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

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1 Molecules 2005, 10, molecules ISS Synthesis and Potent Antimicrobial Activity of Some ovel -(Alkyl)-2-Phenyl-1H-Benzimidazole-5-Carboxamidines Hakan Göker 1, *, Mehmet Alp 1 and Sulhiye Yıldız 2 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara-Turkey; Fax: (+90) Department of Microbiology, Faculty of Pharmacy, Ankara University, Tandogan, Ankara- Turkey * Author to whom correspondence should be addressed goker@pharmacyankaraedutr Received: 7 June 2005; in revised form: 25 October 2005 / Accepted: 26 October 2005 / Published: 30 ovember 2005 Abstract: A series of 22 novel 1,2-disubstituted-1H-benzimidazole--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)-- (2-diethylaminoethyl)-1H-benzimidazole-5-carboxamidine], with a 3,4-dichlorophenyl group at the C-2 position, displayed the greatest activity (MIC = 312 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-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 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

2 Molecules 2005, Figure 1 H I H H II H H III H IV Results and Discussion Chemistry Syntheses of the target benzimidazoles (Tables 1, 2) were achieved by two different methods, as shown in Scheme 1 Scheme 1 Method A C O 2 a C 1-8 O 2 b EtO H O 2 c R'H H 9-18 O 2 d R'H H R R 1 2 R 3 e R'H R 4 c Method B C 2 e C : H 29 : isopropyl 30 : n-butyl 31 : benzyl 32 R 2 R 3 R 4 b EtO H R 2 R 3 R 4 Reagents : a : 2 b : H(g)/EtOH c : R' 2 /EtOH d : H 2 /PdC e : a 2 S 2 O 5 adduct of benzaldehydes

3 Molecules 2005, Method A involved nucleophilic displacement in DMF of the chloro group of 4-chloro-3-nitrobenzonitrile 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 H 2, Pd/C produced (Table 5) Condensation of these derivatives with the a 2 S 2 O 5 adducts of several benzaldehydes afforded the corresponding benzimidazoles 45 52, 55 and [2] Method B involved cyclization of with the a 2 S 2 O 5 adducts of various benzaldehydes to afford 5-cyanobenzimidazoles (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 For its practical advantages this method was used in particular for cyanobenzimidazoles, which have better solubility in EtOH, although the yields were low o Table 1 Formulas and in vitro antibacterial and antifungal activities of R'H R 2 R 3 Substituents Minimal inhibitory concentration, µg/ml R R 2 R 3 R 4 S a MRSA MRSA* E c E f C a 40 >50 >50 >50 >50 >50 >50 41 CH(CH 3 ) 2 >50 >50 >50 >50 >50 >50 42 CH(CH 3 ) 2 F >50 >50 >50 >50 >50 >50 43 (Et) 2 CH 2 CH 2 F >50 >50 >50 >50 >50 >50 44 (Me) 2 CH 2 CH 2 >50 >50 >50 >50 >50 >50 45 (Me) 2 CH 2 CH > CH(CH 3 ) 2 CH 3 C >50 >50 >50 >50 >50 >50 47 CH(CH 3 ) 2 CH 3 OCH 3 OCH 3 >50 >50 >50 >50 >50 >50 48 Cyclopropyl COOCH 3 >50 >50 >50 >50 >50 >50 49 PhCH 2 COOH >50 >50 >50 >50 >50 >50 50 PhCH 2 COOCH > Cyclohexyl COOH >50 >50 >50 >50 >50 >50 52 n-butyl >50 >50 >50 >50 >50 >50 53 (Me) 2 CH 2 CH 2 n-butyl F >50 >50 >50 >50 >50 >50 54 CH(CH 3 ) 2 CH(CH 3 ) 2 F >50 >50 >50 >50 >50 >50 55 (Me) 2 CH 2 CH 2 Ph > (Me) 2 CH 2 CH 2 PhCH > (Et) 2 CH 2 CH 2 PhCH (Me) 2 CH 2 CH 2 PhCH > ,4-di-- 59 (Et) 2 CH 2 CH 2 benzyl (Et) 2 CH 2 CH 2 PhCH 2 CH 2 OCH 3 OCH 3 >50 >50 >50 >50 >50 >50 61 Isobutyl PhCH 2 CH > Ref OH t-butyl Sult R 4

4 Molecules 2005, Table 1 Cont o R R 2 R 3 R 4 S a MRSA MRSA* E c E f C a Amp Cip 039 Flu 156 Sa: 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 Antimicrobial Activity The benzimidazoles 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 DMSOwater (40 %) at a concentration of 400 µg/ml The concentration was adjusted to 100 µg/ml by fourfold 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 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 312 µg/ml More lipophilic substituents on the benzimidazole -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,-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)--(2-diethylaminoethyl)-1Hbenzimidazole-5-carboxamidine (59), having a 3,4-dichlorophenyl at the C-2 position, exhibited the greatest activity, with a MIC value of 312 µg/ml against S aureus and MRSA Detailed mechanistic studies are required to understand the potent activity of this compound

5 Molecules 2005, Acknowledgments This work was supported by Ankara University Research Fund (Project o: ) The Central Lab of the Faculty of Pharmacy of Ankara University provided support for acquisition of the MR, mass spectrometer and elemental analyser used in this work Experimental General Uncorrected melting points were measured on an Electrothermal 9100 capillary melting point apparatus 1 H-MR 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 CHS-932 Some H salts of compounds were prepared by using dry H gas in EtOH or isopropanol All chemicals and solvents were purchased from Aldrich Chemical Co or Fischer Scientific Compounds were synthesized as described in our previous study [2] Table 2 Physical and spectral data for compounds o Mp ( O C) Yield (%) 40 > > > > Formula Calculated Found C 14 H H H 2 O C: 4650 H: 418 : 155 C: 4624 H: 399 : 153 C 17 H H 15H 2 O C: 4566 H: 473 : 125 C: 4567 H: 445 : 124 C 17 H 17 F 4 2H 15H 2 O C: 5152 H: 559 : 141 C: 5190 H: 531 : 141 C 20 H 24 F 5 3H 2H 2 O C: 4815 H: 626 : 141 C: 4801 H: 602 : 1399 *C 18 H H 15H 2 O 05C 2 H 6 O C: 4227 H: 597 : 1297 C: 4254 H: 604 : 1301 C 18 H H 025H 2 O 025C 3 H 8 O C: 4999 H: 514 : 1554 C: 4997 H: 514 : 1523 C 19 H H 2 O 01C 3 H 8 O C: 6615 H: 655 : 1998 C: 6619 H: 597 : 1982 C 20 H 24 4 O 2 18H 2 O C: 6242 H: 722 : 1455 C: 6262 H: 694 : 1421 C 19 H 18 4 O 2 3H 2 O 03C 3 H 8 O C: 5880 H: 654 : 1378 C: 5872 H: 620 : H-MR δ ppm (DMSO-d6) (if not stated otherwise) (arom 7H), 831 (s), 930 (s), 953 (s) 121 (d, 6H), 403 (m, 1H), 76 (m, 2H), 78(d, J=85, 1H), 783 (d, J=2, 1H), 792 (d, J=84, 1H), 805 (d, J=15, 1H), 906 (s), 942 (s), 954 (d) 129 (d, 6H), 408 (m, 1H), 751 (t, 2H), 767 (d, J=83, 1H), 787 (d, J=84, 1H), 808 (s, 1H), 844 (m, 2H), 908 (s), 948 (s), 962 (d) 129 (t, 6H), 326 (4H), 346 (2H), 400 (2H), 756 (t, 2H), 784 (d, J=84, 1H), 792 (d, J=84, 1H), 828 (s, 1H), 852 (m, 2H), 967 (s), 993 (s), 1021 (1H), 1096 (s) (CD 3 OD): 305 (6H), 366 (t, 2H), 403 (t, 2H), 769 (m, 1H), 782 (m, 2H), 799 (d, 1H), 8102 (s, 2H), 854 (s, 1H) 223 (s, 6H), 258 (t, 2H), 353 (t, 2H), 756 (dd, J=84, 16, 1H), 780 (d, J=88, 1H), 786 (d, J=84, 1H), 806 (s, 1H), 825 (dd, J= 84 18, 1H), 850 (d, J=18, 1H) 123 (d, 6H), 395 (m, 1H), 397 (s, 3H), 776 (m, 2H), 809 (m, 5H) (DMSO-d drop D 2 O): 117 (d, 6H), (10H), 715 (d, J=84, 1H), 74 (s, 2H), 762 (m, 2H), 797 (s, 1H) 077 (2H), 091 (d, 2H), 279 (1H), 389 (s, 3H), 753 (d, J=8, 1H), 767 (d, J=84, 1H), 804 (s, 1H), 809 (d, J=78, 2H), 838 (d, J=8, 2H) MS (ESI+) m/z (33) (65) 351 (11) (33) (69) 380 (11) Synthesis Method and Isolation Column Chromatography if not stated otherwise Crys Ethanolic H CH 2 2 : Isopropanol : 3 (100 : 60 : 4) CH 2 2 : Isopropanol : 3 (100 : 60 : 3) CH 2 2 : Isopropanol : 3 (100 : 60 : 3) CH 2 2 : Isopropanol : 3 (100 : 60 : 3) CH 2 2 : Isopropanol : Ethylamine (100 : 50 : 3) CH 2 2 : Isopropanol : Propylamine (100 : 50 : 4) CH 2 2 : Isopropanol : Propylamine (100 : 50 : 3) Cryst Isopropanol

6 Molecules 2005, > C 22 H 18 4 O 2 2H 2 O C: 6501 H: 545 : 1377 C: 6495 H: 523 : 1378 C 23 H 20 4 O 2 025H 2 O C: 7103 H: 531 : 1440 C: 7107 H: 514 : 1432 C 21 H 22 4 O 2 2H 15H 2 O C: 5455 H: 588 : 1212 C: 5426 H: 589 : 1262 C 18 H H 22H 2 O 05C 3 H 8 O C: 5384 H: 704 : 1288 C: 5398 H: *** : 1281 C 22 H 28 F 5 3H 2H 2 O C: 5014 H: 669 : 1329 C: 5034 H: 652 : 1292 C 20 H 23 F 4 3H 125H 2 O C: 5107 H: 611 : 1191 C: 5105 H: 631 : 1155 C 24 H H 125H 2 O C: 4934 H: 491 : 1198 C: 4945 H: *** : 1187 C 25 H H 125H 2 O C: 5018 H: 514 : 117 C: 5006 H: 522 : 1149 C 27 H H 2H 2 O C: 5068 H: 567 : 1094 C: 5066 H: 562 : ** 33 ** C 25 H H C 27 H H H 2 O C: 4664 H: 471 : 1007 C: 4680 H: 482 : 986 C 30 H 37 5 O 2 3H H 2 O C: 5434 H: 699 : 1056 C: 5440 H: 680 : 1045 C 26 H H C: 6004 H: 533 : 1077 C: 6005 H: 550 : 1066 Table 2 Cont 473 (s, 2H), (aromat 12H) (s, 3H), 453 (s, 2H), (m, 7H), (m, 3H), 837 (d, J=84, 2H), (m, 10H), 378 (1H), 765 (d, J=84, 1H), 787 (d, J=84, 1H), 81 (s, 1H), 815 (d, J=8, 2H), 847 (d, J=76, 2H), 919 (s), 950 (s), 963 (d) (Base) : 064 (t, 3H), 101 (m, 2H), 156 (m, 2H), 433 (t, 2H), 756 (m, 3H), 776 (m, 3H), 795 (d, J=86, 1H), 823 (d, J=14, 1H), 918 (s), 941 (s) 078 (t, 3H), 120 (m, 2H), 170 (m, 2H), 290 (s, 6H), 351 (t, 2H), 403 (t, 2H), 445 (t, 2H), 755 (m, 2H), 801 (m, 3H), 810 (d, J=86, 1H), 842 (d, J=13, 1H), 977 (s), 997 (s), 1028 (s) 134 (d, 6H), 166 (d, 6H), 42 (m, 1H), 48 (m, 1H), 754 (m, 2H), 774 (d, J=87, 1H), 786 (m, 2H), 822 (2H), 921 (s), 958 (s), 968 (d) 285 (s, 6H), 344 (t, 2H), 392 (t, 2H), (m, 10H), 841 (d, J=12, 1H), 952 (s), 983 (s), 1007 (s), 1081 (s) 290 (6H), 350 (t, 2H), 403 (q, 2H), 574 (s, 2H), 704 (m, 2H),734 (m, 3H), 782 (m, 4H), 802 (d, J=19, 1H), 842 (d, J=14, 1H), 971 (s), 986 (s), 1015 (d), 1114 (s) 127 (t, 6H), 324 (4H), 345 (2H), 399 (2H), 572 (s, 2H), 698 (m, 2H), 727 (m, 3H), 781 (m, 4H), 801 (d, J=2, 1H), 835 (s, 1H), 961 (s), 981 (s), 1011 (s), 1097 (s) (CD 3 OD): 329 (6H), 363 (t, 2H), 399 (t, 2H), 557 (s, 2H), 703 (m, 2H), 726 (m, 3H), 761 (dd, J=84, 2, 1H), 768 (d, J=81, 1H), 782 (d, J=2, 1H), 795 (m, 2H), 842 (s, 1H) 125 (t, 6H), 323 (m, 4H), 341 (t, 2H), 384 (2H), 571 (s, 2H), 673 (d, J=88, 1H), 730 (dd, J=84 2, 1H), (m, 5H), 793 (d, J=2, 1H), 835 (s, 1H), 964 (s), 983 (s), 1013 (s), 1096 (s) 129 (t, 6H), 308 (t, 2H), 325 (4H), 348 (d, 2H), 384 (s,3h), 389 (s,3h), 404 (q, 2H), 483 (t, 2H), 69 (m, 2H), (m, 6H), 803 (d, J=88, 1H), 827 (d, J=88, 1H), 836 (s, 1H), 987 (s), 1008 (s), 1044 (s), 111 (s) 096 (d,6h), 206 (m, 1H), 293 (t, 2H), 316 (t, 2H), 463 (t, 2H), 667 (d, J=72, 2H), 703 (t, J=72, 2H), 711 (m, 1H), 745 (2H), 770 (m, 2H), 801 (d, J=88, 1H), 812 (s, 1H), 902 (s), 944 (s), 975 (s) (68) 456 (12) (68) 470 (11) (68) 498 (12) (71) 470 (13) 562 (80) (51) 568 (14) (67) 469 (11) * Hygroscopic; ** Very hygroscopic, not measurable; *** o satisfactory result Cryst EtOH CH 2 2 : Isopropanol : Propylamine (100 : 100 : 6) Cryst Ethanolic H CH 2 2 : Isopropanol : 3 (100 : 50 : 4) CH 2 2 : Isopropanol : Ethylamine (100 : 50 : 3) CH 2 2 : Isopropanol : Ethylamine (100 : 50 : 3) CH 2 2 : Isopropanol : 3 (100 : 50 : 15) CH 2 2 : Isopropanol : Ethylamine (100 : 50 : 5) CH 2 2 : Isopropanol : Ethylamine (100 : 50 : 3) CH 2 2 : Isopropanol : 3 (100 : 50 : 05) CH 2 2 : Isopropanol : 3 (100 : 50 : 05) CH 2 2 : Isopropanol : isopropylamine (90 : 30 : 2) CH 2 2 : Isopropanol : ethylamine (100 : 50 : 05) 4-(2,4-Dichlorobenzyl)amino-3-nitrobenzonitrile (7) A mixture of 2,4-dichlorobenzylamine (352 g, 20 mmol) and 4-chloro-3-nitrobenzonitrile (2 g, 1095 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: 192 o C; 1 H-MR (CD 3 ): 466 (d, J=56, 2H), 68

7 Molecules 2005, (d, J=92, 1H), 724 (m, 2H), 747 (d, J=2, 1H), 758 (dd, J o =92, J m =2, 1H), 855 (d, J=2, 1H), 878 (brt, 1H) 4-(2-Phenylethyl)amino-3-nitrobenzonitrile (8) 2-Phenylethylamine (13 g, 108 mmol) and 4-chloro-3-nitrobenzonitrile (1g, 54 mmol) were allowed to react for 2 h, at 100 o C and the product wasisolated as described for 7; yield 79 %; mp: 111 o C; 1 H-MR (CD 3 ): 304 (t, J=71, 2H), 361 (q, J=68, 2H), 689 (d, J=88, 1H), (m, 4H), 758 (dd, J o =88, J m =2, 1H), 844 (brt, 1H), 847 (d, J=2, 1H) Table 3 Formulas and melting points of 1 8 C O 2 Comp Formula Ref 1 H C 7 H 5 3 O 2 Commercial 2 methyl C 8 H 7 3 O 2 Lit [1] 3 iso-propyl C 10 H 11 3 O 2 Lit [2] 4 n-butyl C 11 H 13 3 O 2 Lit [2] 5 phenyl C 13 H 9 3 O 2 lit [5] 126 o C, 126 o C 6 benzyl C 14 H 11 3 O 2 Lit [2] 7 2,4-dichlorobenzyl C 14 H O PhCH 2 CH 2 C 15 H 13 3 O General Procedure for Synthesis of 9 18, 40 44, 53, 54, (45 mmol) and (Table 6, 1 mmol) were suspended in absolute EtOH, cooled in a icesalt bath, and dry H 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 H in absolute EtOH was stirred with corresponding the amines (15-2 fold excess) overnight at o C 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 H salts for the next steps since they were prepared completely pure In contrast, crude products 40 44, 53, 54, were treated with dilute a 2 CO 3 solution, then water Further purification methods are given in Table 2 Table 4 Formulas, spectroscopic data, mp and yields of 9-18 Comp R Formula 9 butyl C 11 H 16 4 O 2 MR δ ppm (DMSO-d 6 ) 0895 (t, 3H), 134 (m, 2H), 156 (m, 2H), 341 (q, 2H), 722 (d, J=92, 1H), 795 (dd, J=92, 24, 1H), 86 (t, 1H), 867 (d, J=24, 1H), 92 (brs) Mass (ESI+) iso-propyl methyl C 11 H 16 4 O 2 Lit [1] mp ( o C) Yield (%)

8 Molecules 2005, (CH 3 ) 2 (CH 2 ) 2 C 11 H 17 5 O 2 Table 4 Cont 224 (s, 6H), 258 (t, 2H), 35 (t, 2H), 714 (d, J=88,1H), 75 (dd, J=88, 2, 1H), 809 (brs, 2H), 848 (d, J=2, 1H) () Lit [6] 12 cyclo-propyl C 10 H 12 4 O 2 Lit [1] 13 cyclo-hexyl C 13 H 18 4 O 2 Lit [1] 14 Benzyl C 14 H 14 4 O 2 Lit [1] 15 (CH 3 ) 2 (CH 2 ) 2 phenyl C 17 H 21 5 O 2 16 iso-butyl PhCH 2 CH 2 C 19 H 24 4 O 2 17 (C 2 H 5 ) 2 (CH 2 ) 2 PhCH 2 CH 2 C 21 H 29 5 O 2 18 (C 2 H 5 ) 2 (CH 2 ) 2 2,4-dichloro benzyl C 20 H O (s, 6H), 269 (2H), 34 (brs), 356 (t, 2H), 712 (d, J=93, 1H), (m, 5H), 785 (dd, J=92, 23, 1H), 861 (d, J=23, 1H), 985 (s, 1H) (CD 3 OD): 105 (d, 6H), 207 (m,1h), 305 (t, 2H), 325 (d, 2H), 372 (m, 2H), 721 (m, 2H), 73 (m, 4H), 779 (dd, J=92 24, 1H), 853 (t, 1H), 859 (d, J=24, 1H) (CD 3 OD): 111 (t, 6H), 268 (q, 4H), 278 (t, 2H), 303 (t, 2H), 357 (t, 2H), 47 (s, 2H), 721 (m, 2H), 73 (m, 4H), 781 (dd, J=88,24, 1H), 862 (d, J=24, 1H) (CD 3 OD): 111 (t, 6H), 268 (q, 4H), 279 (t, 2H), 357 (t, 2H), 478 (t, 2H), 698 (d, J=92, 1H), 730 (dd, J=86 24, 1H), 737 (d, J=88, 1H), 753 (d, J=2, 1H), 778 (dd, J=92, 24, 1H), 868 (d, J=24, 1H) (65) 442 (12) Hygros 110 () Table 5 Formulas, spectroscopic data, mp and yields of R'H H 2 Comp R Formula 19 butyl C 11 H 18 4 MR δ ppm (DMSO-d 6 ) 088 (t, 3H), 136 (m, 2H), 156 (m, 2H), 31 (t, 2H), 501 (brs, 2H), 557 (brs, 2H), 646 (d, J=8, 1H), 69 (s, 1H), 707 (d, J=84, 1H), 858 (s, 2H), 874 (s, 2H) MS (ESI+) mp ( o C) Yield (%) iso-propyl methyl C 11 H 18 4 Lit [1] 21 (CH 3 ) 2 (CH 2 ) 2 C 11 H 19 5 (+ one drop D 2 O): 22 (s, 6H), 254 (t, 2H), 34 (t, 2H), 657 (d, J=8, 1H), 685 (m, 2H) 222 Lit [6] 22 cyclo-propyl C 10 H 14 4 Lit [1] 23 cyclo-hexyl C 13 H 20 4 Lit [1] 24 benzyl C 14 H 16 4 Lit [1] 25 (CH 3 ) 2 (CH 2 ) 2 phenyl C 17 H 23 5 (+ one drop D 2 O) : 227 (s, 6H), 263 (t, 2H), 35 (t, 2H), (8H) 298 * 90

9 Molecules 2005, Table 5 Cont 26 iso-butyl -CH 2 CH 2 Ph C 19 H (C 2 H 5 ) 2 (CH 2 ) 2 -CH 2 CH 2 Ph C 21 H (C 2 H 5 ) 2 (CH 2 ) 2 2,4-dichlorobenzyl C 20 H * o sharp melting point 092 (d, 6H), 196 (m, 1H), 29 (t, 2H), 317 (t, 2H), 331(2H), 439 (t, 1H), 503 (2H), 566 (t,1h), 657 (d, J=84, 1H), 687 (d, J=16, 1H), 698 (dd, J=84 16, 1H), (m, 5H), 857 (s, 1H), 89 (s, 1H) (CD 3 OD): 109 (t, 6H), 265 (q, 4H), 277 (t, 2H), 296 (t, 2H), 347 (t, 2H), 352 (t, 2H), 668 (d, J=8, 1H), 701 (d, J=2, 1H), (m, 6H) (CD 3 OD): 114 (t, 6H), 276 (4H), 289 (2H), 357 (t, 2H), 454 (s, 2H), 637 (d, J=8, 1H), 706 (d, J=2, 2H), 725 (dd, J=82 2, 1H), 731 (d, J=84, 1H), 749 (d, J=2, 1H) * (65) 412(11) * 92 General Procedure for Synthesis of Compound 9 18 (35 mmol) in EtOH (75 ml) was subjected to hydrogenation using 40 psi of H 2 and 10 % Pd-C (40mg) until uptake of H 2 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 H 2 pressure was employed Table 6 Formulas, spectroscopic data, mp and yields of C R 2 R 3 R 4 Comp R 2 R 3 R 4 Formula MR Mass mp δ ppm (CD 3 ) (ESI+) ( o C) 33 C 14 H 8 3 Lit [2] 34 C 14 H Lit [2] 35 F C 14 H 8 F 3 Lit [2] 36 iso-propyl F C 17 H 14 F 3 Lit [2] 37 n-butyl F C 18 H 16 F 3 Lit [2] 38 benzyl C 21 H benzyl C 21 H (s, 2H), 705 (m, 2H), (m,7h), 782 (d, J=2, 1H), 816 (d, J=15, 1H) 527 (s, 2H), 691(m, 2H), (m, 8H), 816 (1H) (50) 380(33) 382(6) (54) 380(31) 382(5) Yield (%) İsolation Cryst EtOHwater Cryst EtOHwater

10 Molecules 2005, General Procedure for Synthesis of 33 39, 45 52, 55, The corresponding benzaldehydes (15 mmol) were dissolved in EtOH (50 ml) and sodium metabisulfite (16 g) in H 2 O (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 and (1 mmol) in DMF (1-2 ml) was heated at 120 o C for 4h The reaction mixture was cooled, poured into H 2 O, and the solid was filtered Purification methods are given in Tables 2 and 6 References 1 Göker, H; Özden, S; Yildiz, S; Boykin, DW Synthesis and potent antibacterial activity against MRSA of some novel 1,2-Disubstituted-1H-Benzimidazole--alkylated-5-carboxamidines Eur J Med Chem 2005, 40, Göker, H; Kus, C; Boykin, DW; Yildiz, S; Altanlar, Synthesis of some new 2-substitutedphenyl-1H-benzimidazole-5-carbonitriles and their potent activity against Candida species Bioorg Med Chem 2002, 10, Weidner-Wells, MA; Ohemeng, KA; guyen, V; Fraga-Spano, S; Macielag, MJ; Werblood, HM; Foleno, BD; Webb, GC; Barrett, JF; Hlasta, DJ Amidino benzimidazole inhibitors of bacterial two-component systems Bioorg Med Chem Lett 2001, 11, a) ational Committee for inical Laboratory Standards Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 5th ed; Approved Standard (M7A5); ational Committee for inical Laboratory Standards: Wayne, PA, 2000; b) Shadomy, S; Pfaller, MA Laboratory Studies with Antifungal Agents: Susceptibility Tests and Quantitation in Body Fluids In Manual of inical Microbiology 5th Ed ; Balows, A; Hausler, WJ; Hermann, KL; Isenberg, HD; Shadomy, HJ, Eds; Washington DC, 1991; Chapter 117, p Ritter, H DE , Roesner, M; Raethner, W DE , 1980 Sample availability : Available from the authors 2005 by MDPI ( Reproduction is permitted for noncommercial purposes