3-Biphenylimidazo[1,2-a]pyridines or [1,2-b]pyridazines and analogues, novel Flaviviridae inhibitors

European Journal of Medicinal Chemistry 64 (2013) 448e463

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European Journal of Medicinal Chemistry

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Original article

3-Biphenylimidazo[1,2-a]pyridines or [1,2-b]pyridazines and

analogues, novel Flaviviridae inhibitors

,

b

a

Cécile Enguehard-Gueifﬁer ^a, Simone Musiu , Nicolas Henry , Jean-Baptiste Véron ,

*

Sylvie Mavel ^c, Johan Neyts ^b, Pieter Leyssen ^b, Jan Paeshuyse ^b, Alain Gueifﬁer ^a

^aUniversité François Rabelais, UMR INRA 1282 Infectiologie et Santé Publique, Recherche et Innovation en Chimie Médicinale, Faculté de Pharmacie,

31 avenue Monge, F-37200 Tours, France

^bDepartment for Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium

^cUniversité François-Rabelais, INSERM U930, CHRU de Tours, 31 avenue Monge, F-37200 Tours, France

a r t i c l e i n f o

a b s t r a c t

Article history:

Using Ttou 84 as starting point, a novel class of biphenyl derivatives of imidazo[1,2-a]pyridine and

imidazo[1,2-b]pyridazine was designed to optimize the inhibitory properties on the replication of the

bovine viral diarrhoea virus (BVDV) and hepatitis C virus (HCV). Three sites of pharmacomodulation were

chosen i.e. positions 2, 3 and 6 on the central heterocyclic core structure. From the 49 analogues tested,

only compound 18j (3-(2⁰-hydroxybiphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-b]pyr-

idazine) showed antiviral activity in the HCV replicon system reminiscent of selective inhibition (60e70%

inhibition). Compound 4f (3-(biphen-3-yl)-2-(4-ﬂuorophenyl)-6-phenylthioimidazo[1,2-a]pyridine)

proved to be the most selective inhibitor of BVDV replication and showed no or only marginal cross-

resistance with known inhibitors of pestivirus replication. The cross-resistance proﬁle of 4f might

indicate that 4f does not interact with the same binding site as BPIP, VP32947, AG110 or LZ37. From 42

analogues tested against both viruses, QSAR studies were discussed in regard to BVDV antiviral activity.

Received 31 January 2013

Received in revised form

21 March 2013

Accepted 24 March 2013

Available online 19 April 2013

Keywords:

Imidazo[1,2-a]pyridine

imidazo[1,2-b]pyridazine

BVDV

HCV

Antiviral agent

1. Introduction

Worldwide, 170 million people are chronic carriers of HCV.

This virus is a major cause of cirrhosis and primary hepatocellular

The bovine viral diarrhoea virus (BVDV) and hepatitis C virus

(HCV), for which BVDV was once considered to be a surrogate [1], are

two viral pathogens that belong to the family of Flaviviridae [2].

BVDV, together with the classical swine fever virus (CSFV), are rep-

resentatives of the genus Pestivirus. These viruses cause important

diseases in livestock (i.e. BVDV in cattle and CSFV in pigs) regardless

of the availability of efﬁcacious vaccines and the implementation of

elaborate eradication or control programs [3e5]. An alternative

approach to combat BVDV and CSFV infections could be the use of

antiviral agents that speciﬁcally inhibit the replication of the virus

[6,7]. Although likely not suited to treat large herds, it may be

important to have selective broad-spectrum anti-Pestivirus com-

pounds at hand, for example as a mean to rapidly control CSFV

outbreaks [6,7].

carcinoma, and the main reason for liver transplantations among

adults in Western countries [8]. The current therapy for hepatitis C

virus infection consists of the combination of pegylated interferon-

a, the nucleoside analogue ribavirin and a virus-speciﬁc protease

inhibitor Telaprevir or Boceprevir [9]. This therapy is only effective

in about 60e70% of patients that suffer from chronic HCV infection,

and is associated with important side effects. A potential concern is

the emergence of drug-resistant virus variants. The ultimate goal in

HCV therapy is to evolve to an all-oral, interferonesparse combi-

nation of direct-acting antivirals against HCV with minimal adverse

effects and against which no drug-resistance develops during

treatment [10,11]. Hence, there is still a need to diversify the

portfolio of direct-acting antiviral drugs, especially drugs with

novel mechanisms of action.

In the course of our studies on the evaluation of the chemical

and pharmacological properties of imidazoazines, we previously

identiﬁed a novel series of compounds with selective antiviral

activity against the human cytomegalovirus (hCMV) and varicella-

Abbreviations: CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2;

CCR5, CC chemokine receptor 5; TLC, thin layer chromatography; BVDV, bovine viral

diarrhoea virus; HCV, hepatitis C virus; EC₅₀, 50% effective concentration; CC₅₀, 50%

cytotoxic concentration; QSAR, quantitative structureeactivity relationship.

* Corresponding author. Tel.: þ33 (0)247 36 71 72.

zoster virus (VZV); viral pathogens that belong to the

-Herpesviridae respectively [12,13]. In our search for selective

inhibitors of virus replication, we decided to evaluate the antiviral

a- and

b

E-mail address: cecile.enguehard-gueif ﬁ er@univ-tours.fr (C. Enguehard-Gueifﬁer).

http://dx.doi.org/10.1016/j.ejmech.2013.03.054

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

449

properties of around 120 compounds from our chemical library

against BVDV and HCV. Five hits were identiﬁed from this library

and a pharmacomodulation studies was initiated on the most

potent compound against HCV (Ttou 84, EC₅₀¼ 2.64

m ,

g mL^ꢀ1

CC₅₀¼ 36

m

g mL^ꢀ1, Scheme 1), leading to a novel series of biphenyl

derivatives of imidazo[1,2-a]pyridine and [1,2-b]pyridazine. The

synthesis of this new series of compounds, its biological evaluation

and a QSAR study is subject of the present article.

2. Chemistry

49 compounds were obtained in this program. From the hit

compound Ttou 84 (Fig. 1), three sites for pharmacomodulation

were chosen at positions 2, 3 and 6 of the heterocycle; the two

series imidazo[1,2-a]pyridine and imidazo[1,2-b]pyridazine were

studied concomitantly. Various groups were introduced at site I

(aryl, amine, thioether) and site II (hydrogen, alkyl, ester, aryl). The

biphenyl part of site III was ﬁrst studied in o, m and p conﬁguration,

and the terminal phenyl subsequently replaced by other biaryl

groups.

Fig. 1. Structure of Ttou 84 and pharmacomodulation sites.

coupling reaction in position 3 in traditional conditions (1.2 eq of

boronic acid, 2 mol% of Pd(PPh₃)₄, 2 eq of Na₂CO₃in DME/H₂O).

Different metallo-catalyzed cross-coupling reactions were per-

formed on position 6 in order to introduce variability at this posi-

tion: Suzuki cross-coupling reactions, and copper-catalyzed cross-

couplings of amines and thiophenol. Two series of compounds 4aef

and 8aeh were obtained.

2.1. Pharmacomodulation at site I

In a ﬁrst approach, we decided to study the inﬂuence of the

substituent in position 6 concomitantly with the evaluation of the

biphenyl substitution in position 3, and this in the two imidazoa-

zines series (Scheme 1). The same synthetic strategy was adopted

in both series with a ﬁrst metallo-catalyzed cross-coupling reaction

in position 6 of the heterocycle, followed by an iodination in posi-

tion 3 (using N-iodosuccinimide in acetonitrile) and a Suzuki cross-

2.2. Pharmacomodulation at site II

Depending on the biological results previously obtained in the

study of site I, we decided to continue the pharmacomodulation

program with a thienyl group in position 6 (Scheme 2). This thienyl

group was introduced directly on the starting aminopyridine or

aminopyridazine. The condensation of these aminothienylazines

N

iii

i

F

N

I

R₆

1

R₃

4a-f

2a-d R₃= H

ii

3a-d

R₃= I

N

iii

i

F

N

Cl

N

R₆

N

R₆

N

R₃

5

8a-h

6a-d R₃= H

7a-d

ii

R₃= I

2a, 3a, 6a, 7a R₆= Ph

2b, 3b, 6b, 7b R₆= morpholin-1-yl

2c, 3c, 6c, 7c R₆= thien-3-yl

2d, 3d

R₆= SPh

R₆= N-methylpiperazin-1-yl

6d,7d

Reagents and conditions: (i) phenyl or thien-3-ylboronic acids (1.2 eq), Pd(PPh₃)₄(2 mol%),

Na₂CO₃(2 eq)/DME, H₂O; or morpholine (1.1 eq), CuI (15 mol%), K₃PO₄(2 eq), ethylene

glycol (2 eq)/2-propanol (1 mL); or thiophenol (1.2 eq), CuI (5 mol%), K₂CO₃(2 eq), N,N’-

dimethylethylenediamine (15 mol%)/2-propanol (1 mL); or amine, reflux. (ii) NIS (1.5

eq)/CH₃CN. (iii) biphenylboronic acids (1.2 eq), Pd(PPh₃)₄(2 mol%), Na₂CO₃(2 eq)/DME,

H₂O.

Scheme 1. Pharmacomodulation in positions 3 and 6 of the 2-(4-ﬂuorophenyl)imidazoazines 1 and 5.

450

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

NH₂R₂

O

N

i

R₂

+

N

X

Br

R₃

S

9a

X = CH

10a-i R₃= H

11a-i R₃= I

ii

9b X = N

iii

N

a X = CH, R₂= H

b X = CH, R₂= tert-but

c X = CH, R₂= CO₂Et

d X = CH, R₂= thien-3-yl

R₂

N

X

e

f

S

X = CH, R₂= 2-CH₃OPh

12a-i

X = CH, R₂= 3-CH₃OPh

X = CH, R₂= 4-CH₃OPh

X = N, R₂= tert-but

g

h

i

X = N, R₂= 2-CH₃OPh

Reagents and conditions: (i) EtOH, reflux (ii) NIS (1.5 eq)/CH₃CN (iii) biphen-3-ylboronic

acid (1,2 eq), Pd(PPh₃)₄(2 mol%), Na₂CO₃(2 eq)/DME,H₂O.

Scheme 2. Pharmacomodulation in position 2 of the 6-thien-3-ylimidazo[1,2-a]pyridine and [1,2-b]pyridazine.

with convenient

a-halogenocarbonyl compounds in reﬂuxing

considered to be selective inhibitors of HCV replication in this

ethanol led to the imidazoazines diversely functionalized in posi-

tion 2. Like previously, an iodination and a Suzuki cross-coupling

reaction in position 3 led to compounds 12aei.

replicon system.

Table 1 summarizes the antiviral data. For all of the compounds,

except for compound 18j, the antiviral activity that was observed in

the Huh 5-2 replicon system clearly could be attributed to an

adverse effect of the compound on the host cell metabolism, quite

frequently yielding low selectivity index values (SI ¼ CC₅₀/EC₅₀).

Therefore, these compounds could not be considered selective in-

hibitors of the replication of HCV, even though an EC₅₀value could

frequently be derived from the doseeresponse curves. For com-

pound 18j, a SI of >40 was obtained and an inhibitory effect of 60e

70% on virus replication could be observed at concentrations that

do not signiﬁcantly affect the host cell metabolism. The SI of 18j was

thus improved compared to the SI of the parent compound Ttou 84.

2.3. Pharmacomodulation at site III

First, we studied the inﬂuence of substituting different positions

of the terminal phenyl in the biphenyl side group. Subsequently, the

terminal phenyl was replaced by various heteroaromatic groups.

This functionalization was performed starting from the 3-(3-

bromophenyl)imidazo[1,2-a]pyridines 15aeb leading to com-

pounds 16aep, or starting from the 3-(3-bromophenyl)-6-

thienylimidazo[1,2-b]pyridazines 17bec leading to compounds

18aej (Scheme 3). The 3-bromophenyl present in the intermediates

15aeb and 17bec could not be introduced in position 3 of the

heterocycle through a Suzuki cross-coupling reaction, as auto-

condensation of the boronic acid on the 3-bromophenyl occurred.

3.2. Anti-BVDV activity

The biphenyl compounds were then evaluated for selective in-

hibition of BVDV (strain NADL)-induced CPE formation as assessed

by the MTS method in MDBK cells (Table 1).

The nature of the biphenyl group appears to largely inﬂuence

the anti-BVDV activity in the two imidazoazines series as follows:

biphen-3-yl > biphen-2-yl > biphen-4-yl (see, for examples,

compounds 4aeb, 8aeb and 8cee). This inﬂuence cannot be

attributed only to the adverse effect on the host cell as seen for

compound 8a with an SI of 20. The study was then pursued using a

biphen-3-yl group in position 3 of the scaffold.

We thus synthesized the convenient

a-halogenocarbonyl com-

pounds 14aec that were condensed to the appropriate amino-

pyridine 13 or 3-amino-6-thienylpyridazine 9b. Finally, the

substitution of the intermediates 15aeb and 17bec was performed

using a Suzuki cross-coupling reaction.

3. Results and discussion

3.1. Anti-HCV activity

The biphenyl compounds were evaluated for selective antiviral

activity in the HCV subgenomic replicon system (genotype 1b) in

Huh 5-2 cells [14]. The adverse effect of the compounds on the host

cell metabolism was quantiﬁed in parallel with the antiviral effect

to allow correct interpretation of the data. As per deﬁnition, only

compounds that produce signiﬁcant inhibition of viral replication

(>>70% inhibition) at concentrations that do not elicit an anti-

metabolic effect on the host cells (cell viability >>90%) can

3.2.1. Pharmacomodulation at site I

For position 6, the thien-3-yl substituted derivatives 4e and 8f

produced EC₅₀values amongst the lowest in the two series of

imidazoazines (EC₅₀¼ 0.74e1.7

rivative 4f and the 6-phenyl compound 8a showed also interesting

g mL^ꢀ1, respectively). The

m

g mL^ꢀ1). The 6-thiophenyl de-

anti-BVDV activities (EC₅₀¼ 2.0 and 1.23

m

best SI were obtained in the imidazo[1,2-a]pyridine series, for the

6-thien-3-yl and 6-thiophenyl derivatives 4eef (SI > 45).

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

451

N

R₂

O

R₂

N

NH₂

ii

i

Br

+

Br

N

R₃

Br

13

14a-b

15a-b

16a-p

14a, 15a R₂= 4-FPh

14b, 15b

R₂= 2-MeOPh

N

R₂

O

R₂

NH₂

N

ii

N

i

N

Br

+

N

Br

S

R₃

Br

9b

14b-c

17b-c

18a-j

14b, 17b

R₂= 2-MeOPh

14c, 17c R₂= tert-But

Reagents and conditions: (i) EtOH, reflux (ii) R₃B(OH)₂(1.2 eq), Pd(PPh₃)₄(2 mol%),

Na₂CO₃(2 eq)/DME, H₂O; or amine (1.2 eq), Pd₂dba₃(0.5 mol%), BINAP (1.5 mol%),

NaOtBu (1.4 eq)/toluene.

Scheme 3. Pharmacomodulation of the biphenyl group in the imidazo[1,2-a]pyridine and imidazo[1,2-b]pyridazine series.

3.2.2. Pharmacomodulation at site II

patterns for CPE reduction, viral RNA synthesis and infectious virus

yield were observed. The doseeresponse effect of the compound

on the number of RNA copies that are released as progeny virions

by treated, infected cells allowed to calculate an EC₅₀of

With regard to the 2-substitution pattern, the study was per-

formed on the 3-(biphen-3-yl)-6-(thien-3-yl)imidazo[1,2-a]pyri-

dine scaffold. A total loss of activity was observed with all the

substituents tested (hydrogen, tertio-butyl, ester, and 2-, 3-, or 4-

methoxyphenyl) in 12aeg, except for compound 12d with a

2.7 ꢁ 0.4

particles, released by treated, infected cells, was about two-fold less

pronounced, i.e. 8.0 ꢁ 0.2 g mL. The concentration of 4f that

reduced the proliferation of exponentially growing MDBK cells by

50% (50% cytostatic concentration or CC₅₀) was >100 g mL

mg mL while the effect on the number of infectious virus

thien-3-yl group in the 2-position (EC₅₀¼ 6.6

m

g mL^ꢀ1). Only two

m

substituents were evaluated in the imidazo[1,2-b]pyridazine series,

both without success.

m

(Fig. 2A). Hence, a SI of 19e37 was calculated. Compound 4f largely

3.2.3. Pharmacomodulation at site III

retained its antiviral potency when evaluated on the replication of

The study was performed on the 6-unsubstituted imidazo[1,2-a]

pyridine and the 6-(thien-3-yl)imidazo[1,2-b]pyridazine series.

Most compounds showed a pronounced cytotoxic effect on the host

cells with SI-values ranging between 1.4 and 7.1. Only two de-

rivatives combined activity with a low cytotoxic effect, e.g. the 2-(4-

ﬂuorophenyl)-3-(4⁰-methoxybiphen-3-yl)imidazo[1,2-a]pyridine

16d and 2-tertio-butyl-3-(3⁰-hydroxybiphen-3-yl)-6-(thien-3-yl)

imidazo[1,2-b]pyridazine 18d (SI of 36 and 26.7, respectively).

BPIP-resistant virus (EC₅₀¼ 6.0 ꢁ 1.1

compared to WT), AG110-resistant virus (EC₅₀¼ 3.9 ꢁ 0.8

a 0.9-fold change compared to WT) and was about 2-fold less active

m

g mL or 1.5-fold change

mg mL or

against LZ37 resistant virus (EC₅₀¼ 8.8 ꢁ 1.9

mg mL or 2.2-fold

change compared to WT).

3.3. QSAR study

The QSAR study described here is based on 42 derivatives pre-

senting an EC₅₀for BVDV. The QSAR software (Discovery Studio_

2.5, Accelrys, Inc., San Diego, CA) presents 179 physicochemical

descriptors: electronic, spatial, shadow, shape and thermodynamic

indices. After several ﬁlters (analyzing the correlation matrix,

eliminating the highly correlated descriptors), and eliminating

descriptors with too wide range of training data (>100), only 26 2D

or 3D descriptors were retained as dependent on EC₅₀for BVDV by

Genetic Function Approximation (GFA) algorithms.

3.2.4. Characterization of the anti-BVDV activity

Out of 42 analogues, 4f (Scheme 4) was identiﬁed as the most

potent and selective inhibitor of BVDV (strain NADL)-induced

cytopathic effect (CPE) formation as assessed by the MTS method.

In a multiple infection cycle assay in MDBK cells, it was shown that

the compound inhibited virus-induced CPE formation in a dose-

dependent manner (Fig. 2A). Based on the data represented in

Fig. 2A, the 50% effective concentration (EC₅₀) was determined to be

4.0 ꢁ 1.3

m

g mL. The anti-BVDV activity of 4f was further conﬁrmed

The QSAR equation with the regression statistics is given in

Fig. 3. The best predictive result is obtained with the lower log EC₅₀.

As the coefﬁcient in the equation is positive for Estate Keys as

“Sums of electrotopological state values for CH₃or CH atoms”

(ES_Sum_sCH₃and _aaCH, respectively), the lower the number of

by means of RT-qPCR to assess its effect at the level of viral RNA

synthesis (Fig. 2A) and determination of infectious virus yield to

quantify the inhibitory effect at the level of the production of

progeny infectious virus particles (Fig. 2B). Comparable inhibition

452

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

Table 1

Inhibitory effects of biphenylimidazoazines and analogues on cytopathogenic effect induction by the BVDV in MDBK cells and on HCV subgenomic replicon replication in Huh

5-2 cells.

N

R₂

N

R₆

X

R₃

Cpd

X

R₆

R₂

R₃

BVDV

HCV

CC₅₀

(m

g mL^ꢀ1

)

EC₅₀

(

m

g mL^ꢀ1

)

SI^c

(

m

g mL^ꢀ1

)

EC₅₀(m )

g mL^ꢀ1

SI^c

a

b

a

b

4a

4b

4c

4d

4e

4f

8a

8b

8c

8d

8e

8f

CH Ph

CH Morpholin-1-yl

CH Thien-3-yl

CH SPh

4-FePh

3-Ph

4-Ph

3-Ph

4-Ph

3-Ph

4-Ph

2-Ph

3-Ph

4-Ph

3-Ph

4-Ph

3-Ph

>50

30

12

>4

>1

>45

>50

20

>1

3

56.8

11.4

11.1

15.6

>112

>106

15.9

45.3

11.1

33.3

13.4

>112

45.3

>142

>123

>118

52.2

4.5

6.7

1.09

2.5

1.7

2.3

>1

1.4

ND

1

>30

>50

0.74

2.0

1.23

>50

11

4

>50

1.7

>50

>100

62.6

>67.7

6.6

68.6

15.0

22.7

37.9

3.4

ND

2.7

3.1

2.8

ND

3.0

3.9

0.98

ND

1.7

2.3

ND

7.55

ND

17.7

>100

1.7

14.2

3.7

>100

ND

>100

1.36

>50

>33

>100

25

>50

31

12

6.7

>112

>106

11.3

ND

11.1

ND

N

Ph

Morpholin-1-yl

Thien-3-yl

ND

1.2

>1

ND

> 1.6

> 2.4

> 2.2

> 1.8

3.3

1.6

> 1.9

2.8

6.6

1.7

2.8

11.8

21.3

5.1

9.7

5.3

2.3

1.9

3.4

5.1

2

>50

13

>1

8

ND

11.2

>112

ND

8g

8h

>50

>100

67.7

>100

10.4

ND

>1

>1.6

>1

N-Methylpiperazin-1-yl 4-FePh

12a CH Thien-3-yl

12b CH Thien-3-yl

12c

12d CH Thien-3-yl

12e

12f

12g CH Thien-3-yl

12h

12i

16a CH

16b CH

16c

16d CH

16e

16f

16g CH

16h CH

16i

16j

16k CH

16l CH

16m CH

16n CH

16o CH

16p CH

18a

18b

18c

18d

18e

18f

18g

18h

18i

H

89.5

42.5

53.1

64.5

17.5

52.4

56.8

22.7

14.7

34.9

10.7

4.3

1.63

13.5

5.9

10.3

20.2

12.8

19.2

11.1

28

21.8

48.9

3.5

18.8

8.6

8.7

5

3.9

30.1

15.1

11.1

2.4

3.8

4.8

tert-But

CO₂Et

Thien-3-yl 3-Ph

2-CH₃OPh 3-Ph

3-CH₃OPh 3-Ph

4-CH₃OPh 3-Ph

CH Thien-3-yl

>15.2 >115

CH Thien-3-yl

>1.5

>6.7

>4.4

>2.6

3.1

ND

3.2

3.7

>36

ND

4.3

1.4

7.1

ND

3.2

3.0

ND

1.9

ND

>5.6

1

2.7

7.1

26.7

1.0

ND

1.0

56.8

85.2

>109

63.2

97.4

60.4

29.7

50.2

34.8

69.5

57.5

54.5

45.5

24.7

66.2

56.7

54.9

60.1

95.8

40.3

43.8

41.3

55.1

15

N

Thien-3-yl

tert-But

2-CH₃OPh 3-Ph

3-Ph

H

4-FePh

2-CH₃OPh 3-(2-OHePh)

2-CH₃OPh 3-(3-OHePh)

2-CH₃OPh 3-(4-OHePh)

tert-But

tert-but

tert-But

3-Ph

3-(2-CH₃OPh)

3-(3-CH₃OPh)

3-(4-CH₃OPh)

3-(4-OHePh)

3-(4-ClePh)

3-(4-FePh)

3-(Pyridin-4-yl)

3-(Fur-2-yl)

3-(Fur-3-yl)

3-(Thien-3-yl)

8.5

CH

H

11.5

>100

ND

12.7

5.4

7.0

ND

5.6

7.0

H

CH

H

CH

H

3-(Benzo[b]fur-2-yl)

3-(N-Methylpiperazin-1-yl) ND

ND

2.8

2

14.4

ND

11.4

2.3

4.8

6.3

3

5.2

2.5

3.6

1.4

9.2

20.8

43

>100

4.5

>100

ND

>100

16.2

N

Thien-3-yl

3-(fur-2-yl)

3-(fur-3-yl)

3-(4-OHePh)

3-(3-OHePh)

3-(2-OHePh)

20

75.3

54.3

15.7

22.1

78.5

2-CH₃OPh 3-(fur-2-yl)

2-CH₃OPh 3-(fur-3-yl)

2-CH₃OPh 3-(4-OHePh)

2-CH₃OPh 3-(3-OHePh)

2-CH₃OPh 3-(2-OHePh)

1.0

12

18j

>205.0

ND ¼ not determined.

a

Minimum cytotoxic concentration required to reduce the overall cell metabolism by 50%.

Minimum concentration required to reduce the virus-produced luciferase reporter gene activity by 50%.

Selectivity index (ratio of CC₅₀to EC₅₀).

b

c

CH₃, and the smaller the structure (with the less number of CH with

two aromatic bonds), the lower the EC₅₀. Concerning the molecular

properties, the pKa of ionisable sites should be as “high” as possible.

Concerning the molecular properties counter, the number of

hydrogen bond accepting group should be also as “high” as

possible. The topological descriptor depending on the connectivity

indices: CHI_V_3_P (Kier and Hall valence-modiﬁed connectivity

index amount of branching, ring structures present, and ﬂexibility)

derives from the 2D topology of the molecule. To reduce the EC₅₀, it

should be as high as possible. “Dipole” 3D descriptors indicate the

strength and the orientation behaviour of a molecule in an elec-

trostatic ﬁeld, and here, the higher is the value, the lower is EC₅₀

.

The equations showed occurrences of 3D Jurs descriptors suggest-

ing the importance of charge distribution (Jurs_RNCG showed that

the relative negative charge should be as small as possible) and

surface areas (Jurs_RNCS and Jurs_WNSA_3 dependant on the total

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

453

Several key features of the 3D-QSAR contour map are predicted

to increase BVDV antiviral activity:

N

F

N

S

- a more positive environment (as alkyl group) at C-7 but also C-

6 (instead of an aromatic substituent in site I) (electronic study)

- reinforce negative environment on para position of the phenyl

group at the C-2 position (for example, a para diaromatic group

in site II) (electronic study)

4f

- a more negative environment on the meta or para phenyl

group at the C-3 position (electronegative group on para or

meta on the terminal phenyl in site III) (electronic study)

- alkyl group on the position C-7 or on C-6 (in site I) (steric study)

- more bulk group near the meta or para position on the 3⁰-

biphenyl group in site III (steric study)

Scheme 4. Structure of 4f.

molecular solvent-accessible surface area, and both should be small

values, and so suggesting compact structure).

The 3D-QSAR model (Discovery Studio^Ò2.5, Accelrys), deﬁnes

the critical regions (steric or electrostatic) of imidazo[1,2-a]pyridine

or [1,2-b]pyridazine derivatives affecting the cytopathogenic effect

induction by the BVDV. It was a Partial Least Squares (PLS) model

built on 400 independent variables (N ¼ 42, R-square ¼ 0.71). A

contour plot of the electrostatic ﬁeld region favourable (in blue) or

unfavourable (in red) against the BVDV is shown in Fig. 4 with the

visible superposition of one active compound 4f and one inactive

18a. The energy grids corresponding to the favourable (in green) or

unfavourable (orange) steric effects against the BVDV are shown in

Fig. 5 with the superposition of 4f and 18a. A good active derivative

should have strong Van der Waals attraction in the green areas and a

polar group in the blue electrostatic potential areas (which are

dominant near the second phenyl ring substituted on C-3).

4. Conclusions

Using hit Ttou 84 as starting point, a novel series of biphenyl

derivatives of imidazo[1,2-a]pyridine and [1,2-b]pyridazine was

synthesized in an attempt to optimize this compound class for

inhibitory properties on the replication of the bovine viral diar-

rhoea virus and hepatitis C virus.

Following evaluation of 49 analogues in the HCV subgenomic

replicon system, all except one compound have proven to be

aspeciﬁc inhibitors of HCV replication. Only compound 18j dis-

played some properties in the HCV replicon system suggesting

selective inhibition of HCV replication. However, the antiviral ac-

tivity was not very pronounced (60e70% inhibition).

Additional optimization of the biphenylimidazo[1,2-a]pyridine

or [1,2-b]pyridazine scaffold might result in a more potent inhibitor

of HCV replication. Given the fact that no cross-resistance was

observed with BPIP-resistant BVDV, this class of compounds has the

potential to inhibit-HCV replication by a yet unknown mechanism.

Namely, we previously demonstrated that BPIP-analogues could be

further optimized to exert speciﬁc anti-HCV inhibitory activity

[15,16]. One analogue of BPIP is currently under clinical investiga-

tion, i.e. GS9190 [17].

The biphenyl compounds were then evaluated for selective in-

hibition of BVDV (strain NADL)-induced CPE formation as assessed

by the MTS method in MDBK cells. Compound 4f proved to be the

most selective inhibitor of BVDV replication as corroborated by

different methods. Furthermore, 4f did not or only marginally

showed cross-resistance with known inhibitors of the Pestivirus

replication. Previously, we and others reported compounds with

very different chemical structures that all appear to interact with a

single drug-binding pocket within the ﬁnger domain region of the

BVDV RdRp with two separate but potentially overlapping binding

sites rather than two distinct drug-binding pockets [18]. The cross-

resistance proﬁle of 4f might indicate that 4f interact with a

potentially new binding site in the RdRp, different from the binding

site of BPIP [19], VP32947 [20], AG110 [21] or LZ37 [18], or could

have a completely different, potentially unknown, target. However,

selection of 4f resistant virus in conjunction with a thorough geno-

and phenotypic analysis are in order to better understand the

mechanism by which 4f exerts its anti-BVDV and anti-HCV activity.

5. Experimental section

5.1. General remarks

All solvents were anhydrous reagents from commercial sources.

Unless otherwise noted, all chemicals and reagents were obtained

commercially and used without puriﬁcation. NMR spectra were

recorded at 200 MHz (¹H) or 50 MHz (¹³C) on a Bruker DPX instru-

Fig. 2. (A) Effect of 4f on BVDV (NADL)-induced CPE formation in MDBK cells (open

bars), on release of extracellular viral RNA (ﬁlled bars) and on the proliferation of

exponentially growing MDBK cells (line). (B) Inhibitory effect of 4f on infectious virus

yield (open bars).

ment. The chemical shifts are reported in parts per million (ppm, d)

454

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Fig. 3. Line plot of predicted log EC₅₀activity versus log EC₅₀of the BVDV of imidazo[1,2-a]pyridine or [1,2-b]pyridazine derivatives with the regression statistics and the QSAR

equation by genetic function approximation (GFA) process and statistics.

relative to residual deuterated solvent peaks. The possible inversion

of two values in the NMR spectra is expressed by an asterisk. Known

compounds were prepared according to literature procedures: 2-(4-

ﬂuorophenyl)-6-iodoimidazo[1,2-a]pyridine (1) [22], 6-chloro-2-(4-

ﬂuorophenyl)imidazo[1,2-b]pyridazine (5) [23], 2-(4-ﬂuorophenyl)-

6-phenylimidazo[1,2-a]pyridine (2a) [24], 2-(4-ﬂuorophenyl)-6-

morpholinoimidazo[1,2-a]pyridine (2b) [22], 2-(4-ﬂuorophenyl)-6-

(thien-3-yl)imidazo[1,2-a]pyridine (2c) [24], 2-(4-ﬂuorophenyl)-

6-phenylimidazo[1,2-b]pyridazine (6a) [23], 2-(4-ﬂuorophenyl)-6-

(thien-3-yl)imidazo[1,2-b]pyridazine (6c) [23], 2-amino-5-(thien-3-

yl)pyridine (9a) [25], 3-amino-6-(thien-3-yl)pyridazine (9b) [26],

6-(thien-3-yl)imidazo[1,2-a]pyridine (10a) [24].

Fig. 4. Isosurface of the 3D-QSAR model coefﬁcients on Electrostatic Potential (EP) grids with positive electrostatic potential in red solid representation and the negative area is

represented in blue triangle mesh representation for the aligned molecular structures of 42 imidazo[1,2-a]pyridine or [1,2-b]pyridazine derivatives. Derivatives 4f and 18a are

shown in yellow stick and blue stick representation, respectively. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this

article.)

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

455

Fig. 5. Isosurface of the 3D-QSAR model coefﬁcients on Van der Waals (VDW) grids. The green triangle mesh representation indicates positive coefﬁcients; the orange solid surface

indicates negative coefﬁcients. Derivatives 4f and 18a are shown in yellow stick and blue stick representation, respectively. (For interpretation of the references to colour in this

ﬁgure legend, the reader is referred to the web version of this article.)

5.2. Chemistry

7.77 (m, 1H, H-8), 7.70e7.45 (m, 6H, Ph, H-7), 7.23 (t, 2H, J ¼ 8.9 Hz,

FePh-3,5).

5.2.1. 2-(4-Fluorophenyl)-6-phenylsulfanylimidazo[1,2-a]pyridine

(2d)

2-(4-Fluorophenyl)-6-iodoimidazo[1,2-a]pyridine 1 (338 mg,

1 mmol), copper(I) iodide (9.5 mg, 5 mol%), and K₂CO₃(198 mg,

2 mmol) were added to a screw-capped test tube. The tube was

evacuated and backﬁlled with argon. N,N⁰-dimethylethylenedi-

5.2.3. 2-(4-Fluorophenyl)-3-iodo-6-(morpholin-1-yl)imidazo[1,2-

a]pyridine (3b)

Method A (88% yield). Mp 233e234 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.06 (dd, 2H, J ¼ 8.9e5.4 Hz, FePh-2,6), 7.64 (d, 1H,

J ¼ 2.2 Hz, H-5), 7.58 (d, 1H, J ¼ 9.7 Hz, H-8), 7.20 (t, 2H, J ¼ 8.9 Hz,

FePh-3,5), 7.19 (dd, 1H, J ¼ 9.7e2.2 Hz, H-7), 3.96 (t, 4H, J ¼ 4.8 Hz,

mor), 3.19 (t, 4H, J ¼ 4.8 Hz, mor).

amine (16 ml, 15 mol%), thiophenol (132 mg, 1.2 mmol), and iso-

propanol (1 mL) were added successively by syringe at room

temperature. The tube was sealed with a Teﬂon-lined cap and the

reaction mixture was heated at 112 ^ꢂC for 48 h. After cooling at

room temperature, the suspension was diluted with CH₂Cl₂and

ﬁltered on Celite^Ò. The solvent was removed with the aid of a rotary

evaporator to give a residue that was puriﬁed by column chroma-

tography on silica gel eluting with CH₂Cl₂eCH₃OH (98/02 v/v) to

give a white product (83% yield). Mp 134e135 ^ꢂC. ¹H NMR

5.2.4. 2-(4-Fluorophenyl)-3-iodo-6-(thien-3-yl)imidazo[1,2-a]

pyridine (3c)

Method A (95% yield). Mp 246e247 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.45 (m, 1H, H-5), 8.10 (dd, 2H, J ¼ 8.8e5.4 Hz, FePh-2,6),

7.74 (d, 1H, J ¼ 9.3 Hz, H-8), 7.63e7.52 (m, 3H, H-7, th-2,4), 7.46

(m, 1H, th-5), 7.23 (t, 2H, J ¼ 8.8 Hz, FePh-3,5).

(200 MHz, CDCl₃)

d

8.31 (dd, 1H, J ¼ 1.7e0.9 Hz, H-5), 7.95 (dd, 2H,

J ¼ 8.8e5.4 Hz, FePh-2,6), 7.82 (s, 1H, H-3), 7.63 (d, 1H, J ¼ 9.4 Hz,

5.2.5. 2-(4-Fluorophenyl)-3-iodo-6-(phenylsulfanyl)imidazo[1,2-a]

pyridine (3d)

H-8), 7.35e7.27 (m, 5H, Ph), 7.23 (dd, 1H, J ¼ 9.4e1.7 Hz, H-7), 7.17

(t, 2H, J ¼ 8.8 Hz, FePh-3,5). ¹³C NMR (50 MHz, CDCl₃)

d

163.4

Method A. The residue was puriﬁed by chromatography (neutral

(J ¼ 245 Hz), 146.2, 145.2, 136.5, 130.5, 130.2 (J ¼ 1 Hz), 129.9, 129.8,

129.2, 128.4 (J ¼ 8.1 Hz), 127.6, 119.9, 118.3, 116.3 (J ¼ 21.5 Hz), 108.7.

alumina, CH₂Cl₂) to obtain 3d in 58% yield. Mp 173e175 ^ꢂC. ¹H NMR

(200 MHz, CDCl₃)

FePh-2,6), 7.61 (d, 1H, J ¼ 9.4 Hz, H-8), 7.37e7.26 (m, 5H, Ph), 7.29

(d, 1H, J ¼ 9.4 Hz, H-7), 7.22 (t, 2H, J ¼ 8.7 Hz, FePh-3,5).

d

8.42 (s, 1H, H-5), 8.08 (dd, 2H, J ¼ 8.7e5.4 Hz,

5.2.2. 2-(4-Fluorophenyl)-3-iodo-6-phenylimidazo[1,2-a]pyridine

(3a)

Method A: A mixture of the conveniently substituted 2-(4-

ﬂuorophenyl)imidazo[1,2-a]pyridine (0.4 mmol), N-iodosuccini-

mide (0.6 mmol), and acetonitrile (1 mL) was stirred 3 h at room

temperature. The resulting solid was ﬁltered off and washed with

10 mL of acetonitrile. The compound was obtained in 77% yield and

used without further puriﬁcation. Mp > 260 ^ꢂC. ¹H NMR (200 MHz,

5.2.6. 3-(Biphen-3-yl)-2-(4-ﬂuorophenyl)-6-phenylimidazo[1,2-a]

pyridine (4a)

Method B: A screw-cap test tube was charged, under argon, with

the conveniently substituted 3-iodoimidazo[1,2-a]pyridine or 3-(3-

bromophenyl)imidazo[1,2-a]pyridine or 3-(3-bromophenyl)imidazo

[1,2-b]pyridazine (0.5 mmol), biphenylboronic acid (0.6 mmol),

tetrakis(triphenylphosphine)palladium (2 mol %), and Na₂CO₃

CDCl₃)

d

8.42 (m, 1H, H-5), 8.12 (dd, 2H, J ¼ 8.9e5.4 Hz, FePh-2,6),

456

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

(1.05 mmol) in 1,2-dimethoxyethane (2 mL) and water (1 mL). The

screw-cap test tube was sealed with a cap and the reaction mixture

was stirred magnetically at 100 ^ꢂC for 4 h. After cooling, the sus-

pension was taken up in water, extracted with CH₂Cl₂three times.

The organic layer was dried with MgSO₄, and evaporated to dryness.

The residue was chromatographed on neutral alumina eluting with

CH₂Cl₂to give 98% of 4a. Mp 153e154 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

5.2.11. 3-(Biphen-3-yl)-2-(4-ﬂuorophenyl)-6-(phenylsulfanyl)

imidazo[1,2-a]pyridine (4f)

Method B. The residue was chromatographed on silica gel eluting

with CH₂Cl₂epetroleum spirit (50/50 v/v) to give 4f (52% yield). Mp

153e154 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d 8.22 (s, 1H, H-5), 7.80e7.66

(m, 6H, FePh-2,6, biPh), 7.62e7.41 (m, 6H, H-8, biPh), 7.30e7.20 (6H,

H-7, Ph), 7.04 (t, 2H, J ¼ 8.7 Hz, FePh-3,5). ¹³C NMR (50 MHz, CDCl₃)

d

8.20 (dd, 1H, J ¼ 1.8e1 Hz, H-5), 7.84 (dd, 1H, J ¼ 9.3e1 Hz, H-8),

d

162.7 (J ¼ 246 Hz), 143.8, 142.9, 140.2, 136.0, 130.3, 130.1, 129.9

7.82e7.69 (m, 5H, FePh-2,6, biPh), 7.63e7.30 (m, 12H, H-7, Ph, biPh),

(J ¼ 8 Hz), 129.6, 129.4, 129.3, 129.2, 129.0, 128.2, 127.9, 127.2, 127.0,

126.4, 121.2, 119.5, 117.9, 115.5 (J ¼ 21 Hz). Anal. Calcd for C₃₁H₂₁FN₂S:

C, 78.79; H, 4.48; N, 5.93. Found: C, 78.97; H, 4.49; N, 6.04.

7.05 (t, 2H, J ¼ 8.9 Hz, FePh-3,5). ¹³C NMR (50 MHz, CDCl₃)

d 163.1

(J ¼ 246 Hz), 144.5, 143.4, 142.8, 140.6, 137.9, 133.7, 130.8, 130.5, 130.3

(J ¼ 8 Hz), 130.0, 129.7, 129.5, 128.6, 128.5, 128.4, 127.9, 127.7, 127.5,

126.6, 121.1, 117.8, 115.9 (J ¼ 21.3 Hz). Anal. Calcd for C₃₁H₂₁FN₂: C,

84.52; H, 4.81; N, 6.36. Found: C, 84.77; H, 4.62; N, 6.51.

5.2.12. 2-(4-Fluorophenyl)-6-(morpholin-1-yl)imidazo[1,2-b]

pyridazine (6b)

A

mixture of 6-chloro-2-(4-ﬂuorophenyl)imidazo[1,2-b]pyr-

5.2.7. 3-(Biphen-4-yl)-2-(4-ﬂuorophenyl)-6-phenylimidazo[1,2-a]

pyridine (4b)

idazine (4 mmol) and morpholine (10 mL) was reﬂuxed for 24 h. After

cooling, the mixture was diluted with water (50 mL), the resulting

solid wasﬁltered offand washed withwatertogive6b(87%yield). Mp

Method B. The residue was chromatographed on silica gel

eluting with CH₂Cl₂eCH₃OH (99.5/0.5 v/v) to give 4b (98% yield).

202e203 ^ꢂC.¹HNMR (200 MHz, CDCl₃)

d 7.98 (s,1H, H-3), 7.91(dd, 2H,

Mp 215e216 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.23 (m, 1H, H-5),

J ¼ 8.7e5.4 Hz, FePh-2,6), 7.79 (d, 1H, J ¼ 9.8 Hz, H-8), 7.16 (t, 2H,

7.86e7.71 (m, 7H, FePh-2,6, H-8, biPh), 7.62e7.41 (m, 11H, Ph, biPh,

J¼ 8.7 Hz, FePh-3,5), 6.84(d,1H, J ¼ 9.8Hz, H-7), 3.90(t, 4H, J¼ 4.9Hz,

H-7), 7.05 (t, 2H, J ¼ 8.7 Hz, FePh-3,5). ¹³C NMR (50 MHz, CDCl₃)

mor), 3.52 (t, 4H, J ¼ 4.9 Hz, mor). ¹³C NMR (50 MHz, CDCl₃)

d 163.0

d

163.0 (J ¼ 245 Hz), 144.8, 142.8, 142.3, 140.6, 138.0, 131.6, 130.8

(J ¼ 245 Hz, FePh-4), 155.4 (C-6), 143.6 (C-2), 137.2 (C-8a), 130.4

(FePh-1), 127.6 (J ¼ 8 Hz, FePh-2,6), 125.9 (C-8), 116.1 (J ¼ 21 Hz,

FePh-3,5), 112.8 (C-3), 110.2 (C-7), 66.9 (mor), 46.9 (mor).

(J ¼ 3.3 Hz), 130.4 (J ¼ 8 Hz), 129.7, 129.6, 128.9, 128.9, 128.4, 127.6,

126.3, 121.5, 121.1, 117.9, 115.9 (J ¼ 21 Hz). Anal. Calcd for C₃₁H₂₁FN₂:

C, 84.52; H, 4.81; N, 6.36. Found: C, 84.38; H, 5.02; N, 6.39.

5.2.13. 2-(4-Fluorophenyl)-6-(N-methylpiperazin-1-yl)imidazo[1,2-

5.2.8. 3-(Biphen-3-yl)-2-(4-ﬂuorophenyl)-6-(morpholin-1-yl)

imidazo[1,2-a]pyridine (4c)

b]pyridazine (6d)

A

mixture of 6-chloro-2-(4-ﬂuorophenyl)imidazo[1,2-b]pyr-

Method B. The residue was chromatographed on silica gel

eluting with CH₂Cl₂eCH₃OH (99.5/0.5 v/v) to give 4c (49% yield).

idazine (4 mmol) and N-methylpiperazine (25 mL) was reﬂuxed for

24 h. After cooling, the mixture was diluted with water (150 mL), the

resulting solid was ﬁltered off and washed with water to give 6d (78%

Mp 198e199 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d 7.77 (m, 1H, biPh),

7.72e7.57 (m, 7H, FePh-2,6, H-8, biPh), 7.53e7.41 (m, 5H, H-5,

biPh), 7.17 (dd,1H, J ¼ 9.7e2.2 Hz, H-7), 7.01 (t, 2H, J ¼ 8.8 Hz, FePh-

3,5), 3.87 (m, 4H, mor), 3.04 (m, 4H, mor). ¹³C NMR (50 MHz, CDCl₃)

yield). Mp 190e191 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d 7.96 (s, 1H, H-3),

7.90(dd,2H, J¼ 9e5.4Hz, FePh-2,6), 7.74(d,1H,J¼ 9.9Hz,H-8), 7.15(t,

2H, J ¼ 9 Hz, FePh-3,5), 6.85 (d, 1H, J ¼ 9.9 Hz, H-7), 3.57 (m, 4H, pip),

d

162.4 (J ¼ 245 Hz), 142.8, 142.0, 141.7, 140.3, 130.6, 130.5

2.59 (m, 4H, pip), 2.40 (s, 3H, CH₃). ¹³C NMR (50 MHz, CDCl₃)

d 162.9

(J ¼ 3.5 Hz), 130.3, 129.8, 129.7, 129.5 (J ¼ 7.6 Hz), 129.4, 129.2, 129.1,

128.0, 127.8, 127.2, 121.4, 117.6, 115.4 (J ¼ 21.3 Hz), 108.9, 66.8, 50.7.

Anal. Calcd for C₂₉H₂₄FN₃O: C, 77.49; H, 5.38; N, 9.35. Found: C,

77.86; H, 5.16; N, 9.36.

(J ¼ 246 Hz, FePh-4), 155.3 (C-6), 143.3 (C-2), 137.1 (C-8a), 130.5

(J ¼ 3 Hz, FePh-1), 127.5 (J ¼ 8 Hz, FePh-2,6), 125.7 (C-8), 116.0

(J¼ 21 Hz, FePh-3,5),112.8(C-3),110.6(C-7), 56.8(pip), 46.5(pip, CH₃).

5.2.14. 2-(4-Fluorophenyl)-3-iodo-6-phenylimidazo[1,2-b]

pyridazine (7a)

5.2.9. 3-(Biphen-4-yl)-2-(4-ﬂuorophenyl)-6-(morpholin-1-yl)

imidazo[1,2-a]pyridine (4d)

Method A (96% yield). Mp 265e266 ^ꢂC. ¹H NMR (200 MHz,

Method B. The residue was chromatographed on silica gel

eluting with CH₂Cl₂eCH₃OH (99.5/0.5 v/v) to give 4d (63% yield).

CDCl₃)

d

8.22e8.10 (m, 4H, Ph-2,6, FePh-2,6), 8.02 (d, 1H, J ¼ 9.4 Hz,

H-8), 7.65 (d, 1H, J ¼ 9.4 Hz, H-7), 7.63e7.58 (m, 3H, Ph-3,4,5), 7.25

Mp 191e192 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d 7.85e7.65 (m, 7H, Fe

(t, 2H, J ¼ 8.7 Hz, FePh-3,5).

Ph-2,6, H-8, biPh), 7.58e7.30 (m, 6H, H-5, biPh), 7.18 (dd, 1H,

J ¼ 9.6e2.2 Hz, H-7), 7.03 (t, 2H, J ¼ 8.9 Hz, FePh-3,5), 3.89 (m, 4H,

5.2.15. 2-(4-Fluorophenyl)-3-iodo-6-(morpholin-1-yl)imidazo[1,2-

b]pyridazine (7b)

mor), 3.05 (m, 4H, mor). ¹³C NMR (50 MHz, CDCl₃)

d 162.7

(J ¼ 245 Hz), 142.3, 141.9, 140.5, 140.4, 131.2, 130.7, 130.0 (J ¼ 8 Hz),

129.4,129.0,128.6,128.3,127.4,121.8,121.5,117.8,115.6 (J ¼ 21.5 Hz),

109.1, 67.0, 51.0. Anal. Calcd for C₂₉H₂₄FN₃O: C, 77.49; H, 5.38; N,

9.35. Found: C, 77.36; H, 5.49; N, 9.28.

Method A (93% yield). Mp 235e236 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.11 (dd, 2H, J ¼ 8.9e5.4 Hz, FePh-2,6), 7.73 (d, 1H,

J ¼ 9.8 Hz, H-8), 7.19 (t, 2H, J ¼ 8.9 Hz, FePh-3,5), 6.88 (d, 1H,

J ¼ 9.8 Hz, H-7), 3.92 (4H, mor), 3.61 (4H, mor).

5.2.10. 3-(Biphen-3-yl)-2-(4-ﬂuorophenyl)-6-(thien-3-yl)imidazo

[1,2-a]pyridine (4e)

5.2.16. 2-(4-Fluorophenyl)-3-iodo-6-(thien-3-yl)imidazo[1,2-b]

pyridazine (7c)

Method B. (95% yield). Mp 164e166 ^ꢂC. ¹H NMR (200 MHz,

Method A (78% yield). Mp > 260 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

CDCl₃)

d

8.21 (dd, 1H, J ¼ 0.7 Hz, H-5), 7.78e7.71 (m, 5H, FePh-2,6,

d

8.47 (m, 1H, Th-2), 8.20 (d, 1H, J ¼ 9.6 Hz, H-8), 8.19 (m, 2H, FePh-

H-8, biPh), 7.61e7.58 (m, 3H, th-2,4, biPh), 7.53e7.43 (m, 7H, H-7,

biPh), 7.29 (m, 1H, th-5), 7.04 (t, 2H, J ¼ 8.8 Hz, FePh-3,5). ¹³C NMR

2,6), 7.92 (d, 1H, J ¼ 9.6 Hz, H-7), 7.90e7.79 (m, 2H, Th-4,5), 7.40 (t,

2H, J ¼ 8.9 Hz, FePh-3,5).

(50 MHz, CDCl₃)

d

162.6 (J ¼ 245 Hz), 144.0, 142.9, 142.0, 140.2,

138.3, 130.3, 130.2, 130.1 (J ¼ 3 Hz), 129.8 (J ¼ 8 Hz), 129.5, 129.0,

128.0, 127.9, 127.2, 127.1, 125.9, 125.5, 122.3, 121.2, 121.0, 119.9, 117.5,

115.4 (J ¼ 21 Hz). Anal. Calcd for C₂₉H₁₉FN₂S: C, 78.00; H, 4.29; N,

6.27. Found: C, 77.82; H, 4.59; N, 6.14.

5.2.17. 2-(4-Fluorophenyl)-3-iodo-6-(N-methylpiperazin-1-yl)

imidazo[1,2-b]pyridazine (7d)

Method A (62% yield). Mp 234e235 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

8.11 (dd, 2H, J ¼ 8.7e5.4 Hz, FePh-2,6), 7.69 (d, 1H, J ¼ 9.7 Hz, H-8),

d

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

457

7.19 (t, 2H, J ¼ 8.7 Hz, FePh-3,5), 6.89 (d, 1H, J ¼ 9.7 Hz, H-7), 3.67

J ¼ 3e1.3 Hz, Th-2), 7.79 (dd, 2H, J ¼ 8.8e5.4 Hz, FePh-2,6), 7.76e

7.68 (m, 3H, th-4, biPh), 7.63e7.58 (m, 3H, biPh), 7.55 (d, 1H,

J ¼ 9.4 Hz, H-7), 7.51e7.35 (m, 4H, Th-5, biPh), 7.10 (t, 2H, J ¼ 8.8 Hz,

(m, 4H, pip), 2.61 (m, 4H, pip), 2.45 (s, 3H, CH₃).

5.2.18. 3-(Biphen-3-yl)-2-(4-ﬂuorophenyl)-6-phenylimidazo[1,2-b]

pyridazine (8a)

FePh-3,5). ¹³C NMR (50 MHz, CDCl₃)

d

162.8 (J ¼ 246 Hz), 154.3,

147.6, 142.8, 141.5, 140.7, 138.2, 138.1, 130.3 (J ¼ 8.1 Hz), 129.3, 129.2,

129.1, 128.9, 127.6, 127.4, 127.1, 127.0, 126.1, 125.3, 124.9, 124.7, 116.5,

115.6 (J ¼ 21 Hz). Anal. Calcd for C₂₈H₁₈FN₃S: C, 75.15; H, 4.05; N,

9.39. Found: C, 74.98; H, 4.13; N, 9.46.

Method B (75% yield). Mp 190e191 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.21 (d, 1H, J ¼ 9.5 Hz, H-8), 8.04e7.99 (m, 3H, Ph-2,6,

biPh), 7.83 (dd, 2H, J ¼ 8.9e5.4 Hz, FePh-2,6), 7.75e7.35 (m, 12H,

H-7, Ph-3,4,5, biPh), 7.12 (t, 2H, J ¼ 8.9 Hz, FePh-3,5). ¹³C NMR

(50 MHz, CDCl₃)

d

162.9 (J ¼ 246 Hz),151.4, 142.8,141.6, 140.8,138.2,

5.2.24. 3-(Biphen-4-yl)-2-(4-ﬂuorophenyl)-6-(thien-3-yl)imidazo

[1,2-b]pyridazine (8g)

135.7, 130.4 (J ¼ 8 Hz), 130.2, 130.1, 129.4, 129.3, 129.1, 129.1, 128.9,

127.6, 127.5, 127.2, 127.0, 125.3, 125.1, 116.5, 115.6 (J ¼ 21 Hz). Anal.

Calcd for C₃₀H₂₀FN₃: C, 81.61; H, 4.57; N, 9.52. Found: C, 81.74; H,

4.33; N, 9.64.

Method B (84% yield). Mp 218e219 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.07 (d, 1H, J ¼ 9.4 Hz, H-8), 7.90 (dd, 1H, J ¼ 3e1.3 Hz, Th-

2), 7.82e7.73 (m, 8H, FePh-2,6, biPh), 7.70 (dd, 1H, J ¼ 5.1e1.3 Hz,

Th-4), 7.58e7.43 (m, 3H, biPh), 7.52 (d, 1H, J ¼ 9.4 Hz, H-7), 7.46 (dd,

1H, J ¼ 5.1e3 Hz, Th-5), 7.10 (t, 2H, J ¼ 8.8 Hz, FePh-3,5). ¹³C NMR

5.2.19. 3-(Biphen-4-yl)-2-(4-ﬂuorophenyl)-6-phenylimidazo[1,2-b]

pyridazine (8b)

(50 MHz, CDCl₃)

d

163.1 (J ¼ 246 Hz), 147.8, 143.1, 141.4, 140.8, 138.5,

Method B (98% yield). Mp 233e234 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

131.1, 130.7, 130.6 (J ¼ 8 Hz), 129.3, 128.0, 127.6, 127.5, 127.3, 126.5,

125.6, 125.1, 116.7, 115.9 (J ¼ 21 Hz). Anal. Calcd for C₂₈H₁₈FN₃S: C,

75.15; H, 4.05; N, 9.39. Found: C, 75.26; H, 4.03; N, 9.24.

d

8.13 (d, 1H, J ¼ 9.5 Hz, H-8), 8.01 (m, 2H, Ph-2,6), 7.85e7.73 (m, 8H,

biPh, FePh-2,6), 7.62 (d, 1H, J ¼ 9.5 Hz, H-7), 7.57e7.43 (m, 6H, Ph-

3,4,5, biPh), 7.11 (t, 2H, J ¼ 8.8 Hz, FePh-3,5). ¹³C NMR (50 MHz,

CDCl₃)

d

162.5 (J ¼ 246 Hz), 151.7, 143.3, 141.5, 140.9, 138.6, 136.0,

5.2.25. 3-(Biphen-4-yl)-2-(4-ﬂuorophenyl)-6-(4-methylpiperazin-

1-yl)imidazo[1,2-b]pyridazine (8h)

131.2, 130.7 (J ¼ 7.5 Hz), 130.4, 129.4, 129.3, 128.0, 127.6, 127.5, 127.4,

125.7, 125.3, 116.7, 115.9 (J ¼ 21.5 Hz). Anal. Calcd for C₃₀H₂₀FN₃: C,

81.61; H, 4.57; N, 9.52. Found: C, 81.94; H, 4.41; N, 9.55.

Method B (92% yield). Mp 242e243 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

7.82 (d, 1H, J ¼ 9.8 Hz, H-8), 7.78e7.69 (m, 8H, FePh-2,6,

biPh), 7.51 (m, 2H, biPh), 7.43 (m,1H, biPh), 7.06 (t, 2H, J ¼ 8.8 Hz, Fe

Ph-3,5), 6.91 (d, 1H, J ¼ 9.8 Hz, H-7), 3.56 (t, 2H, J ¼ 4.9 Hz, pip), 2.58

(t, 2H, J ¼ 4.9 Hz, pip), 2.39 (s, 3H, CH₃). ¹³C NMR (50 MHz, CDCl₃)

5.2.20. 3-(Biphen-2-yl)-2-(4-ﬂuorophenyl)-6-(morpholin-1-yl)

imidazo[1,2-b]pyridazine (8c)

Method B (30% yield). Mp 179e180 ^ꢂC. ¹H NMR (200 MHz,

d

162.8 (J ¼ 245 Hz), 154.8, 141.5, 140.8, 136.6, 131.4, 130.9, 130.4

CDCl₃)

d

7.76 (d, 1H, J ¼ 9.8 Hz, H-8), 7.61e7.44 (m, 6H, FePh-2,6,

(J ¼ 8 Hz), 129.2, 128.6, 127.9, 127.4, 127.3, 126.0, 124.7, 115.7

(J ¼ 21 Hz), 110.2, 54.9, 46.4. Anal. Calcd for C₂₉H₂₆FN₅: C, 75.14; H,

5.65; N, 15.11. Found: C, 45.46; H, 5.37; N, 14.82.

biPh), 7.10e6.90 (m, 7H, FePh-3,5, biPh), 6.75 (d, 1H, J ¼ 9.8 Hz, H-

7), 3.79 (m, 4H, mor), 3.33 (m, 4H, mor). ¹³C NMR (50 MHz, CDCl₃)

d

162.6 (J ¼ 245 Hz), 154.7, 143.5, 141.3, 141.0, 135.9, 132.7, 130.7,

130.6, 129.8, 129.3 (J ¼ 8 Hz), 128.3, 128.0, 127.9, 127.1, 125.5, 125.0,

115.5 (J ¼ 21 Hz), 109.6, 66.8, 46.7. Anal. Calcd for C₂₈H₂₃FN₄O: C,

74.65; H, 5.15; N, 12.44. Found: C, 74.52; H, 5.33; N, 12.80.

5.2.26. 2-tert-Butyl-6-(thien-3-yl)imidazo[1,2-a]pyridine (10b)

Method C:

A

mixture of 2-amino-5-(thien-3-yl)pyridine

-halogen-

(2.27 mmol), the conveniently substituted compound

a

ocarbonyle (2.73 mmol), and 1,2-dimethoxyethane (5 mL) was stir-

red overnight at room temperature. The solvent was removed under

reduced pressure, the resulting residue was then dissolved in ethanol

(5 mL) and reﬂuxed overnight. After cooling and concentration, the

residue was suspended in water, make alkaline with Na₂CO₃and

extracted with CH₂Cl₂. After drying with MgSO₄, the organic layers

were evaporated to dryness. The residue was chromatographed on

neutral alumina eluting with CH₂Cl₂to give 10b in 94% yield. Mp

5.2.21. 3-(Biphen-3-yl)-2-(4-ﬂuorophenyl)-6-(morpholin-1-yl)

imidazo[1,2-b]pyridazine (8d)

Method B (48% yield). Mp 125e126 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

7.92 (s, 1H, biPh), 7.90 (d, 1H, J ¼ 9.8 Hz, H-8), 7.72 (dd, 2H,

J ¼ 8.8e5.4 Hz, FePh-2,6), 7.61 (m, 5H, biPh), 7.51e7.38 (m, 3H,

biPh), 7.07 (t, 2H, J ¼ 8.8 Hz, FePh-3,5), 6.91 (d, 1H, J ¼ 9.8 Hz, H-7),

3.85 (m, 4H, mor), 3.50 (m, 4H, mor). ¹³C NMR (50 MHz, CDCl₃)

d

162.9 (J ¼ 246 Hz), 155.0, 141.6, 141.2, 141.1, 136.4, 130.9, 130.4

119e120 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

1H, J ¼ 9.3 Hz, H-8), 7.43e7.12 (m, 5H, Th-2,4,5, H-7, H-3),1.35 (m, 9H,

3CH₃). ¹³C NMR (50 MHz, CDCl₃)

158.0 (C-8a), 144.8 (C-2), 138.6

d 8.06 (m, 1H, H-5), 7.45 (d,

(J ¼ 8 Hz), 129.8, 129.4, 129.2, 127.9, 127.3, 126.1, 125.0, 115.8

(J ¼ 21 Hz),110.2, 66.8, 46.8. Anal. Calcd for C₂₈H₂₃FN₄O: C, 74.65; H,

5.15; N, 12.44. Found: C, 74.71; H, 5.29; N, 12.46.

d

(Th-3), 127.2 (Th-2), 125.9 (Th-5), 124.3 (C-7), 122.4 (C-5), 121.4 (C-6),

120.6 (Th-4), 117.2 (C-8), 107.6 (C-3), 32.7 (C-^tBu), 30.6 (3 CH₃).

5.2.22. 3-(Biphen-4-yl)-2-(4-ﬂuorophenyl)-6-(morpholin-1-yl)

imidazo[1,2-b]pyridazine (8e)

5.2.27. Ethyl6-(thien-3-yl)imidazo[1,2-a]pyridin-2-carboxylate(10c)

Method C (69% yield). Mp 125e126 ^ꢂC. ¹H NMR (200 MHz,

Method B (92% yield). Mp 213e214 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

7.88 (d, 1H, J ¼ 9.9 Hz, H-8), 7.75e7.68 (m, 8H, FePh-2,6,

CDCl₃)

d

8.28 (s, 1H, H-5), 8.15 (s, 1H, H-3), 7.61 (d, 1H, J ¼ 9.5 Hz, H-

biPh), 7.52 (m, 2H, biPh), 7.43 (m, 1H, biPh), 7.07 (t, 2H, J ¼ 8.8 Hz,

FePh-3,5), 6.90 (d, 1H, J ¼ 9.9 Hz, H-7), 3.87 (m, 4H, mor), 3.50 (m,

8), 7.40 (m, 3H, Th-2,4, H-7), 7.27 (dd, 1H, J ¼ 1.2e4.9 Hz, Th-5), 4.38

(q, J ¼ 7.1 Hz, 2H, CH₂),1.37 (t, 3H, J ¼ 7.1 Hz, CH₃). ¹³C NMR (50 MHz,

4H, mor). ¹³C NMR (50 MHz, CDCl₃)

d

162.8 (J ¼ 245 Hz),155.0,141.4,

CDCl₃) d 163.6 (C]O), 144.7 (C-8a), 137.7 (C-2), 137.5 (Th-3), 127.7

141.0, 140.8, 136.6, 131.0, 130.9, 130.4 (J ¼ 8 Hz), 129.3, 128.4, 127.9,

127.4, 127.3, 126.1, 124.8, 115.7 (J ¼ 21 Hz), 110.0, 66.8, 46.8. Anal.

Calcd for C₂₈H₂₃FN₄O: C, 74.65; H, 5.15; N, 12.44. Found: C, 74.80; H,

5.37; N, 12.31.

(Th-2), 126.9 (Th-5), 125.9 (C-7), 123.5 (C-6), 122.9 (C-5), 121.8

(Th-4), 119.1 (C-3), 117.8 (C-8), 61.5 (CH₂), 14.8 (CH₃).

5.2.28. 2,6-di(Thien-3-yl)imidazo[1,2-a]pyridine (10d)

Method C (66% yield). Mp > 300 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

5.2.23. 3-(Biphen-3-yl)-2-(4-ﬂuorophenyl)-6-(thien-3-yl)imidazo

[1,2-b]pyridazine (8f)

d

8.38 (s,1H, H-5), 7.95 (d,1H, J ¼ 2.9 Hz, Th-2), 7.82 (s,1H, H-3), 7.75

(d, 1H, J ¼ 9.5 Hz, H-8), 7.58e7.49 (m, 4H, H-7, Th-2,4, Th-4⁰), 7.45e

Method B (83% yield). Mp 197e198 ^ꢂC. ¹H NMR (200 MHz,

7.37 (m, 1H, Th-5, Th-5⁰). ¹³C NMR (50 MHz, CDCl₃)

d 145.8, 142.2,

CDCl₃)

d

8.07 (d, 1H, J ¼ 9.4 Hz, H-8), 7.95 (m, 1H, BiPh), 7.89 (dd, 1H,

136.6, 127.6, 126.7, 126.3, 126.1, 122.6, 122.4, 121.4, 121.1, 117.4, 113.7.

458

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

5.2.29. 2-(2-Methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-a]pyridine

(10e)

7.60e7.50 (m, 2H, Th-2,5), 7.43 (dd, 1H, J ¼ 5.1e1.2 Hz, Th-4), 4.53

(q, 2H, J ¼ 7.1 Hz, CH₂), 1.54 (t, 3H, J ¼ 7.1 Hz, CH₃).

Method C (54% yield). Mp 94e95 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.48 (d,1H, J ¼ 7.7 Hz, Ph-6), 8.23 (s,1H, H-5), 8.17 (s,1H, H-3), 7.61

5.2.37. 3-Iodo-2,6-di(thien-3-yl)imidazo[1,2-a]pyridine (11d)

Method A (71% yield). Mp > 300 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

(d, 1H, J ¼ 9.3 Hz, H-8), 7.40 (m, 2H, Th-2,4), 7.35 (dd, 1H, J ¼ 9.3e

1.6 Hz, H-7), 7.29 (m, 2H, Th-5, Ph-4), 7.14 (t, 1H, J ¼ 7.7 Hz, Ph-5),

6.98 (d, 1H, J ¼ 7.7 Hz, Ph-3), 3.95 (s, 3H, CH₃). ¹³C NMR (50 MHz,

d

8.41 (s, 1H, H-5), 8.16 (m, 1H, J ¼ 2.9e1.3 Hz, Th-2⁰), 7.95 (m, 1H,

J ¼ 5.0e1.3 Hz, Th-4⁰), 7.69 (d, 1H, J ¼ 9.3 Hz, H-8), 7.59e7.43 (m, 5H,

CDCl₃)

d

157.2, 144.0, 142.0, 138.6, 129.1, 127.3, 126.0, 125.0, 122.7,

Th-5⁰, H-7, Th-2,4,5).

122.5, 121.7, 121.4, 120.8, 117.4, 113.5, 111.3, 55.8.

5.2.38. 3-Iodo-2-(2-methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-a]

pyridine (11e)

5.2.30. 2-(3-Methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-a]pyridine

(10f)

Method A (99% yield). Mp > 95 ^ꢂC degradation. ¹H NMR

Method C (47% yield). Mp 130e131 ^ꢂC. ¹H NMR (200 MHz,

(200 MHz, CDCl₃)

d

8.39 (m,1H, J ¼ 1.7 Hz, H-5), 7.82 (dd,1H, J ¼ 9.3e

CDCl₃)

d

8.32 (dd,1H, J ¼ 1.7e0.9 Hz, H-5), 7.89 (s,1H, H-3), 7.68 (dd,

0.9 Hz, H-8), 7.60 (dd, 1H, J ¼ 9.3e1.7 Hz, H-7), 7.58e7.46 (m, 4H, Th-

2,5, Ph-4,6), 7.44 (dd, 1H, J ¼ 5.0e1.4 Hz, Th-4), 7.12 (td, 1H, J ¼ 6.9e

1 Hz, Ph-5), 7.08 (d, 1H, J ¼ 7.8 Hz, Ph-3), 3.93 (s, 3H, CH₃).

1H, J ¼ 9.4e0.9 Hz, H-8), 7.59 (m, 1H, Ph-2), 7.55 (m, 1H, Ph-6),

7.50e7.34 (m, 5H, H-7, Th-2,4,5, Ph-5), 6.93 (m, 1H, Ph-4), 3.93 (s,

3H, CH₃). ¹³C NMR (50 MHz, CDCl₃)

d 160.5 (Ph-3), 146.5 (C-8a),

145.2 (C-2), 138.4 (Th-3), 135.5 (Ph-1), 130.2 (Ph-5), 127.5 (Th-2),

126.1 (Th-5), 125.4 (C-7), 122.5 (C-5), 122.3 (C-6), 121.1 (Th-4), 118.9

(Ph-6), 117.8 (C-8), 114.6 (Ph-4), 111.3 (Ph-2), 109.2 (C-3), 55.8 (CH₃).

5.2.39. 3-Iodo-2-(3-methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-a]

pyridine (11f)

Method A (99% yield). Mp 168e169 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.42 (m,1H, H-5), 7.83 (d,1H, J ¼ 9.1 Hz, H-8), 7.65e7.40 (m,

5.2.31. 2-(4-Methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-a]pyridine

(10g)

7H, H-7, Th-2,4,5, Ph-2,5,6), 7.02 (m, 1H, Ph-4), 3.96 (s, 3H, CH₃).

Method C (75% yield). Mp 212e213 ^ꢂC. ¹H NMR (200 MHz,

5.2.40. 3-Iodo-2-(4-methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-a]

pyridine (11g)

CDCl₃)

d

8.35 (dd, 1H, J ¼ 1.6e0.9 Hz, H-5), 7.93 (m, 2H, Ph-2,6), 7.84

(s, 1H, H-3), 7.68 (d, 1H, J ¼ 9.3 Hz, H-8), 7.48 (m, 3H, H-7, Th-2,4),

7.38 (dd, 1H, J ¼ 3.7e2.7 Hz, Th-5), 7.02 (m, 2H, Ph-3,5), 3.89 (s, 3H,

Method A (99% yield). Mp 185e186 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d 8.41 (m, 1H, H-5), 7.95 (m, 2H, Ph-2,6), 7.78 (dd, 1H,

CH₃). ¹³C NMR (50 MHz, CDCl₃)

d

160.2 (Ph-4), 145.6 (C-8a), 144.6

J ¼ 9.2e0.8 Hz, H-8), 7.62e7.51 (m, 3H, H-7, Th-2,5), 7.44 (dd, 1H,

(C-2), 138.3 (Th-3), 132.8 (Ph-1), 127.8 (Ph-2,6), 127.5 (Th-2), 126.1

(Th-5), 125.9 (C-7), 122.7 (C-5, C-6), 121.3 (Th-4), 117.1 (C-8), 114.6

(Ph-3,5), 108.1 (C-3), 55.8 (CH₃).

J ¼ 5.0e1.4 Hz, Th-4), 7.07 (m, 2H, Ph-3,5), 3.92 (s, 3H, CH₃).

5.2.41. 3-Iodo-2-tert-butyl-6-(thien-3-yl)imidazo[1,2-b]pyridazine

(11h)

5.2.32. 2-tert-Butyl-6-(thien-3-yl)imidazo[1,2-b]pyridazine (10h)

Method C (82% yield). Mp 97e98 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

Method A (58% yield). Mp 159e160 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

7.97 (dd, 1H, J ¼ 2.9e1.3 Hz, Th-2), 7.91 (d, 1H, J ¼ 9.3 Hz,

d

7.88 (d,1H, J ¼ 9.4 Hz, H-8), 7.82 (dd,1H, J ¼ 2.9e1.3 Hz, Th-2), 7.76

H-8), 7.85 (dd, 1H, J ¼ 5.1e1.3 Hz, Th-4), 7.50 (dd, 1H, J ¼ 5.1e2.9 Hz,

(s, 1H, H-3), 7.66 (dd, 1H, J ¼ 5.1e1.3 Hz, Th-4), 7.42 (dd, 1H, J ¼ 5.1e

Th-5), 7.46 (d, 1H, J ¼ 9.3 Hz, H-7), 1.62 (s, 9H, 3 CH₃).

2.9 Hz, Th-5), 7.32 (d, 1H, J ¼ 9.4 Hz, H-7), 1.44 (s, 9H, 3 CH₃). ¹³

C

NMR (50 MHz, CDCl₃)

d

158.2 (C-6), 147.8 (C-2), 138.7 (C-8a), 138.3

5.2.42. 3-Iodo-2-(2-methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-b]

pyridazine (11i)

(Th-3), 127.5 (Th-5), 126.5 (Th-4), 125.3 (C-8), 124.8 (Th-2), 116.1

(C-7), 112.3 (C-3), 33.1 (C-^tBu), 30.7 (3 CH₃).

Method A (54% yield). Mp 166e167 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.16 (d,1H, J ¼ 9.5 Hz, H-8), 8.05 (m,1H, MeOPh-6), 7.86 (m,

5.2.33. 2-(2-Methoxyphenyl)-6-(thien-3-yl)imidazo[1,2-b]

pyridazine (10i)

1H, Th-2), 7.63 (d, 1H, J ¼ 9.5 Hz, H-7), 7.56e7.48 (m, 3H, MeOPh-4,

Th-4,5), 7.19e7.08 (m, 2H, MeOPh-3,5), 3.95 (s, 3H, CH₃).

Method C. The compound was not isolated and used directly in

reaction.

5.2.43. 3-(Biphen-3-yl)-6-(thien-3-yl)imidazo[1,2-a]pyridine (12a)

Method B (82% yield). Mp ¼ 75e76 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

5.2.34. 3-Iodo-6-(thien-3-yl)imidazo[1,2-a]pyridine (11a)

d

8.59 (s, 1H, H-5), 7.82 (m, 2H, biPh), 7.77 (d, 1H, J ¼ 9.3 Hz, H-8),

Method A (98% yield). Mp 128e129 ^ꢂC. ¹H NMR (200 MHz,

7.73e7.30 (m, 11H, BiPh, H-7, H-2, Th-2,4,5). ¹³C NMR (50 MHz,

CDCl₃) 143.0, 140.8, 138.7, 133.5, 130.2, 129.4, 128.2, 127.6, 127.5,

127.2, 126.3, 125.2, 122.7, 121.4, 120.4, 118.6. Anal. Calcd for

₂₃H₁₆N₂S: C, 78.38; H, 4.58; N, 7.95. Found: C, 78.59; H, 4.61; N, 7.85.

CDCl₃)

d

8.33 (dd, 1H, J ¼ 1.6e0.9 Hz, H-5), 7.74 (s, 1H, H-2), 7.72

d

(dd, 1H, J ¼ 9.3e0.9 Hz, H-8), 7.59e7.50 (m, 3H, Th-2,5, H-7), 7.43

(dd, 1H, J ¼ 4.9e1.4 Hz, Th-5).

C

5.2.35. 3-Iodo-2-tert-butyl-6-(thien-3-yl)imidazo[1,2-a]pyridine

(11b)

5.2.44. 3-(Biphen-3-yl)-2-tert-butyl-6-(thien-3-yl)imidazo[1,2-a]

pyridine (12b)

Method A (65% yield). Mp 129e130 ^ꢂC. ¹H NMR (200 MHz,

Method B (62% yield). Mp 160e161 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

CDCl₃)

(m, 3H, H-7, Th-2,5), 7.29 (dd, 1H, J ¼ 4.7e1.4 Hz, Th-4), 1.56 (m, 9H,

3CH₃). ¹³C NMR (50 MHz, CDCl₃)

157.2 (C-8a), 145.8 (C-2a), 138.5

d

8.31 (m, 1H, H-5), 7.55 (dd, 1H, J ¼ 9.2e0.5 Hz, H-8), 7.36

d

7.83e7.34 (m, 14H, H-7, H-8, Th-2,5, BiPh), 7.20 (dd, 1H, J ¼ 4.8e

1.5 Hz, Th-4), 1.41 (s, 9H, 3CH₃). ¹³C NMR (50 MHz, CDCl₃)

d

153.4,

d

142.8, 142.4, 140.6, 139.0, 132.3, 131.3, 131.1, 130.0, 129.4, 128.2, 127.6,

127.2, 126.3, 124.6, 121.8, 120.9, 120.6, 120.4, 117.3. Anal. Calcd for

C₂₇H₂₄N₂S: C, 79.37; H, 5.92; N, 6.86. Found: C, 79.11; H, 5.97; N, 6.96.

(Th-3), 127.4 (Th-2), 126.2 (Th-5), 125.1 (C-7), 122.8 (C-5), 122.6

(C-6), 121.3 (Th-4), 117.5 (C-8), 57.9 (C-3), 33.8 (Ce^tBu), 30.6 (3 CH₃).

5.2.36. Ethyl 3-iodo-6-(thien-3-yl)imidazo[1,2-a]pyridin-2-

5.2.45. Ethyl 3-(biphen-3-yl)-6-(thien-3-yl)imidazo[1,2-a]pyridine-

2-carboxylate (12c)

carboxylate (11c)

Method A (96% yield). ¹H NMR (200 MHz, CDCl₃)

d

8.43 (s, 1H,

Method B (48% yield). Mp 83e84 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

H-5), 7.83 (d, 1H, J ¼ 9.5 Hz, H-8), 7.65 (dd, 1H, J ¼ 9.5e1.6 Hz, H-7),

d

8.19 (s, 1H, H-5), 7.88e7.40 (m, 13H, H-8, H-7, Th-2,5, BiPh), 7.26

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

459

(dd, 1H, J ¼ 4.4e1.5 Hz, Th-4), 4.40 (q, 2H, J ¼ 7.1 Hz, CH₂), 1.35 (t, 3H,

7.82e7.30 (m, 14H, H-7, Th-4,5, BiPh, MeOPh-4,6), 7.13 (t, 1H,

J ¼ 7.4 Hz, MeOPh-5), 6.96 (d, 1H, J ¼ 8.4 Hz, MeOPh-3), 3.45 (s, 3H,

J ¼ 7.1 Hz, CH₃). ¹³C NMR (50 MHz, CDCl₃)

d 163.6, 144.0, 142.3, 140.7,

138.0, 134.0, 130.0, 129.9, 129.8, 129.3, 128.9, 128.7, 128.1, 127.6, 127.2,

126.1,123.8,121.9,120.7,119.4, 61.4,14.7. Anal. Calcd for C₂₆H₂₀N₂O₂S:

C, 73.56; H, 4.75; N, 6.60. Found: C, 73.84; H, 4.62; N, 6.54.

CH₃). ¹³C NMR (50 MHz, CDCl₃)

d 157.5, 147.9, 142.5, 141.5, 141.3,

138.9, 138.5, 132.5, 130.9, 130.4, 129.4, 129.0, 128.2, 128.1, 127.9,

127.6, 127.5, 126.9, 126.8, 126.7, 126.1, 125.2, 124.5, 121.6, 116.3, 111.8,

55.6. Anal. Calcd for C₂₉H₂₁N₃OS: C, 75.79; H, 4.61; N, 9.14. Found: C,

75.68; H, 4.77; N, 9.19.

5.2.46. 3-(Biphen-3-yl)-2,6-di(thien-3-yl)imidazo[1,2-a]pyridine

(12d)

Method B (81% yield). Mp 90e91 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

5.2.52. 2-Bromo-2-(3-bromophenyl)-4-ﬂuoroacetophenone (14a)

d

8.15 (s, 1H, H-5), 7.84e7.25 (m, 17H, H-8, H-7, Th, Th⁰, BiPh), 7.56e

Method D. A mixture of 2-(3-bromophenyl)-4-ﬂuoroaceto-

7.48 (m, 4H), 7.45e7.36 (m, 4H), 7.30e7.25 (m, 2H). ¹³C NMR

(50 MHz, CDCl₃) 143.7, 142.8, 140.1, 139.8,138.2, 135.1, 130.3, 129.9,

phenone (2.6 g, 8.9 mmol) and 30% aqueous solution of HBr

(0.1 mL) in CHCl₃(25 mL) is added dropwise with bromine (0.46 mL,

8.9 mmol) in CHCl₃(2.5 mL). After stirring at room temperature

overnight, the reaction mixture is washed with a 5% aqueous solu-

tion of sodium thiosulfate and then with water. The organic phase is

dried with MgSO₄, ﬁltered and evaporated to dryness. The residue is

puriﬁed by column chromatography on silica gel eluting with

CH₂Cl₂epetroleum ether (10/90 v/v) to give 14a in 72% yield. Mp

d

129.7, 129.1, 128.3, 128.0, 127.2, 127.1, 127.0, 125.9,125.6,125.5, 123.1,

122.3, 121.0, 120.8, 120.0, 117.2. Anal. Calcd for C₂₇H₁₈N₂S₂: C, 74.62;

H, 4.17; N, 6.45. Found: C, 74.77; H, 4.12; N, 6.76.

5.2.47. 3-(Biphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-3-yl)

imidazo[1,2-a]pyridine (12e)

Method B (36% yield). Mp 110e111 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

102e103 ^ꢂC.¹H NMR (200 MHz, CDCl₃)

FePh-2,6), 7.74 (t, 1H, J ¼ 1.9 Hz, BrePh-2), 7.53e7.48 (m, 2H, BrePh-

4,6), 7.28 (dd,1H, J ¼ 7.7e8 Hz, BrePh-5), 7.18 (t, 2H, J ¼ 8.8 Hz, FePh-

3,5), 6.28 (s, 1H, CH).

d

8.07 (dd, 2H, J ¼ 8.8e5.3 Hz,

d

8.51 (s, 1H, H-5), 7.79 (dd, 1H, J ¼ 9.3e0.8 Hz, H-8), 7.69 (dd, 1H,

J ¼ 7.5e1.7 Hz, MeOPh-6), 7.66e7.34 (m, 13H, H-7, Th-2,5, MeOPh-4,

BiPh), 7.31 (dd, 1H, J ¼ 5.6e1.4 Hz, Th-4), 7.08 (td, 1H, J ¼ 7.5e1.0 Hz,

MeOPh-5), 6.88 (d, 1H, J ¼ 8.2 Hz, MeOPh-3), 3.41 (s, 3H, CH₃). ¹³

C

NMR (50 MHz, CDCl₃)

d

157.1, 144.5, 142.3, 141.9, 141.0, 139.0, 132.5,

5.2.53. 2-Bromo-2-(3-bromophenyl)-2-methoxyacetophenone

131.6, 129.8, 129.3, 128.3, 128.0, 127.5, 127.4, 127.1, 126.3, 125.0,

124.0, 123.4, 122.3, 121.1, 120.1, 118.2, 111.4, 55.2. Anal. Calcd for

(14b)

Method D (56% yield). The compound was not isolated and used

directly in reaction.

C

₃₀H₂₂N₂OS: C, 78.57; H, 4.84; N, 6.11. Found: C, 78.46; H, 4.92; N,

6.31.

5.2.54. 1-Bromo-1-(3-bromophenyl)-3,3-dimethylbutan-2-one

5.2.48. 3-(Biphen-3-yl)-2-(3-methoxyphenyl)-6-(thien-3-yl)

imidazo[1,2-a]pyridine (12f)

(14c)

Method D (93% yield). Oil. ¹H NMR (200 MHz, CDCl₃)

d 7.74 (m,

Method B (16% yield). Mp 127e128 ^ꢂC. ¹H NMR (200 MHz,

1H, BrePh-2), 7.50e7.42 (m, 2H, BrePh-4,6), 7.21 (t, 1H, J ¼ 7.9 Hz,

CDCl₃)

BiPh, MeOPh-2,5,6), 6.86 (m, 1H, MeOPh-4), 3.74 (s, 3H, CH₃). ¹³C

NMR (50 MHz, CDCl₃) 160.0, 144.4, 143.3, 143.1, 140.6, 138.7, 135.8,

d 8.21 (s, 1H, H-5), 7.82e7.75 (m, 17H, H-8, H-7, Th-2,4,5,

BrePh-5), 5.81 (s, 1H, CH), 1.22 (s, 9H, 3 CH₃).

d

5.2.55. 3-(3-Bromophenyl)-2-(4-ﬂuorophenyl)imidazo[1,2-a]

pyridine (15a)

130.8, 130.6, 130.0, 129.8, 129.4, 128.2, 127.5, 127.4, 126.3, 125.5,

122.4, 121.9, 121.2, 120.9, 120.3, 118.0, 114.8, 112.9, 55.5. Anal. Calcd

for C₃₀H₂₂N₂OS: C, 78.57; H, 4.84; N, 6.11. Found: C, 78.80; H, 4.77;

N, 6.28.

Method C (70% yield). Mp 123e124 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

7.99 (d, 1H, J ¼ 7.0 Hz, H-5), 7.73e7.61 (m, 5H, FePh-2,6, H-

8, BrePh-2,4), 7.50e7.37 (m, 2H, BrePh-5,6), 7.27 (ddd, 1H, J ¼ 8.9e

7.0e1.2 Hz, H-7), 7.00 (t, 2H, J ¼ 8.8 Hz, FePh-3,5), 6.82 (t, 1H,

5.2.49. 3-(Biphen-3-yl)-2-(4-methoxyphenyl)-6-(thien-3-yl)

imidazo[1,2-a]pyridine (12g)

J ¼ 7.0e1.0 Hz, H-6). ¹³C NMR (50 MHz, CDCl₃)

163.1 (J ¼ 246 Hz),

d

145.6, 142.7, 133.8, 132.7, 131.8, 134.4 (J ¼ 8 Hz), 130.1, 128.2

(J ¼ 3 Hz), 125.8, 124.1, 123.7, 119.8, 118.2, 115.7 (J ¼ 20 Hz), 113.3.

Method B (50% yield). Mp 205e206 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

MeOPh-2,6, BiPh), 7.29 (m, 1H, Th-4), 6.88 (d, 2H, J ¼ 8.9 Hz,

MeOPh-3,5), 3.84 (s, 3H, CH₃). ¹³C NMR (50 MHz, CDCl₃)

159.7,

d 8.21 (m, 1H, H-5), 7.80e7.40 (m, 15H, H-8, H-7, Th-2,5,

5.2.56. 3-(3-Bromophenyl)-2-(2-methoxyphenyl)imidazo[1,2-a]

d

pyridine (15b)

144.5, 143.4, 143.0, 140.6, 138.8, 131.0, 130.6, 129.9, 129.8, 129.7,

129.4, 128.2, 128.1, 127.6, 127.4, 127.1, 126.2, 125.3, 122.2, 121.1, 121.0,

120.1, 117.7, 114.3, 55.7. Anal. Calcd for C₃₀H₂₂N₂OS: C, 78.57; H,

4.84; N, 6.11. Found: C, 78.62; H, 4.85; N, 6.18.

Method C (17% yield). Oil. ¹H NMR (200 MHz, CDCl₃)

d 8.18 (d, 1H,

J ¼ 6.9 Hz, H-5), 7.71 (d, 1H, J ¼ 8.9 Hz, H-8), 7.64 (dd, 1H, J ¼ 7.5e

1.4 Hz, MeOPh-6), 7.58 (s,1H, BrPh-2), 7.49 (m,1H, BrPh-4), 7.38e7.18

(m, 4H, H-7, BrePh-5,6, MeOPh-4), 7.04 (t, 1H, J ¼ 7.4 Hz, MeOPh-5),

6.86e6.77 (m, 2H, H-6, MeOPh-3), 3.40 (s, 3H, CH₃). ¹³C NMR

5.2.50. 3-(Biphen-3-yl)-2-tert-butyl-6-(thien-3-yl)imidazo[1,2-b]

pyridazine (12h)

(50 MHz, CDCl₃) d 157.0, 145.5, 141.7, 133.5, 132.6, 132.2, 131.4, 131.0,

130.2, 128.4, 125.2, 123.7, 124.5, 123.2, 121.3, 118.5, 113.2, 111.5, 55.3.

Method B (69% yield). Mp 171e172 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.01 (d, 1H, J ¼ 9.4 Hz, H-8), 7.79e7.39 (m, 12H, H-7, Th-2,4, BiPh),

5.2.57. 3-(Biphen-3-yl)-2-(4-ﬂuorophenyl)imidazo[1,2-a]pyridine

(16a)

7.34 (dd, 1H, J ¼ 5.1e2.9 Hz, Th-5), 1.44 (s, 9H, 3 CH₃). ¹³C NMR

(50 MHz, CDCl₃)

d

154.1, 147.5, 141.5, 141.4, 139.0, 136.7, 132.1, 131.4,

Method B (81% yield). Mp 64e65 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

131.2, 129.5, 129.2, 128.1, 127.8, 127.7, 127.2, 126.7, 125.3, 124.7, 115.8,

34.7, 31.8. Anal. Calcd for C₂₆H₂₃N₃S: C, 76.25; H, 5.66; N, 10.26.

Found: C, 76.34; H, 5.74; N, 10.19.

d

8.06 (d, 1H, J ¼ 6.9 Hz, H-5), 7.79e7.71 (m, 5H, FePh-2,6, H-8,

biPh), 7.68e7.58 (m, 3H, BiPh), 7.53e7.40 (m, 4H, BiPh), 7.26 (m, 1H,

J ¼ 9.0e6.9e1.2 Hz, H-7), 7.03 (m, 2H, J ¼ 8.8 Hz, FePh-3,5), 6.79 (td,

1H, J ¼ 6.9e1.2 Hz, H-6). ¹³C NMR (50 MHz, CDCl₃)

d 163.0

5.2.51. 3-(Biphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-3-yl)

imidazo[1,2-b]pyridazine (12i)

(J ¼ 245 Hz), 145.4, 143.2, 142.2, 140.8, 130.9 (J ¼ 3 Hz), 130.8, 130.7,

130.4 (J ¼ 8 Hz), 130.1, 129.8, 129.6, 128.4, 128.3, 127.7, 125.6, 124.0,

121.3, 118.1, 115.9 (J ¼ 21 Hz), 113.1. Anal. Calcd for C₂₅H₁₇FN₂: C,

82.40; H, 4.70; N, 7.69. Found: C, 82.59; H, 4.77; N, 7.58.

Method B (38% yield). Mp 174e175 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.08 (d, 1H, J ¼ 9.4 Hz, H-8), 7.91 (dd, 1H, J ¼ 3.0e1.3 Hz, Th-2),

460

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

5.2.58. 2-(4-Fluorophenyl)-3-(2⁰-methoxybiphen-3-yl)imidazo[1,2-

a]pyridine (16b)

BiPh), 7.57 (dd, 2H, J ¼ 8.8e5.3 Hz, BiPh-2⁰,6⁰), 7.47 (m, 1H, BiPh),

7.26 (dd, J ¼ 9.0e6.8 Hz, H-7), 7.16 (t, 2H, J ¼ 8.8 Hz, BiPh-3⁰-5⁰), 7.02

Method B (89% yield). Mp 146e147 ^ꢂC. ¹H NMR (200 MHz,

(t, 2H, J ¼ 8.8 Hz, FePh-3,5), 6.80 (td, 1H, J ¼ 6.8e1.1 Hz, H-6). ¹³

C

CDCl₃)

d

8.14 (dd, 1H, J ¼ 6.9e1.2 Hz, H-5), 7.81e7.66 (m, 5H, FePh-

NMR (50 MHz, CDCl₃)

d

163.3 (J ¼ 246 Hz), 163.0 (J ¼ 245 Hz), 145.5,

2,6, H-8, BiPh), 7.60 (td, 1H, J ¼ 7.4e0.7 Hz, BiPh-5), 7.43e7.36 (m,

3H, BiPh), 7.26 (m, 1H, J ¼ 9.5e6.8e1.2 Hz, H-7), 7.12e7.00 (m, 4H,

FePh-3,5, BiPh-3⁰,5⁰), 6.81 (td, 1H, J ¼ 6.9e6.8e1.2 Hz, H-6), 3.82 (s,

142.2, 142.1, 136.9 (J ¼ 3 Hz), 130.9, 130.8, 130.3 (J ¼ 8 Hz), 130.0,

129.7, 129.3 (J ¼ 8 Hz), 128.2, 125.5, 123.9, 121.2, 118.1, 116.4

(J ¼ 21 Hz), 115.9 (J ¼ 21 Hz), 113.1. Anal. Calcd for C₂₅H₁₆F₂N₂: C,

78.52; H, 4.22; N, 7.33. Found: C, 78.64; H, 4.18; N, 7.40.

3H, CH₃). ¹³C NMR (50 MHz, CDCl₃)

d

162.9 (J ¼ 245 Hz),156.9,150.2,

145.4, 142.0, 140.4, 132.5, 131.3, 130.9 (J ¼ 3 Hz), 130.6, 130.5, 130.4,

130.1, 129.9, 129.7 (J ¼ 8 Hz), 129.6, 125.5, 124.1, 121.6, 118.0, 115.8

(J ¼ 21 Hz), 112.9, 111.8, 56.1. Anal. Calcd for C₂₆H₁₉FN₂O: C, 79.17; H,

4.86; N, 7.10. Found: C, 79.35; H, 4.92; N, 7.28.

5.2.64. 3-[3-(Pyridin-4-yl)phenyl]-2-(4-ﬂuorophenyl)imidazo[1,2-

a]pyridine (16h)

Method B (81% yield). Mp 119e120 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.59 (d, 2H, J ¼ 5.3 Hz, Py-3,5), 7.96 (dd, 1H, J ¼ 6.8e1.1 Hz,

5.2.59. 2-(4-Fluorophenyl)-3-(3⁰-methoxybiphen-3-yl)imidazo[1,2-

a]pyridine (16c)

H-5), 7.72e7.57 (m, 6H, FePh-2,6, H-8, Ph), 7.47 (dd, 1H, J ¼ 7.4e

1.1 Hz, Ph), 7.41 (d, 2H, J ¼ 5.3 Hz, Py-2,6), 7.16 (ddd, 1H, J ¼ 9.0e

6.8e1.1 Hz, H-7), 6.96 (t, 2H, J ¼ 8.8 Hz, FePh-3,5), 6.71 (t, 1H,

Method B (71% yield). Mp 69e70 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.06 (d,1H, J ¼ 6.9 Hz, H-5), 7.78e7.70 (m, 5H, H-8, FePh-2,6, BiPh),

J ¼ 6.8 Hz, H-6). ¹³C NMR (50 MHz, CDCl₃)

d

162.9 (J ¼ 246 Hz),

7.62 (t, 1H, J ¼ 7.5 Hz, BiPh-5), 7.43 (m, 1H, BiPh-5⁰), 7.35 (d, 1H,

150.9, 147.8, 145.5, 142.4, 140.1, 131.7, 131.2, 131.1, 130.7 (J ¼ 3 Hz),

130.3 (J ¼ 8 Hz), 129.7, 128.1, 125.6, 123.7, 122.1, 120.7, 118.1, 115.9

(J ¼ 21 Hz), 113.2. Anal. Calcd for C₂₄H₁₆FN₃: C, 78.89; H, 4.41; N,

11.50. Found: C, 78.94; H, 4.37; N, 11.58.

J¼ 7.7Hz,BiPh),7.27e6.91(m, 6H, H-7, BiPh-2⁰,4⁰,6⁰, FePh-2,5), 6.77(t,

1H, J ¼ 6.9 Hz, H-6), 3.85 (s, 3H, CH₃).¹³C NMR (50 MHz, CDCl₃)

d 163.0

(J ¼ 245 Hz), 160.6, 145.5, 143.1, 142.3, 142.2, 130.9 (J ¼ 3 Hz), 130.8,

130.7, 130.6, 130.5, 130.4, 130.0 (J ¼ 8 Hz), 128.3, 125.5, 123.9, 121.3,

120.1, 118.1, 115.9 (J ¼ 21 Hz), 113.8, 113.4, 113.1, 55.9. Anal. Calcd for

5.2.65. 3-[3-(Fur-2-yl)phenyl]-2-(4-ﬂuorophenyl)imidazo[1,2-a]

pyridine (16i)

C

₂₆H₁₉FN₂O: C, 79.17; H, 4.86; N, 7.10. Found: C, 79.28; H, 4.85; N, 7.16.

Method B (82% yield). Oil. ¹H NMR (200 MHz, CDCl₃)

d 8.01 (dd,

5.2.60. 2-(4-Fluorophenyl)-3-(4⁰-methoxybiphen-3-yl)imidazo[1,2-

a]pyridine (16d)

1H, J ¼ 6.8e1.2 Hz, H-5), 7.85e7.81 (m, 2H, BiPh), 7.75e7.68 (m, 3H,

H-8, FePh-2,4), 7.58 (dd, 1H, J ¼ 8.6e7.7 Hz, Ph-5), 7.51 (dd, 1H,

J ¼ 1.8e0.7 Hz, Fur-5), 7.33 (dt, 1H, J ¼ 7.7e1.3 Hz, Ph), 7.26 (dd, 1H,

J ¼ 9.1e6.8 Hz, H-7), 7.01 (t, 2H, J ¼ 8.9 Hz, FePh-3,5), 6.79 (td, 1H,

J ¼ 6.8e1.2 Hz, H-6), 6.72 (dd, 1H, J ¼ 3.4e0.7 Hz, Fur-3), 6.52 (dd,

Method B (77% yield). Mp 175e176 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.04 (d, 1H, J ¼ 6.8 Hz, H-5), 7.78e7.67 (m, 5H, H-8, FePh-2,6,

BiPh-2⁰,6⁰), 7.63 (d, 1H, J ¼ 7.5 Hz, BiPh-5), 7.53 (m, 2H, BiPh), 7.43

(dt, 1H, J ¼ 7.5e1.5 Hz, BiPh), 7.24 (ddd, 1H, J ¼ 8.9e6.8e1.2 Hz,

H-7), 7.07e6.96 (m, 4H, FePh-3,5, BiPh-3⁰,5⁰), 6.77 (td, 1H, J ¼ 6.8e

1H, J ¼ 3.4e1.8 Hz, Fur-4). ¹³C NMR (50 MHz, CDCl₃)

d 162.9

(J ¼ 245 Hz), 153.6, 145.4, 143.2, 142.1, 132.9, 130.8, 130.7, 130.3

(J ¼ 8 Hz), 130.2, 126.3, 125.6, 124.9, 124.0, 121.1, 118.0, 115.9

(J ¼ 21 Hz), 113.1, 112.5, 106.5. Anal. Calcd for C₂₃H₁₅FN₂O: C, 77.95;

H, 4.27; N, 7.90. Found: C, 78.04; H, 4.21; N, 7.88.

1.2 Hz, H-6), 3.86 (s, 3H, CH₃). ¹³C NMR (50 MHz, CDCl₃)

d 163.0

(J ¼ 245 Hz), 160.1, 145.4, 142.8, 142.2, 133.2, 130.9 (J ¼ 3 Hz), 130.7,

130.5 (J ¼ 8 Hz), 129.4, 129.3, 128.7, 127.8, 125.5, 124.0, 121.4, 118.1,

115.8 (J ¼ 21 Hz), 114.9, 113.0, 55.9. Anal. Calcd for C₂₆H₁₉FN₂O: C,

79.17; H, 4.86; N, 7.10. Found: C, 79.33; H, 4.91; N, 7.08.

5.2.66. 3-[3-(Fur-3-yl)phenyl]-2-(4-ﬂuorophenyl)imidazo[1,2-a]

pyridine (16j)

5.2.61. 2-(4-Fluorophenyl)-3-(4⁰-hydroxybiphen-3-yl)imidazo[1,2-

a]pyridine (16e)

Method B (94% yield). Mp 121e122 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

7.98 (d, 1H, J ¼ 6.9 Hz, H-5), 7.75e7.64 (m, 5H, FePh-2,6, H-

Method B (65% yield). Mp 262e263 ^ꢂC.¹H NMR (200 MHz, DMSO)

8, Ph), 7.60 (m, 1H, Fur-2), 7.54 (t, 1H, J ¼ 7.7 Hz, Ph-5), 7.48 (m, 1H,

Fur-4), 7.33 (dt, 1H, J ¼ 7.3e1.6 Hz, Ph), 7.21 (ddd, 1H, J ¼ 9.0e6.9e

0.8 Hz, H-7), 6.99 (t, 2H, J ¼ 8.7 Hz, FePh-3,5), 6.75 (t, 1H, J ¼ 6.9 Hz,

H-6), 6.68 (dd, 1H, J ¼ 1.8e0.9 Hz, Fur-5). ¹³C NMR (50 MHz, CDCl₃)

d

9.63 (s, 1H, OH), 8.11 (d, 1H, J ¼ 6.3 Hz, H-5), 7.79e7.53 (m, 8H, H-8,

FePh-2,6, BiPh), 7.41e7.30 (m, 2H, H-7, BiPh), 7.17 (t, 2H, J ¼ 8.5 Hz,

FePh-3,5), 6.95e6.84 (m, 3H, H-6, BiPh-3⁰,5⁰). ¹³C NMR (50 MHz,

DMSO)

d

162.6 (J ¼ 243 Hz),158.5,145.1,142.6,141.4,131.8,131.3,131.1,

d

162.7 (J ¼ 245 Hz), 145.2, 144.4, 142.0, 139.3, 134.3, 130.7, 130.6,

130.8, 130.4 (J ¼ 8 Hz), 129.6, 129.0, 128.9, 127.7, 126.4, 125.0, 121.7,

117.9, 116.8, 116.3 (J ¼ 21 Hz), 113.9. Anal. Calcd for C₂₅H₁₇FN₂O: C,

78.93; H, 4.50; N, 7.36. Found: C, 79.09; H, 4.52; N, 7.28.

130.1 (J ¼ 8 Hz), 129.6, 128.2, 126.8, 126.1, 125.3, 123.7, 120.9, 117.9,

115.7 (J ¼ 21 Hz), 112.8, 109.0. Anal. Calcd for C₂₃H₁₅FN₂O: C, 77.95;

H, 4.27; N, 7.90. Found: C, 78.12; H, 4.28; N, 7.91.

5.2.62. 3-(4⁰-Chlorobiphen-3-yl)-2-(4-ﬂuorophenyl)imidazo[1,2-a]

pyridine (16f)

5.2.67. 3-[3-(Thien-3-yl)phenyl]-2-(4-ﬂuorophenyl)imidazo[1,2-a]

pyridine (16k)

Method B (82% yield). Mp 116e117 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

Method B (89% yield). Mp 159e160 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.01 (d, 1H, J ¼ 6.8 Hz, H-5), 7.74e7.50 (m, 7H, H-8, FePh-2,6,

d

8.04 (d, 1H, J ¼ 6.9 Hz, H-5), 7.79e7.69 (m, 5H, FePh-2,6, H-8, Ph),

BiPh), 7.48 (d, 2H, J ¼ 8.6 Hz, BiPh-2⁰,6⁰), 7.38 (m, 2H, J ¼ 8.6 Hz,

BiPh-3⁰,5⁰), 7.22 (dd, 1H, J ¼ 9e6.8 Hz, H-7), 6.99 (t, 2H, J ¼ 8.7 Hz,

FePh-3,5), 6.76 (t, 1H, J ¼ 6.8 Hz, H-6). ¹³C NMR (50 MHz, CDCl₃)

7.61 (t, 1H, J ¼ 7.5 Hz, Ph-5), 7.51 (dd, 1H, J ¼ 2.7e1.2 Hz, Th-2), 7.47

(m, 3H, Th-4,5, Ph), 7.28 (ddd, 1H, J ¼ 9.0e6.9e1.3 Hz, H-7), 7.02 (t,

2H, J ¼ 8.9 Hz, FePh-3,5), 6.81 (td, 1H, J ¼ 6.9e1.1 Hz, H-6). ¹³C NMR

d

163.0 (J ¼ 246 Hz), 145.5, 142.3, 142.0, 139.2, 134.5, 131.0, 130.8,

(50 MHz, CDCl₃)

d

162.9 (J ¼ 245 Hz), 145.4, 142.1, 141.8, 137.8, 130.9

130.4 (J ¼ 8 Hz), 130.3, 129.7, 129.6, 128.9, 128.1, 125.6, 123.8, 121.1,

118.1, 115.9 (J ¼ 21 Hz), 113.1. Anal. Calcd for C₂₅H₁₆ClFN₂: C, 75.28;

H, 4.04; N, 7.02. Found: C, 75.41; H, 4.23; N, 6.94.

(J ¼ 3 Hz), 130.8, 130.7, 130.3 (J ¼ 8 Hz), 129.9, 127.5, 127.3, 126.6,

125.5, 123.9, 121.6, 121.2, 118.0, 115.8 (J ¼ 21 Hz), 113.0. Anal. Calcd for

C

₂₃H₁₅FN₂S: C, 74.57; H, 4.08; N, 7.56. Found: C, 74.76; H, 4.16; N, 7.48.

5.2.63. 3-(4⁰-Fluorobiphen-3-yl)-2-(4-ﬂuorophenyl)imidazo[1,2-a]

pyridine (16g)

5.2.68. 3-[3-(Benzo[b]fur-2-yl)phenyl]-2-(4-ﬂuorophenyl)imidazo

[1,2-a]pyridine (16l)

Method B (58% yield). Mp 114e115 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

Method B (76% yield). Mp 121e122 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.04 (d, 1H, J ¼ 6.8 Hz, H-5), 7.76e7.60 (m, 6H, H-8, FePh-2,6,

d 8.06e8.01 (m, 3H, H-5, Ph-2,4), 7.77e7.60 (m, 5H, FePh-2,6, H-8,

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

461

Bzfur-4,7), 7.54 (m, 1H, Ph), 7.44 (dt, 1H, J ¼ 7.7e1.3 Hz, Ph-6), 7.38e

5.2.73. 3-(3-Bromophenyl)-2-(2-methoxyphenyl)-6-(thien-3-yl)

imidazo[1,2-b]pyridazine (17b)

Method C (19% yield). Mp 199e200 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

7.24 (m, 3H, H-7, Bzfur-5,6), 7.09 (d, 1H, J ¼ 0.8 Hz, Fur-3), 7.00 (t, 2H,

J ¼ 8.7 Hz, FePh-3,5), 6.83 (dd, 1H, J ¼ 6.8e1.1 Hz, H-6). ¹³C NMR

(50 MHz, CDCl₃)

d

163.0 (J ¼ 246 Hz), 155.6, 155.4, 145.5, 142.3, 132.6,

d

8.00 (m, 1H, MeOePh-6), 7.99 (d, 1H, J ¼ 9.4 Hz, H-8), 7.84

131.4, 131.0, 131.9, 130.7 (J ¼ 3 Hz),130.3 (J ¼ 8 Hz),129.6,127.4,126.0,

125.7, 125.3, 124.0, 123.8, 121.7, 120.8, 118.1, 115.9 (J ¼ 21 Hz), 113.2,

111.9, 102.9. Anal. Calcd for C₂₇H₁₇FN₂O: C, 80.18; H, 4.24; N, 6.93.

Found: C, 80.46; H, 4.14; N, 7.12.

(dd, 1H, J ¼ 2.8e0.9 Hz, Th-2), 7.71e7.66 (m, 2H, Th-4, BrePh-2),

7.53 (m, 1H, Th-5), 7.49e7.37 (m, 4H, H-7, BrePh-4,6, MeOPh-4),

7.23 (t, 1H, J ¼ 7.9 Hz, BrePh-5), 7.11 (t, 1H, J ¼ 7.4e7.1 Hz,

MeOPh-5), 6.91 (d, 1H, J ¼ 8.2 Hz, MeOPh-3), 3.43 (s, 3H, CH₃). ¹³

C

NMR (50 MHz, CDCl₃) d 157.2, 147.9, 142.5, 138.6, 132.6, 132.4, 132.0,

5.2.69. 3-[3-(4-Methylpiperazin-1-yl)phenyl]-2-(4-ﬂuorophenyl)

imidazo[1,2-a]pyridine (16m)

130.9, 130.7, 130.0,127.8, 127.6, 125.3, 124.0, 122.4, 121.6, 116.6, 111.8,

55.5.

A screw-cap test tube was charged under argon, with 15a

(184 mg, 0.5 mmol), Pd₂(dba)₃(2.3 mg, 0.5 mol %), BINAP (5 mg,

1.5 mol %), and NaO^tBu (67 mg, 0.7 mmol). A Teﬂon septum was

attached, and the tube was evacuated and backﬁlled with argon.

The evacuated/backﬁeld sequence was repeated an additional time.

5.2.74. 3-(3-Bromophenyl)-2-tert-butyl-6-(thien-3-yl)imidazo[1,2-

b]pyridazine (17c)

Method C (64% yield). Mp 135e136 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

7.84 (d, 1H, J ¼ 9.4 Hz, H-8), 7.60e7.57 (m, 2H, Th-2, BrPh-

Then, N-methylpiperazine (67

ml, 0.6 mmol) and toluene (2 mL)

2), 7.52 (dd, 1H, J ¼ 7.3e1.7 Hz, BrPh-6), 7.35 (dd, 1H, J ¼ 5.0e1.2 Hz,

Th-4), 7.33e7.24 (m, 3H, H-7, BrPh-4,5), 7.18 (dd, 1H, J ¼ 5.0e2.9 Hz,

were added by syringe under argon. The screw-cap test tube was

sealed with a cap and the reaction mixture was stirred magnetically

at 80 ^ꢂC for 6 days. After cooling, the suspension was taken up in

water, extracted with dichloromethane three times. The organic

layer was dried with MgSO₄, and evaporated to dryness. The res-

idue was chromatographed on neutral alumina with CH₂Cl₂to give

Th-5), 1.31 (s, 9H, 3 CH₃). ¹³C NMR (50 MHz, CDCl₃)

d 153.4, 146.7,

137.9, 135.9, 134.5, 133.0, 131.4, 130.5, 129.4, 126.4, 125.7, 124.5,

124.1, 122.9, 121.8, 115.3, 33.8, 31.0.

5.2.75. 3-[3-(Fur-2-yl)phenyl]-2-tert-butyl-6-(thien-3-yl)imidazo

[1,2-b]pyridazine (18a)

81% of 16m. Mp 132e133 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d 7.96 (dd,

1H, J ¼ 6.9e1.3 Hz, H-5), 7.71 (dd, 2H, J ¼ 8.9e5.5 Hz, FePh-2,6),

7.68 (dd, 1H, J ¼ 9 Hz, H-8), 7.44 (t, 1H, J ¼ 7.8 Hz, Ph-5), 7.17 (ddd,

1H, J ¼ 9e6.7e1.3 Hz, H-7), 7.08e6.88 (m, 5H, FePh-3,5, Ph), 6.70

(ddd, 1H, J ¼ 6.9e6.7e1.1 Hz, H-6), 3.20 (t, 4H, J ¼ 5.2e4.9 Hz, Pi-

2,6), 2.54 (t, 4H, J ¼ 5.2e4.9 Hz, Pi-3,5), 2.32 (s, 3H, CH₃). ¹³C

Method B (97% yield). Mp 152e153 ^ꢂC. ¹H NMR (200 MHz, CDCl₃)

d

8.01 (d, 1H, J ¼ 9.4 Hz, H-8), 7.85e7.81 (m, 2H, Ph-2,4), 7.73 (dd, 1H,

J ¼ 2.9e1.2 Hz, Th-2), 7.58 (t, 1H, J ¼ 7.4 Hz, Ph-5), 7.51 (d,1H, J ¼ 1.8 Hz,

Fur-5), 7.49 (dd, 1H, J ¼ 5.1e1.2 Hz, Th-4), 7.42 (d, 1H, J ¼ 9.4 Hz, H-7),

7.39 (d,1H, J ¼ 7.4 Hz, Ph-6), 7.32 (dd,1H, J ¼ 5.1e2.9 Hz, Th-5), 6.73 (d,

1H, J ¼ 3.3 Hz, Fur-3), 6.52 (dd,1H, J ¼ 3.3e1.8 Hz, Fur-4),1.43 (s, 9H, 3

NMR (50 MHz, CDCl₃)

d

162.8 (J ¼ 245 Hz), 152.7, 145.2, 141.7, 131.0,

130.9, 130.9, 130.2 (J ¼ 8 Hz), 125.3, 124.1, 122.0, 121.9, 118.3, 118.0,

CH₃).¹³C NMR (50 MHz, CDCl₃)

d 153.5, 153.2, 146.8, 142.1, 138.1, 135.9,

117.0, 115.8 (J ¼ 21 Hz), 112.9, 55.4, 49.2, 46.6. Anal. Calcd for

131.3, 130.7, 128.4, 127.1, 126.4, 126.0, 124.5, 124.4, 124.0, 123.7, 115.2,

111.6,105.2, 33.9, 31.1. Anal. Calcd for C₂₄H₂₁N₃OS: C, 72.15; H, 5.30; N,

10.52. Found: C, 72.42; H, 5.23; N, 10.62.

C

₂₄H₂₃FN₄: C, 74.59; H, 6.00; N, 14.50. Found: C, 74.41; H, 6.28; N,

14.64.

5.2.70. 3-(2⁰-Hydroxybiphen-3-yl)-2-(2-methoxyphenyl)imidazo

[1,2-a]pyridine (16n)

5.2.76. 3-[3-(Fur-3-yl)phenyl]-2-tert-butyl-6-(thien-3-yl)imidazo

[1,2-b]pyridazine (18b)

Method B (75% yield). Mp 234e235 ^ꢂC. ¹H NMR (200 MHz,

Method B (99% yield). Mp 144e145 ^ꢂC. ¹H NMR (200 MHz,

MeOH)

H-7), 7.07e6.85 (m, 4H), 6.78e6.65 (m, 4H, H-6, BiPh-3⁰,5⁰, MeOPh-

3), 3.22 (s, 3H, CH₃). ¹³C NMR (50 MHz, MeOH)

156.8, 154.1, 144.5,

139.6, 139.5, 131.7, 130.4, 129.8, 129.6, 129.5, 128.9, 128.7, 128.5,

127.8, 127.2, 125.2, 123.6, 123.1, 122.9, 120.5, 119.8, 116.5, 115.8, 112.7,

111.1, 54.4. Anal. Calcd for C₂₆H₂₀N₂O₂: C, 79.57; H, 5.14; N, 8.15.

Found: C, 79.74; H, 5.22; N, 8.37.

d

8.26 (d, 1H, J ¼ 6.9 Hz, H-5), 7.52e7.26 (m, 6H), 7.14 (m, 1H,

CDCl₃)

d

7.96 (d, 1H, J ¼ 9.4 Hz, H-8), 7.79 (s, 1H, Fur-2), 7.68 (dd, 1H,

J ¼ 2.9e1.2 Hz, Th-2), 7.63e7.58 (m, 2H, Ph-2,4), 7.51 (t, 1H,

J ¼ 7.4 Hz, Ph-5), 7.48 (m,1H, Fur-5), 7.44 (dd,1H, J ¼ 5.0e1.2 Hz, Th-

4), 7.37 (dt, 1H, J ¼ 7.4e1.5 Hz, Ph-6), 7.36 (d, 1H, J ¼ 9.4 Hz, H-7),

7.26 (dd, 1H, J ¼ 5.0e2.9 Hz, Th-5), 6.74 (dd, 1H, J ¼ 1.8e0.9 Hz, Fur-

d

4),1.41, (s, 9H, 3 CH₃). ¹³C NMR (50 MHz, CDCl₃)

d 153.2,146.7, 143.6,

138.5, 138.1, 135.9, 132.1, 131.5, 130.4, 129.1, 128.5, 126.4, 126.0,

125.9, 125.8, 124.5, 124.4, 124.0, 115.1, 108.6, 33.9, 31.1. Anal. Calcd

for C₂₄H₂₁N₃OS: C, 72.15; H, 5.30; N, 10.52. Found: C, 72.28; H, 5.29;

N, 10.57.

5.2.71. 3-(3⁰-Hydroxybiphen-3-yl)-2-(2-methoxyphenyl)imidazo

[1,2-a]pyridine (16o)

Method B (75% yield). Mp 274e275 ^ꢂC.¹H NMR (200 MHz, MeOH)

d

8.11 (d, 1H, J ¼ 6.9 Hz, H-5), 7.42 (d, 1H, J ¼ 9.1 Hz, H-8), 7.38e6.97

(m, 9H), 6.85e6.57 (m, 6H), 3.13 (s, 3H, CH₃). ¹³C NMR (50 MHz,

MeOH) 157.9, 157.2, 145.1, 142.5, 142.4, 140.4, 132.3, 131.0, 130.4,

5.2.77. 3-(4⁰-Hydroxybiphen-3-yl)-2-tert-butyl-6-(thien-3-yl)

imidazo[1,2-b]pyridazine (18c)

d

Method B (19% yield). Mp > 300 ^ꢂC. ¹H NMR (200 MHz, DMSO)

130.3, 129.8, 128.3, 128.1, 127.3, 125.8, 124.0, 123.4, 123.3, 121.2, 118.9,

117.2, 115.0, 114.4, 113.4, 111.6, 55.1. Anal. Calcd for C₂₆H₂₀N₂O₂: C,

79.57; H, 5.14; N, 8.15. Found: C, 79.82; H, 5.37; N, 8.12.

d

9.60 (s, 1H, OH), 8.20 (m, 1H, Th-2), 8.17 (d, 1H, J ¼ 9.5 Hz, H-8),

7.77 (d, 1H, J ¼ 9.5 Hz, H-7), 7.77e7.69 (m, 2H, Ph), 7.63 (dd, 1H,

J ¼ 4.9e3 Hz, Th-5), 7.59 (m, 1H, Ph), 7.57 (d, 2H, J ¼ 8.4 Hz, HOPh-

2,6), 7.43e7.39 (m, 2H, Th-4, Ph), 6.86 (d, 1H, J ¼ 8.4 Hz, HOPh-3,5),

5.2.72. 3-(4⁰-Hydroxybiphen-3-yl)-2-(2-methoxyphenyl)imidazo

[1,2-a]pyridine (16p)

1.32 (s, 9H, 3 CH₃). ¹³C NMR (50 MHz, DMSO)

d 157.6, 152.6, 147.1,

140.2, 138.1, 135.7, 131.5, 130.6, 130.0, 129.4, 129.0, 128.0, 126.4,

125.9, 125.3, 124.2, 116.1, 34.1, 31.3. Anal. Calcd for C₂₆H₂₃N₃OS: C,

73.38; H, 5.45; N, 9.87. Found: C, 73.51; H, 5.39; N, 10.02.

Method B (71% yield). Mp 275e276 ^ꢂC. ¹H NMR (200 MHz,

MeOH)

(m, 5H), 6.79e6.54 (m, 5H), 3.08 (s, 3H, CH₃). ¹³C NMR (50 MHz,

MeOH) 157.4, 145.0, 142.4, 132.2, 130.3, 129.8, 128.5, 127.7, 127.2,

d

8.09 (d, 1H, J ¼ 6.9 Hz, H-5), 7.38e7.15 (m, 6H), 7.10e7.00

d

5.2.78. 3-(3⁰-Hydroxybiphen-3-yl)-2-tert-butyl-6-(thien-3-yl)

imidazo[1,2-b]pyridazine (18d)

126.8, 126.1, 124.1, 123.6, 121.1, 117.0, 116.1, 113.6, 111.6, 55.0. Anal.

Calcd for C₂₆H₂₀N₂O₂: C, 79.57; H, 5.14; N, 8.15. Found: C, 79.74; H,

5.07; N, 8.11.

Method B (83% yield). Mp 268e269 ^ꢂC. ¹H NMR (200 MHz,

DMSO) d 9.59 (s, 1H, OH), 8.17 (m, 2H, Th-2, H-8), 7.79e7.10 (m, 10H,

462

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

H-7, Th-4,5, BiPh), 6.79 (d, 1H, J ¼ 8.0 Hz, BiPh-4⁰), 1.33 (s, 9H, 3

131.7, 130.0, 129.7, 129.3, 128.3, 127.5, 126.8, 126.4, 126.3, 125.8,

124.8, 124.6, 122.4, 120.7, 118.2, 116.3, 114.8, 114.2, 111.2, 54.7. Anal.

Calcd for C₂₉H₂₁N₃O₂S: C, 73.24; H, 4.45; N, 8.84. Found: C, 73.38; H,

4.35; N, 8.92.

CH₃). ¹³C NMR (50 MHz, DMSO)

d 158.2, 152.7, 147.1, 141.4, 140.3,

138.1, 135.8, 131.6, 131.0, 130.4, 130.1, 129.1, 128.0, 127.1, 125.9, 125.3,

124.0, 117.7, 116.2, 115.0, 113.7, 34.1, 31.3. Anal. Calcd for

C

9.98.

₂₆H₂₃N₃OS: C, 73.38; H, 5.45; N, 9.87. Found: C, 73.45; H, 5.51; N,

5.2.84. 3-(2⁰-Hydroxybiphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-

3-yl)imidazo[1,2-b]pyridazine (18j)

5.2.79. 3-(2⁰-Hydroxybiphen-3-yl)-2-tert-butyl-6-(thien-3-yl)

imidazo[1,2-b]pyridazine (18e)

Method B (67% yield). Mp 257e258 ^ꢂC. ¹H NMR (200 MHz,

DMSO)

d

9.52 (s, 1H, OH), 8.37 (m, 1H, Th-2), 8.23 (d, 1H, J ¼ 9.4 Hz,

Method B (61% yield). Mp 271e272 ^ꢂC. ¹H NMR (200 MHz,

H-8), 7.85 (d, 1H, J ¼ 9.4 Hz, H-7), 7.71e7.33 (m, 8H), 7.11e6.79 (m,

DMSO)

d

9.69 (s,1H, OH), 8.22 (s,1H, Th-2), 8.16 (d,1H, J ¼ 9.4 Hz, H-

6H, MeOPh-3,4,5, HOPh-3,4,5), 3.29 (s, 3H, CH₃). ¹³C NMR (50 MHz,

8), 7.75 (d, 1H, J ¼ 9.4 Hz, H-7), 7.71e7.55 (m, 4H, Th-5, HOPh-6, Ph-

2,4), 7.49e7.42 (m, 2H, Th-4, Ph-5), 7.35 (d,1H, J ¼ 8.2 Hz, Ph-6), 7.19

(t, 1H, J ¼ 7.2 Hz, HOPh-4), 6.99e6.86 (m, 2H, HOPh-3,5), 1.34 (s, 9H,

DMSO) d 156.9, 154.6, 147.5, 141.3, 138.7, 138.1, 137.5, 131.7, 130.5,

130.2, 129.5, 129.2, 128.9, 128.2, 127.9, 127.8, 126.7, 126.5, 126.1,

126.0, 125.9, 124.1, 120.8, 119.7, 116.6, 116.3, 112.0, 55.0. Anal. Calcd

for C₂₉H₂₁N₃O₂S: C, 73.24; H, 4.45; N, 8.84. Found: C, 73.49; H, 4.51;

N, 8.74.

3CH₃). ¹³C NMR (50 MHz, DMSO)

d 158.7, 152.7, 147.0, 138.6, 138.1,

135.7, 132.6, 130.6, 130.4, 130.0, 129.4, 129.0, 128.2, 128.0, 127.3,

126.0,125.9,125.3,124.3,119.9,116.4,116.1, 34.1, 31.3. Anal. Calcd for

C

₂₆H₂₃N₃OS: C, 73.38; H, 5.45; N, 9.87. Found: C, 73.55; H, 5.28; N,

5.3. Biology

9.91.

5.3.1. Cells and viruses

5.2.80. 3-[3-(Fur-2-yl)phenyl]-2-(2-methoxyphenyl)-6-(thien-3-yl)

imidazo[1,2-b]pyridazine (18f)

MadineDarby bovine kidney (MDBK) cells were grown in MEM

(Gibco) supplemented with 5% heat-inactivated FCS (Integro).

BVDV strain NADL was provided by Dr P Kerkhofs (CODA/SERVA,

Ukkel, Belgium) and Huh-5-2 HCV subgenomic replicon-containing

cells were provided by Prof R Bartenschlager (University of Hei-

delberg, Heidelberg, Germany). BPIP-resistant virus, AG110-

resistant virus, LZ37-resistant virus were selected as described

previously [18,19,21].

Method B (94% yield). Mp ¼ 195e196 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.19 (m, 1H, MeOPh-6), 8.10 (d, 1H, J ¼ 9.4 Hz, H-8), 7.92

(dd,1H, J ¼ 2.9e1.3 Hz, Th-2), 7.76 (dd,1H, J ¼ 5.1e1.3 Hz, Th-4), 7.68

(m, 2H, Fur-5, Ph), 7.51 (d, 1H, J ¼ 9.4 Hz, H-7), 7.56e7.38 (m, 5H,

MeOPh-4, Ph), 7.11 (t, 1H, J ¼ 7.4 Hz, MeOPh-5), 6.93 (d, 1H,

J ¼ 8.2 Hz, MeOPh-3), 6.58 (d, 1H, J ¼ 3.4 Hz, Fur-3), 6.50 (dd, 1H,

J ¼ 3.4e1.8 Hz, Fur-4), 3.46 (s, 3H, CH₃). ¹³C NMR (50 MHz, CDCl₃)

d

156.7, 153.8, 147.2, 141.9, 138.1, 137.7, 131.7, 130.4, 130.0, 129.8,

5.3.2. Anti-BVDV assay

128.2, 127.5, 126.7, 126.0, 125.6, 125.3, 124.5, 124.0, 123.4, 122.8,

120.8, 115.7, 111.6, 111.1, 104.8, 55.9. Anal. Calcd for C₂₇H₁₉N₃O₂S: C,

72.14; H, 4.26; N, 9.35. Found: C, 72.33; H, 4.12; N, 9.41.

Assays were performed as described previously [18,19,21]. In

brief, MDBK cells were seeded at a density of 5 ꢃ 10³per well in 96-

well cell culture plates in MEM-FCS. Following 24 h incubation at

37 ^ꢂC and 5% CO₂, medium was removed and 5-fold serial dilutions

5.2.81. 3-[3-(Fur-3-yl)phenyl]-2-(2-methoxyphenyl)-6-(thien-3-yl)

imidazo[1,2-b]pyridazine (18g)

of the test compounds were added in a total volume of 100 ml, after

which the BVDV inoculum (MOI ¼ 2) was added to each well. This

inoculum resulted in a greater than 90% destruction of the cell

monolayer after 3 days of incubation at 37 ^ꢂC. Uninfected cells and

cells receiving virus without compound were included in each

Method B (99% yield). Mp ¼ 196e197 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

d

8.07 (d, 1H, J ¼ 9.4 Hz, H-8), 7.86 (m, 2H, Th-2, MeOePh-6),

7.73e7.62 (m, 4H, Ph-2,4, Th-4, Fur-2), 7.50e7.40 (m, 6H, H-7, Th-5,

Fur-5, Ph-5,6, MeOPh-4), 7.12 (t, 1H, J ¼ 7.5 Hz, MeOPh-5), 6.93 (d,

assay plate. After 5 days, medium was removed, and 90

ml of MEM-

1H, J ¼ 8.2 Hz, MeOPh-3), 6.60 (s, 1H, Fur-4), 3.44 (s, 3H, CH₃). ¹³

C

FCS supplemented with 10 l of 3-(4,5-dimethylthiazol-2-yl)-5-(3-

m

NMR (50 MHz, CDCl₃)

d

156.7, 147.2, 143.6, 141.5, 138.3, 138.1, 137.7,

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phe-

nazinemethosulfate (MTS/PMS) solution (Promega, Leiden, The

Netherlands) was added to each well. Following a 2 h incubation

period at 37 ^ꢂC, the optical density of each well was read at 490 nm

in a microplate reader (signal to noise ratio ¼ 5). The percentage

CPE was calculated. The 50% effective concentration was derived

from the doseeresponse curves as the concentration of compound

that offered 50% protection of the cells against virus-induced

cytopathic effect and was calculated using linear interpolation.

131.9, 131.7, 130.1, 129.8, 128.3, 127.0, 126.8, 126.2, 126.0, 125.9,

125.3, 124.8, 124.4, 123.7, 120.8, 115.6, 111.1, 108.5, 54.9. Anal. Calcd

for C₂₇H₁₉N₃O₂S: C, 72.14; H, 4.26; N, 9.35. Found: C, 72.46; H, 4.08;

N, 9.52.

5.2.82. 3-(4⁰-Hydroxybiphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-

3-yl)imidazo[1,2-b]pyridazine (18h)

Method B (53% yield). Mp 223e224 ^ꢂC.¹H NMR (200 MHz, DMSO)

d

9.61 (s,1H, OH), 8.42 (m,1H, Ph-6), 8.29 (d,1H, J ¼ 9.5 Hz, H-8), 7.91

(d, 1H, J ¼ 9.5 Hz, H-7), 7.77e7.42 (m, 8H, MeOPh-4, Ph-2,3,4,5, Th-

2,4,5), 7.31 (d, 2H, J ¼ 8.4 Hz, HOPh-2,6), 7.09 (m, 2H, MeOPh-3,5),

6.81(d, 2H, J ¼ 8.4 Hz, HOPh-3,5), 3.35 (s, 3H, CH₃). ¹³C NMR

5.3.3. Anti-HCV assay in Huh 5-2 cells

The anti-HCV assay was performed as described previously

[27,28]. In brief, Huh 5-2 cells were seeded at a density of 5 ꢃ 10³

per well in a 96-well white View plate (Packard, Canberra, Canada)

(50 MHz, DMSO) d 158.2, 157.7, 148.3, 142.2, 140.9, 138.8, 138.4, 132.5,

131.7, 131.0, 130.8, 129.7, 129.1, 128.6, 127.3, 127.1, 126.9, 126.7, 126.4,

126.3,124.9,121.7,117.5,116.8,112.7, 55.8. Anal. Calcd for C₂₆H₂₃N₃OS:

C, 73.24; H, 4.45; N, 8.84. Found: C, 73.46; H, 4.39; N, 8.91.

in complete DMEM supplemented with 250 mg mL G418. Following

incubation for 24 h at 37 ^ꢂC (5% CO₂), medium was removed and 3-

fold serial dilutions in complete DMEM (without G418) of the test

compounds were added in a total volume of 100 ml. After 4 days of

5.2.83. 3-(3⁰-Hydroxybiphen-3-yl)-2-(2-methoxyphenyl)-6-(thien-

3-yl)imidazo[1,2-b]pyridazine (18i)

incubation at 37 ^ꢂC, cell culture medium was removed and lucif-

erase activity was quantiﬁed using the Steady-Glo luciferase assay

system (Promega, Leiden, The Netherlands); the luciferase signal

was measured using a Luminoskan Ascent (Thermo, Vantaa,

Finland). The 50% effective concentration was deﬁned as the con-

centration of compound that reduced the luciferase signal by 50%.

Method B (89% yield). Mp 155e156 ^ꢂC. ¹H NMR (200 MHz,

CDCl₃)

7.61e7.16 (m, 10H), 6.98e6.82 (m, 5H), 3.36 (s, 3H, CH₃). ¹³C NMR

(50 MHz, CDCl₃) 157.3, 156.8, 147.5, 141.9, 140.5, 140.4, 137.6, 137.3,

d

8.08 (d, 1H, J ¼ 9.6 Hz, H-8), 7.76 (m, 2H, MeOPh-6, Th-2),

d

C. Enguehard-Gueifﬁer et al. / European Journal of Medicinal Chemistry 64 (2013) 448e463

463

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5.3.4. Cytotoxicity assay

MDBK or Huh 5-2 cells were seeded at a density of 5 ꢃ 10³cells

per well of a 96-well plate in MEM-FCS; 24 h later, serial dilutions of

the test compounds were added. Cells were allowed to proliferate

for 3 days at 37 ^ꢂC, after which the overall cell metabolic activity,

which is representative for the number of cells, was quantiﬁed by

means of the MTS/PMS method (Promega). The 50% cytostatic

concentration was deﬁned as the concentration that inhibited the

proliferation of exponentially growing cells by 50% and was

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5.3.5. Molecular modelling

Computational results were obtained using software programs

from Accelrys Software Inc. The molecules were built and mini-

mized in molecular package (Discovery Studio^Ò2.5.5, Accelrys, San

Diego, CA) by CHARMm with CFF partial charge estimation method.

The 3D structures were generated and optimized using a CHARMm

forceﬁeld with a root mean squared (RMS) difference of energy

gradient of 0.1 kcal/mol. The GFA model in QSAR protocol was used

with a population size of 100 and 5000 maximum generations, in

linear model form. The descriptors correlation matrix is given in

Supplementary data.

For the 3D-QSAR model, all the structures were superimposed

by choosing imidazo-pyridine/pyridazine scaffold, with N-1, N-4,

and C-7 as tethers to superimpose. The grid spacing was 2.5 A. All

ꢀ

the parameters have been left to the system defaults.

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Clercq, A. Gueifﬁer, J.C. Teulade, P. Herdewijn, G. Puerstinger, F. Koenen,

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of bovine viral diarrhea virus replication that targets the viral RNA-dependent

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Acknowledgement

[19] J. Paeshuyse, P. Leyssen, E. Mabery, N. Boddeker, R. Vrancken, M. Froeyen,

I.H. Ansari, H. Dutartre, J. Rozenski, L.H. Gil, C. Letellier, R. Lanford, B. Canard,

F. Koenen, P. Kerkhofs, R.O. Donis, P. Herdewijn, J. Watson, E. De Clercq,

We thank Carolien De Keyzer, Stijn Delmotte and Katrien Geerts

for excellent technical assistance. The authors thank SAVIT, Tours

(France) for NMR spectrometry. This work was supported by a

postdoctoral fellowship from the Research Foundation Flanders-

FWO to Jan Paeshuyse, the IWT-SBO project #100042, KU Leuven

grant (GOA/10/014) and by grant G.0728.09N of the Research

Foundation Flanders-FWO.

G. Puerstinger, J. Neyts,

A novel, highly selective inhibitor of pestivirus

replication that targets the viral RNA-dependent RNA polymerase, J. Virol. 80

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Appendix A. Supplementary material

Supplementary materials related to this article can be found at

http://dx.doi.org/10.1016/j.ejmech.2013.03.054.

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3-Biphenylimidazo[1,2-a]pyridines or [1,2-b]pyridazines and analogues, novel Flaviviridae inhibitors

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