*

(17,79)

4,75

(1,49)

8,50

(2,78)

41,30*

(5,16)

36,41

(7,33)

РК11195

(10,0 mg/kg)

+ 1a (0,5

24,63*#

(19,71)

149,75#

(26,91)

2,00#

(1,69)

8,88

(2,47)

14,28*#

(12,68)

18,09#

(15,36)

mg/kg)

CI +

86,88*

(32,37)

125,88

(28,60)

6,50

(4,07)

8,50

(3,25)

40,59*

(14,86)

40,53*

(13,34)

Diazepam

(1,0 mg/kg)

РК11195

Diazepam

(10,0 mg/kg)

+ Diazepam

(1,0 mg/kg)

90,13*

(22,41)

99,50*

(19,91)

7,38*

(3,07)

8,25

(3,01)

47,28*

(4,10)

46,84*

(4,52)

p (ANOVA)

<0,00001 <0,00001 <0,00001 0,9621 <0,00001 <0,00001

Notes:

The data are represented as M (SD), where M is the arithmetic mean; SD-standard deviation;

CI - control injection of distilled water

* – statistically significant differences (P < 0.05) compared with the control group under the

Mann-Whitney test.

# – statistically significant differences (P < 0.05) between "CI+1a (0,5 mg/kg)" and "РК11195 (10,0 mg/kg)

+ 1a (0,5 mg/kg)" groups under the Mann-Whitney test.

17

2.3.4. In vitro binding studies

The most active compounds 1a and 1b were tested for their potential activity in

inhibiting the specific binding of [³H]-PK11195 to membrane homogenates of human

glioblastoma U-118 MG cells in comparison with unlabeled PK11195. Both compounds

demonstrated high TSPO affinities comparable with that of PK11195. 1a showed 5.4*10^-8

M

(5.2*10^-8M) and 1b - 5.6*10^-7M (5.3*10^-7M) IC₅₀(K_i) values. PK11195 in the same experiment

demonstrated IC₅₀1.1*10^-8M and K_i1.1*10^-8M.

Compound 1a was tested for its ability to displace [³H]-flunitrazepam binding to

membrane homogenates prepared from rat cerebral cortex to prove the selectivity of this

compound for TSPO over benzodiazepine site of the GABA_Areceptor (GABA_AR). Only at

1*10^-5M 21% inhibition of GABA_AR benzodiazepine site was detected. The reference compound

diazepam in the same experiment demonstrated IC₅₀9.1*10^-9M and K_i7.6*10^-9M.

3. Conclusion

In conclusion, a series of novel 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides have been

designed and synthesized, and their anxiolytic-like activity has been evaluated. In general, some

of compounds displayed high level of anxiolytic-like efficacy comparable with that of diazepam.

The involvement of TSPO receptor in the mechanism of the anxiolytic-like activity of the new

compounds was proved by antagonism of the most active compound 1a with TSPO selective

inhibitor PK11195. In vitro binding studies demonstrated high TSPO affinities for compounds 1a

and 1b and very low GABA_AR benzodiazepine site affinity for compound 1a.

18

4. Experimental

4.1. Chemistry

The chemical reagents used in the synthesis were obtained from commercial supplier

and used without purification. TLC was performed on silica gel F₂₅₄plates (Merck). Silica gel

(130-270 mesh, Sigma-Aldrich) was used for column chromatographic purification. Melting

points are uncorrected and were measured in open capillary tubes, using OptiMelt melting

1

point apparatus. H NMR spectral data were recorded on Bruker AC-250 spectrometer (250

MHz) in DMSO-d6 and CDCl₃using the signals of the residual protons of the solvents (δ 2.50 and

7.25, respectively) as the internal standard.

4.1.1. Phenyl(1H-pyrrol-2-yl)methanone (2a)

This compound was synthesized from benzoyl chloride 4a, morpholine and pyrrole

according to procedure reported earlier [33, 34]. Yield: 90% (lit. [34] 88%). White crystals. M.p.

77-79°C (lit. [34] m.p. 77.5-79.5°C). ¹Н NMR (СDCl₃, δ, ppm): 6.33 (m, 1 H, H(4)); 6.89 (m, 1 H,

H(3)); 7.15 (m, 1 H, H(5)); 7.39-7.60 and 7.87-7.94 (two m, 5 H, Ph); 10.01 (c, 1 H, NH).

4.1.2. Pyrrole(2-halophenyl)ketones (2b-d)

These compounds were synthesized from 2-halobenzoyl chlorides 4b-d and pyrrole

according to procedure reported earlier using Grignard reaction [34, 36].

4.1.2.1. (2-fluorophenyl)(1H-pyrrol-2-yl)methanone (2b)

Yield: 30% (lit. [34] 35%). Pale pink crystals. B.p. 174-178°С (1.5 Torr). M.p. 76-78°C (lit.

1

[34] m.p. 76-78°C). Н NMR (СDCl₃, δ, ppm): 6.31 (m, 1 H, H(4)); 6.76 (m, 1 H, H(3)); 7.13-7.64

(m, 5 H, H(5) and Ph); 9.38 (c, 1 H, NH).

4.1.2.2. (2-chlorophenyl)(1H-pyrrol-2-yl)methanone (2c)

Yield: 31% (lit. [34] 48%). Pale pink crystals. B.p. 178-182°С (1.5 Torr). M.p. 92-93°C (lit.

[34] m.p. 93-94°C).¹Н NMR (СDCl₃, δ, ppm): 6.28 (m, 1 H, H(4)); 6.60 (m, 1 H, H(3)); 7.20 (m, 1 H,

H(5)); 7.25-7.60 (m, 4 H, Ph); 10.40 (c, 1 H, NH).

4.1.2.3. (2-bromophenyl)(1H-pyrrol-2-yl)methanone (2d) [35]

1

Yield: 35%. Pink oil. B.p. 180-185°С (1.5 Torr). Н NMR (СDCl₃, δ, ppm): 6.29 (m, 1 H,

H(4)); 6.68 (m, 1 H, H(3)); 7.20 (m, 1 H, H(5)); 7.25-7.67 (m, 4 H, Ph); 10.29 (c, 1 H, NH).

19

4.1.3. Synthesis of acrylamides 11

Solution of 150 mmol (13.58 g) of acryloyl chloride in 40 ml of dry dichloromethane was

added dropwise to the stirred solution of 150 mmol of appropriate amine 10 and 195 mmol

(19.69 g) of triethylamine in 60 ml of dry dichloromethane at -5°С. The mixture was stirred at

room temperature for 1 hour, and organic solution was washed with 70 ml of water, 50 ml of

10% HCl, 50 ml of 5% NaOH and 50 ml of water, dried over paper filter and evaporated in

vacuum to give the desired product.

4.1.3.1. N-benzyl-N-methylacrylamide (11a) [39]

1

Yield: 67%. Yellow oil. Н NMR (СDCl₃, δ, ppm): 2.98, 2.99 (two s, 3 Н, СН₃); 4.58, 4.67

(two s, 2 Н, СН₂); 5.69 (m, 1 Н, Н(3а)); 6.37 (m, 1 Н, Н(3b)); 6.59 (m, 1 Н, Н(2а)); 7.12-7.38 (m, 5

Н, Ph). Found (%):C, 75.55; H, 7.55; N, 7.83. С₁₁H₁₃NO. Calcd. (%): C, 75.40; H, 7.48; N, 7.99.

4.1.3.2. N-butyl-N-methylacrylamide (11b) [40]

3

Yield: 80%. Yellow oil. ¹Н NMR (СDCl₃, δ, ppm): 0.90 (t, 3 Н, СН₂СН₃, J = 7.3); 1.29 (m, 2

Н, СН₂СН₃); 1.51 (m, 2 Н, СН₂СН₂СН₃); 2.95, 3.01 (two s, 3 Н, NСН₃); 3.30, 3.39 (two t, 2 Н, NСН₂,

³J = 7.4); 5.62 (m, 1 Н, Н(3а)); 6.28 (m, 1 Н, Н(3b)); 6.53 (m, 1 Н, Н(2а)). Found (%): C, 67.90; H,

10.74; N, 10.15. С₈H₁₅NO. Calcd. (%): C, 68.04; H, 10.71; N, 9.92.

4.1.3.3. N-(sec-butyl)-N-methylacrylamide (11c) [41]

3

Yield: 73%. Yellow oil. ¹Н NMR (СDCl₃, δ, ppm): 0.82 (t, 3 Н, СН₂СН₃, J = 7.3); 1.07, 1.15

3

(two d, 3 Н, СНСН₃, J = 6.8); 1.47 (m, 2 Н, СН₂СН₃); 2.78, 2.82 (two s, 3 Н, NСН₃); 3.87, 4.67

(two m, 1 Н, СНСН₃); 5.62 (m, 1 Н, Н(3а)); 6.23 (m, 1 Н, Н(3b)); 6.56 (m, 1 Н, Н(2а)). Found (%):

C, 68.12; H, 10.73; N, 9.88. С₈H₁₅NO. Calcd. (%): C, 68.04; H, 10.71; N, 9.92.

4.1.4. Synthesis of 2,3-dibromopropanamides 12

45 mmol (7.19 g) of bromine was added dropwise to the stirred solution of 44 mmol of

the appropriate arcrylamide 11 in 100 ml of dry dichloromethane at 0°C. The mixture was

stirred at room temperature for 2 hours and then washed with solution of 5 g of Na₂S₂O₄in 60

ml of water and twice with 40 ml of water. The organic phase was dried over paper filter and

evaporated in vacuum to give the desired product.

20

4.1.4.1. N-benzyl-2,3-dibromo-N-methylpropanamide (12a)

Yield: 83%. Yellow oil. ¹Н NMR (СDCl₃, δ, ppm): 2.99, 3.02 (two s, 3 Н, СН₃); 3.68 (m, 1 Н,

Н(3а)); 4.22 (m, 1 Н, Н(3b)); 4.38-4.84 (m, 3 Н, Н(2), СН₂Рh); 7.15-7.43 (m, 5 Н, Ph). Found (%):

C, 39.55; H, 3.78; N, 4.11; Br, 47.67. С₁₁H₁₃Br₂NO. Calcd. (%): C, 39.43; H, 3.91; N, 4.18; Br,

47.70.

4.1.4.2. 2,3-dibromo-N-butyl-N-methylpropanamide (12b)

1

3

Yield: 81%. Yellow oil. НNMR (СDCl₃, δ, ppm): 0.91, 0.95 (two t, 3 Н, СН₂СН₃, J = 7.3);

1.33, 1.53 (two m, 2 Н, СН₂СН₃); 1.62, 3.18 (two m, 2 Н, СН₂СН₂СН₃); 2.99, 3.05 (two s, 3 Н,

NСН₃); 3.42 (m, 2 Н, NСН₂); 3.65 (m, 1 Н, Н(3а)); 4.14 (m, 1 Н, Н(3b)); 4.62 (m, 1 Н, Н(2)). Found

(%): C, 31.91; H, 5.18; N, 4.67; Br, 53.00. С₈H₁₅Br₂NO. Calcd. (%): C, 31.92; H, 5.02; N, 4.65; Br,

53.09.

4.1.4.3. 2,3-dibromo-N-(sec-butyl)-N-methylpropanamide (12c)

Yield: 80%. Yellow oil.¹Н NMR (СDCl₃, δ, ppm): 0.77-0.95 (m, 3 Н, СН₂СН₃); 1.03-1.24 (m,

3 Н, СНСН₃); 1.39-1,61 (m, 2 Н, СН₂СН₃); 2.77-2.90 (m, 3 Н, NСН₃); 3.59-4.79 (three m, 4 Н,

СНСН₃, BrCH₂CH). Found (%): C, 31.80; H, 4.98; N, 4.71; Br, 52.96. С₈H₁₅Br₂NO. Calcd. (%): C,

31.92; H, 5.02; N, 4.65; Br, 53.09.

4.1.5. Synthesis of 2-azidoacrylamides 3

Solution of 20 mmol of 2,3-dibromopropanamide 12 in 30 ml of dry DMF was added to

the suspension of 44 mmol (2.86 g) of sodium azide in 50 ml of dry DMF at 75°C. The mixture

was stirred at 75°C for 25 min followed by cooling to the room temperature and diluting with

350 ml of water. The product was extracted with 3*100 ml of ether, the combined fractions

were washed with 80 ml of water, filtered over paper filter and evaporated to dryness. The

residue was chromatographed on silica gel using a mixture of ethyl acetate and hexane (1:3) as

eluent. Appropriate fractions were evaporated to dryness to give the desired product.

4.1.5.1. 2-azido-N-benzyl-N-methylacrylamide (3a)

1

Yield: 78%. Yellow oil. НNMR (СDCl₃, δ, ppm): 2.90, 2.98 (two s, 3 Н, СН₃); 4.62 (s, 2 Н,

СН₂); 5.08 (m, 2 Н, СН₂=); 7.10-7.43 (m, 5 Н, Ph). Found C, 61.03; H, 5.54; N, 25.80. С₁₁H₁₂N₄O.

Calcd. (%): C, 61.10; H, 5.59; N, 25.91.

21

4.1.5.2. 2-azido-N-butyl-N-methylacrylamide (3b)

3

Yield: 79%. Yellow oil. ¹Н NMR (СDCl₃, δ, ppm): 0.92 (t, 3 Н, СН₂СН₃, J = 7.3); 1.30 (m, 2

Н, СН₂СН₃); 1.55 (m, 2 Н, СН₂СН₂СН₃); 2.94, 3.05 (two s, 3 Н, NСН₃); 3.39 (t, 2 Н, NСН₂, ³J = 7.3);

4.95, 5.02 (two m, each 1 Н, СН₂=). Found C, 52.80; H, 7.80; N, 30.74. С₈H₁₄N₄O. Calcd. (%): C,

52.73; H, 7.74; N, 30.75.

4.1.5.3. 2-azido-N-(sec-butyl)-N-methylacrylamide (3c)

Yield: 77%. Yellow oil. ¹Н NMR (СDCl₃, δ, ppm): 0.84 (m, 3 Н, СН₂СН₃); 1.12, 1.17 (two d,

3 Н, СНСН₃, ³J = 6.6); 1.51 (m, 2 Н, СН₂СН₃); 2.77, 2.87 (two s, 3 Н, NСН₃); 3.94 (m, 1 Н, СНСН₃);

4.92, 5.01 (two m, each 1 Н, СН₂=). Found C, 52.68; H, 7.79; N, 30.66. С₈H₁₄N₄O. Calcd. (%): C,

52.73; H, 7.74; N, 30.75.

4.1.6.

Synthesis

of

1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides

1

from

arylpyrroleketones 2 and 2-azidoacrylamides 3

Solution of 2 mmol of 2-azidoacrylamide 3, 2.2 mmol (0.72 g) of Cs₂CO₃and 2 mmol of

arylpyrroleketone 2 in 6 ml of dry DMF was stirred for 24 h at room temperature. The mixture

was diluted with 30 ml of water and extracted with 4*20 ml of EtOAc. The combined extracts

were washed with 20 ml of water and filtered over paper filter and evaporated to dryness. The

residue was chromatographed on silica gel using a mixture of ethyl acetate and hexane (1:3) as

eluent. Appropriate fractions were evaporated to dryness and the residue was recrystallized

from ethanol to give the desired product.

4.1.6.1. N-benzyl-N-methyl-1-phenylpyrrolo[1,2-a]pyrazine-3-carboxamide (1a, the

ratio of rotamers is 1:1.5)

Yield: 61%. Yellow crystals. M.p. 101-103°С. IR (KBr): 3097, 2911 (CH₃, CH₂, CH), 1639

1

(C=O) cm^-1. НNMR (СDCl₃, δ, ppm) (the signals of minor rotamer are shown in italics): 3.06,

3.29 (two s, 3 Н, СН₃); 4.76, 5.03 (two s, 2 Н, СН₂); 6.73 (m, 1 Н, Н(8)); 6.95 (m, 1 Н, Н(7)); 7.26-

8.02 (m, 11 Н, 2 Ph, Н(6)); 8.52 (s, 1 Н, Н(4)). Found (%): C, 77.63; H, 5.65; N, 12.47. С₂₂H₁₉N₃O.

Calcd. (%): C, 77.40; H, 5.61; N, 12.31.

4.1.6.2. N-butyl-N-methyl-1-phenylpyrrolo[1,2-a]pyrazine-3-carboxamide (1b, the ratio

of rotamers is 1:1.4)

22

Yield: 57%. Yellow crystals. M.p. 85-87°С.IR (KBr): 3101, 2956, 2873 (CH₃, CH₂, CH), 1621

1

(C=O) cm^-1. НNMR (СDCl₃, δ, ppm) (the signals of minor rotamer are shown in italics): 0.81,

3

0.97 (two t, 3 Н, СН₂СН₃, J = 7.3); 1.19, 1.39 (two m, 2 Н, СН₂СН₃); 1.67 (m, 2 Н, СН₂СН₂СН₃);

3

3.11, 3.32 (two s, 3 Н, NСН₃); 3.54, 3.69 (two t, 2 Н, NСН₂, J = 7.4); 6.90-7.05 (m, 2 Н, Н(7),

Н(8)); 7.40-8.05 (m, 6 Н, Ph, Н(6)); 8.41 (s, 1 Н, Н(4)). Found (%): C, 74.21; H, 6.70; N, 13.50.

С₁₉H₂₁N₃O. Calcd. (%): C, 74.24; H, 6.89; N, 13.67.

4.1.6.3. N-butyl-1-(2-fluorophenyl)-N-methylpyrrolo[1,2-a]pyrazine-3-carboxamide (1c,

the ratio of rotamers is 1:1.4)

Yield: 54%. Yellow crystals. M.p. 91-93°С. IR (KBr): 3064, 2953, 2870 (CH₃, CH₂, CH), 1624

1

(C=O) cm^-1. НNMR (СDCl₃, δ, ppm) (the signals of minor rotamer are shown in italics): 0.78,

3

0.96 (two t, 3 Н, СН₂СН₃, J = 7.3); 1.18, 1.38 (two m, 2 Н, СН₂СН₃); 1.64 (m, 2 Н, СН₂СН₂СН₃);

3

3.09, 3.27 (two s, 3 Н, NСН₃); 3.52, 3.68 (two t, 2 Н, NСН₂, J = 7.4); 6.70, 6.72 (two m, 1 Н,

Н(8)); 6.94, 6.96 (two m, 1 Н, Н(7)); 7.16-7.71 (m, 5 Н, Ar, Н(6)); 8.44 (s, 1 Н, Н(4)).Found (%): C,

70.21; H, 6.00; N, 12.92; F, 5.87. С₁₉H₂₀FN₃O. Calcd. (%): C, 70.13; H, 6.20; N, 12.91; F, 5.84.

4.1.6.4.

N-butyl-1-(2-chlorophenyl)-N-methylpyrrolo[1,2-a]pyrazine-3-carboxamide

(1d, the ratio of rotamers is 1:1.4)

Yield: 50%. Yellow crystals. M.p. 61-63°С. IR (KBr): 3062, 2948, 2867 (CH₃, CH₂, CH), 1639

1

(C=O) cm^-1. НNMR (СDCl₃, δ, ppm) (the signals of minor rotamer are shown in italics): 0.76,

3

0.95 (two t, 3 Н, СН₂СН₃, J = 7.3); 1.16, 1.38 (two m, 2 Н, СН₂СН₃); 1.60 (m, 2 Н, СН₂СН₂СН₃);

3

3.08, 3.25 (two s, 3 Н, NСН₃); 3.50, 3.69 (two t, 2 Н, NСН₂, J = 7.4); 6.50, 6.52 (two m, 1 Н,

Н(8)); 6.91, 6.92 (two m, 1 Н, Н(7)); 7.32-7.58 (m, 5 Н, Ar, Н(6)); 8.42 (s, 1 Н, Н(4)).Found (%): C,

66.46; H, 5.84; N, 12.59; Cl, 10.28. С₁₉H₂₀N₃OCl. Calcd. (%): C, 66.76; H, 5.90; N, 12.29; Cl, 10.37.

4.1.6.5.

1-(2-bromophenyl)-N-butyl-N-methylpyrrolo[1,2-a]pyrazine-3-carboxamide

(1e, the ratio of rotamers is 1:1.4)

Yield: 66%. White crystals. M.p. 57-59°С. IR (KBr): 3112, 2955, 2870 (CH₃, CH₂, CH), 1624

1

(C=O) cm^-1. НNMR (СDCl₃, δ, ppm) (the signals of minor rotamer are shown in italics): 0.76,

3

0.95 (two t, 3 Н, СН₂СН₃, J = 7.3); 1.18, 1.39 (two m, 2 Н, СН₂СН₃); 1.62 (m, 2 Н, СН₂СН₂СН₃);

3

3.08, 3.26 (two s, 3 Н, NСН₃); 3.50, 3.70 (two t, 2 Н, NСН₂, J = 7.4); 6.48, 6.49 (two m, 1 Н,

Н(8)); 6.92, 6.93 (two m, 1 Н, Н(7)); 7.24-7.75 (m, 5 Н, Ar, Н(6)); 8.42 (s, 1 Н, Н(4)).Found (%): C,

23

58.88; H, 5.20; N, 10.93; Br, 20.57. С₁₉H₂₀BrN₃O. Calcd. (%): C, 59.08; H, 5.22; N, 10.88; Br,

20.68.

4.1.6.6. N-(sec-butyl)-N-methyl-1-phenylpyrrolo[1,2-a]pyrazine-3-carboxamide (1f, the

ratio of rotamers is 1:1.8)

Yield: 68%. Yellow crystals. M.p. 117-119°С. IR (KBr): 3129, 3069, 2960 (CH₃, CH₂, CH),

1

1621 (C=O) cm^-1. НNMR (СDCl₃, δ, ppm) (the signals of minor rotamer are shown in italics):

3

0.81, 0.96 (two t, 3 Н, СН₂СН₃, J = 7.3); 1.22, 1.30 (two d, 3 Н, СНСН₃, ³J = 6.6); 1.40-1.70 (m, 2

Н, СН₂СН₃); 2.94, 3.10 (two s, 3 Н, NСН₃); 4.38, 4.72 (two m, 1 Н, СНСН₃); 6.95 (m, 1 Н, Н(8));

6.99 (m, 1 Н, Н(7)); 7.46-8.01 (m, 6 Н, Ph, Н(6)); 8.33 (s, 1 Н, Н(4)). Found (%): C, 74.36; H, 7.13;

N, 13.80. С₁₉H₂₁N₃O. Calcd. (%): C, 74.24; H, 6.89; N, 13.67.

4.1.7. Ethyl 2-azidoacrylate (14) [43]

The solution of 40 mmol (10.4 g) of ethyl 2,3-dibromopropionate in 20 ml of dry DMF

was added to the stirred suspension of 100 mmol (6.2 g) of sodium azide in 60 ml of dry DMF at

60°C. The mixture was stirred at 60°C for 90 min, cooled to the room temperature and diluted

with 350 ml of water. The product was extracted with 3*60 ml of dichloromethane and

combined organic solutions were washed with 60 ml of water, filtered over paper filter and

evaporated to dryness. The residue was chromatographed on silica gel using a mixture of ethyl

acetate and hexane (1:5) as eluent. Appropriate fractions were evaporated to yield 4.7 g of

1

product as a pale yellow oil (yield: 83%). Н NMR (СDCl₃, δ, ppm): 1.31 (t, 3 H, CH₃, ³J =7.2); 4.26

(q, 2 H, OCH₂, ³J =7.2); 5.31, 5.81 (two s, each 1 H, CH₂=).

4.1.8. Synthesis of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxylic acids (16) from

arylpyrroleketones 2 and ethyl 2-azidoacrylate 14

Solution of 44 mmol (5.6 g) of methyl 2-azidoacrylate 14, 53 mmol (13.3 g) of Cs₂CO₃

and 34 mmol of arylpyrroleketone 2 in 80 ml of dry DMF was stirred for 24 h at room

temperature. The mixture was evaporated, diluted with 200 ml of water and 50 ml of

methanol. 68 mmol (2.7 g) of NaOH was added to the solution and the mixture was refluxed

until complete ester 15 saponification that was monitored by TLC. The mixture was cooled,

diluted with 100 ml of water and acidified with 36% HCl to pH~5.The mixture was extraxted

with 4*40 ml of ethyl acetate. All organic fractions were combined, filtered over paper filter

and evaporated to dryness to give the desired product.

24

4.1.8.1. 1-Phenylpyrrolo[1,2-a]pyrazine-3-carboxylic acid (16a)

Yield: 48%. Yellow crystals. M.p. 155-157°C. ¹Н NMR (СDCl₃, δ, ppm): 7.08 (m, 1 Н, Н(8));

7.14 (m, 1 Н, Н(7)); 7.51-8.03 (m, 6 Н, Ph, Н(6)); 8.80 (s, 1 Н, Н(4)); 11.5 (s, 1 Н, СООН).Found

(%): C, 70.52; H, 4.36; N, 11.86. С₁₄H₁₀N₂O₂. Calcd. (%): C, 70.58; H, 4.23; N, 11.76.

4.1.8.2. 1-(2-Chlorophenyl)pyrrolo[1,2-a]pyrazine-3-carboxylic acid (16b)

1

Yield: 45%. Yellow crystals. M.p. 173-175°C. Н NMR (DMSO-d6, δ, ppm): 6.48 (m, 1 Н,

Н(8)); 7.04 (m, 1 Н, Н(7)); 7.45-7.70 (m, 5 Н, Ph, Н(6)); 8.80 (s, 1 Н, Н(4)); 11.5 (s, 1 Н, СООН).

Found (%): C, 61.84; H, 3.22; N, 10.22; Cl, 13.04. С₁₄H₉ClN₂O₂. Calcd. (%): C, 61.67; H, 3.33; N,

10.27; Cl, 13.00.

4.1.9. Synthesis of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides

phenylpyrrolo[1,2-a]pyrazine-3-carboxylic acids (16) and amines (10)

1

from 1-

57 mmol (0.9 g) of POCl₃, 5 ml of triethylamine and 1 drop of DMF were added to the

solution of 4.4 mmol (1.1 g) of 1-phenylpyrrolo[1,2-a]pyrazine-3-carboxylic acid (16) in 40 ml of

dichloromethane and the mixture was stirred for 25 min at room temperature. 5.7 mmol of

amine (10) was added to the reaction and the mixture was stirred for 15 min at room

temperature. The reaction was diluted with 40 ml of water and acidified with 1M HCl to acid

reaction. The product was extracted with 3*30 ml of dichloromethane, the combined fractions

were washed with 30 ml of water, filtered over paper filter and evaporated to dryness. The

residue was recrystallized from ethanol to give the desired product.

4.1.9.1. N-methyl-1-phenylpyrrolo[1,2-a]pyrazine-3-carboxamide (1g)

Yield: 45%. Pale pink crystals. M.p. 167-169°С. IR (KBr): 3383 (NH), 3127, 3081, 2926

1

(CH₃, CH), 1651 (C=O) cm^-1. Н NMR (СDCl₃, δ, ppm): 3.03, 3.05 (two s, 3 Н, СН₃); 6.95 (m, 1 Н,

Н(8)); 7.00 (m, 1 Н, Н(7)); 7.49-8.02 (m, 7 Н, Ph, Н(6), NH); 8.73 (s, 1 Н, Н(4)). Found (%): C,

71.86; H, 5.22; N, 16.90. С₁₅H₁₃N₃O. Calcd. (%): C, 71.70; H, 5.21; N, 16.72.

4.1.9.2. N-benzyl-1-phenylpyrrolo[1,2-a]pyrazine-3-carboxamide (1h)

Yield: 53%. Yellow crystals. M.p. 173-175°С. IR (KBr): 3256 (NH), 3064, 2920 (CH₂, CH),

1

3

1644 (C=O) cm^-1. Н NMR (СDCl₃, δ, ppm): 4.71 (d, 2 Н, СН₂, J = 6.2); 6.98 (m, 1 Н, Н(8)); 7.01

25

3

(m, 1 Н, Н(7)); 7.25-7.99 (m, 11 Н, 2 Ph, Н(6)); 8.35 (t, 1 Н, NH, J = 6.2); 8.77 (s, 1 Н, Н(4)).

Found (%): C, 77.31; H, 5.27; N, 13.15. С₂₁H₁₇N₃O. Calcd. (%): C, 77.04; H, 5.23; N, 12.84.

4.1.9.3.

N-butyl-1-(2-chlorophenyl)-N-methylpyrrolo[1,2-a]pyrazine-3-carboxamide

(1d)

Yield: 54%.Compound is identical to that received in 4.1.6.4.

4.2. Pharmacology

4.2.1. Animals

Adult male inbred Balb/c mice and CD-1 mice weighing 19 - 25 g were used in this study.

The animals were obtained from the nursery of laboratory animals of the Shemyakin and

Ovchinnikov Institute of bioorganic chemistry of the Russian Academy of Sciences and adapted

for 2 weeks prior to testing in the Zakusov Institute of Pharmacology vivarium. The mice were

maintained in an environmentally controlled facility with a 12 h light-dark cycle (lights on at

08:00 hours), a temperature of 22˚C ± 2˚C and humidity (60 ± 10%). All mice were maintained

on a standard diet with food and water available ad libitum. The animals were randomized in

groups of 8 animals by body weight with a deviation from mean value no more than ± 10%. All

the animals were kept for 24 hours in the experimental room at the "home" cages before

experiments. The experimental protocols were in compliance with the European Communities

Council Directive (86/609/EEC).

4.2.2. Open field test (OFT)

The OFT apparatus consisted of a white round arena with a diameter of 1 m with white

boards 50 cm high. Arena is illuminated by four 75 W shadowless lamps fixed at 1 m above the

floor. The arena of the open field is divided by 4 concentric circles, which in turn are divided

into the sectors by radii in a way that the peripheral circle consists of 16 identical curvilinear

squares.

Adult male Balb/c mice were used in this experiment. After 30 min of intraperitoneal

administration of vehicle and compounds as a suspension in 1% Tween 80 (v/v) the animals was

placed for 1 minute in a dark chamber, and then – on the one of the peripheral squares of

arena. The number of squares crossed in the periphery (SCP), in the central regions (SCC), the

number of entries into the center (C) and number of rearing (R) were measured during 3 min.

The total number of crossed squares with number of rearing was taken as the total locomotor

26

activity (TLA). After each individual test session, the equipment was washed with soap and

water, cleaned with ethanol 70% and dried. Then, the subsequent mouse was tested.

4.2.3. Elevated plus-maze test (EPM)

The EPM consisted of four arms elevated 40 cm above the floor, with each arm

positioned at 90° relative to the adjacent arms. Two of the arms were enclosed with high walls

(65× 5 × 15 cm), and the other arms were connected via a central area (5 × 5 cm) to form a plus

sign. The maze floor and the walls of enclosed arms were painted black. The room was

illuminated with a 40-W lamp at the central platform.

Adult male CD-1 mice were used in this experiment. The animals were treated with

vehicle and compounds as a suspension in 1% Tween 80 (v/v) intraperitoneally, 30 min prior to

the test. The experiment was performed between 09:00 and 14:00 hours. Each mouse was

individually placed on the central platform facing toward an open arm. The frequency and

duration of entries into the open and closed arms were observed for 5 min. An entry was

counted when all four paws of the mouse entered an open or closed arm. The percentage of

time spent (duration) in the open arms (100 × open/[open + enclosed]) and percentage of the

number of open arm entries (frequency, 100 × open/total entries) were calculated for each

animal. The apparatus was thoroughly cleaned after each trial.

4.2.4. PK11195 influence in EPM test

Experiment was carried out on CD-1 mice, divided into four groups, 8 mice in each

group. Group 1: control animals; distilled water was administered twice: 60 min and 30 min

before testing. Group 2: PK11195 (10.0 mg/kg) was administered 60 min before and distilled

water 30 min before testing. Group 3: distilled water was administered 60 min before and

compound 1a (0.5 mg/kg) 30 min before testing. Group 4: PK11195 (10.0 mg/kg) was

administered 60 min before and compound 1a (0.5 mg/kg) 30 min before testing. Animal

behavior was evaluated in EPM test. The dose of PK11195 was chosen from the literature [46].

The animals were treated with vehicle and compounds as a suspension in 1% Tween 80 (v/v)

intraperitoneally.

4.2.5. The influence on locomotor activity

27

To study the influence of compounds on spontaneous locomotor activity infrared

actimetry (Panlab, Spain) was used. 1a and 1b in the dose range of 0.1-5.0 mg/kg did not

change locomotor activity parameters in outbred CD-1 mice compared with control group.

4.2.6. Statistical analysis

Statistical processing of the results was performed using ANOVA (Kruskal-Wallis test)

and non-parametric analysis for independent variables (U-Mann-Whitney test).

4.2.7. TSPO-binding assay

Binding studies were carried out by CEREP (France) according to Kugler et al. [53].

Membrane homogenates of human glioblastoma U-118 MG cells (100 μg protein) were

incubated for 15 min at 22°C with 0.2 nM [³H]-PK11195 in the absence or presence of the test

compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.02 mg/ml aprotinine

and 0.1 mg/ml bacitracine. Nonspecific binding was determined in the presence of 10 μM

PK11195. Following incubation, the samples were filtered rapidly under vacuum through glass

fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50

mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters were dried then

counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation

cocktail (Microscint 0, Packard).

Six concentrations of each compound were assayed, each performed in triplicate. The

standard reference compound was PK11195. Specific binding was determined by subtracting

nonspecific binding from total binding. The IC₅₀values for each test compound were

determined by non-linear regression analysis of the competition curves generated with mean

replicate values using Hill equation curve fitting. This analysis was performed using software

developed at Cerep (Hill software) and validated by comparison with data generated by the

constants (K_i) were calculated according to the following formula: K_i= IC₅₀/(1 + [L]/K_D), where

[L] and K_Dare the concentration of [³H]-PK11195 and the dissociation constant of PK11195

calculated by Scatchard analysis, respectively.

4.2.8. GABA_AR benzodiazepine site binding assay

Binding studies were carried out by CEREP (France) according to Speth et al. [54].

Membrane homogenates of cerebral cortex (80 μg protein) are incubated for 60 min at 4°C

28

with 0.4 nM [³H]-flunitrazepam in the absence or presence of the test compound in a buffer

containing 50 mM Tris-HCl (pH 7.7). Nonspecific binding is determined in the presence of 3 μM

diazepam. Following incubation, the samples are filtered rapidly under vacuum through glass

fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50

mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then

counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation

cocktail (Microscint 0, Packard).

Two concentrations of compound 1a were assayed, each performed in duplicate. The

standard reference compound was diazepam. Specific binding was determined by subtracting

nonspecific binding from total binding. The IC₅₀and K_ivalues for diazepam was determined in

the same manner as in 4.2.7.

Acknowledgements

This work was supported by the Basic Research Program of the Presidium of the Russian

Academy of Sciences (The project “Design, synthesis and pharmacological properties of the

original ligands of the mitochondrial protein TSPO”).

29

Article Doi

Design, synthesis and anxiolytic-like activity of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides

DOI: 10.1016/j.bmc.2015.04.049

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2015.04.049

Full text of DOI:10.1016/j.bmc.2015.04.049

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