Introduction

A wide-ranging array of microbes including bacteria, viruses and fungi are becoming resistant to the standard marketed drugs used to treat infections. This resistance is a major hurdle for the treatment of highly infectious diseases globally. The World Health Organization (WHO) has considered this antimicrobial drug resistance and the dwindling number of active antimicrobial drugs to be one of the greatest threats to human health. Moreover, problems of multi-drug resistance of bacteria and fungi such as Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Staphylococcus aureus (VRSA), Vancomycin resistant enterococcus (VRE) and Fluconazole resistant Candida have reached an alarming level becoming a serious medical problem worldwide (Mishra et al. 2013; Cetinkaya et al. 2000; Gulshan and Moye-Rowley 2007). Therefore, it is necessary to develop antimicrobial agents with improved potency and the combat is still on to discover novel and potent antimicrobial agents for medicinal chemists (Moellering 2011). The structural modifications in existing drugs have shown astounding results in the field of drug discovery. Newly investigational drugs or trial drugs which utilize this strategy are safer with a broader spectrum of activity. Figure 1 represents potent molecules either in phase II or phase III of clinical trials from the existing NCE’S (Butler et al. 2013).

Fig. 1
figure 1

Chemical remodeling of existing antibacterial drugs from potent molecules either in phase II or phase III of clinical trials

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Searching for structure with significant bioactivity, we focused onto the development of molecules through combination of different active pharmacophores like quinolines, pyrazoline and pyridine into one core structure. That may lead to compounds with improved antimicrobial activity. Among the important heterocyclic moieties of biological and pharmacological attention, the quinoline ring is endowed with various activities such as antimicrobial (Desai et al. 2014), antiplasmodial (Vandekerckhove et al. 2015), antituberculosis (Keri and Patil 2014), anti-inflammatory (El-Feky et al. 2015), antimalarial (Vandekerckhove and D’hooghe 2015), anticancer (Spanò et al. 2015), antioxidant (Vivekanand et al. 2015), and anti-HIV (Ahmed et al. 2010). Furthermore, quinolines are used as an inhibitor in tyrokinase PDGF-RTK inhibiting agents (Maguire et al. 1994), SH2-containing inositol 5′-Phosphatase (SHIP) (Russo et al. 2015) and Mycobacterium tuberculosis DNA gyraseB inhibitors (Medapi et al. 2015).

2-Pyrazoline derivatives have been reported to exhibit various pharmacological activities such as antimicrobial (Karthikeyan et al. 2007), anti-inflammatory (Barsoum et al. 2006), antihypertensive, anti-tumor (Lin et al. 2007), anticancer (Gowramma et al. 2009) and anticonvulsant (Ozdemir et al. 2007). In addition, pyrazolines are also reported to possess cytotoxic properties against human lung tumor cell line (A549) (Greenlee et al. 2001). Nowadays some newly steroidal pyrazoline are also synthesized for finding a novel drug molecule (Shamsuzzaman and Mohd 2012). Pyridine is a versatile bioactive heterocycle having its wide presence in many synthetic drugs such as rosiglitazone (antidiabetic), pioglitazone (hypoglycemic), lansoprazole (proton-pump inhibitor), and etoricoxib (COX-2 selective inhibitor). The compounds containing pyridine scaffold exhibit anticancer (Kadayat et al. 2015), anti-inflammatory, anti-ulcer (Mohareb et al. 2015), anti-mycobacterial (Jose et al. 2015b) and antimicrobial (Jose et al. 2015a) activities.

Prompted by the above-mentioned observations, we have focused onto construct some new antimicrobial derivatives bearing quinoline, pyrazoline and pyridine scaffolds and study the structure activity relationship due to substituent variations. Earlier, our research group had synthesized quinoline and other bioactive derivatives as potential antimicrobial agents (Desai et al. 2013a, b, 2015a, b). In continuation to this, (5-(2-chloroquinolin-3-yl)-3-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanones (4a–o) have been synthesized and screened for their antimicrobial activity. In addition, cytotoxicity studies were also conducted on HeLa cell lines to assess the ability of these compounds to inhibit the cell growth. The newly synthesized compounds (4a–o) were elucidated by IR, NMR and mass spectroscopy for their structural characterization.

Results and discussion

Chemistry

Synthetic strategies adopted to achieve the target compounds are depicted in Scheme 1. Here, scaffold 4 is a part of synthesis of new chemical entities in the form of antimicrobial agents. According to Scheme 1, the key chalcone derivatives 3-(2-chloroquinolin-3-yl)-1-(aryl)prop-2-en-1-ones (3a–o) are used as precursors for the synthesis of title compounds (4a–o). Compounds (3a–o) were synthesized through the Claisen–Schmidt condensation of equimolar amounts of different acetophenone derivatives (2a–o) and compound 2-chloroquinoline-3-carbaldehyde 1 by stirring the reactants in aqueous alcoholic solution containing sodium hydroxide at room temperature. Compound 1 is Vilsmeier–Haack reactions adduct; it provides a vital and efficient intermediate for the synthesis of several new substituted heterocyclic analogs. Chalcone compounds (3a–o) on cyclocondensation with isoniazid yielded final compounds (5-(2-chloroquinolin-3-yl)-3-aryl-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanones (4a–o). Further it has been observed that compounds (4a–o) have pyrazoline core nucleus and were generated through cyclization, migration of α, β unsaturated double bonds, and diminishing of carbonyl group.

Scheme 1
scheme 1

Synthetic pathway of novel compounds 4a–o

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Designed series of molecules (4a–o) were characterized by IR, 1H NMR, 13C NMR, and mass spectrometry techniques. IR spectrums of title compounds (4a–o) gave stretching vibrations at 3035–3059 cm−1 due to aromatic C–H stretching vibrations corresponds to methylene group that appears over the range at 2872–2926 cm−1. The strong intensity absorption bands at 1645–1696 cm−1 is due to stretching vibrations of C=O group. The characteristic signals in 1H NMR of compounds (4a–o) were of three pyrazoline protons which displayed doublet of doublet. Out of three, two pyrazoline protons (C-13) H a and H b give AB system. Part one of AB system displayed a signal at δ = 3.29–3.40 ppm as doublet of doublet with coupling constants 17.42–17.55 and 3.04–3.10 Hz and part two of AB system appeared as doublet of doublet at δ = 3.74–3.82 ppm with coupling constants 17.40–17.52 and 11.01–11.16 Hz. Proton Hc of pyrazoline (C-11, methine proton) also appeared as doublet of doublet at δ = 6.02–6.09 ppm with coupling constants in range of 11.08–11.22 and 3.08–3.17 Hz. Moreover, 13C NMR confirmed the final compounds structure by the appearance of signals at δ = 171.2–172.9 ppm due to carbonyl carbon of isoniazid directly attached to pyrazoline nitrogen. Compounds (4a–o) showed signal at δ = 39.1–39.9 ppm corresponding to carbon of methylene group present in pyrazoline nucleus and methine carbon showed a chemical shift at δ = 60.0–66.6 ppm. Furthermore, the mass spectrum of (4a–o) showed a molecular ion peak corresponding to molecular formula (4a–o) along with of other fragment peaks, which supported the proposed structure of compounds. The detail discussion of characterization data are given in experimental section. The structure and carbon numbering of compound (4a–o) as an example are described in Fig. 2.

Fig. 2
figure 2

Carbon numbering of the final compounds 4a–o

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Discussion of antibacterial screening

Amongst synthesized compounds (4a–o), several compounds exhibited antimicrobial potency that diverged from good to excellent. On the basis of antibacterial screening results from Table 1, compound 4o (-2,4-(Cl)2) displayed good activity against E. coli, while compounds 4f (-4-F) and 4n (-4-NO2) possessed very good activity against E. coli at 50 µg ml−1 MIC. It is noteworthy that compound 4e (-4-Cl) showed highest inhibition at MIC = 12.5 µg ml−1 against the same Gram-negative bacterium, i.e. E. coli. In the case of P. aeruginosa, compound 4o (-2,4-(Cl)2) showed same inhibition as to the standard drug. While compounds 4e (-4-Cl) and 4n (-4-NO2) inhibited bacteria at MIC = 50 µg ml−1 (very good activity) and maximum inhibition (MIC = 12.5 µg ml−1) was shown by compound 4f (-4-F) against P. aeruginosa. Moreover, compound 4e (-4-Cl) displayed good activity and compounds 4f (-4-F) and 4n (-4-NO2) showed very good activity at 250 and 100 µg ml−1 MIC values against S. aureus. Compound 4o (-2,4-(Cl)2) exhibited inhibition at MIC = 12.5 µg ml−1 against Gram-positive bacterium, i.e. S. aureus. In case of Gram-negative bacterium (S. pyogenes), compounds 4e (-4-Cl) and 4o (-2,4-(Cl)2) exhibited good activity at MIC = 50 µg ml−1 and compound 4f (-4-F) displayed very good activity against S. pyogenes. Compound 4n (-4-NO2) had shown MIC 12.5 µg ml−1 which observed as the highest inhibition against S. pyogenes. The remaining compounds of the series possessed feeble antibacterial activity as shown in Table 1. Tabular data revealed that the presence of functional group at para position improved antibacterial activity as compared to ortho and meta substituted compounds.

Table 1 Antimicrobial screening of the compounds 4a–o
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Discussion of antifungal screening

Minimum inhibitory concentration (MIC) values of antifungal activity spotted against Candida albicans, Aspergillus niger and Aspergillus clavatus were by conventional broth micro dilution method. We have discussed and compared antifungal activities based on standard drug Griseofulvin shown in Table 1. Compounds 4m (-3-NO2), 4n (-4-NO2) and 4o (-2,4-(Cl)2) exhibited same MIC value as standard drug against C. albicans. Excellent and very good activity possessed against C. albicans by compounds 4d (-2-Cl) and 4g (-4-Br) at MIC = 50 and 100 µg ml−1, respectively. Same compounds 4d and 4g also displayed very good activity against A. niger, while highest inhibition (12.5 µg ml−1) exists in di-chloro substituted compounds, i.e. 4o (-2,4-(Cl)2). Furthermore, compounds 4g and 4o having -4-Br, -2,4-(Cl)2 functional groups respectively, possessed very good activity against A. clavatus, while excellent activity against A. clavatus was demonstrated by -2-Cl (4d) derivative. Compound 4o having electron withdrawing groups in structure proves its significance to almost all bacterial and fungal strains that makes a great imaginary impact in antimicrobial activity.

Discussion of in vitro cytotoxicity studies

In vitro cytotoxic activity of newly synthesized compounds (4a–o) was evaluated against human cervical cancer cell line (HeLa) by the MTT colorimetric assay (Mosmann 1983), which measures the reduction of tetrazolium bromide salt into a formazan dye by mitochondrial dehydrogenases in treated vs. untreated cells. The IC50 values achieved for these compounds were shown in Table 2. Cytotoxicity results displayed that the derivatives 4e (4-Cl), 4f (4-F), 4g (4-Br), 4n (4-NO2) and 4o (2,4-(Cl)2) accounted no toxicity at concentration of 100 µg/ml (IC50 > 100 µg/ml), while other derivatives exhibited moderate toxicity against HeLa cell lines.

Table 2 Levels of cytotoxicity prompted by compounds 4a–o on HeLa cells
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Experimental

Melting points were noted on Gallenkamp apparatus and were left uncorrected. The completion of reaction and the purity of all compounds was checked on aluminum-coated TLC plates 60, F245 (E. Merck) using various solvent systems as mobile phase and visualized under ultraviolet (UV) light, or iodine vapor. Perkin-Elmer 2400 CHN analyzer was used for elemental analysis (% C, H, N). IR spectra were recorded on Perkin Elmer FT-IR spectrophotometer. 1H NMR spectra were recorded on a Bruker Avance II 400 MHz while 13C NMR spectra on Varian Mercury-400, 100 MHz in CDCl3 as a solvent and tetramethylsilane (TMS) as an internal standard using 5 mm tube. Chemical shifts were reported in ppm units with use of δ scale. Mass spectra were also scanned on a Shimadzu LCMS 2010 spectrometer. The essential chemicals were purchased from E. Merck. Buchi Rota vapor instrument was used for distillation purpose.

Preparation of 2-chloroquinoline-3-carbaldehyde (1)

To a solution of acetanilide (5 mmol) in dry DMF (15 mmol) at 0–5 °C with stirring phosphoryl chloride (POCl3) (60 mmol) was added drop-wise and the mixture stirred at 100 °C for 16–20 h. The mixture was poured into crushed ice, stirred for 5 min and the resulting solid filtered, washed well with water and dried. The yellow crystals formed were filtered, washed with water and crystallized using ethyl acetate (Meth-Cohn 1993). 1H NMR (CDCl3, 400 MHz): δ = 7.59 (t, 1H, Ar–H), 7.69 (t, 1H, Ar–H), 8.01 (d, 1H, Ar–H), 8.17 (d, 1H, Ar–H), 8.55 (s, 1H, Ar–H), 9.61 (s, 1H, Ar–H); anal. calcd. for C10H6ClNO: C, 62.68; H, 3.16; N, 7.31; found: C, 62.73; H, 3.24; N, 7.36.

General procedure of 3-(2-chloroquinolin-3-yl)-1-(aryl)prop-2-en-1-ones (3a–o)

Compounds 3-(2-chloroquinolin-3-yl)-1-(aryl)prop-2-en-1-ones (3a–o) were prepared according to the literature method (Elgazwy 2008). All other derivatives were prepared by the same method and monitored by thin layer chromatography (TLC). 1H NMR (CDCl3, 400 MHz): δ = 7.31–7.74 (m, 7H, Ar–H), 7.78 (d, 1H), 7.97 (d, 3H), 8.21 (s, 1H, quinoline-H4); anal. calcd. for C18H12ClNO: C, 73.60; H, 4.12; N, 4.77; found: C, 73.69; H, 4.19; N, 4.70.

General procedure of (5-(2-chloroquinolin-3-yl)-3-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanones (4a–o)

A mixture of differently substituted quinolinyl chalcones 3a–o (0.01 mol) and isoniazid (0.02 mol) were taken in 20 ml glacial acetic acid and refluxed at 120 °C over a period of 8 h. The mixture was concentrated under vacuum and diluted with ice cold water. The separated solid was filtered, dried, and crystallized from ethanol (95 %).

(5-(2-Chloroquinolin-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4a)

Yellow crystals; yield: 73 %; mp 118–120 °C; IR (KBr) ν max 3064 (C–H, aromatic), 2871 (C–H stretching, –CH2-group), 1692 (C=O stretching), 1579 (C=N), 1513 (C=C), 735 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.29 (dd, J ab = 17.45 Hz, J ac = 3.05 Hz, 1H, Ha), 3.78 (dd, J ab = 17.41 Hz, J bc = 11.02 Hz, 1H, Hb), 6.06 (dd, J ac = 3.09 Hz, J bc = 11.08 Hz, 1H, Hc), 7.06–8.32 (m, 14H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.6 (C-12, CH2 of pyrazoline), 62.7 (C-11, CH–N of pyrazoline), 122.1–148.2 (Ar–C), 152.1 (C-8, C–Cl of quinoline), 156.6 (C-13, C=N of pyrazoline), 167.3 (C-23, C=O); LCMS: m/z 412.11 [M+]; anal. calcd. for C24H17ClN4O: C, 69.82; H, 4.15; N, 13.57; Found: C, 69.86; H, 4.14; N, 13.66.

(5-(2-Chloroquinolin-3-yl)-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4b)

Cream crystals; yield: 67 %; mp 184–185 °C; IR (KBr) ν max 3065 (C–H, aromatic), 2922, 2875 (C–H stretching, –CH3 group, –CH2-group), 1690 (C=O stretching), 1584 (C=N), 1516 (C=C), 736 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 2.30 (s, 3H, Ar–CH3), 3.38 (dd, J ab = 17.44 Hz, J ac = 3.07 Hz, 1H, Ha), 3.74 (dd, J ab = 17.43 Hz, J bc = 11.04 Hz, 1H, Hb), 6.05 (dd, J ac = 3.10 Hz, J bc = 11.09 Hz, 1H, Hc), 7.10–8.38 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 20.1 (C-15, Ar–CH3), 43.3 (C-12, CH2 of pyrazoline), 62.5 (C-11, CH–N of pyrazoline), 122.2–148.4 (Ar–C), 152.2 (C-8, C–Cl of quinoline), 156.7 (C-13, C=N of pyrazoline), 167.4 (C-23, C=O); LCMS: m/z 426.12 [M+]; anal. calcd. for C25H19ClN4O: C, 70.34; H, 4.49; N, 13.12; found: C, 70.42; H, 4.46; N, 13.20.

(5-(2-Chloroquinolin-3-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4c)

Light cream crystals; yield: 65 %; mp 172–174 °C; IR (KBr) ν max 3063 (C–H, aromatic), 2920, 2878 (C–H stretching, –CH2-group, –CH3 group), 1670 (C=O stretching), 1582 (C=N), 1518 (C=C), 734 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 2.25 (s, 3H, Ar–CH3), 3.30 (dd, J ab = 17.43 Hz, J ac = 3.06 Hz, 1H, Ha), 3.78 (dd, J ab = 17.43 Hz, J bc = 11.03 Hz, 1H, Hb), 6.04 (dd, J ac = 3.09 Hz, J bc = 11.10 Hz, 1H, Hc), 7.10–8.32 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 21.5 (C-17, Ar–CH3), 43.4 (C-12, CH2 of pyrazoline), 62.7 (C-11, CH–N of pyrazoline), 122.0–148.6 (Ar–C), 152.1 (C-8, C–Cl of quinoline), 156.5 (C-13, C=N of pyrazoline), 167.2 (C-23, C=O); LCMS: m/z 426.12 [M+]; anal. calcd. for C25H19ClN4O: C, 70.34; H, 4.49; N, 13.12; found: C, 70.40; H, 4.42; N, 13.16.

(3-(2-Chlorophenyl)-5-(2-chloroquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4d)

Light yellow crystals; yield: 69 %; mp 164–166 °C; IR (KBr) ν max 3066 (C–H, aromatic), 2900 (C–H stretching, –CH2-group), 1690 (C=O stretching), 1585 (C=N), 1520 (C=C), 745 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.40 (dd, J ab = 17.45 Hz, J ac = 3.06 Hz, 1H, Ha), 3.78 (dd, J ab = 17.43 Hz, J bc = 11.06 Hz, 1H, Hb), 6.08 (dd, J ac = 3.11 Hz, J bc = 11.12 Hz, 1H, Hc), 7.18–8.43 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.1 (C-12, CH2 of pyrazoline), 62.6 (C-11, CH–N of pyrazoline), 122.2–148.9 (Ar–C), 128.6 (C-15, Ar–C–Cl), 152.5 (C-8, C–Cl of quinoline), 156.3 (C-13, C=N of pyrazoline), 167.4 (C-23, C=O); LCMS: m/z 446.07 [M+]; anal. calcd. for C24H16Cl2N4O: C, 64.44; H, 3.61; N, 12.53; found: C, 64.49; H, 3.62; N, 12.58.

(3-(4-Chlorophenyl)-5-(2-chloroquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4e)

Yellow crystals; yield: 67 %; mp 122–124 °C; IR (KBr) ν max 3059 (C–H, aromatic), 2926 (C–H stretching, –CH2-group), 1645 (C=O stretching), 1587 (C=N), 1568 (C=C), 750 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.33 (dd, J ab = 17.46 Hz, J ac = 3.05 Hz, 1H, Ha), 3.77 (dd, J ab = 17.45 Hz, J bc = 11.07 Hz, 1H, Hb), 6.04 (dd, J ac = 3.10 Hz, J bc = 11.13 Hz, 1H, Hc), 7.22–8.42 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.3 (C-12, CH2 of pyrazoline), 62.7 (C-11, CH–N of pyrazoline), 122.1–148.8 (Ar–C), 136.2 (C-17, Ar–C–Cl), 152.2 (C-8, C–Cl of quinoline), 156.4 (C-13, C=N of pyrazoline), 167.2 (C-23, C=O); LCMS: m/z 446.07 [M+]; anal. calcd. for C24H16Cl2N4O: C, 64.44; H, 3.61; N, 12.53; Found: C, 64.53; H, 3.69; N, 12.60.

(5-(2-Chloroquinolin-3-yl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4f)

Dark yellow crystals; yield: 71 %; mp 150–152 °C; IR (KBr) ν max 3060 (C–H, aromatic), 2874 (C–H stretching, –CH2-group), 1695 (C=O stretching), 1584 (C=N), 1528 (C=C), 740 (C–Cl stretching), 1152 (C–F stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.40 (dd, J ab = 17.48 Hz, J ac = 3.08 Hz, 1H, Ha), 3.81 (dd, J ab = 17.42 Hz, J bc = 11.10 Hz, 1H, Hb), 6.02 (dd, J ac = 3.12 Hz, J bc = 11.20 Hz, 1H, Hc), 7.13–8.30 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.6 (C-12, CH2 of pyrazoline), 62.6 (C-11, CH–N of pyrazoline), 121.9–147.5 (Ar–C), 152.3 (C-8, C–Cl of quinoline), 156.8 (C-13, C=N of pyrazoline), 164.5 (C-17, Ar–C–F), 167.8 (C-23, C=O); LCMS: m/z 430.10 [M+]; anal. calcd. for C24H16ClFN4O: C, 66.90; H, 3.74; N, 13.00; found: C, 64.86; H, 3.65; N, 13.10.

(3-(4-Bromophenyl)-5-(2-chloroquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4g)

Gray crystals; yield: 65 %; mp 141–143 °C; IR (KBr) ν max 3064 (C–H, aromatic), 2879 (C–H stretching, –CH2-group), 1693 (C=O stretching), 1585 (C=N), 1530 (C=C), 734 (C–Cl stretching), 663 (C–Br stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.39 (dd, J ab = 17.50 Hz, J ac = 3.05 Hz, 1H, Ha), 3.77 (dd, J ab = 17.48 Hz, J bc = 11.08 Hz, 1H, Hb), 6.08 (dd, J ac = 3.14 Hz, J bc = 11.22 Hz, 1H, Hc), 7.16–8.44 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.8 (C-12, CH2 of pyrazoline), 62.7 (C-11, CH–N of pyrazoline), 122.1–147.5 (Ar–C), 126.2 (C-17, Ar–C–Br), 152.6 (C-8, C–Cl of quinoline), 157.0 (C-13, C=N of pyrazoline), 167.5 (C-23, C=O); LCMS: m/z 490.02 [M+]; anal. calcd. for C24H16ClBrN4O: C, 58.62; H, 3.28; N, 11.39; found: C, 58.70; H, 3.19; N, 11.46.

(5-(2-Chloroquinolin-3-yl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4h)

Light brown crystals; yield: 62 %; mp 195–197 °C; IR (KBr) ν max 3066 (C–H, aromatic), 2937, 2879 (C–H stretching, –OCH3 group, –CH2-group), 1692 (C=O stretching), 1584 (C=N), 1531 (C=C), 736 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.35 (dd, J ab = 17.54 Hz, J ac = 3.10 Hz, 1H, Ha), 3.67 (s, 3H, –OCH3), 3.80 (dd, J ab = 17.52 Hz, J bc = 11.15 Hz, 1H, Hb), 6.03 (dd, J ac = 3.17 Hz, J bc = 11.20 Hz, 1H, Hc), 7.17–8.48 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.9 (C-12, CH2 of pyrazoline), 56.2 (Ar–OCH3), 62.5 (C-11, CH–N of pyrazoline), 122.0–147.7 (Ar–C), 152.8 (C-8, C–Cl of quinoline), 156.7 (C-13, C=N of pyrazoline), 167.7 (C-23, C=O); LCMS: m/z 442.12 [M+]; anal. calcd. for C25H19ClN4O2: C, 67.80; H, 4.32; N, 12.65; found: C, 67.91; H, 4.41; N, 12.58.

(5-(2-Chloroquinolin-3-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4i)

Brownish crystals; yield: 63 %; mp 166–168 °C; IR (KBr) ν max 3064 (C–H, aromatic), 2942, 2877 (C–H stretching, –OCH3 group, –CH2-group), 1694 (C=O stretching), 1585 (C=N), 1528 (C=C), 732 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.34 (dd, J ab = 17.50 Hz, J ac = 3.08 Hz, 1H, Ha), 3.61 (s, 3H, –OCH3), 3.82 (dd, J ab = 17.48 Hz, J bc = 11.12 Hz, 1H, Hb), 6.08 (dd, J ac = 3.14 Hz, J bc = 11.18 Hz, 1H, Hc), 7.10–8.44 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.6 (C-12, CH2 of pyrazoline), 55.5 (Ar–OCH3), 62.4 (C-11, CH–N of pyrazoline), 121.8–148.1 (Ar–C), 152.6 (C-8, C–Cl of quinoline), 156.6 (C-13, C=N of pyrazoline), 167.4 (C-23, C=O); LCMS: m/z 442.12 [M+]; anal. calcd. for C25H19ClN4O2: C, 67.80; H, 4.32; N, 12.65; found: C, 67.83; H, 4.39; N, 12.62.

(5-(2-Chloroquinolin-3-yl)-3-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4j)

Yellow crystals; yield: 62 %; mp 155–157 °C; IR (KBr) ν max 3407 (–OH), 3062 (C–H, aromatic), 2875 (C–H stretching, –CH2-group), 1695 (C=O stretching), 1586 (C=N), 1527 (C=C), 745 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.36 (dd, J ab = 17.48 Hz, J ac = 3.06 Hz, 1H, Ha), 3.77 (dd, J ab = 17.44 Hz, J bc = 11.07 Hz, 1H, Hb), 6.07 (dd, J ac = 3.10 Hz, J bc = 11.11 Hz, 1H, Hc), 7.12–8.48 (m, 13H, Ar–H), 9.17 (s, 1H, OH); 13C NMR (CDCl3, 100 MHz): δ = 43.8 (C-12, CH2 of pyrazoline), 62.3 (C-11, CH–N of pyrazoline), 121.7–148.5 (Ar–C), 152.5 (C-8, C–Cl of quinoline), 156.4 (C-13, C=N of pyrazoline), 162.5 (C-15, Ar–C–OH), 167.2 (C-23, C=O); LCMS: m/z 428.10 [M+]; anal. calcd. for C24H17ClN4O: C, 67.21; H, 4.00; N, 13.06; found: C, 67.29; H, 4.08; N, 13.10.

(5-(2-Chloroquinolin-3-yl)-3-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4k)

Dark yellow crystals; yield: 60 %; mp 219–221 °C; IR (KBr) ν max 3410 (–OH), 3060 (C–H, aromatic), 2878 (C–H stretching, –CH2-group), 1693 (C=O stretching), 1588 (C=N), 1524 (C=C), 740 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.37 (dd, J ab = 17.44 Hz, J ac = 3.05 Hz, 1H, Ha), 3.75 (dd, J ab = 17.46 Hz, J bc = 11.09 Hz, 1H, Hb), 6.08 (dd, J ac = 3.11 Hz, J bc = 11.13 Hz, 1H, Hc), 7.14–8.49 (m, 13H, Ar–H), 9.07 (s, 1H, OH); 13C NMR (CDCl3, 100 MHz): δ = 43.6 (C-12, CH2 of pyrazoline), 62.2 (C-11, CH–N of pyrazoline), 121.5–148.8 (Ar–C), 152.6 (C-8, C–Cl of quinoline), 156.6 (C-13, C=N of pyrazoline), 160.2 (C-16, Ar–C–OH), 167.4 (C-23, C=O); LCMS: m/z 428.10 [M+]; anal. calcd. for C24H17ClN4O: C, 67.21; H, 4.00; N, 13.06; found: C, 67.18; H, 4.08; N, 13.03.

(5-(2-Chloroquinolin-3-yl)-3-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4l)

Light yellow crystals; yield: 70 %; mp 227–229 °C; IR (KBr) ν max 3415 (–OH), 3063 (C–H, aromatic), 2879 (C–H stretching, –CH2-group), 1696 (C=O stretching), 1585 (C=N), 1520 (C=C), 736 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.40 (dd, J ab = 17.46 Hz, J ac = 3.06 Hz, 1H, Ha), 3.77 (dd, J ab = 17.47 Hz, J bc = 11.07 Hz, 1H, Hb), 6.07 (dd, J ac = 3.13 Hz, J bc = 11.12 Hz, 1H, Hc), 7.20–8.47 (m, 13H, Ar–H), 9.14 (s, 1H, OH); 13C NMR (CDCl3, 100 MHz): δ = 43.8 (C-12, CH2 of pyrazoline), 62.3 (C-11, CH–N of pyrazoline), 121.4–148.7 (Ar–C), 152.5 (C-8, C–Cl of quinoline), 156.7 (C-13, C=N of pyrazoline), 161.1 (C-17, Ar–C–OH), 167.5 (C-23, C=O); LCMS: m/z 428.10 [M+]; Anal. Calcd. for C24H17ClN4O: C, 67.21; H, 4.00; N, 13.06; found: C, 67.29; H, 4.05; N, 13.12.

(5-(2-Chloroquinolin-3-yl)-3-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4m)

Brown crystals; yield: 61 %; mp 221–223 °C; IR (KBr) ν max 3065 (C–H, aromatic), 2877 (C–H stretching, –CH2-group), 1694 (C=O stretching), 1589 (C=N), 1518 (C=C), 1486, 1353 (N–O asymmetric, symmetric stretching, –NO2 group), 735 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.38 (dd, J ab = 17.43 Hz, J ac = 3.05 Hz, 1H, Ha), 3.74 (dd, J ab = 17.49 Hz, J bc = 11.06 Hz, 1H, Hb), 6.07 (dd, J ac = 3.15 Hz, J bc = 11.17 Hz, 1H, Hc), 7.25–8.56 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.5 (C-12, CH2 of pyrazoline), 62.7 (C-11, CH–N of pyrazoline), 121.5–149.1 (Ar–C), 140.2 (C-15, Ar–C–NO2), 152.8 (C-8, C–Cl of quinoline), 155.8 (C-13, C=N of pyrazoline), 167.2 (C-23, C=O); LCMS: m/z 457.09 [M+]; anal. calcd. for C24H16ClN5O3: C, 62.96; H, 3.52; N, 15.30; found: C, 62.93; H, 3.48; N, 15.26.

(5-(2-Chloroquinolin-3-yl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4n)

Orange yellow crystals; yield: 65 %; mp 211–213 °C; IR (KBr) ν max 3063 (C–H, aromatic), 2876 (C–H stretching, –CH2-group), 1693 (C=O stretching), 1592 (C=N), 1519 (C=C), 1481, 1348 (N–O asymmetric, symmetric stretching, –NO2 group), 731 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.40 (dd, J ab = 17.44 Hz, J ac = 3.07 Hz, 1H, Ha), 3.76 (dd, J ab = 17.42 Hz, J bc = 11.08 Hz, 1H, Hb), 6.07 (dd, J ac = 3.12 Hz, J bc = 11.09 Hz, 1H, Hc), 7.26–8.59 (m, 13H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.6 (C-12, CH2 of pyrazoline), 62.6 (C-11, CH–N of pyrazoline), 121.4–149.3 (Ar–C), 150.1 (C-17, Ar–C–NO2), 152.7 (C-8, C–Cl of quinoline), 155.6 (C-13, C=N of pyrazoline), 167.4 (C-23, C=O); LCMS: m/z 457.09 [M+]; anal. calcd. for C24H16ClN5O3: C, 62.96; H, 3.52; N, 15.30; found: C, 62.91; H, 3.48; N, 15.31.

(5-(2-Chloroquinolin-3-yl)-3-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone (4o)

White yellow crystals; yield: 68 %; mp 231–233 °C; IR (KBr) ν max 3068 (C–H, aromatic), 2872 (C–H stretching, –CH2-group), 1690 (C=O stretching), 1591 (C=N), 1523 (C=C), 734 (C–Cl stretching) cm−1; 1H NMR (CDCl3, 400 MHz): δ = 3.37 (dd, J ab = 17.49 Hz, J ac = 3.09 Hz, 1H, Ha), 3.79 (dd, J ab = 17.46 Hz, J bc = 11.09 Hz, 1H, Hb), 6.09 (dd, J ac = 3.15 Hz, J bc = 11.12 Hz, 1H, Hc), 7.16–8.48 (m, 12H, Ar–H); 13C NMR (CDCl3, 100 MHz): δ = 43.8 (C-12, CH2 of pyrazoline), 62.7 (C-11, CH–N of pyrazoline), 121.5–149.7 (Ar–C), 125.3, 127.8 (C-15, C-17, Ar–C–Cl), 152.7 (C-8, C–Cl of quinoline), 155.4 (C-13, C=N of pyrazoline), 167.6 (C-23, C=O); LCMS: m/z 480.03 [M+]; anal. calcd. for C24H15Cl3N4O: C, 59.84; H, 3.14; N, 11.63; found: C, 62.89; H, 3.17; N, 11.67.

Biological assay

Antibacterial bioassay

The newly synthesized compounds (4a–o) were screened for their antibacterial activity against Gram positive [Staphylococcus aureus (MTCC-96), Streptococcus pyogenes (MTCC-442)], and Gram-negative bacteria [Escherichia coli (MTCC-443), Pseudomonas aeruginosa (MTCC-1688)] at different concentrations of 1000, 500, 200, 100, 50, 25, 12.5 µg ml−1. Several of the newly synthesized compounds (4a–o) were found to exhibit moderate to excellent antimicrobial activity. Antibacterial activity was carried out as per National Committee for Clinical Laboratory Standards (NCCLS) protocol using serial broth dilution method and all the standard strains used for the antimicrobial activity were procured from Institute of Microbial Technology (IMTECH), Chandigarh. “The drugs which were found to be active in primary screening were similarly diluted to obtain 100, 50, 25 and 12.5 µg ml−1 concentrations for secondary screening to test in a second set of dilution against all microorganisms. Inoculum size for test strain was adjusted to 106 CFU/ml (Colony Forming Unit per milliliter) by likening the turbidity. Mueller–Hinton Broth was used as a nutrient medium to grow and dilute the synthesized compound suspension for test organisms. 2 % DMSO was used as a diluent/vehicle to acquire the desired concentration of synthesized compounds and standard drugs to test upon standard microbial strains. Diluted 1000 µg/ml concentrated solution of synthesized compounds were used as stock solution. The control tube containing not any antibiotic was instantly subcultured [before inoculation] by spreading a loopful evenly over quarter of a plate of medium suitable for the growth of test organisms. The culture tubes were then incubated for 24 h at 37 °C, and the growth was observed visually and spectrophotometrically. After that, 10 μg ml−1 suspensions were further inoculated on appropriate media and development was noted after 24 and 48 h. The lowest concentration preventing appearance of turbidity was considered as minimum inhibitory concentration (MIC, μg ml−1) i.e., the amount of growth from the control tube before incubation (which represents the original inoculum) was compared. Solvent had no influence on strain growth and the result of this was greatly affected by the size of inoculum. DMSO and sterilized distilled water were used as negative control while ‘ampicillin’ antibiotic (1 U strength) was used as positive control. Standard drug ‘ampicillin’ was used in this study for evaluating antibacterial activity which showed 100, 100, 250 and 100 μg ml−1 MIC against E. coli, P. aeruginosa, S. aureus, and S. pyogene, respectively as shown in Table 1”.

Antifungal bioassay

“The same newly synthesized compounds (4a–o) were screened for their antifungal activity against in six sets against C. albicans (MTCC-227), A. niger (MTCC-282) and A. clavatus (MTCC-1323) at various primary concentrations of 1000, 500 and 250 μg ml−1. The primary screen active compounds were similarly diluted to obtain 200, 125, 100, 62.5, 50, 25 and 12.5 μg ml−1 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 showed 500, 100 and 100 μg ml−1 MIC against C. albicans, A. niger and A. clavatus, respectively (Table 1). For growth of fungi, in the present procedure, we have used Sabourauds dextrose broth at 28 °C in aerobic condition for 48 h. DMSO and sterilized distilled water used as negative controls while ‘griseofulvin’ (1 U strength) was used as a positive control”.

MTT assay for cytotoxic activity

In vitro cytotoxicity activity of compounds (4a–o) was measured by means of the IC50 using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay process. “The IC50 determination was achieved according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. All synthesized compounds were dissolved in 0.1 % DMSO with the stock concentration of 10 g/l and diluted with medium freshly before drug administration. Cell lines were sowed into 96-well plates at density of 8000 cells/ well. After seeding it for 24 h, each compound dilution was added in duplicate, and cultivation continued at 37 °C in a moistened atmosphere containing 10 % FBS, 1 % glutamine, and 50 µM/ml gentamicin sulfate in a 5 CO2 and 95 % air. After 24 h, 20 µL MTT reagent at 5 mg/ml in PBS (filter sterilized, light protected and stored at 4 °C) per well was added, and after 4 h of incubation at 37 °C, MTT is changed to a blue formazan product by mitochondrial succinate dehydrogenase. This product was eluted from cells by addition of 150 ml of DMSO. The absorbance at 570 nm was determined by an ELISA using ELX800 micro plate spectrophotometer”.

SAR studies

Structure–activity relationship (SAR) studies revealed that the antimicrobial activity in heterocyclic class of quinoline, pyrazoline and pyridine molecules depend on the nature of the peripheral substituents and their spatial relationship within this skeleton. The pattern of substitution for the derivatives is carefully selected to confer different electronic environment of the molecules. The electronic nature of the substituent groups lead to significant discrepancy in antimicrobial activity. The presence of chloro, fluoro and nitro substituents at para position on aromatic ring system has amplified the antibacterial activity of compounds compared to those of electron donating substituents. By replacing substitution position to ortho and meta, compound tends to lost its potency (Chart 1 and 3). Incorporation of electron donating groups such as methyl, methoxy and hydroxy diminished the antibacterial property (Chart 2 and 4). The presence of lipophilic substituent on phenyl ring provides a positive effect on antifungal activity. In rationality with the above results, electron withdrawing groups like chloro and bromo on ortho and para substituted position showed optimal activity. It is concluded from Table 1 that, a compound without any substitution does not display antimicrobial activity against a panel of all microorganisms. Figure 3 showed antimicrobial activity in the form of bar chart.

Fig. 3
figure 3

Antibacterial and antifungal activity are expressed in the form of bar chart (EW electron withdrawing, ED electron donating)

Full size image

Conclusion

We have synthesized compounds (4a–o) by conventional method with enlargement in yield of reactions. The synthesized compounds were screened for their in vitro antibacterial and antifungal activity against various bacterial and fungal strains. It is concluded from biological activity table that structural and electronic diversity of these products influences their biological activities. Compounds 4d, 4e, 4f, 4g, 4n and 4o are the most characteristic derivatives identified in present study because of their notable in vitro antimicrobial potency. SAR studies revealed that when electron withdrawing groups like fluoro, chloro, bromo and nitro present in structure demonstrated effective antimicrobial activity. Likewise di-substituted chloro group (compound 4o) shows optimum inhibition over all strains of bacteria and fungi. It may be considered as a promising lead for further design and development of new lead molecules. As discussed from toxicity studies, we were encouraged to make modifications in electronic diversity on the basic structure of the final compounds (4a–o) for generation of non-toxic antimicrobial agents. It may be concluded that the presence of three different pharmacophore scaffolds enriched the biological activity.