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. 2005 Nov 30;10(11):1377–1386. doi: 10.3390/10111377

Synthesis and Potent Antimicrobial Activity of Some Novel N-(Alkyl)-2-Phenyl-1H-Benzimidazole-5-Carboxamidines

Hakan Göker 1,*, Mehmet Alp 1, Sulhiye Yıldız 2
PMCID: PMC6147528  PMID: 18007533

Abstract

A series of 22 novel 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidine derivatives were synthesized and evaluated for in vitro antibacterial activity against S. aureus and methicillin resistant S. aureus (MRSA), E. coli, E. faecalis and for antifungal activity against C. albicans. Compound 59 [1-(2,4-dichlorobenzyl)-N-(2-diethylaminoethyl)-1H-benzimidazole-5-carboxamidine], with a 3,4-dichlorophenyl group at the C-2 position, displayed the greatest activity (MIC = 3.12 μg/mL against both some bacteria and the fungus C. albicans).

Keywords: 1H-Benzimidazole carboxamidines, methicillin-resistant S. aureus (MRSA), antibacterial activity and antifungal activity

Introduction

We have already reported the synthesis of a series of 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidine derivatives and their very potent antibacterial activities against S. aureus and methicillin resistant S. aureus [1]. The study revealed that compounds I–IV (Figure 1) exhibited the best activity, with MIC values of 0.78 - 0.39 μg/mL against these species. As part of a continuing program focused on development of new antimicrobial benzimidazole carboxamidines, we planned to modify the structure of compounds I–IV.

Figure 1.

Figure 1

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Results and Discussion

Chemistry

Syntheses of the target benzimidazoles (Table 1, Table 2) were achieved by two different methods, as shown in Scheme 1.

Table 1.

Formulas and in vitro antibacterial and antifungal activities of 40 - 61.

graphic file with name molecules-10-01377-i001.jpg

No. Substituents Minimal inhibitory concentration, μg/mL
R’ R1 R2 R3 R4 S .a. MRSA MRSA* E. c. E. f. C. a.
40 Cl >50 >50 >50 >50 >50 >50
41 CH(CH3)2 Cl Cl >50 >50 >50 >50 >50 >50
42 CH(CH3)2 F >50 >50 >50 >50 >50 >50
43 (Et)2NCH2CH2 F >50 >50 >50 >50 >50 >50
44 (Me)2NCH2CH2 Cl >50 >50 >50 >50 >50 >50
45 (Me)2NCH2CH2 Cl Cl 12.5 12.5 12.5 >50 50 12.5
46 CH(CH3)2 CH3 CN >50 >50 >50 >50 >50 >50
47 CH(CH3)2 CH3 OCH3 OCH3 >50 >50 >50 >50 >50 >50
48 Cyclopropyl COOCH3 >50 >50 >50 >50 >50 >50
49 PhCH2 COOH >50 >50 >50 >50 >50 >50
50 PhCH2 COOCH3 12.5 25 3.12 >50 50 25
51 Cyclohexyl COOH >50 >50 >50 >50 >50 >50
52 n-butyl >50 >50 >50 >50 >50 >50
53 (Me)2NCH2CH2 n-butyl F >50 >50 >50 >50 >50 >50
54 CH(CH3)2 CH(CH3)2 F >50 >50 >50 >50 >50 >50
55 (Me)2NCH2CH2 Ph Cl Cl 12.5 3.12 12.5 >50 25 12.5
56 (Me)2NCH2CH2 PhCH2 Cl Cl 12.5 12.5 12.5 >50 25 12.5
57 (Et)2NCH2CH2 PhCH2 Cl Cl 12.5 12.5 6.25 50 12.5 12.5
58 (Me)2NCH2CH2 PhCH2 Cl Cl 50 12.5 12.5 >50 50 25
59 (Et)2NCH2CH2 2,4-di-Cl- benzyl Cl Cl 3.12 3.12 3.12 12.5 6.25 3.12
60 (Et)2NCH2CH2 PhCH2CH2 OCH3 OCH3 >50 >50 >50 >50 >50 >50
61 Isobutyl PhCH2CH2 Cl Cl 6.25 6.25 6.25 >50 12.5 12.5
Ref OH t-butyl 0.78 0.78 0.78
Sult 0.39 25 25 1.56
Amp 0.78 50 0.78
Cip 0.39
Flu 1.56
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S.a.: Staphylococcus aureus (ATCC 25923); MRSA (methicillin resistant Staphylococcus aureus, ATCC 43300); MRSA* (Methicillin resistant Staphylococcus aureus, clinical isolate); E. c.: Escherichia coli (ATCC 25922); E. f.: E. faecalis (ATCC 29212); C. a.: Candida albicans (ATCC 10231); Ref: this compound was found to be the most active compound against S. aureus by Weidner-Wells et al [3]; Sult: Sultamicillin; Amp: Ampicillin; Cip: Ciprofloxacin; Flu: Fluconazole

Table 2.

Physical and spectral data for compounds 40 – 61.


No
Mp
(OC)
Yield
(%)		 Formula
Calculated
Found		 1H-NMR
δ ppm (DMSO-d6)
(if not stated otherwise)
MS
(ESI+) m/z		 Synthesis Method and Isolation
Column Chromatography
if not stated otherwise
40 >300 35 C14H11ClN4. 2HCl . H2O
C: 46.50 H: 4.18 N: 15.5
C: 46.24 H: 3.99 N: 15.3		 7.59 - 7.97 (arom. 7H), 8.31 (s), 9.30 (s), 9.53 (s) 271 (100)
273 (33)		 (B)
Crys. Ethanolic HCl
41 >290 40 C17H16Cl2N4. 2HCl . 1.5H2O
C: 45.66 H: 4.73 N: 12.5
C: 45.67 H: 4.45 N: 12.4		 1.21 (d, 6H), 4.03 (m, 1H), 7.6 (m, 2H), 7.8(d, J=8.5, 1H), 7.83 (d, J=2, 1H), 7.92 (d, J=8.4, 1H), 8.05 (d, J=1.5, 1H), 9.06 (s), 9.42 (s), 9.54 (d) 347 (100)
349 (65)
351 (11)		 (B)
CH2Cl2 : Isopropanol : NH3 (100 : 60 : 4)
42 >300 28 C17H17FN4. 2HCl . 1.5H2O
C: 51.52 H: 5.59 N: 14.1
C: 51.90 H: 5.31 N: 14.1		 1.29 (d, 6H), 4.08 (m, 1H), 7.51 (t, 2H), 7.67 (d, J=8.3, 1H), 7.87 (d, J=8.4, 1H), 8.08 (s, 1H), 8.44 (m, 2H), 9.08 (s), 9.48 (s), 9.62 (d) 297 (100) (B)
CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
43 >300 41 C20H24FN5. 3HCl . 2H2O
C: 48.15 H: 6.26 N: 14.1
C: 48.01 H: 6.02 N: 13.99		 1.29 (t, 6H), 3.26 (4H), 3.46 (2H), 4.00 (2H), 7.56 (t, 2H), 7.84 (d, J=8.4, 1H), 7.92 (d, J=8.4, 1H), 8.28 (s, 1H), 8.52 (m, 2H), 9.67 (s), 9.93 (s), 10.21 (1H), 10.96 (s) 354 (100) (B)
CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
44 100-110
bubb
37		 * C18H20ClN5. 4HCl . 1.5H2O.
0.5C2H6O
C: 42.27 H: 5.97 N: 12.97
C: 42.54 H: 6.04 N: 13.01		 (CD3OD): 3.05 (6H), 3.66 (t, 2H), 4.03 (t, 2H), 7.69 (m, 1H), 7.82 (m, 2H), 7.99 (d, 1H), 8.102 (s, 2H), 8.54 (s, 1H) 342 (100)
344 (33)		 (B)
CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
45 95-100
bubb		 18 C18H19Cl2N5. 1.5HCl 0.25H2O . 0.25C3H8O
C: 49.99 H: 5.14 N: 15.54
C: 49.97 H: 5.14 N: 15.23		 2.23 (s, 6H), 2.58 (t, 2H), 3.53 (t, 2H), 7.56 (dd, J=8.4, 1.6, 1H), 7.80 (d, J=8.8, 1H), 7.86 (d, J=8.4, 1H), 8.06 (s, 1H), 8.25 (dd, J= 8.4 1.8, 1H), 8.50 (d, J=1.8, 1H) 376 (100)
378 (69)
380 (11)		 (A)
CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
46 200-210
bubb		 30 C19H19N5. 1.5H2O . 0.1C3H8O
C: 66.15 H: 6.55 N: 19.98
C: 66.19 H: 5.97 N: 19.82		 1.23 (d, 6H), 3.95 (m, 1H), 3.97 (s, 3H), 7.76 (m, 2H), 8.09 (m, 5H) 318 (100) (A)
CH2Cl2 : Isopropanol : Propylamine(100 : 50 : 4)
47 100-110
bubb		 26 C20H24N4O2 . 1.8H2O
C: 62.42 H: 7.22 N: 14.55
C: 62.62 H: 6.94 N: 14.21		 (DMSO-d6 + 1 drop D2O): 1.17 (d, 6H), 3.85-3.90 (10H), 7.15 (d, J=8.4, 1H), 7.4 (s, 2H), 7.62 (m, 2H), 7.97 (s, 1H) 353 (100) (A)
CH2Cl2 : Isopropanol : Propylamine (100 : 50 : 3)
48 285-290
bubb		 60 C19H18N4O2. 3H2O . 0.3C3H8O
C: 58.80 H: 6.54 N: 13.78
C: 58.72 H: 6.20 N: 13.36		 0.77 (2H), 0.91 (d, 2H), 2.79 (1H), 3.89 (s, 3H), 7.53 (d, J=8, 1H), 7.67 (d, J=8.4, 1H), 8.04 (s, 1H), 8.09 (d, J=7.8, 2H), 8.38 (d, J=8, 2H) 335 (100) (A)
Cryst. Isopropanol
49 260-270
bubb		 34 C22H18N4O2 . 2H2O
C: 65.01 H: 5.45 N: 13.77
C: 64.95 H: 5.23 N: 13.78
4.73 (s, 2H), 7.35-8.30 (aromat. 12H)
371 (100) (A)
Cryst. EtOH
50 265-275
bubb		 52 C23H20N4O2 . 0.25H2O
C: 71.03 H: 5.31 N: 14.40
C: 71.07 H: 5.14 N: 14.32		 3.88 (s, 3H), 4.53 (s, 2H), 7.27-7.56 (m, 7H), 8.03-8.06 (m, 3H), 8.37 (d, J=8.4, 2H), 385 (100) (A)
CH2Cl2 : Isopropanol : Propylamine
(100 : 100 : 6)
51 >300 64 C21H22N4O2 . 2HCl . 1.5H2O
C: 54.55 H: 5.88 N: 12.12
C: 54.26 H: 5.89 N: 12.62		 1.21-1.42-1.63-1.77-1.98 (m, 10H), 3.78 (1H), 7.65 (d, J=8.4, 1H), 7.87 (d, J=8.4, 1H), 8.1 (s, 1H), 8.15 (d, J=8, 2H), 8.47 (d, J=7.6, 2H), 9.19 (s), 9.50 (s), 9.63 (d) 363 (100) (A)
Cryst. Ethanolic HCl
52 150-155
bubb		 22 C18H20N4. 2HCl . 2.2H2O 0.5C3H8O
C: 53.84 H: 7.04 N: 12.88
C: 53.98 H: *** N: 12.81		 (Base) : 0.64 (t, 3H), 1.01 (m, 2H), 1.56 (m, 2H), 4.33 (t, 2H), 7.56 (m, 3H), 7.76 (m, 3H), 7.95 (d, J=8.6, 1H), 8.23 (d, J=1.4, 1H), 9.18 (s), 9.41 (s) 293 (100) (A)
CH2Cl2 : Isopropanol : NH3 (100 : 50 : 4)
53 205-210 bubb 32 C22H28FN5 . 3HCl . 2H2O
C: 50.14 H: 6.69 N: 13.29
C: 50.34 H: 6.52 N: 12.92		 0.78 (t, 3H), 1.20 (m, 2H), 1.70 (m, 2H), 2.90 (s, 6H), 3.51 (t, 2H), 4.03 (t, 2H), 4.45 (t, 2H), 7.55 (m, 2H), 8.01 (m, 3H), 8.10 (d, J=8.6, 1H), 8.42 (d, J=1.3, 1H), 9.77 (s), 9.97 (s), 10.28 (s) 382 (100) (B)
CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
54 115-120
bubb		 20 C20H23FN4 . 3HCl . 1.25H2O
C: 51.07 H: 6.11 N: 11.91
C: 51.05 H: 6.31 N: 11.55		 1.34 (d, 6H), 1.66 (d, 6H), 4.2 (m, 1H), 4.8 (m, 1H), 7.54 (m, 2H), 7.74 (d, J=8.7, 1H), 7.86 (m, 2H), 8.22 (2H), 9.21 (s), 9.58 (s), 9.68 (d) 339 (100) (B)
CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
55 295-300
bubb		 30 C24H23Cl2N5. 3HCl . 1.25H2O
C: 49.34 H: 4.91 N: 11.98
C: 49.45 H: *** N: 11.87		 2.85 (s, 6H), 3.44 (t, 2H), 3.92 (t, 2H), 7.37-7.80 (m, 10H), 8.41 (d, J=1.2, 1H), 9.52 (s), 9.83 (s), 10.07 (s), 10.81 (s) 452 (100)
454 (68)
456 (12)		 (A)
CH2Cl2 : Isopropanol : NH3 (100 : 50 : 15)
56 130-135
bubb		 49 C25H25Cl2N5 . 3HCl . 1.25H2O
C: 50.18 H: 5.14 N: 11.7
C: 50.06 H: 5.22 N: 11.49		 2.90 (6H), 3.50 (t, 2H), 4.03 (q, 2H), 5.74 (s, 2H), 7.04 (m, 2H),7.34 (m, 3H), 7.82 (m, 4H), 8.02 (d, J=1.9, 1H), 8.42 (d, J=1.4, 1H), 9.71 (s), 9.86 (s), 10.15 (d), 11.14 (s) 466 (100)
468 (68)
470 (11)		 (B)
CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 5)
57 120-130
bubb		 46 C27H29Cl2N5 . 3HCl . 2H2O
C: 50.68 H: 5.67 N: 10.94
C: 50.66 H: 5.62 N: 10.92		 1.27 (t, 6H), 3.24 (4H), 3.45 (2H), 3.99 (2H), 5.72 (s, 2H), 6.98 (m, 2H), 7.27 (m, 3H), 7.81 (m, 4H), 8.01 (d, J=2, 1H), 8.35 (s, 1H), 9.61 (s), 9.81 (s), 10.11 (s), 10.97 (s) 494 (100)
496 (68)
498 (12)		 (B)
CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
58 ** 33 ** C25H25Cl2N5 . HCl (CD3OD): 3.29 (6H), 3.63 (t, 2H), 3.99 (t, 2H), 5.57 (s, 2H), 7.03 (m, 2H), 7.26 (m, 3H), 7.61 (dd, J=8.4 , 2, 1H), 7.68 (d, J=8.1, 1H), 7.82 (d, J=2, 1H), 7.95 (m, 2H), 8.42 (s, 1H) 466 (100)
468 (71)
470 (13)		 (B)
CH2Cl2 : Isopropanol : NH3 (100 : 50 : 0.5)
59 120-125
bubb		 35 C27H27Cl4N5 . 3HCl . H2O
C: 46.64 H: 4.71 N: 10.07
C: 46.80 H: 4.82 N: 9.86		 1.25 (t, 6H), 3.23 (m, 4H), 3.41 (t, 2H), 3.84 (2H), 5.71 (s, 2H), 6.73 (d, J=8.8, 1H), 7.30 (dd, J=8.4 2, 1H), 7.62-7.81 (m, 5H), 7.93 (d, J=2, 1H), 8.35 (s, 1H), 9.64 (s), 9.83 (s), 10.13 (s), 10.96 (s) 562 (80)
564 (100)
566 (51)
568 (14)		 (A)
CH2Cl2 : Isopropanol : NH3 (100 : 50 : 0.5)
60 127-130
bubb.		 30 C30H37N5O2 . 3HCl . H2O
C: 54.34 H: 6.99 N: 10.56
C: 54.40 H: 6.80 N: 10.45		 1.29 (t, 6H), 3.08 (t, 2H), 3.25 (4H), 3.48 (d, 2H), 3.84 (s,3H), 3.89 (s,3H), 4.04 (q, 2H), 4.83 (t, 2H), 6.9 (m, 2H), 7.12-7.31 (m, 6H), 8.03 (d, J=8.8, 1H), 8.27 (d, J=8.8, 1H), 8.36 (s, 1H), 9.87 (s), 10.08 (s), 10.44 (s), 11.1 (s) 500 (100) (A)
CH2Cl2 : Isopropanol : iso-propylamine(90 : 30 : 2)
61 288-290 19 C26H26Cl2N4 . 1.5HCl
C: 60.04 H: 5.33 N: 10.77
C: 60.05 H: 5.50 N: 10.66		 0.96 (d,6H), 2.06 (m, 1H), 2.93 (t, 2H), 3.16 (t, 2H), 4.63 (t, 2H), 6.67 (d, J=7.2, 2H), 7.03 (t, J=7.2, 2H), 7.11 (m, 1H), 7.45 (2H), 7.70 (m, 2H), 8.01 (d, J=8.8, 1H), 8.12 (s, 1H), 9.02 (s), 9.44 (s), 9.75 (s) 465 (100)
467 (67)
469 (11)		 (A)
CH2Cl2 : Isopropanol : ethylamine(100 : 50 : 0.5)
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* Hygroscopic; ** Very hygroscopic, not measurable; *** No satisfactory result.

Scheme 1.

Scheme 1

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Method A involved nucleophilic displacement in DMF of the chloro group of 4-chloro-3-nitro-benzonitrile by reaction with several amines to give 1–8 (Table 3). The cyano group was then converted into the imidate ester, using a modified Pinner method [1], and the imidate esters were used directly to make the corresponding benzamidines 9–18 (Table 4). Their reduction with H2, Pd/C produced 19–28 (Table 5). Condensation of these derivatives with the Na2S2O5 adducts of several benzaldehydes afforded the corresponding benzimidazoles 45–52, 55 and 59–61 [2]. Method B involved cyclization of 29–32 with the Na2S2O5 adducts of various benzaldehydes to afford 5-cyano-benzimidazoles 33–39 (Table 6), following this, the cyano groups were converted into the imidate esters, as in method A, and these were used to prepared the corresponding amidine compounds 40–44, 53, 54 and 56–58. For its practical advantages this method was used in particular for cyano-benzimidazoles, which have better solubility in EtOH, although the yields were low.

Table 3.

Formulas and melting points of 1–8.

graphic file with name molecules-10-01377-i002.jpg

Comp R1 Formula Ref.
1 H C7H5N3O2 Commercial
2 methyl C8H7N3O2 Lit [1]
3 iso-propyl C10H11N3O2 Lit [2]
4 n-butyl C11H13N3O2 Lit [2]
5 phenyl C13H9N3O2 lit [5] 126oC, 126 oC
6 benzyl C14H11N3O2 Lit [2]
7 2,4-dichlorobenzyl C14H9Cl2N3O2 ---
8 PhCH2CH2 C15H13N3O2 ---
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Table 4.

Formulas, spectroscopic data, m.p. and yields of 9 - 18.

Comp R’ R1 Formula NMR
δ ppm (DMSO-d6)		 Mass
(ESI+) 		mp
(oC)		 Yield (%)
9 butyl C11H16N4O2 0.895 (t, 3H), 1.34 (m, 2H), 1.56 (m, 2H), 3.41 (q, 2H), 7.22 (d, J=9.2, 1H), 7.95 (dd, J=9.2, 2.4, 1H), 8.6 (t, 1H), 8.67 (d, J=2.4, 1H), 9.2 (br.s) 237
(100)		 200-4 62
10 iso-propyl methyl C11H16N4O2 Lit [1]
11 (CH3)2N(CH2)2 C11H17N5O2 2.24 (s, 6H), 2.58 (t, 2H), 3.5 (t, 2H), 7.14 (d, J=8.8,1H), 7.5 (dd, J=8.8, 2, 1H), 8.09 (br.s, 2H), 8.48 (d, J=2, 1H). 252
(100)		 220-230
(bubb)
Lit [6]
12 cyclo-propyl C10H12N4O2 Lit [1]
13 cyclo-hexyl C13H18N4O2 Lit [1]
14 Benzyl C14H14N4O2 Lit [1]
15 (CH3)2N(CH2)2 phenyl C17H21N5O2 2.32 (s, 6H), 2.69 (2H), 3.4 (br.s), 3.56 (t, 2H), 7.12 (d, J=9..3, 1H), 7.28-7.49 (m, 5H), 7.85 (dd, J=9.2, 2.3, 1H), 8.61 (d, J=2.3, 1H), 9.85 (s, 1H) 328
(100)		 Hygros.
110
(bubb)		 87
16 iso-butyl PhCH2CH2 C19H24N4O2 (CD3OD): 1.05 (d, 6H), 2.07 (m,1H), 3.05 (t, 2H), 3.25 (d, 2H), 3.72 (m, 2H), 7.21 (m, 2H), 7.3 (m, 4H), 7.79 (dd, J=9.2 2.4, 1H), 8.53 (t, 1H), 8.59 (d, J=2.4, 1H) 341
(100)		 247-9 75
17 (C2H5)2N(CH2)2 PhCH2CH2 C21H29N5O2 (CD3OD): 1.11 (t, 6H), 2.68 (q, 4H), 2.78 (t, 2H), 3.03 (t, 2H), 3.57 (t, 2H), 4.7 (s, 2H), 7.21 (m, 2H), 7.3 (m, 4H), 7.81 (dd, J=8.8 ,2.4, 1H), 8.62 (d, J=2.4, 1H). 384
(100)		 236-9 85
18 (C2H5)2N(CH2)2 2,4-dichloro benzyl C20H25Cl2N5O2 (CD3OD): 1.11 (t, 6H), 2.68 (q, 4H), 2.79 (t, 2H), 3.57 (t, 2H), 4.78 (t, 2H), 6.98 (d, J=9.2, 1H), 7.30 (dd, J=8.6 2.4, 1H), 7.37 (d, J=8.8, 1H), 7.53 (d, J=2, 1H), 7.78 (dd, J=9.2, 2.4, 1H), 8.68 (d, J=2.4, 1H) 438(100)
440 (65)
442 (12)		 203-5 89
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Table 5.

Formulas, spectroscopic data, mp and yields of 19 - 28.

graphic file with name molecules-10-01377-i003.jpg

Comp R’ R1 Formula NMR
δ ppm (DMSO-d6)		 MS
(ESI+)		 mp
(oC)		 Yield
(%)
19 butyl C11H18N4 0.88 (t, 3H), 1.36 (m, 2H), 1.56 (m, 2H), 3.1 (t, 2H), 5.01 (br.s, 2H), 5.57 (br.s, 2H), 6.46 (d, J=8, 1H), 6.9 (s, 1H), 7.07 (d, J=8.4, 1H), 8.58 (s, 2H), 8.74 (s, 2H). 207(100) 285 94
20 iso-propyl methyl C11H18N4 Lit [1]
21 (CH3)2N(CH2)2 C11H19N5 (+ one drop D2O): 2.2 (s, 6H), 2.54 (t, 2H), 3.4 (t, 2H), 6.57 (d, J=8, 1H), 6.85 (m, 2H) 222(100) Lit [6]
22 cyclo-propyl C10H14N4 Lit [1]
23 cyclo-hexyl C13H20N4 Lit [1]
24 benzyl C14H16N4 Lit [1]
25 (CH3)2N(CH2)2 phenyl C17H23N5 (+ one drop D2O) : 2.27 (s, 6H), 2.63 (t, 2H), 3.5 (t, 2H), 6.85-7.26 (8H) 298(100) * 90
26 iso-butyl -CH2CH2 Ph C19H26N4 0.92 (d, 6H), 1.96 (m, 1H), 2.9 (t, 2H), 3.17 (t, 2H), 3.31(2H), 4.39 (t, 1H), 5.03 (2H), 5.66 (t,1H), 6.57 (d, J=8.4, 1H), 6.87 (d, J=1.6, 1H), 6.98 (dd, J=8.4 1.6, 1H), 7.2-7.3 (m, 5H), 8.57 (s, 1H), 8.9 (s, 1H) 311(100) 98-100 94
27 (C2H5)2N(CH2)2 -CH2CH2 Ph C21H31N5 (CD3OD): 1.09 (t, 6H), 2.65 (q, 4H), 2.77 (t, 2H), 2.96 (t, 2H), 3.47 (t, 2H), 3.52 (t, 2H), 6.68 (d, J=8, 1H), 7.01 (d, J=2, 1H), 7.14-7.3 (m, 6H) 354(100) * 95
28 (C2H5)2N(CH2)2 2,4-dichloro-benzyl C20H27Cl2N5 (CD3OD): 1.14 (t, 6H), 2.76 (4H), 2.89 (2H), 3.57 (t, 2H), 4.54 (s, 2H), 6.37 (d, J=8, 1H), 7.06 (d, J=2, 2H), 7.25 (dd, J=8.2 2, 1H), 7.31 (d, J=8.4, 1H), 7.49 (d, J=2, 1H) 408(100)
410(65)
412(11)		 * 92
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* No sharp melting point.

Table 6.

Formulas, spectroscopic data, mp and yields of 33 - 39.

graphic file with name molecules-10-01377-i004.jpg

Comp R1 R2 R3 R4 Formula NMR
δ ppm (CDCl3)		 Mass
(ESI+) 		mp
(oC)		 Yield
(%)		 İsolation
33 Cl C14H8ClN3 Lit [2]
34 Cl Cl C14H7Cl2N3 Lit [2]
35 F C14H8FN3 Lit [2]
36 iso-propyl F C17H14FN3 Lit [2]
37 n-butyl F C18H16FN3 Lit [2]
38 benzyl Cl Cl C21H13Cl2N3 5.47 (s, 2H), 7.05 (m, 2H), 7.3-7.55 (m,7H),7.82 (d, J=2, 1H), 8.16 (d, J=1.5, 1H) 91(100)
378(50)
380(33)
382(6)		 192 75 Cryst.
EtOH-water
39 benzyl Cl Cl C21H13Cl2N3 5.27 (s, 2H), 6.91(m, 2H), 7.26-7.57 (m, 8H), 8.16 (1H) 91(100)
378(54)
380(31)
382(5)		 195-6 73 Cryst.
EtOH-water
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Antimicrobial Activity

The benzimidazoles 40–61 were tested by the macro-broth dilution [4] assay for in vitro antibacterial activity against Gram positive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA, a clinical isolate from a wound), Enterococcus faecalis and Gram negative Escherichia coli and for antifungal activity against Candida albicans. The MIC values are listed in Table 1. The synthesized compounds and reference drugs were dissolved in water or DMSO-water (40 %) at a concentration of 400 μg/mL. The concentration was adjusted to 100 μg/mL by four- fold dilution with media culture and bacteria solution at the first tube. Data was not taken for the initial solution because of the high DMSO concentration (10 %). We have already reported that 3,4-dichloro substitution on the 2-phenyl group of amidinobenzimidazoles plays an important role in their antibacterial activity [1]. Thus, the most active compound, 59, and less active compounds 45, and 55– 57 all have a 3,4-dichlorophenyl group at the C-2 position. Replacement of 3,4-dichloro substitution with other functions such as fluoro, cyano, methoxy, carboxyl or methyl ester caused a reduction in inhibitory activities, and only compound 50, having a methyl ester group, exhibited moderate activity against MRSA with a MIC of 3.12 μg/mL. More lipophilic substituents on the benzimidazole N-atom such as phenyl, benzyl and 2,4-dichlorobenzyl do lead to quite active compounds (55–59), however substitution with methyl, butyl and isopropyl (cf. 46, 47, 52–54) gave no significant activity. Introduction of N,N-diethylaminoethyl substitution on the cationic amidine 59 led to inhibitory activity against E. coli and C. albicans. This is a very important result, as it represents the first example to date of inhibitory activity against E. coli with these amidinobenzimidazoles. Except for compound 59, none of the compounds showed important inhibitory activity against E. faecalis and C. albicans.

Conclusions

Introduction of aromatic amidine groups into the benzimidazole system gives a good profile of Gram-positive antibacterial activity. In particular, 1-(2,4-dichlorobenzyl)-N-(2-diethylaminoethyl)-1H-benzimidazole-5-carboxamidine (59), having a 3,4-dichlorophenyl at the C-2 position, exhibited the greatest activity, with a MIC value of 3.12 μg/mL against S. aureus and MRSA. Detailed mechanistic studies are required to understand the potent activity of this compound.

Experimental

General

Uncorrected melting points were measured on an Electrothermal 9100 capillary melting point apparatus. 1H-NMR spectra were recorded employing a Varian Mercury 400 MHz FT spectrometer, chemical shifts (δ) are in ppm relative to TMS, and coupling constants (J) are reported in Hertz. Mass spectra were taken on a Waters Micromass ZQ using the ESI(+) method. Microanalyses were performed by Leco CHNS-932. Some HCl salts of compounds 40–61 were prepared by using dry HCl gas in EtOH or isopropanol. All chemicals and solvents were purchased from Aldrich Chemical Co. or Fischer Scientific. Compounds 29–32 were synthesized as described in our previous study [2]

4-(2,4-Dichlorobenzyl)amino-3-nitrobenzonitrile (7)

A mixture of 2,4-dichlorobenzylamine (3.52 g, 20 mmol) and 4-chloro-3-nitrobenzonitrile (2 g, 10.95 mmol) in DMF (3 mL) was heated under reflux for 4h at 120°C. The mixture was allowed to cool and EtOH was added. The resultant yellow precipitate was filtered, washed with water and crystallised from EtOAc-n-hexane; yield 57 %; mp: 192oC; 1H-NMR (CDCl3): 4.66 (d, J=5.6, 2H), 6.8 (d, J=9.2, 1H), 7.24 (m, 2H), 7.47 (d, J=2, 1H), 7.58 (dd, Jo=9.2, Jm=2, 1H), 8.55 (d, J=2, 1H), 8.78 (br.t, 1H).

4-(2-Phenylethyl)amino-3-nitrobenzonitrile (8)

2-Phenylethylamine (1.3 g, 10.8 mmol) and 4-chloro-3-nitrobenzonitrile (1g, 5.4 mmol) were allowed to react for 2 h, at 100oC and the product wasisolated as described for 7; yield 79 %; mp: 111oC; 1H-NMR (CDCl3): 3.04 (t, J=7.1, 2H), 3.61 (q, J=6.8, 2H), 6.89 (d, J=8.8, 1H), 7.25-7.37 (m, 4H), 7.58 (dd, Jo=8.8, Jm=2, 1H), 8.44 (br.t, 1H), 8.47 (d, J=2, 1H).

General Procedure for Synthesis of 9–18, 40–44, 53, 54, 56–58

1–8 (4.5 mmol) and 33–39 (Table 6, 1 mmol) were suspended in absolute EtOH, cooled in a ice-salt bath, and dry HCl gas was passed through the solution for 40 min. The solution was stirred in a stoppered flask at room temperature for 3 days and then diluted with dry ether. The imidate esters precipitated as yellow solids, which were washed with ether then dried under vacuum at room temperature. All imidate esters were used directly without characterisation. A suspension of imidate ester HCl in absolute EtOH was stirred with corresponding the amines (1.5 - 2 fold excess) overnight at 25-30oC. The reaction mixture was evaporated and diluted with ether, the precipitate was filtered, washed with ether, then dried. Compounds 9–18 (Table 4) were used without purification as HCl salts for the next steps since they were prepared completely pure. In contrast, crude products 40–44, 53, 54, 56–58 were treated with dilute Na2CO3 solution, then water. Further purification methods are given in Table 2.

General Procedure for Synthesis of 19 – 28

Compound 9–18 (3.5 mmol) in EtOH (75 mL) was subjected to hydrogenation using 40 psi of H2 and 10 % Pd-C (40mg) until uptake of H2 ceased. The catalyst was filtered on a bed of Celite, washed with EtOH, and the filtrate was concentrated in vacuo. The crude o-phenylenediamines (grey–purple– black in colour) were used for the subsequent steps without crystallisation (Table 5). In order to prevent halogen reduction of compound 28, 15 psi of H2 pressure was employed.

General Procedure for Synthesis of 33–39, 45–52, 55, 59–61

The corresponding benzaldehydes (15 mmol) were dissolved in EtOH (50 mL) and sodium metabisulfite (1.6 g) in H2O (10 mL) was added in portions. The reaction mixture was stirred vigorously and more EtOH was added. The mixture was kept in a refrigerator for a several hours. The precipitate was filtered and dried (yields over 93 %). The mixture of these salts (1 mmol) and 19–28 and 29–32 (1 mmol) in DMF (1-2 mL) was heated at 120oC for 4h. The reaction mixture was cooled, poured into H2O, and the solid was filtered. Purification methods are given in Table 2 and Table 6.

Acknowledgments

This work was supported by Ankara University Research Fund (Project No: 0000018-2003). The Central Lab. of the Faculty of Pharmacy of Ankara University provided support for acquisition of the NMR, mass spectrometer and elemental analyser used in this work.

Footnotes

Sample Availability : Available from the authors.

References

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