Design, synthesis, binding and docking-based 3D-QSAR studies of 2-pyridylbenzimidazoles - A new family of high affinity CB1 cannabinoid ligands

Molecules 2013, 18, 3972-4001; doi:10.3390/molecules18043972

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molecules

ISSN 1420-3049

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Article

Design, Synthesis, Binding and Docking-Based 3D-QSAR

Studies of 2-Pyridylbenzimidazoles—A New Family of High

Affinity CB1 Cannabinoid Ligands

Jaime A. Mella-Raipán ¹, Carlos F. Lagos ², Gonzalo Recabarren-Gajardo ²,

Christian Espinosa-Bustos ², Javier Romero-Parra ², Hernán Pessoa-Mahana ³,

Patricio Iturriaga-Vásquez ⁴and Carlos David Pessoa-Mahana ^2,*

1

Departamento de Química y Bioquímica, Facultad de Ciencias, Universidad de Valparaíso,

Casilla 5030, Avda. Gran Bretaña 1111, Playa Ancha, Valparaíso, Chile

2

Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile,

Casilla 306, 22, Santiago, Chile

3

Departamento de Química Orgánica y Físicoquímica, Facultad de Química y Ciencias

Farmacéuticas, Universidad de Chile, Casilla 233, Santiago, Chile

4

Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 233, Santiago, Chile

* Author to whom correspondence should be addressed; E-Mail: cpessoa@uc.cl;

Tel.: +56-2-2354-4041; Fax: +56-2-2354-4744.

Received: 26 February 2013; in revised form: 24 March 2013 / Accepted: 1 April 2013 /

Published: 4 April 2013

Abstract: A series of novel 2-pyridylbenzimidazole derivatives was rationally designed

and synthesized based on our previous studies on benzimidazole 14, a CB1 agonist used as

a template for optimization. In the present series, 21 compounds displayed high affinities

with K_ivalues in the nanomolar range. JM-39 (compound 39) was the most active of the

series (K_iCB1= 0.53 nM), while compounds 31 and 44 exhibited similar affinities to WIN

55212-2. CoMFA analysis was performed based on the biological data obtained and

resulted in a statistically significant CoMFA model with high predictive value (q²= 0.710,

r²= 0.998, r²_pred= 0.823).

Keywords: cannabinoid, CB1 receptor, binding, docking, 3D-QSAR

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1. Introduction

The regulation of the endocannabinoid system (ECS) has been studied extensively because of its

great potential in the treatment of many conditions. CB1 agonists has been associated to the treatment

of pain, [1], cancer [2], and emesis [3] among others. Otherwise, CB1 antagonists have been mainly

used in the treatment of obesity [4,5]. Its physiological effects are mediated through the so-called

cannabinoid receptors, which are attractive targets for current drug development [6,7]. These proteins

belong to the G protein-coupled receptor (GPCR) family and have been the focus of much recent work

since the discovery of the CB1 and CB2 receptor subtypes [8,9]. Among the reported and non-classical

cannabinergic ligands [10–12], several compounds contain a central heterocyclic scaffold such as

indole or benzofuran, displaying a range of different pharmacological profiles [13,14]. For example,

the aminoalkylindole derivative WIN55212-2 has been reported to be a potent agonist of the CB1

receptor (K_i= 9.4 nM) [15], AM679 is a non-selective CB1 and CB2 agonist (K_i= 13.5 nM CB1) [16],

JWH-007 has been reported as a non selective CB1/CB2 cannabinoid agonist (K_i= 9.5 nM) [17], while

the benzofuran derivative LY320135 is a selective CB1 antagonist (K_i= 131 nM) [18,19] (Figure 1).

In the field of cannabimimetic heterocycles, benzimidazoles have been recently reported as CB2

agonist with good CNS penetration by Pfizer [20]. By other hand, Abbott Laboratories have developed

azaindoles with agonist activity over the CB1 and CB2 receptors [21], while imidazopyridines from

Merck have been reported to be selective CB2 agonists [22] (Figure 1).

Figure 1. Previously and recently reported cannabinoid ligands, prototype 14 (JM-6), and

39 (JM-39), the most active compound of the present series. K_iCB1 data for 14 (JM-6) and

39 (JM-39) reported herein.

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For many years our research group has been interested in the synthesis of benzimidazole derivatives

as ligands of the type 1 cannabinoid receptor. In recent screening tests using mice brain synaptosomes [23],

compound 14 (JM-6) was identified as a high affinity CB1 agonist (K_i= 98.2 nM). Considering the

structural resemblance of 14 to the reported ligands (Figure 1), we defined a strategy to develop new

and more active CB1 ligand derivatives. Docking simulation studies of 14 with our reported CB1

model [24] allowed us to propose the existence of three major interaction regions: (a) a hydrogen bond

between the pyridine ring and the quaternary amino group of the side chain of residue K3.28(192);

(b) the naphthalene moiety establishing hydrophobic and aromatic interactions with the side-chain

residues F2.57(170), F2.61(174), L3.32(196), I5.31(267), F7.35(379), A7.36(380); and (c) the benzene

ring of the benzimidazole core which displayed hydrophobic and aromatic interactions with the side

chain residues F2.57(170), F3.36(200), W6.48(356) and L6.51(359).

In order to explore SAR requirements of benzimidazoles and optimize the affinity with the CB1

receptor, compound 14 was subjected to the following changes (Figure 2A): (i) Region I: replacement

of the 3-pyridyl ring by 2- and 4-pyridyl isomers to study the influence of the position of the nitrogen

atom on hydrogen bond interactions. (ii) Region II and linker: the 1-naphthyl moiety was replaced by a

2-naphthyl group and the carbonyl function exchanged for a methylene group aimed at providing an

improved orientation and a more favorable conformation in the receptor cavity. (iii) Region III.

Finally, substitution on the benzimidazole ring (C-5 and C-6) with fluorine and methyl groups was

thought to increase π-stacking interactions. Accordingly, we report the design, synthesis and biological

evaluation of novel series of 1 and 2-naphthyl, and 1 and 2-naphthoyl -2-pyridyl-benzimidazole

derivatives (Figure 2B) targeting the CB1 receptor. Structure-activity relationships were explored by

molecular docking in an updated CB1 receptor model and a 3D-QSAR (CoMFA) was developed using

the docking-based alignment.

Figure 2. A. Regions for modification on compound 14. B. General structure of

synthesized compounds.

2. Results and Discussion

2.1. Chemistry

The synthetic sequence for obtaining the target compounds is shown in Scheme 1. A series

of substituted o-phenylenediamines (benzene-1,2-diamine; 4,5-dimethylbenzene-1,2-diamine and

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4-fluorobenzene-1,2-diamine) were reacted with 2-, 3- and 4-pyridinecarbaldehyde in ethanol in the

presence of cerium (IV) ammonium nitrate (CAN) and H₂O₂as oxidizing agents [25], yielding

benzimidazole derivatives 1–9. The obtained benzimidazoles 1–9 were finally acylated [26,27] with 1-

or 2-naphthoyl chloride or alkylated with 1- or 2-(bromomethyl)naphthalene to yield compounds 10–56.

Scheme 1. Synthesis of Compounds 1–9 and 10–56.

Reagents and Conditions: (i) Ce(NH₄)₂(NO₃)₆(CAN), H₂O₂, 50 °C 1 h. (ii) THF, NaH, 1- or

2-naphthoyl chloride, or 1- or 2-(bromomethyl)naphthalene.

Since the benzimidazole ring exists in tautomeric forms, the acylation or alkylation of 2-pyridin-5-

fluoro-1H-benzo[d]imidazoles generate two regioisomers whose structures have been definitively

assigned by HMBC experiments which showed an HMBC correlation between C-3a (Figure 3) and the

hydrogen at C-7 in the 5-fluoro isomer, and with the hydrogens at C-5 and C-7 in the 6-fluoro isomer.

Figure 3. HMBC correlations in regioisomeric products.

2.2. CB1 Binding Affinities, Docking and SAR

In our efforts to obtain novel CB1 ligands with improved affinities and to gain experimental

evidence concerning the mode of interaction of cannabimimetic benzimidazoles with this receptor, we

have designed and synthesized a series of benzimidazoles 10–56. The CB1 receptor affinity values for

these compounds are shown in Table 1.

Significant affinity (nanomolar range) was observed for 21 of the 47 synthesized compounds, while

another eight compounds had affinities in the micromolar range. Benzimidazoles 23, 36 and 51

exhibited IC₅₀values around 20 nM and four molecules (21, 31, 39, 44) exhibited an exceptional

affinity with IC₅₀values under 10 nM. Docking results showed that all the synthesized compounds

adopt a Y-like conformation within the CB1 active site, with the pyridine and naphthalenyl or

naphthoyl groups as the arms, and the benzimidazole scaffold as the body (Figure 4A,B). The binding

site of the ligands is located amidst transmembrane helices 2, 3, 6 and 7. The binding mode of 39, the

most active compound of the series, is represented in Figure 4C.

Molecules 2013, 18

Table 1. Radioligand displacement results for 2-arylbenzimidazoles 10–56 ^a.

3976

CB1

(K_i,nM)

n.a

Comp. X

Y

Z

R

R¹R²

R³

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

N

CH CH C=O

H

F

H

F

1-naphtyl

n.a

N

H

n.a

N

CH CH C=O CH₃CH₃1-naphtyl

n.a

CH

N

CH C=O

H

F

H

F

1-naphtyl 98.24 ± 20.0

1-naphtyl

534

n.a

H

CH C=O CH₃CH₃1-naphtyl

CH CH

N

C=O

H

F

H

F

1-naphtyl 37.81 ± 5.3

1-naphtyl

n.a

H

C=O CH₃CH₃1-naphtyl 3.95 ± 0.8

N

CH CH C=O

H

F

2-naphtyl

n.a

2-naphtyl 11.41 ± 0.9

N

CH CH C=O CH₃CH₃2-naphtyl 28.08 ± 2.3

CH

N

CH C=O

H

F

H

F

2-naphtyl

n.a

2-naphtyl 5700 ± 200

2-naphtyl 963 ± 155

H

CH C=O CH₃CH₃2-naphtyl 567 ± 27.8

CH CH

N

C=O

H

F

H

F

2-naphtyl 4487 ± 133

2-naphtyl 1028 ± 57.9

2-naphtyl 5.55 ± 0.9

H

C=O CH₃CH₃2-naphtyl

n.a

N

CH CH CH₂

H

F

H

F

1-naphtyl

N

H

N

CH CH CH₂CH₃CH₃1-naphtyl 13.04 ± 1.1

CH

N

CH CH₂

H

F

H

F

1-naphtyl 75.87 ± 16.6

1-naphtyl 98.18 ± 33.5

1-naphtyl 0.53 ± 0.9

H

CH CH₂CH₃CH₃1-naphtyl

n.a

1-naphtyl 149 ± 1.9

1-naphtyl n.a

1-naphtyl 614 ± 54.7

CH CH

N

CH₂

H

F

H

F

H

CH₂CH₃CH₃1-naphtyl 4.84 ± 2.0

N

CH CH CH₂

H

F

H

F

2-naphtyl 31.82 ± 12.0

2-naphtyl

n.a

H

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Table 1. Cont.

CB1

Comp. X

Y

Z

R

R¹R²

R³

(K_i,nM)

48

49

50

51

52

53

54

55

56

N

CH CH CH₂CH₃CH₃2-naphtyl 47.39 ± 11.1

CH

N

CH CH₂

H

F

H

F

2-naphtyl 1844 ± 79.8

2-naphtyl 327 ± 60.0

2-naphtyl 10.68 ± 2.8

H

CH CH₂CH₃CH₃2-naphtyl 140 ± 10.1

CH CH

N

CH₂

H

F

H

F

2-naphtyl 464 ± 30.3

2-naphtyl 44.42 ± 5.5

2-naphtyl 567 ± 7.0

H

CH₂CH₃CH₃2-naphtyl 241 ± 10.0

WIN55212

CP55940

9.40 ^b

0.60 ^b

a

Data are means ± SEM of n = 3 separate experiments and are expressed as K_i(nM).

n.a. = compounds which did not displace [³H]CP-55,940 by more than 50% at 1 µM in the

preliminary screen. ^bKi values obtained from technical data sheet of Perkin Elmer.

In order to obtain useful structure-activity relationships concerning the mode of interaction of the

benzimidazole series, we have focused our analysis on the molecular regions defined in Figure 2A.

Figure 4. Superimposition of top-scored binding models for the ligands within the CB1

receptor binding site. (A) Side view, and (B) top view from the extracellular side; the loops

have been omitted for greater clarity. (C) Schematic representation of the binding mode of

the most active compound of the series (39). The 2D representation was obtained using

LigandScout v3.03. The main interactions are: (1) Two hydrogen bonds, one between the

pyridine nitrogen and K3.28(192) and the other between the benzimidazole N-3 and

T3.33(197); (2) Hydrophobic and aromatic interactions of the naphthalene ring with the

side-chains of F2.61(174), F7.35(379), A7.36 (380), I5.31(267), between the pyridine ring

and L3.29(193), Y5.39(275), M6.55(363), F7.35(379) and of the benzimidazole with

F2.57(170), F3.36(200), L6.51(359) and W6.48(356).

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Figure 4. Cont.

Region I

First, we assessed the effect on affinity of replacing the 3-pyridyl moiety. According to our docking

results, this region is crucial for ligand stabilization through an H-bond with the amino group of

Lys192. The remainder of the ring presents hydrophobic interactions with the side chains of residues

L3.29(193), Y5.39(275) and L6.51(359). Sixteen benzimidazoles were synthesized with a 3-pyridyl

group at C-2. Besides the reference compound 14, four derivatives displayed affinities in the

nanomolar range (<200 nM, 39, 51, 37, 38, Table 1).

As expected, variation of the N position in the pyridinyl substituent to generate the corresponding

2-pyridyl homologs (compounds 35, 47, 33, 34, 10) had a dramatic effect on CB1 affinities (Table 1).

The inactivity of these compounds might be explained in terms of an unfavorable distance between

Lys 192 and the nitrogen to establish a hydrogen bond.

The 4-pyridyl analogs (compounds 43, 55, 41, 42, 56) showed modest affinities compared with the

3-pyridyl derivatives (Table 1). Formation of a weaker H-bond between the nitrogen atom and Lys192

compared to the 3-pyridyl compounds seems to be a reasonable explanation. The increased distance

between the pyridine nitrogen and Lys192 on going from 3- to 4-pyridyl derivatives observed in our

docking models (2 Å and 2.5 Å respectively) corroborates this. However, the activity/inactivity seems

actually to be related to the still unknown combined effect of more than one modification. That could

explain some exemptions to the observed SAR trends as when homologous compounds 13, 17 and 21

are compared. In summary, 3- or 4-pyridyl seems to be a better substituent than 2-pyridyl in region I.

Region II

According to our docking studies, the naphthyl ring interacts with the CB1 receptor via

hydrophobic and aromatic interactions with the side-chains of hydrophobic residues. A comparison of

the most active 1-naphthyl derivatives (39, 44, 36, 37, 38 and 41) with their 2-naphthyl congeners (51,

56, 48, 49, 50 and 53) was carried out. In general, the 2-naphthyl derivatives displayed lower CB1

receptor affinities than their 1-naphthyl congeners (Table 1). For benzimidazoles bearing a 2-naphthyl

moiety, the naphthalene ring is oriented in a less favourable spatial region in order to interact with the

hydrophobic area delimited by Phe379, Phe174, Val196, Ala 380, Ile267 and Phe170.

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Linker

The methylene bridge does not present any important interaction with the protein, but ligands

containing a keto group as linker can establish, according to our docking analysis, hydrogen bonds

with Ser383. However, when the carbonyl group of prototype 14 (K_i= 98.24 nM) was replaced by a

methylene linker (compound 37, K_i= 75.87 nM) no significant variation of CB1 receptor affinity was

observed. In fact, derivatives with both carbonyl and methylene are among the most active compounds

of the series (Table 1).

There appears to be no a priori explanation for this experimental evidence. In order to gain some

insight into the origin of these unexpected differences in receptor affinity, a comparative molecular

modeling and receptor docking study was carried out for the naphthylmethyl and the corresponding

naphthoyl congeners. No significant differences were seen in the angular arrangement of the

naphthalene ring and the pose of the benzimidazole was maintained. Discarding a different spatial

orientation induced by the linker as the explanation for the loss in affinity, we focused our analysis on

the penalty imposed by the ligand desolvation factor. As described in the literature [28], desolvation of

the ligand plays a significant role in the binding affinity in the cases where the ligand must be entirely

desolvated to perform its function. This is of special relevance in membrane proteins because the

ligand has to be transferred from the aqueous environment to the transmembrane binding site.

Replacement of the carbonyl bridge by a methylene group could enhance the CB1 receptor binding

affinities due to a significant decrease in the desolvation penalty, facilitating the entrance of the ligands

into the binding site and compensating for the weaker ligand-receptor interaction. This was confirmed

by semiempirical AM1-RHF solvation energy calculations (VAMP software) which displayed an

increase in the amount of heat required to raise the entropy of carbonyl compounds.

Region III

In order to study the effect of C5 and C6 substitution of the benzimidazole ring on CB1 affinity, the

substitution pattern at these positions for the most potent compounds of the series was analyzed. As

already mentioned, according to our docking studies the phenyl ring of the benzimidazole core

displayed hydrophobic and aromatic interactions with the side chain of residues Phe170, Trp356 and

Leu359. Besides, the basic nitrogen of the benzimidazole nucleus forms a hydrogen bond with the side

chain of Thr197 for all the compounds.

In general, substitution at the C5 and C6 positions of the phenyl ring of the benzimidazoles with

methyl or fluorine seems to be beneficial for CB1 receptor affinity. Indeed, just two of the most potent

derivatives displayed in Table 1 (compounds 45 and 18) are not substituted on this ring. The

enhancement of π-stacking interactions between the benzimidazole ring and Phe170 and Trp356 is

suggested from our docking analysis. The most potent derivatives are monofluorinated at C6 (39, 31,

51, 23). On the other hand, an important decrease in affinity is seen when the fluorine is at C5, keeping

the rest unchanged (38, 30, 50). Our docking analysis revealed a possible solvation sphere of water

mediating a H bond between the fluorine atom at C6 fluorine and the TM2 Phe170 backbone, which

might be crucial to enhance affinity. The structure-activity relationships for the complete series are

summarized in Figure 5.

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Figure 5. Structure-activity relationships.

2.3. 3D-QSAR (CoMFA)

Active conformation selection is a key step for CoMFA analysis [29]. On the other hand, structural

alignment is viewed as one of the most sensitive parameters in CoMFA analysis [30]. The predictive

accuracy of CoMFA models and the reliability of the contours are directly dependent on the structural

alignment rule [30]. In this case this problem was solved using the conformations obtained by docking

studies and active compounds were aligned according to the flexible-ligand docking solutions. As can

be seen on Figure 6, there are some molecules that are not well overlayed with respect to many of the

other derivatives. Specifically, compounds 21, 36 and 44 display a different docking conformation.

Derivatives 21 and 44 are directing the pyridine ring to the same region as the rest of the series

compounds. It is noteworthy that compounds 21, 36 and 44 are dimethylated at C5 and C6 positions of

the benzimidazole and localize the dimethylated ring towards the same region where the other

compounds of the series direct the naphthalene ring. Indeed, this region on the active site favors

hydrophobic interactions with bulky substituents. Finally Gasteiger-Hückel charges [31,32] were

assigned to all the molecules.

Figure 6. (A) Alignment of the 29 studied molecules. (B) Docking conformation of

compounds 21, 36 and 44.

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A 3D-QSAR model was obtained from CoMFA analysis and its statistical parameters are listed in

Table 2. For a reliable predictive model, the square q²of the cross-validation coefficient should be

greater than 0.5 [33]. This model has high r²(0.998), F (944.13), and q²(0.71), as well as a small SEE

(0.057), suggesting that it is reliable and predictive. The steric and electrostatic contributions were

found to be 35% and 65% respectively.

Table 2. Statistical parameters of the CoMFA model ^a.

Statistic Index Summary of CoMFA

Contribution

N

q²

r²

SEE

SD Press

F

pred

Steric Electrostatic

7

0.71 0.998 0.823 0.057 5.18 0.92 944.1

35

65

Number of Components Analysis

SEP 0.781 0.707 0.660 0.614 0.619 0.624 0.624 0.649 0.675

q²0.376 0.511 0.594 0.667 0.679 0.692 0.710 0.706 0.703

0.706

0.698

10

N

1

2

3

4

5

6

7

8

9

^aN is the optimal number of components, q²is the square of the LOO cross-validation (CV) coefficient, r²is

the square of non cross-validation coefficient, r²is the predictive r²based only on the test set molecules,

pred

SEE is the standard error of estimation of non CV analysis, SEP is the standard error of prediction of LOO

analysis, SD is the sum of the squared deviations between the biological activities of molecules in the test set

and the mean activity of the training-set molecules, PRESS is the sum of the squared deviations between

predicted and actual biological activity values for every molecule in the test set, and F is the F-test value.

The activity values of compounds predicted by CoMFA are listed in Table 3. The plot of the

predicted pIC₅₀values versus the experimental ones for CoMFA analysis is also shown in Figure 7, in

which most points are evenly distributed along the line Y = X, suggesting that the quality of the

3D-QSAR model is good. The steric and electrostatic contour maps of CoMFA are displayed in Figure 8.

Table 3. Experimental and predicted activity for training and test set.

Experimental activity Predicted activity

Compd.

Residual

(pIC₅₀, M)

Training Set

(pIC₅₀, M)

39

21

44

51

23

36

24

45

54

48

37

38

14

41

56

50

53

9.009

8.140

8.052

7.708

7.679

7.621

7.288

7.234

7.089

7.061

6.857

6.745

6.563

6.355

6.222

6.070

9.015

8.126

8.076

7.767

7.624

7.622

7.300

7.237

7.067

6.954

6.686

6.693

6.575

6.351

6.208

6.009

−0.006

0.013

−0.024

−0.059

0.056

0.000

−0.011

−0.003

0.022

−0.006

−0.097

0.059

0.052

−0.012

0.004

0.014

0.061

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Table 3. Cont.

Experimental activity Predicted activity

Compd.

Residual

(pIC₅₀, M)

6.009

(pIC₅₀, M)

6.000

15

28

55

27

30

49

26

0.008

−0.022

−0.015

0.061

−0.001

0.042

5.983

6.005

5.983

5.998

5.753

5.692

5.725

5.726

5.471

5.429

4.981

5.115

−0.134

Test Set

31

18

52

43

29

7.992

7.159

6.592

5.949

5.085

8.433

7.032

6.777

6.703

5.409

−0.441

0.128

−0.185

−0.755

−0.325

Figure 7. Experimental versus predicted activity for A. Training Set and B. Test Set.

Figure 8. Left: CoMFA steric contour map for the most active compound 39. The green

contour indicates where a bulky group favors activity, while the yellow contour indicates

where a small group favors activity. Right: CoMFA electrostatic contour map for the most

active compound 39. The blue contour indicates where positive charge favors activity,

while a red contour indicates where negative charge favors activity.

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Region I

The CoMFA steric contour map shows two yellow polyhedra near the pyridine ring suggesting that

substitution or replacement of this ring with bulky groups is detrimental for activity. An optimal

distance for the H-bond with Lys192 is a very important steric restriction in this region. On the other

hand, the CoMFA electrostatic contour map displays red areas above and below the pyridine ring,

which means that electronegative atoms are required on this ring for optimal activity. This is consistent

with the hydrogen bonding between the pyridine nitrogen and the Lys192 residue observed in our

docking analysis. Moreover, next to the red areas two blue areas can be seen. A detailed analysis of the

active site reveals the presence of amino acids like tyrosine and threonine, available for H-bonding

interactions, beyond Lys192, suggesting that replacement of the pyridine with extended hydrogen-bonding

donor groups able to reach these residues might also generate active compounds.

Linker and Region II

For region II the CoMFA steric contour map shows large green polyhedra around the naphthyl

group, suggesting that bulky groups at this position favor affinity. This result is coherent with our

docking results, which displayed the naphthyl group oriented toward the lumen of the receptor without

any spatial restrictions. With regard to the CoMFA electrostatic contour map, high electron density

rings would increase affinity in the region of the naphthyl ring. Not only surfaces provide valuable

information, but empty areas too. That is clear analyzing the corresponding CoMFA electrostatic

contour map for the linker region, which from the standpoint of the CoMFA seems not to be involved

in any H-bonding interaction.

Region III

The CoMFA steric contour map reveals a small green surface near the fluorine atom at C6,

supporting the synthesis of new compounds substituted at that position, for example with chlorine or

bromine. The other positions of the benzimidazole moiety (C4 and C5) do not display any steric

restrictions. With respect to the CoMFA electrostatic contour map, the C5 and C6 positions of the

benzimidazole ring do not seen to be implicated in relevant electrostatic interactions.

3. Experimental

3.1. General

All organic solvents used for the syntheses were analytical grade. Melting points were determined

on a Stuart Scientific SMP3 apparatus and are uncorrected. IR spectra were recorded on a Bruker

1

13

Vector 22 spectrophotometer using KBr discs. H and C-NMR spectra were obtained on a Bruker

AM-400 spectrometer. The chemical shifts are expressed in ppm (δ scale) downfield from TMS,

J values are given in Hertz for solutions in CDCl₃unless otherwise indicated. Column chromatography

was performed on Merck silica gel 60 (70–230 mesh). Thin layer chromatographic separations were

performed on Merck silica gel 60 (70–230 mesh) chromatofoils. Purity of all final derivatives for

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biological testing was confirmed to be >95% as determined using elemental analysis carried out on a

FISONS EA 1108 CHNS-O analyzer.

General Method for 2-Pyridinyl-1H-benzo[d]imidazoles 1–9

A mixture of an o-phenylenediamine (benzene-1,2-diamine; 4,5-dimethylbenzene-1,2-diamine or

4-fluorobenzene-1,2-diamine, 1 mmol), pyridinecarbaldehyde (pyridine-2-carbaldehyde, pyridine-3-

carbaldehyde or pyridine-4-carbaldehyde, 1 mmol), H₂O₂(30%, 4 mmol, 0.4 mL), and NH₄Ce(NO₃)₆

(0.1 mmol, 0.0548 g) was heated at 50 °C for 1 h. After completion of the reaction, the reaction

mixture was dissolved in EtOH (10 mL) and then poured into ice-water (30 mL). The solid product

was ﬁltered, washed with ice-water, and dried. Purification of the crude material by column

chromatography on silica gel with dichloromethane as the eluent yielded target compounds 1–9 as pale

yellow solids.

2-(Pyridin-2-yl)-1H-benzo[d]imidazole (1). Yield = 95%. Mp. 238–239 °C. ¹H-NMR (CDCl₃+ DMSO-d₆)

δ 12.50 (s, 1H), 8.59 (d, J = 4.3 Hz, 1H), 8.29 (d, J = 7.9 Hz, 1H), 7.79 (t, J = 7.2 Hz, 1H), 7.63–7.52

(m, 2H), 7.31 (dd, J₁= 6.8, J₂=5.4 Hz, 1H), 7.14 (dd, J₁= 6.0 Hz, J ₂=3.1 Hz, 2H). ¹³C-NMR (CDCl₃+

DMSO-d₆) δ 151.2, 149.3 (2C), 149.0, 137.2 (2C), 124.5 (2C), 122.8 (2C), 121.6 (2C). IR (KBr) 3400,

1430 cm⁻¹. Anal. Calcd. For C₁₂H₉N₃(MW 195.22): C, 73.83%; H, 4.65%; N, 21.52%. Found: C,

73.50%; H, 4.60%; N, 21.90%.

1

5-Fluoro-2-(pyridin-2-yl)-1H-benzo[d]imidazole (2). Yield = 96%. Mp. >300 °C. H-NMR (CDCl₃+

DMSO-d₆) δ 12.95 (s, 1H), 8.67 (d, J = 4.2 Hz, 1H), 8.35 (d, J = 7.8 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H), 7.61

(s, broad, 1H), 7.40 (dd, J₁= 6.5 Hz, J₂= 4.2 Hz, 1H), 7.33 (s, broad, 1H), 6.99 (t, J = 8.4 Hz, 1H).

¹³C-NMR (CDCl₃+ DMSO-d₆) δ 151.9 (d, J = 108.2 Hz), 149.2 (2C), 148.7, 137.3 (3C), 124.6 (2C),

121.7, 110.8 (d, J = 11.8 Hz), 98.6 (d, J = 25.7 Hz). IR (KBr) 3425, 1400 cm⁻¹. Anal. Calcd. For

C₁₂H₈FN₃(MW 213.21): C, 67.60%; H, 3.78%; N, 19.71%. Found: C, 67.55%; H, 3.38%; N, 19.43%.

5,6-Dimethyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole (3). Yield = 97%. Mp. >300 °C. ¹H-NMR (CDCl₃+

DMSO-d₆) δ 11.20 (s, 1H), 8.50 (ddd, J₁= 0.7 Hz, J₂= 1.7 Hz, J₃= 4.8 Hz, 1H), 7.51 (dt, J₁= 1,7 Hz,

J₂= 7.7 Hz, 1H), 7.29 (dd, J₁= 4,8 Hz, J₂= 7.7 Hz, 1H), 6.77 (d, J₁= 7.7 Hz, 1H), 6.22 (s, 2H), 2.36

13

(s, 3H, CH₃), 2.37 (s, 3H, CH₃). C-NMR (CDCl₃+ DMSO-d₆) δ 157.8, 150.1, 149.1 (2C), 136.8,

124.4, 121.5, 120.1, 120.9 (2C), 110.8 (2C), 20.7, 20.4. IR (KBr) 3430, 1444 cm⁻¹. Anal. Calcd. For

C₁₄H₁₃N₃(MW 223.27): C, 75.31%; H, 5.87%; N, 18.82%. Found: C, 75.91%; H, 5.86%; N, 18.46%.

1

2-(Pyridin-3-yl)-1H-benzo[d]imidazole (4). Yield = 85%. Mp: 255–256 °C. H-NMR (CDCl₃) δ

12.58–12.70 (s, 1H), 9.43 (dd, J₁= 2.3 Hz, J₂= 0.8 Hz, 1H), 8.66 (dd, J₁= 4.8 Hz, J₂= 1.5 Hz, 1H),

8.54–8.51 (m, 1H), 7.82–7.76 (m, 1H), 7.48–7.56 (m, 1H), 7.44 (dd, J₁= 8.1 Hz, J₂= 4.9 Hz, 1H),

13

7.21–7.31 (m, 2H). C-NMR (CDCl₃) δ 150.8, 149.8, 138.2 (2C), 131.3, 126.7, 125.8, 125.5, 120.8

(2C), 113.5 (2C). IR (KBr) 3420, 1446 cm⁻¹. Anal. Calcd. For C₁₂H₉N₃(MW 195.22): C, 73.83%; H,

4.65%; N, 21.52%. Found: C, 73.51%; H, 4.60%; N, 21.90%.

1

5-Fluoro-2-(pyridin-3-yl)-1H-benzo[d]imidazole (5). Yield = 80%. Mp. 221,5–221,7 °C. H-NMR

(CDCl₃+ DMSO-d₆) δ 12.89 (s, 1H), 9.39 (s, 1H), 8.66 (d, J = 4.2 Hz, 1H), 8.49 (d, J = 7.9 Hz, 1H),

Molecules 2013, 18

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7.82–7.50 (m, 1H), 7.43 (dd, J₁= 7.9, J₂= 4.9 Hz, 1H), 7.37–7.14 (m, 1H), 7.00 (dt, J₁= 9.8 Hz,

13

J₂= 9.5 Hz, J₃= 2.0 Hz, 1H). C-NMR (CDCl₃+ DMSO-d₆) δ 150.3, 149 (d, J = 254.6 Hz), 147.7,

134.0 (2C), 126.4, 126.3, 123.5 (2C), 110.9 (d, J = 8.4 Hz), 108.3, 104.2. IR (KBr) 3045, 1450,

707 cm⁻¹. Anal. Calcd. For For C₁₂H₈N₃F (MW 213.21): C, 67.60%; H, 3.78%; N, 19.71%. Found: C,

67.32%; H, 4.02%; N, 19.38%.

5,6-Dimethyl-2-(pyridin-3-yl)-1H-benzo[d]imidazole (6). Yield = 88%. Mp. 254.8–254.9 °C. ¹H-NMR

(CDCl₃+ DMSO-d₆) δ 12.63 (s, 1H), 9.36 (d, J = 1.7 Hz, 1H), 8.60 (dd, J₁= 4.7 Hz, J₂= 1.3 Hz, 1H),

8.46 (d, J = 8.0 Hz, 1H), 7.44 (dd, J₁= 8.0 Hz, J₂= 4.9 Hz, 1H), 7.38 (s, 2H), 2.36 (s, 6H, 2 × CH₃).

¹³C-NMR (CDCl₃+ DMSO-d₆) δ 152.6, 150.5, 150.0, 149.7 (2C), 148.0, 147.5, 133.5 (2C), 131.2,

123.4 (2C), 20.1 (2C). IR (KBr) 3067, 1609 cm⁻¹. Anal. Calcd. For C₁₄H₁₃N₃(MW 223.27): C,

75.31%; H, 5.87%; N, 18.82%. Found: C, 73.71%; H, 5.86%; N, 18.85%.

2-(Pyridin-4-yl)-1H-benzo[d]imidazole (7). Yield = 98%. Mp. 207.3–208 °C. ¹H-NMR (CDCl₃+ DMSO-d₆)

δ 12.74 (s, 1H), 8.73 (d, J = 6.0 Hz, 2H), 8.12 (d, J = 6.0 Hz, 2H), 7.74–7.59 (m, 2H), 7.28 (dd, J₁= 6.0,

13

J₂= 3.1 Hz, 2H). C-NMR (CDCl₃) δ 150.2, 155.0, 149.1 (2C), 141.7 (2C), 137.6 (2C), 123.0 (2C),

120.6 (2C). IR (KBr) 3448, 1610 cm⁻¹. Anal. Calcd. For C₁₂H₉N₃(MW 195.92): C, 73.83%; H,

4.65%; N, 21.52%. Found: C, 73.77%; H, 4.50%; N, 21.03%.

5-Fluoro-2-(pyridin-4-yl)-1H-benzo[d]imidazole (8). Yield = 90%. Mp >300. ¹H-NMR (CDCl₃+ DMSO-d₆)

δ 13.20 (s, 1H), 8.84–8.65 (m, 2H), 8.20–8.08 (m, 2H), 7.68–7.59 (m, 1H), 7.35 (d, J = 7.2 Hz, 1H),

7.02 (t, J = 9.2 Hz, 1H). ¹³C-NMR (CDCl₃+ DMSO-d₆) δ 155.3, 150.0 (2C) 142.3 (2C), 125.7,

125.6(2C), 120.3 (2C), 116.2 (d, J = 14.4 Hz), 110.8. IR (KBr) 3449, 1612, 832 cm⁻¹. Anal. Calcd. For

C₁₂H₈FN₃(MW 213.21): C, 67.60%; H, 3.78%; N, 19.71%. Found: C, 67.54%; H, 3.39%; N, 19.31%.

1

5,6-Dimethyl-2-(pyridin-4-yl)-1H-benzo[d]imidazole (9). Yield = 98%. Mp >300. H-NMR (CDCl₃+

DMSO-d₆) δ 8.85–8.60 (m, 2H), 8.22–8.05 (m, 2H), 7.50–7.36 (m, 2H), 2.37 (s, 6H, 2 × CH₃). ¹³C-NMR

(CDCl₃+ DMSO-d₆) δ 153.0, 150.0(2C), 147.9, 137.8, 131.8, 130.5 (2C), 120.4 (2C), 110.5 (2C), 20.1

(2C). IR (KBr) 3418, 1609 cm⁻¹. Anal. Calcd. For C₁₄H₁₃N₃(MW 223.27): C, 75.31%; H, 5.87%; N,

18.82%. Found: C, 75.81%; H, 5.46%; N, 18.36%.

General Method for the Synthesis of (2-Pyridinyl-1H-benzo[d]imidazol-1-yl)(naphthalenyl)methanones

10–32 and 2-Pyridinyl-1-(naphthylmethyl)-1H-benzo[d]imidazoles 33–56.

A solution of 2-pyridinyl-1H-benzo[d]imidazole (1 mmol) in anhydrous THF (50 mL) containing NaH

(1 mmol) and the corresponding 1 or 2-naphthoyl chloride (1 mmol), or 1 or 2-(bromomethyl)naphthalene

(1 mmol) was stirred at room temperature for 20 min under a nitrogen atmosphere. The solvent was

evaporated under reduced pressure, and the residue was purified by column chromatography on silica

gel with dichloromethane as eluent to yield target compounds 10–56 as pale yellow solids.

Naphthalen-1-yl(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)methanone (10). Yield = 77%. Mp.

1

138.0–138.1 °C. H-NMR (CDCl₃) δ 8.80 (d, J = 8.6 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.93 (d,

J = 7.1 Hz, 1H), 7.88–7.79 (m, 3H), 7.78–7.70 (m, 2H), 7.64–7.52 (m, 2H), 7.42 (pd, J₁= 7.3 Hz,

J₂= 1.4 Hz, 2H), 7.20–7.08 (m, 2H), 6.88 (ddd, J₁= 7.5 Hz, J₂= 4.8 Hz, J₃= 1.2 Hz, 1H). ¹³C-NMR

Molecules 2013, 18

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(CDCl₃) δ 169.0, 152.3, 148.4, 148.2, 142.8, 136.4, 135.6, 133.6, 133.3, 132.9, 131.3, 128.3, 128.3, 126.8,

125.9, 125.5, 124.5, 124.0, 123.5, 123.1(2C), 120.5, 112.9. IR (KBr) 1720 cm⁻¹. Anal. Calcd. For

C₂₃H₁₅N₃O (MW 349.38): C, 79.07%; H, 4.33%; N, 12.03%. Found: C, 79.51%; H, 4.35%; N, 12.02%.

(5-Fluoro-2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (11). Yield = 70%.

Mp. 167.2–167.9 °C. ¹H-NMR (CDCl₃) δ 8.76 (d, J = 8.5 Hz, 1H), 8.03 (t, J = 7.0 Hz, 1H), 7.85–7.79

(m, 3H), 7.77–7.69 (m, 2H), 7.64–7.54 (m, 3H), 7.19 (dt, J₁= 9.1, J₂= 2.5 Hz, 1H), 7.16–7.10 (m,

13

2H), 6.88 (dd, J₁= 7.7 Hz, J₂= 4.1 Hz, 1H). C-NMR (CDCl₃) δ 168.9, 160.6 (d, J = 241.0 Hz),

153.8, 148.4, 148.2, 136.7, 133.7 (d, J = 7.8 Hz), 132.9, 132.2, 131.5, 128.5, 128.4, 127.1, 126.0,

124.1, 123.9, 123.4, 121.6 (d, J = 10.4 Hz), 114.0, 113.8, 113.7, 106.6 (d, J = 24.0 Hz), 100.2 (d,

J = 28.9 Hz). IR (KBr) 1735 cm⁻¹. Anal. Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H, 3.84%;

N, 11.44%. Found: C, 75.59%; H, 4.30%; N, 11.04%.

(6-Fluoro-2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (12). Yield = 64%.

Mp. 177.2–178.0 °C. ¹H-NMR (CDCl₃) δ 8.76 (d, J = 8.6 Hz, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.86 (dd,

J₁= 9.0 Hz, J₂= 5.1 Hz 1H), 7.84–7.79 (m, 3H), 7.74 (t, J = 7.7 Hz, 1H), 7.64–7.52 (m, 2H), 7.48 (dd,

J₁= 8.8 Hz, J₂= 2.4 Hz, 1H), 7.19 (dt, J₁= 9.2 Hz, J₂= 2.5 Hz, 1H), 7.17–7.12 (m, 2H), 6.89 (J₁= 7.0 Hz,

13

J₂= 4.7 Hz, 1H). C-NMR (CDCl₃) δ 168.7, 161.1 (d, J = 243.5 Hz), 152.8, 148.2 (d, J = 19.1 Hz),

139.1, 136.5, 133.6, 133.6 (d, J = 5.5 Hz), 131.3, 128.4, 128.2, 126.9, 125.8, 123.9, 123.6, 122.9,

121.4, 121.3,113.8, 113.6, 113.0 (d, J = 25.3 Hz), 110.0, 100.0 (d, J = 29.2 Hz). IR (KBr) 1740 cm⁻¹.

Anal. Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H, 3.84%; N, 11.44%. Found: C, 75.49%; H,

4.35%; N, 11.94%.

(5,6-Dimethyl-2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (13). Yield = 93%.

1

Mp. 167.2–168.0 °C. H-NMR (CDCl₃) δ 8.63 (d, J = 4.5 Hz, 1H), 8.57 (d, J = 4.3 Hz, 1H), 8.47 (d,

J = 8.0 Hz, 1H), 7.84 (td, J₁= 7.7 Hz, J₂= 1.8 Hz, 1H), 7.65 (s broad, 1H), 7.51 (td, J₁= 7.7 Hz,

J₂= 1.8 Hz, 1H), 7.36–7.26 (m, 2H), 7.22–7.11 (m, 2H), 6.87 (d, J = 7.9 Hz, 1H), 6.28 (s, 2H), 2.41 (s,

3H, CH₃), 2.37 (s, 3H, CH₃). ¹³C-NMR (CDCl₃) δ 158.0, 150.9, 149.3, 148.8, 144.0, 141.6, 139.6,

137.1, 137.0, 135.7, 133.4, 132.2, 131.6(2C), 128.7, 126.5, 125.7, 125.0, 124.5, 123.8, 114.7, 113.1,

110.9, 18.8(2C). IR (KBr) 1635 cm⁻¹. Anal. Calcd. For C₂₅H₁₉N₃O (MW 377.44): C, 79.55%; H,

5.07%; N, 11.13%; Found: C, 79.49%; H, 5.35%; N, 11.54%.

Naphthalen-1-yl-(2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)methanone (14). Yield = 79%. Mp.

1

148.0–148.1 °C. H-NMR (CDCl₃) δ 8.67 (s, 1H), 8.33 (s, 1H), 7.98 (d, J = 7.97 Hz, 1H), 7.88 (d,

J = 3.65 Hz, 1H), 7.86 (d, J = 3.98 Hz, 1H), 7.80 (d, J = 7.20 Hz, 1H), 7.66 (d, J = 8.10 Hz, 1H), 7.60

(d, J = 7.27 Hz, 2H), 7.54 (t, J₁= 7.08 Hz, J₂= 7.08 Hz, 2H), 7.42 (t, J₁= 7.58 Hz, J₂= 7.58 Hz, 1H),

13

7.38–7.28 (m, 2H), 6.90 (s, 1H). C-NMR (CDCl₃) δ 151.4, 150.1, 149.2, 142.9, 135.7, 133.9 (2C),

133.5, 130.7 (2C), 130.1 (2C), 128.7, 127.1, 125.7, 125.2, 124.5, 124.2 (2C), 122.17, 120.6, 114.2

(2C). IR (KBr) 1708 cm⁻¹. Anal. Calcd. For C₂₃H₁₅N₃O (MW 349.38): C, 79.07%; H, 4.33%; N,

12.03%. Found: C, 79.64%; H, 4.34%; N, 12.12%.

(5-Fluoro-2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (15). Yield = 60%.

Mp. 169.7–170.7 °C. ¹H-NMR (CDCl₃) δ 8.60 (d, J = 1.3 Hz, 1H), 8.30 (dd, J₁= 1.3 Hz, J₂= 4.9 Hz

Molecules 2013, 18

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1H), 7.99 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.71 (dd, J₁= 9.0 Hz,

J₂= 4.7 Hz, 1H), 7.63–7.52 (m, 5H), 7.31 (t, J = 7.7 Hz, 1H), 7.13 (dt, J₁= 9.1 Hz, J₂= 2.4 Hz, 1H),

6.87 (dd, J₁= 7.7 Hz, J₂= 4.9 Hz, 1H).¹³C-NMR (CDCl₃) δ 168.2, 160.8 (d, J = 242.1 Hz), 152.9,

150.4, 149.3, 143.7 (d, J = 12.4 Hz), 139.3, 135.8, 134.2, 133.6, 131.2, 130.7 (d, J = 21.6 Hz), 130.2,

128.97, 129.0, 128.3, 126.9, 124.6, 124.2, 122.3, 115.2 (d, J = 9.5 Hz), 113.9 (d, J = 25.5 Hz), 106.7

(d, J = 24.4 Hz). IR (KBr) 1735 cm⁻¹. Anal. Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H,

3.84%; N, 11.44%. Found: C, 75.19%; H, 3.35%; N, 11.34%.

(6-Fluoro-2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (16). Yield = 55%.

Mp. 168.1–168.7 °C. ¹H-NMR (CDCl₃) δ 8.60 (d, J = 1.6 Hz, 1H), 8.29 (dd, J₁= 4.9 Hz, J₂= 1.7 Hz,

1H), 7.99 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.85–7.79 (m, 2H) 7.63–7.53 (m, 4H), 7.49 (dd,

J₁= 9.0 Hz, J₂= 2.4 Hz, 1H), 7.33 (dd, J₁= 8.2 Hz, J₂= 7.3 Hz, 1H), 7.20 (td, J₂= 9.0 Hz, J₂= 2.5 Hz,

1H), 6.87 (dd, J₁= 7.8 Hz, J₂= 4.9 Hz, 1H).¹³C-NMR (CDCl₃) δ 168.3, 161.2 (d, J = 243.6 Hz), 151.9,

151.9, 150.2, 149.2, 139.4, 135.8, 135.0 (d, J = 13.8 Hz), 134.3, 133.6, 130.8, 130.5, 130.3, 129.0 (d,

J = 3.1 Hz), 127.4, 127.0, 124.61, 124.2, 122.4, 121.5 (d, J = 10.1 Hz), 113.7 (d, J = 24.9 Hz), 101.9

(d, J = 29.4 Hz). IR (KBr) 1740 cm⁻¹Anal. Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H,

3.84%; N, 11.44%. Found: C, 75.20%; H, 3.95%; N, 11.92%.

(5,6-Dimethyl-2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (17). Yield = 73%.

1

Mp. 177.2–178.0 °C. H-NMR (CDCl₃/DMSO-d₆) δ 8.51 (s, 1H), 8.25 (s, 1H), 8.03–7.95 (m, 1H),

7.95–7.88 (m, 2H), 7.74–7.66 (m, 2H), 7.65–7.51 (m, 3H), 7.50–7.45 (m, 1H), 7.39 (t, J = 7.72 Hz,

13

1H), 7.02–6.89 (m, 1H), 2.39 (s, 3H, CH₃), 2.32 (s, 3H, CH₃). C-NMR (CDCl₃/DMSO-d₆) δ 188.8,

168.3, 150.7, 150.0, 149.0, 141.5, 136.2, 134.7, 134.0, 133.6, 133.3, 133.0, 131.0, 130.3, 129.0, 128.7,

127.4, 127.2, 125.0, 124.3, 122.5, 120.5, 114.6, 20.7, 20.3. IR (KBr) 1637 cm⁻¹. Anal. Calcd. For

C₂₅H₁₉N₃O (MW 377.44): C, 79.55%; H, 5.07%; N, 11.13%; Found: C, 80.00%; H, 5.57%; N, 11.62%.

Naphthalen-1-yl-(2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)methanone (18). Yield = 65%. Mp.

1

155.0–156.1 °C. H-NMR (DMSO-d₆) δ 7.55–7.43 (m, 3H), 7.22–7.07 (m, 3H), 6.97–6.90 (m, 2H),

6.88–6.83 (m, 1H), 6.82–6.76 (m, 1H), 6.72–6.65 (m, 1H), 6.65–6.53 (m, 2H), 6.52–6.46 (m, 2H).

¹³C-NMR (CDCl₃) δ 166.6, 150.7(2C), 148.2, 142.3, 137.1, 134.3, 133.0, 132.7, 130.5, 130.4, 129.3,

128.0, 127.8, 126.2, 124.8, 124.1, 124.0, 123.4, 121.8(2C), 120.2, 113.3. IR (KBr) 1720 cm⁻¹. Anal.

Calcd. For C₂₃H₁₅N₃O (MW 349.38): C, 79.07%; H, 4.33%; N, 12.03%. Found: C, 79.04%; H, 4.84%;

N, 12.10%.

(5-Fluoro-2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (19). Yield = 62%.

Mp. 180.7–181.7°C. ¹H-NMR (CDCl₃) δ 8.30–8.26 (m, 2H), 8.11 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.2 Hz,

1H), 7.83 (d, J = 7.7 Hz, 1H), 7.69 (dd, J₁= 9.0 Hz, J₂= 4.7 Hz, 1H), 7.63 (dt, J₁= 8.5 Hz, J₂= 1.6 Hz,

1H), 7.60–7.55 (m, 2H), 7.54 (d, J = 7.0 Hz, 1H), 7.29 (dd, J₁= 7.6 Hz, J₂= 7.9 Hz 1H), 7.26–7.23 (m,

2H), 7.15 (td, J₁= 9.1 Hz, J₂= 2.5 Hz, 1H). ¹³C-NMR (CDCl₃) δ 168.0, 160.8 (d, J = 242.8 Hz), 153.1,

149.1, 143.5 (d, J = 12.4 Hz), 138.3, 134.6, 133.6, 133.5, 131.3, 131.0, 130.5, 130.4, 129.1(2C), 127.8,

126.3, 124.3 (d, J = 12.1 Hz), 122.9(2C), 115.2 (d, J = 9.6 Hz), 114.4 (d, J = 25.4 Hz), 107.0 (d,

J = 24.5 Hz). IR (KBr) 1710 cm⁻¹. Anal. Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H, 3.84%;

N, 11.44%. Found: C, 75.69%; H, 4.25%; N, 11.92%.

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(6-Fluoro-2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (20). Yield = 63%.

Mp. 190.4–190.9 °C. ¹H-NMR (CDCl₃) δ 8.29–8.24 (m, 2H), 8.12 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 8.5

Hz, 1H), 7.87–7.81 (m, 2H), 7.67–7.57 (m, 2H), 7.54 (dd, J₁= 7.2 Hz, J₂= 1.3 Hz, 1H) 7.47 (dd,

J₁= 8.9 Hz, J₂= 2.5 Hz, 1H), 7.30 (dd, J₁= 8.3 Hz, J₂= 7.2 Hz, 1H), 7.25–7.21 (m, 2H), 7.19 (dd,

J₁= 9.0 Hz, J₂= 2.5 Hz, 1H).¹³C-NMR (CDCl₃) δ 168.0, 161.4 (d, J = 244.4 Hz), 152.2 (d, J = 3.6 Hz),

150.5, 149.2(2C), 139.3, 138.2, 135.2, 135.1, 134.7, 133.6, 131.0, 130.4, 130.4, 129.1, 127.4, 124.3 (d,

J = 12.4 Hz), 122.8(2C), 121.8 (d, J = 10.2 Hz), 113.9 (d, J = 25.0 Hz), 101.8 (d, J = 29.4 Hz). IR

(KBr) 1733 cm⁻¹. Anal. Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H, 3.84%; N, 11.44%.

Found: C, 74.98%; H, 4.33%; N, 11.01%.

(5,6-Dimethyl-2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-1-yl)methanone (21). Yield = 77%.

1

Mp. 167.2–168.0 °C. H-NMR (CDCl₃) δ 8.20 (d, J = 5.9 Hz, 2H), 8.07 (d, J = 8.3 Hz, 1H), 7.86 (d,

J = 8.2 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H), 7.62–7.48 (m, 4H), 7.30–7.23 (m, 1H), 7.17 (d,

J = 5.9 Hz, 2H), 2.41 (s, 3H, CH₃), 2.36 (s, 3H, CH₃).¹³C-NMR (CDCl₃) δ 168.5, 151.1, 149.3(2C),

141.5, 138.6, 135.8, 134.6, 134.2, 133.6, 133.4, 131.2, 131.1, 130.3, 129.0, 128.8, 127.2, 124.5, 124.4,

122.8(2C), 120.9, 114.7, 20.9, 20.5. IR (KBr) 1617 cm⁻¹. Anal. Calcd. For C₂₅H₁₉N₃O (MW 377.44): C,

79.55%; H, 5.07%; N, 11.13%; Found: C, 79.05%; H, 5.52%; N, 10.98%.

Naphthalen-2-yl(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)methanone (22). Yield = 82%. Mp.

1

145.0–146.0 °C. H-NMR (CDCl₃) δ 8.29 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 4.7 Hz, 1H), 8.05 (s, 1H),

7.97–7.91 (m, 2H), 7.86–7.81 (m, 2H), 7.74 (d, J = 8.2 Hz, 1H), 7.69 (td, J₁= 7.7 Hz, J₂= 1.7 Hz, 1H),

7.56 (td, J₁= 8.1 Hz, J₂= 1.2 Hz, 1H), 7.53–7.46 (m, 2H), 7.41 (td, J₁= 8.1 Hz, J₂= 7.7 Hz, J₃= 1.3 Hz,

1H), 7.35 (d, J = 8.2 Hz, 1H), 7.05 (ddd, J₁= 7.6 Hz, J₂= 4.8 Hz, J₃= 1.2 Hz, 1H). ¹³C-NMR (CDCl₃)

δ 169.4, 152.0, 148.5, 148.1, 143.0, 136.7, 135.6, 135.5, 132.2, 131.8, 131.5, 129.5, 128.9, 128.8,

127.8, 127.0, 125.3, 125.1, 124.2, 124.0, 123.3, 120.5, 112.3. IR (KBr) 1715 cm⁻¹. Anal. Calcd. For

C₂₃H₁₅N₃O (MW 349.38): C, 79.07%; H, 4.33%; N, 12.03%. Found: C, 79..57%; H, 4.83%; N, 12.01%.

(6-Fluoro-2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (23). Yield = 66%.

1

Mp. 195.7–195.9 °C. H-NMR (CDCl₃) δ 8.78 (s, 1H), 8.61 (d, J = 4.8 Hz, 1H), 8.54 (d, J = 7.9 Hz,

1H), 8.21 (d, J = 8.6 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.92 (t, J = 9.0 Hz, 3H), 7.66 (dd, J₁= 8.7 Hz,

J₂= 4.7 Hz, 1H),7.63–7.53 (m, 2H), 7.45–7.34 (m, 2H), 7.06 (td, J₁= 9.2 Hz, J₂= 2.4 Hz, 1H).

¹³C-NMR (CDCl₃) δ 171.0, 160.1 (d, J = 239.6 Hz), 151.3, 148.6, 147.8, 139.0, 138.1, 135.8, 132.6,

131.8, 129.5, 128.4 (d, J = 18.9 Hz), 127.8, 127.6, 127.4, 126.7, 125.7, 124.9, 123.2, 122.6, 116.8,

112.1 (d, J = 25.5 Hz), 101.6 (d, J = 40.1 Hz). IR (KBr) 1723 cm⁻¹. Anal. Calcd. For C₂₃H₁₄FN₃O

(MW 367,38): C, 75.19%; H, 3.84%; N, 11.44%. Found: C, 75.20%; H, 4.26%; N, 11.00%.

(5,6-Dimethyl-2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (24). Yield = 85%.

1

Mp. 168.7–169.6 °C. H-NMR (CDCl₃) δ 8.59 (d, J = 4.9 Hz, 1H), 8.53 (d, J = 5.0 Hz, 1H), 8.45 (d,

J = 8.0 Hz, 1H), 7.81 (td, J₁= 7.7 Hz, J₂= 1.8 Hz, 1H), 7.62 (s, 1H), 7.48 (td, J₁= 7.7 Hz, J₂= 1.9 Hz,

1H), 7.31–7.22 (m, 2H), 7.19–7.04 (m, 2H), 6.84 (d, J = 7.8 Hz, 1H), 6.25 (s, 2H), 2.38 (s, 3H, CH₃),

13

2.33 (s, 3H, CH₃). C-NMR (CDCl₃) δ 157.8, 153.1, 150.6, 149.1, 148.6, 148.5, 147.9, 147.8, 141.4,

139.0, 136.9, 136.8, 135.4, 133.2, 132.0, 124.3, 123.5, 122.2, 120.8, 120.1, 119.0, 118.4, 110.7, 20.6,

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20.3. IR (KBr) 1629 cm⁻¹. Anal. Calcd. For C₂₅H₁₉N₃O (MW 377.44): C, 79.55%; H, 5.07%; N,

11.13%; Found: C, 80.04%; H, 5.57%; N, 10.84%.

Naphthalen-2-yl(2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)methanone (25). Yield = 90%. Mp.

1

148.0–148.6 °C. H-NMR (CDCl₃) δ 8.92 (s, 1H), 8.46 (d, J = 3.3 Hz, 1H), 8.27 (s, 1H), 7.97 (dt,

J₁= 7.9 Hz, J₂= 1.8 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.90–7.86 (m, 2H), 7.85–7.80 (m, 2H), 7.65 (dt,

J₁= 7.6 Hz, J₂= 1.1 Hz, 1H), 7.56 (dt, J₁= 7.5 Hz, J₂= 1.0 Hz, 1H), 7.41 (dt, J₁= 7.6 Hz, J₂= 1.2 Hz, 1H),

7.36 (d, J = 7.9 Hz, 1H), 7.31–7.26 (m, 1H), 7.19 (dd, J₁= 7.7 Hz, J₂= 4.9 Hz, 1H). ¹³C-NMR (CDCl₃) δ

168.7, 151.4, 150.5, 149.7, 143.0, 136.2, 136.0, 135.0, 133.1, 132.2, 129.8, 129.6, 129.5, 129.3, 128.0,

127.5, 127.0, 125.3, 125.2, 124.8, 123.0, 120.6, 113.3. IR (KBr) 1755 cm⁻¹. Anal. Calcd. For C₂₃H₁₅N₃O

(MW 349.38): C, 79.07%; H, 4.33%; N, 12.03%. Found: C, 78..61%; H, 4.87%; N, 12.02%.

(5-Fluoro-2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (26). Yield = 50%.

Mp. 177.7–118.6 °C. ¹H-NMR (CDCl₃) δ 8.63 (s, 1H), 8.51 (d, J = 8.1 Hz, 1H), 8.25 (s, 1H), 8.09 (d,

J = 8.6 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.91–7.82 (m, 3H), 7.79 (dd, J₁= 8.6 Hz, J₂= 1.8 Hz, 1H),

7.63–7.51 (m, 3H), 7.35 (dd, J₁= 9.0 Hz, J₂= 4.6 Hz, 1H), 7.07 (dt, J₁= 9.1 Hz, J₂= 2.5 Hz, 1H).

¹³C-NMR (CDCl₃) δ 168.3, 160.2 (d, J = 241.6 Hz), 152.7, 150.5, 150.4, 149.5, 147.9, 143.5 (d,

J = 12.1 Hz), 136.2, 135.9, 135.4, 133.0, 132.0, 131.4, 129.7, 129.4 (d, J = 3.8 Hz), 128.0, 127.6, 126.5,

125.0, 114.0 (d, J = 9.7 Hz), 113.2 (d, J = 26.0 Hz), 106.4 (d, J = 23.6 Hz). IR (KBr) 1715 cm⁻¹. Anal.

Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H, 3.84%; N, 11.44%. Found: C, 74.89%; H,

4.27%; N, 11.42%.

(6-Fluoro-2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (27). Yield = 50%.

Mp. 167.2–167.7 °C. ¹H-NMR (CDCl₃) δ 8.74 (s, 1H), 8.46 (d, J = 8.5 Hz, 1H), 8.26 (s, 1H), 8.17 (d,

J = 8.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.95–7.76 (m, 3H), 7.70–7.54 (m, 2H), 7.49 (dd, J₁= 8.8 Hz,

13

J₂= 4.6 Hz, 1H), 7.24–7.11 (m, 2H), 6.96 (t, J = 8.3 Hz, 1H). C-NMR (CDCl₃) δ 168.4, 160.4 (d,

J = 158.9 Hz), 151.6, 150.3, 149.4, 139.3, 136.4, 136.0, 135.7, 135.3, 134.9, 132.1, 129.4 (d, J = 10.9 Hz),

129.3, 128.4, 128.0, 127.8, 127.7, 126.7, 125.1, 121.4 (d, J = 10.2 Hz), 113.2 (d, J = 25.4 Hz), 111.9

(d, J = 25.4 Hz). IR (KBr) 1739 cm⁻¹. Anal. Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H,

3.84%; N, 11.44%. Found: C, 74.70%; H, 4.34%; N, 11.93%.

(5,6-Dimethyl-2-(pyridin-3-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (28). Yield = 75%.

Mp. 166.7–166.9 °C. ¹H-NMR (CDCl₃) δ 8.98 (s broad, 1H), 8.51 (s broad, 1H), 8.32 (s, 1H), 8.02 (dt,

J₁= 7.9 Hz, J₂= 1.9 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.94–7.87 (m, 2H), 7.77 (s, 1H), 7.73 (td,

J₁= 7.6 Hz, J₂= 1.4 Hz, 1H), 7.65 (td, J₁= 7.5 Hz, J₂= 1.2 Hz, 1H), 7.34 (s, 1H), 7.24 (d, J = 5.2 Hz,

13

1H), 2.51 (s, 3H, CH₃), 2.40 (s, 3H, CH₃). C-NMR (CDCl₃) δ 169.1, 150.3, 149.8, 148.1, 144.0,

141.7, 136.2, 136.0, 134.9, 134.0, 133.7, 133.2, 132.3, 130.2, 129.6(2C), 129.3, 128.1, 127.6, 125.5,

123.1, 120.7, 113.7, 20.8, 20.5. IR (KBr) 1639 cm⁻¹. Anal. Calcd. For C₂₅H₁₉N₃O (MW 377.44): C,

79.55%; H, 5.07%; N, 11.13%; Found: C, 80.04%; H, 5.56%; N, 10.64%.

Naphthalen-2-yl(2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)methanone (29). Yield = 75%. Mp.

145.0–146.1 °C. ¹H-NMR (CDCl₃) δ 8.57 (s broad, 2H), 8.27 (s, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.90 (s

broad, 1H), 7.88–7.86 (m, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.61–7.53 (m, 3H),

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13

7.42 (t, J = 7.4 Hz, 1H), 7.35–7.26 (m, 2H), 7.88–7.86 (m, 1H). C-NMR (CDCl₃) δ 168.7, 151.8,

150.1, 143.1, 138.2, 136.2, 135.3, 133.3, 132.3, 129.9, 129.9, 129.6, 129.5, 128.2, 127.8(2C), 125.7,

125.4(2C), 125.0, 123.2, 121.0, 113.4. IR (KBr) 1731 cm⁻¹. Anal. Calcd. For C₂₃H₁₅N₃O (MW

349.38): C, 79.07%; H, 4.33%; N, 12.03%. Found: C, 79.57%; H, 4.33%; N, 12.01%.

(5-Fluoro-2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (30). Yield = 40%.

1

Mp. 195.7–200.6 °C. H-NMR (CDCl₃) δ 8.52 (d, J = 5.5 Hz, 2H), 8.22 (s, 1H), 7.88 (t, J = 8.4 Hz,

2H), 7.84–7.79 (m, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.59 (d, J₁= 8.35 Hz, J₂= 2.5 Hz, 1H), 7.57–7.55 (m,

1H), 7.53 (d, J = 5.5 Hz, 2H), 7.31 (dd, J₁= 9.0 Hz, J₂= 4.6 Hz, 1H), 7.05 (td, J₁= 9.1 Hz, J₂= 2.5 Hz,

13

1H). C-NMR (CDCl₃) δ 168.4, 160.5 (d, J = 242.0 Hz), 153.1, 150.0(2C), 143.6 (d, J = 12.6 Hz),

137.9, 136.2, 133.2, 132.1, 131.7, 130.0, 129.5(3C), 128.1, 127.8, 125.2, 123.1(2C), 114.1, 113.9 (d,

J = 26.2 Hz), 106.8 (d, J = 24.4 Hz). IR (KBr) 1755 cm⁻¹. Anal. Calcd. For C₂₃H₁₄FN₃O (MW

367.38): C, 75.19%; H, 3.84%; N, 11.44%. Found: C, 75.69%; H, 3.37%; N, 11.40%.

(6-Fluoro-2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (31). Yield = 55%.

1

Mp. 187.3–188.2°C. H-NMR (CDCl₃) δ 8.52 (d, J = 6.1 Hz, 2H), 8.23 (s, 1H), 7.95–7.80 (m, 5H),

7.67 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.53 (d, J = 6.1 Hz, 2H), 7.18 (td, J₁= 9.0 Hz,

J₂= 2.4 Hz, 1H), 7.12 (dd, J₁= 9.0 Hz, J₂= 2.5 Hz, 1H).¹³C-NMR (CDCl₃) δ 170.3, 161.6 (d,

J = 245.0 Hz), 159.3, 152.2, 150.0 (2C), 143.7 (d, J = 11.9 Hz), 143.0, 138.0, 136.0, 133.2, 132.2,

131.7, 130.1, 129.5, 128.0, 127.8, 125.2, 123.0(2C), 114.0, 113.8 (d, J = 26.2 Hz), 106.7 (d, J = 24.3 Hz).

IR (KBr) 1754 cm⁻¹. Anal. Calcd. For C₂₃H₁₄FN₃O (MW 367.38): C, 75.19%; H, 3.84%; N, 11.44%.

Found: C, 75.01%; H, 3.94%; N, 11.43%.

(5,6-Dimethyl-2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (32). Yield = 85%.

1

Mp. 160.4–160.9 °C. H-NMR (CDCl₃) δ 8.49 (s broad, 1H), 8.21 (s, 1H), 7.94–7.77 (m, 4H),

7.70–7.64 (m, 2H), 7.60 (dd, J₁= 3.8 Hz, J₂= 1.4 Hz, 1H), 7.57–7.48 (m, 3H), 7.20 (s, 1H), 2.41 (s,

3H, CH₃), 2.29 (s, 3H, CH₃). ¹³C-NMR (CDCl₃) δ 168.8, 149.8, 141.5, 138.3, 136.0, 135.2, 134.0,

133.0(2C), 132.0, 129.9, 129.6, 129.4(2C), 129.2, 128.0(2C), 127.5, 125.2, 120.7, 120.0, 113.4, 113.4,

20.7, 20.3. IR (KBr) 1616 cm⁻¹. Anal. Calcd. For C₂₅H₁₉N₃O (MW 377.44): C, 79.55%; H, 5.07%; N,

11.13%; Found: C, 79.54%; H, 5.49%; N, 11.62%.

1-(Naphthalen-1-ylmethyl)-2-(pyridin-2-yl)-1H-benzo[d]imidazole (33). Yield: 70%. Mp. 150.7–151.6 °C.

¹H-NMR (CDCl₃) δ 8.48 (d, J = 8.0 Hz, 1H), 8.44 (d, J = 4.2 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.95

(d, J = 8.1 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.80 (td, J₁= 7.8 Hz, J₂= 1.4 Hz, 1H), 7.72 (d, J = 8.2 Hz,

1H), 7.63 (t, J = 7.2 Hz, 1H), 7.57 (t, J = 7.3 Hz, 1H), 7.37–7.32 (m, 1H), 7.26–7.17 (m, 4H), 6.70 (s,

2H, CH₂), 6.63 (d, J = 7.1 Hz, 1H). ¹³C-NMR (CDCl₃) δ 150.5, 148.7, 142.8, 139.9, 137.1, 136.8,

133.6, 132.8, 130.5, 129.0, 127.6, 126.4, 125.9, 125.6, 124.5, 123.8, 123.7, 123.0, 122.9, 122.5, 120.3,

110.8, 46.8. IR (KBr) 1447 cm⁻¹. Anal. Calcd. For C₂₃H₁₇N₃(MW 335.40): C, 82.36%; H, 5.11%; N,

12.53%; Found: C, 82.86%; H, 5.10%; N, 12.07%.

5-Fluoro-1-(naphthalen-1-ylmethyl)-2-(pyridin-2-yl)-1H-benzo[d]imidazole (34). Yield: 55%. Mp.

155.7–155.9 °C. ¹H-NMR (CDCl₃) δ 8.50–8.41 (m, 2H), 8.16 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 7.9 Hz,

1H), 7.81 (td, J₁= 7.7 Hz, J₂= 1.8 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.64–7.54 (m, 3H), 7.27–7.19 (m,

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2H), 7.10 (dd, J₁= 8.9 Hz, J₂= 4.6 Hz, 1H), 6.96 (td, J₁= 9.1 Hz, J₂= 2.4 Hz, 1H), 6.68 (s, 2H, CH₂),

13

6.61 (d, J = 7.2 Hz, 1H). C-NMR (CDCl₃) δ 159.8 (d, J = 238.2 Hz), 151.7, 150.2, 148.8, 143.1 (d,

J = 12.5 Hz), 136.9, 133.6, 132.5, 130.5, 129.0, 127.8, 127.7, 126.5, 126.0, 125.6, 124.6, 124.0, 122.9,

122.4, 114.0, 112.2 (d, J = 26.3 Hz), 111.2 (d, J = 10.2 Hz), 105.7 (d, J = 24.0 Hz), 47.0. IR (KBr)

1456 cm⁻¹. Anal. Calcd. For C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N, 11.89% Found: C,

78.07%; H, 5.01%; N, 12.02%.

6-Fluoro-1-(naphthalen-1-ylmethyl)-2-(pyridin-2-yl)-1H-benzo[d]imidazole (35). Yield = 50%. Mp.

160.7–161.1 °C. ¹H-NMR (CDCl₃) δ 8.49–8.40 (m, 2H), 8.16 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.0 Hz,

1H),7.84 (dd, J₁= 9.0 Hz, J₂= 4.9 Hz, 1H), 7.79 (dt, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.74 (d, J = 8.2 Hz,

1H), 7.63 (t, J₂= 7.7 Hz, J₂= 1.5 Hz, 1H), 7.57 (t, J = 7.2 Hz, 1H), 7.28–7.19 (m, 2H), 7.07 (td,

J₁= 9.2 Hz, J₂= 2.4 Hz, 1H), 6.88 (dd, J₁= 8.8 Hz, J₂= 2.5 Hz, 1H), 6.65 (s, 2H, CH₂), 6.63 (d,

13

J = 6.9 Hz, 1H). C-NMR (CDCl₃) δ 160.3 (d, J = 241.5 Hz), 151.2, 150.3, 148.8, 139.2, 137.4 (d,

J = 13.4 Hz), 136.9, 133.7, 132.3, 130.5, 129.0, 127.8, 126.5, 126.0, 125.6, 124.3, 123.9, 122.9,

122.42, 121.1 (d, J = 10.3 Hz), 111.6 (d, J = 25.1 Hz), 97.4 (d, J = 27.3 Hz), 47.1. IR (KBr) cm⁻¹:

1446. Anal. Calcd. For C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N, 11.89%. Found: C,

78.56%; H, 4.58%; N, 11.42%.

5,6-Dimethyl-1-(naphthalen-1-ylmethyl)-2-(pyridin-2-yl)-1H-benzo[d]imidazole (36). Yield = 85%.

1

Mp. 155.5–156.1 °C. H-NMR (CDCl₃) δ 8.44 (d, J = 7.8 Hz, 1H), 8.40 (d, J = 3.8 Hz, 1H), 8.19 (d,

J = 8.4 Hz, 1H), 7.92 (d, J = 7.5 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.73–7.67 (m, 2H), 7.67–7.61 (m,

1H), 7.60–7.55 (m, 1H), 7.23–7.16 (m, 2H), 6.99 (s, 1H), 6.65 (s, 2H, CH₂), 6.58 (d, J = 7.1 Hz, 1H),

13

2.40 (s, 3H, CH₃), 2.28 (s, 3H, CH₃). C-NMR (CDCl₃) δ 150.7, 148.6, 141.5, 136.7, 135.7, 133.6,

133.2, 133.0, 132.0, 130.5, 129.0, 127.4, 126.3, 125.8, 125.7, 124.3, 123.5, 122.7, 122.5, 120.9, 120.2,

110.7, 46.7, 20.6, 20.4. IR (KBr) 1446 cm⁻¹. Anal. Calcd. For C₂₅H₂₁N₃(MW 363.45): C, 82.61%; H,

5.82%; N, 11.56%. Found: C, 82.21%; H, 6.31%; N, 11.55%.

1-(Naphthalen-1-ylmethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole (37). Yield = 80%. Mp. 160.2–161.1 °C.

¹H-NMR (CDCl₃) δ 8.88–8.86 (s, 1H), 8.58 (d, J = 4.8 Hz, 1H), 7.94 (dt, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H),

7.91–7.84 (m, 3H), 7.77 (d, J = 8.3 Hz, 1H), 7.56–7.50 (m, 2H), 7.33–7.16 (m, 4H), 7.12 (d, J = 7.9 Hz,

1H), 6.79 (d, J = 7.1 Hz, 1H), 5.84 (s, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 151.3, 150.9, 149.6, 143.3,

136.4, 136.4, 133.8, 130.9, 130, 129.3, 128.6, 127.0, 126.4, 126.3, 125.7, 123.8, 123.6, 123.2, 122.9,

121.9, 120.3, 110.6, 46.6. IR (KBr) 1450 cm⁻¹. Anal. Calcd. For C₂₃H₁₇N₃(MW 335.40): C, 82.36%;

H, 5.11%; N, 12.53%. Found: C, 82.16%; H, 5.60%; N, 12.67%.

5-Fluoro-1-(naphthalen-1-ylmethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole (38). Yield = 58%. Mp.

163.4–163.9 °C. ¹H-NMR (CDCl₃) δ 8.94 (d, J = 0.01Hz, 1H), 8.66 (dd, J₁= 4.9 Hz, J₂= 1.7 Hz, 1H),

8.04–7.96 (m, 2H), 7.95–7.91 (m, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.64–7.58 (m, 3H), 7.40–7.30 (m,

2H), 7.10 (dd, J₁= 8.9 Hz, J₂= 4.6 Hz, 1H), 7.01 (td, J₁= 9.1, J₂= 2.4 Hz, 1H), 6.85 (dd, J₁= 7.2 Hz,

J₂= 1.3 Hz, 1H), 5.91 (s, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 159.9 (d, J = 238.7 Hz), 152.7, 151.0, 149.5,

143.7 (d, J = 13.0 Hz), 136.3, 133.9, 132.9, 130.6, 129.3, 128.8, 127.8, 127.0, 126.5, 125.7, 123.7,

122.8, 121.8, 114.1, 112.2 (d, J = 26.1 Hz), 111.1 (d, J = 10.4 Hz), 106.0 (d, J = 24.0 Hz), 46.8. IR

Molecules 2013, 18

3992

(KBr) 1476 cm⁻¹. Anal. Calcd. For C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N, 11.89%.

Found: C, 78.27%; H, 5.05%; N, 12.32%.

6-Fluoro-1-(naphthalen-1-ylmethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole (39). Yield = 56%. Mp.

1

156.1–157.0 °C. H-NMR (CDCl₃) δ 8.93 (s, 1H), 8.64 (dd, J₁= 4.9 Hz, J₂= 1.7 Hz, 1H), 8.01–7.94

(m, 2H), 7.93–7.89 (m, 1H), 7.89–7.82 (m, 2H), 7.64–7.57 (m, 2H), 7.35 (t, J = 7.7 Hz, 1H), 7.30 (dd,

J₁= 8.0 Hz, J₂= 4.9 Hz, 1H), 7.09 (dt, J₁= 9.2 Hz, J₂= 2.4 Hz, 1H), 6.89–6.84 (m, 2H), 5.86 (s, 2H,

CH₂). ¹³C-NMR (CDCl₃) δ 160.2 (d, J = 241.8 Hz), 156.4, 152.0 (d, J = 3.5 Hz), 150.9, 149.5, 139.7,

136.7 (d, J = 13.0 Hz), 136.2, 133.9, 130.4, 129.3, 128.8, 127.0, 126.5, 125.6, 123.6, 122.8, 121.8,

121.2 (d, J = 10.0 Hz), 114.1, 111.7 (d, J = 25.1 Hz), 97.3 (d, J = 27.8 Hz), 46.8. IR (KBr) 1480 cm⁻¹.

Anal. Calcd. For For C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N, 11.89%. Found: C, 78.61%;

H, 4.44%; N, 11.79%.

5,6-Dimethyl-1-(naphthalen-1-ylmethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole (40). Yield = 90%.

1

Mp. 141.4–142.1 °C. H-NMR (CDCl₃) δ 8.88 (s, 1H), 8.59 (d, J = 4.4 Hz, 1H), 7.99–7.90 (m, 3H),

7.83 (d, J = 8.2 Hz, 1H), 7.69 (s, 1H), 7.64–7.57 (m, 2H), 7.32 (t, J = 7.7 Hz, 1H), 7.27–7.21 (m, 1H),

6.94 (s, 1H), 6.85 (d, J = 7.1 Hz, 1H), 5.81 (s, 2H, CH₂), 2.40 (s, 3H, CH₃), 2.27 (s, 3H, CH₃).

¹³C-NMR (CDCl₃) δ 150.5, 150.4, 149.4, 141.9, 136.2, 135.1, 133.8, 133.2, 132.2, 131.2, 130.0, 129.2,

128.5, 126.9, 126.5, 126.4, 125.8, 123.5, 122.9, 122.0, 120.2, 110.6, 46.5, 20.6, 20.4. IR (KBr) 1440

cm⁻¹. Anal. Calcd. For C₂₅H₂₁N₃(MW 363.45): C, 82.61%; H, 5.82%; N, 11.56%. Found: C, 83.11%;

H, 5.92%; N, 11.57%.

1-(Naphthalen-1-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole (41). Yield = 93%. Mp. 163.3–163.8 °C.

¹H-NMR (CDCl₃) δ 8.56 (d, J = 6.1 Hz, 2H), 7.97–7.88 (m, 3H), 7.81 (d, J = 8.3 Hz, 1H), 7.62–7.54

(m, 4H), 7.33 (t, J = 7.6 Hz, 1H), 7.28–7.24 (m, 1H), 7.22–7.16 (m, 1H), 7.11 (d, J = 8.1 Hz, 1H), 6.81

13

(d, J = 7.1 Hz, 1H), 5.84 (s, 2H, CH₂). C-NMR (CDCl₃) δ 151.3, 150.5(2C), 143.2, 137.5, 136.6,

133.9, 130.9, 129.9, 129.4, 129.3, 128.7, 126.5, 125.7, 124.1, 123.6, 123.4, 122.9(2C), 121.9, 120.6,

110.7, 46.6. IR (KBr) 1449 cm⁻¹. Anal. Calcd. For C₂₃H₁₇N₃(MW 335.40): C, 82.36%; H, 5.11%; N,

12.53%. Found: C, 82.86%; H, 5.20%; N, 12.77%.

5-Fluoro-1-(naphthalen-1-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole (42). Yield = 53%. Mp.

151.5–152.5 °C. ¹H-NMR (DMSO-d6) δ 8.65 (d, J = 5.2 Hz, 2H), 8.05–7.93 (m, 2H), 7.89 (d, J = 8.4 Hz,

1H), 7.70–7.55 (m, 5H), 7.38 (t, J = 7.7 Hz, 1H), 7.12 (dd, J₁= 8.8 Hz, J₂= 4.6 Hz, 1H), 7.03 (td,

J₁= 8.9 Hz, J₂= 2.4 Hz, 1H), 6.86 (d, J = 7.2 Hz, 1H), 5.93 (s, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 160.0

(d, J = 238.6 Hz), 152.7, 150.6(2C), 143.6 (d, J = 12.8 Hz), 137.2, 133.9, 133.1, 130.5, 129.9, 129.4,

128.9, 127.8 (d, J = 5.1 Hz), 127.1, 126.6, 125.7, 122.8(2C), 121.8, 112.7 (d, J = 26.3 Hz), 111.2 (d,

J = 10.0 Hz), 106.2 (d, J = 24.2 Hz), 46.8. IR (KBr) 1471 cm⁻¹. Anal. Calcd. For C₂₃H₁₆FN₃(MW

353.39): C, 78.17%; H, 4.56%; N, 11.89%. Found: C, 78.67%; H, 4.55%; N, 12.30%.

6-Fluoro-1-(naphthalen-1-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole (43). Yield = 60%. Mp.

1

167.4–168.1°C. H-NMR (CDCl₃) δ 8.63 (d, J = 5.6 Hz, 2H), 8.04–7.96 (m, 1H), 7.98–7.91 (m, 1H),

7.91–7.84 (m, 2H), 7.74–7.45 (m, 4H), 7.38 (t, J = 7.7 Hz, 1H), 7.12 (td, J₁= 8.9 Hz, J₂= 2.4 Hz, 1H),

6.91–6.84 (m, 2H), 5.89 (s, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 160.4 (d, J = 241.6 Hz), 152.0, 150.5(2C),

Molecules 2013, 18

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139.6, 137.2, 133.9, 130.3, 129.9, 129.4, 128.9, 127.8 (d, J = 6.7 Hz), 127.1, 126.6, 125.7, 123.1,

122.8(2C), 121.8, 121.5 (d, J = 9.8 Hz), 112.0 (d, J = 25.3 Hz), 97.3 (d, J = 27.5 Hz), 46.8. IR (KBr)

1431 cm⁻¹. Anal. Calcd. For C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N, 11.89%. Found: C,

78.38%; H, 5.05%; N, 12.21%.

5,6-Dimethyl-1-(naphthalen-1-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole (44). Yield = 92%.

1

Mp. 156.8–157.6 °C. H-NMR (CDCl₃) δ 8.59 (s broad, 2H), 7.98 (s broad, 2H), 7.86 (d, J = 7.0 Hz,

1H), 7.74–7.53 (m, 5H), 7.39–7.31 (m, 1H), 6.96 (s, 1H), 6.86 (d, J = 6.8 Hz, 1H), 5.87 (s, 2H, CH₂),

13

2.41 (s, 3H, CH₃), 2.29 (s, 3H, CH₃). C-NMR (CDCl₃) δ 150.4(2C), 141.9, 137.7, 135.3, 133.9,

133.7, 132.5, 131.1, 129.9, 128.6, 127.9, 127.0, 126.5, 125.8, 123.1, 122.9, 122.8(2C), 122.0, 120.5,

110.6, 46.6, 20.6, 20.4. IR (KBr) 1455 cm⁻¹. Anal. Calcd. For C₂₅H₂₁N₃(MW 363.45): C, 82.61%; H,

5.82%; N, 11.56%. Found: C, 83.07%; H, 6.30%; N, 12.01%.

1-(Naphthalen-2-ylmethyl)-2-(pyridin-2-yl)-1H-benzo[d]imidazole (45). Yield = 87%. Mp. 150.7–151.8 °C.

¹H-NMR (CDCl₃) δ 8.74 (d, J = 4.2 Hz, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.95

(td, J = 7.9, 1.7 Hz, 1H), 7.89 (dd, J₁= 6.0 Hz, J₂= 3.4 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.80 (dd,

J₁= 6.0 Hz, J₂= 3.4 Hz, 1H), 7.67 (s, 1H), 7.56–7.51 (m, 2H), 7.51–7.45 (m, 2H), 7.44–7.40 (m, 2H),

7.37 (d, J = 6.6 Hz, 1H), 6.48 (s, 2H, CH₂).¹³C-NMR (CDCl₃) δ 150.6, 150.1, 148.7, 142.8, 136.9,

136.9, 135.0, 133.3, 132.7, 128.5, 127.8, 127.7, 126.2, 125.9, 125.4, 124.9, 124.7, 123.9, 123.7, 122.9,

120.2, 110.8, 49.2. IR (KBr) 1466 cm⁻¹. Anal. Calcd. For C₂₃H₁₇N₃(MW 335.40): C, 82.36%; H,

5.11%; N, 12.53%. Found: C, 82.06%; H, 5.61%; N, 12.05%.

5-Fluoro-1-(naphthalen-2-ylmethyl)-2-(pyridin-2-yl)-1H-benzo[d]imidazole (46). Yield = 62%. Mp.

1

165.7–166.4 °C. H-NMR (CDCl₃) δ 8.65 (d, J = 3.9 Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H), 7.87 (td,

J₁= 7.9 Hz, J₂= 1.9 Hz, 1H), 7.83–7.76 (m, 2H), 7.72 (dd, J₁= 6.2 Hz, J₂= 3.3 Hz, 1H), 7.61–7.54

(m, 2H), 7.46 (d, J = 6.3 Hz, 1H), 7.45 (d, J = 6.2 Hz, 1H), 7.39–7.32 (m, 2H), 7.29 (dd, J₁= 9.0 Hz,

J₂= 4.4 Hz, 1H), 7.02 (td, J₁= 9.1 Hz, J₂= 2.4 Hz, 1H), 6.37 (s, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 159.8

(d, J = 238.1 Hz), 151.3, 150.3, 148.8, 143.2, 143.1, 137.0, 134.7, 133.4, 133.3, 132.8, 128.6, 127.8 (d,

J = 9.7 Hz), 126.3, 126.0, 125.8, 125.4, 124.8 (d, J = 2.8 Hz), 124.1, 112.1 (d, J = 26.4 Hz), 111.3 (d,

J = 10.2 Hz), 105.7 (d, J = 23.9 Hz), 49.4. IR (KBr) 1484 cm⁻¹. Anal. Calcd. For C₂₃H₁₆FN₃(MW

353.39): C, 78.17%; H, 4.56%; N, 11.89%. Found: C, 77.95%; H, 5.02%; N, 12.35%.

6-Fluoro-1-(naphthalen-2-ylmethyl)-2-(pyridin-2-yl)-1H-benzo[d]imidazole (47). Yield = 55%. Mp.

1

170.5–171.3 °C. H-NMR (CDCl₃) δ 8.65 (d, J = 5.8 Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 7.9–7.76 (m,

4H), 7.73 (dd, J₁= 6.2 Hz, J₂= 3.4 Hz, 1H), 7.58 (s, 1H), 7.47 (d, J = 6.3 Hz, 1H), 7.46 (d, J = 6.2 Hz,

1H), 7.40–7.31 (m, 2H), 7.13–7.02 (m, 2H), 6.34 (s, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 160.3 (d,

J = 211.9 Hz), 150.4, 148.7, 145.1, 139.2, 137.2, 137.0, 134.5, 133.3, 132.8, 128.6, 128.1, 127.8 (d,

J = 12.1 Hz), 126.3, 126.0, 125.5, 124.8, 124.5, 124.0, 121.0 (d, J = 9.8 Hz), 111.5 (d, J = 25.1 Hz),

97.4 (d, J = 27.9 Hz), 49.4. IR (KBr) 1464 cm⁻¹. Anal. Calcd. For C₂₃H₁₆FN₃(MW 353.39): C,

78.17%; H, 4.56%; N, 11.89%. Found: C, 78.15%; H, 4.52%; N, 12.33%.

5,6-Dimethyl-1-(naphthalen-2-ylmethyl)-2-(pyridin-2-yl)-1H-benzo[d]imidazole (48). Yield = 77%.

Mp. 147.7–148.5 °C. ¹H-NMR (CDCl₃) δ 8.61 (d, J = 4.3 Hz, 1H), 8.45 (d, J = 7.9 Hz, 1H), 7.89–7.75

Molecules 2013, 18

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(m, 3H), 7.67 (s, 1H), 7.58–7.49 (m, 2H), 7.48–7.41 (m, 2H), 7.36 (d, J = 8.5 Hz, 1H), 7.31–7.26 (m,

1H), 7.15 (s, 1H), 6.33 (s, 2H, CH₂), 2.41 (s, 3H, CH₃), 2.35 (s, 3H, CH₃). ¹³C-NMR (CDCl₃) δ 150.8,

148.6, 141.5, 136.8, 135.6, 135.3, 133.3, 133.1, 132.7, 131.9, 128.4, 127.8, 127.7, 126.2, 125.8, 125.4,

125.2, 124.9, 124.5, 123.6, 120.1, 110.8, 49.1, 20.8, 20.4. IR (KBr) 1440 cm⁻¹. Anal. Calcd. For

C₂₅H₂₁N₃(MW 363.45): C, 82.61%; H, 5.82%; N, 11.56%. Found: C, 82.97%; H, 6.15%; N, 11.50%.

1-(Naphthalen-2-ylmethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole (49). Yield = 86%. Mp. 146.3–147.0 °C.

¹H-NMR (CDCl₃) δ 9.02 (s broad, 1H), 8.74 (s broad, 1H), 8.06 (d, J = 7.3 Hz, 1H), 7.97 (d, J = 7.6 Hz,

1H), 7.92–7.82 (m, 2H), 7.72 (d, J = 7.6 Hz, 1H), 7.57–7.46 (m, 3H), 7.44–7.37 (m, 2H), 7.35–7.24

(m, 3H), 5.65 (s, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 150.9, 136.8, 136.3, 135.0, 133.4, 133.3, 133.0,

131.7, 129.3, 127.9, 127.8, 126.8, 126.5, 124.6, 123.8, 123.6, 123.2, 120.3, 114.1, 112.8, 110.7, 110.7,

48.6. IR (KBr) 1459.5 cm⁻¹. Anal. Calcd. For C₂₃H₁₇N₃(MW 335.40): C, 82.36%; H, 5.11%; N,

12.53%. Found: C, 81.99%; H, 5.54%; N, 12.55%.

5-Fluoro-1-(naphthalen-2-ylmethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole (50). Yield = 66%. Mp.

175.7–176.8 °C ¹H-NMR (CDCl₃) δ 9.00 (s, 1H), 8.74 (s broad, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.93–7.80

(m, 3H), 7.73 (d, J = 7.9 Hz, 1H), 7.56–7.50 (m, 2H), 7.48–7.46 (m, 1H), 7.43–7.37 (m, 1H), 7.26 (d,

J = 8.5 Hz, 1H), 7.13 (td, J₁= 9.2 Hz, J₂= 2.3 Hz, 1H), 6.99 (dd, J₁= 8.5 Hz, J₂= 2.4 Hz, 1H), 5.62 (d,

J = 14.7 Hz, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 160.1 (d, J = 234.2 Hz), 151.9, 151.1, 149.7, 139.7, 136.6

(d, J = 8.2 Hz), 133.4, 133.0, 132.8, 129.5, 127.9 (d, J = 4.8 Hz), 126.8, 126.6, 126.3, 124.6, 123.6,

123.5, 121.2 (d, J = 10.1 Hz), 112.3, 111.7 (d, J = 25.7 Hz), 105.9, 97.4 (d, J = 27.5 Hz), 48.8. IR

(KBr) 1478 cm⁻¹. Anal. Calcd. For C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N, 11.89%.

Found: C, 77.88%; H, 4.96%; N, 11.39%.

6-Fluoro-1-(naphthalen-2-ylmethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole (51). Yield = 62%. Mp.

1

166.8–167.4 °C. H-NMR (CDCl₃) δ 9.00 (s, 1H), 8.75 (d, J = 4.8 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H),

7.94–7.83 (m, 2H), 7.77–7.70 (m, 1H), 7.61 (dd, J₁= 9.1 Hz, J₂= 2.4 Hz, 1H),7.56–7.50 (m, 2H), 7.48

(s, 1H), 7.42 (dd, J₁= 8.0 Hz, J₂= 4.8 Hz, 1H), 7.31–7.24 (m, 1H), 7.22 (dd, J₁= 8.8 Hz, J₂= 4.5 Hz, 1H),

7.06 (td, J₁= 9.0 Hz, J₂= 2.3 Hz, 1H), 5.65 (s, 2H, CH₂).¹³C-NMR (CDCl₃) δ 159.9 (d, J = 238.9 Hz),

151.1, 149.7, 144.4, 143.7, 136.7, 133.4, 133.0, 130.0, 129.4, 129.4, 127.8 (d, J = 4.5 Hz), 126.9, 126.6,

125.9, 124.6, 123.5, 118.4, 116.1, 112.2 (d, J = 26.3 Hz), 111.1 (d, J = 10.2 Hz), 106.0 (d, J = 24.3 Hz),

48.8. IR (KBr) 1500 cm⁻¹. Anal. Calcd. For C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N,

11.89%. Found: C, 78.30%; H, 4.59%; N, 11.92%.

5,6-Dimethyl-1-(naphthalen-2-ylmethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole (52). Yield = 96%.

1

Mp. 138.5–139.5 °C. H-NMR (CDCl₃) δ 8.99 (s broad, 1H), 8.70 (s broad, 1H), 8.03 (d, J = 7.5 Hz,

1H), 7.94–7.82 (m, 2H), 7.77–7.66 (m, 2H), 7.56–7.44 (m, 3H), 7.36 (s broad, 1H), 7.29 (d, J = 6.5 Hz,

1H), 7.08 (s, 1H), 5.60 (s, 2H, CH₂), 2.44 (s, 3H, CH₃), 2.35 (s, 3H, CH₃). ¹³C-NMR (CDCl₃) δ 150.7,

142.0, 136.5, 135.0, 133.7, 133.5, 133.1, 132.9, 132.2, 129.3, 127.9, 127.8, 126.7, 126.4, 124.5, 123.6,

123.5, 120.3, 110.6, 107.1, 103.8, 103.0, 48.5, 20.6, 20.4. IR (KBr) 1447 cm⁻¹. Anal. Calcd. For

C₂₅H₂₁N₃(MW 363.45): C, 82.61%; H, 5.82%; N, 11.56%. Found: C, 82.66%; H, 5.92%; N, 11.66%.

Molecules 2013, 18

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1-(Naphthalen-2-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole (53). Yield = 94%. Mp. 133.0–131.0 °C.

¹H-NMR (CDCl₃) δ 8.73 (d, J = 5.9 Hz, 2H), 7.97 (d, J = 8.1 Hz, 1H), 7.94–7.86 (m, 2H), 7.73 (d,

J = 6.6 Hz, 1H), 7.67 (d, J = 5.9 Hz, 2H), 7.58–7.48 (m, 3H) 7.45–7.37 (m, 1H), 7.36–7.28 (m, 3H),

13

5.68 (s, 2H, CH₂). C-NMR (CDCl₃) δ 150.5(2C), 143.2, 137.7, 136.5, 133.3, 129.4, 127.9, 127.8,

126.8, 126.5, 124.6, 124.2, 123.5, 123.4, 123.2(2C), 120.6, 119.5, 119.3, 118.7, 110.7, 48.7. IR (KBr)

1437 cm⁻¹. Anal. Calcd. For C₂₃H₁₇N₃(MW 335.40): C, 82.36%; H, 5.11%; N, 12.53%. Found: C,

81.22%; H, 5.60%; N, 12.68%.

5-Fluoro-1-(naphthalen-2-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole (54). Yield = 50%. Mp.

1

177.8–178.5 °C. H-NMR (CDCl₃) δ 8.76 (s broad, 2H), 7.95–7.85 (m, 2H), 7.77–7.71 (m, 1H), 7.67

(s broad, 2H), 7.62 (dd, J₁= 9.2 Hz, J₂= 2.4 Hz, 1H), 7.58–7.51 (m, 2H), 7.48 (s, 1H), 7.32–7.26 (m,

1H), 7.22 (dd, J₁= 8.8 Hz, J₂= 4.4 Hz, 1H), 7.06 (td, J₁= 9.1 Hz, J₂= 2.4 Hz, 1H), 5.66 (s, 2H, CH₂).

¹³C-NMR (CDCl₃) δ 158.7, 151.5 (d, J = 214.6 Hz), 143.5, 138.7, 137.4, 133.3 (d, J = 12.0 Hz), 133.0,

132.9(2C), 131.7, 129.5, 127.8(2C), 127.7, 126.9, 126.6, 125.1, 124.6, 123.4, 119.1, 112.6 (d, J = 26.5

Hz), 111.2 (d, J = 11.0 Hz), 106.2 (d, J = 24.3 Hz), 48.8. IR (KBr) 1585 cm⁻¹. Anal. Calcd. For

C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N, 11.89%. Found: C, 78.66%; H, 4.57%; N, 11.79%.

6-Fluoro-1-(naphthalen-2-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole (55). Yield = 53%. Mp.

1

163.5–164.1 °C. H-NMR (CDCl₃) δ 8.79 (s broad, 2H), 8.00–7.82 (m, 3H), 7.80–7.60 (m, 2H),

7.59–7.44 (m, 3H), 7.37–7.25 (m, 2H), 7.15 (td, J₁= 9.2 Hz, J₂= 2.4 Hz, 1H), 6.99 (dd, J₁= 8.5 Hz,

J₂= 2.4 Hz, 1H), 5.64 (s, 2H, CH₂). ¹³C-NMR (CDCl₃) δ 153.3 (d, J = 227.6 Hz), 150.4, 147.3, 144.0,

136.0, 134.2, 133.4, 132.7, 132.7, 129.6, 127.9(2C), 126.9, 126.6, 124.6, 124.1, 123.4(2C), 121.5 (d,

J = 8.1 Hz), 120.5, 112.0 (d, J = 23.3 Hz), 97.4 (d, J = 26.9 Hz), 48.9. IR (KBr) 1497 cm⁻¹. Anal.

Calcd. For C₂₃H₁₆FN₃(MW 353.39): C, 78.17%; H, 4.56%; N, 11.89%. Found: C, 78.18%; H, 4.99%;

N, 11.55%.

5,6-Dimethyl-1-(naphthalen-2-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole (56). Yield = 90%.

1

Mp. 133.7–134.5 °C. H-NMR (CDCl₃) δ 8.69 (d, J = 6.0 Hz, 2H), 7.96–7.85 (m, 2H), 7.77–7.69 (m,

2H), 7.64 (d, J = 6.0 Hz, 2H), 7.58–7.51 (m, 2H), 7.49 (s, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.08 (s, 1H),

13

5.64 (s, 2H, CH₂), 2.44 (s, 3H, CH₃), 2.35 (s, 3H, CH₃). C-NMR (CDCl₃) δ 162.2, 161.3, 150.5,

150.4(2C), 142.2, 133.7, 129.4, 127.9, 127.8, 126.8, 126.5, 126.5, 124.8, 124.4, 123.6, 123.1(2C),

120.4, 115.5, 112.8, 110.7, 109.1, 96.5, 50.2. IR (KBr) 1450 cm⁻¹. Anal. Calcd. For C₂₅H₂₁N₃(MW

363.45): C, 82.61%; H, 5.82%; N, 11.56%. Found: C, 83.06%; H, 5.88%; N, 11.78%.

3.2. Pharmacological Evaluation Binding Assays

For the CB1 receptor binding assays the new compounds were first subjected to a preliminary

screen carried out using three concentrations of test compounds (1, 0.1 and 0.01 µM), human

cannabinoid CB1 receptor (PerkinElmer Inc., Waltham, MA, USA, Product No.: 6110129400UA, Lot

No.: 509–845-A) and [³H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxy-

propyl)cyclohexanol ([³H]CP-55,940; K_d= 500 pM) as the high affinity ligand as described by the

manufacturer (Perkin-Elmer). Compounds which displaced [³H]CP-55,940 by more than 50% at

1 µM were further analyzed. Displacement curves were generated by incubating drugs with 0.5 nM of

Molecules 2013, 18

3996

[³H]CP-55,940. In all cases, K_ivalues were calculated by applying the Cheng-Prusoff equation [34] to

the IC₅₀values (obtained by GraphPad, GraphPad Software, Inc., La Jolla, CA, USA) for the

displacement of the bound radioligand by increasing concentrations of the test compounds (after three

separate experiments for each compound).

3.3. Construction of the hCB1 Receptor Homology Model

The sequence of the human CB1 receptor was retrieved from UniProt database with accession

number P21554. The crystal structure of human adenosine A_2Areceptor (PDB ID: 3EML) [35], was

identified from the Protein Data Bank (PDB) and selected as the template for homology modeling

according to the results of a BLAST search. The CB1R model was generated by comparative modeling

using MODELLER v9.10 [36,37]. The selection of the template was reinforced with evidence from

molecular phylogenetic reports that indicate that this receptor is more evolutionarily related to CB1

than the other members of family A GPCRs for which a crystal structure exists. The Clustal obtained

alignment was manually modified to preserve functionally important residues and the disulfide bridge

between residues C257 and C264. The residues corresponding to segment 306–324 of intracellular

loop 3 were not considered in the modeling. The final alignment obtained between both sequences

presents 25.4% and 49.2% of sequence identity and similarity, respectively. Most conserved residues

within each helix are denoted using the Ballesteros-Weinstein notation [38]. A set of 100 models was

generated, and the best model according to the internal scoring function of the program (PDF score)

was subjected to an energy minimization protocol in order to relax the structure and optimize bond

geometry. Model validation was performed using the tools available at the NIH SAVES server

(http://nihserver.mbi.ucla.edu/SAVES/).

The model presents an alpha-carbon root-mean square deviation of 0.64 Å against the template

structure (Figure 9A). The final model contains residues 113–420, with a disulfide bridge between

C257 and C264, and presents more than 97% of the residues in allowed regions according to

Ramachandran plot analysis [39] (Figure 9B).

3.4. Docking of the Library of Compounds

Molecular docking of the compounds was performed using FRED v2.2.5 (OpenEye Scientific

Software, Santa Fe, NM, USA). The CB1 model binding site was prepared using the FRED receptor,

considering the residues involved in the interaction of CB1 with WIN55212, as our group has reported

previously [24]. A multiconformer library was prepared using OMEGA v2.4.3 (OpenEye Scientific

Software). Candidate poses of the ligands within the receptor site (100) were obtained and optimized

using the Chemgauss3 scoring function. Consensus structures of the poses returned from exhaustive

docking and optimization were obtained by consensus scoring using the PLP, Chemscore and

Chemgauss3 scoring functions. Finally, the top ranked binding modes for each compound were

minimized using the CHARMM22 force-field in Discovery Studio v2.1 (Accelrys Inc., San Diego,

CA, USA). The minimization protocol allows movement of the side chains of residues positioning

within a radius of 6 Å from the mass centroid of all docked ligands, using the conjugate gradient

algorithm to a convergence criterion of 0.001 kcal/mol Å for the RMS of energy gradient. Energy

evaluation was performed for each obtained complex using the PLP, LigScore, PMF, and LUDI

Molecules 2013, 18

3997

scoring functions. Additional binding energy calculations were performed using the PBSA (Poisson

Boltzmann with non-polar Surface Area) implicit solvent model [40], with a dielectric constant of 4 in

order to simulate the membrane environment for the complexes.

Figure 9. (A) Superimposition of the obtained CB1R model (red) with the A_2AR template

(blue). The plasma membrane is delimited by the red and blue dotted lines (B)

Ramachandran plot for the CB1R model.

3.5. Generation of CoMFA and Partial Least Squares (PLS) Analysis

CoMFA studies were performed using SYBYL-X 1.2 molecular modeling software (Tripos Inc., St.

Louis, MO, USA). Models of steric and electrostatic fields for CoMFA were based on both Lennard–

Jones and Coulombic potentials [41]. Steric and electrostatic energies were calculated using an sp³

carbon atom with a van der Waals radius of 0.152 nm, +1 charge, and 0.2 nm grid spacing. The

CoMFA cutoff values were set to 125.46 kJ/mol for both steric and electrostatic fields.

PLS analysis was used to construct a linear correlation between the CoMFA descriptors

(independent variables) and the affinity values (dependent variables) [42]. To select the best model, the

cross-validation analysis was performed using the LOO method (and SAMPLS), which deduces the

square of the cross-validation coefficient (q²) and the optimal number of components N. The non-

cross-validation was performed with a column filter value of 2.0 to speed up the analysis and reduce

the noise. The predictive r²(r²_pred) [43,44] was based only on molecules not included in the training set

and was obtained according to the following equation: r²_pred= (SD − PRESS)/SD (Table 2).

Molecules 2013, 18

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4. Conclusions

In summary, we have discovered a series of novel and potent CB1 receptor ligands. The

benzimidazole derivatives were rationally designed based on chemical modifications of the prototype

compound 14. A major focus of the optimization effort was to increase CB1 affinity.

Biological characterization of these compounds in radioligand binding assays established that the

position of the nitrogen atom on the pyridine ring is crucial for activity. Both 3- and 4-pyridyl groups

yield active compounds, but 3-pyridyl is a much better substituent than 4-pyridyl and the 2-pyridyl

seems useless. These results suggest possible hydrogen bonding with K3.28(192). No significant

differences in affinity were observed when the carbonyl linker was replaced by a methylene group.

Changing the position of the naphthyl ring from α to β resulted in a decrease of CB1 receptor

affinities. Only two of the most potent derivatives are not substituted on the benzimidazole ring,

suggesting that substitution at the C5 and/or C6 positions is beneficial for CB1 affinity, probably by

enhancing -stacking interactions in which F2.57(170), F3.36(200) and W6.48(356) may be involved.

The QSAR model gave good statistical results in terms of q²and r²values. Our CoMFA analysis

based on binding affinity data of benzimidazole ligands for the CB1 receptor allowed us to approach

optimal substitution in the regions labeled I, II and III. The analysis of the contour maps indicates that

the activity of the compounds is due largely to their electronic properties rather than to steric or

lipophilic contributions, defining two key areas for further chemical modification: the naphthalene and

the pyridine rings. The information gained in this work should lead to the development of additional

high-affinity CB1 receptor ligands.

Acknowledgments

Financial support under FONDECYT Projects 1100493 and 1100542 to CDPM and PIV is

gratefully acknowledged. CFL, CEB and JRP are CONICYT PhD fellows. CFL thanks OpenEye

Scientific Software and Inteligand for academic software licenses. S. D. G.

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Design, synthesis, binding and docking-based 3D-QSAR studies of 2-pyridylbenzimidazoles - A new family of high affinity CB1 cannabinoid ligands

DOI: 10.3390/molecules18043972

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