- Email: [email protected]
Synthesis and evaluation of in vitro antimycobacterial activity of novel 1H-benzo[d]imidazole derivatives and analogues
Accepted Manuscript Synthesis and evaluation of in vitro antimycobacterial activity of novel 1Hbenzo[d]imidazole derivatives and analogues Katarzyna Gobis, Henryk Foks, Marcin Serocki, Ewa Augustynowicz-Kopeć, Agnieszka Napiórkowska PII:
S0223-5234(14)00958-1
DOI:
10.1016/j.ejmech.2014.10.031
Reference:
EJMECH 7438
To appear in:
European Journal of Medicinal Chemistry
Received Date: 20 December 2013 Revised Date:
10 September 2014
Accepted Date: 12 October 2014
Please cite this article as: K. Gobis, H. Foks, M. Serocki, E. Augustynowicz-Kopeć, A. Napiórkowska, Synthesis and evaluation of in vitro antimycobacterial activity of novel 1H-benzo[d]imidazole derivatives and analogues, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.10.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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We synthesized a new series of benzimidazoles. Compounds were tested against M. tuberculosis and M. bovis in vitro. Some compounds exhibited excellent tuberculostatic activity.
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Two derivatives were characterized by high therapeutic index in vitro.
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Cytotoxic activity towards eukaryotic cells was determined.
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European Journal of Medicinal Chemistry Original article
Synthesis and evaluation of in vitro antimycobacterial activity
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of novel 1H-benzo[d]imidazole derivatives and analogues Katarzyna Gobisa,*, Henryk Foksa, Marcin Serockib,
Ewa Augustynowicz-Kopećc, Agnieszka Napiórkowskac a
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Department of Organic Chemistry, Medical University of Gdańsk, 107 Gen. Hallera Ave., 80-416 Gdańsk, Poland b Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 11/12 G. Narutowicza Str., 80-233 Gdańsk, Poland c Department of Microbiology, Institute of Tuberculosis and Pulmonary Diseases, 26 Płocka Str., 01-138 Warsaw, Poland
Abstract
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A series of novel 1H-benzo[d]imidazole derivatives and analogues (1-25) have been synthesized and evaluated for tuberculostatic activity. Benzimidazoles substituted at the C-2 position with cyclohexylethyl, cyclohexylpropyl and phenylpropyl moiety or 4-phenylpyridine system were obtained. Compounds 3, 4, 6 and 7 bearing halogen atoms or methyl groups at the benzimidazole system and cyclohexylethyl substituent at the C-2 position showed an excellent tuberculostatic activity against M. tuberculosis and M. bovis strains with MIC values ranging from 0.75 to 1.5 µg/mL. More importantly, derivatives 4 (5-Bromo-2-(2-cyclohexylethyl)-1Hbenzo[d]imidazole) and 6 (2-(2-cyclohexylethyl)-5,6-dimethyl-1H-benzo[d]imidazole) appeared selective for M. tuberculosis and M. bovis as compared with non-malignant eukaryotic cells (LLC-PK1 pig kidney epithelial cell line). These compounds may thus represent a novel, selective class of anti-tubercular agents.
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Keywords: Benzimidazole; synthesis; tuberculostatic activity; cytotoxicity; SAR-study
*Corresponding author. Tel.: +48 58 349 16 47; fax: +48 58 349 12 77. E-mail address: [email protected] (K. Gobis)
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1.
Introduction Infections caused by Mycobacterium tuberculosis are the reason of one of the
most serious contagious diseases [1]. Despite the passage of time, money spent and
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the hard work of many research laboratories and social organizations, tuberculosis still causes about 1.3 million deaths per annum [2]. Particularly dangerous is its
variety resistant to applied chemotherapeutic agents (MDR-TB), especially when it
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concerns patients with a compromised immune system [3]. Human tuberculosis mainly results from the human-to-human transmission of Mycobacterium tuberculosis
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[4,5] with a few cases being caused by other closely related species: M. bovis [6], M. caprae [7,8], M. microti [9,10] and M. pinipedii [11].
Due to the continuous rise of the number of cases caused by resistant strains, a need for new anti-tuberculosis drugs that are active towards the broad spectrum of species of the Mycobacterium genus becomes obvious [12]. This need is even greater
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because of the generally small number of effective anti-tuberculosis chemotherapeutic agents. Isoniazid (INH) [13], pyrazinamide (PZA) [14] and ethionamide (ETA) [15] should be mentioned among the leading drugs of first and second line therapy.
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However, these agents cause a number of side effects [13-16]. The anti-tuberculosis activity of other nitrogen heterocyclic compounds, e.g. derivatives of 1H-
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benzo[d]imidazole has been also reported. In particular, compounds substituted at the C-2 position of this system appear to be interesting [17]. Significant activity showed 2-styrene [18], 2-polyfluoroalkyl [19] and 2-(1,2,4-triazole)-phenyl [20] derivatives. Previously our research group has also described the high activity of 2cyclohexylethyl derivatives [21] and benzimidazole type systems (naphthoimidazole, imidazopyridine, imidazophenazine) (Figure 1) [22]. The activity of these compounds was at the level corresponding to the activity of anti-tuberculosis drugs (MIC 1.5-3.1 2
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µg/mL). Continuing this research we have taken in the present work the synthesis of benzimidazoles
substituted
at
the
C-2
position
with
cyclohexylethyl,
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cyclohexylpropyl, phenylpropyl or 4-phenylpyridine moiety. Syntheses were designed to receive compounds with different substituents at the benzimidazole system, and, at the C-2 position, the cyclohexane or benzene ring distant from the benzimidazole
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system by two or three methylene groups. Compounds with the pyridine ring
substituted directly to benzimidazole were also obtained. That choice of the structural
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parameters would enable assessment of the effect of the substituent at the C-2 structure, the chain length and the degree of the six-member ring saturation at its end, for tuberculostatic activity of the compounds. Furthermore, derivatives substituted with the phenylsulfonyl or methylsulfonyl group at the N-1 position have been
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synthesized for one of the benzimidazoles obtained.
2
Results and discussion
2.1
Chemistry
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We synthesized structures in which the cyclohexylethyl, cyclohexylpropyl, phenylpropyl or 4-phenylpyridine-2-yl moiety is connected to the benzimidazole
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system. The structures with different substituents at the benzene ring of the benzimidazole system, including the methyl or nitro group, fluorine, chlorine or bromine atoms were obtained. The synthesized compounds have been screened for their tuberculostatic and cytotoxic activities. Finally, target structures were planned in order to investigate the preliminary structure–activity relationship, with the emphasis on the crucial role of various substituents at the benzene ring of the benzimidazole system and at its C-2 or N-1 position. The synthetic route of the studies was outlined 3
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in Schemes 1 and 2. Syntheses of the target benzimidazole derivatives 1–25 were achieved in 37–96% yields by the procedures described below. The starting compounds for a series of syntheses were the commercially available derivatives and of
o-phenylenediamine:
3,4-diaminotoluene,
4-fluoro-1,2-
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analogues
phenylenediamine, 4-chloro-1,2-phenylenediamine, 4-bromo-1,2-phenylenediamine, 4-nitro-1,2-phenylenediamine, 4,5-dimethyl-1,2-phenylenediamine, 4,5-dichloro-1,2-
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phenylenediamine, 2,3-diaminopyridine, 5-bromo-2,3-diaminopyridine and 3,4-
diaminopyridine. Compounds 1–7 (Scheme 1) were synthesized by the heating of 3-
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cyclohexylpropanoic acid with appropriate diamines according to the method described by Algul et al. [23] and based on the use of polyphosphoric acid (PPA) as a solvent with strong acidic properties (method A). Compound 7 has been already obtained by Li and co-workers as anti-hepatitis B virus agent but its activity was not significant [24]. The above method for the synthesis was tested first, but much better
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yields of benzimidazoles 8-14 were obtained by fusion of the starting materials at 160-180°C without solvent, the process of which was elaborated earlier by our team (method B) [21]. We used 4-cyclohexylbutanoic and 4-phenylbutanoic acid. In the
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next step benzimidazole 12 reacted with methane- and benzenesulfonyl chloride in anhydrous dioxane in the presence of triethylamine (TEA), giving the corresponding
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sulfonamide derivatives 15 and 16. Compounds 17-25 were synthesized from 4-phenylpicolinothioamide (Scheme
2), which was obtained from starting carbonitrile via methyl carbimidate in the reaction with DBU and ammonium polysulfide according to the modified method reported by Mukkala et al. [25]. Then, upon treatment of appropriate diamines in ethylene glycol, 4-phenylpicolinothioamide gave benzimidazoles 17-22 and imidazopyridines 23-25. Until now the synthesis of benzimidazoles from thioamides 4
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has not been reported. Compounds 17 and 23 have been already described by Case [26], although synthesized directly from 4-phenylpicolinonitrile. All the newly synthesized compounds were characterized by IR and 1H NMR
structures were also confirmed by
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spectra as well as the elemental analysis listed in the experimental section. Some C NMR. The spectral analyses were in
accordance with the assigned structures. Biological activity
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2.2
All of the obtained benzimidazole derivatives 1-25 were evaluated for their in
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vitro tuberculostatic activity against the Mycobacterium tuberculosis H37Rv strain and two “wild” strains isolated from tuberculosis patients: one (Spec. 210) resistant to paminosalicylic acid (PAS), isonicotinic acid hydrazide (INH), ethambutol (ETB) and rifampicin (RMP) and the other (Spec. 192) fully sensitive to the administrated tuberculostatics. The MIC values were determined as the minimum concentration
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inhibiting the growth of tested tuberculosis strains in relation to the probe with no tested compound. INH, PZA and RMP were used as reference drugs. Some compounds were also tested against the Mycobacterium bovis strain. The most active
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compounds of the benzimidazole structure 3, 4, 6 and 7 which exhibited the highest tuberculostatic activity were then tested for effect on the proliferation of two
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eukaryotic cell lines: human non-small lung cancer cell line A549 and pig kidney epithelial cell line LLC-PK1. The bioactive data were summarized in Tables 1 and 2. 2.2.1 Tuberculostatic activity The results of tuberculostatic activity indicated that most of the synthesized
compounds exhibited moderate to excellent activity against M. tuberculosis and M. bovis strains in vitro. As seen in Table 1, the tested derivatives showed high tuberculostatic activity. Generally, compounds possessing the cyclohexylethyl 5
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substituent at the C-2 position were more active (MICs 0.75-3.1 µg/mL) than those with the longer chain linker (MICs 6.2-25 µg/mL). Derivatives with the 4phenylpyridin-2-yl moiety were also less active. The activity of four benzimidazoles
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3, 4, 6 and 7 was excellent (MICs 0.75 µg/mL) and much higher than the activity determined for clinically applied INH (12.5-25 µg/mL). Among the most active
compounds were derivatives possessing one or two halogen atoms (3, 4, 7) or two
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methyl groups (6) at the benzimidazole system. Derivatives 15 and 16 with bulky
substituents, methylsulfonyl or phenylsulfonyl group, at the N-1 position were
(MICs 12.5-50 µg/mL). The
obtained
derivatives
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definitely less active (MICs 50-100 µg/mL) than unsubstituted benzimidazole 12
and
analogs
of
2-(4-phenylpyridin-2-yl)-1H-
benzo[d]imidazole exhibited different tuberculostatic activity with MIC values in the range of 3.1-25 µg/mL. Compound 18 possessing a methyl group at the C-5 position
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of the benzimidazole system showed very good activity at low concentration of 3.16.2 µg/mL. Derivatives 19-21 with halogen atoms at the C-5 position of the benzimidazole system exhibited good tuberculostatic activity with MIC values
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ranging from 6.2-12.5 µg/mL. Three of the obtained compounds 22, 23 and 25 showed moderate activity with MIC values in the range of 12.5-25 µg/mL. Their
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activity against sensitive and resistant strains were comparable. Compound 24 possessing the imidazopyridine system substituted with the bromine atom at the C-6 position showed the weakest tuberculostatic activity (MICs 25 µg/mL) against all the strains tested. In the case of compounds 18-21 and 25, higher activity was observed against the resistant strain (Spec.210) than the sensitive one (Spec.192). 2.2.3 Cytotoxic activity
6
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Benzimidazoles 3, 4, 6 and 7, the most active toward M. tuberculosis strains, were tested for cytotoxicity against human non-small lung cancer cell line A549 and pig kidney epithelial cell line LLC-PK1 (Figure 2, Table 2). All values are means of at
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least two independent experiments, each done in duplicate. Cisplatin (Cis-Pt) and doxorubicin (Dox) were used as control cytostatic drugs. IC50 values determined in the A549 cell line were 0.63 ± 0.018 µM (Cis-Pt) and 0.008 ± 0.0011 µM (Dox). For
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tumour cells (A549) the IC50 values did not differ significantly for the individual compounds (IC50 11-14 µM), but for the immortalized kidney cells (LLC-PK1), the
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IC50 values were much more diverse. Chlorine-substituted benzimidazoles 3 and 7 were much more toxic for these cells (IC50 ca. 10 µM) than those substituted with bromine (IC50 ca. 33 µM) or methyl groups (IC50 ca. 44 µM). However, the IC50 value (9.62 µM) obtained for derivative 7 substituted with two chlorine atoms was four times higher than MICs (0.75 µg/mL). In the case of compound 6, the least cytotoxic,
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IC50 value (43.94 µM) was fifteen times higher than MICs (0.75 µg/mL). The differences in toxicity towards both lines are not surprising given the higher
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sensitivity of tumour cells.
Conclusions
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In conclusion, a series of novel 3-cyclohexylpropanoic acid derivatives were successfully synthesized. All these new compounds were confirmed by IR and NMR spectra as well as an elemental analysis. Their tuberculostatic activity in vitro was evaluated against M. tuberculosis H37Rv, Spec. 192 and Spec. 210 strains and M. bovis strain, using the two-fold serial dilution MIC method. The results showed that most of the synthesized derivatives exhibited moderate tuberculostatic activity; however compounds 3, 4, 6 and 7 bearing halogen atoms or two methyl groups at the 7
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benzimidazole system and cyclohexylethyl moiety at its C-2 position exhibited excellent tuberculostatic activity with MIC values ranging from 0.75 to 1.5 µg/mL. More importantly, active compound 6 exhibited a very low cytotoxic effect on the
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proliferation of eukaryotic LLC-PK1 pig kidney epithelial cell line. These findings demonstrated that among 2-cyclohexylethylbenzimidazoles one can find compounds with interesting therapeutic properties that could become a new group of potential
Experimental
4.1
Chemistry
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4
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tuberculostatic agents.
All materials and solvents were of analytical reagent grade. Thin-layer chromatography was performed on Merck silica gel 60F254 plates and visualized with UV. The results of elemental analyses (%C, H, N) for all of obtained compounds were in agreement with the calculated values within ± 0.3 % range. 1H and
13
C NMR
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spectra in CDCl3 or DMSO-d6 were recorded on Varian Unity Plus (500 MHz) and Varian Gemini (200 MHz) instruments. IR Spectra (KBr) were determined as KBr pellets of the solids on a Satellite FT-IR spectrophotometer. Melting points were
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determined on a Stuart SMP30 apparatus and were uncorrected. 4.1.1 General procedure for the synthesis of benzimidazoles (1-14)
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Method A (for compounds 1-7). 3-Cyclohexylpropanoic acid (2.35 mL, 15 mmol), appropriate diamine (10 mmol) and PPA (5 mL) were stirred at 180-200 ºC for 5 h, then the mixture was allowed to cool to an ambient temperature and poured into cold water (50 mL). The mixture was neutralized with a saturated solution of sodium hydrogen carbonate and the resulting precipitate was filtered off, washed several times with water and recrystallized from a methanol/water mixture (1:1) with the addition of activated carbon. 8
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Method B (for compounds 8-14). 4-Phenylbutanoic or 4-cyclohexylbutanoic acid (15 mmol) and appropriate diamine (10 mmol) were heated on Wood's metal bath at 160-180°C for 1 h. While cooling down, a 10% NaOH solution (10 mL) was
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added and the reaction mixture was stirred at room temperature for 24 h. Then the precipitated benzimidazoles were filtered off, washed with water to the neutral reaction, dried and purified by crystallization.
2-(2-Cyclohexylethyl)-5-methyl-1H-benzo[d]imidazole
(1).
Starting
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4.1.1.1
from 3,4-diaminotoluene (1.22 g), the title compound 1 was obtained by method A as
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colourless crystals (1.53 g, 63%): m.p. 245-248 °C; IR (KBr): 2925, 2852 (ν C-H), 1575 (δ N-H), 1450 (ν C=C), 1231 (ν C-C), 1072, 960 (δ C-H), 523 (ν N-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 0.86-0.97(m, 2H, CH2), 1.06-1.21 (m, 4H, 2CH2), 1.62-1.90 (m, 7H, 6H 3CH2 and 1H CH), 2.37 (s, 3H, CH3), 2.80 (t, 2H, CH2, J = 7.8 Hz), 6.84-7.01 (m, 1H, ArH), 7.13-7.48 (m, 2H, ArH), 7.65 (br s, 1H, NH + D2O 13
C NMR (200 MHz, DMSO-d6): δ 21.5, 25.7, 26.0 (2C), 26.3,
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exchangeable) ppm;
32.7 (2C), 35.1, 36.8, 113.9, 114.3, 123.6, 131.4, 135.6, 137.2, 155.1 ppm; Anal.
N, 11.47. 4.1.1.2
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Calcd for C16H22N2 (242.36): C, 79.29; H, 9.15; N, 11.56; Found: C, 79.35; H, 9.18;
2-(2-Cyclohexylethyl)-5-fluoro-1H-benzo[d]imidazole
(2).
Starting
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from 4-fluoro-1,2-phenylenediamine (1.26 g), the title compound 2 was obtained by method A as colourless crystals (1.33 g, 54%): m.p. 236-240 ºC; IR (KBr): 2925, 2853 (ν C-H), 1578 (δ N-H), 1458 (ν C=C), 1220 (ν C-C), 1073, 958 (δ C-H), 526 (γ N-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 0.86-0.97 (m, 2H, CH2), 1.06-1.21 (m, 4H, 2CH2), 1.62-1.90 (m, 7H, 6H 3CH2 and 1H CH), 2.80 (t, 2H, CH2, J = 7.8 Hz), 6.84-7.01 (m, 1H, ArH), 7.13-7.48 (m, 2H, ArH), 7.65 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C15H19FN2 (246.32): C, 73.14; H, 7.77; N, 11.37; 9
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Found: C, 73.18; H, 7.79; N, 11.36. 4.1.1.3
5-Chloro-2-(2-cyclohexylethyl)-1H-benzo[d]imidazole
(3).
Starting
from 4-chloro-1,2-phenylenediamine (1.43 g), the title compound 3 was obtained by
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method A as white solid (1.73 g, 66%): m.p. 117-120 ºC; IR (KBr): 2923, 2851 (ν CH), 1539 (δ N-H), 1448, 1420 (ν C=C), 1276 (ν C-C), 1058, 924 (δ C-H), 805 (γ C-
H) 599 (γ N-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 0.81-0.96 (m, 2H, CH2),
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1.06-1.20 (m, 4H, 2CH2), 1.58-1.74 (m, 7H, 6H 3CH2 and 1H CH), 2.79 (t, 2H, CH2, J = 7.9 Hz), 7.10 (d, 1H, ArH, J = 7.4 Hz), 7.13-7.45 (m, 2H, ArH), 12.34 (br s, 1H,
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NH + D2O exchangeable) ppm; Anal. Calcd for C15H19ClN2 (262.78): C, 68.56; H, 7.29; N, 10.66; Found: C, 68.51; H, 7.21; N, 10.71. 4.1.1.4
5-Bromo-2-(2-cyclohexylethyl)-1H-benzo[d]imidazole
(4).
Starting
from 4-bromo-1,2-phenylenediamine (1.87 g), the title compound 4 was obtained by method A as colourless crystals (1.57 g, 51%): m.p. 157-159 ºC; IR (KBr): 2920,
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2850 (ν C-H), 1539 (ν N-H), 1446, 1420 (ν C=C), 1273 (ν C-C), 1000, 913 (δ C-H), 750 (γ C-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 0.82-0.97 (m, 2H CH2) 1.111.20 (m, 4H, 2CH2), 1.63-1.76 (m, 7H, 6H 3CH2 and 1H CH), 2.79 (t, 2H, CH2, J =
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7.8 Hz), 7.06-7.10 (m, 1H, ArH), 7.41-7.45 (m, 2H, ArH), 12.16 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C15H19BrN2 (307.23): C, 58.64; H, 6.23; N, 9.12;
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Found: C, 58.61; H, 6.26; N, 9.08. 4.1.1.5
2-(2-Cyclohexylethyl)-5-nitro-1H-benzo[d]imidazole (5). Starting from
4-nitro-1,2-phenylenediamine (1.53 g), the title compound 5 was obtained by method A as beige crystals (1.75 g, 64%): m.p. 266-269 ºC; IR (KBr): 2926, 2853 (ν C-H), 1574 (δ N-H), 1522 (ν NO2), 1486, 1463 (ν C=C), 1268 (ν C-C), 1025, 958 (δ C-H), 854 (γ C-H), 535 (γ N-H) cm -1; 1H NMR (200 MHz, DMSO-d6): δ 0.85-0.96 (m, 2H, CH2), 1.06-1.21 (m, 4H, 2CH2), 1.46-1.74 (m, 7H, 6H 3CH2 and 1H CH), 2.78 (t, 2H, 10
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CH2, J = 7.9 Hz), 6.89-6.99 (m, 1H, ArH), 7.22-7.27 (m, 1H, ArH), 7.39-7.46 (m, 1H, ArH), 8.50 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C15H19N3O2 (273.33): C, 65.91; H, 7.01; N, 15.37; Found: C, 65.89; H, 7.06; N, 15.42. 2-(2-Cyclohexylethyl)-5,6-dimethyl-1H-benzo[d]imidazole (6). Starting
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4.1.1.6
from 4,5-dimethyl-1,2-phenylenediamine (1.36 g), the title compound 6 was obtained
by method A as beige solid (2.01 g, 79%): m.p. 193-194 ºC; IR (KBr): 2920, 2853 (ν
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C-H), 1543 (δ N-H), 1451, 1420 (ν C=C), 1308 (ν C-C), 1013, 1001 (δ C-H), 851 (γ
C-H) cm-1; 1H NMR (200 MHz, CDCl3): 0.82-0.93 (m, 2H, CH2), 1.10-1.27 (m, 4H,
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2CH2), 1.64-1.79 (m, 7H, 6H 3CH2 and 1H CH), 2.35 (s, 6H, 2CH3), 2.92 (t, 2H, CH2, J = 8.1 Hz), 7.33 (s, 2H, ArH), 10.48 (br s, 1H, NH + D2O exchangeable) ppm; 13C NMR (200 MHz, DMSO-d6): δ 26.0 (2C), 26.4, 32.8 (2C), 35.3, 36.8, 101.1, 109.4, 115.1, 135.2, 156.1, 157.2, 160.7 ppm; Anal. Calcd for C17H24N2 (256.39): C, 79.64; H, 9.44; N, 10.93; Found: C, 79.56; H, 9.49; N, 10.95.
5,6-Dichloro-2-(2-cyclohexylethyl)-1H-benzo[d]imidazole (7). Starting
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4.1.1.7
from 4,5-dichloro-1,2-phenylenediamine (1.77 g), the title compound 7 was obtained by method A as colourless crystals (2.41 g, 81%): m.p. 254-256 ºC; IR (KBr): 2925,
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2851 (ν C-H), 1561 (δ N-H), 1455 (ν C=C), 1199 (ν C-C), 1071, 960 (δ C-H), 882 (γ C-H) 523 (γ N-H) cm-1; 1H NMR (200 MHz, DMSO-d6): 0.85-0.96 (m, 2H, CH2),
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1.09-1.20 (m, 4H, 2CH2), 1.58-1.73 (m, 7H, 6H 3CH2 and 1H CH), 2.80 (t, 2H, CH2, J = 7.9 Hz), 7.71 (s, 2H, ArH), 8.20 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C15H18Cl2N2 (297.22): C, 60.61; H, 6.10; N, 9.43; Found: C, 60.69; H, 6.17; N, 9.37. All data consistent with the literature description [24]. 4.1.1.8
2-(3-Cyclohexylpropyl)-5-fluoro-1H-benzo[d]imidazole (8). Starting
from 4-cyclohexylbutanoic acid (2.55 g) and 4-fluoro-1,2-phenylenediamine (1.26 g), the title compound 8 was obtained by method B as beige solid (1.54 g, 59%): m.p. 11
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131-133 ºC (cyclohexane); IR (KBr): 2926, 2852 (ν C-H), 1487, 1448, 1421 (ν C=C), 1138 (δ C-H), 845, 804 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): 0.75-0.82 (m, 2H, CH2), 1.03-1.26 (m, 6H, 3CH2), 1.59-1.63 (m, 5H, 4H 2CH2 and 1H CH), 1.82-1.88
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(m, 2H, CH2), 2.91 (t, 2H, CH2, J = 7.8 Hz), 6.95-6.99 (m, 1H, ArH), 7.22 (dd, 1H,
ArH, J1 = 9.0 Hz, J2 = 2.4 Hz), 7.44-7.47 (m, 1H, ArH), 10.64 (br s, 1H, NH + D2O
exchangeable) ppm; Anal. Calcd for C16H21N2 (260.35): C, 73.81; H, 8.13; N, 10.76;
4.1.1.9
2-(3-Cyclohexylpropyl)-5,6-dimethyl-1H-benzo[d]imidazole from
4-cyclohexylbutanoic
acid
(2.55
g)
and
(9).
4,5-dimethyl-1,2-
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Starting
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Found: C, 73.74; H, 8.09; N, 10.82.
phenylenediamine (1.36 g), the title compound 9 was obtained by method B as greyish white solid (1.84 g, 68%): m.p. 145-146 °C (methanol/water 1:1); IR (KBr): 2920, 2849 (ν C-H), 1542 (δ N-H), 1445, 1417 (ν C=C), 1308, 1025, 999 (δ C-H), 854 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): 0.78-0.81 (m, 2H, CH2), 1.08-1.17
TE D
(m, 4H, 2CH2), 1.19-1.24 (m, 2H, CH2), 1.60-1.62 (m, 5H, 4H 2CH2 and 1H CH), 1.80-1.86 (m, 2H, CH2), 2.34 (s, 6H, 2CH3), 2.89 (t, 2H, CH2, J = 7.8 Hz), 7.33 (s, 2H, ArH), 8.16 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C18H26N2
4.1.1.10
5,6-Dichloro-2-(3-cyclohexylpropyl)-1H-benzo[d]imidazole from
4-cyclohexylbutanoic
AC C
Starting
EP
(270.41): C, 79.95; H, 9.69; N, 10.36; Found: C, 80.08; H, 9.62; N, 10.30.
acid
(2.55
g)
and
(10).
4,5-dichloro-1,2-
phenylenediamine (1.77 g), the title compound 10 was obtained by method B as beige solid (2.02 g, 65%): m.p. 117-118 °C (methanol/water 1:1); IR (KBr): 2921, 2849 (ν C-H), 1540 (δ N-H), 1446, 1401 (ν C=C), 1285, 1096 (δ C-H), 859 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): 0.69-0.85 (m, 2H, CH2), 1.02-1.28 (m, 6H, 3CH2), 1.191.60-1.62 (m, 5H, 4H 2CH2 and 1H CH), 1.76-1.92 (m, 2H, CH2), 2.87-2.98 (m, 2H, CH2), 7.63 (s, 2H, ArH), 10.60 (br s, 1H, NH + D2O exchangeable) ppm; 12
13
C NMR
ACCEPTED MANUSCRIPT
(200 MHz, DMSO-d6): δ 25.0, 26.1 (2C), 26.4, 29.0, 33.0 (2C), 36.7, 37.0, 121.5 (2C), 123.7 (2C), 158.5 ppm; Anal. Calcd for C16H20N2 (311.25): C, 61.74; H, 6.48; N, 9.00; Found: C, 61.81; H, 6.42; N, 9.18. 5-Methyl-2-(3-phenylpropyl)-1H-benzo[d]imidazole (11). Starting from
RI PT
4.1.1.11
4-phenylbutanoic acid (2.46 g) and 3,4-diaminotoluene (1.22 g), the title compound 11 was obtained by method B as light brown solid (1.65 g, 66%): m.p. 115-117 °C
SC
(cyclohexane); IR (KBr): 3023, 2938, 2869, 2778 (ν C-H), 1551 (δ N-H), 1494, 1449,
1420 (ν C=C), 1278, 1027 (δ C-H), 806, 739 (γ C-H), 600 (γ N-H) cm-1; 1H NMR
M AN U
(200 MHz, CDCl3): 2.12-2.19 (m, 2H, CH2), 2.46 (s, 3H, CH3), 2.68 (t, 2H, CH2, J = 7.5 Hz), 2.88 (t, 2H, CH2, J = 7.7 Hz), 7.02-7.46 (m, 8H, ArH), 8.40 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C17H18N2 (250.34): C, 81.56; H, 7.25; N, 11.19; Found: C, 81.62; H, 7.28; N, 11.10. 4.1.1.12
5-Fluoro-2-(3-phenylpropyl)-1H-benzo[d]imidazole (12). Starting from
TE D
4-phenylbutanoic acid (2.55 g) and 4-fluoro-1,2-phenylenediamine (1.26 g), the title compound 12 was obtained by method B as shiny beige solid (1.61 g, 63%): m.p. 111-112 °C (methanol/water 1:1); IR (KBr): 3023, 2945, 2856 (ν C-H), 1543 (δ N-H),
EP
1486, 1449, 1414 (ν C=C), 1133 (δ C-H), 847, 803, 752, 699 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): 2.08-2.23 (m, 2H, CH2), 2.65 (t, 2H, CH2, J = 7.4 Hz), 2.91 (t,
AC C
2H, CH2, J = 7.7 Hz), 6.93-7.26 (m, 7H, ArH), 7.41-7.48 (m, 1H, ArH), 10.83 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C16H15N2 (311.25): C, 75.57; H, 5.95; N, 11.02; Found: C, 75.48; H, 5.90; N, 11.09. 4.1.1.13
5,6-Dimethyl-2-(3-phenylpropyl)-1H-benzo[d]imidazole (13). Starting
from 4-phenylbutanoic acid (2.46 g) and 4,5-dimethyl-1,2-phenylenediamine (1.36 g), the title compound 13 was obtained by method B as yellow solid (1.77 g, 67%): m.p. 119-121 °C (cyclohexane); IR (KBr): 3030, 2927, 2856 (ν C-H), 1540 (δ N-H), 1464, 13
ACCEPTED MANUSCRIPT
1435, 1414 (ν C=C), 1305, 1032, 1018, 999 (δ C-H), 860, 744, 697 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): 2.14-2.18 (m, 2H, CH2), 2.34 (s, 6H, 2CH3), 2.63 (t, 2H, CH2, J = 7.5 Hz), 2.90 (t, 2H, CH2, J = 7.7 Hz), 7.02-7.07 (m, 2H, ArH), 7.15-7.19
RI PT
(m, 3H, ArH), 7.34 (s, 2H, ArH), 10.20 (br s, 1H, NH + D2O exchangeable) ppm; 13C NMR (200 MHz, DMSO-d6): δ 20.2 (2C), 28.3, 29.6, 34.9, 126.0, 128.5 (2C), 128.6
(2C), 141.9, 154.0 ppm; Anal. Calcd for C18H20N2 (264.36): C, 81.78; H, 7.63; N,
4.1.1.14
SC
10.60; Found: C, 81.81; H, 7.58; N, 10.56.
5,6-Dichloro-2-(3-phenylpropyl)-1H-benzo[d]imidazole (14). Starting
M AN U
from 4-phenylbutanoic acid (2.55 g) and 4,5-dichloro-1,2-phenylenediamine (1.77 g), the title compound 14 was obtained by method B as brown solid (2.01 g, 66%): m.p. 115-117 °C (dioxane/water 2:1); IR (KBr): 2925, 2854 (ν C-H), 1492, 1442, 1403 (ν C=C), 1198, 1106 (δ C-H), 872, 827 (γ C-H) cm-1; 1H NMR (200 MHz, DMSO-d6): 2.03-2.08 (m, 2H, CH2), 2.66 (t, 2H, CH2, J = 7.3 Hz), 2.78-2.86 (m, 2H, CH2), 6.53-
TE D
6.89 (m, 2H, ArH), 7.20-7.30 (m, 3H, ArH), 8.12 (s, 2H, ArH), 12.70 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C16H14Cl2N2 (305.20): C, 62.97; H, 4.62; N, 9.18; Found: C, 62.78; H, 4.58; N, 9.23.
EP
4.1.2 General method for the synthesis of 1-methyl- and 1-phenylsulfonyl-2substituted benzimidazoles 15, 16.
AC C
Benzimidazole 12 (5 mmol) was dissolved in anhydrous dioxane (10 mL) and
treated with TEA (2 mL, 15 mmol). Then methane- or benzenesulfonyl chloride (10 mmol) was added drop-wise and the reaction mixture was stirred at 50°C for 5 h and then at an ambient temperature for another 12 h. The solvent was evaporated under reduced pressure and 20 mL of water with ice was added. After a few hours the precipitated compounds 15 and 16 were filtered off, washed with water, and recrystallized. 14
ACCEPTED MANUSCRIPT
4.1.2.1
5-Fluoro-1-(methylsulfonyl)-2-(3-phenylpropyl)-1H-
benzo[d]imidazole (15). Starting from methanesulfonyl chloride (0.78 ml), the title compound 15 was obtained as light brown solid (1.38 g, 83%): m.p. 108-110 ºC
RI PT
(MeOH/water 1:1); IR (KBr): 3027, 2931, 2860 (ν C-H), 1542, 1476, 1449 (ν C=C), 1365, 1163 (ν SO2), 1051, 965, (δ C-H), 804, 770, 696 (γ C-H) cm-1; 1H NMR (200
MHz, CDCl3): δ 2.19-2.34 (m, 2H, CH2), 2.79 (t, 2H, CH2, J = 7.6 Hz), 3.04-3.16 (m,
SC
5H, 3H CH3 and 2H CH2), 7.05-7.44 (m, 7H, ArH), 7.56-7.82 (m, 1H, ArH) ppm; 13C NMR (200 MHz, DMSO-d6): δ 26.1, 33.1, 37.1, 42.2, 100.5, 101.1, 105.6, 106.1,
M AN U
112.1 (2C), 112.6 (2C), 114.2, 114.4, 120.6, 120.8, 157.2 ppm; Anal. Calcd for C17H17FN2O2S (332.39): C, 61.43; H, 5.16; N, 8.43; Found: C, 61.38; H, 5.19; N, 8.47. 4.1.2.2
5-Fluoro-2-(3-phenylpropyl)-1-(phenylsulfonyl)-1H-
benzo[d]imidazole (16). Starting from benzenesulfonyl chloride (1.28 mL), the title
TE D
compound 16 was obtained as beige solid (1.89 g, 96%): m.p. 82-85 ºC (dioxane/water 1:1); IR (KBr): 2926, 2859 (ν C-H), 1541, 1477, 1448 (ν C=C), 1376, 1172 (ν SO2), 1088, 1044 (δ C-H), 736, 685 (γ C-H) cm-1; 1H NMR (200 MHz,
EP
CDCl3): δ 2.15-2.30 (m, 2H, CH2), 2.79 (t, 2H, CH2, J = 7.4 Hz), 3.07-3.16 (m, 2H, CH2), 7.03-7.15 (m, 1H, ArH), 7.20-7.40 (m, 5H, ArH), 7.43-7.51 (m, 2H, ArH),
AC C
7.57-7.66 (m, 2H, ArH), 7.76-7.81 (m, 2H, ArH), 7.96-8.03 (m, 1H, ArH) ppm; Anal. Calcd for C22H19FN2O2S (394.46): C, 66.99; H, 4.85; N, 7.10; Found: C, 66.87; H, 4.83; N, 7.19.
4.1.3 Procedure for the preparation of 4-phenylpicolinothioamide 4-Phenylpicolinonitrile (3.6 g, 20 mmol) was dissolved in 15 mL of methanol, DBU (0.5 mL, 3 mmol) was added and reaction mixture was refluxed for 15 min. After cooling down, ammonium polysulfide (5 mL, ~44 mmol) was added and the 15
ACCEPTED MANUSCRIPT
mixture was allowed to stand for 2 h. Then water (50 mL) was added and the precipitate of crude product was collected by filtration. The product was purified by recrystallization from ethanol yielding 3.1 g (72%) of yellow needles; m.p. 129-130
RI PT
ºC; IR (KBr): 3317, 3246 (ν N-H), 3150 (ν C-H), 1616, 1592 (δ N-H), 1464, 1420 (ν C=C), 1253 (ν C=S), 918, 759, 698 (γ C-H), 524 (γ N-H) cm-1; 1H NMR (200 MHz, CDCl3): δ 4.75 (s, 2H NH2 + D2O exchangeable), 7.43-7.74 (m, 6H, 5H ArH and 1H
SC
pyridine), 8.55 (d, 1H, pyridine J = 4.8 Hz), 8.97 (s, 1H, pyridine) ppm; Anal. Calcd for C12H10N2S (214.29): C, 67.26; H, 4.70; N, 13.07; Found: C, 67.37; H, 4.73; N,
M AN U
13.19.
4.1.4 Procedure for the synthesis of arenoimidazoles (17-25). 4-Phenylpicolinothioamide (0.214 g, 1 mmol) and the appropriate diamine (1.3 mmol) were refluxed in ethylene glycol (3 mL) until hydrogen sulfide evolution ceased (3-5 h). Then ice (20 g) was added and the precipitated product was collected
TE D
by filtration and purified by recrystallization with the addition of activated carbon. In this manner the following arenoimidazoles were obtained. 4.1.4.1
2-(4-Phenylpyridin-2-yl)-1H-benzo[d]imidazole (17). Starting from o-
EP
phenylenediamine (0.141 g), the title compound 17 was obtained as beige solid (0.271 g, 95%): m.p. 215-217 ºC (benzene); IR (KBr): 3050, 2918 (ν C-H), 1602, 1537,
AC C
1469, 1429 (ν C=C), 1247, 1128 (δ C-H), 758, 738, 695 (γ C-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 7.21-7.30 (m, 2H, ArH), 7.55-7.94 (m, 8H, 7H ArH and 1H pyridine), 8.59 (s, 1H, pyridine), 8.79 (d, 2H, pyridine, J = 5.3 Hz), 13.18 (br s, 1H, NH + D2O exchangeable) ppm. All data consistent with the literature description [26]. 4.1.4.2
5-Methyl-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole (18). Starting
from 3,4-diaminotoluene (0.159 g), the title compound 18 was obtained as beige solid (0.271 g, 95%): m.p. 178-180 ºC (ethanol/water 1:1); IR (KBr): 3048, 2922 (ν C-H), 16
ACCEPTED MANUSCRIPT
1599, 1545, 1469, 1430, 1353 (ν C=C), 1144 (δ C-H), 845, 758, 696 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): δ 2.52 (s, 3H, CH3), 7.17 (d, 1H, ArH, J = 8.2 Hz), 7.497.53 (m, 5H, ArH), 7.54-7.62 (m, 2H, 1H ArH and 1H pyridine), 7.81 (d, 1H, ArH, J
NH + D2O exchangeable) ppm;
RI PT
= 7.1 Hz), 8.66 (d, 1H, pyridine, J = 5.2 Hz), 8.81 (s, 1H, pyridine), 13.03 (br s, 1H, C NMR (200 MHz, DMSO-d6): δ 21.7, 111.9,
13
112.0, 118.6, 119.2, 122.1, 123.9, 125.0, 127.2 (2C), 129.6 (2C), 129.9, 132.7, 137.1,
5.30; N, 14.73; Found: C, 79.83; H, 5.28; N, 14.74.
5-Fluoro-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole (19). Starting
M AN U
4.1.4.3
SC
142.3, 148.6, 150.3, 150.5 ppm; Anal. Calcd for C19H15N3 (285.34): C, 79.98; H,
from 4-fluoro-1,2-phenylenediamine (0.164 g), the title compound 19 was obtained as beige solid (0.260 g, 90%): m.p. 216-218-161 ºC (benzene/diethyl ether 2:1); IR (KBr): 3049, 2922 (ν C-H), 1599, 1545, 1469, 1430, 1353 (ν C=C), 1144 (δ C-H), 845, 758, 696 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): δ 7.08-7.12 (m, 1H, ArH),
TE D
7.36-7.39 (m, 2H, 1H ArH and 1H NH + D2O exchangeable), 7.52-7.56 (m, 3H, 2H ArH and 1H pyridine), 7.64-7.66 (m, 2H, ArH), 7.81 (d, 2H, ArH, J = 6.9 Hz), 8.68 (d, 1H, pyridine, J = 5.2 Hz), 8.77 (s, 1H, pyridine) ppm; Anal. Calcd for C18H12FN3
4.1.4.4
EP
(289.31): C, 74.73; H, 4.18; N, 14.52; Found: C, 74.71; H, 4.22; N, 14.58. 5-Chloro-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole (20). Starting
AC C
from 4-chloro-1,2-phenylenediamine (0.186 g), the title compound 20 was obtained as white solid (0.294 g, 96%): m.p. 187-189 °C (ethanol/water 1:1); IR (KBr): 3045, 2925 (ν C-H), 1604, 1471, 1429, 1374 (ν C=C), 758, 697 (γ C-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 7.24-7.30 (m, 1H, ArH), 7.50-7.94 (m, 8H, 7H ArH and 1H pyridine), 8.58 (s, 1H, pyridine), 8.90 (d, 1H, pyridine, J = 5.1 Hz), 13.35 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C18H12ClN3 (305.76): C, 70.71; H, 3.96; N, 13.74; Found: C, 70.65; H, 3.94; N, 13.82. 17
ACCEPTED MANUSCRIPT
4.1.4.5
5-Bromo-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole (21). Starting
from 4-bromo-1,2-phenylenediamine (0.243 g), the title compound 21 was obtained as beige solid (0.130 g, 37%): m.p. 163-165 ºC (ethanol); IR (KBr): 3055, 2925 (ν C-
RI PT
H), 1606, 1548, 1429, 1373 (ν C=C), 915, 761, 696 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): δ 7.42 (d, 1H, ArH, J = 8.8 Hz), 7.49-7.58 (m, 4H, ArH), 7.64-7.65 (m, 1H, ArH), 7.74 (d, 1H, ArH, J = 6.8 Hz), 7.79 (d, 1H, ArH, J = 7.3 Hz), 7.85 (s,
SC
1H, pyridine), 8.66 (d, 1H, pyridine, J = 4.9 Hz), 8.75 (m, 1H, pyridine), 9.00 (br s,
1H, NH + D2O exchangeable) ppm; 13C NMR (200 MHz, DMSO-d6): δ 118.9, 122.7,
M AN U
125.1, 126.7, 127.1, 127.2 (2C), 127.4, 129.6 (3C), 129.9, 130.5, 136.9, 148.7, 149.0, 150.5, 152.0 ppm; Anal. Calcd for C18H12BrN3 (350.21): C, 61.73; H, 3.45; N, 12.00; Found: C, 61.81; H, 3.43; N, 12.05. 4.1.4.6
5,6-Dimethyl-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole
(22).
Starting from 4,5-dimethyl-1,2-phenylenediamine (0.177 g), the title compound 22
TE D
was obtained as beige solid (0.228 g, 76%); m.p. 200-202 ºC. IR (KBr): 3062, 2926 2857 (ν C-H), 1601, 1437 (ν C=C), 1034 (δ C-H), 852, 765, 689 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): δ 2.36 (s, 6H, 2CH3), 7.42-7.53 (m, 6H, ArH), 7.72-7.80
EP
(m, 2H, 1H ArH and 1H pyridine), 8.56-8.62 (m, 1H, pyridine), 8.66-8.68 (m, 1H, pyridine), 12.93 (br s, 1H, NH) ppm;
C NMR (200 MHz, DMSO-d6): δ 20.3 (2C),
13
AC C
118.5 (2C), 122.0, 127.2 (2C), 127.4, 129.6 (3C), 129.8 (2C), 137.1, 148.5, 149.7, 150.1, 150.2 (2C), 150.6, 152.0 ppm; Anal. Calcd for C20H17N3 (299.37): C, 80.24; H, 5.72; N, 14.04; Found: C, 80.31; H, 5.67; N, 14.02. 4.1.4.4
2-(4-Phenylpyridin-2-yl)-3H-imidazo[4,5-b]pyridine (23). Starting from
2,3-diaminopyridine (0.142 g), the title compound 23 was obtained as shiny beige crystals (0.240 g, 88%): m.p. 282-283 ºC (dioxane); IR (KBr): 3059, 2960, 2865 (ν CH), 1603, 1544, 1467, 1433, 1408, 1355 (ν C=C), 1271 (δ C-H), 767 (γ C-H) cm-1; 1H 18
ACCEPTED MANUSCRIPT
NMR (200 MHz, DMSO-d6): δ 7.49-7.58 (m, 4H, 3H ArH and 1H pyridine), 7.887.93 (m, 3H, 2H ArH and 1H pyridine), 8.36 (d, 1H, pyridine, J = 5.6 Hz), 8.64 (s, 1H, pyridine), 8.80 (d, 1H, pyridine, J = 5.1 Hz), 9.06 (s, 1H, pyridine), 13.61 (br s,
RI PT
1H, NH + D2O exchangeable) ppm. All data consistent with the literature description [26]. 4.1.4.8
6-Bromo-2-(4-phenylpyridin-2-yl)-3H-imidazo[4,5-b]pyridine
(24).
SC
Starting from 2,3-diamino-5-bromopyridine (0.244 g), the title compound 24 was obtained as shiny gray crystals (0.320 g, 91%): m.p. >260 ºC (decomp.)
M AN U
(ethanol/water 1:1); IR (KBr): 3317 (ν N-H), 3170, 2922 (ν C-H), 1592, 1473, 1418 (ν C=C), 1318, 1239 (δ C-H), 918, 862, 761, 696 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): δ 7.42-7.57 (m, 4H, 3H ArH and 1H pyridine), 7.59-7.74 (m, 4H, 2H ArH and 2H pyridine), 8.55 (d, 1H, pyridine, J = 4.5 Hz), 8.96-8.97 (m, 1H pyridine), 9.60 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C17H11BrN4 (351.20): C,
4.1.4.9
TE D
58.14; H, 3.16; N, 15.95; Found: C, 58.18; H, 3.19; N, 15.81. 2-(4-Phenylpyridin-2-yl)-3H-imidazo[4,5-c]pyridine (25). Starting from
3,4-diaminopyridine (0.142 g), the title compound 25 was obtained as yellow solid
EP
(0.237 g, 87%): m.p. 248-250 °C (ethanol); IR (KBr): 3242 (ν N-H), 3054, 2957, 2890, 2851 (ν C-H), 1605, 1547, 1472, 1427, 1295 (ν C=C), 1025, 960 (δ C-H), 814,
AC C
756, 698, 614 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): δ 7.24-7.28 (m, 1H, ArH), 7.51-7.54 (m, 3H, ArH), 7.66 (d, 1H pyridine, J = 4.3 Hz), 7.76-7.80 (m, 2H, 1H ArH and 1H pyridine), 8.49 (d, 1H, pyridine, J = 5.4 Hz), 8.70-9.73 (m, 2H, pyridine), 9.15 (s, 1H, pyridine Hz), 10.95 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C17H12N4 (272.30): C, 74.98; H, 4.44; N, 20.58; Found: C, 75.02; H, 4.48; N, 20.50. 4.2
Biological assays
4.2.1 Antimycobacterial activity assay 19
ACCEPTED MANUSCRIPT
The synthesized compounds were examined in vitro for their tuberculostatic activity against the Mycobacterium tuberculosis H37Rv strain and two “wild” strains isolated from tuberculosis patients: one (Spec. 210) resistant to p-aminosalicylic acid
RI PT
(PAS), isonicotinic acid hydrazide (INH), ethambutol (ETB) and rifampicine (RMP) and the another (Spec. 192) fully sensitive to the administrated tuberculostatics. Investigations were performed by a classical test-tube method of successive dilution
SC
in Youmans’ modification of the Proskauer and Beck liquid medium containing 10%
of bovine serum [27, 28]. Bacterial suspensions were prepared from 14 days old
M AN U
cultures of slowly growing strains and from 48 h old cultures of saprophytic strains [29, 30]. Solutions of compounds in ethylene glycol were tested. Stock solutions contained 10 mg of compounds in 1 millilitre. Dilutions (in geometric progression) were prepared in Youmans’ medium. The medium containing no investigated substances and containing isoniazid (INH), pyrazinamide (PZA) or rifampicin (RMP)
TE D
as reference drugs were used for comparison. Incubation was performed at a temperature of 37 °C. The MIC values were determined as minimum concentration inhibiting the growth of tested tuberculosis strains in relation to the probe with no
EP
tested compound. The influence of the compound on the growth of bacteria at a certain concentration, 0.75, 1.5, 3.1, 6.2, 12.5, 25, 50 and 100 µg/mL were evaluated.
AC C
For compounds that showed the highest activity studies have been extended to a Mycobacterium bovis strain. The tests were performed according to the procedure described above.
4.2.3 Cytotoxicity assay Stock solutions were freshly prepared by dissolving compounds in DMSO. A549 cells are adenocarcinomic human alveolar basal epithelial cells. This line was initiated in 1972 by D. J. Giard, et al. [31] through explant culture of lung 20
ACCEPTED MANUSCRIPT
carcinomatous tissue from a 58-year-old Caucasian male. LLC-PK1 are normal, epithelial, adherent cells isolated from the kidney of a 3-4 weeks old male pig. All cells were from ATCC and showed no mycoplasma contamination as revealed by
RI PT
Roche ELISA-based test [32]. Multiwell (24–well) plates were seeded at 1.25x104 LLC-PK1 cells/well in Medium 199 supplemented with 3% FBS and antibiotics (penicillin/streptomycin) or 4x103
A549 cells/well in RPMI1640 medium
SC
supplemented with 10% FBS and antibiotics (penicillin/streptomycin). Cells were
allowed to attach overnight. Drugs were added to wells in 10 µL aliquots of 200-times
M AN U
concentrated drug solutions dissolved in DMSO, in duplicates. To control wells, 10 µL of DMSO was added. Cells were incubated with studied compounds for 120 h at 37oC and 95%/5% CO2 atmosphere [33]. To all wells 200 µL of MTT solution in PBS (4 mg/mL) was added and incubated further for 4 h in 37oC. Absorbance was measured after solubilization of formazan crystals in 1 mL DMSO and using a
TE D
multiwell plate reader (Victor3, Perkin-Wallac) at λ = 540 nm. Cytotoxicity was determined compared to non-treated cells (% control).
EP
Acknowledgments
This project was funded by the National Science Centre (Cracow, Poland) on the basis
AC C
of decision number DEC-2011/01/B/NZ4/01187.
References
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[10] G. Panteix, M.C. Gutierrez, M.L. Boschiroli, M. Rouviere, A. Plaidy, D. Pressac, H. Porcheret, G. Chyderiotis, M. Ponsada, K. Van Oortegem, S. Salloum, S. Cabuzel, A.L. Bañuls, P. Van de Perre, S. Godreuil, Pulmonary tuberculosis due
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[19] G.R. Jadhav, M.U. Shaikh, R.P. Kale, M.R. Shiradkar, C.H. Gill, SAR study of clubbed [1,2,4]-triazolyl with fluorobenzimidazoles as antimicrobial and antituberculosis agents, Eur. J. Med. Chem. 44 (2009) 2930-2935. [20] V. Klimešova, J. Koči, K. Waisser, J. Kaustova, New benzimidazole derivatives 23
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new benzimidazole derivatives, Chem. Heterocyc. Compd. 42 (2006) 611-614. [22] K. Gobis, H. Foks, K. Bojanowski, E. Augustynowicz-Kopeć, A. Napiórkowska, Synthesis of novel 3-cyclohexylpropanoic acid-derived nitrogen heterocyclic
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[25] V.-M. Mukkala, P. Liitti, I. Hemmila, H. Takalo, C. Matachescu, J. Kankare, Novel thiazole-containing complexing agents and luminescence of their
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europium(III) and terbium(III) chelates, Helv. Chim. Acta 79 (1996) 295-306. [26] F.H. Case, The preparation of 2-substituted benzimidazole derivatives containing the ferroin group (1), J. Het. Chem. 4 (1967) 157-159.
[27] G.P. Youmans, Test tube evaluation of tuberculostatic agents, Am. Rev. Tuberc. 56 (1947) 376. [28] G.P. Youmans, A.S. Youmans, A method for the determination of the rate of growth of tubercle bacilli by the use of small inocula, J. Bactriol. 58 (1949) 24724
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255. [29] R.M. Atlas, J.W. Snyder, Handbook of Media for Clinical Microbiology, second ed., CRC Press Book, Boca Raton, 1995.
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[30] H. Foks, M. Buraczewska, W. Manowska, J. Sawlewicz, Investigation on pyrazine derivatives, Dissert. Pharm. Pharmacol. 1971, 23, 49-58.
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[32] M. Welti, K. Jaton, M. Altweg, R. Sahli, A. Wenger, J. Bille, Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions, Diagn. Microbiol. Infect. Dis. 45 (2003) 85–95. [33] M.M. Wintrobe, J.P. Greer, Wintrobe’s clinical hematology, twelfth ed., Wolters
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Kluwer Health/Lippincott Williams & Wilkins, Philadelphia, 2009.
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Table 1 In vitro tuberculostatic activity of compounds 1-25.a, b, c MIC [µg/mL] Compds. M. tuberculosis M. bovis H37Rv Spec. 192 Spec. 210 1.5 1.5 1.5 12.5 1 3.1 3.1 3.1 3.1 2 3 0.75 0.75 0.75 0.75 4 0.75 0.75 0.75 0.75 3.1 3.1 3.1 3.1 5 6 0.75 0.75 0.75 0.75 7 0.75 0.75 0.75 1.5 6.2 6.2 6.2 6.2 8 25 6.2 6.2 9 12.5 6.2 6.2 25 10 25 6.2 100 25 11 12.5 50 50 12 6.2 6.2 6.2 6.2 13 6.2 6.2 6.2 3.1 14 50 50 50 15 100 100 50 16 12.5 6.2 6.2 17 3.1 6.2 3.1 25 18 6.2 12.5 6.2 19 12.5 12.5 6.2 20 12.5 12.5 6.5 21 25 12.5 12.5 22 12.5 12.5 12.5 23 25 25 25 24 12.5 12.5 3.1 25 12.5 12.5 25 INH 25 25 >400 PZA 1.2 1.2 2.5 RMP
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Minimum inhibitory concentrations for bacterial strains were determined by twofold serial dilution method for microdilution plates and for mycobacterial strains by two-fold classical test-tube method of successive dilution. b INH isoniazid; PZA pyrazinamide; RMP rifampicin. c M. tuberculosis H37Rv, Spec. 192, Spec. 210, M. bovis
Table 2 Cytotoxicity of compounds 3, 4, 6, and 7 Compds. IC50a [µM] b A549 10.90 (±0.57) 3 13.36 (±0.90) 4 11.15 (±0.30) 6 11.05 (±1.05) 7
LLC-PK1c 17.11 (±0.30) 32.57 (±2.15) 43.94 (±0.51) 9.62 (±1.10)
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a Concentrations inhibiting the cell growth by 50% were determined by the MTT method with expotentially growing cells and continuous drug exposure (120 h); b A549 – human non-small lung cancer cell line; c LLC-PK1 – pig kidney epithelial cell line.
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Figure Captions Figure 1. Structure of benzimidazole derivatives and analogues of tuberculostatic activity Scheme 1. Synthesis of benzimidazole derivatives 1–16. Reagents and conditions: (i) method
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A (compounds 1-7): 3-cyclohexylpropanoic acid (1.5 equiv.), diamine (1 equiv.), PPA, 180– 200 °C; NaHCO3/H2O; method B (compounds 8-14): 4-cyclohexylbutanoic or 4phenylbutanoic acid (1.5 equiv.), diamine (1 equiv.), 160-180 °C; NaOH/H2O; (ii)
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benzimidazole 12 (1 eqiuv.), TEA (3 equiv.), methane or benzenesulfonyl chloride (2 equiv.), anhydrous dioxane, 50 °C; H2O.
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Scheme 2. Synthesis of benzimidazole derivatives and analogues 17-25. Reagents and conditions: (i) 4-phenylpicolinonitrile (1 equiv.), DBU (0.15 equiv.), methanol, reflux; (ii) ammonium polysulfide (2.2 equiv.), methanol, room temperature; H2O; (iii) 4phenylpicolinothioamide (1 equiv.), diamine (1.3 equiv.), ethylene glycol, reflux; ice. Figure 2. Effect of compounds 3, 4, 6, and 7 on A549 (a) and LLC-PK1 eukaryotic cell lines
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S0223-5234(14)00958-1
DOI:
10.1016/j.ejmech.2014.10.031
Reference:
EJMECH 7438
To appear in:
European Journal of Medicinal Chemistry
Received Date: 20 December 2013 Revised Date:
10 September 2014
Accepted Date: 12 October 2014
Please cite this article as: K. Gobis, H. Foks, M. Serocki, E. Augustynowicz-Kopeć, A. Napiórkowska, Synthesis and evaluation of in vitro antimycobacterial activity of novel 1H-benzo[d]imidazole derivatives and analogues, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.10.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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We synthesized a new series of benzimidazoles. Compounds were tested against M. tuberculosis and M. bovis in vitro. Some compounds exhibited excellent tuberculostatic activity.
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Two derivatives were characterized by high therapeutic index in vitro.
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Cytotoxic activity towards eukaryotic cells was determined.
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European Journal of Medicinal Chemistry Original article
Synthesis and evaluation of in vitro antimycobacterial activity
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of novel 1H-benzo[d]imidazole derivatives and analogues Katarzyna Gobisa,*, Henryk Foksa, Marcin Serockib,
Ewa Augustynowicz-Kopećc, Agnieszka Napiórkowskac a
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Department of Organic Chemistry, Medical University of Gdańsk, 107 Gen. Hallera Ave., 80-416 Gdańsk, Poland b Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 11/12 G. Narutowicza Str., 80-233 Gdańsk, Poland c Department of Microbiology, Institute of Tuberculosis and Pulmonary Diseases, 26 Płocka Str., 01-138 Warsaw, Poland
Abstract
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A series of novel 1H-benzo[d]imidazole derivatives and analogues (1-25) have been synthesized and evaluated for tuberculostatic activity. Benzimidazoles substituted at the C-2 position with cyclohexylethyl, cyclohexylpropyl and phenylpropyl moiety or 4-phenylpyridine system were obtained. Compounds 3, 4, 6 and 7 bearing halogen atoms or methyl groups at the benzimidazole system and cyclohexylethyl substituent at the C-2 position showed an excellent tuberculostatic activity against M. tuberculosis and M. bovis strains with MIC values ranging from 0.75 to 1.5 µg/mL. More importantly, derivatives 4 (5-Bromo-2-(2-cyclohexylethyl)-1Hbenzo[d]imidazole) and 6 (2-(2-cyclohexylethyl)-5,6-dimethyl-1H-benzo[d]imidazole) appeared selective for M. tuberculosis and M. bovis as compared with non-malignant eukaryotic cells (LLC-PK1 pig kidney epithelial cell line). These compounds may thus represent a novel, selective class of anti-tubercular agents.
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Keywords: Benzimidazole; synthesis; tuberculostatic activity; cytotoxicity; SAR-study
*Corresponding author. Tel.: +48 58 349 16 47; fax: +48 58 349 12 77. E-mail address: [email protected] (K. Gobis)
1
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1.
Introduction Infections caused by Mycobacterium tuberculosis are the reason of one of the
most serious contagious diseases [1]. Despite the passage of time, money spent and
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the hard work of many research laboratories and social organizations, tuberculosis still causes about 1.3 million deaths per annum [2]. Particularly dangerous is its
variety resistant to applied chemotherapeutic agents (MDR-TB), especially when it
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concerns patients with a compromised immune system [3]. Human tuberculosis mainly results from the human-to-human transmission of Mycobacterium tuberculosis
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[4,5] with a few cases being caused by other closely related species: M. bovis [6], M. caprae [7,8], M. microti [9,10] and M. pinipedii [11].
Due to the continuous rise of the number of cases caused by resistant strains, a need for new anti-tuberculosis drugs that are active towards the broad spectrum of species of the Mycobacterium genus becomes obvious [12]. This need is even greater
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because of the generally small number of effective anti-tuberculosis chemotherapeutic agents. Isoniazid (INH) [13], pyrazinamide (PZA) [14] and ethionamide (ETA) [15] should be mentioned among the leading drugs of first and second line therapy.
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However, these agents cause a number of side effects [13-16]. The anti-tuberculosis activity of other nitrogen heterocyclic compounds, e.g. derivatives of 1H-
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benzo[d]imidazole has been also reported. In particular, compounds substituted at the C-2 position of this system appear to be interesting [17]. Significant activity showed 2-styrene [18], 2-polyfluoroalkyl [19] and 2-(1,2,4-triazole)-phenyl [20] derivatives. Previously our research group has also described the high activity of 2cyclohexylethyl derivatives [21] and benzimidazole type systems (naphthoimidazole, imidazopyridine, imidazophenazine) (Figure 1) [22]. The activity of these compounds was at the level corresponding to the activity of anti-tuberculosis drugs (MIC 1.5-3.1 2
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µg/mL). Continuing this research we have taken in the present work the synthesis of benzimidazoles
substituted
at
the
C-2
position
with
cyclohexylethyl,
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cyclohexylpropyl, phenylpropyl or 4-phenylpyridine moiety. Syntheses were designed to receive compounds with different substituents at the benzimidazole system, and, at the C-2 position, the cyclohexane or benzene ring distant from the benzimidazole
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system by two or three methylene groups. Compounds with the pyridine ring
substituted directly to benzimidazole were also obtained. That choice of the structural
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parameters would enable assessment of the effect of the substituent at the C-2 structure, the chain length and the degree of the six-member ring saturation at its end, for tuberculostatic activity of the compounds. Furthermore, derivatives substituted with the phenylsulfonyl or methylsulfonyl group at the N-1 position have been
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synthesized for one of the benzimidazoles obtained.
2
Results and discussion
2.1
Chemistry
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We synthesized structures in which the cyclohexylethyl, cyclohexylpropyl, phenylpropyl or 4-phenylpyridine-2-yl moiety is connected to the benzimidazole
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system. The structures with different substituents at the benzene ring of the benzimidazole system, including the methyl or nitro group, fluorine, chlorine or bromine atoms were obtained. The synthesized compounds have been screened for their tuberculostatic and cytotoxic activities. Finally, target structures were planned in order to investigate the preliminary structure–activity relationship, with the emphasis on the crucial role of various substituents at the benzene ring of the benzimidazole system and at its C-2 or N-1 position. The synthetic route of the studies was outlined 3
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in Schemes 1 and 2. Syntheses of the target benzimidazole derivatives 1–25 were achieved in 37–96% yields by the procedures described below. The starting compounds for a series of syntheses were the commercially available derivatives and of
o-phenylenediamine:
3,4-diaminotoluene,
4-fluoro-1,2-
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analogues
phenylenediamine, 4-chloro-1,2-phenylenediamine, 4-bromo-1,2-phenylenediamine, 4-nitro-1,2-phenylenediamine, 4,5-dimethyl-1,2-phenylenediamine, 4,5-dichloro-1,2-
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phenylenediamine, 2,3-diaminopyridine, 5-bromo-2,3-diaminopyridine and 3,4-
diaminopyridine. Compounds 1–7 (Scheme 1) were synthesized by the heating of 3-
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cyclohexylpropanoic acid with appropriate diamines according to the method described by Algul et al. [23] and based on the use of polyphosphoric acid (PPA) as a solvent with strong acidic properties (method A). Compound 7 has been already obtained by Li and co-workers as anti-hepatitis B virus agent but its activity was not significant [24]. The above method for the synthesis was tested first, but much better
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yields of benzimidazoles 8-14 were obtained by fusion of the starting materials at 160-180°C without solvent, the process of which was elaborated earlier by our team (method B) [21]. We used 4-cyclohexylbutanoic and 4-phenylbutanoic acid. In the
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next step benzimidazole 12 reacted with methane- and benzenesulfonyl chloride in anhydrous dioxane in the presence of triethylamine (TEA), giving the corresponding
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sulfonamide derivatives 15 and 16. Compounds 17-25 were synthesized from 4-phenylpicolinothioamide (Scheme
2), which was obtained from starting carbonitrile via methyl carbimidate in the reaction with DBU and ammonium polysulfide according to the modified method reported by Mukkala et al. [25]. Then, upon treatment of appropriate diamines in ethylene glycol, 4-phenylpicolinothioamide gave benzimidazoles 17-22 and imidazopyridines 23-25. Until now the synthesis of benzimidazoles from thioamides 4
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has not been reported. Compounds 17 and 23 have been already described by Case [26], although synthesized directly from 4-phenylpicolinonitrile. All the newly synthesized compounds were characterized by IR and 1H NMR
structures were also confirmed by
13
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spectra as well as the elemental analysis listed in the experimental section. Some C NMR. The spectral analyses were in
accordance with the assigned structures. Biological activity
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2.2
All of the obtained benzimidazole derivatives 1-25 were evaluated for their in
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vitro tuberculostatic activity against the Mycobacterium tuberculosis H37Rv strain and two “wild” strains isolated from tuberculosis patients: one (Spec. 210) resistant to paminosalicylic acid (PAS), isonicotinic acid hydrazide (INH), ethambutol (ETB) and rifampicin (RMP) and the other (Spec. 192) fully sensitive to the administrated tuberculostatics. The MIC values were determined as the minimum concentration
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inhibiting the growth of tested tuberculosis strains in relation to the probe with no tested compound. INH, PZA and RMP were used as reference drugs. Some compounds were also tested against the Mycobacterium bovis strain. The most active
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compounds of the benzimidazole structure 3, 4, 6 and 7 which exhibited the highest tuberculostatic activity were then tested for effect on the proliferation of two
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eukaryotic cell lines: human non-small lung cancer cell line A549 and pig kidney epithelial cell line LLC-PK1. The bioactive data were summarized in Tables 1 and 2. 2.2.1 Tuberculostatic activity The results of tuberculostatic activity indicated that most of the synthesized
compounds exhibited moderate to excellent activity against M. tuberculosis and M. bovis strains in vitro. As seen in Table 1, the tested derivatives showed high tuberculostatic activity. Generally, compounds possessing the cyclohexylethyl 5
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substituent at the C-2 position were more active (MICs 0.75-3.1 µg/mL) than those with the longer chain linker (MICs 6.2-25 µg/mL). Derivatives with the 4phenylpyridin-2-yl moiety were also less active. The activity of four benzimidazoles
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3, 4, 6 and 7 was excellent (MICs 0.75 µg/mL) and much higher than the activity determined for clinically applied INH (12.5-25 µg/mL). Among the most active
compounds were derivatives possessing one or two halogen atoms (3, 4, 7) or two
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methyl groups (6) at the benzimidazole system. Derivatives 15 and 16 with bulky
substituents, methylsulfonyl or phenylsulfonyl group, at the N-1 position were
(MICs 12.5-50 µg/mL). The
obtained
derivatives
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definitely less active (MICs 50-100 µg/mL) than unsubstituted benzimidazole 12
and
analogs
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2-(4-phenylpyridin-2-yl)-1H-
benzo[d]imidazole exhibited different tuberculostatic activity with MIC values in the range of 3.1-25 µg/mL. Compound 18 possessing a methyl group at the C-5 position
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of the benzimidazole system showed very good activity at low concentration of 3.16.2 µg/mL. Derivatives 19-21 with halogen atoms at the C-5 position of the benzimidazole system exhibited good tuberculostatic activity with MIC values
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ranging from 6.2-12.5 µg/mL. Three of the obtained compounds 22, 23 and 25 showed moderate activity with MIC values in the range of 12.5-25 µg/mL. Their
AC C
activity against sensitive and resistant strains were comparable. Compound 24 possessing the imidazopyridine system substituted with the bromine atom at the C-6 position showed the weakest tuberculostatic activity (MICs 25 µg/mL) against all the strains tested. In the case of compounds 18-21 and 25, higher activity was observed against the resistant strain (Spec.210) than the sensitive one (Spec.192). 2.2.3 Cytotoxic activity
6
ACCEPTED MANUSCRIPT
Benzimidazoles 3, 4, 6 and 7, the most active toward M. tuberculosis strains, were tested for cytotoxicity against human non-small lung cancer cell line A549 and pig kidney epithelial cell line LLC-PK1 (Figure 2, Table 2). All values are means of at
RI PT
least two independent experiments, each done in duplicate. Cisplatin (Cis-Pt) and doxorubicin (Dox) were used as control cytostatic drugs. IC50 values determined in the A549 cell line were 0.63 ± 0.018 µM (Cis-Pt) and 0.008 ± 0.0011 µM (Dox). For
SC
tumour cells (A549) the IC50 values did not differ significantly for the individual compounds (IC50 11-14 µM), but for the immortalized kidney cells (LLC-PK1), the
M AN U
IC50 values were much more diverse. Chlorine-substituted benzimidazoles 3 and 7 were much more toxic for these cells (IC50 ca. 10 µM) than those substituted with bromine (IC50 ca. 33 µM) or methyl groups (IC50 ca. 44 µM). However, the IC50 value (9.62 µM) obtained for derivative 7 substituted with two chlorine atoms was four times higher than MICs (0.75 µg/mL). In the case of compound 6, the least cytotoxic,
TE D
IC50 value (43.94 µM) was fifteen times higher than MICs (0.75 µg/mL). The differences in toxicity towards both lines are not surprising given the higher
3
EP
sensitivity of tumour cells.
Conclusions
AC C
In conclusion, a series of novel 3-cyclohexylpropanoic acid derivatives were successfully synthesized. All these new compounds were confirmed by IR and NMR spectra as well as an elemental analysis. Their tuberculostatic activity in vitro was evaluated against M. tuberculosis H37Rv, Spec. 192 and Spec. 210 strains and M. bovis strain, using the two-fold serial dilution MIC method. The results showed that most of the synthesized derivatives exhibited moderate tuberculostatic activity; however compounds 3, 4, 6 and 7 bearing halogen atoms or two methyl groups at the 7
ACCEPTED MANUSCRIPT
benzimidazole system and cyclohexylethyl moiety at its C-2 position exhibited excellent tuberculostatic activity with MIC values ranging from 0.75 to 1.5 µg/mL. More importantly, active compound 6 exhibited a very low cytotoxic effect on the
RI PT
proliferation of eukaryotic LLC-PK1 pig kidney epithelial cell line. These findings demonstrated that among 2-cyclohexylethylbenzimidazoles one can find compounds with interesting therapeutic properties that could become a new group of potential
Experimental
4.1
Chemistry
M AN U
4
SC
tuberculostatic agents.
All materials and solvents were of analytical reagent grade. Thin-layer chromatography was performed on Merck silica gel 60F254 plates and visualized with UV. The results of elemental analyses (%C, H, N) for all of obtained compounds were in agreement with the calculated values within ± 0.3 % range. 1H and
13
C NMR
TE D
spectra in CDCl3 or DMSO-d6 were recorded on Varian Unity Plus (500 MHz) and Varian Gemini (200 MHz) instruments. IR Spectra (KBr) were determined as KBr pellets of the solids on a Satellite FT-IR spectrophotometer. Melting points were
EP
determined on a Stuart SMP30 apparatus and were uncorrected. 4.1.1 General procedure for the synthesis of benzimidazoles (1-14)
AC C
Method A (for compounds 1-7). 3-Cyclohexylpropanoic acid (2.35 mL, 15 mmol), appropriate diamine (10 mmol) and PPA (5 mL) were stirred at 180-200 ºC for 5 h, then the mixture was allowed to cool to an ambient temperature and poured into cold water (50 mL). The mixture was neutralized with a saturated solution of sodium hydrogen carbonate and the resulting precipitate was filtered off, washed several times with water and recrystallized from a methanol/water mixture (1:1) with the addition of activated carbon. 8
ACCEPTED MANUSCRIPT
Method B (for compounds 8-14). 4-Phenylbutanoic or 4-cyclohexylbutanoic acid (15 mmol) and appropriate diamine (10 mmol) were heated on Wood's metal bath at 160-180°C for 1 h. While cooling down, a 10% NaOH solution (10 mL) was
RI PT
added and the reaction mixture was stirred at room temperature for 24 h. Then the precipitated benzimidazoles were filtered off, washed with water to the neutral reaction, dried and purified by crystallization.
2-(2-Cyclohexylethyl)-5-methyl-1H-benzo[d]imidazole
(1).
Starting
SC
4.1.1.1
from 3,4-diaminotoluene (1.22 g), the title compound 1 was obtained by method A as
M AN U
colourless crystals (1.53 g, 63%): m.p. 245-248 °C; IR (KBr): 2925, 2852 (ν C-H), 1575 (δ N-H), 1450 (ν C=C), 1231 (ν C-C), 1072, 960 (δ C-H), 523 (ν N-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 0.86-0.97(m, 2H, CH2), 1.06-1.21 (m, 4H, 2CH2), 1.62-1.90 (m, 7H, 6H 3CH2 and 1H CH), 2.37 (s, 3H, CH3), 2.80 (t, 2H, CH2, J = 7.8 Hz), 6.84-7.01 (m, 1H, ArH), 7.13-7.48 (m, 2H, ArH), 7.65 (br s, 1H, NH + D2O 13
C NMR (200 MHz, DMSO-d6): δ 21.5, 25.7, 26.0 (2C), 26.3,
TE D
exchangeable) ppm;
32.7 (2C), 35.1, 36.8, 113.9, 114.3, 123.6, 131.4, 135.6, 137.2, 155.1 ppm; Anal.
N, 11.47. 4.1.1.2
EP
Calcd for C16H22N2 (242.36): C, 79.29; H, 9.15; N, 11.56; Found: C, 79.35; H, 9.18;
2-(2-Cyclohexylethyl)-5-fluoro-1H-benzo[d]imidazole
(2).
Starting
AC C
from 4-fluoro-1,2-phenylenediamine (1.26 g), the title compound 2 was obtained by method A as colourless crystals (1.33 g, 54%): m.p. 236-240 ºC; IR (KBr): 2925, 2853 (ν C-H), 1578 (δ N-H), 1458 (ν C=C), 1220 (ν C-C), 1073, 958 (δ C-H), 526 (γ N-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 0.86-0.97 (m, 2H, CH2), 1.06-1.21 (m, 4H, 2CH2), 1.62-1.90 (m, 7H, 6H 3CH2 and 1H CH), 2.80 (t, 2H, CH2, J = 7.8 Hz), 6.84-7.01 (m, 1H, ArH), 7.13-7.48 (m, 2H, ArH), 7.65 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C15H19FN2 (246.32): C, 73.14; H, 7.77; N, 11.37; 9
ACCEPTED MANUSCRIPT
Found: C, 73.18; H, 7.79; N, 11.36. 4.1.1.3
5-Chloro-2-(2-cyclohexylethyl)-1H-benzo[d]imidazole
(3).
Starting
from 4-chloro-1,2-phenylenediamine (1.43 g), the title compound 3 was obtained by
RI PT
method A as white solid (1.73 g, 66%): m.p. 117-120 ºC; IR (KBr): 2923, 2851 (ν CH), 1539 (δ N-H), 1448, 1420 (ν C=C), 1276 (ν C-C), 1058, 924 (δ C-H), 805 (γ C-
H) 599 (γ N-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 0.81-0.96 (m, 2H, CH2),
SC
1.06-1.20 (m, 4H, 2CH2), 1.58-1.74 (m, 7H, 6H 3CH2 and 1H CH), 2.79 (t, 2H, CH2, J = 7.9 Hz), 7.10 (d, 1H, ArH, J = 7.4 Hz), 7.13-7.45 (m, 2H, ArH), 12.34 (br s, 1H,
M AN U
NH + D2O exchangeable) ppm; Anal. Calcd for C15H19ClN2 (262.78): C, 68.56; H, 7.29; N, 10.66; Found: C, 68.51; H, 7.21; N, 10.71. 4.1.1.4
5-Bromo-2-(2-cyclohexylethyl)-1H-benzo[d]imidazole
(4).
Starting
from 4-bromo-1,2-phenylenediamine (1.87 g), the title compound 4 was obtained by method A as colourless crystals (1.57 g, 51%): m.p. 157-159 ºC; IR (KBr): 2920,
TE D
2850 (ν C-H), 1539 (ν N-H), 1446, 1420 (ν C=C), 1273 (ν C-C), 1000, 913 (δ C-H), 750 (γ C-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 0.82-0.97 (m, 2H CH2) 1.111.20 (m, 4H, 2CH2), 1.63-1.76 (m, 7H, 6H 3CH2 and 1H CH), 2.79 (t, 2H, CH2, J =
EP
7.8 Hz), 7.06-7.10 (m, 1H, ArH), 7.41-7.45 (m, 2H, ArH), 12.16 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C15H19BrN2 (307.23): C, 58.64; H, 6.23; N, 9.12;
AC C
Found: C, 58.61; H, 6.26; N, 9.08. 4.1.1.5
2-(2-Cyclohexylethyl)-5-nitro-1H-benzo[d]imidazole (5). Starting from
4-nitro-1,2-phenylenediamine (1.53 g), the title compound 5 was obtained by method A as beige crystals (1.75 g, 64%): m.p. 266-269 ºC; IR (KBr): 2926, 2853 (ν C-H), 1574 (δ N-H), 1522 (ν NO2), 1486, 1463 (ν C=C), 1268 (ν C-C), 1025, 958 (δ C-H), 854 (γ C-H), 535 (γ N-H) cm -1; 1H NMR (200 MHz, DMSO-d6): δ 0.85-0.96 (m, 2H, CH2), 1.06-1.21 (m, 4H, 2CH2), 1.46-1.74 (m, 7H, 6H 3CH2 and 1H CH), 2.78 (t, 2H, 10
ACCEPTED MANUSCRIPT
CH2, J = 7.9 Hz), 6.89-6.99 (m, 1H, ArH), 7.22-7.27 (m, 1H, ArH), 7.39-7.46 (m, 1H, ArH), 8.50 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C15H19N3O2 (273.33): C, 65.91; H, 7.01; N, 15.37; Found: C, 65.89; H, 7.06; N, 15.42. 2-(2-Cyclohexylethyl)-5,6-dimethyl-1H-benzo[d]imidazole (6). Starting
RI PT
4.1.1.6
from 4,5-dimethyl-1,2-phenylenediamine (1.36 g), the title compound 6 was obtained
by method A as beige solid (2.01 g, 79%): m.p. 193-194 ºC; IR (KBr): 2920, 2853 (ν
SC
C-H), 1543 (δ N-H), 1451, 1420 (ν C=C), 1308 (ν C-C), 1013, 1001 (δ C-H), 851 (γ
C-H) cm-1; 1H NMR (200 MHz, CDCl3): 0.82-0.93 (m, 2H, CH2), 1.10-1.27 (m, 4H,
M AN U
2CH2), 1.64-1.79 (m, 7H, 6H 3CH2 and 1H CH), 2.35 (s, 6H, 2CH3), 2.92 (t, 2H, CH2, J = 8.1 Hz), 7.33 (s, 2H, ArH), 10.48 (br s, 1H, NH + D2O exchangeable) ppm; 13C NMR (200 MHz, DMSO-d6): δ 26.0 (2C), 26.4, 32.8 (2C), 35.3, 36.8, 101.1, 109.4, 115.1, 135.2, 156.1, 157.2, 160.7 ppm; Anal. Calcd for C17H24N2 (256.39): C, 79.64; H, 9.44; N, 10.93; Found: C, 79.56; H, 9.49; N, 10.95.
5,6-Dichloro-2-(2-cyclohexylethyl)-1H-benzo[d]imidazole (7). Starting
TE D
4.1.1.7
from 4,5-dichloro-1,2-phenylenediamine (1.77 g), the title compound 7 was obtained by method A as colourless crystals (2.41 g, 81%): m.p. 254-256 ºC; IR (KBr): 2925,
EP
2851 (ν C-H), 1561 (δ N-H), 1455 (ν C=C), 1199 (ν C-C), 1071, 960 (δ C-H), 882 (γ C-H) 523 (γ N-H) cm-1; 1H NMR (200 MHz, DMSO-d6): 0.85-0.96 (m, 2H, CH2),
AC C
1.09-1.20 (m, 4H, 2CH2), 1.58-1.73 (m, 7H, 6H 3CH2 and 1H CH), 2.80 (t, 2H, CH2, J = 7.9 Hz), 7.71 (s, 2H, ArH), 8.20 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C15H18Cl2N2 (297.22): C, 60.61; H, 6.10; N, 9.43; Found: C, 60.69; H, 6.17; N, 9.37. All data consistent with the literature description [24]. 4.1.1.8
2-(3-Cyclohexylpropyl)-5-fluoro-1H-benzo[d]imidazole (8). Starting
from 4-cyclohexylbutanoic acid (2.55 g) and 4-fluoro-1,2-phenylenediamine (1.26 g), the title compound 8 was obtained by method B as beige solid (1.54 g, 59%): m.p. 11
ACCEPTED MANUSCRIPT
131-133 ºC (cyclohexane); IR (KBr): 2926, 2852 (ν C-H), 1487, 1448, 1421 (ν C=C), 1138 (δ C-H), 845, 804 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): 0.75-0.82 (m, 2H, CH2), 1.03-1.26 (m, 6H, 3CH2), 1.59-1.63 (m, 5H, 4H 2CH2 and 1H CH), 1.82-1.88
RI PT
(m, 2H, CH2), 2.91 (t, 2H, CH2, J = 7.8 Hz), 6.95-6.99 (m, 1H, ArH), 7.22 (dd, 1H,
ArH, J1 = 9.0 Hz, J2 = 2.4 Hz), 7.44-7.47 (m, 1H, ArH), 10.64 (br s, 1H, NH + D2O
exchangeable) ppm; Anal. Calcd for C16H21N2 (260.35): C, 73.81; H, 8.13; N, 10.76;
4.1.1.9
2-(3-Cyclohexylpropyl)-5,6-dimethyl-1H-benzo[d]imidazole from
4-cyclohexylbutanoic
acid
(2.55
g)
and
(9).
4,5-dimethyl-1,2-
M AN U
Starting
SC
Found: C, 73.74; H, 8.09; N, 10.82.
phenylenediamine (1.36 g), the title compound 9 was obtained by method B as greyish white solid (1.84 g, 68%): m.p. 145-146 °C (methanol/water 1:1); IR (KBr): 2920, 2849 (ν C-H), 1542 (δ N-H), 1445, 1417 (ν C=C), 1308, 1025, 999 (δ C-H), 854 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): 0.78-0.81 (m, 2H, CH2), 1.08-1.17
TE D
(m, 4H, 2CH2), 1.19-1.24 (m, 2H, CH2), 1.60-1.62 (m, 5H, 4H 2CH2 and 1H CH), 1.80-1.86 (m, 2H, CH2), 2.34 (s, 6H, 2CH3), 2.89 (t, 2H, CH2, J = 7.8 Hz), 7.33 (s, 2H, ArH), 8.16 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C18H26N2
4.1.1.10
5,6-Dichloro-2-(3-cyclohexylpropyl)-1H-benzo[d]imidazole from
4-cyclohexylbutanoic
AC C
Starting
EP
(270.41): C, 79.95; H, 9.69; N, 10.36; Found: C, 80.08; H, 9.62; N, 10.30.
acid
(2.55
g)
and
(10).
4,5-dichloro-1,2-
phenylenediamine (1.77 g), the title compound 10 was obtained by method B as beige solid (2.02 g, 65%): m.p. 117-118 °C (methanol/water 1:1); IR (KBr): 2921, 2849 (ν C-H), 1540 (δ N-H), 1446, 1401 (ν C=C), 1285, 1096 (δ C-H), 859 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): 0.69-0.85 (m, 2H, CH2), 1.02-1.28 (m, 6H, 3CH2), 1.191.60-1.62 (m, 5H, 4H 2CH2 and 1H CH), 1.76-1.92 (m, 2H, CH2), 2.87-2.98 (m, 2H, CH2), 7.63 (s, 2H, ArH), 10.60 (br s, 1H, NH + D2O exchangeable) ppm; 12
13
C NMR
ACCEPTED MANUSCRIPT
(200 MHz, DMSO-d6): δ 25.0, 26.1 (2C), 26.4, 29.0, 33.0 (2C), 36.7, 37.0, 121.5 (2C), 123.7 (2C), 158.5 ppm; Anal. Calcd for C16H20N2 (311.25): C, 61.74; H, 6.48; N, 9.00; Found: C, 61.81; H, 6.42; N, 9.18. 5-Methyl-2-(3-phenylpropyl)-1H-benzo[d]imidazole (11). Starting from
RI PT
4.1.1.11
4-phenylbutanoic acid (2.46 g) and 3,4-diaminotoluene (1.22 g), the title compound 11 was obtained by method B as light brown solid (1.65 g, 66%): m.p. 115-117 °C
SC
(cyclohexane); IR (KBr): 3023, 2938, 2869, 2778 (ν C-H), 1551 (δ N-H), 1494, 1449,
1420 (ν C=C), 1278, 1027 (δ C-H), 806, 739 (γ C-H), 600 (γ N-H) cm-1; 1H NMR
M AN U
(200 MHz, CDCl3): 2.12-2.19 (m, 2H, CH2), 2.46 (s, 3H, CH3), 2.68 (t, 2H, CH2, J = 7.5 Hz), 2.88 (t, 2H, CH2, J = 7.7 Hz), 7.02-7.46 (m, 8H, ArH), 8.40 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C17H18N2 (250.34): C, 81.56; H, 7.25; N, 11.19; Found: C, 81.62; H, 7.28; N, 11.10. 4.1.1.12
5-Fluoro-2-(3-phenylpropyl)-1H-benzo[d]imidazole (12). Starting from
TE D
4-phenylbutanoic acid (2.55 g) and 4-fluoro-1,2-phenylenediamine (1.26 g), the title compound 12 was obtained by method B as shiny beige solid (1.61 g, 63%): m.p. 111-112 °C (methanol/water 1:1); IR (KBr): 3023, 2945, 2856 (ν C-H), 1543 (δ N-H),
EP
1486, 1449, 1414 (ν C=C), 1133 (δ C-H), 847, 803, 752, 699 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): 2.08-2.23 (m, 2H, CH2), 2.65 (t, 2H, CH2, J = 7.4 Hz), 2.91 (t,
AC C
2H, CH2, J = 7.7 Hz), 6.93-7.26 (m, 7H, ArH), 7.41-7.48 (m, 1H, ArH), 10.83 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C16H15N2 (311.25): C, 75.57; H, 5.95; N, 11.02; Found: C, 75.48; H, 5.90; N, 11.09. 4.1.1.13
5,6-Dimethyl-2-(3-phenylpropyl)-1H-benzo[d]imidazole (13). Starting
from 4-phenylbutanoic acid (2.46 g) and 4,5-dimethyl-1,2-phenylenediamine (1.36 g), the title compound 13 was obtained by method B as yellow solid (1.77 g, 67%): m.p. 119-121 °C (cyclohexane); IR (KBr): 3030, 2927, 2856 (ν C-H), 1540 (δ N-H), 1464, 13
ACCEPTED MANUSCRIPT
1435, 1414 (ν C=C), 1305, 1032, 1018, 999 (δ C-H), 860, 744, 697 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): 2.14-2.18 (m, 2H, CH2), 2.34 (s, 6H, 2CH3), 2.63 (t, 2H, CH2, J = 7.5 Hz), 2.90 (t, 2H, CH2, J = 7.7 Hz), 7.02-7.07 (m, 2H, ArH), 7.15-7.19
RI PT
(m, 3H, ArH), 7.34 (s, 2H, ArH), 10.20 (br s, 1H, NH + D2O exchangeable) ppm; 13C NMR (200 MHz, DMSO-d6): δ 20.2 (2C), 28.3, 29.6, 34.9, 126.0, 128.5 (2C), 128.6
(2C), 141.9, 154.0 ppm; Anal. Calcd for C18H20N2 (264.36): C, 81.78; H, 7.63; N,
4.1.1.14
SC
10.60; Found: C, 81.81; H, 7.58; N, 10.56.
5,6-Dichloro-2-(3-phenylpropyl)-1H-benzo[d]imidazole (14). Starting
M AN U
from 4-phenylbutanoic acid (2.55 g) and 4,5-dichloro-1,2-phenylenediamine (1.77 g), the title compound 14 was obtained by method B as brown solid (2.01 g, 66%): m.p. 115-117 °C (dioxane/water 2:1); IR (KBr): 2925, 2854 (ν C-H), 1492, 1442, 1403 (ν C=C), 1198, 1106 (δ C-H), 872, 827 (γ C-H) cm-1; 1H NMR (200 MHz, DMSO-d6): 2.03-2.08 (m, 2H, CH2), 2.66 (t, 2H, CH2, J = 7.3 Hz), 2.78-2.86 (m, 2H, CH2), 6.53-
TE D
6.89 (m, 2H, ArH), 7.20-7.30 (m, 3H, ArH), 8.12 (s, 2H, ArH), 12.70 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C16H14Cl2N2 (305.20): C, 62.97; H, 4.62; N, 9.18; Found: C, 62.78; H, 4.58; N, 9.23.
EP
4.1.2 General method for the synthesis of 1-methyl- and 1-phenylsulfonyl-2substituted benzimidazoles 15, 16.
AC C
Benzimidazole 12 (5 mmol) was dissolved in anhydrous dioxane (10 mL) and
treated with TEA (2 mL, 15 mmol). Then methane- or benzenesulfonyl chloride (10 mmol) was added drop-wise and the reaction mixture was stirred at 50°C for 5 h and then at an ambient temperature for another 12 h. The solvent was evaporated under reduced pressure and 20 mL of water with ice was added. After a few hours the precipitated compounds 15 and 16 were filtered off, washed with water, and recrystallized. 14
ACCEPTED MANUSCRIPT
4.1.2.1
5-Fluoro-1-(methylsulfonyl)-2-(3-phenylpropyl)-1H-
benzo[d]imidazole (15). Starting from methanesulfonyl chloride (0.78 ml), the title compound 15 was obtained as light brown solid (1.38 g, 83%): m.p. 108-110 ºC
RI PT
(MeOH/water 1:1); IR (KBr): 3027, 2931, 2860 (ν C-H), 1542, 1476, 1449 (ν C=C), 1365, 1163 (ν SO2), 1051, 965, (δ C-H), 804, 770, 696 (γ C-H) cm-1; 1H NMR (200
MHz, CDCl3): δ 2.19-2.34 (m, 2H, CH2), 2.79 (t, 2H, CH2, J = 7.6 Hz), 3.04-3.16 (m,
SC
5H, 3H CH3 and 2H CH2), 7.05-7.44 (m, 7H, ArH), 7.56-7.82 (m, 1H, ArH) ppm; 13C NMR (200 MHz, DMSO-d6): δ 26.1, 33.1, 37.1, 42.2, 100.5, 101.1, 105.6, 106.1,
M AN U
112.1 (2C), 112.6 (2C), 114.2, 114.4, 120.6, 120.8, 157.2 ppm; Anal. Calcd for C17H17FN2O2S (332.39): C, 61.43; H, 5.16; N, 8.43; Found: C, 61.38; H, 5.19; N, 8.47. 4.1.2.2
5-Fluoro-2-(3-phenylpropyl)-1-(phenylsulfonyl)-1H-
benzo[d]imidazole (16). Starting from benzenesulfonyl chloride (1.28 mL), the title
TE D
compound 16 was obtained as beige solid (1.89 g, 96%): m.p. 82-85 ºC (dioxane/water 1:1); IR (KBr): 2926, 2859 (ν C-H), 1541, 1477, 1448 (ν C=C), 1376, 1172 (ν SO2), 1088, 1044 (δ C-H), 736, 685 (γ C-H) cm-1; 1H NMR (200 MHz,
EP
CDCl3): δ 2.15-2.30 (m, 2H, CH2), 2.79 (t, 2H, CH2, J = 7.4 Hz), 3.07-3.16 (m, 2H, CH2), 7.03-7.15 (m, 1H, ArH), 7.20-7.40 (m, 5H, ArH), 7.43-7.51 (m, 2H, ArH),
AC C
7.57-7.66 (m, 2H, ArH), 7.76-7.81 (m, 2H, ArH), 7.96-8.03 (m, 1H, ArH) ppm; Anal. Calcd for C22H19FN2O2S (394.46): C, 66.99; H, 4.85; N, 7.10; Found: C, 66.87; H, 4.83; N, 7.19.
4.1.3 Procedure for the preparation of 4-phenylpicolinothioamide 4-Phenylpicolinonitrile (3.6 g, 20 mmol) was dissolved in 15 mL of methanol, DBU (0.5 mL, 3 mmol) was added and reaction mixture was refluxed for 15 min. After cooling down, ammonium polysulfide (5 mL, ~44 mmol) was added and the 15
ACCEPTED MANUSCRIPT
mixture was allowed to stand for 2 h. Then water (50 mL) was added and the precipitate of crude product was collected by filtration. The product was purified by recrystallization from ethanol yielding 3.1 g (72%) of yellow needles; m.p. 129-130
RI PT
ºC; IR (KBr): 3317, 3246 (ν N-H), 3150 (ν C-H), 1616, 1592 (δ N-H), 1464, 1420 (ν C=C), 1253 (ν C=S), 918, 759, 698 (γ C-H), 524 (γ N-H) cm-1; 1H NMR (200 MHz, CDCl3): δ 4.75 (s, 2H NH2 + D2O exchangeable), 7.43-7.74 (m, 6H, 5H ArH and 1H
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pyridine), 8.55 (d, 1H, pyridine J = 4.8 Hz), 8.97 (s, 1H, pyridine) ppm; Anal. Calcd for C12H10N2S (214.29): C, 67.26; H, 4.70; N, 13.07; Found: C, 67.37; H, 4.73; N,
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13.19.
4.1.4 Procedure for the synthesis of arenoimidazoles (17-25). 4-Phenylpicolinothioamide (0.214 g, 1 mmol) and the appropriate diamine (1.3 mmol) were refluxed in ethylene glycol (3 mL) until hydrogen sulfide evolution ceased (3-5 h). Then ice (20 g) was added and the precipitated product was collected
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by filtration and purified by recrystallization with the addition of activated carbon. In this manner the following arenoimidazoles were obtained. 4.1.4.1
2-(4-Phenylpyridin-2-yl)-1H-benzo[d]imidazole (17). Starting from o-
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phenylenediamine (0.141 g), the title compound 17 was obtained as beige solid (0.271 g, 95%): m.p. 215-217 ºC (benzene); IR (KBr): 3050, 2918 (ν C-H), 1602, 1537,
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1469, 1429 (ν C=C), 1247, 1128 (δ C-H), 758, 738, 695 (γ C-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 7.21-7.30 (m, 2H, ArH), 7.55-7.94 (m, 8H, 7H ArH and 1H pyridine), 8.59 (s, 1H, pyridine), 8.79 (d, 2H, pyridine, J = 5.3 Hz), 13.18 (br s, 1H, NH + D2O exchangeable) ppm. All data consistent with the literature description [26]. 4.1.4.2
5-Methyl-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole (18). Starting
from 3,4-diaminotoluene (0.159 g), the title compound 18 was obtained as beige solid (0.271 g, 95%): m.p. 178-180 ºC (ethanol/water 1:1); IR (KBr): 3048, 2922 (ν C-H), 16
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1599, 1545, 1469, 1430, 1353 (ν C=C), 1144 (δ C-H), 845, 758, 696 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): δ 2.52 (s, 3H, CH3), 7.17 (d, 1H, ArH, J = 8.2 Hz), 7.497.53 (m, 5H, ArH), 7.54-7.62 (m, 2H, 1H ArH and 1H pyridine), 7.81 (d, 1H, ArH, J
NH + D2O exchangeable) ppm;
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= 7.1 Hz), 8.66 (d, 1H, pyridine, J = 5.2 Hz), 8.81 (s, 1H, pyridine), 13.03 (br s, 1H, C NMR (200 MHz, DMSO-d6): δ 21.7, 111.9,
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112.0, 118.6, 119.2, 122.1, 123.9, 125.0, 127.2 (2C), 129.6 (2C), 129.9, 132.7, 137.1,
5.30; N, 14.73; Found: C, 79.83; H, 5.28; N, 14.74.
5-Fluoro-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole (19). Starting
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4.1.4.3
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142.3, 148.6, 150.3, 150.5 ppm; Anal. Calcd for C19H15N3 (285.34): C, 79.98; H,
from 4-fluoro-1,2-phenylenediamine (0.164 g), the title compound 19 was obtained as beige solid (0.260 g, 90%): m.p. 216-218-161 ºC (benzene/diethyl ether 2:1); IR (KBr): 3049, 2922 (ν C-H), 1599, 1545, 1469, 1430, 1353 (ν C=C), 1144 (δ C-H), 845, 758, 696 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): δ 7.08-7.12 (m, 1H, ArH),
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7.36-7.39 (m, 2H, 1H ArH and 1H NH + D2O exchangeable), 7.52-7.56 (m, 3H, 2H ArH and 1H pyridine), 7.64-7.66 (m, 2H, ArH), 7.81 (d, 2H, ArH, J = 6.9 Hz), 8.68 (d, 1H, pyridine, J = 5.2 Hz), 8.77 (s, 1H, pyridine) ppm; Anal. Calcd for C18H12FN3
4.1.4.4
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(289.31): C, 74.73; H, 4.18; N, 14.52; Found: C, 74.71; H, 4.22; N, 14.58. 5-Chloro-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole (20). Starting
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from 4-chloro-1,2-phenylenediamine (0.186 g), the title compound 20 was obtained as white solid (0.294 g, 96%): m.p. 187-189 °C (ethanol/water 1:1); IR (KBr): 3045, 2925 (ν C-H), 1604, 1471, 1429, 1374 (ν C=C), 758, 697 (γ C-H) cm-1; 1H NMR (200 MHz, DMSO-d6): δ 7.24-7.30 (m, 1H, ArH), 7.50-7.94 (m, 8H, 7H ArH and 1H pyridine), 8.58 (s, 1H, pyridine), 8.90 (d, 1H, pyridine, J = 5.1 Hz), 13.35 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C18H12ClN3 (305.76): C, 70.71; H, 3.96; N, 13.74; Found: C, 70.65; H, 3.94; N, 13.82. 17
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4.1.4.5
5-Bromo-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole (21). Starting
from 4-bromo-1,2-phenylenediamine (0.243 g), the title compound 21 was obtained as beige solid (0.130 g, 37%): m.p. 163-165 ºC (ethanol); IR (KBr): 3055, 2925 (ν C-
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H), 1606, 1548, 1429, 1373 (ν C=C), 915, 761, 696 (γ C-H) cm-1; 1H NMR (500 MHz, CDCl3): δ 7.42 (d, 1H, ArH, J = 8.8 Hz), 7.49-7.58 (m, 4H, ArH), 7.64-7.65 (m, 1H, ArH), 7.74 (d, 1H, ArH, J = 6.8 Hz), 7.79 (d, 1H, ArH, J = 7.3 Hz), 7.85 (s,
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1H, pyridine), 8.66 (d, 1H, pyridine, J = 4.9 Hz), 8.75 (m, 1H, pyridine), 9.00 (br s,
1H, NH + D2O exchangeable) ppm; 13C NMR (200 MHz, DMSO-d6): δ 118.9, 122.7,
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125.1, 126.7, 127.1, 127.2 (2C), 127.4, 129.6 (3C), 129.9, 130.5, 136.9, 148.7, 149.0, 150.5, 152.0 ppm; Anal. Calcd for C18H12BrN3 (350.21): C, 61.73; H, 3.45; N, 12.00; Found: C, 61.81; H, 3.43; N, 12.05. 4.1.4.6
5,6-Dimethyl-2-(4-phenylpyridin-2-yl)-1H-benzo[d]imidazole
(22).
Starting from 4,5-dimethyl-1,2-phenylenediamine (0.177 g), the title compound 22
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was obtained as beige solid (0.228 g, 76%); m.p. 200-202 ºC. IR (KBr): 3062, 2926 2857 (ν C-H), 1601, 1437 (ν C=C), 1034 (δ C-H), 852, 765, 689 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): δ 2.36 (s, 6H, 2CH3), 7.42-7.53 (m, 6H, ArH), 7.72-7.80
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(m, 2H, 1H ArH and 1H pyridine), 8.56-8.62 (m, 1H, pyridine), 8.66-8.68 (m, 1H, pyridine), 12.93 (br s, 1H, NH) ppm;
C NMR (200 MHz, DMSO-d6): δ 20.3 (2C),
13
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118.5 (2C), 122.0, 127.2 (2C), 127.4, 129.6 (3C), 129.8 (2C), 137.1, 148.5, 149.7, 150.1, 150.2 (2C), 150.6, 152.0 ppm; Anal. Calcd for C20H17N3 (299.37): C, 80.24; H, 5.72; N, 14.04; Found: C, 80.31; H, 5.67; N, 14.02. 4.1.4.4
2-(4-Phenylpyridin-2-yl)-3H-imidazo[4,5-b]pyridine (23). Starting from
2,3-diaminopyridine (0.142 g), the title compound 23 was obtained as shiny beige crystals (0.240 g, 88%): m.p. 282-283 ºC (dioxane); IR (KBr): 3059, 2960, 2865 (ν CH), 1603, 1544, 1467, 1433, 1408, 1355 (ν C=C), 1271 (δ C-H), 767 (γ C-H) cm-1; 1H 18
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NMR (200 MHz, DMSO-d6): δ 7.49-7.58 (m, 4H, 3H ArH and 1H pyridine), 7.887.93 (m, 3H, 2H ArH and 1H pyridine), 8.36 (d, 1H, pyridine, J = 5.6 Hz), 8.64 (s, 1H, pyridine), 8.80 (d, 1H, pyridine, J = 5.1 Hz), 9.06 (s, 1H, pyridine), 13.61 (br s,
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1H, NH + D2O exchangeable) ppm. All data consistent with the literature description [26]. 4.1.4.8
6-Bromo-2-(4-phenylpyridin-2-yl)-3H-imidazo[4,5-b]pyridine
(24).
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Starting from 2,3-diamino-5-bromopyridine (0.244 g), the title compound 24 was obtained as shiny gray crystals (0.320 g, 91%): m.p. >260 ºC (decomp.)
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(ethanol/water 1:1); IR (KBr): 3317 (ν N-H), 3170, 2922 (ν C-H), 1592, 1473, 1418 (ν C=C), 1318, 1239 (δ C-H), 918, 862, 761, 696 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): δ 7.42-7.57 (m, 4H, 3H ArH and 1H pyridine), 7.59-7.74 (m, 4H, 2H ArH and 2H pyridine), 8.55 (d, 1H, pyridine, J = 4.5 Hz), 8.96-8.97 (m, 1H pyridine), 9.60 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C17H11BrN4 (351.20): C,
4.1.4.9
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58.14; H, 3.16; N, 15.95; Found: C, 58.18; H, 3.19; N, 15.81. 2-(4-Phenylpyridin-2-yl)-3H-imidazo[4,5-c]pyridine (25). Starting from
3,4-diaminopyridine (0.142 g), the title compound 25 was obtained as yellow solid
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(0.237 g, 87%): m.p. 248-250 °C (ethanol); IR (KBr): 3242 (ν N-H), 3054, 2957, 2890, 2851 (ν C-H), 1605, 1547, 1472, 1427, 1295 (ν C=C), 1025, 960 (δ C-H), 814,
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756, 698, 614 (γ C-H) cm-1; 1H NMR (200 MHz, CDCl3): δ 7.24-7.28 (m, 1H, ArH), 7.51-7.54 (m, 3H, ArH), 7.66 (d, 1H pyridine, J = 4.3 Hz), 7.76-7.80 (m, 2H, 1H ArH and 1H pyridine), 8.49 (d, 1H, pyridine, J = 5.4 Hz), 8.70-9.73 (m, 2H, pyridine), 9.15 (s, 1H, pyridine Hz), 10.95 (br s, 1H, NH + D2O exchangeable) ppm; Anal. Calcd for C17H12N4 (272.30): C, 74.98; H, 4.44; N, 20.58; Found: C, 75.02; H, 4.48; N, 20.50. 4.2
Biological assays
4.2.1 Antimycobacterial activity assay 19
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The synthesized compounds were examined in vitro for their tuberculostatic activity against the Mycobacterium tuberculosis H37Rv strain and two “wild” strains isolated from tuberculosis patients: one (Spec. 210) resistant to p-aminosalicylic acid
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(PAS), isonicotinic acid hydrazide (INH), ethambutol (ETB) and rifampicine (RMP) and the another (Spec. 192) fully sensitive to the administrated tuberculostatics. Investigations were performed by a classical test-tube method of successive dilution
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in Youmans’ modification of the Proskauer and Beck liquid medium containing 10%
of bovine serum [27, 28]. Bacterial suspensions were prepared from 14 days old
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cultures of slowly growing strains and from 48 h old cultures of saprophytic strains [29, 30]. Solutions of compounds in ethylene glycol were tested. Stock solutions contained 10 mg of compounds in 1 millilitre. Dilutions (in geometric progression) were prepared in Youmans’ medium. The medium containing no investigated substances and containing isoniazid (INH), pyrazinamide (PZA) or rifampicin (RMP)
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as reference drugs were used for comparison. Incubation was performed at a temperature of 37 °C. The MIC values were determined as minimum concentration inhibiting the growth of tested tuberculosis strains in relation to the probe with no
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tested compound. The influence of the compound on the growth of bacteria at a certain concentration, 0.75, 1.5, 3.1, 6.2, 12.5, 25, 50 and 100 µg/mL were evaluated.
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For compounds that showed the highest activity studies have been extended to a Mycobacterium bovis strain. The tests were performed according to the procedure described above.
4.2.3 Cytotoxicity assay Stock solutions were freshly prepared by dissolving compounds in DMSO. A549 cells are adenocarcinomic human alveolar basal epithelial cells. This line was initiated in 1972 by D. J. Giard, et al. [31] through explant culture of lung 20
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carcinomatous tissue from a 58-year-old Caucasian male. LLC-PK1 are normal, epithelial, adherent cells isolated from the kidney of a 3-4 weeks old male pig. All cells were from ATCC and showed no mycoplasma contamination as revealed by
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Roche ELISA-based test [32]. Multiwell (24–well) plates were seeded at 1.25x104 LLC-PK1 cells/well in Medium 199 supplemented with 3% FBS and antibiotics (penicillin/streptomycin) or 4x103
A549 cells/well in RPMI1640 medium
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supplemented with 10% FBS and antibiotics (penicillin/streptomycin). Cells were
allowed to attach overnight. Drugs were added to wells in 10 µL aliquots of 200-times
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concentrated drug solutions dissolved in DMSO, in duplicates. To control wells, 10 µL of DMSO was added. Cells were incubated with studied compounds for 120 h at 37oC and 95%/5% CO2 atmosphere [33]. To all wells 200 µL of MTT solution in PBS (4 mg/mL) was added and incubated further for 4 h in 37oC. Absorbance was measured after solubilization of formazan crystals in 1 mL DMSO and using a
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multiwell plate reader (Victor3, Perkin-Wallac) at λ = 540 nm. Cytotoxicity was determined compared to non-treated cells (% control).
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Acknowledgments
This project was funded by the National Science Centre (Cracow, Poland) on the basis
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of decision number DEC-2011/01/B/NZ4/01187.
References
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[25] V.-M. Mukkala, P. Liitti, I. Hemmila, H. Takalo, C. Matachescu, J. Kankare, Novel thiazole-containing complexing agents and luminescence of their
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255. [29] R.M. Atlas, J.W. Snyder, Handbook of Media for Clinical Microbiology, second ed., CRC Press Book, Boca Raton, 1995.
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[30] H. Foks, M. Buraczewska, W. Manowska, J. Sawlewicz, Investigation on pyrazine derivatives, Dissert. Pharm. Pharmacol. 1971, 23, 49-58.
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[32] M. Welti, K. Jaton, M. Altweg, R. Sahli, A. Wenger, J. Bille, Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions, Diagn. Microbiol. Infect. Dis. 45 (2003) 85–95. [33] M.M. Wintrobe, J.P. Greer, Wintrobe’s clinical hematology, twelfth ed., Wolters
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Kluwer Health/Lippincott Williams & Wilkins, Philadelphia, 2009.
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a
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Table 1 In vitro tuberculostatic activity of compounds 1-25.a, b, c MIC [µg/mL] Compds. M. tuberculosis M. bovis H37Rv Spec. 192 Spec. 210 1.5 1.5 1.5 12.5 1 3.1 3.1 3.1 3.1 2 3 0.75 0.75 0.75 0.75 4 0.75 0.75 0.75 0.75 3.1 3.1 3.1 3.1 5 6 0.75 0.75 0.75 0.75 7 0.75 0.75 0.75 1.5 6.2 6.2 6.2 6.2 8 25 6.2 6.2 9 12.5 6.2 6.2 25 10 25 6.2 100 25 11 12.5 50 50 12 6.2 6.2 6.2 6.2 13 6.2 6.2 6.2 3.1 14 50 50 50 15 100 100 50 16 12.5 6.2 6.2 17 3.1 6.2 3.1 25 18 6.2 12.5 6.2 19 12.5 12.5 6.2 20 12.5 12.5 6.5 21 25 12.5 12.5 22 12.5 12.5 12.5 23 25 25 25 24 12.5 12.5 3.1 25 12.5 12.5 25 INH 25 25 >400 PZA 1.2 1.2 2.5 RMP
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Minimum inhibitory concentrations for bacterial strains were determined by twofold serial dilution method for microdilution plates and for mycobacterial strains by two-fold classical test-tube method of successive dilution. b INH isoniazid; PZA pyrazinamide; RMP rifampicin. c M. tuberculosis H37Rv, Spec. 192, Spec. 210, M. bovis
Table 2 Cytotoxicity of compounds 3, 4, 6, and 7 Compds. IC50a [µM] b A549 10.90 (±0.57) 3 13.36 (±0.90) 4 11.15 (±0.30) 6 11.05 (±1.05) 7
LLC-PK1c 17.11 (±0.30) 32.57 (±2.15) 43.94 (±0.51) 9.62 (±1.10)
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a Concentrations inhibiting the cell growth by 50% were determined by the MTT method with expotentially growing cells and continuous drug exposure (120 h); b A549 – human non-small lung cancer cell line; c LLC-PK1 – pig kidney epithelial cell line.
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Figure Captions Figure 1. Structure of benzimidazole derivatives and analogues of tuberculostatic activity Scheme 1. Synthesis of benzimidazole derivatives 1–16. Reagents and conditions: (i) method
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A (compounds 1-7): 3-cyclohexylpropanoic acid (1.5 equiv.), diamine (1 equiv.), PPA, 180– 200 °C; NaHCO3/H2O; method B (compounds 8-14): 4-cyclohexylbutanoic or 4phenylbutanoic acid (1.5 equiv.), diamine (1 equiv.), 160-180 °C; NaOH/H2O; (ii)
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benzimidazole 12 (1 eqiuv.), TEA (3 equiv.), methane or benzenesulfonyl chloride (2 equiv.), anhydrous dioxane, 50 °C; H2O.
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Scheme 2. Synthesis of benzimidazole derivatives and analogues 17-25. Reagents and conditions: (i) 4-phenylpicolinonitrile (1 equiv.), DBU (0.15 equiv.), methanol, reflux; (ii) ammonium polysulfide (2.2 equiv.), methanol, room temperature; H2O; (iii) 4phenylpicolinothioamide (1 equiv.), diamine (1.3 equiv.), ethylene glycol, reflux; ice. Figure 2. Effect of compounds 3, 4, 6, and 7 on A549 (a) and LLC-PK1 eukaryotic cell lines
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