Medicinal Chemistry Research

, Volume 21, Issue 12, pp 4463–4472

Synthesis, characterization and antimicrobial screening of hybrid molecules containing benzimidazole-pyrazole and pyridine nucleus

Authors

    • Division of Medicinal Chemistry, Department of ChemistryMahatma Gandhi Campus, Bhavnagar University
  • D. D. Pandya
    • Division of Medicinal Chemistry, Department of ChemistryMahatma Gandhi Campus, Bhavnagar University
  • V. V. Joshi
    • Division of Medicinal Chemistry, Department of ChemistryMahatma Gandhi Campus, Bhavnagar University
  • K. M. Rajpara
    • Division of Medicinal Chemistry, Department of ChemistryMahatma Gandhi Campus, Bhavnagar University
  • H. V. Vaghani
    • Division of Medicinal Chemistry, Department of ChemistryMahatma Gandhi Campus, Bhavnagar University
  • H. M. Satodiya
    • Division of Medicinal Chemistry, Department of ChemistryMahatma Gandhi Campus, Bhavnagar University
Original Research

DOI: 10.1007/s00044-012-9990-4

Cite this article as:
Desai, N.C., Pandya, D.D., Joshi, V.V. et al. Med Chem Res (2012) 21: 4463. doi:10.1007/s00044-012-9990-4

Abstract

The rising prevalence of multi-drug resistant Gram-positive, Gram-negative bacteria and fungi continues to provide momentum for search and development of novel active antimicrobial agents against these pathogens. In continuation to this, the present article deals with the synthesis and antimicrobial activity of a novel series of (3-(1H-benzo[d]imidazol-2-yl)-5-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanones 4an, synthesized by the reaction of benzimidazolyl chalcones with isoniazide 3. The structures of synthesized compounds were elucidated on the basis of IR, NMR, and mass spectra. The newly synthesized compounds 4an were screened for their antimicrobial activity against different strains of bacteria and fungi using serial broth dilution method. Compounds 4d, 4f, 4h, and 4g were found to be most active with MIC of 25 μg/mL against selected bacterial strains. Compounds 4c, 4g, and 4n were found to be most active with MIC of 100 μg/mL against selected fungal strains.

Keywords

Benzimidazole-pyrazolePyridineAntibacterial screeningAntifungal screeningMIC

Introduction

A wide array of microorganisms including bacteria, viruses, protozoa, and fungi are becoming resistant to drugs used to treat infections. This resistance is a major obstacle for the treatment of infectious diseases worldwide. Faced with the extent of antimicrobial drug resistance and the dwindling number of effective antimicrobial drugs, the World Health Organization (WHO) has considered it to be one of the greatest threats to human health. Moreover, problems of multi-drug resistant microorganisms have reached an alarming level in many countries around the world. Therefore, it is necessary to have antimicrobial agents with improved potency. In the present research study, we have incorporated designs of new compounds and development of hybrid molecules through the combination of different pharmacophores in one structure, which may lead to compounds with increased antimicrobial activity.

Heterocyclic compounds are the commonly used scaffolds on which pharmacophores are arranged to provide potent and selective drugs (Krchnak and Holladay, 2002; Nfzi et al., 1997; Terrett et al., 1995). This is especially true for nitrogen containing heterocyclic compounds, which serve as the core components of a large number of substances that possess a wide range of interesting biological activities (Desai et al., 2011a). Benzimidazole scaffold is a useful structural motif for displaying chemical functionality in biologically active molecules (Desai et al., 2011d). Moreover, benzimidazoles have been used as “privileged” scaffolds to produce substances of interest in numerous therapeutic areas, such as antimicrobial (Goker et al., 2002), antioxidant (Kus et al., 2004), anthelmintic, fungicide (Mavrova et al., 2007), antihypertensive (Starcevic et al., 2007), antifungal (Jat et al., 2006), antiinflammatory (Lazer et al., 1987), anti HIV(Rao et al., 2002), anticancer (Refaat, 2010), antiparasitic (Navarette-Vazquez et al., 2001), anti-giardial (Xiao et al., 1996), and antiproliferative (Garuti et al., 2000). Moreover, other lead compounds bearing this nucleus is used in a wide range of therapeutic areas such as casein kinase (Pagano et al., 2004), factor Xa (Ueno et al., 2004), hepatitis C virus (Beaulieu et al., 2004) etc. Due to this reason benzimidazole has been an important pharmacophore in medicinal chemistry. Optimization of benzimidazole-based structures has resulted in marketed medicines such as omeprazole, thiabendazol, triclabendazole, mebendazole, chlormidazole, and enviroxime.

Esomeprazole and lansoprazole are blockbuster drugs containing two heterocyclic moieties i.e., benzimidazole and pyridine (Fig. 1). Esomeprazole and lansoprazole are gastric parietal cell proton inhibitors (PPIs), which are widely used for the treatment of acid related gastric diseases due to their ability to inhibit acid secretion (Gatta et al., 2003).
https://static-content.springer.com/image/art%3A10.1007%2Fs00044-012-9990-4/MediaObjects/44_2012_9990_Fig1_HTML.gif
Fig. 1

Commercially available drugs bearing benzimidazole and pyridine

The pyridine nucleus is prevalent in numerous natural products and is extremely important in chemistry of biological systems. In many enzymes of living organisms it is the prosthetic pyridine nucleotide (NADP) that is involved in various oxidation–reduction processes. Other evidence of potent activity of pyridine in biological systems is its presence in important vitamins such as niacin and pyridoxine (vitamin B6). The primary use of pyridine derivatives is as an intermediate in manufacture of pharmaceuticals particularly; anti-histamines and piroxican. Lornoxican and Tenoxican are considered new non-steroidal anti-inflammatory drugs of oxicam class inhibiting cyclooxygenase, the key enzyme of prostaglandin biosynthesis at the site of inflammation (Olkkola et al., 1994). Pyridine containing derivatives such as streptonigrin, streptonigrone, and lavendamycin are reported as anticancer drugs and cerivastatin is reported as HMG-CoA enzyme inhibitor (Bringmann et al., 2004). Moreover, substituted pyridines are reported as leukotriene B-4 antagonists (Taylor et al., 1991).

Moreover, pyrazole derivatives are undoubtedly one of the most important classes of heterocycles. The compounds containing pyrazole scaffold have been shown to exhibit HIV-1 reverse transcriptase and IL-1 synthesis inhibition, as well as antihyperglycemic (Kees et al., 1996), antibacterial, sedative-hypnotic, anti-inflammatory (Zelenin et al., 1999; Bekhit et al., 2005), antipyretic and analgesic activity (Amir and Kumar, 2005). Recent success of pyrazole COX-2 inhibitor has further highlighted the importance of these heterocycles in medicinal chemistry. As a result of remarkable pharmacological efficiency of benzimidazole, pyrazole, and pyridine derivatives, intensive research has been focused on biological activity of pyrimidine nucleus. In continuation to this, it is our ongoing project to synthesize bioactive heterocyclic compounds (Desai et al., 2010a, 2011b, c, e). We have synthesized (3-(1H-benzo[d]imidazol-2-yl)-5-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanones 4an derivatives and screened for their antimicrobial activity.

Looking to all the above facts, our studies have been focused toward the synthesis and bio-evaluation of novel benzimidazole, pyrazole, and pyridine nucleus by hybrid approach as possible bioactive molecules. The synthesized compounds 4an were characterized by IR, NMR, and mass spectra. These synthesized compounds were screened for their antimicrobial activity.

Results and discussion

Chemistry

Synthetic strategies adopted to obtain the target compounds were depicted in Scheme 1. Present scaffold 4 is a part of synthesis of new chemical entities in the form of antimicrobial agents. Scaffolds 2an were prepared in an excellent yield into one consequent step by Claisen–Schmidt condensation of compound 1 with different substituted aldehydes in ethanolic KOH. To investigate the structure activity relationship with respect to antimicrobial properties, compounds 2an on cyclocondensation reaction with isoniazide 3 yields final compounds (3-(1H-benzo[d]imidazol-2-yl)-5-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanones 4an in good yield.
https://static-content.springer.com/image/art%3A10.1007%2Fs00044-012-9990-4/MediaObjects/44_2012_9990_Sch1_HTML.gif
Scheme 1

Synthetic pathway of novel compounds 4an

Characterization

IR spectrum of title compound 4a (molecular formula C22H17N5O, mw 367) has given stretching vibration at 3,055 cm−1 over the range, which shows multiple weak intensity absorption peaks corresponding to Het–H and Ar–H. The high frequency region of IR spectra of this compound contains –NH stretching vibration at 3,460 cm−1. Stretching vibration at 2,872 cm−1 over the range corresponds to methylene group while absorption peak at 1,458 cm−1 is due to bending vibration corresponding to methylene group. The strong intensity absorption at 1,692 cm−1 is due to stretching vibration of C=O group. The weak intensity absorption band at 1,610 cm−1 corresponds to C=N stretching vibration, while C=C shows medium intensity absorption stretching vibration band at 1,515 and 1,565 cm−1.

In 1H NMR spectra, it has been observed from the chemical structure of compound 4a that different pairs of carbons e.g., C-1 and C-2, C-3 and C-6, C-4 and C-5, C-14 and C-17, C-15 and C-16, C-18 and C-22, C-19 and C-21 are attached to chemically equivalent protons, which appeared at δ = 7.13–8.79 ppm, respectively. Proton of secondary amine appeared as a singlet at δ = 10.17 ppm. Pyrazoline protons Ha and Hb give AB system. A part of AB system at δ = 3.34 ppm as doublet of doublet with coupling constant Jab = 16.50 Hz and Jac = 3.02 Hz and B part of AB system at δ = 3.73 ppm as doublet of doublet with coupling constant Jab = 16.49 Hz and Jbc = 11.01 Hz. Proton Hc of pyrazoline appeared as double of doublet at δ = 6.45 ppm with coupling constant Jac = 3.02 Hz and Jbc = 11.00 Hz. Proton attached to C-20 appeared as a multiplet at δ = 7.24 ppm.

Looking to 13C NMR, chemical shifts of final compound 4a vary from δ = 171.3–41.3 ppm. Carbon nuclei under the influence of a strong electronegative environment appeared downfield e.g., carbonyl C-11, has a chemical shift value of δ = 171.3 ppm. Carbons C-4 and C-5 directly attached to nitrogen atom appeared at the same value of δ = 132.6 ppm. Carbon C-7 showed a chemical shift at δ = 152.6 ppm which is directly attached to nitrogen atom on both sides. Carbons of benzimidazole ring C-1 and C-2, C-3, and C-6 which are chemically equivalent carbons have shown chemical shift at δ = 121.4 and 115.2 ppm, respectively. Carbons of pyridine ring C-14 and C-17, C-15 and C-16, which are equivalent carbons showed a chemical shift at δ = 123.2 and 149.8 ppm, respectively. The chemically equivalent carbons of phenyl ring C-18 and C-22, C-19 and C-21 appeared at δ = 127.2 and 128.4 ppm, respectively. Carbon of pyridine ring C-13 appeared at a chemical shift value of δ = 139.5 ppm due to the influence of carbonyl group. Carbons of phenyl ring C-12 and C-20 showed a chemical shift at δ = 141.3 and 126.6 ppm, respectively. Carbons of benzimidazole ring C-8 and C-10 appeared at a chemical shift value of δ = 157.5 and 66.5 ppm due to attachment to nitrogen atom. Carbon of benzimidazole ring C-9 showed a chemical shift at δ = 41.3 ppm. The structure and carbon numbering of compound 4a is described in Fig. 2.
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Fig. 2

Carbon numbering of the final compound-4a

Antimicrobial activity

Compounds 4an were evaluated against Gram-positive, Gram-negative bacteria, and fungi strains. The individual minimum inhibitory concentration (MIC, μg/mL) obtained for compounds 4an are presented in (Table 1). It is observed that compounds 4c (2-OH), 4d (3-OH), 4f (4-OH-3-OCH3), 4g (4-OH), 4h (4-CH3), and 4n (4-N(CH3)2) are the most active compounds. For antibacterial activity, compounds 4b, 4d, 4e, and 4i possessed good activity against E. coli. When we replace hydrogen by 4-NO2 group in 4a, it will lead to compound 4m and it possessed very good activity against E. coli. It is our observation that by introducing 4-OH group in 4g and 4-CH3 group in 4h, the activity is enhanced and it possessed excellent activity against E. coli. Compounds 4c (2-OH), 4e (3-NO2), 4l (4-F), and 4n (4-N(CH3)2) possessed good activity, while compounds 4h (4-CH3) and 4i (4-OCH3) possessed very good activity against P. aeruginosa. When we replaced hydrogen by hydroxy group in compound 4a, it leads to compound 4d and the activity enhanced and it possessed excellent activity against P. aeruginosa. Compounds 4a (–H), 4b (2-Cl), 4g (4-OH), 4j (4-Cl), 4k (4-Br), and 4n (4-N(CH3)2) possessed good activity against S. aureus. Compounds 4c (2-OH) and 4i (4-OCH3) possessed very good activity and compounds 4d (3-OH), 4f (4-OH-3-OCH3), and 4h (4-CH3) possessed excellent activity against S. aureus. Compounds 4a (–H), 4c (2-OH), 4f (4-OH-3-OCH3) and 4l (4-F) possessed good activity against S. pyogenes. At the same time if we replace hydrogen by 3-hydroxy group in compound 4a, it led to 4d and it possessed very good activity against S. pyogenes. For antifungal activity, compounds 4b (2-Cl), 4d (3-OH), 4e (3-NO2), 4f (4-OH3-OCH3), 4i (4-OCH3), 4l (4-F), and 4m (4-NO2) possessed good activity against C. albicans. Compound 4a possessed very good activity against C. albicans. Furthermore, replacement with hydroxy group at 2 and 4-position in 4a i.e., 4c and 4g are formed respectively, the activity enhanced and it possessed excellent activity against C. albicans. Compounds 4d (3-OH) and 4f (4-OH-3-OCH3) possessed good activity against A. niger. Replacement of hydrogen by -4-N(CH3)2 group in 4a, showed excellent activity against A. niger. Compounds 4c (2-OH), 4e (3-NO2), 4 m (4-NO2), and 4n (4-N(CH3)2) possessed good activity against A. clavatus. The minimum inhibitory concentration (MIC) values were determined by comparison with ampicillin and griseofulvin as reference drugs.
Table 1

Antimicrobial activity of finally synthesized compounds 4an

Compd.

–R

Minimum inhibitory concentration for bacteria μg/mL ± SD

Minimum inhibitory concentration for fungi μg/mL ± SD

Gram-negative

Gram-positive

 

E. coli

P. aeruginosa

S. aureus

S. pyogenes

C. albicans

A. niger

A. clavatus

MTCC-443

MTCC-1688

MTCC-96

MTCC-442

MTCC-227

MTCC-282

MTCC-1323

4a

–H

250 ± 1

500 ± 2.06

250 ± 3***

100 ± 3.05***

250 ± 4.16**

200 ± 3.60*

500 ± 3.21**

4b

–2-Cl

100 ± 2.08***

500 ± 3.51**

250 ± 4.04

500 ± 3.51

500 ± 3.05

1000 ± 2.64

1000 ± 3.24

4c

–2-OH

500 ± 2.34

100 ± 1***

125 ± 3.60**

100 ± 4.16*

100 ± 2.05**

1000 ± 3.05

100 ± 3.21***

4d

–3-OH

100 ± 3.05

25 ± 4***

100 ± 2.64

50 ± 2.64***

500 ± 2.08

100 ± 3.51**

500 ± 3.46

4e

–3-NO2

100 ± 3.51*

100 ± 2.08*

500 ± 3.05

500 ± 4.58

500 ± 4.04

200 ± 3.60

100 ± 3.51

4f

–4-OH-3-OCH3

250 ± 2.64*

500 ± 1.52

100 ± 3.60**

100 ± 2.51

500 ± 4.04

100 ± 3.05

200 ± 3.21*

4g

–4-OH

25 ± 1***

250 ± 2.64

250 ± 4.16

250 ± 3.05*

100 ± 3.51*

1000 ± 2.08*

1000 ± 3.05

4h

–4-CH3

25 ± 1.51**

50 ± 1.50**

100 ± 1.16*

250 ± 4.93

1000 ± 3

1000 ± 3.05

1000 ± 3.51

4i

–4-OCH3

100 ± 2.64

50 ± 2.64

125 ± 3.60**

200 ± 4.72

500 ± 3.51

200 ± 3.51**

500 ± 3*

4j

–4-Cl

500 ± 1.52

500 ± 1.52

250 ± 2.64

250 ± 3.05

1000 ± 3.51*

1000 ± 2.08

1000 ± 3.05

4k

–4-Br

250 ± 2.64

250 ± 2.64**

250 ± 3.51**

250 ± 4.93**

1000 ± 3

1000 ± 3.05

1000 ± 3.51

4l

–4-F

500 ± 2.64

100 ± 3.50

500 ± 3.60

100 ± 1***

500 ± 3.51

200 ± 3.51*

500 ± 3*

4m

–4-NO2

50 ± 1.24**

500 ± 2.08

500 ± 3.05

500 ± 4.58

500 ± 4.58

200 ± 3.60

100 ± 3.51**

4n

–4-N(CH3)2

250 ± 4.72

100 ± 2.51**

250 ± 3.21

250 ± 3

1000 ± 3.78

25 ± 1.24**

100 ± 3.60

Ampicillin

100 ± 4

100 ± 4.04

250 ± 4.04

100 ± 3.51

Griseofulvin

500 ± 2.64

100 ± 3

100 ± 3.46

All values are presented as mean of 6 experiments (n = 6). All significant differences are considered from control value 0.00

SD Standard deviation

*** P < 0.001 extremely significant

** P < 0.01 moderately significant

P < 0.05 significant

Structure activity relationship (SAR)

The substitution pattern of the benzimidazole-pyridine based pyrazole derivatives is carefully selected to confer different electronic environment of the molecules. Electron-donating groups on aromatic ring, such as methyl, methoxy, hydroxy and dimethylamine and electron withdrawing groups from aromatic ring, such as nitro and halogen are chosen as substituents on the chemical structure of the target compounds. Compounds 4c, 4d, 4f, 4g, 4h, and 4n containing electron-donating groups showed lower MIC value then reference drug against different microbial strains (Table 1). It may be concluded that methyl, hydroxy, methoxy, and dimethylamine substituents bearing derivatives are the most suitable compounds for achieving the best antimicrobial spectrum. It has been reported that electron-donating groups increase the electron density which makes the compounds effective against microorganisms and enhances the antimicrobial activity. Thus, for a compound, an optimum electron density is inevitable so as to gain a significant antimicrobial activity. As a result it can be thought that electron donating ability of methyl, hydroxy, methoxy, and dimethylamine groups contribute to compounds 4c, 4d, 4f, 4g, 4h, and 4n to reach an optimum electron density which is important for antimicrobial activity.

Biological screening

Antibacterial assay

The newly synthesized compounds (4an) were screened for their antibacterial activity against Gram-positive bacteria (Staphylococcus aureus (MTCC-96), Streptococcus pyogenes (MTCC-442)) and Gram-negative (Escherichia coli (MTCC-443), Pseudomonas aeruginosa (MTCC-1688)). All MTCC cultures were collected from Institute of Microbial Technology, Chandigarh. The activity of compounds was determined as per National Committee for Clinical Laboratory Standards (NCCLS) protocol using Mueller Hinton Broth (Becton Dickinson, USA) (Al-Bayati and Al-Mola, 2008; Desai et al., 2010b; Finegold and Garrod, 1995). Compounds were screened for their antibacterial activity as primary screening in six sets against E. coli, S. aureus, P. aeruginosa, and S. pyogenes at different concentrations of 1,000, 500, 250 μg/mL. The compounds found to be active in primary screening were similarly diluted to obtain 200, 125, 100, 62.5, 50, and 25 μg/mL concentrations for secondary screening to test in a second set of dilution against all microorganisms. Inoculum size for test strain is adjusted to 108 CFU/mL (Colony Forming Unit per milliliter) by comparing the turbidity (turbidimetric method). Mueller Hinton Broth is used as nutrient medium to grow and dilute the compound suspension for test bacteria. Two percentage DMSO is used as a diluent/vehicle to obtain the desired concentration of synthesized compounds and standard drugs to test upon standard microbial strains. Synthesized compounds were diluted to 1,000 μg/mL concentration, as a stock solution. The control tube containing no antibiotic was immediately subcultured [before inoculation] by spreading a loopful evenly over a quarter of plate of medium suitable for the growth of test organisms. The tubes are then put for incubation at 37 °C for 24 h for bacteria. Ten microgram/millilitre suspensions were further inoculated on an appropriate media and growth is noted after 24–48 h. The highest dilution (lowest concentration) preventing appearance of turbidity was considered as minimum inhibitory concentration (MIC, μg/mL) i.e., the amount of growth from the control tube before incubation (which represents the original inoculum) was compared. A set of tubes containing only seeded broth and solvent controls were maintained under identical conditions so as to make sure that the solvent had no influence on strain growth. The result of this is greatly affected by the size of the inoculum. The test mixture should contain 108 CFU/mL organisms. Standard drug used in the present study is “ampicillin” for evaluating antibacterial activity which shows 100, 100, 250, and 100 μg/mL MIC against E. coli, P. aeruginosa, S. aureus and S. pyogenes, respectively.

Antifungal assay

The same compounds (4an) were tested for antifungal activity as primary screening in six sets against Candida albicans, Aspergillus niger, and Aspergillus clavatus at various concentrations of 1,000, 500, 250 μg/mL. The compounds found to be active in primary screening were similarly diluted to obtain 200, 125, 100, 62.5, 50, and 25 μg/mL concentrations for secondary screening to test in a second set of dilution against all microorganisms. Griseofulvin was used as a standard drug for antifungal activity, which shows 500, 100, and 100 μg/mL MIC against C. albicans, A. niger and A. clavatus, respectively. For fungal growth, in the present protocol, we have used Sabourauds dextrose broth at 22°C in an aerobic condition for 72 h.

Statistical analysis

Standard deviation value is expressed in terms of ±SD. On the basis of calculated value by using one-way ANOVA method followed by independent two sample t test, it has been observed that differences below 0.001 level are considered as statistically significant.

Materials and methods

Melting points are determined on an electro thermal melting point apparatus and are reported uncorrected. Completion of reaction and purity of all compounds is checked on aluminum coated TLC plates 60 F245 (E. Merck) using n-hexane:ethyl acetate (7.5:2.5 V/V) as mobile phase and visualized under ultraviolet (UV) light, or iodine vapor. Elemental analysis (%C, H, N) is carried out by a Perkin-Elmer 2400 CHN analyzer. IR spectra of all compounds has been recorded on Perkin-Elmer FT-IR spectrophotometer in KBr. 1H-NMR and 13C-NMR spectra are recorded on Bruker (400 MHz) and (100 MHz) spectrometer, respectively, using DMSO-d6 as a solvent and TMS as an internal standard. Chemical shifts are reported in parts per million (ppm). Mass spectra was scanned on Shimadzu LCMS 2010 spectrometer. Anhydrous reactions are carried out in oven-dried glassware in nitrogen atmosphere. In the conventional method, compounds are synthesized by using Random synthesizer.

Experimental

General preparation of 1-(1H-benzo[d]imidazol-2-yl)-3-(aryl)prop-2-en-1-ones (2an)

Compounds 1-(1H-benzo[d]imidazol-2-yl)-3-(aryl)prop-2-en-1-ones 2an are prepared according to the literature method (Kalirajan et al.,2011; Ouattara et al.,2011; Shaharyar et al.,2010).

All other derivatives are prepared by the same method and are monitored by thin layer chromatography.

General preparation of (3-(1H-benzo[d]imidazol-2-yl)-5-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanones (4an)

A mixture of differently substituted benzimidazolyl chalcones 2an (0.01 mol) and isoniazide 3 (0.02 mol) is taken in 20 mL glacial acetic acid and refluxed at 130 °C over a period of 6 h. The mixture is concentrated under vacuum and diluted with ice cold water. The separated solid is filtered, dried, and crystallized from chloroform.

Physical constants and characterization of synthesized compounds (4an)

(3-(1H-Benzo[d]imidazol-2-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)-methanone (4a)

Yield: 70%; brown crystal; m.p.: 184–186 °C; IR (KBr) cm−1: 3460 (N–H stretching, secondary amine), 3055 (C–H stretching, aromatic ring), 2872 (C–H stretching, –CH2–group), 1692 (C=O stretching), 1515, 1565, 1610 (C=C, C=N, stretching, aromatic ring), 1458 (C–H bending, –CH2–group); 1H NMR (400 MHz, DMSO-d6,δ, ppm): 10.17 (s, 1H, –NH–), 3.34 (dd, Jab = 16.50 Hz, Jac = 3.02 Hz, 1H, Ha), 3.73 (dd, Jab = 16.49 Hz, Jbc = 11.01 Hz, 1H, Hb), 6.45 (dd, Jac = 3.02 Hz, Jbc = 11.00 Hz, 1H, Hc), 7.13–8.79 (m, 13H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.3, 66.5, 115.2, 121.4, 123.2, 126.6, 127.2, 128.4, 132.6, 139.5, 141.3, 149.8, 152.6, 157.5, 171.3; LCMS (m/z): 367 (M+); Anal. Calcd. for C22H17N5O: C-71.92, H-4.66, N-19.06; Found: C-71.88, H-4.62, N-19.01%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4b)

Yield: 68%; light yellow crystal; m.p.: 209–211 °C; IR (KBr) cm−1: 3466 (N–H stretching, secondary amine), 3072 (C–H stretching, aromatic ring), 2880 (C–H stretching, –CH2– group), 1698 (C=O stretching), 1511, 1578, 1603 (C=C, C=N, stretching, aromatic ring), 1463 (C–H bending, –CH2-group), 748 (C–Cl stretching); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.23 (s, 1H, –NH–), 3.36 (dd, Jab = 16.51 Hz, Jac = 3.03 Hz, 1H, Ha), 3.75 (dd, Jab = 16.51 Hz, Jbc = 11.05 Hz, 1H, Hb), 6.52 (dd, Jac = 3.02 Hz, Jbc = 11.05 Hz, 1H, Hc), 7.15–8.83 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.6, 66.3, 115.4, 121.7, 123.4, 126.4, 128.1, 128.3, 128.7, 132.5, 133.8, 139.5, 139.8, 149.6, 152.5, 157.3, 171.5; LCMS (m/z): 401 (M+); Anal. Calcd. for C22H16ClN5O: C-65.76, H-4.01, N-17.43; Found: C-65.71, H-3.97, N-17.38%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridine-4-yl)methanone (4c)

Yield: 71%; light brown crystal; m.p.: 199–201 °C; IR (KBr) cm−1: 3459 (N–H stretching, secondary amine), 3415 (O–H stretching), 3068 (C–H stretching, aromatic ring), 2891 (C–H stretching, –CH2–group), 1702 (C=O stretching), 1520, 1568, 1611 (C=C, C=N, stretching, aromatic ring), 1456 (C–H bending, –CH2– group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.32 (s, 1H, –NH–), 9.45 (s, 1H, Ar–OH), 3.41 (dd, Jab = 16.51 Hz, Jac = 3.07 Hz, 1H, Ha), 3.78 (dd, Jab = 16.50 Hz, Jbc = 11.04 Hz, 1H, Hb), 6.57 (dd, Jac = 3.07 Hz, Jbc = 11.04 Hz, 1H, Hc), 7.17–8.82 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.2, 66.6, 115.3, 115.6, 121.1, 121.7, 123.5, 126.8, 129.8, 128.6, 132.7, 139.4, 149.9, 152.4, 154.6, 157.8, 171.2; LCMS: (m/z): 383 (M+); Anal. Calcd. for C22H17N5O2: C-68.92, H-4.47, N-18.27; Found: C-68.90, H-4.41, N-18.23%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4d)

Yield, 65%; light reddish crystal; m.p.: 173–175 °C; IR (KBr) cm−1: 3464 (N–H stretching, secondary amine), 3401 (O–H stretching), 3073 (C–H stretching, aromatic ring), 2903 (C–H stretching, –CH2–group), 1691 (C=O stretching), 1512, 1572, 1623 (C=C, C=N, stretching, aromatic ring), 1448 (C–H bending, –CH2–group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.27 (s, 1H, –NH–), 9.53 (s, 1H, Ar–OH), 3.39 (dd, Jab = 16.50 Hz, Jac = 3.02 Hz, 1H, Ha), 3.75 (dd, Jab = 16.50 Hz, Jbc = 11.05 Hz, 1H, Hb), 6.55 (dd, Jac = 3.02 Hz, Jbc = 11.04 Hz, 1H, Hc), 7.15–8.84 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.5, 66.2, 114.1, 114.3, 115.6, 119.8, 121.2, 123.5, 129.6, 132.4, 139.7, 142.7, 149.5, 152.3, 155.4, 157.7, 171.1; LCMS (m/z): 383 (M+); Anal. Calcd. for C22H17N5O2: C-68.92, H-4.47, N-18.27; Found: C-68.87, H-4.44, N-18.22%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4e)

Yield; 73%; yellow crystal; m.p.: 170–172 °C; IR (KBr) cm−1: 3471 (N–H stretching, secondary amine), 3083 (C–H stretching, aromatic ring), 2897 (C–H stretching, –CH2–group), 1695 (C=O stretching), 1514, 1575, 1623 (C=C, C=N, stretching, aromatic ring), 1442 (C–H bending, –CH2–group), 1469 (N–O asymmetric stretching, –NO2 group), 1351 (N–O symmetric stretching, –NO2 group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.38 (s, 1H, –NH–), 3.45 (dd, Jab = 16.53 Hz, Jac = 3.01 Hz, 1H, Ha), 3.81 (dd, Jab = 16.52 Hz, Jbc = 11.02 Hz, 1H, Hb), 6.60 (dd, Jac = 3.01 Hz, Jbc = 11.02 Hz, 1H, Hc), 7.20–8.82 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.6, 66.8, 115.5, 121.3, 121.6, 122.3, 123.7, 129.1, 132.4, 133.2, 139.2, 144.2, 148.1, 149.7, 152.3, 157.6, 171.5; LCMS (m/z): 412 (M+); Anal. Calcd. for C22H16N6O3: C-64.07, H-3.91, N-20.38; Found: C-64.02, H-3.88, N-20.33%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4-hydoxy-3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4f)

Yield; 67%; dark brown crystal; m.p.: 176–178 °C; IR (KBr) cm−1: 3453 (N–H stretching, secondary amine), 3401 (O–H stretching), 3069 (C–H stretching, aromatic ring), 2903, 2938 (C–H stretching, –CH2–group, –OCH3 group), 1679 (C=O stretching), 1521, 1583, 1610 (C=C, C=N, stretching, aromatic ring), 1417, 1448 (C–H bending, –OCH3 group, –CH2–group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.29 (s, 1H, –NH–), 9.47 (s, 1H, Ar–OH), 3.52 (s, 3H, –OCH3), 3.42 (dd, Jab = 16.55 Hz, Jac = 3.02 Hz, 1H, Ha), 3.77 (dd, Jab = 16.55 Hz, Jbc = 11.00 Hz, 1H, Hb), 6.49 (dd, Jac = 3.01 Hz, Jbc = 11.00 Hz, 1H, Hc), 7.17–8.83 (m, 11H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.4, 53.6, 66.3, 113.4, 115.2, 115.7, 121.5, 121.7, 123.4, 132.4, 134.2, 139.8, 145.3, 146.7, 149.4, 152.7, 157.4, 171.3; LCMS (m/z): 413 (M+); Anal. Calcd. for C23H19N5O3: C-66.82, H-4.63, N-16.94; Found: C-66.87, H-4.70, N-16.88%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-(pyridin-4-yl)methanone (4g)

Yield: 69%; light grey crystal; m.p. 210–212 °C; IR (KBr) cm−1: 3464 (N–H stretching, secondary amine), 3420 (O–H stretching), 3057 (C–H stretching, aromatic ring), 2893 (C–H stretching, –CH2–group), 1689 (C=O stretching), 1524, 1585, 1610 (C=C, C=N, stretching, aromatic ring), 1462 (C–H bending, –CH2–group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.23 (s, 1H, –NH–), 9.53 (s, 1H, Ar–OH), 3.38 (dd, Jab = 16.54 Hz, Jac = 3.00 Hz, 1H, Ha), 3.75 (dd, Jab = 16.54 Hz, Jbc = 11.03 Hz, 1H, Hb), 6.48 (dd, Jac = 3.00 Hz, Jbc = 11.02 Hz, 1H, Hc), 7.14–8.81 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.2, 66.4, 115.6, 115.9, 121.4, 123.3, 127.6, 132.7, 134.1, 139.5, 149.4, 152.5, 154.8, 157.9, 171.1; LCMS (m/z): 383 (M+); Anal. Calcd. for C22H17N5O2: C-68.92, H-4.47, N-18.27; Found: C-68.89, H-4.42, N-18.20%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4-methylphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-(pyridin-4-yl)methanone (4h)

Yield: 75%; light pink crystal; m.p.: 217–219 °C; IR (KBr) cm−1: 3451 (N–H stretching, secondary amine), 3059 (C–H stretching, aromatic ring), 2878, 2921 (C–H stretching, –CH2–group, –CH3 group), 1684 (C=O stretching), 1516, 1575, 1624 (C=C, C=N, stretching, aromatic ring), 1371, 1452 (C–H bending, –CH3 group, –CH2–group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.17 (s, 1H, –NH–), 2.41 (s, 3H, Ar–CH3), 3.31 (dd, Jab = 16.50 Hz, Jac = 3.05 Hz, 1H, Ha), 3.70 (dd, Jab = 16.50 Hz, Jbc = 11.01 Hz, 1H, Hb), 6.39 (dd, Jac = 3.04 Hz, Jbc = 11.01 Hz, 1H, Hc), 7.09–8.77 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 19.6, 41.1, 66.6, 115.6, 121.7, 123.1, 126.2, 128.1, 132.3, 134.3, 138.4, 139.4, 149.6, 152.2, 157.4, 171.0; LCMS (m/z): 381 (M+); Anal. Calcd. for C23H19N5O: C-72.42, H-5.02, N-18.36; Found: C-72.37, H-5.00, N-18.31%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-(pyridin-4-yl)methanone (4i)

Yield: 72%; light brown cryatal; m.p.: 169–171 °C; IR (KBr) cm−1: 3462 (N–H stretching, secondary amine), 3072 (C–H stretching, aromatic ring), 2883, 2939 (C–H stretching, –CH2–group, –OCH3 group), 1692 (C=O stretching), 1523, 1583, 1613 (C=C, C=N, stretching, aromatic ring), 1412, 1463 (C–H bending, –OCH3 group, –CH2–group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.25 (s, 1H, –NH–), 3.57 (s, 3H, Ar–OCH3), 3.38 (dd, Jab = 16.49 Hz, Jac = 3.08 Hz, 1H, Ha), 3.78 (dd, Jab = 16.49 Hz, Jbc = 11.03 Hz, 1H, Hb), 6.44 (dd, Jac = 3.07 Hz, Jbc = 11.03 Hz, 1H, Hc), 7.16–8.85 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.4, 53.2, 66.3, 113.8, 115.6, 121.2, 123.7, 126.8, 132.2, 133.4, 139.4, 149.5, 152.8, 157.4, 157.6, 171.4; LCMS (m/z): 397 (M+); Anal. Calcd. for C23H19N5O2: C-69.51, H-4.82, N-17.62; Found: C-69.46, H-4.77, N-17.55%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4j)

Yield: 71%; yellow crystal; m.p.: 189–191 °C; IR (KBr) cm−1: 3460 (N–H stretching, secondary amine), 3067 (C–H stretching, aromatic ring), 2873 (C–H stretching, –CH2–group), 1691 (C=O stretching), 1509, 1582, 1612 (C=C, C=N, stretching, aromatic ring), 1459 (C–H bending, –CH2–group), 757 (C–Cl stretching); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.21 (s, 1H, –NH–), 3.34 (dd, Jab = 16.53 Hz, Jac = 3.03 Hz, 1H, Ha), 3.77 (dd, Jab = 16.53 Hz, Jbc = 11.01 Hz, 1H, Hb), 6.51 (dd, Jac = 3.03 Hz, Jbc = 11.02 Hz, 1H, Hc), 7.13–8.81 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.5, 66.3, 115.5, 121.3, 123.6, 127.5, 128.6, 132.4, 132.6, 138.7, 139.7, 149.5, 152.8, 157.3, 171.4; LCMS (m/z): 401 (M+); Anal. Calcd. for C22H16ClN5O: C-65.76, H-4.01, N-17.43; Found: C-65.69, H-4.07, N-17.47%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4k)

Yield: 69%; light grey crystal; m.p.: 179–181 °C; IR (KBr) cm−1: 3467 (N–H stretching, secondary amine), 3071 (C–H stretching, aromatic ring), 2877 (C–H stretching, –CH2–group), 1701 (C=O stretching), 1517, 1582, 1614 (C=C, C=N, stretching, aromatic ring), 1456 (C–H bending, –CH2–group), 662 (C–Br stretching); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.26 (s, 1H, –NH–), 3.40 (dd, Jab = 16.50 Hz, Jac = 3.02 Hz, 1H, Ha), 3.81 (dd, Jab = 16.51 Hz, Jbc = 11.07 Hz, 1H, Hb), 6.55 (dd, Jac = 3.02 Hz, Jbc = 11.07 Hz, 1H, Hc), 7.20–8.86 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.4, 66.6, 115.7, 120.6, 121.3, 123.4, 127.4, 131.5, 132.7, 139.8, 140.8, 149.5, 152.2, 157.7, 171.6; LCMS (m/z): 445 (M+); Anal. Calcd. for C22H16BrN5O: C-59.21, H-3.61, N-15.69; Found: C-59.26, H-3.66, N-15.63%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4l)

Yield: 74%; light brown crystal; m.p.: 223–225 °C; IR (KBr) cm−1: 3472 (N–H stretching, secondary amine), 3079 (C–H stretching, aromatic ring), 2882 (C–H stretching, –CH2–group), 1707 (C=O stretching), 1523, 1587, 1619 (C=C, C=N, stretching, aromatic ring), 1460 (C–H bending, –CH2–group), 1154 (C–F stretching); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.29 (s, 1H, –NH–), 3.44 (dd, Jab = 16.53 Hz, Jac = 3.07 Hz, 1H, Ha), 3.83 (dd, Jab = 16.53 Hz, Jbc = 11.04 Hz, 1H, Hb), 6.57 (dd, Jac = 3.07 Hz, Jbc = 11.04 Hz, 1H, Hc), 7.26–8.89 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.7, 66.3, 115.1, 115.5, 121.7, 123.6, 128.3, 132.7, 137.6, 139.5, 149.6, 152.3, 157.7, 161.1, 171.6; LCMS (m/z): 385 (M+); Anal. Calcd. for C22H16FN5O: C-68.56, H-4.18, N-18.17; Found: C-68.61, H-4.12, N-18.25%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4m)

Yield; 70%; dark yellow crystal; m.p.: 201–203 °C; IR (KBr) cm−1: 3469 (N–H stretching, secondary amine), 3082 (C–H stretching, aromatic ring), 2893 (C–H stretching, –CH2–group), 1698 (C=O stretching), 1520, 1581, 1626 (C=C, C=N, stretching, aromatic ring), 1466 (C–H bending, –CH2–group), 1472 (N–O asymmetric stretching, –NO2 group), 1352 (N–O symmetric stretching, –NO2 group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.33 (s, 1H, –NH–), 3.56 (dd, Jab = 16.54 Hz, Jac = 3.00 Hz, 1H, Ha), 3.79 (dd, Jab = 16.54 Hz, Jbc = 11.01 Hz, 1H, Hb), 6.61 (dd, Jac = 3.01 Hz, Jbc = 11.01 Hz, 1H, Hc), 7.30–8.87 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.6, 66.8, 115.5, 121.7, 123.2, 123.4, 123.6, 132.7, 139.4, 145.8, 147.4, 149.6, 152.8, 157.4, 171.8; LCMS (m/z): 412 (M+); Anal. Calcd. for C22H16N6O3: C-64.07, H- 3.91, N-20.38; Found: C-64.12, H-3.97, N-20.45%.

(3-(1H-Benzo[d]imidazol-2-yl)-5-(4(dimethylamino)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4n)

Yield; 69%; light yellow crystal; m.p. 179–181 °C; IR (KBr) cm−1: 3447 (N–H stretching, secondary amine), 3068 (C–H stretching, aromatic ring), 2893, 2927 (C–H stretching, –CH2–group, –CH3 group), 1681 (C=O stretching), 1514, 1582, 1617 (C=C, C=N, stretching, aromatic ring), 1379, 1457 (C–H bending, –CH3 group, –CH2-group); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.14 (s, 1H, –NH–), 2.17 (s, 6H, –N–(CH3)2), 3.35 (dd, Jab = 16.56 Hz, Jac = 3.08 Hz, 1H, Ha), 3.74 (dd, Jab = 16.55 Hz, Jbc = 11.07 Hz, 1H, Hb), 6.43 (dd, Jac = 3.08 Hz, Jbc = 11.07 Hz, 1H, Hc), 7.12-8.86 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 41.2, 42.6, 66.4, 112.4, 115.7, 121.7, 123.1, 128.7, 131.5, 132.5, 139.7, 148.7, 149.3, 152.4, 157.3, 171.5; LCMS (m/z): 410 (M+); Anal. Calcd. for C24H22N6O: C-70.23, H-5.40, N-20.47; Found: C-70.19, H-5.34, N-20.45%.

Conclusion

As a part of our research on 2-substituted benzimidazole ring systems and attempts to identify novel lead compounds by compiling pyrazole and pyridine heterocycles, we have found that compounds 4d, 4f, 4h, and 4g exhibit potential activity against bacterial strains and 4c, 4g, and 4n exhibit potential activity against fungal strains as compared to the commercially available drugs. Structure activity correlation of the obtained results showed that incorporation of electron donating hydroxy, methyl and methoxy groups as a substituent increases antibacterial as well as antifungal activities. Efforts are currently being taken up to optimize the lead structure and results of which will be the basis of our future research endeavor. Further investigations are currently in progress to verify the susceptibility of other bacteria and fungi to these compounds and to outline their pharmacokinetic profile.

Acknowledgments

The authors are thankful to the Head, Department of Chemistry, Mahatma Gandhi Campus, Bhavnagar University, Bhavnagar for providing laboratory facilities.

Copyright information

© Springer Science+Business Media, LLC 2012