Design, synthesis and antimicrobial evaluation of novel benzimidazole type of Fluconazole analogues and their synergistic effects with Chloromycin, Norfloxacin and Fluconazole

European Journal of Medicinal Chemistry 64 (2013) 329e344

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Design, synthesis and antimicrobial evaluation of novel benzimidazole

type of Fluconazole analogues and their synergistic effects with

Chloromycin, Norﬂoxacin and Fluconazole

1

*

Hui-Zhen Zhang, Guri L.V. Damu , Gui-Xin Cai , Cheng-He Zhou

Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing 400715,

People’s Republic of China

a r t i c l e i n f o

a b s t r a c t

A novel series of benzimidazole type of Fluconazole analogues were synthesized and characterized by ¹

H

Article history:

NMR, ¹³C NMR, IR, MS and HRMS spectra. All the new compounds were screened for their antimicrobial

activities in vitro by two-fold serial dilution technique. The bioactive evaluation showed that 3,5-

bis(triﬂuoromethyl)phenyl benzimidazoles gave comparable or even stronger antibacterial and anti-

fungal efﬁciency in comparison with reference drugs Chloromycin, Norﬂoxacin and Fluconazole. The

combination of 2,4-diﬂuorobenzyl benzimidazole derivative 5m and its hydrochloride 7 respectively

with antibacterial Chloromycin, Norﬂoxacin or antifungal Fluconazole showed better antimicrobial ef-

ﬁciency with less dosage and broader antimicrobial spectrum than the separated use of them alone.

Notably, these combined systems were more sensitive to Fluconazole-insensitive Aspergillus ﬂavus and

methicillin-resistant MRSA.

Received 25 January 2013

Received in revised form

20 March 2013

Accepted 24 March 2013

Available online 9 April 2013

Keywords:

Benzimidazole

Antibacterial

Antifungal

Fluconazole

Norﬂoxacin

Chloromycin

1. Introduction

new more effective antifungal agents with broader antimicrobial

spectrum and better therapeutic indexes [2e5]. Currently, the

Fluconazole is a well known ﬁrst-line antifungal drug recom-

mended by World Health Organization and is prevalently used to

treat fungal infections by Candida albicans and Cryptococcus neo-

formans with an exceptional therapeutic record including good

activity and favourable pharmacokinetic characteristics. It is

commonly considered that the triazole ring in Fluconazole could

efﬁciently coordinate with the iron(II) ion of heme to restrain the

biosynthesis of ergosterol and thus inhibiting the growth of fungi.

However, this action generally leads to severe toxicity and pre-

cludes its application in the treatment of deep-seated mycoses and

life-threatening systemic infections [1]. Moreover, its increasingly

serious drug resistance, narrow antifungal spectrum and low ac-

tivity against non-Candida fungi have attracted a large amount of

effort towards further researches of Fluconazole in order to develop

related work is mainly involved in the development of its prodrugs,

structural modiﬁcation of the side chains and exploitation of its

analogues with new structural skeletons. Especially, the explora-

tion of new leading compounds with wholly new structural mol-

ecules similar to the basic backbone of Fluconazole has become one

of the predominant directions [4,6,7]. Some Fluconazole analogues

such as Ravuconazole, Albaconazole and Isavuconazole have been

successfully developed as clinical drugs for the treatment of fungal

infections [8,9].

It is well known that tertiary amine moiety is an important

structural fragment in bioactive compounds. This type of molecular

fragment is able to form hydrogen bonds, coordinate with metal

ions and accept protons and perform quaternization, and could not

only regulate physicochemical properties of desired molecules but

also interact with various enzymes and receptors in biological

system which exerts broad bioactive spectrum. Therefore, tertiary

amine fragment is widely present in clinical drugs like analgesic

Methadone Hydrochloride and Fentanyl Citrate, antiallergic Cetir-

izine, antitumour Melphalan and Cyclophosphamide and so on

[10]. Recently, a tertiary amine type of Fluconazole analogues has

been found to show large potential as new type of antimicrobial

* Corresponding authors. Tel./fax: þ86 23 68254967.

E-mail addresses: gxcai@swu.edu.cn (G.-X. Cai), zhouch@swu.edu.cn,

zhouch6848@sina.com (C.-H. Zhou).

1

Postdoctoral fellow from Indian Institute of Chemical Technology (IICT),

Hyderabad 500 607, India.

http://dx.doi.org/10.1016/j.ejmech.2013.03.049

330

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

agents in which the tertiary alcohol moiety in Fluconazole was

replaced by a tertiary amine fragment that may exert the same

function with the active site residue H310 as the tertiary alcohol

moiety in Fluconazole [11,12]. Like Fluconazole, this tertiary amine

type of bis-triazole and bis-imidazole derivatives exhibited good

antifungal activity. Interestingly, they also gave promising anti-

bacterial efﬁciency [13,14]. This compels us with overwhelming

interest to continuously investigate this tertiary amine type of

special Fluconazole analogues. Benzimidazole is a fused ring of

imidazole with benzene having larger conjugated system and

electron richer properties than either triazole or imidazole, this

unique structure endows its derivatives to possess extensive po-

tential in medicinal chemistry especially antimicrobial aspect [15].

Herein we would like to develop this tertiary amine type of benz-

imidazole derivatives (Fig. 1) as wholly new structure type of

antimicrobial agents which were expected to play large roles in the

treatment of disease-caused fungi and bacteria.

number of researches have been directing towards benzimidazole-

based medicinal drugs and recently a lot of work has shown that

benzimidazoles possess much potential to inhibit the growth of

bacterial and fungal strains [20]. Benzimidazole is structurally

similar to purine, and its derivatives could compete with purines,

resulting in the distinct inhibition of the synthesis of nucleic acids

and proteins inside the bacterial cell wall, thereby killing the bac-

terial strains or inhibiting their growth. These observations showed

that the mechanism of benzimidazole type of antibacterial agents

was different from that of well known classes of antibacterial drugs,

thus development of benzimidazole derivatives as novel antibac-

terial agents may be served as a good strategy to overcome bacterial

resistances. Notably, benzimidazole ring has been actively

employed to modify natural antibiotics like macrocyclic lactones

and

b-lactams and synthetic antibacterial drugs [15]. The

benzimidazole-modiﬁed derivatives usually exhibited much better

antibacterial efﬁcacy than their corresponding precursors, espe-

cially benzimidazole-modiﬁed antibacterial ﬂuoroquinolone

(BMAF) with piperazine type of tertiary amine fragment also

showed signiﬁcant activity against C. albicans [21]. More impor-

tantly, some structurally simple benzimidazole compounds like

benzimidazole amines BA and ZSL gave strong antimicrobial efﬁ-

cacy [19,22,23]. Especially high anti-Klebsiella pneumoniae activity

The special benzene-fused imidazole ring could readily interact

with various active targets in biological system via diverse non-

covalent interactions like hydrogen bonds, coordination, ion-

dipole, cationep, pep stacking and hydrophobic effect as well as

van der Waals force, therefore benzimidazole-based derivatives

exhibit various bioactivities [16e19]. So far many benzimidazole

compounds have been successfully developed as clinical drugs such

as antihistaminic Astemizole, anti-anabrotic Omeprazole, antipar-

asitic Albendazole and antihypertensive Candesartan, and have

been prevalently used in treatment of various diseases. A large

was observed for compound BA with MIC value of 0.5

comparable to the reference drug Cefoperazone, and compound

ZSL gave strong inhibitory activity against MRSA (MIC ¼ 2 g/mL)

(Fig. 2). All these clearly point out that benzimidazole derivatives

mg/mL, nearly

m

Enhance the binding ability

between the drugs and the active

sites of target enzymes

NH

R⁴

N

Bind with the

R

prosthetic group

iron(II) ion of

N

cytochrome P450

N N

R¹

R²

N N

Form hydrogen

bond with H₃₁₀

in the active site

OH

R³

F

R = alkyl, aralkyl

R¹, R², R³, R⁴=

Regulate

physicochemical

properties

H, F, Cl, CH₃, CF₃, NO₂

Target compounds

F

Clinical Fluconazole

N

Z N

Z

N

X¹

X²

Z = N, CH,

X¹, X², X³= H, F, Cl

X³

The reported tertiary amine bis-azoles

Fig. 1. Design of benzimidazole tertiary amine type of Fluconazole analogues.

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

331

N

H

O

N

O

N

Bu-n

N

H

N

F

i-Pr

H₃C

HO

CH₃

8

S

O

H₃C

8

N

O

NH

OH

HN

N

H

N

H

BA

BMAF

ZSL

Fig. 2. Structures of some antimicrobial benzimidazole derivatives.

possess large potential as new antibacterial and antifungal agents.

Reasonably, benzimidazole tertiary amine type of Fluconazole an-

alogues has attracted our special interest.

diamine via cyclization to produce intermediate 2-chloromethyl-

benzimidazole 2 in excellent yield of 91.2%, and then further treated

by a series of anilines in reﬂuxing ethanol to give selectively the

corresponding secondary amines 3aei with high yields ranging

from 70.4% to 83.7%. The N-alkylation of compounds 3aei with a

series of alkyl bromides in acetonitrile at 70 ^ꢀC using potassium

carbonate as base respectively afforded the target alkyl benzimid-

azole compounds 4aef in 40.6e45.1% yields which showed that the

length of aliphatic chain in alkyl bromides exhibited slight effect on

the formation of target compounds. However, halobenzyl halides

and intermediates 3aei underwent N-alkylation to produce com-

pounds 5aen with a large difference in yields of 5.9e59.5%, this

suggested that substituents should have remarkable inﬂuences in

preparation of desired compounds. The quite low yields for com-

pounds 5hen especially 3,5-bis(triﬂuoromethyl)phenyl derivatives

5m and 5n (6.7% and 5.9% respectively) demonstrated that the

electron-withdrawing groups were unfavourable for the formation

of target compounds probably due to the low electric density of the

nitrogen atom in the secondary amines. Some representative

compounds 4 and 5 could be further readily converted into the

corresponding hydrochlorides 6 and 7 in good yields (74.3% and

77.8% respectively).

As shown in Scheme 2, coumarin-derived benzimidazoles were

prepared from commercial resorcinol. The cyclization of resorcinol

8 with ethyl acetoacetate efﬁciently provided hydroxyl coumarin 9

in the presence of oxalic acid or piperidine, and the latter was

subsequently reacted with alkyl or aralkyl dibromides in acetone to

produce coumarin bromides 10aeb in high yields of 71.2e84.1%.

The further N-alkylation with intermediate 3 afforded the target

tertiary amine type of coumarin benzimidazole derivatives in 39.6e

40.1% yields. Carbazole bromide 13 was prepared by the reaction of

carbazole with 1,4-dibromobutane at room temperature in the

presence of sodium hydride, and successfully N-alkylated with in-

termediate 3 in acetonitrile to yield tertiary amine type of carbazole

benzimidazole 14 in a yield of 39.2%. It was specially pointed out

that the feeding sequence highly affected the yields of target ter-

tiary amine type of benzimidazoles, and only when potassium

carbonate was added to the reaction system of compounds 3aei

and halides, the desired tertiary amine type of benzimidazole

compounds would be produced.

In our continuous development of tertiary amine type of target

molecules, benzimidazole ring was introduced into the tertiary

amine type of Fluconazole analogue skeleton to replace ﬁve-

membered imidazole or triazole ring with the aim to investigate

the effect of benzene-fused imidazole ring on biological activities. A

series of different lengths of aliphatic chains were incorporated into

target molecules in order to investigate the suitable length of chain

in exerting biological potency because a lot of work showed that

alkyl chain was able to regulate the lipidewater partition co-

efﬁcients [24e26]. Various substituted phenyl moieties including

chloro, ﬂuoro, triﬂuoromethyl, methyl and nitro groups were

introduced into target compounds due to the reason that they could

usually improve the pharmacological properties by enhancing the

rate of absorption and transport of drugs in vivo [27e29]. Moreover,

coumarin [30,31] and carbazole [32,33] fragments with large con-

jugated systems possess desirable electronic and charge-transport

properties, and these characteristics result in the extensive poten-

tial applications in antimicrobial aspects. Thereby, they were also

incorporated into target compounds to study their contribution to

the antimicrobial activities (Fig. 1).

In view of the above considerations, a series of novel tertiary

amine type of benzimidazole derivatives as Fluconazole analogues

were designed and prepared from commercial o-phenylene

diamine and chloroacetic acid according to the synthetic routes

shown in Schemes 1 and 2. The newly prepared compounds were

evaluated for antimicrobial activities including antibacterial and

antifungal ones. Numerous researches have shown that the com-

bination therapy in clinic with two or more agents might improve

the efﬁciency and bioavailability, reduce or even eliminate side

effects and allergic reactions and overcome multi-drug resistances

as well as treat mixed diseases which cannot be cured by single

drug [34,35]. In this work, the synergistic effects of representative

benzimidazole compounds with clinical antibacterial Chloromycin,

Norﬂoxacin or antifungal Fluconazole were evaluated in vitro

against three Gram-positive bacteria, three Gram-negative bacteria

as well as ﬁve fungi.

All the structures of target benzimidazoles including alkyl

compound 4, aralkyl derivatives involving halobenzyl one 5,

coumarin compound 11 and carbazole derivative 14 as well as their

corresponding salts 6 and 7 were shown in Schemes 1 and 2.

2.2. Analysis of spectra

All the new compounds were characterized by IR, ¹H NMR, ¹³

C

2. Results and discussion

NMR, MS and HRMS spectra. Their spectral analyses were consis-

tent with the assigned structures and listed in the experimental

section. The mass spectra for benzimidazole compounds gave a

major fragment of [M þ H]^þaccording to their molecular formula.

2.1. Chemistry

The target benzimidazole tertiary amine type of Fluconazole

analogues was prepared from commercial o-phenylene diamine

and chloroacetic acid. Their synthetic routes were outlined in

Schemes 1 and 2. Chloroacetic acid was reacted with o-phenylene

2.2.1. IR spectra

In IR spectra, all the synthesized benzimidazole compounds 4e

5, 11 and 14 gave broad absorption in 3439e3415 cm^ꢁ1which

332

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

Scheme 1. Synthesis of tertiary amine type of benzimidazole compounds 4e7.

indicated the presence of NH group in benzimidazole ring, whereas

two broad absorption between 3435 and 3415 and 3317e

3301 cm^ꢁ1were attributed to the NH group in compounds 3aei. In

addition, two sharp and strong peaks at 2931e2900 and 2882e

2838 cm^ꢁ1in compounds 4e5, 11 and 14 were ascribed to the vi-

bration of CeH bond of CH₂and CH₃. The characteristic C]N bands

of benzimidazole ring in all benzimidazole derivatives appeared in

the region between 1635 and 1604 cm^ꢁ1. All the other absorption

bands were also observed at the expected regions.

protons in 11b gave a large d value at 5.19 ppm. This phenomenon

might be ascribed to the electron-withdrawing effects of their

adjacent coumarin and benzene ring and thus causing downﬁeld

shifts of protons in target compounds. It was also observed that the

peaks of protons 4,7-H and 5,6-H in benzimidazole ring for benz-

imidazole derivatives 3e5, 11 and 14 appeared at

d 7.59e7.50 and

7.28e7.22 ppm. In addition, all the other aromatic and aliphatic

protons appeared at the appropriate chemical shifts and integral

values.

It was particularly noticed that the methylene protons in the

prepared salts 6 and 7 gave large downﬁeld shifts which were

attributed to the presence of positive charges. These evidences

proved the structure of hydrochloric salts, and the positive charges

on nitrogen atoms resulted in obvious downﬁeld shifts of methy-

lene protons.

2.2.2. ¹H NMR spectra

In ¹H NMR spectra, compounds 3e5, 11 and 14 gave singlets at

4.75e4.50 ppm assigned to the CH₂protons linked with benz-

imidazole ring, while the NeCH₂protons of alkyl chain in com-

pound

4 gave lower shift signals at 3.43e3.35 ppm. The

substitution of alkyl group in compound 4 by halobenzyl moiety to

yield compound 5 led to downﬁeld shifts of NeCH₂protons up to

4.96e4.66 ppm, which were higher than those of NeCH₂protons

attached benzimidazole ring because of the stronger electron-

withdrawing ability of halophenyl moieties in comparison with

benzimidazole ring. In contrast to alkyl compounds 4aef, the cor-

responding coumarin and carbazole derivatives 11 and 14 gave

2.2.3. ¹³C NMR spectra

The ¹³C NMR spectral analyses were in accordance with the

assigned structures. The conversion of secondary amine in-

termediates 3aei into tertiary amines 4e5, 11 and 14 resulted in

downﬁeld ¹³C chemical shifts

d 56.31e52.98 ppm for the methylene

carbons which were connected with benzimidazole ring. The sig-

nals at 51.07e49.21 ppm in compounds 5aen were assigned to the

methylene carbon in halobenzyl group. However, no large

remarkable downﬁeld chemical shifts at

d 5.19e4.36 ppm for pro-

tons coumarineOCH₂and carbazoleeNCH₂. Especially the OCH₂

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

333

Scheme 2. Synthesis of tertiary amine type of benzimidazole-based coumarin 11 and carbazole 14.

differences were observed in ¹³C chemical shifts of the benzimid-

azole ring. Similarly, the carbon signals of NeCH₂group of alkyl

chain in compound 4 were resonated at 49.04e48.77 ppm. It was

noticeable that the transformation of tertiary amines 4 and 5 into

the corresponding salts 6 and 7 resulted in downﬁeld ¹³C shifts

with 4.39e3.87 ppm and 6.09e5.59 ppm for the carbons of

methylene groups due to the incorporation of positive charge on

the aniline and benzimidazole nitrogen atoms, while all the other

carbons gave ¹³C peaks at the expected regions.

2.3.1. Antibacterial activity

The in vitro antibacterial evaluation demonstrated that the

newly prepared compounds could effectively, to some extent,

inhibit the growth of all the tested microorganisms and give broad

antimicrobial spectrum and potent antibacterial activity except that

intermediate N-((benzimidazol-2-yl)methyl)-4-methylaniline 3f

d

showed no obvious activity with a MIC value of 512

mg/mL against

M. luteus.

Table 1 showed the signiﬁcant effects of the types of side chains

and substituents on the aniline ring on biological activity. Most of

alkyl tertiary amine benzimidazoles 4aef displayed moderate

antibacterial activity against the tested bacteria. In comparison

with other bacteria, they were relatively less sensitive to E. coli.

Among all the alkyl derivatives, compound 4c with octyl group gave

best anti-B. subtilis and anti-P. aeruginosa activity with MIC value of

2.3. Biological activity

The in vitro antimicrobial screening for all the synthesized

compounds was evaluated for four Gram-positive bacteria (Staph-

ylococcus aureus ATCC 6538, Methicillin-resistant S. aureus N315

(MRSA), Micrococcus luteus ATCC 4698 and Bacillus subtilis ATCC

21216), four Gram-negative bacteria (Escherichia coli ATCC 8099,

Pseudomonas aeruginosa ATCC 27853, Bacillus proteus ATCC13315

and Bacillus proteus ATCC 13315) and ﬁve fungi (C. albicans ATCC

76615, Candida mycoderma, Candida utilis, Saccharomyces cerevisiae

and Aspergillus ﬂavus) using two fold serial dilution technique

recommended by National Committee for Clinical Laboratory

Standards (NCCLS) with the positive control of clinically antimi-

crobial drugs Chloromycin, Norﬂoxacin and Fluconazole [36]. The

combination studies were screened by microdilution checkerboard

method as described by Fivelman and co-workers (FIC (fractional

inhibitory concentration) ¼ MIC of compound A in mixture/MIC of

compound A alone þ MIC of compound B in mixture/MIC of com-

pound B alone, FIC ꢂ 1 represents synergistic effect, FIC > 1 and <2

represents additive interaction, FIC > 2 represents antagonistic

effect) [37e39]. The values of clog P, a partition coefﬁcient as a kind

of measurement for hydrophobicity/lipophilicity, were calculated

using ChemDraw Ultra 10.0 software integrated with Cambridge

soft Software (Cambridge Soft Corporation). The antibacterial and

antifungal data as well as clog P values were depicted in Tables 1e5.

16

(MIC ¼ 16

ment, which yielded compound 4b, resulted in low activity towards

the tested strains (MIC ¼ 64e256 g/mL). Moreover, when the alkyl

mg/mL which has equivalent potency to Chloromycin

mg/mL). The replacement of octyl group by butyl frag-

m

substituents were extended to decyl, dodecyl and octadecyl groups,

compounds 4def gave relatively weaker inhibitory activity. These

results suggested the noticeable effects of alkyl chain lengths on

biological activities. The short alkyl chains with poor lipophilicity

and long alkyl chains with good lipophilicity in these compounds

might make them unfavourable for being delivered to the binding

sites.

In comparison with alkyl benzimidazole compounds 4aef, most

of halobenzyl benzimidazole derivatives 5aen exerted relatively

better activities in inhibiting the growth of tested strains. Notably,

in this series of halobenzyl derivatives, compound 5m with 2,4-

diﬂuorobenzyl group gave the best antibacterial efﬁciencies with

MIC values of 8e32

Noticeably, its anti-M. luteus and anti-P. aeruginosa activities

(MIC ¼ 8 and 16 g/mL respectively) were equivalent to Chlor-

omycin. The replacement of 2,4-diﬂuorobenzyl moiety by 2,4-

mg/mL towards the corresponding bacteria.

m

334

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

m

g/mL) for compounds 3e14.^a,b,c

Table 1

clog P values and antibacterial data as MIC (

Compds

clog P

Gram-positive bacteria

Gram-negative bacteria

S. aureus

MRSA

B. subtilis

M. luteus

B. proteus

E. coli

P. aeruginosa

B. typhi

3a

3b

3c

3d

3e

3f

3g

3h

3i

4a

4b

4c

4d

4e

4f

5a

5b

5c

5d

5e

5f

5g

5h

5i

5j

5k

5l

5m

5n

6

7

11a

2.96

3.92

2.96

3.53

3.94

3.01

3.21

4.35

4.99

5.06

6.64

7.70

8.76

9.82

12.99

6.79

7.77

6.91

6.38

5.41

5.95

7.01

7.11

7.62

8.76

7.72

7.33

7.48

6.53

8.16

ꢁ1.09

0.58

16

32

64

32

16

64

2

128

32

128

64

128

64

32

64

32

16

64

32

64

8

32

16

64

32

64

32

4

64

128

16

64

128

64

32

64

32

128

64

16

64

128

8

64

32

64

16

32

64

16

64

2

32

64

32

64

16

32

2

128

256

128

256

32

64

128

32

64

128

32

64

32

64

16

64

128

32

128

16

128

64

128

64

4

256

64

32

128

64

32

128

2

128

64

128

64

128

32

64

16

64

32

64

128

32

64

16

64

128

32

128

64

16

256

128

64

128

32

128

512

64

8

128

64

128

64

256

128

32

128

256

64

256

64

32

128

32

64

32

128

32

16

32

64

16

64

256

64

32

4

16

128

64

32

64

32

64

32

64

128

64

128

16

128

16

128

64

128

64

256

16

64

128

64

32

64

128

16

128

64

128

16

128

64

128

64

128

8

128

64

8

32

128

64

8

16

64

32

2

128

8

11b

14

Chloromycin

Norﬂoxacin

16

1

8

1

a

Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates.

S. aureus, Staphylococcus aureus (ATCC 25923); MRSA, Methicillin-resistant Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus (ATCC

b

4698); B. proteus, Bacillus proteus (ATCC 13315); E. coli, Escherichia coli (JM109); P. aeruginosa, Pseudomonas aeruginosa; B. typhi, Bacillus typhi; C. albicans, Candida albicans

(ATCC 76615); C. mycoderma, Candida mycoderma; C. utilis, Candida utilis; S. cerevisiae, Saccharomyces cerevisiae; A. ﬂavus, Aspergillus ﬂavus.

c

clog P values were calculated by ChemDraw Ultra 10.0.

dichlorobenzyl group which gave compound 5n resulted in much

weaker potency against all the tested strains at the concentrations

the corresponding bacteria. Especially, it was more sensitive to

S. aureus and M. luteus species with MIC value of 8 g/mL, which

m

of 32e128

m

g/mL. The substitution of chlorine atom in compound

was almost equipotent to the reference drug Chloromycin.

It was unexpected that intermediate compounds 3aei, as shown

in Table 1, exhibited moderate to excellent activities against all the

tested bacterial strains in comparison with reference drugs Chlor-

5f by nitro group which gave derivative 5g was not beneﬁcial for

the antibacterial activities.

Many researches revealed that introduction of coumarin or

carbazole ring was helpful to improve antimicrobial potency

[30,33]. In our work, it was also observed that the coumarin de-

rivative 11a remarkably improved antimicrobial activities against

omycin (MIC ¼ 8e32

Particularly, compound 3i gave the most potent antimicrobial ef-

ﬁciencies with MIC value of 2 g/mL against S. aureus, which was 4-

mg/mL) and Norﬂoxacin (MIC ¼ 1e8

mg/mL).

m

some of the tested strains (MIC ¼ 16e128

m

g/mL) in comparison

fold more active than the reference drugs Chloromycin and Nor-

with the corresponding compound 4b, however, when the (CH₂)₆

linker in 11a became into the phenylene spacer in 11b, the anti-

bacterial efﬁcacy would decrease. The carbazole incorporated

benzimidazole derivative 14 also improved the antibacterial po-

tency of some tested bacteria.

ﬂoxacin. For E. coli and P. aeruginosa strains, compound 3i (MIC ¼ 2

and 4 mg/mL, respectively) displayed 8- and 4-fold more inhibitory

activity than Chloromycin. Further study is necessary to elucidate

their mechanisms and structureeactivity relationships.

Some organic hydrochloride salts with improved water solubi-

lity have been proved to be favourable for enhancing the bioactivity.

Compounds 6 and 7 were also found to exhibit better inhibitory

activity than their corresponding precursors. Among all target

tertiary amine type of benzimidazole derivatives including alkyl

compound 4, aralkyl derivatives involving halobenzyl one 5,

coumarin compound 11 and carbazole derivative 14 as well as their

corresponding salts, compound 7 displayed the best antibacterial

2.3.2. Antifungal activity

The antifungal evaluation in vitro showed that all target tertiary

amine type of benzimidazole derivatives displayed better activities

against Fluconazole-insensitive A. ﬂavus and compounds 4c and 5m

gave comparable efﬁcacy to Fluconazole against S. cerevisiae. These

prepared compounds were less sensitive towards other fungi than

reference drug. The length of aliphatic chain exhibited obvious ef-

fects on antifungal activity. The suitable length of alkyl chain to

exert the best antifungal efﬁcacy was observed to be (CH₂)₈, the

activity at the concentrations ranging from 8 to 32 mg/mL against

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

335

Table 2

octyl benzimidazole 4c gave generally better activity in contrast

with other alkyl derivatives with shorter or longer chain length.

Halophenyl fragments also showed effects on antifungal potency.

Bis(triﬂuoromethyl) phenyl derivative 5m gave the highest anti-

,b,c

Antifungal data as MIC (m

g/mL) for compounds 3e14.^a

Compds

C. albicans C. mycoderma C. utilis S. cerevisiae

A. ﬂavus

3a

3b

3c

3d

3e

3f

3g

3h

3i

4a

4b

4c

4d

4e

4f

5a

5b

5c

5d

5e

5f

5g

5h

5i

32

64

32

64

32

2

32

16

32

64

32

64

32

8

32

64

32

64

2

128

64

16

32

64

32

64

128

64

128

32

16

32

64

8

32

16

32

fungal activity (MIC ¼ 16e32

mg/mL) among these prepared tertiary

amine type of benzimidazole compounds, and specially towards

64

C. albicans, S. cerevisiae and A. ﬂavus fungi, this compound exerted

128

256

32

2

128

64

8

128

64

32

64

128

32

128

64

16

64

128

8

better bioactivity with low MIC value of 16

mg/mL. Moreover, salts 6

and 7 with MIC values ranging from 8 to 128

mg/mL exerted higher

activity than their precursors, which might be attributed to the

improved water solubility.

Unexpectedly, compound 3i gave strong bioactivity against

C. mycoderma and S. cerevisiae strains with MIC value of 2 mg/mL,

which were 2- and 8-fold more potent than the reference drug

Fluconazole respectively. This showed that this compound could be

served as further researches as a new antifungal agent.

128

64

16

64

32

64

32

64

128

32

64

128

32

16

64

8

128

32

128

16

32

64

32

64

128

32

64

32

64

32

64

32

64

32

64

16

64

32

8

2.3.3. Synergistic effects

The combination use of target benzimidazole compounds with

antibacterial Chloromycin or Norﬂoxacin against microorganism

were described in Tables 3 and 4. All the combinations of the tested

benzimidazoles and clinical drugs showed excellent antimicrobial

efﬁcacies with less dosage and broad antimicrobial spectrum. The

FIC index was no more than 0.5 which showed that the combina-

tions of benzimidazoles with Chloromycin or Norﬂoxacin had

excellent synergistic effects. It was worthy to note that combination

32

128

64

32

64

128

16

32

128

64

5j

5k

5l

5m

5n

6

of compound 5m or 7 with Chloromycin (2

mg/mL) or Norﬂoxacin

(0.25 g/mL) together could effectively inhibit the growth of MRSA,

m

7

which was 8- or 4-fold than themselves alone. These results

manifested that the combination of benzimidazoles with Chlor-

omycin or Norﬂoxacin could enhance antibacterial activity, over-

come drug resistance and broaden antimicrobial spectrum, and this

might be attributed to the different binding sites of these com-

pounds towards the tested bacteria.

As demonstrated in Table 5, combinations of Fluconazole with

compound 5m or 7 also displayed good activity against all the

tested fungi especially C. albicans and C. mycoderma with low MIC

11a

11b

14

Fluconazole

16

64

32

1

32

64

16

32

64

256

4

a

Minimal inhibitory concentrations were determined by micro broth dilution

method for microdilution plates.

b

S. aureus, Staphylococcus aureus (ATCC 25923); MRSA, Methicillin-resistant

Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis; M. luteus, Micrococcus

luteus (ATCC 4698); B. proteus, Bacillus proteus (ATCC 13315); E. coli, Escherichia coli

(JM109); P. aeruginosa, Pseudomonas aeruginosa; B. typhi, Bacillus typhi; C. albicans,

Candida albicans (ATCC 76615); C. mycoderma, Candida mycoderma; C. utilis, Candida

utilis; S. cerevisiae, Saccharomyces cerevisiae; A. ﬂavus, Aspergillus ﬂavus.

values of 0.25e0.5

mg/mL. Intriguingly, these combinations gave

high activity with less dosage against Fluconazole-insensitive

A. ﬂavus. It clearly showed that combination of Fluconazole with

benzimidazoles broaden antifungal spectrum.

c

clog P values were calculated by ChemDraw Ultra 10.0.

Table 3

,b,c

Synergistic effects of compounds 5m and 7 with antibacterial Chloromycin.^a

Bacteria

Compds

MIC

FIC index

0.250

Effect

Compds

MIC

FIC index

0.375

Effect

S. aureus

Chloromycin

5m

Chloromycin

5m

Chloromycin

5m

Chloromycin

5m

Chloromycin

5m

Chloromycin

5m

Chloromycin

5m

Chloromycin

5m

1

2

8

4

2

4

2

1

2

1

4

Synergistic

Chloromycin

7

Chloromycin

7

Chloromycin

7

Chloromycin

7

Chloromycin

7

Chloromycin

7

Chloromycin

7

Chloromycin

7

1

2

4

8

2

1

2

4

2

8

2

4

Synergistic

MRSA

0.375

0.500

0.250

Synergistic

0.375

0.500

0.375

Synergistic

B. subtilis

M. luteus

B. proteus

E. coli

P. aeruginosa

B. typhi

a

Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates.

S. aureus, Staphylococcus aureus (ATCC 25923); MRSA, Methicillin-resistant Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus (ATCC

b

4698); B. proteus, Bacillus proteus (ATCC 13315); E. coli, Escherichia coli (JM109); P. aeruginosa, Pseudomonas aeruginosa; B. typhi, Bacillus typhi; C. albicans, Candida albicans

(ATCC 76615); C. mycoderma, Candida mycoderma; C. utilis, Candida utilis; S. cerevisiae, Saccharomyces cerevisiae; A. ﬂavus, Aspergillus ﬂavus.

c

clog P values were calculated by ChemDraw Ultra 10.0.

336

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

Table 4

,b,c

Synergistic effects of compounds 5m and 7 with antibacterial Norﬂoxacin.^a

Bacteria

Compds

MIC

FIC index

0.250

Effect

Compds

MIC

FIC index

0.250

Effect

S. aureus

Norﬂoxacin

5m

1

Synergistic

Norﬂoxacin

7

1

Synergistic

MRSA

Norﬂoxacin

5m

Norﬂoxacin

5m

Norﬂoxacin

5m

Norﬂoxacin

5m

Norﬂoxacin

5m

Norﬂoxacin

5m

Norﬂoxacin

5m

0.25

4

0.25

2

0.125

2

0.5

2

0.25

2

0.25

2

0.125

4

0.375

0.250

0.375

0.250

0.375

0.250

Synergistic

Norﬂoxacin

7

Norﬂoxacin

7

Norﬂoxacin

7

Norﬂoxacin

7

Norﬂoxacin

7

Norﬂoxacin

7

Norﬂoxacin

7

0.25

2

0.25

1

0.125

1

0.5

2

0.25

4

0.125

2

0.25

2

0.375

0.250

0.375

0.250

0.375

Synergistic

B. subtilis

M. luteus

B. proteus

E. coli

P. aeruginosa

B. typhi

a

Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates.

S. aureus, Staphylococcus aureus (ATCC 25923); MRSA, Methicillin-resistant Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus (ATCC

b

4698); B. proteus, Bacillus proteus (ATCC 13315); E. coli, Escherichia coli (JM109); P. aeruginosa, Pseudomonas aeruginosa; B. typhi, Bacillus typhi; C. albicans, Candida albicans

(ATCC 76615); C. mycoderma, Candida mycoderma; C. utilis, Candida utilis; S. cerevisiae, Saccharomyces cerevisiae; A. ﬂavus, Aspergillus ﬂavus.

c

clog P values were calculated by ChemDraw Ultra 10.0.

2.3.4. Effect of clog P values on antimicrobial activity

commercial o-phenylene diamine, chloroacetic acid and anilines.

All new compounds were conﬁrmed by ¹H NMR, ¹³C NMR, IR, MS

and HRMS spectra. The in vitro antimicrobial activities revealed that

most title compounds showed moderate to good bioactivities

against the tested strains and some displayed equipotent or supe-

rior activities to the clinical drugs Chloromycin, Norﬂoxacin and

Fluconazole. Unexpectedly, intermediate 3i gave the most potent

Hydrophobic/lipophilic properties played an important role in

exerting biological activity [40,41]. The clog P values as one of the

most important factors have been widely employed to predict the

bioactivity of target molecules. The calculated liposome/water

partition coefﬁcients (clog P) for all tested compounds were shown

in Table 1. The results demonstrated that compounds with lower

values of clog P showed better antimicrobial activities, and these

compounds possessing comparable clog P values with respect to

the reference drugs exhibited equivalent potency. As seen from

Table 1, the clog P values of compounds 4aef generally increased

with the length of the alkyl groups increasing, and the enhance-

ment of the antimicrobial activities was observed in compounds

4aec, but the bioactivities were decreased in compounds 4def.

These might be explained by the possibility that higher lipophilic

compounds were unfavourable for being delivered to the binding

sites in organism, and indicated the signiﬁcant role of suitable

lipophilicity in drug design.

antimicrobial efﬁciency with MIC values of 2e16 mg/mL among all

the tested compounds. The halobenzyl benzimidazole derivatives

exhibited better bioactivity than the alkyl ones, especially bis(tri-

ﬂuoromethyl)benzyl compound 5m showed good activity against

S. aureus and M. luteus strains (MIC ¼ 8

mg/mL). These results

manifested that bis(triﬂuoromethyl)benzyl containing compounds

3i and 5m should be worthy to be further investigated as potential

antimicrobial agents. Moreover, combination of benzimidazoles

with antibacterial Chloromycin, Norﬂoxacin or antifungal Flucon-

azole showed notable enhanced antimicrobial efﬁciency in 4- to 8-

fold with less dosage as well as broader antimicrobial spectrum

than the separated use of them alone. More importantly, these

combined systems were more sensitive to Fluconazole-insensitive

A. ﬂavus and methicillin-resistant MRSA. These results manifested

that the synthesized tertiary amine type of benzimidazoles had

broad antimicrobial spectrum, and they not only exerted good

3. Conclusion

In summary, a new series of benzimidazole type of Fluconazole

analogues have been successfully synthesized starting from

Table 5

,b,c

Synergistic effects of compounds 5m and 7 with antifungal Fluconazole.^a

Fungi

Compds

MIC (

mg/mL)

FIC index

0.250

Effect

Compds

MIC (

m

g/mL)

FIC index

0.375

Effect

C. albicans

Fluconazole

5m

0.25

4

Synergistic

Fluconazole

7

0.25

1

Synergistic

C. mycoderma

C. utilis

Fluconazole

5m

Fluconazole

5m

Fluconazole

5m

Fluconazole

5m

0.5

4

2

8

2

4

32

4

0.250

0.500

0.375

Synergistic

Fluconazole

7

Fluconazole

7

Fluconazole

7

Fluconazole

7

0.5

2

1

4

1

2

32

2

0.250

0.375

Synergistic

S. cerevisiae

A. ﬂavus

a

Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates.

S. aureus, Staphylococcus aureus (ATCC 25923); MRSA, Methicillin-resistant Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus (ATCC

b

4698); B. proteus, Bacillus proteus (ATCC 13315); E. coli, Escherichia coli (JM109); P. aeruginosa, Pseudomonas aeruginosa; B. typhi, Bacillus typhi; C. albicans, Candida albicans

(ATCC 76615); C. mycoderma, Candida mycoderma; C. utilis, Candida utilis; S. cerevisiae, Saccharomyces cerevisiae; A. ﬂavus, Aspergillus ﬂavus.

c

clog P values were calculated by ChemDraw Ultra 10.0.

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

337

antifungal activity but also gave antibacterial activity, which made

a good starting point for the exploration of antimicrobial agents.

122.89 (Bim-6,7-C),129.87 (CF₃eC),131.34 (3-CF₃Ph-5-C),131.76 (3-

CF₃Ph-3-C), 138.31 (Bim-4,9-C), 147.28 (Bim-2-C), 152.49 (3-CF₃Ph-

1-C) ppm; ESI-MS (m/z): 292 [M þ H]^þ; HRMS (ESI) calcd. for

C₁₅H₁₂F₃N₃[M þ H]^þ, 292.1062; found, 292.1063.

4. Experimental

4.1. General methods

4.1.4. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

ﬂuoroaniline (3c)

Melting points were recorded on X-6 melting point apparatus

and uncorrected. TLC analysis was done using pre-coated silica gel

plates. FT-IR spectra were carried out on Bruker RFS100/S spec-

trophotometer (Bio-Rad, Cambridge, MA, USA) using KBr pellets in

the 400e4000 cm^ꢁ1range. ¹H NMR and ¹³C NMR spectra were

recorded on a Bruker AV 300 spectrometer using TMS as an internal

standard. The following abbreviations were used to designate aryl

groups: Bim ¼ benzimidazolyl, Ph ¼ phenyl. The chemical shifts

were reported in parts per million (ppm), the coupling constants (J)

were expressed in hertz (Hz) and signals were described as singlet

(s), doublet (d), triplet (t) as well as multiplet (m). The mass spectra

were recorded on LCMS-2010A and HRMS. All chemicals and sol-

vents were commercially available and were used without further

puriﬁcation.

Compound 3c was prepared the same way as the general pro-

cedure described for compound 3a, starting from compound 2

(1.670 g, 0.01 mol), 4-ﬂuoroaniline (1.116 g, 0.01 mol) and sodium

carbonate (1.272 g, 0.012 mol). The pure product 3c (1.785 g) was

obtained as yellow solid. Yield: 74.1%; mp: 168e169 ^ꢀC; IR (KBr)

n:

3420 (BimeNH), 3308 (anilineeNH), 3020 (aromatic CeH), 2908

(CH₂), 1604 (C]N), 1593, 1574, 1507, 1453 (aromatic frame),

823 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃)

; d: 4.63 (s, 2H, BimeCH₂),

7.05e7.03 (m, 2H, 4-FPh-3,5-H), 7.11e7.08 (m, 2H, 4-FPh-2,6-H),

7.27e7.24 (m, 2H, Bim-6,7-H), 7.56e7.54 (m, 2H, Bim-5,8-H) ppm;

¹³C NMR (75 MHz, CDCl₃)

d: 42.74 (BimeCH₂), 115.42 (Bim-5,8-C),

116.31 (4-FPh-3,5-C), 119.83 (4-FPh-2,6-C), 122.79 (Bim-6,7-C),

138.24 (Bim-4,9-C), 146.87 (Bim-2-C), 148.23 (4-FPh-1-C), 155.43

(4-FPh-4-C) ppm; ESI-MS (m/z): 242 [M þ H]^þ; HRMS (ESI) calcd.

for C₁₄H₁₂FN₃[M þ H]^þ, 242.1094; found, 242.1092.

4.1.1. Synthesis of 2-(chloromethyl)-1H-benzo[d]imidazole (2)

2-(Chloromethyl)-1H-benzimidazole 2 was prepared according

to the literature procedure, starting from o-phenylene diamine

(5.412 g, 0.05 mol) and chloroacetic acid (7.215 g, 0.075 mol). Yield:

91.2%; mp: 216e218 ^ꢀC. (literature [42] mp: 218e219 ^ꢀC).

4.1.5. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

chloroaniline (3d)

Compound 3d was prepared according to the procedure

described for compound 3a, starting from compound 2 (1.667 g,

0.01 mol), 4-chloroaniline (1.312 g, 0.01 mol) and sodium carbonate

(1.280 g, 0.012 mol). The pure product 3d (1.948 g) was obtained as

4.1.2. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-2-

ﬂuoroaniline (3a)

yellow solid. Yield: 75.6%; mp: 208e210 ^ꢀC; IR (KBr)

n

: 3421 (Bime

NH), 3310 (anilineeNH), 3022 (aromatic CeH), 2909 (CH₂), 1607

(C]N), 1595, 1576, 1508 (aromatic frame), 829 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃)

A mixture of compound 2 (1.669 g, 0.01 mol), 2-ﬂuoroaniline

(1.110 g, 0.01 mol) and sodium carbonate (1.276 g, 0.012 mol) in

ethanol (20 mL) was reﬂuxed for 24 h. Upon completion of the

reaction, the solvent was evaporated. The residue was extracted

with ethyl acetate (3 ꢃ 20 mL), and the organic phase was dried

over anhydrous sodium sulphate and further puriﬁed by silica gel

column chromatography (eluent, petroleum ether/ethyl acetate (5/

1, V/V)) to afford the desired compound 3a as yellow solid. Yield:

;

d

: 4.62 (s, 2H, Bim-CH₂), 6.64 (d, 2H, J ¼ 6 Hz, 4-

ClPh-2,6-H), 7.25e7.23 (m, 2H, Bim-6,7-H), 7.28 (d, 2H, J ¼ 6 Hz, 4-

ClPh-3,5-H), 7.55 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 42.82 (BimeCH₂), 114.35 (4-ClPh-2,6-C), 115.48 (Bim-5,8-C),

122.91 (Bim-6,7-C), 126.47 (4-ClPh-4-C), 129.78 (4-ClPh-3,5-C),

138.37 (Bim-4,9-C), 147.02 (Bim-2-C), 151.09 (4-ClPh-1-C) ppm; ESI-

MS (m/z): 258 [M þ H]^þ; HRMS (ESI) calcd. for C₁₄H₁₂ClN₃[M þ H]^þ,

258.0798; found, 257.0799.

73.1%; mp: 164e165 ^ꢀC; IR (KBr)

NH), 3017 (aromatic CeH), 2903 (CH₂), 1604 (C]N), 1591, 1502,

1458 (aromatic frame), 753 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

: 4.61

n: 3415 (BimeNH), 3305 (anilinee

d

(s, 2H, BimeCH₂), 6.74e6.71 (d, H, Ph-6-H), 6.82 (s, H, Ph-4-H),

7.00e6.97 (d, H, Ph-5-H), 7.21e7.19 (d, H, Ph-3-H), 7.27e7.24 (m, 2H,

Bim-6,7-H), 7.58e7.55 (m, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz,

4.1.6. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

iodoaniline (3e)

Compound 3e was prepared according to the procedure depic-

ted for compound 3a, starting from compound 2 (1.654 g, 0.01 mol),

4-iodoaniline (2.079 g, 0.01 mol) and sodium carbonate (1.273 g,

0.012 mol). The pure product 3e (2.458 g) was obtained as yellow

CDCl₃) d: 42.63 (BimeCH₂), 109.67 (2-FPh-6-C), 114.98 (Bim-5,8-C),

115.03 (2-FPh-3-C), 115.33 (2-FPh-4-C), 122.89 (Bim-6,7-C), 129.93

(2-FPh-5-C), 131.34 (2-FPh-1-C), 138.31 (Bim-4,9-C), 147.35 (Bim-2-

C), 153.89 (2-FPh-2-C) ppm; ESI-MS (m/z): 242 [M þ H]^þ; HRMS

(ESI) calcd. for C₁₄H₁₂FN₃[M þ H]^þ, 242.1094; found, 242.1093.

syrup. Yield: 70.4%; IR (KBr)

3025 (aromatic CeH), 2911 (CH₂), 1608 (C]N), 1596, 1576, 1508

(aromatic frame), 834 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃)

: 4.63 (s,

n: 3423 (BimeNH), 3313 (anilineeNH),

;

d

4.1.3. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-3-

(triﬂuoromethyl)aniline (3b)

2H, Bim-CH₂), 6.61 (d, 2H, J ¼ 3 Hz, 4-IPh-2,6-H), 7.24e7.23 (m, 2H,

Bim-6,7-H), 7.48 (d, 2H, J ¼ 3 Hz, 4-IPh-3,5-H), 7.55 (s, 2H, Bim-5,8-

Compound 3b was prepared according to the procedure

described for compound 3a, starting from compound 2 (1.665 g,

0.01 mol), 3-(triﬂuoromethyl)aniline (1.607 g, 0.01 mol) and so-

dium carbonate (1.278 g, 0.012 mol). The pure product 3b (2.068 g)

was obtained as yellow solid. Yield: 71.2%; mp: 155e156 ^ꢀC; IR

H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 43.02 (BimeCH₂), 81.12 (4-

IPh-4-C), 114.73 (4-IPh-2,6-C), 115.45 (Bim-5,8-C), 122.95 (Bim-

6,7-C), 137.32 (4-IPh-3,5-C), 138.31 (Bim-4,9-C), 146.13 (Bim-2-C),

147.61 (4-IPh-1-C) ppm; ESI-MS (m/z): 350 [M þ H]^þ; HRMS (ESI)

calcd. for C₁₄H₁₂IN₃[M þ H]^þ, 350.0154; found, 350.0152.

(KBr)

2911 (CH₂), 1608 (C]N), 1595, 1573, 1507, 1459 (aromatic frame),

786, 698 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

: 4.61 (s, 2H, BimeCH₂),

n: 3423 (BimeNH), 3310 (anilineeNH), 3019 (aromatic CeH),

4.1.7. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

methylaniline (3f)

d

6.74 (d, H, J ¼ 9 Hz, 3-CF₃Ph-6-H), 6.82 (s, H, 3-CF₃Ph-2-H), 7.00e

6.97 (d, H, J ¼ 9 Hz, 3-CF₃Ph-4-H), 7.21 (d, H, J ¼ 6 Hz, 3-CF₃Ph-5-H),

7.27e7.24 (m, 2H, Bim-6,7-H), 7.58e7.55 (m, 2H, Bim-5,8-H) ppm;

Compound 3f was prepared according to the experimental pro-

cedure described for compound 3a, starting from compound 2

(1.659 g, 0.01 mol), 4-methylaniline (1.282 g, 0.01 mol) and sodium

carbonate (1.271 g, 0.012 mol). The pure product 3f (1.987 g) was

obtained as yellow syrup. Yield: 83.7%; mp: 198e200 ^ꢀC (literature

¹³C NMR (75 MHz, CDCl₃)

d: 42.69 (BimeCH₂), 109.67 (3-CF₃Ph-2-

C), 115.03 (3-CF₃Ph-4-C), 115.44 (Bim-5,8-C), 115.55 (3-CF₃Ph-6-C),

338

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

[43] mp: 175e177 ^ꢀC); IR (KBr)

n: 3413 (BimeNH), 3301 (anilinee

4-C), 112.96 (3,5-(CF₃)₂Ph-2,6-C), 114.23 (Bim-5,8-C), 123.35 (Bim-

NH), 3015 (aromatic CeH), 2901 (CH₂),1603 (C]N),1590,1501,1454

6,7-C), 124.89 (CF₃-2C), 132.94 (3,5-(CF₃)₂Ph-3,5-C), 138.53 (Bim-

4,9-C), 149.95 (Bim-2-C), 152.46 (3,5-(CF₃)₂Ph-1-C) ppm; ESI-MS

(m/z): 360 [M þ H]^þ; HRMS (ESI) calcd. for C₁₆H₁₁F₆N₃[M þ H]^þ,

360.0935; found, 360.0936.

(aromatic frame), 1388 (CH₃), 817 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

d

: 2.23 (s, 3H, CH₃), 4.63 (s, 2H, Bim-CH₂), 6.57 (d, 2H, J ¼ 9.0 Hz, 4-

CH₃Ph ꢁ2,6-H), 6.98 (d, 2H, J ¼ 6 Hz, 4-CH₃Ph-3,5-H), 7.26e7.23 (m,

2H, Bim-6,7-H), 7.56e7.53 (m, 2H, Bim-5,8-H) ppm; ¹³C NMR

(75 MHz, CDCl₃)

d

: 20.29 (CH₃), 42.75 (BimeCH₂), 109.21 (4-CH₃Ph-

4.1.11. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

methyl-N-propylaniline (4a)

2,6-C), 114.89 (Bim-5,8-C), 122.56 (Bim-6,7-C), 127.87 (4-CH₃Ph-4-

C), 128.10 (4-CH₃Ph-3,5-C), 138.23 (Bim-4,9-C), 146.02 (Bim-2-C),

147.32 (4-CH₃Ph-1-C) ppm; ESI-MS (m/z): 238 [M þ H]^þ; HRMS (ESI)

calcd. for C₁₅H₁₅N₃[M þ H]^þ, 238.1344; found, 238.1343.

A suspension of compound 3f (0.475 g, 0.002 mol) and 1-

bromopropane (0.247 g, 0.002 mol) was stirred in acetonitrile

(30 mL) at 70 ^ꢀC. After 0.5 h, potassium carbonate (0.410 g,

0.003 mol) was added. After the reaction was completed (moni-

tored by TLC, eluent, petroleum ether/ethyl acetate, 4/1, V/V), the

solvent was removed and the residue was exacted with ethyl ace-

tate (3 ꢃ 20 mL), dried over anhydrous sodium sulphate and pu-

riﬁed by silica gel column chromatography (eluent, petroleum

ether/ethyl acetate (5/1, V/V)) to afford compound 4a as yellow oil.

4.1.8. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-2,4-

diﬂuoroaniline (3g)

Compound 3g was prepared according to the procedure

described for compound 3a, starting from compound 2 (1.665 g,

0.01 mol), 2,4-diﬂuoroaniline (1.290 g, 0.01 mol) and sodium car-

bonate (1.282 g, 0.012 mol). The pure product 3g (1.987 g) was

Yield: 41.5%; IR (KBr)

2913, 2841 (CH₂, CH₃), 1611 (C]N), 1599, 1573, 1509, 1459 (aro-

matic frame), 1383, 1379 (CH₃), 817 cm^ꢁ^{1 1}H NMR (300 MHz,

CDCl₃)

n: 3419 (BimeNH), 3035 (aromatic CeH),

obtained as yellow solid. Yield: 79.7%; mp: 166e167 ^ꢀC; IR (KBr)

n:

3425 (BimeNH), 3312 (anilineeNH), 3022 (aromatic CeH), 2908

(CH₂), 1607 (C]N), 1596, 1576, 1506, 1462 (aromatic frame),

;

d

: 1.08e1.06 (t, 3H, J ¼ 3.0 Hz, CH₂CH₃), 1.34 (m, 2H,

826 cm^ꢁ1

;

¹H NMR (300 MHz, CDCl₃)

d: 4.64 (s, 2H, BimeCH₂),

CH₂CH₂CH₃), 2.23 (s, 3H, 4-CH₃Ph), 3.40e3.35 (t, 2H, J ¼ 7.5 Hz,

CH₂CH₂CH₃), 4.69 (s, 2H, BimeCH₂), 6.71 (d, 2H, J ¼ 9.0 Hz, 4-

CH₃Ph-2,6-H), 7.03 (d, 2H, J ¼ 6.0 Hz, 4-CH₃Ph-3,5-H), 7.25 (s, 2H,

Bim-6,7-H), 7.56 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

6.56e6.48 (m, 1H, 2,4-F₂Ph-3-H), 6.67e6.61 (m, 1H, 2,4-F₂Ph-5-H),

6.81e6.74 (m, 1H, 2,4-F₂Ph-6-H), 7.27e7.25 (m, 2H, Bim-6,7-H),

7.58e7.54 (m, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d:

43.03 (BimeCH₂), 103.55 (2,4-F₂Ph-3-C), 110.72 (2,4-F₂Ph-5-C),

111.00 (Bim-5,8-C), 112.63 (2,4-F₂Ph-6-C), 122.67 (Bim-6,7-C),

132.24 (2,4-F₂Ph-1-C), 138.39 (Bim-4,9-C), 149.18 (Bim-2-C), 152.70

(2,4-F₂Ph-4-C), 156.62 (2,4-F₂Ph-2-C) ppm; ESI-MS (m/z): 260

[M þ H]^þ; HRMS (ESI) calcd. for C₁₄H₁₁F₂N₃[M þ H]^þ, 260.0999;

found, 260.0997.

d: 11.54 (CH₂CH₃), 19.89 (CH₂CH₂CH₃), 20.16 (4-CH₃Ph), 49.04

(CH₂CH₂CH₃), 54.96 (BimeCH₂), 114.06 (4-CH₃Ph-2,6-C), 114.09

(Bim-5,8-C), 122.69 (Bim-6,7-C), 128.86 (4-CH₃Ph-3,5-C), 130.66 (4-

CH₃Ph-4-C), 138.19 (Bim-4,9-C), 145.64 (Bim-2-C), 152.04 (4-

CH₃Ph-1-C) ppm; MS (m/z): 280 [M þ H]^þ; HRMS (ESI) calcd. for

C₁₈H₂₂N₃[M þ H]^þ, 280.1814; found, 280.1812.

4.1.9. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-2,4-

dichloroaniline (3h)

4.1.12. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-

hexyl-4-methylaniline (4b)

Compound 3h was prepared according to the procedure

described for compound 3a, starting from compound 2 (1.669 g,

0.01 mol), 2,4-dichoroaniline (1.615 g, 0.01 mol) and sodium car-

bonate (1.272 g, 0.012 mol). The pure product 3h (1.987 g) was

Pure compound 4b was obtained in process of synthesizing

compound 4a as yellow oil. Yield: 40.6%; IR (KBr) n: 3417 (BimeNH),

3034 (aromatic CeH), 2913, 2840 (CH₂, CH₃), 1610 (C]N), 1599,

1573, 1509, 1459 (aromatic frame), 1383, 1378 (CH₃), 817 cm^ꢁ1; ¹H

obtained as yellow solid. Yield: 72.1%; mp: 163e164 ^ꢀC; IR (KBr)

n

:

NMR (300 MHz, CDCl₃)

d

: 0.87e0.85 (t, 3H, J ¼ 3.0 Hz, CH₂CH₃),

3429 (BimeNH), 3313 (anilineeNH), 3022 (aromatic CeH), 2910

(CH₂), 1609 (C]N), 1598, 1576, 1508, 1464 (aromatic frame),

1.32e1.30 (m, 8H, CH₂(CH₂)₄CH₃), 2.23 (s, 3H, 4-CH₃Ph), 3.39e3.35

(t, 2H, J ¼ 6.0 Hz, CH₂(CH₂)₄CH₃), 4.70 (s, 2H, BimeCH₂), 6.70 (d, 2H,

J ¼ 6.0 Hz, 4-CH₃Ph-2,6-H), 7.02 (d, 2H, J ¼ 3.0 Hz, 4-CH₃Ph-3,5-H),

7.24 (s, 2H, Bim-6,7-H), 7.55 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR

831 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

d: 4.63 (s, 2H, BimeCH₂), 6.54

(d,1H, J ¼ 9.0 Hz, 2,4-Cl₂Ph-6-H), 7.18 (d,1H, J ¼ 9.0 Hz, 2,4-Cl₂Ph-5-

H), 7.27e7.25 (m, 2H, Bim-6,7-H), 7.59e7.54 (m, 2H, Bim-5,8-H),

7.85 (d, 1H, J ¼ 9.0 Hz, 2,4-Cl₂Ph-3-H) ppm; ¹³C NMR (75 MHz,

(75 MHz, CDCl₃)

d: 13.95 (CH₂CH₃), 20.17 (4-CH₃Ph), 21.34

(CH₂CH₃), 26.58 (CH₂(CH₂)₂CH₃), 28.47 (CH₂(CH₂)₃CH₃), 31.03

(CH₂CH₂CH₃), 48.79 (CH₂(CH₂)₄CH₃), 54.95 (BimeCH₂), 114.05 (4-

CH₃Ph-2,6-C), 114.08 (Bim-5,8-C), 122.69 (Bim-6,7-C), 128.85 (4-

CH₃Ph-3,5-C), 130.64 (4-CH₃Ph-4-C), 138.18 (Bim-4,9-C), 145.63

(Bim-2-C), 152.03 (4-CH₃Ph-1-C) ppm; MS (m/z): 322 [M þ H]^þ;

HRMS (ESI) calcd. for C₂₁H₂₈N₃[M þ H]^þ, 322.2283; found,

322.2282.

CDCl₃) d: 42.97 (BimeCH₂), 114.02 (Bim-5,8-C), 115.32e115.28 (2,4-

Cl₂Ph-6-C), 123.11 (Bim-6,7-C), 123.89 (2,4-Cl₂Ph-2-C), 125.21 (2,4-

Cl₂Ph-4-C), 128.04 (2,4-Cl₂Ph-5-C), 130.96 (2,4-Cl₂Ph-3-C), 138.43

(Bim-4,9-C), 149.82 (Bim-2-C), 151.34 (2,4-Cl₂Ph-1-C) ppm; ESI-MS

(m/z): 292 [M þ H]^þ; HRMS (ESI) calcd. for C₁₄H₁₁Cl₂N₃[M þ H]^þ,

292.0408; found, 292.0407.

4.1.10. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-3,5-

bis(triﬂuoromethyl)aniline (3i)

4.1.13. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

methyl-N-octylaniline (4c)

Compound 3i was prepared according to the procedure

described for compound 3a, starting from compound 2 (1.662 g,

0.01 mol), 3,5-bis(triﬂuoromethyl)aniline (2.234 g, 0.01 mol) and

sodium carbonate (1.279 g, 0.012 mol). The pure product 3i

(1.987 g) was obtained as white solid. Yield: 82.5%; mp: 212e

Pure compound 4c was obtained in process of synthesizing

compound 4a as yellow oil. Yield: 42.1%; IR (KBr) n: 3416 (BimeNH),

3034 (aromatic CeH), 2913, 2839 (CH₂, CH₃), 1608 (C]N), 1599,

1573, 1509, 1459 (aromatic frame), 1382, 1378 (CH₃), 817 cm^ꢁ1; ¹H

NMR (300 MHz, CDCl₃)

d

: 0.88e0.86 (t, 3H, J ¼ 3.0 Hz, CH₂CH₃),

214 ^ꢀC; IR (KBr)

matic CeH), 2913 (CH₂), 1615 (C]N), 1603, 1581, 1513, 1473 (aro-

matic frame), 736 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃)

: 4.63 (d, 2H,

n

: 3435 (BimeNH), 3317 (anilineeNH), 3025 (aro-

1.30e1.28 (m, 12H, (CH₂)₆CH₃), 2.23 (s, 3H, 4-CH₃Ph), 3.41e3.38 (t,

2H, J ¼ 4.5 Hz, CH₂(CH₂)₆CH₃), 4.72 (s, 2H, BimeCH₂), 6.71 (d, 2H,

J ¼ 9.0 Hz, 4-CH₃Ph-2,6-H), 7.04 (d, 2H, J ¼ 9.0 Hz, 4-CH₃Ph-3,5-H),

7.23 (s, 2H, Bim-6,7-H), 7.54 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR

;

d

J ¼ 3.0 Hz, BimeCH₂), 6.95 (s, 2H, 3,5-(CF₃)₂Ph-2,6-H), 7.18 (s, H, 3,5-

(CF₃)₂Ph-4-H), 7.26 (s, 2H, Bim-6,7-H), 7.57 (s, 2H, Bim-5,8-H) ppm;

(75 MHz, CDCl₃)

d: 13.95 (CH₂CH₃), 20.17 (4-CH₃Ph), 21.32

¹³C NMR (75 MHz, CDCl₃)

d: 43.13 (BimeCH₂), 112.04 (3,5-(CF₃)₂Ph-

(CH₂CH₃), 26.75 (CH₂(CH₂)₄CH₃), 28.13 (CH₂(CH₂)₃CH₃), 28.21

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

339

(CH₂(CH₂)₂CH₃), 28.45 (CH₂(CH₂)₅CH₃), 31.32 (CH₂CH₂CH₃), 48.78

(CH₂(CH₂)₆CH₃), 54.96 (Bim-CH₂), 114.05 (4-CH₃Ph-2,6-C), 114.09

(Bim-5,8-C), 122.69 (Bim-6,7-C), 128.85 (4-CH₃Ph-3,5-C), 130.64 (4-

CH₃Ph-4-C), 132.08 (Bim-4,9-C), 145.64 (Bim-2-C), 152.03 (4-

CH₃Ph-1-C) ppm; MS (m/z): 350 [M þ H]^þ; HRMS (ESI) calcd. for

C₂₃H₃₁N₃[M þ H]^þ, 350.2596; found, 350.3597.

CH₃Ph-3,5-C), 130.64 (4-CH₃Ph-4-C), 138.18 (Bim-4,9-C), 145.63

(Bim-2-C), 152.02 (4-CH₃Ph-1-C) ppm; MS (m/z): 490 [M þ H]^þ;

HRMS (ESI) calcd. for C₃₃H₅₁N₃[M þ H]^þ, 490.4161; found, 490.4160.

4.1.17. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(3,4-

dichlorobenzyl)-2-ﬂuoroaniline (5a)

Pure compound 5a was obtained in process of synthesizing

4.1.14. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-

decyl-4-methylaniline (4d)

compound 4a aswhitesolid. Yield:31.2%; mp:128e129 ^ꢀC;IR(KBr)

n:

3422 (BimeNH), 3021 (aromatic CeH), 2905 (CH₂), 2863 (3,4-Cl₂Ph-

CH₂),1607 (C]N), 1593,1505,1461 (aromatic frame), 831, 758 cm^ꢁ1

¹H NMR (300 MHz, CDCl₃)

: 4.65 (s, 2H, Bim-CH₂), 4.70 (s, 2H,

Pure compound 4d was obtained in process of synthesizing

;

compound 4a as yellow oil. Yield: 45.1%; IR (KBr)

3034 (aromatic CeH), 2913, 2838 (CH₂, CH₃),1608 (C]N),1599,1573,

1509, 1459 (aromatic frame), 1382, 1379 (CH₃), 815 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃)

n: 3416 (BimeNH),

d

dichlorobenzyleCH₂), 6.68e6.64 (m, H, 2-FPh-4-H), 6.93e6.92 (d, H,

J ¼ 3.0 Hz, 2-FPh-6-H), 7.02 (d, 2H, J ¼ 3.0 Hz, 2-FPh-3,5-H), 7.23 (s,1H,

3,4-Cl₂Ph-6-H), 7.26(d, 2H,J¼ 3Hz, Bim-6,7-H), 7.28(s,1H, 3,4-Cl₂Ph-

2-H), 7.52 (d, 2H, J ¼ 6.0 Hz, Bim-5,8-H), 7.80 (d, 1H, J ¼ 9.0 Hz, 3,4-

;

d

: 0.84e0.81 (t, 3H, J ¼ 4.5 Hz, CH₂CH₃), 1.30e1.28

(m,14H, (CH₂)₇CH₃),1.76e1.74 (m, 2H, CH₂(CH₂)₇CH₃), 2.23 (s, 3H, 4-

CH₃Ph), 3.40e3.38 (t, 2H, J ¼ 3.0 Hz, CH₂(CH₂)₈CH₃), 4.69 (s, 2H, Bime

CH₂), 6.68 (d, 2H, J ¼ 9.0 Hz, 4-CH₃Ph-2,6-H), 7.02 (d, 2H, J ¼ 9.0 Hz, 4-

CH₃Ph-3,5-H), 7.25 (s, 2H, Bim-6,7-H), 7.53 (s, 2H, Bim-5,8-H) ppm;

Cl₂Ph-5-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 50.32 (3,4-Cl₂Ph-

CH₂), 54.76 (BimeCH₂),114.10 (Bim-5,8-C),114.23 (2-FPh-6-C),115.04

(2-FPh-3-C),122.95(2-FPh-4-C),122.97(Bim-6,7-C),124.32(2-FPh-5-

C), 126.59 (3,4-Cl₂Ph-6-C),128.63 (3,4-Cl₂Ph-3-C), 128.75 (3,4-Cl₂Ph-

2-C), 130.25 (3,4-Cl₂Ph-5-C), 131.25 (3,4-Cl₂Ph-4-C), 132.91 (3,4-

Cl₂Ph-1-C), 134.67 (2-FPh-1-C), 138.20 (Bim-4,9-C), 145.67 (Bim-2-

C), 154.04 (2-FPh-2-C) ppm; MS (m/z): 400 [M þ H]^þ; HRMS (ESI)

calcd. for C₂₁H₁₆Cl₂FN₃[M þ H]^þ, 400.0784; found, 400.0783.

¹³C NMR (75 MHz, CDCl₃)

d: 13.95 (CH₂CH₃), 20.17 (4-CH₃Ph), 21.33

(CH₂CH₃), 26.74 (CH₂(CH₂)₆CH₃), 28.14 (CH₂(CH₂)₅CH₃), 28.18

(CH₂(CH₂)₂CH₃), 28.45 (CH₂(CH₂)₄CH₃), 28.51 (CH₂(CH₂)₃CH₃), 31.32

(CH₂CH₂CH₃), 48.79 (CH₂(CH₂)₈CH₃), 54.96 (Bim-CH₂), 114.05 (4-

CH₃Ph-2,6-C), 114.09 (Bim-5,8-C), 122.69 (Bim-6,7-C), 128.85 (4-

CH₃Ph-3,5-C), 130.64 (4-CH₃Ph-4-C), 138.18 (Bim-4,9-C), 145.64

(Bim-2-C), 152.02 (4-CH₃Ph-1-C) ppm; MS (m/z): 378 [M þ H]^þ;

HRMS (ESI) calcd. for C₂₅H₃₅N₃[M þ H]^þ, 378.2909; found, 378.2911.

4.1.18. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2,4-

dichlorobenzyl)-3-(triﬂuoromethyl) aniline (5b)

Compound 5b was synthesized according to the experimental

procedure reported for compound 4a, starting from compound 3b

(1.179 g, 0.004 mol) and 2,4-dichloro-1-(chloromethyl)benzene

(0.832 g, 0.004 mol). The crude product was obtained and puriﬁed

via silica gel column chromatography (eluent, petroleum ether/

ethyl acetate, 5/1, V/V) to give pure compound 4b (0.522 g) as

4.1.15. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-

dodecyl-4-methylaniline (4e)

Pure compound 4e was obtained in process of synthesizing

compound 4a as yellow oil. Yield: 43.2%; IR (KBr) n: 3416 (Bime

NH), 3034 (aromatic CeH), 2913, 2838 (CH₂, CH₃), 1607 (C]N),

1599, 1573, 1509, 1459 (aromatic frame), 1381, 1379 (CH₃),

yellow solid. Yield: 48.7%; mp: 134e135 ^ꢀC; IR (KBr)

n: 3425 (Bime

815 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

d

: 0.87e0.84 (t, 3H, J ¼ 4.5 Hz,

NH), 3022 (aromatic CeH), 2913 (CH₂), 2868 (2,4-Cl₂Ph-CH₂), 1609

(C]N), 1596, 1575, 1508, 1462 (aromatic frame), 837, 833, 789,

CH₂CH₃), 1.36e1.30 (m, 16H, (CH₂)₈CH₃), 1.79e1.75 (m, 4H,

(CH₂)₂(CH₂)₈CH₃), 2.23 (s, 3H, 4-CH₃Ph), 3.43e3.41 (t, 2H,

J ¼ 3.0 Hz, CH₂(CH₂)₁₀CH₃), 4.65 (s, 2H, BimeCH₂), 6.67 (d, 2H,

J ¼ 4.5 Hz, 4-CH₃Ph-2,6-H), 7.04 (d, 2H, J ¼ 4.5 Hz, 4-CH₃Ph-3,5-H),

7.23 (s, 2H, Bim-6,7-H), 7.53 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR

688 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

d: 4.69 (s, 2H, BimeCH₂), 4.77

(s, 2H, 2,4-Cl₂Ph-CH₂), 6.85 (d, H, J ¼ 9.0 Hz, 3-CF₃Ph-6-H), 6.92 (s,

H, 3-CF₃Ph-4-H), 7.01 (d, 2H, J ¼ 6.0 Hz, 2,4-Cl₂Ph-6-H, 3-CF₃Ph-2-

H), 7.19e7.16 (m, H, 3-CF₃Ph-5-H), 7.25 (d, 1H, J ¼ 3.0 Hz, 2,4-Cl₂Ph-

5-H), 7.26 (d, 2H, J ¼ 9.0 Hz, Bim-6,7-H), 7.43 (d, 1H, 2,4-Cl₂Ph-3-H),

(75 MHz, CDCl₃)

(CH₂CH₃), 26.74 (CH₂(CH₂)₈CH₃), 28.13 (CH₂(CH₂)₇CH₃), 28.19

(CH₂(CH₂)₂CH₃), 28.44e28.27 ((CH₂)₄(CH₂)₃CH₃), 31.32

d: 13.95 (CH₂CH₃), 20.16 (4-CH₃Ph), 21.32

7.54 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 48.85

(2,4-Cl₂Ph-CH₂), 52.98 (BimeCH₂), 109.03 (3-CF₃Ph-5-C), 114.76 (3-

CF₃Ph-4-C), 115.18 (Bim-5,8-C), 115.86 (3-CF₃Ph-6-C), 123.97 (3-

CF₃Ph-2-C), 125.47 (Bim-6,7-C), 128.37 (CF₃eC), 129.83 (2,4-Cl₂Ph-

5-C), 130.18 (2,4-Cl₂Ph-3-C), 131.66 (2,4-Cl₂Ph-6-C), 131.98 (3-

CF₃Ph-3-C), 132.08 (2,4-Cl₂Ph-4-C), 133.60 (2,4-Cl₂Ph-2-C), 134.04

(2,4-Cl₂Ph-1-C), 136.10 (Bim-4,9-C), 147.36 (Bim-2-C), 150.89 (3-

CF₃Ph-1-C) ppm; MS (m/z): 450 [M þ H]^þ; HRMS (ESI) calcd. for

C₂₂H₁₆Cl₂F₃N₃[M þ H]^þ, 450.0752; found, 450.0751.

(CH₂CH₂CH₃), 48.78 (CH₂(CH₂)₁₀CH₃), 54.96 (BimeCH₂), 114.03 (4-

CH₃Ph-2,6-C), 114.09 (Bim-5,8-C), 122.69 (Bim-6,7-C), 128.85 (4-

CH₃Ph-3,5-C), 130.64 (4-CH₃Ph-4-C), 138.18 (Bim-4,9-C), 145.62

(Bim-2-C), 152.02 (4-CH₃Ph-1-C) ppm; MS (m/z): 406 [M þ H]^þ;

HRMS (ESI) calcd. for C₂₇H₃₉N₃[M þ H]^þ, 406.3222; found,

406.3223.

4.1.16. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

methyl-N-octadecylaniline (4f)

4.1.19. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2,4-

dichlorobenzyl)-4-ﬂuoroaniline (5c)

Compound 4f was obtained as brown syrup. Yield: 40.7%; IR

(KBr)

CH₃), 1607 (C]N), 1599, 1573, 1509, 1459 (aromatic frame), 1382,

1379 (CH₃), 813 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

: 0.87e0.85 (t, 3H,

J ¼ 3.0 Hz, CH₂CH₃), 1.38e1.35 (m, 28H, (CH₂)₁₄CH₃), 1.75e1.72 (m,

4H, (CH₂)₂(CH₂)₁₄CH₃), 2.23 (s, 3H, 4-CH₃Ph), 3.40e3.35 (t, 2H,

J ¼ 7.5 Hz, CH₂(CH₂)₁₆CH₃), 4.69 (s, 2H, BimeCH₂), 6.70 (d, 2H,

J ¼ 4.5 Hz, 4-CH₃Ph-2,6-H), 7.04 (d, 2H, J ¼ 4.5 Hz, 4-CH₃Ph-3,5-H),

7.22 (s, 2H, Bim-6,7-H), 7.52 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR

n

: 3416 (BimeNH), 3034 (aromatic CeH), 2913, 2838 (CH₂,

Prepared according to the general procedure described for

compound 4a, starting from compound 3c (0.970 g, 0.004 mol) and

2,4-dichloro-1-(chloromethyl)benzene (0.804 g, 0.004 mol), the

pure compound 5c (0.482 g) was obtained as yellow solid. Yield:

d

32.3%; mp: 152e153 ^ꢀC; IR (KBr)

n

: 3423 (BimeNH), 3021 (aromatic

CeH), 2910 (CH₂), 2865 (2,4-Cl₂Ph-CH₂), 1606 (C]N), 1594, 1575,

1508, 1456 (aromatic frame), 830, 821 cm^ꢁ^{1 1}H NMR (300 MHz,

CDCl₃) : 4.66 (s, 2H, BimeCH₂), 4.72 (s, 2H, 2,4-Cl₂Ph-CH₂), 6.72e

;

d

(75 MHz, CDCl₃)

26.73 (CH₂(CH₂)₁₄CH₃),

(CH₂(CH₂)₂CH₃), 28.43e28.37

(CH₂CH₂CH₃), 48.77 (CH₂(CH₂)₁₆CH₃), 54.97 (BimeCH₂), 114.04 (4-

CH₃Ph-2,6-C), 114.09 (Bim-5,8-C), 122.69 (Bim-6,7-C), 128.85 (4-

d

: 13.94 (CH₂CH₃), 20.17 (4-CH₃Ph), 21.32 (CH₂CH₃),

6.68 (m, 2H, 4-FPh-2,6-H), 7.05e7.01 (m, 2H, 4-FPh-3,5-2H), 7.14 (s,

1H, 2,4-Cl₂Ph-6-H), 7.27 (d, 2H, J ¼ 9.0 Hz, Bim-6,7-H), 7.30 (s, 1H,

2,4-Cl₂Ph-5-H), 7.54 (d, 2H, J ¼ 9.0 Hz, Bim-5,8-H), 7.82e7.79 (s, 1H,

28.13

(CH₂(CH₂)₁₃CH₃),

28.21

31.32

((CH₂)₁₀(CH₂)₃CH₃),

2,4-Cl₂Ph-3-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 49.21 (2,4-Cl₂Ph-

CH₂), 54.56 (BimeCH₂), 114.21 (Bim-5,8-C), 114.32 (4-FPh-2,6-C),

340

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

115.25 (4-FPh-3,5-C), 123.04 (Bim-6,7-C), 125.47 (2,4-Cl₂Ph-5-C),

130.56 (2,4-Cl₂Ph-3-C), 130.97 (2,4-Cl₂Ph-6-C), 133.89 (2,4-Cl₂Ph-4-

C), 134.12 (2,4-Cl₂Ph-2-C), 135.35 (2,4-Cl₂Ph-1-C), 138.26 (Bim-4,9-

C), 145.61 (Bim-2-C), 149.15 (4-FPh-1-C), 154.23 (4-FPh-4-C) ppm;

MS (m/z): 400 [M þ H]^þ; HRMS (ESI) calcd. for C₂₁H₁₆Cl₂FN₃

[M þ H]^þ, 400.0784; found, 400.0782.

(d, 2H, J ¼ 6.0 Hz, 4-ClPh-2,6-H), 7.41 (s, 2H, 4-ClPh-3,5-H), 7.50 (s, 2H,

Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

: 20.32 (CH₃), 49.58 (4-

d

ClPh-CH₂), 54.68 (BimeCH₂), 109.55 (4-CH₃Ph-2,6-C), 113.47 (Bim-

5,8-C), 122.45 (Bim-6,7-C), 123.10 (4-ClPh-3,5-C), 123.10 (4-CH₃Ph-

3,5-C), 127.65 (4-ClPh-2,6-C), 127.73 (4-CH₃Ph-4-C), 128.51 (4-ClPh-

4-C), 129.81 (4-ClPh-1-C), 137.83 (Bim-4,9-C), 145.02 (Bim-2-C),

151.76 (4-CH₃Ph-1-C) ppm; MS (m/z): 362 [M þ H]^þ; HRMS (ESI)

calcd. for C₂₂H₂₀ClN₃[M þ H]^þ, 362.1424; found, 362.1422.

4.1.20. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2-

chlorobenzyl)-4-methylaniline (5d)

Compound 5d was synthesized according to the experimental

procedure reported for compound 4a, starting from compound 3f

(0.234 g, 0.001 mol) and 1-chloro-3-(chloromethyl)benzene

(0.174 g, 0.001 mol). The crude product was obtained and puriﬁed

via silica gel column chromatography (eluent, ethyl acetate/meth-

anol, 5/1, V/V) to give pure compound 5d (0.182 g) as white solid.

4.1.23. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

methyl-N-(4-nitrobenzyl)aniline (5g)

Compound 5g was prepared according to the experimental

procedure described for compound 4a, starting from compound 3a

(0.474 g, 2.0 mmol) and 1-(bromomethyl)-4-nitrobenzene (0.442 g,

2.0 mmol). The pure product 5g was obtained as white solid. Yield:

Yield: 50.1%; mp: 158e160 ^ꢀC; IR (KBr)

(aromatic CeH), 2900 (CH₂), 2849 (2-ClPh-CH₂), 1605 (C]N), 1592,

1571, 1504, 1454 (aromatic frame), 1387 (CH₃), 820, 753 cm^ꢁ^{1 1}H

NMR (300 MHz, CDCl₃) : 2.23 (s, 3H, eCH₃), 4.51 (s, 2H, BimeCH₂),

n

: 3421 (BimeNH), 3019

59.5%; mp: 123e125 ^ꢀC; IR (KBr)

n

: 3423 (BimeNH), 3020 (aromatic

CeH), 2903 (CH₂), 2857 (4-NO₂Ph-CH₂), 1606 (C]N), 1597, 1572,

1507, 1455 (aromatic frame), 1388 (CH₃), 843, 824 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃) : 2.38 (s, 3H, eCH₃), 4.52 (s, 2H, BimeCH₂), 4.84

;

d

4.69 (s, 2H, 2-ClPh-CH₂), 6.70 (d, 2H, J ¼ 3.0 Hz, 4-CH₃Ph-2,6-H),

7.05 (d, 3H, J ¼ 9.0 Hz, 4-CH₃Ph-3,5-H 2-ClPh-6-H), 7.25e7.23 (m,

4H, Bim-6,7-H, 2-ClPh-4,5-H), 7.36 (d, 1H, J ¼ 6.0 Hz, 2-ClPh-5-H),

(s, 2H, 4-NO₂Ph-CH₂), 6.74 (d, 2H, J ¼ 3.0 Hz, 4-CH₃Ph-2,6-H), 7.09

(d, 2H, J ¼ 6.0 Hz, 4-CH₃Ph-3,5-H), 7.25e7.24 (m, 2H, Bim-6,7-H),

7.52 (s, 2H, Bim-5,8-H), 7.58 (d, 2H, J ¼ 6.0 Hz, 4-NO₂Ph-2,6-H),

7.52 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 20.34

7.86e7.85 (s, 2H, 4-NO₂Ph-3,5-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d:

(methyl-C), 50.23 (2-ClPh-CH₂), 54.68 (BimeCH₂), 109.46 (4-

CH₃Ph-2,6-C), 113.51 (Bim-5,8-C), 122.48 (Bim-6,7-C), 122.78 (2-

ClPh-5-C), 122.81 (2-ClPh-4-C), 122.87 (2-ClPh-3-C), 122.99 (2-

ClPh-6-C), 123.11 (4-CH₃Ph-3,5-C), 127.68 (4-CH₃Ph-4-C), 129.04

(2-ClPh-2-C), 132.32 (2-ClPh-1-C), 137.89 (Bim-4,9-C), 145.05 (Bim-

2-C), 151.58 (4-CH₃Ph-1-C) ppm; MS (m/z): 362 [M þ H]^þ; HRMS

(ESI) calcd. for C₂₀H₂₀ClN₃[M þ H]^þ, 362.1424; found, 362.1422.

20.30 (CH₃), 49.62 (4-NO₂Ph-CH₂), 54.67 (Bim-CH₂), 109.54 (4-

CH₃Ph-2,6-C), 113.49 (Bim-5,8-C), 122.47 (Bim-6,7-C), 122.79 (4-

NO₂Ph-3,5-C), 122.97 (4-NO₂Ph-2,6-C), 123.13 (4-CH₃Ph-3,5-C),

127.74 (4-CH₃Ph-4-C), 137.85 (Bim-4,9-C), 145.04 (Bim-2-C), 149.03

(4-NO₂Ph-1-C), 151.34 (4-NO₂Ph-4-C), 151.78 (4-CH₃Ph-1-C) ppm;

MS (m/z): 373 [M þ H]^þ; HRMS (ESI) calcd. for C₂₂H₂₀N₄O₂

[M þ H]^þ, 373.1665; found, 373.1664.

4.1.21. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(3-

chlorobenzyl)-4-methylaniline (5e)

4.1.24. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2,4-

diﬂuorobenzyl)-4-methylaniline (5h)

Prepared the same way as the general procedure described for

compound 4a, starting from compound 3f (0.234 g, 0.001 mol) and

1-chloro-3-(chloromethyl)benzene (0.161 g, 0.001 mol), pure

compound 5e (0.176 g) was synthesized as white solid. Yield: 48.7%;

Compound 5h was prepared according to the procedure

depicted for compound 4a, starting from compound 3f (0.330 g,

1.4 mmol) and 1-(bromomethyl)-2,4-diﬂuorobenzene (0.302 g,

1.5 mmol). The pure product 5h was obtained as white solid. Yield:

mp: 115e117 ^ꢀC; IR (KBr)

n

: 3421 (BimeNH), 3020 (aromatic CeH),

2901 (CH₂), 2851 (3-ClPh-CH₂), 1605 (C]N), 1593, 1571, 1504, 1454

(aromatic frame), 1387 (CH₃), 821, 768, 683 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃) : 2.23 (s, 3H, eCH₃), 4.52 (s, 2H, BimeCH₂), 4.71

51.2%; mp: 155e156 ^ꢀC; IR (KBr)

n

: 3422 (BimeNH), 3019 (aromatic

CeH), 2906 (CH₂), 2851 (2,4-F₂Ph-CH₂), 1604 (C]N), 1595, 1569,

1506, 1453 (aromatic frame), 1386 (CH₃), 830, 821 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃) : 2.23 (s, 3H, eCH₃), 4.53 (s, 2H, BimeCH₂), 4.66

;

d

(s, 2H, 3-ClPh-CH₂), 6.70 (d, 2H, J ¼ 3.0 Hz, 4-CH₃Ph-2,6-H), 7.05 (d,

3H, J ¼ 9.0 Hz, 4-CH₃Ph-3,5-H, 3-ClPh-6-H), 7.25e7.24 (m, 2H, Bim-

6,7-H), 7.33 (d, 2H, J ¼ 3.0 Hz, 3-ClPh-4,5-H), 7.42 (s, 1H, 3-ClPh-2-

(s, 2H, 2,4-F₂Ph-CH₂), 6.63e6.61 (m, 1H, 2,4-F₂Ph-3-H), 6.65 (d, 2H,

J ¼ 3.0 Hz, 4-CH₃Ph-2,6-H), 6.69e6.67 (m, 1H, 2,4-F₂Ph-5-H), 6.74

(d, 2H, J ¼ 9.0 Hz, 4-CH₃Ph-3,5-H), 7.20e7.18 (m, 1H, 2,4-F₂Ph-6-H),

7.25 (d, 2H, J ¼ 9.0 Hz, Bim-6,7-H), 7.50 (s, 2H, Bim-5,8-H) ppm; ¹³C

H), 7.52 (s, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 20.31

(CH₃), 50.19 (3-ClPh-CH₂), 54.75 (BimeCH₂), 109.50 (4-CH₃Ph-2,6-

C), 113.59 (Bim-5,8-C), 122.46 (Bim-6,7-C), 122.65 (3-ClPh-6-C),

122.73 (3-ClPh-5-C), 122.80 (3-ClPh-4-C), 123.09 (4-CH₃Ph-3,5-C),

127.72 (4-CH₃Ph-4-C), 128.02 (3-ClPh-2-C), 128.02 (3-ClPh-3-C),

132.67 (3-ClPh-1-C), 137.86 (Bim-4,9-C), 145.12 (Bim-2-C), 151.55

(4-CH₃Ph-1-C) ppm; MS (m/z): 362 [M þ H]^þ; HRMS (ESI) calcd. for

C₂₂H₂₀ClN₃[M þ H]^þ, 362.1424; found, 362.1423.

NMR (75 MHz, CDCl₃) d: 20.20 (CH₃), 49.22 (2,4-F₂Ph-CH₂), 54.92

(BimeCH₂), 107.21 (2,4-F₂Ph-3-C), 110.54 (2,4-F₂Ph-5-C), 114.08 (4-

CH₃Ph-2,6-C), 114.11 (Bim-5,8-C), 119.82 (2,4-F₂Ph-5-C), 122.68

(Bim-6,7-C), 128.89 (4-CH₃Ph-3,5-C), 130.65 (4-CH₃Ph-4-C), 131.13

(2,4-F₂Ph-6-C), 138.17 (Bim-4,9-C), 145.65 (Bim-2-C), 152.07 (4-

CH₃Ph-1-C), 155.13 (2,4-F₂Ph-4-C), 160.04 (2,4-F₂Ph-2-C) ppm;

MS (m/z): 364 [M þ H]^þ; HRMS (ESI) calcd. for C₂₂H₁₉F₂N₃

[M þ H]^þ, 364.1625; found, 364.1624.

4.1.22. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(4-

chlorobenzyl)-4-methylaniline (5f)

4.1.25. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2,4-

dichlorobenzyl)-4-methylaniline (5i)

Compound 5f was prepared according to the experimental pro-

cedure reported for compound 4a, starting from compound 3f

(0.158 g, 0.7 mmol) and 1-chloro-4-(chloromethyl)benzene (0.130 g,

0.7 mmol). The desired pure compound 5f (0.15 g) was obtained as

Compound 5i was prepared according to the experimental

procedure reported for compound 4a, starting from compound 3f

(0.956 g, 4.0 mmol) and 2,4-dichloro-1-(chloromethyl)benzene

(0.902 g, 4.3 mmol). The pure product 5i was obtained as white

white solid. Yield: 51.4%; mp: 145e147 ^ꢀC; IR (KBr)

n

: 3421 (BimeNH),

3019 (aromatic CeH), 2900 (CH₂), 2850 (4-ClPh-CH₂), 1605 (C]N),

1593, 1571, 1505, 1454 (aromatic frame), 1386 (CH₃), 835, 820 cm^ꢁ1

¹H NMR (300 MHz, CDCl₃)

: 2.23 (s, 3H, eCH₃), 4.51 (s, 2H, BimeCH₂),

solid. Yield: 47.6%; mp: 125e127 ^ꢀC; IR (KBr)

n: 3423 (BimeNH),

;

3021 (aromatic CeH), 2907 (CH₂), 2853 (2,4-Cl₂Ph-CH₂), 1605

(C]N), 1597, 1571, 1508, 1455 (aromatic frame), 1389 (CH₃), 834,

d

4.70 (s, 2H, 4-ClPh-CH₂), 6.71 (d, 2H, J ¼ 6.0 Hz, 4-CH₃Ph-2,6-H), 7.07

823 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

d: 2.24 (s, 3H, eCH₃), 4.51 (s,

(d, 2H, J ¼ 6.0 Hz, 4-CH₃Ph-3,5-H), 7.25e7.24 (m, 2H, Bim-6,7-H), 7.34

2H, BimeCH₂), 4.68 (s, 2H, 2,4-Cl₂Ph-CH₂), 6.65 (d, 2H, J ¼ 6.0 Hz, 4-

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

341

CH₃Ph-2,6-H), 6.75 (d, 2H, J ¼ 9.0 Hz, 4-CH₃Ph-3,5-H), 6.89e6.87

(m, 1H, 2,4-Cl₂Ph-6-H), 7.25 (d, 3H, J ¼ 9.0 Hz, Bim-6,7-H, 2,4-Cl₂Ph-

5-H), 7.51 (s, 2H, Bim-5,8-H), 7.56e7.52 (s, 1H, 2,4-Cl₂Ph-3-H) ppm;

F₂Ph-CH₂), 6.65 (m, H, 2,4-F₂Ph-4-H), 6.68 (d, H, J ¼ 3.0 Hz, 2,4-

Cl₂Ph-6-H), 7.10e7.06 (m, H, 2,4-F₂Ph-5-H), 7.18 (m, 1H, 2,4-

F₂Ph-6-H), 7.22 (d, H, J ¼ 6.0 Hz, 2,4-Cl₂Ph-5-H), 7.27e7.24 (m,

2H, Bim-6,7-H), 7.53 (s, 2H, Bim-5,8-H), 7.84 (d, 1H, J ¼ 6.0 Hz, 2,4-

¹³C NMR (75 MHz, CDCl₃)

d: 20.17 (CH₃), 48.64 (2,4-Cl₂Ph-CH₂),

54.96 (BimeCH₂), 114.04 (4-CH₃Ph-2,6-C), 114.11 (Bim-5,8-C),

122.67 (Bim-6,7-C),124.34 (2,4-Cl₂Ph-5-C),128.86 (4-CH₃Ph-3,5-C),

129.21 (2,4-Cl₂Ph-3-C), 130.64 (4-CH₃Ph-4-C), 130.69 (2,4-Cl₂Ph-6-

C), 131.75 (2,4-Cl₂Ph-4-C), 132.03 (2,4-Cl₂Ph-2-C), 132.69 (2,4-

Cl₂Ph-1-C), 138.15 (Bim-4,9-C), 145.63 (Bim-2-C), 152.05 (4-

CH₃Ph-1-C) ppm; MS (m/z): 396 [M þ H]^þ; HRMS (ESI) calcd. for

C₂₂H₁₉Cl₂N₃[M þ H]^þ, 396.1034; found, 396.1033.

Cl₂Ph-3-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 49.26 (2,4-F₂Ph-

CH₂), 54.23 (BimeCH₂), 103.85 (2,4-F₂Ph-3-C), 109.74 (2,4-F₂Ph-5-

C), 114.13 (Bim-5,8-C), 115.23 (2,4-Cl₂Ph-6-C), 121.43 (2,4-F₂Ph-1-

C), 122.68 (Bim-6,7-C), 123.95 (2,4-Cl₂Ph-2-C), 124.89 (2,4-Cl₂Ph-4-

C), 127.12 (2,4-Cl₂Ph-5-C), 131.24 (2,4-F₂Ph-6-C), 131.43 (2,4-Cl₂Ph-

3-C), 138.13 (Bim-4,9-C), 145.63 (Bim-2-C), 148.45 (2,4-Cl₂Ph-1-C),

159.54 (2,4-F₂Ph-4-C), 162.11 (2,4-F₂Ph-2-C) ppm; MS (m/z): 418

[M þ H]^þ; HRMS (ESI) calcd. for C₂₁H₁₆Cl₂F₂N₃[M þ H]^þ,

418.0689; found, 418.0687.

4.1.26. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(3,4-

dichlorobenzyl)-4-methylaniline (5j)

Pure compound 5j was obtained in process of synthesizing

4.1.29. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2,4-

diﬂuorobenzyl)-3,5-bis (triﬂuoromethyl)aniline (5m)

compound 4a as white solid. Yield: 43.5%; mp: 161e163 ^ꢀC; IR (KBr)

n

: 3423 (BimeNH), 3022 (aromatic CeH), 2907 (CH₂), 2854 (3,4-

Cl₂Ph-CH₂), 1605 (C]N), 1596, 1571, 1507, 1455 (aromatic frame),

1388 (CH₃), 836, 823 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

: 2.22 (s, 3H,

Compound 5m was prepared according to the experimental

procedure reported for compound 4a, starting from compound 3i

(1.079 g, 0.003 mol) and 1-(bromomethyl)-2,4-diﬂuorobenzene

(0.635 g, 0.003 mol). The pure product 5m was obtained as white

d

eCH₃), 4.50 (s, 2H, BimeCH₂), 4.70 (s, 2H, 3,4-Cl₂Ph-CH₂), 6.71 (d,

2H, J ¼ 9.0 Hz, 4-CH₃Ph-2,6-H), 7.02 (d, 3H, J ¼ 9.0 Hz, 4-CH₃Ph-3,5-

H 3,4-Cl₂Ph-6-H), 7.24e7.23 (m, 2H, Bim-6,7-H), 7.28 (s, 1H, 3,4-

Cl₂Ph-2-H), 7.35 (d, 1H, J ¼ 9.0 Hz, 3,4-Cl₂Ph-5-H), 7.52 (s, 2H,

solid. Yield: 6.7%; mp: 128e130 ^ꢀC; IR (KBr)

n: 3431 (BimeNH),

3033 (aromatic CeH), 2915 (CH₂), 2869 (2,4-F₂Ph-CH₂), 1621

(C]N), 1598, 1571, 1509, 1459 (aromatic frame), 886, 842 cm^ꢁ1; ¹H

Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 20.19 (CH₃), 50.08

NMR (300 MHz, CDCl₃) d: 4.73 (s, 2H, BimeCH₂), 4.86 (s, 2H, 2,4-

(3,4-Cl₂Ph-CH₂), 54.91 (BimeCH₂), 114.07 (4-CH₃Ph-2,6-C), 114.10

(Bim-5,8-C), 122.69 (Bim-6,7-C), 126.29 (3,4-Cl₂Ph-6-C), 128.58

(3,4-Cl₂Ph-3-C), 128.64 (3,4-Cl₂Ph-2-C), 128.88 (4-CH₃Ph-3,5-C),

130.24 (3,4-Cl₂Ph-5-C), 130.66 (4-CH₃Ph-4-C), 131.25 (3,4-Cl₂Ph-4-

C), 132.85 (3,4-Cl₂Ph-1-C), 138.18 (Bim-4,9-C), 145.64 (Bim-2-C),

152.06 (4-CH₃Ph-1-C) ppm; MS (m/z): 396 [M þ H]^þ; HRMS (ESI)

calcd. for C₂₂H₁₉Cl₂N₃[M þ H]^þ, 396.1034; found, 396.1032.

F₂Ph-CH₂), 6.82 (m, 1H, 2,4-F₂Ph-3-H), 6.88e6.84 (m, 1H, 2,4-

F₂Ph-5-H), 7.15e7.10 (m, 3H, 2,4-F₂Ph-6-H, 3,5-(CF₃)₂Ph-2,6-H),

7.26e7.23 (s, 2H, Bim-6,7-H), 7.29 (s, 1H, 3,5-(CF₃)₂Ph-4-H), 7.53 (s,

2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 50.14 (2,4-F₂Ph-

CH₂), 56.27 (BimeCH₂), 106.11 (2,4-F₂Ph-3-C), 109.53 (2,4-F₂Ph-5-

C), 113.04 (3,5-(CF₃)₂Ph-4-C), 115.55 (Bim-5,8-C), 116.87 (2,4-F₂Ph-

1-C), 122.53 (3,5-(CF₃)₂Ph-2,6-C), 125.85 (Bim-6,7-C), 129.55 (CF₃-

2C), 133.58 (2,4-F₂Ph-6-C), 134.96 (3,5-(CF₃)₂Ph-3,5-C), 136.07

(Bim-4,9-C), 147.89 (Bim-2-C), 157.78 (3,5-(CF₃)₂Ph-1-C), 163.97

(2,4-F₂Ph-4-C), 167.48 (2,4-F₂Ph-2-C) ppm; MS (m/z): 486

[M þ H]^þ; HRMS (ESI) calcd. for C₂₃H₁₆F₈N₃[M þ H]^þ, 486.1216;

found, 486.1212.

4.1.27. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2,4-

dichlorobenzyl)-2,4-diﬂuoroaniline (5k)

Compound 5k was prepared according to the experimental

procedure described for compound 4a, starting from compound 3g

(1.044 g, 0.004 mol) and 2,4-dichloro-1-(chloromethyl)benzene

(0.803 g, 0.004 mol). The pure product 5k was obtained as yellow

4.1.30. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2,4-

dichlorobenzyl)-3,5-bis(triﬂuoromethyl) aniline (5n)

Prepared according to the general procedure described for 4a,

starting from compound 3i (1.049 g, 0.003 mol) and 2,4-dichloro-1-

(chloromethyl)benzene (0.603 g, 0.003 mol), pure compound 5n

was obtained as white solid. Yield: 5.9%; mp: 132e133 ^ꢀC; IR (KBr)

syrup. Yield: 21.3%; IR (KBr)

H), 2909 (CH₂), 2862 (2,4-Cl₂Ph-CH₂), 1607 (C]N), 1595, 1569,

1505, 1457 (aromatic frame), 837, 832 cm^ꢁ^{1 1}H NMR (300 MHz,

CDCl₃) : 4.54 (s, 2H, BimeCH₂), 4.74 (s, 2H, 2,4-Cl₂Ph-CH₂), 6.55e

n: 3426 (BimeNH), 3027 (aromatic Ce

;

d

6.48 (m, 1H, 2,4-F₂Ph-3-H), 6.67e6.60 (m, 1H, 2,4-F₂Ph-6-H), 6.82e

6.74 (m, 1H, 2,4-F₂Ph-5-H), 7.27e7.25 (m, 2H, Bim-6,7-H), 7.28 (s, H,

2,4-Cl₂Ph-5-H), 7.52 (s, 2H, Bim-5,8-H), 7.71 (s, 1H, 2,4-Cl₂Ph-3-H)

n

: 3439 (BimeNH), 3035 (aromatic CeH), 2919 (CH₂), 2873 (2,4-

Cl₂Ph-CH₂), 1628 (C]N), 1599, 1573, 1509, 1459 (aromatic frame),

892, 847 cm^ꢁ1; ¹H NMR (300 MHz, CDCl₃)

: 4.75 (s, 2H, BimeCH₂),

ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 49.69 (2,4-Cl₂Ph-CH₂), 54.99

d

(BimeCH₂), 104.46 (2,4-F₂Ph-3-C), 111.24 (2,4-F₂Ph-5-C), 114.12

(Bim-5,8-C), 115.36 (2,4-F₂Ph-6-C), 122.69 (Bim-6,7-C), 124.32 (2,4-

Cl₂Ph-5-C), 129.22 (2,4-Cl₂Ph-3-C), 130.72 (2,4-Cl₂Ph-6-C), 131.09

(2,4-F₂Ph-1-C), 131.74 (2,4-Cl₂Ph-4-C), 132.01 (2,4-Cl₂Ph-2-C),

132.67 (2,4-Cl₂Ph-1-C), 138.14 (Bim-4,9-C), 145.62 (Bim-2-C),

154.38 (2,4-F₂Ph-4-C), 157.69 (2,4-F₂Ph-2-C) ppm; MS (m/z): 418

[M þ H]^þ; HRMS (ESI) calcd. for C₂₁H₁₆Cl₂F₂N₃[M þ H]^þ, 418.0689;

found, 418.0688.

4.88 (s, 2H, 2,4-Cl₂Ph-CH₂), 6.98 (s, 1H, 2,4-Cl₂Ph-6-H), 7.12e7.04

(m, 2H, 3,5-(CF₃)₂Ph-2,6-H), 7.26e7.23 (s, 2H, Bim-6,7-H), 7.28 (s,

1H, 2,4-Cl₂Ph-5-H), 7.29 (s, 1H, 3,5-(CF₃)₂Ph-4-H), 7.53 (s, 2H, Bim-

5,8-H), 7.65 (s, 2H, 2,4-Cl₂Ph-3-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d:

51.07 (2,4-Cl₂Ph-CH₂), 56.31 (Bim-CH₂), 113.04 (3,5-(CF₃)₂Ph-4-C),

115.56 (Bim-5,8-C), 122.53 (3,5-(CF₃)₂Ph-2,6-C), 125.86 (Bim-6,7-C),

129.55 (CF₃-2C), 129.89 (2,4-Cl₂Ph-3-C), 134.39 (2,4-Cl₂Ph-5-C),

134.96 (3,5-(CF₃)₂Ph-3,5-C), 131.14 (2,4-Cl₂Ph-4-C), 131.87 (2,4-

Cl₂Ph-2-C), 132.23 (2,4-Cl₂Ph-1-C), 136.09 (Bim-4,9-C), 147.93

(Bim-2-C), 157.80 (3,5-(CF₃)₂Ph-1-C) ppm; MS (m/z): 518 [M þ H]^þ;

HRMS (ESI) calcd. for C₂₃H₁₆Cl₂F₆N₃[M þ H]^þ, 518.0625; found,

518.0623.

4.1.28. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-2,4-

dichloro-N-(2,4-diﬂuorobenzyl)aniline (5l)

Compound 5l was prepared according to the experimental

procedure depicted for compound 4a, starting from compound 3h

(0.475 g, 0.004 mol) and 1-(bromomethyl)-2,4-diﬂuorobenzene

(0.621 g, 0.004 mol). The pure product 5l was obtained as yel-

4.1.31. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-4-

methyl-N-octylaniline hydrochloride (6)

To a solution of compound 4c (98 mg, 0.3 mmol) in ethyl ether

(10 mL) was added hydrochloride acid (10 mL, 4 mol/L). The

mixture was stirred at room temperature for 5e6 h. After the re-

action was completed (monitored by TLC, eluent, ethyl acetate/

low oil. Yield: 33.2%; mp: 128e129 ^ꢀC; IR (KBr)

n

: 3428 (BimeNH),

3029 (aromatic CeH), 2911 (CH₂), 2859 (2,4-F₂Ph-CH₂), 1608

(C]N), 1596, 1569, 1507, 1458 (aromatic frame), 838, 831 cm^ꢁ1; ¹H

NMR (300 MHz, CDCl₃) d: 4.55 (s, 2H, BimeCH₂), 4.69 (s, 2H, 2,4-

342

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

petroleum ether, 1/1, V/V), the formed precipitate was ﬁltered, and

washed with chloroform, dried by freeze drying oven to afford

hydrochloride hydrochloride 6 (88 mg) as yellow solid. Yield:

10.0 mmol), 1,4-dibromohexane (2.91 g, 12.0 mmol) and potassium

carbonate (2.14 g, 15.1 mmol). The desired compound 10a (2.92 g)

was obtained as white solid. Yield: 84.1%; mp: 56e58 ^ꢀC in agree-

ment with the literature [33] (mp: 56e57 ^ꢀC).

74.3%; mp: 125e126 ^ꢀC; IR (KBr)

n

: 3440 (BimeNH), 3039 (aromatic

CeH), 2930, 2853 (CH₂, CH₃), 1623 (C]N), 1601, 1582, 1521, 1469

(aromatic frame), 1383, 1379 (CH₃), 825 cm^ꢁ^{1 1}H NMR (300 MHz,

CDCl₃)

;

4.1.36. Synthesis of intermediate 3-acetyl-7-4-(bromomethyl)

benzyloxy-2H-chromen-2-one (10b)

d

: 0.88e0.86 (t, 3H, J ¼ 3.0 Hz, CH₂CH₃), 1.31e1.28 (m, 12H,

(CH₂)₆CH₃), 2.33 (s, 3H, 4-CH₃Ph-CH₃), 3.55e3.54 (t, 2H, J ¼ 3.0 Hz,

CH₂(CH₂)₆CH₃), 5.28 (s, 2H, BimeCH₂), 7.25e7.23 (m, 2H, 4-CH₃Ph-

3,5-H), 7.54e7.51 (m, 2H, Bim-6,7-H), 7.74 (m, 2H, 4-CH₃Ph-2,6-H),

Compound 10b was prepared according to the experimental

procedure reported for compound 10a, starting from compound 9b

(2.07 g, 10.0 mmol), 1,4-bis(bromomethyl)benzene (3.18 g,

12.0 mmol) and potassium carbonate (2.15 g, 15.1 mmol). The

desired compound 10b (2.76 g) was obtained as white solid. Yield:

71.2%; mp: 68e69 ^ꢀC in agreement with the literature [33] (mp:

66e67 ^ꢀC).

7.91e7.89 (m, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d:

13.95 (CH₂CH₃), 20.17 (4-CH₃Ph), 21.33 (CH₂CH₃), 21.79

(CH₂(CH₂)₅CH₃), 25.92 (CH₂(CH₂)₄CH₃), 28.15 (CH₂(CH₂)₃CH₃),

28.29 (CH₂(CH₂)₂CH₃), 31.33 (CH₂CH₂CH₃), 54.37 (CH₂(CH₂)₆CH₃),

59.35 (BimeCH₂), 120.89 (4-CH₃Ph-2,6-C), 123.14 (Bim-5,8-C),

131.12 (Bim-6,7-C), 138.18 (Bim-4,9-C), 133.21 (4-CH₃Ph-3,5-C),

151.64 (Bim-2-C), 151.83 (4-CH₃Ph-4-C), 159.42 (4-CH₃Ph-1-C)

ppm; MS (m/z): 350 [Me2HCl þ H]^þ; HRMS (ESI) calcd. for

C₂₃H₃₂ClN₃[Me2HCl þ H]^þ, 350.2596; found, 350.2597.

4.1.37. Synthesis of 7-(6-(((1H-benzo[d]imidazol-2-yl)methyl)(4-

ﬂuorophenyl)amino)hexyloxy)-4-methyl-2H-chromen-2-one (11a)

Compound 11a was obtained as yellow solid. Yield: 39.6%; mp:

128e130 ^ꢀC; IR (KBr)

n

: 3432 (BimeNH), 3034 (aromatic CeH), 2913,

2838 (CH₂, CH₃), 1707 (C]O), 1611 (C]N), 1599, 1573, 1509, 1459

(aromatic frame), 1381 (CH₃), 813, 767, 721, 631 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃) : 1.52e1.42 (m, 4H, coumarin-O(CH₂)₂(CH₂)₂

4.1.32. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(2,4-

diﬂuorobenzyl)-3,5-bis (triﬂuoromethyl)aniline hydrochloride (7)

;

d

Compound

7

was prepared according to the procedure

(CH₂)₂), 1.80e1.78 (m, 4H, coumarin-OCH₂CH₂(CH₂)₂CH₂), 2.39 (s,

3H, coumarineCH₃), 3.52e3.49 (m, 2H, coumarin-O(CH₂)₅CH₂),

4.39e4.36 (t, 2H, J ¼ 9.0 Hz, coumarineOCH₂), 4.65 (s, 2H, BimeCH₂),

6.13 (s, 1H, coumarin-3-H), 6.80e6.74 (m, 2H, 4-FPh-2,6-H), 6.99e

6.94 (m, 2H, coumarin-7,9-H), 7.13e7.07 (m, 2H, 4-FPh-3,5-H), 7.28e

7.27 (m, 2H, Bim-6,7-H), 7.50e7.45 (m, 2H, Bim-5,8-H) ppm, 7.77 (d,

depicted for compound 6, starting from compound 4m (101 mg,

0.2 mmol). The product 7 (89 mg) was obtained as yellow solid.

Yield: 77.8%; mp: 137e138 ^ꢀC; IR (KBr)

n

: 3443 (BimeNH), 3040

(aromatic CeH), 2931 (CH₂), 2882 (2,4-F₂Ph-CH₂), 1635 (C]N),

1605, 1579, 1517, 1464 (aromatic frame), 892, 853 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃) : 5.34 (s, 2H, BimeCH₂), 5.47 (s, 2H, 2,4-F₂Ph-

;

d

1H, J ¼ 9.0 Hz, coumarin-6-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 19.03

CH₂), 6.90 (m, 1H, 2,4-F₂Ph-3-H), 6.98e6.95 (m, 1H, 2,4-F₂Ph-5-

H), 7.18 (m, 1H, 2,4-F₂Ph-6-H), 7.35e7.33 (m, 2H, 3,5-(CF₃)₂Ph-

2,6-H), 7.56e7.54 (m, 2H, Bim-6,7-H), 7.61 (s, 1H, 3,5-(CF₃)₂Ph-

4-H), 7.94e7.92 (m, 2H, Bim-5,8-H) ppm; ¹³C NMR (75 MHz,

(coumarineCH₃), 24.31 (coumarineO(CH₂)₃CH₂), 26.68 (coumarine

O(CH₂)₂CH₂), 28.44 (coumarineO(CH₂)₄CH₂), 28.53 (coumarine

OCH₂CH₂), 48.79 (coumarineOCH₂), 54.58 (BimeCH₂), 68.45

(coumarineOC₅H₁₀CH₂), 109.32 (coumarin-9-C), 115.04 (coumarin-

7-C),115.16 (coumarin-5-C),116.54 (coumarin-3-C),114.20 (Bim-5,8-

C), 114.32 (4-FPh-2,6-C), 115.25 (4-FPh-3,5-C), 123.04 (Bim-6,7-C),

125.37 (coumarin-6-C), 138.26 (Bim-4,9-C), 145.59 (Bim-2-C), 149.17

(4-FPh-1-C), 155.45 (coumarin-5-C), 156.67 (coumarin-10-C), 154.23

(4-FPh-4-C),156.75 (coumarin-8-C),158.06 (coumarin-2-C) ppm;MS

(m/z): 500 [M þ H]^þ; HRMS (ESI) calcd. for C₃₀H₃₀FN₃O₃[M þ H]^þ,

500.2349; found, 500.2348.

CDCl₃) d: 56.23 (2,4-F₂Ph-CH₂), 60.14 (BimeCH₂), 106.89 (2,4-

F₂Ph-3-C), 110.25 (2,4-F₂Ph-5-C), 118.08 (2,4-F₂Ph-1-C), 122.34

(Bim-5,8-C), 126.07 (Bim-6,7-C), 126.22 (3,5-(CF₃)₂Ph-4-C),

129.62 (CF₃eC), 129.78 (3,5-(CF₃)₂Ph-2,6-C), 133.86 (3,5-

(CF₃)₂Ph-3,5-C), 134.04 (2,4-F₂Ph-6-C), 140.16 (Bim-4,9-C),

151.85 (Bim-2-C), 162.84 (3,5-(CF₃)₂Ph-1-C), 163.89 (2,4-F₂Ph-4-

C), 168.04 (2,4-F₂Ph-2-C) ppm; MS (m/z): 486 [Me2HCl þ H]^þ;

HRMS (ESI) calcd. for C₂₃H₁₆ClF₈N₃[Me2HCl þ H]^þ, 486.1216;

found, 486.1215.

4.1.38. Synthesis of 7-(4-((((1H-benzo[d]imidazol-2-yl)methyl)(p-

tolyl)amino)methyl)benzyloxy)-3-acetyl-2H-chromen-2-one (11b)

Compound 11b was obtained as brown syrup. Yield: 40.1%; IR

4.1.33. Synthesis of intermediate 7-hydroxy-4-methyl-2H-

chromen-2-one (9a)

(KBr)

CH₃), 1723, 1708 (C]O), 1615 (C]N), 1599, 1573, 1509, 1459 (aro-

matic frame), 1383, 1379 (CH₃), 813, 769, 723, 635 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃) : 2.17 (s, 3H, coumarineCH₃), 2.23 (s, 3H, 4-

n: 3437 (BimeNH), 3034 (aromatic CeH), 2917, 2843 (CH₂,

Compound 9a was prepared according to the procedure

described in literature [33] starting from compound 8a (11.02 g,

100.0 mmol), ethyl acetoacetate (13.61 g, 100.0 mmol) and oxalic

acid (5.24 g, 50.0 mmol). The desired compound 9a (15.46 g) was

obtained as white solid. Yield: 95.0%; mp: 191e193 ^ꢀC in agreement

with the literature (mp: 192e193 ^ꢀC).

;

d

CH₃Ph-CH₃), 4.64 (s, 2H, Bim-CH₂), 4.96 (s, 2H, PheCH₂), 5.19 (s,

2H, coumarineOCH₂), 6.65 (d, 2H, J ¼ 9.0 Hz, 4-CH₃Ph-2,6-H), 7.06e

7.01 (m, 2H, coumarin-6,8-H), 7.13 (d, 2H, J ¼ 9.0 Hz, 4-CH₃Ph-3,5-

H), 7.18e7.13 (m, 4H, PheH), 7.26e7.24 (m, 2H, Bim-6,7-H), 7.58e

7.54 (m, 2H, Bim-5,8-H), 7.82 (d, 1H, J ¼ 9.0 Hz, coumarin-5-H), 8.91

4.1.34. Synthesis of intermediate 3-acetyl-7-hydroxy-2H-chromen-

2-one (9b)

(s, 1H, coumarin-4-H) ppm; ¹³C NMR (75 MHz, CDCl₃)

d: 20.20 (4-

Compound 9b was synthesized according to the experimental

procedure reported for compound 9a, starting from compound 8b

(13.75 g, 100.0 mmol), ethyl acetoacetate (13.61 g, 100.0 mmol) and

oxalic acid (5.21 g, 50.0 mmol). The desired compound 9b (17.73 g)

was obtained as white solid. Yield: 86.7%; mp: 212e213 ^ꢀC (liter-

ature [33] mp: 192e193 ^ꢀC).

CH₃Ph), 28.56 (coumarin-COCH₃), 50.96 (Ph-CH₂), 54.99 (Bim-CH₂),

69.07 (coumarineOCH₂), 109.32 (coumarin-9-C), 114.21 (coumarin-

5-C), 115.12 (coumarin-7-C), 114.07 (4-CH₃Ph-2,6-C), 114.11 (Bim-

5,8-C), 122.69 (Bim-6,7-C), 125.76 (Ph-2,6-CeCH₂O), 128.45 (Ph-

3,5-CeCH₂), 128.89 (4-CH₃Ph-3,5-C), 130.66 (4-CH₃Ph-4-C), 131.12

(coumarin-3-C), 134.78 (Ph-1-C), 136.87 (coumarin-4-C), 137.03

(Ph-6-C), 138.18 (Bim-4,9-C), 145.65 (Bim-2-C), 152.06 (4-CH₃Ph-1-

C), 157.04 (coumarin-10-C), 157.87 (coumarin-2-C), 158.75

(coumarin-8-C), 196.34 (coumarineCOCH₃) ppm; MS (m/z): 544

[M þ H]^þ; HRMS (ESI) calcd. for C₃₄H₂₉N₃O₄[M þ H]^þ, 544.2236;

found, 544.2237.

4.1.35. Synthesis of intermediate 7-(6-bromohexyloxy)-4-methyl-

2H-chromen-2-one (10a)

Compound 10a was prepared according to the procedure

described in literature starting from compound 9a (1.62 g,

H.-Z. Zhang et al. / European Journal of Medicinal Chemistry 64 (2013) 329e344

343

4.1.39. Synthesis of 9-(4-bromobutyl)-9H-carbazole (13)

4.2.2. Antifungal assays

Compound 13 was prepared according to the procedure

described in literature starting from 9H-carbazole (4.99 g,

30.0 mmol), 1,4-dibromobutane (6.03 g, 30.1 mmol) and sodium

hydride (1.22 g, 50.0 mmol). The desired compound 13 (7.05 g) was

obtained as white solid. Yield: 81.5%; mp: 102e103 ^ꢀC in agreement

with the literature [31] (mp: 103e104 ^ꢀC).

The newly synthesized compounds 3e7, 11 and 14 were evalu-

ated for their antifungal activities against C. albicans ATCC 76615,

Aspergillus fumigatus ATCC 96918, C. utilis, S. cerevisiae and A. ﬂavus.

A spore suspension in sterile distilled water was prepared from one

day old culture of the fungi growing on Sabouraud agar (SA) media.

The ﬁnal spore concentration was 1e5 ꢃ 10³spore mL^ꢁ1. From the

stock solutions of the tested compounds and reference antifungal

drug Fluconazole, dilutions in sterile RPMI 1640 medium (Neu-

ronbc Laboraton Technology CO., Ltd, Beijing, China) were made

4.1.40. Synthesis of N-((1H-benzo[d]imidazol-2-yl)methyl)-N-(4-

(9H-carbazol-9-yl)butyl)-2-ﬂuoroaniline (14)

Compound 14 was obtained as brown syrup. Yield: 39.2%; mp:

resulting in eleven wanted concentrations (0.25e256 mg/mL) of

143e145 ^ꢀC; IR (KBr)

n

: 3415 (BimeNH), 3305 (anilineeNH), 3051,

3017 (aromatic CeH), 2915, 2845 (CH₂, CH₃), 1609 (C]N), 1591,

1502, 1458 (aromatic frame), 757, 748, 719, 561 cm^ꢁ^{1 1}H NMR

(300 MHz, CDCl₃) : 1.95e1.86 (m, 2H, carbazolee(CH₂)₂CH₂), 2.11e

each tested compound. These dilutions were inoculated and incu-

bated at 35 ^ꢀC for 24 h.

;

d

Acknowledgements

2.01 (m, 2H, carbazoleeCH₂CH₂), 3.56e3.53 (t, 2H, J ¼ 9.0 Hz,

carbazolee(CH₂)₃CH₂), 4.26e4.24 (t, 2H, J ¼ 6.0 Hz, carbazolee

CH₂), 4.61 (s, 2H, BimeCH₂), 6.72e6.66 (m, H, 2-FPh-4-H), 6.83e

6.78 (m, H, 2-FPh-6-H), 6.99e6.96 (m, H, 2-FPh-3-H), 7.03e7.00 (m,

H, 2-FPh-5-H), 7.16e7.13 (m, 1H, carbazole-6-H), 7.26e7.24 (m, 2H,

Bim-6,7-H), 7.32 (d, 2H, J ¼ 9.0 Hz, carbazole-3,6-H), 7.59e7.52 (m,

4H, carbazole-2,7-H, Bim-5,8-H), 7.76e7.73 (m, 2H, carbazole-1,8-

H), 8.11 (d, 2H, J ¼ 6.0 Hz, carbazole-4,5-H) ppm; ¹³C NMR (75 MHz,

This work was partially supported by National Natural Science

Foundation of China [No. 21172181, 81250110089, 81250110554

(The Research Fellowship for International Young Scientists from

International (Regional) Cooperation and Exchange Program)], the

key program from Natural Science Foundation of Chongqing

(CSTC2012jjB10026), the Specialized Research Fund for the

Doctoral Program of Higher Education of China (SRFDP

20110182110007).

CDCl₃) d: 24.53 (carbazolee(CH₂)₂CH₂), 27.12 (carbazoleeCH₂CH₂),

48.89 (carbazolee(CH₂)₃CH₂), 53.24 (carbazoleeCH₂), 55.17 (Bime

CH₂), 108.56 (carbazole-2,11-C), 114.10 (Bim-5,8-C), 114.22 (2-FPh-

6-C), 115.04 (2-FPh-3-C), 118.97 (carbazole-4,9-C), 120.45 (carba-

zole-5,8-C), 122.88 (carbazole-3,10-C), 122.91 (carbazole-6,7-C),

122.93 (2-FPh-4-C), 122.97 (Bim-6,7-C), 124.32 (2-FPh-5-C), 134.55

(carbazole-1,12-C), 134.68 (2-FPh-1-C), 138.20 (Bim-4,9-C), 145.69

(Bim-2-C), 154.05 (2-FPh-2-C) ppm; MS (m/z): 463 [M þ H]^þ; HRMS

(ESI) calcd. for C₃₀H₂₇FN₄[M þ H]^þ, 463.2298; found, 463.2297.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2013.03.049.

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Article Doi

Design, synthesis and antimicrobial evaluation of novel benzimidazole type of Fluconazole analogues and their synergistic effects with Chloromycin, Norfloxacin and Fluconazole

DOI: 10.1016/j.ejmech.2013.03.049

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