Abstract--Pyrazolo[5,1-b]quinazolines are known to be versatile heterocyclic compounds with different biological properties because of a large number of derivatives with different structural modifications. Therefore, some new Pyrazolo quinazoline heterocycles were synthesized from chalcones and their structures were confirmed by IR, ¹H NMR, ¹³C NMR and Mass spectral data. All these synthesized compounds were tested in vitro for their antimicrobial potential against Gram positive, Gram negative strains of bacteria as well as fungal strains in dimethylsulfoxide. Among the screened compounds, KC-10 showed the most potent antimicrobial activity. None of the compound shows antifungal activity. This may be due to the fact that fungal cells are complex organisms as compared to bacterial cell. Furthermore, the presence of S-CH₃ group in the synthesized compounds may cause inactive behavior of these compounds against the studied fungal strains.

Keywords-- pyrazolo quinazolines, antibacterial activity, antifungal activity, agar-well diffusion method, dimethylsulfoxide

I. INTRODUCTION

Quinazoline derivatives have been of much interest in organic chemistry for a long time because of their wide pharmacological activities (Dangi et al., 2011) such as antibacterial (Bedi et al., 2004; Khalil et al., 2009; Yassin, 2009), anticancer (Ali et al., 2007; Joseph et al., 2010; Zhang et al.,2014), anti-inflammatory (Chandrika et al,; 2008; Kumar et al., 2012), antimicrobial (Patel and Bharat, 2010; Patil et al., 2012; Trivedi et al., 1993), antifungal (Pandey et al., 2005; Pattanaik et al., 1991; Shivananda and Shivarama, 2011) activities, etc. Furthermore, the pyrazolo quinazolines fragment is present in various drug molecules and some biologically active natural products (Kumari et al., 2012). Literature survey shows that quinazoline derivatives also play an important role in drug discovery (Eweas et al., 2013; Helali et al., 2014). Due to these biological properties of quinazoline derivatives, in the present work, some new fused pyrazolo[5,1-b] quinazoline derivatives are synthesized from (E)-2-benzylidene-3,4-dihydro naphthalen-1(2H)-one and their characterization was done by IR, NMR and mass spectral data. The screening of antimicrobial activity of these synthesized compounds was done in vitro against some Gram positive and Gram negative strains of bacteria as well as fungal strains in dimethylsulfoxide (DMSO). The Gram positive bacteria studied were Staphylococcus aureus ATCC29737 (SA), Corynebacterium rubrum ATCC 14898 (CR), Listeria monocytogenes ATCC19112 (LM), Bacillus cereus ATCC11778 (BC); Gram negative bacteria were Pseudomonas aeruginosa ATCC 27853 (PA), Escherichia coli NCIM2931 (EC), Klebsiella pneumoniae NCIM2719 (KP), Salmonella typhimurium ATCC23564 (ST); and Fungi were Candida albicans ATCC2091 (CA), Cryptococcus neoformans NCIM3542 (CN), Candida glabrata NCIM3448 (CG), Candida epicola NCIM3367 (CE).

II. EXPERIMENTAL

A. Synthesis.

Synthesis of (E)-2-benzylidene-3,4dihydro naphthale ne-1(2H)-one:

Equimolar mixtures (not equal volume) of α-tetralone and different substituted benzaldehydes in methanol were stirred for 1.5 h in presence of catalytic amount of potassium hydroxide (4%). All the synthesized compounds (KC-1 to KC-10) contain different substituted benzaldehydes such as 4-Chloro benzaldehyde, 4-methoxy benzaldehyde, 4-Floro benzaldehyde, 4-Bromo benzaldehyde, 3,4-dimethoxy benzaldehyde, 4-cyano benzaldehyde, 3-Chloro benzaldehyde, 3-methoxy benzaldehyde, 3-Bromo benzaldehyde and 4-methyl benzaldehyde. The completion of reaction was confirmed by analytical thin layer chromatography (TLC) (Performed on aluminum coated plates Gel 60F254 (E. Merck)) using 7:3 of Hexane:Ethyl acetate as mobile phase. After completion of reaction, the reaction mass was cooled and the resulting solid was filtered, washed with water and dried under vacuum to give a crude product. The obtained crude product was purified by adding suitable solvent (diethyl ether) to remove colored, nonpolar impurities by scratching/stirring. The product was then allowed to settle down and the above solution was decanted. The procedure was repeated 3-4 times to remove impurities (trituration). The purity of the resulting product was (99.5 %) 0.995 in mole fraction as determined by gas chromatography.

Synthesis of (5-amino-3-(methylthio)-1H-pyrazole-4-carbonitrile:

A mixture of malanonitrile (0.01 mmol) and dry K₂CO₃ (0.012 mmol) were stirred in dry DMF at room temperature for 30 min. To this reaction mixture, 0.02 mole of carbon disulphide was added drop wise and the resulting solution was stirred for 2.5 h at room temperature. The solution was then cooled at 0 to 5⁰C. To this cooled solution, 0.02 mole dimethyl sulphate was added and the solution was again stirred for 5-6 h at room temperature. The progress of the reaction was monitored by thin layer chromatography. After completion of the reaction, it was poured into crushed ice to give solid product. The resulting solid was filtered, washed with cold water and dried under vacuum to give crude product.

Equimolar solution of this crude product and hydrazine hydrate in isopropyl alcohol (IPA) was refluxed for 30 min. The reaction mixture was then poured into crushed ice. The resulting solid was filtered, washed with water and dried under vacuum to give product. In order to remove non polar impurities, the obtained product was purified by trituration with hexane and was used in the next step without further purification.

Synthesis of pyrazolo[5,1-b] quinazolines:

An equimolar mixture of (E)-2-benzylidene-3,4-dihydronaphthalen-1(2H)-one and (5-amino-3-(methylthio)-1H-pyrazole-4-carbonitrile) were refluxed in n-butanol for 4-5 hrs. The completion of reaction was confirmed by Thin Layer Chromatography using (6:4- Hexane: Ethyl acetate) as a mobile phase. The reaction mixture was then allowed to cool and the resulting solid was filtered, washed with diethyl ether to remove impurities. The procedure was repeated 3-4 times to free the product from impurities. The reaction scheme is given in Fig. 1.

B. Spectroscopic study.

The characterization of all these compounds was done by IR, mass, ¹NMR and ¹³C NMR spectral data. The IR spectra were recorded on Shimadzu FT-IR-8400 instrument using KBr pellet method. The Mass spectra were recorded on Shimadzu GC-MS-QP-2010 model using direct inlet probe technique. ¹H NMR was determined in DMSO solution on a Bruker Ac 400 MHz spectrometer and shown in Fig. 2 for compound KC-2. ¹³C NMR was also determined in DMSO solution on a Bruker Ac 400 MHz spectrometer by using TMS as standard, and it is shown in Fig. 3 for compound KC-2.

These synthesized compounds were recrystallized before use. The solvent DMSO was also purified before use by standard methods (Riddick et al., 1986).

C. Microorganisms tested.

The studied microorganisms were obtained from the National Chemical Laboratory (NCL), Pune, India. The microorganisms were maintained at 4°C. The Gram positive bacteria studied were Staphylococcus aureus ATCC29737 (SA), Corynebacterium rubrum ATCC 14898 (CR), Listeria monocytogenes ATCC19112 (LM), Bacillus cereus ATCC11778 (BC); the Gram negative bacteria were Pseudomonas aeruginosa ATCC27853 (PA), Escherichia coli NCIM2931 (EC), Klebsiella pneumoniae NCIM2719 (KP), Salmonella typhimurium ATCC23564 (ST); and the Fungi were Candida albicans ATCC2091 (CA), Cryptococcus neoformans NCIM3542 (CN), Candida glabrata

Fig. 1: Reaction scheme, where R is KC-1 = 4-Cl benzaldehyde, KC-2 = 4-OCH₃ benzaldehyde, KC-3 = 4-F benzaldehyde, KC-4 = 4-Br benzaldehyde, KC-5 = 3,4-diOCH₃ benzaldehyde, KC-6 = 4-CN benzaldehyde, KC-7 = 3-Cl benzaldehyde, KC-8 = 3-OCH₃ benzaldehyde, KC-9 = 3-Br benzaldehyde, KC-10 = 4-CH₃ benzaldehyde.

NCIM3448 (CG), Candida epicola NCIM3367 (CE). The organisms were maintained on nutrient agar and MGYP medium (Hi Media, India) for bacteria and fungi, respectively, at 4°C and sub-cultured before use. The microorganisms studied are clinically important ones causing several infections and food spoilage. The selected Gram positive and Gram negative bacteria are common pathogenic bacteria and their study is clinically important, causing several infections, food borne diseases, spoilages, skin infection and it is essential to overcome them through some active therapeutic agents. Both Gram positive and Gram negative were selected to study a broad spectrum of activity of synthesized compounds.

C. Preparation of compounds solutions.

For all the compounds, DMSO was used for screening of antimicrobial activity. A solution of 20 mg/ml concentration was prepared for all compounds.

Agar well diffusion method:

In vitro antimicrobial activity of the different Pyrazolo quinazolines was studied against pathogenic microbial strains by the Agar well diffusion method. (Perez et al., 1990; Parekh et al., 2005) Mueller Hinton No. 2 / Sabouraud dextrose agar (Hi-media) was used for the antibacterial and antifungal susceptibility test, respectively. The Mueller Hinton agar and Sabouraud dextrose agar were melted and cooled to 48-50°C, and a standardized inoculum (1.5 × 10⁸ CFU/ ml, 0.5 McFarland) was then added aseptically to the molten agar and poured into sterile Petri dishes; wells (8.5 mm) were prepared in the seeded agar plates. The test compound (100 µl) was introduced into the well. The plates were incubated overnight at 37°C and 28°C for 24 h and 48 h respectively, for bacteria and fungi. DMSO was used as negative control. The microbial growth was determined by measuring the diameter of the zone of inhibition and the mean values are considered.

III. RESULTS AND DISCUSSION

A total of 10 compounds were synthesized (KC-1 to KC-10). The physical constants of all the synthesized compounds are given in Table 1.The IR, NMR and Mass spectral data confirmed their molecular structure.

A. Spectral Data.

KC-1: 7-(4-chlorophenyl)-10-(methylthio)-5,6,7,9-tetra hydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3475.85 (-NH (sec.) str.), 3049.56 (Ar-H str.), 2924.18(-CH₂ sym. str.), 2227.86 (-CN str.), 1664.62(C=C str. α, β unsaturated 6-member ring), 1604.83(-NH bending vib. Secondary amine), 1381.08 (-CH bending.), 1315.50 (C-N (sec) bending.), 1242-1010 (C-H in plane bending, phenyl ring), 767.69 (C-H str. 5-adjecent c atoms), 767.69(C-Cl str.),

¹H NMR (DMSO-d₆) δ(ppm): 2.40 (3H, singlet, -CH₃), 1.79-2.75 (4H, multiplet, C-H), 6.07 (1H, singlet, C-H), 7.22-7.70 (8H, multiplet C-H), 10.14 (1H, singlet, -NH).

Elemental analysis: %C = 65.29 (65.34), %H = 4.21 (4.20), %N = 13.88 (13.86), %S = 7.92 (7.92).

Mass: (m/z) =404.09

KC-2: 7-(4-methoxyphenyl)-10-(methylthio)-5,6,7,9-tetrahydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3077.11 (Ar-H asym. str.), 1658.38 (C=N str), 2945.40-2843.17 (CH₂ str. Of cyclohexanone ring), 1597.11(C=C str.), 1090.95 (C-O-C sym. str. 954.80 (ring str. in cyclohexanone), 1210.45 (C-O-C asym. Str.) 2814.11(C-H str. Alkane)

¹H NMR (DMSO-d₆) δ(ppm):

2.44 (3H, singlet,-CH₃), 3.70 (3H, singlet-OCH₃), 1.80-2.76 (4H, multiplet, C-H), 5.94 (1H, singlet, C-H), 6.90-7.70 (8H, multiplet C-H), 10.01 (1H, singlet, N-H).

Elemental analysis: %C = 68.92 (69.00), %H = 5.06 (5.00), %N = 13.97 (14.00), %S = 8.03 (8.00), %O = 4.02 (4.00).

Mass: (m/z) =400.50

KC-3: 7-(4-fluorophenyl)-10-(methylthio)-5,6,7,9-tetrahydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3479.70 (-NH (sec.) str.), 3037.99 (Ar-H str.), 2918.40(-CH₂ sym. str.), 2227.86 (-CN str.), 1666.55 (C=C str. α, β unsaturated 6-member ring), 1599.04 (-NH bending vib. Secondary amine), 1381.08 (-CH bending.), 1319.08 (C-N (sec) bending.), 1242-1010(C-H in plane bending, phenyl ring), 1093.67 (C-F str.), 725.26 (C-H str. 5-adjecent c atoms),

¹H NMR (DMSO-d₆) δ(ppm): 2.42 (3H, singlet, -CH₃), 1.80-2.77 (4H, multiplet, C-H), 5.99 (1H, singlet, C-H), 7.15-7.69 (8H, multiplet C-H), 10.25 (1H, singlet, -NH).

Elemental analysis: %C = 68.07 (68.04), %H = 4.42 (4.38), %N = 14.41 (14.43), %S = 8.25 (8.24).

Mass: (m/z) =388.46

KC-4: 7-(4-bromophenyl)-10-(methylthio)-5,6,7,9-tetrahydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3257.88 (-NH (sec.) str.), 3047.63 (Ar-H str.), 2929.97 (-CH₂ sym. str.), 2227.86 (-CN str.), 1653.05 (C=C str. α, β unsaturated 6-member ring), 1604.83 (-NH bending vib. Secondary amine), 1383.01 (-CH bending.), 1315.50 (C-N (sec) bending.), 1242-1010(C-H in plane bending, phenyl ring), 723.33 (C-H str. 5-adjecent c atoms), 582.52 (C-Br str.),

¹H NMR (DMSO-d₆) δ(ppm): 2.42 (3H, singlet, -CH₃), 1.80-2.79 (4H, multiplet, C-H), 5.99 (1H, singlet, C-H), 7.21-7.82 (8H, multiplet C-H), 10.25 (1H, singlet, -NH).

Elemental analysis: %C = 58.87 (58.93), %H = 3.79 (3.80), %N = 12.45 (12.50), %S = 7.07 (7.14).

Mass: (m/z) =448.37

KC-5: 7-(3,4-dimethoxyphenyl)-10-(methylthio)-5,6, 7,9-tetrahydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3236.66 (-NH (sec.) str.), 3007.12 (Ar-H str.), 2929.97 (-CH₂ sym. str.), 2224.40 (-CN str.), 1666.55(C=C str. α, β unsaturated 6-member ring), 1604.83(-NH bending vib. Secondary amine), 1383.09 (-CH bending.), 1334.78 (C-N (sec) bending.), 1242-1010(C-H in plane bending, phenyl ring), 702.11 (C-H str. 5-adjecent c atoms), 731.05(C-H in plane bending),

¹H NMR (DMSO-d₆) δ(ppm): 2.45 (3H, singlet,-CH₃), 3.71 (3H, singlet-OCH₃), 4.02 (3H, singlet –OCH₃), 1.80-2.76 (4H, multiplet, C-H), 5.98 (1H, singlet, C-H), 7.00-7.76 (8H, multiplet C-H), 10.09 (1H, singlet, -NH).

Elemental analysis: %C = 66.91 (66.97), %H = 5.21 (5.12), %N = 13.08 (13.02), %S = 7.49 (7.44), %O = 7.31 (7.44).

Mass: (m/z) =430.52

KC-6: 7-(4-cyanophenyl)-10-(methylthio)-5,6,7,9-tetrahydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3310.18 (-NH (sec.) str.), 2990.17 (Ar-H str.), 2896.57 (-CH₂ sym. str.), 2227.12 (-CN str.), 1666.55 (C=C str. α, β unsaturated 6-member ring), 1597.64 (-NH bending vib. Secondary amine), 1381.13 (-CH bending.), 1289.74 (C-N (sec) bending.), 1242-1010(C-H in plane bending, phenyl ring), 712.87 (C-H str. 5-adjecent c atoms), 729.67(C-H in plane bending),

¹H NMR (DMSO-d₆) δ(ppm): 2.43 (3H, singlet,-CH₃), 1.71-2.72 (4H, multiplet, C-H), 5.60 (1H, singlet, C-H), 7.22-7.95 (8H, multiplet C-H), 10.14 (1H, singlet, -NH).

Elemental analysis: %C = 69.81 (69.87), %H = 4.39 (4.30), %N = 17.68 (17.72), %S = 8.12 (8.10).

Mass: (m/z) =395.48

KC-7: 7-(3-chlorophenyl)-10-(methylthio)-5,6,7,9-tetrahydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3387.87 (-NH (sec.) str.), 2967.46 (Ar-H str.), 2894.57 (-CH₂ sym. str.), 2224.40 (-CN str.), 1667.99(C=C str. α, β unsaturated 6-member ring), 1590.12(-NH bending vib. Secondary amine), 1357.23 (-CH bending.), 1309.47 (C-N (sec) bending.), 1242-1010(C-H in plane bending, phenyl ring), 699.24 (C-H str. 5-adjecent c atoms), 724.51(C-H in plane bending), 767.69(C-Cl str.),

¹H NMR (DMSO-d₆) δ(ppm): 2.40 (3H, singlet,-CH₃), 1.79-2.75 (4H, multiplet, C-H), 6.07 (1H, singlet, C-H), 7.22-7.70 (8H, multiplet C-H), 10.14 (1H, singlet, -NH).

Elemental analysis: %C = 65.22 (65.34), %H = 4.21 (4.21), %N = 13.78 (13.86), %S = 8.00 (7.92).

Mass: (m/z) =404.92

KC-8: 7-(3-methoxyphenyl)-10-(methylthio)-5,6,7,9-tetrahydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3337.71 (-NH (sec.) str.), 3015.87 (Ar-H str.), 2897.12 (-CH₂ sym. str.), 2227.97 (-CN str.), 1666.55 (C=C str. α, β unsaturated 6-member ring), 1615.92 (-NH bending vib. Secondary amine), 1397.54 (-CH bending.), 1337.56 (C-N (sec) bending.), 1242-1010(C-H in plane bending, phenyl ring), 709.57 (C-H str. 5-adjecent c atoms), 724.69(C-H in plane bending),

¹H NMR (DMSO-d₆) δ(ppm): 2.45 (3H, singlet,-CH₃), 3.70 (3H, singlet-OCH₃), 1.77-2.81 (4H, multiplet, C-H), 6.02 (1H, singlet, C-H), 7.09-7.83 (8H, multiplet C-H), 10.11 (1H, singlet, -NH).

Elemental analysis: %C = 68.92 (69.00), %H = 5.01 (5.00), %N = 13.93 (14.00), %S = 8.12 (8.00), %O = 4.02 (4.00).

Mass: (m/z) =400.14

KC-9: 7-(3-bromophenyl)-10-(methylthio)-5,6,7,9-tetrahydrobenzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3337.69 (-NH (sec.) str.), 2967.43 (Ar-H str.), 2937.65 (-CH₂ sym. str.), 2229.70 (-CN str.), 1666.55(C=C str. α, β unsaturated 6-member ring), 1591.25(-NH bending vib. Secondary amine), 1403.17 (-CH bending.), 1312.36 (C-N (sec) bending.), 1242-1010(C-H in plane bending, phenyl ring), 699.14 (C-H str. 5-adjecent c atoms), 737.45(C-H in plane bending),

¹H NMR (DMSO-d₆) δ(ppm): 2.40 (3H, singlet,-CH₃), 1.70-2.70 (4H, multiplet, C-H), 5.95 (1H, singlet, C-H), 6.99-7.72 (8H, multiplet C-H), 10.12 (1H, singlet, -NH).

Elemental analysis: %C = 58.86 (58.93), %H = 3.87 (3.80), %N = 12.45 912.50), %S = 7.11 (7.14).

Mass: (m/z) =448.37

KC-10: 10-(methylthio)-7-(p-tolyl)-5,6,7,9 tetrahydro benzo[h]pyrazolo[5,1-b]quinazoline-11-carbonitrile

IR(cm^-1, KBr): 3314.25 (-NH (sec.) str.), 2987.34 (Ar-H str.), 2945.67 (-CH₂ sym. str.), 227.64 (-CN str.), 1666.55(C=C str. α, β unsaturated 6-member ring), 1593.14(-NH bending vib. Secondary amine), 1374.28 (-CH bending.), 1340.05 (C-N (sec) bending.), 1242-1010(C-H in plane bending, phenyl ring), 698.47 (C-H str. 5-adjecent c atoms), 742.36(C-H in plane bending),

¹H NMR (DMSO-d₆) δ(ppm): 2.43 (3H, singlet,-CH₃), 3.83 (3H, singlet-CH₃), 1.80-2.80 (4H, multiplet, C-H), 6.02 (1H, singlet, C-H), 7.11-7.81 (8H, multiplet C-H), 10.07 (1H, singlet, -NH).

Elemental analysis: %C = 71.89 (71.88), %H = 5.28 (5.21), %N = 14.52 (14.58), %S = 8.31 (8.33).

Mass: (m/z) =384.50

B. Antimicrobial Activity.

Figure 4 shows the antimicrobial activity of all the synthesized compounds in DMSO against Gram positive

Fig. 2: ¹H NMR spectrum of KC-02

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Fig. 3: ¹³C NMR spectrum of KC-2

Table 1: Physical constants of compounds

Compound Code	Substitution R	Molecular Formula	Mol. weight	Yield (%)
KC-1	-4-Cl	C₂₂H₁₇ClN₄S	404.09	78
KC-2	-4-OCH₃	C₂₃H₂₀N₄OS	400.14	79
KC-3	-4-F	C₂₂H₁₇FN₄S	388.46	78
KC-4	-4-Br	C₂₂H₁₇BrN₄S	449.37	76
KC-5	-3,4-diOCH₃	C₂₄H₂₂N4O₂S	430.52	71
KC-6	-4-CN	C₂₃H₁₇N₅S	395.48	60
KC-7	-3-Cl	C₂₂H₁₇ClN₄S	404.92	77
KC-8	-3-OCH₃	C₂₃H₂₀N₄OS	400.14	76
KC-9	-3-Br	C₂₂H₁₇BrN₄S	449.37	71
KC-10	-4-CH₃	C₂₃H₂₀N₄S	384.50	64

bacteria. It is observed from Fig. 3 that KC-10 inhibited 100% while KC-3 and KC-7 inhibited 75% tested Gram positive bacteria. KC-3 showed maximum zone of inhibition against Bacillus cereus (BC).

For Staphylococcus aureus (SA), KC-4 showed maximum inhibition and is followed by KC-3 and KC-10. KC-6 and KC-3 exhibited maximum inhibition against Corynebacterium rubrum (CR) and Listeria monocytogenes (LM) strains, respectively. KC-2, KC-5 and KC-8 could not inhibit any of the Gram positive bacteria. Furthermore, KC-10 could inhibit all these Gram positive bacteria. In all the 10 compounds, the central moiety is Pyrazolo quinazoline with different side chains. There are reports that with same central moiety and with different side chains, the antibacterial activity varies (Baluja et al., 2012; Vaghasiya et al., 2004). Similar results are also observed in the present study. KC-2, KC-5 and KC-8 contain methoxy groups whereas KC-6 and KC-10 contain cyano and methyl groups, respectively, at para positions. The rest of the compounds contain halogen groups (-F, -Cl and –Br) at meta or para positions. Thus, mostly halogen and methyl containing groups are effective in inhibiting these selected Gram positive bacteria. Rai et al. (2008) have reported maximum inhibition due to the presence of methyl and halogen groups. Similar results have also been reported by some other workers (Jantova et al., 2000; Zaranappa et al., 2012). These workers have also observed that in some cases, the presence of halogen groups increases the inhibition. Ibrahim et al. (1997) have also reported that the presence of the –CH₃ group as a side chain exhibits a good biological activity. Bacillus cereus (BC) was the most susceptible and Staphylococcus aureus (SA) was the most resistant Gram positive bacteria.

Figure 5 shows antimicrobial activity of compounds against Gram negative bacterial strains. Out of four selected Gram negative bacteria, Escherichia coli (EC) was the most resistant Gram negative bacteria not getting inhibited by any of the tested compounds. For the other three bacterias, KC-9 exhibited maximum inhibition. Against Klebsiella pneumoniae (KP) and Salmonella typhimurium (ST), minimum inhibition is shown by KC-4. Furthermore, KC-5 showed inhibition against Klebsiella pneumoniae (KP) and Salmonella typhimurium (ST) whereas KC-2 could inhibit Pseudomonas aeruginosa (PA).

Again, KC-8 could not inhibit any of these Gram negative bacterial strains. Although all the three compounds, KC-2, KC-5 and KC-8 contain methoxy groups, their positions are different. In KC-2, it is at para position whereas in KC-8, it is at meta position. KC-5 contains two methoxy groups at meta and para positions. Thus, the position of groups also affects the inhibition. This is further supported by comparing inhibition of KC-9 and KC-4. Both of these compounds contain the bromine group but their positions are different. In KC-9, it is at meta position whereas in KC-4, it is at para position. Thus, the position of the group is also important in the bacteria inhibition. Overall, against these selected Gram negative bacteria, the bromine group at meta position is most effective. Thus, Escherichia coli (EC) was the most resistant while Pseudomonas aeroginosa (PA) was the most susceptible bacteria.

For all the selected fungal strains, none of the compound showed antifungal activity. In the studied compounds, S-CH₃ group is present in the moiety. This may be the reason of why these compounds are inactive in exhibiting fungal strains. Literature survey shows that most of the Pyrazolo quinazoline derivatives containing amide or other groups (but not S-CH₃ group) showed

Fig. 4: Antimicrobial activity against Gram positive bacteria.

Fig. 5: Antimicrobial activity against Gram negative bacteria.

significant antifungal inhibition (Abdel-Aal et al. 2010; Antipenko et al., 2009; Gouda et al., 2010)

Such screening of various organic compounds and the identification of the active agents is essential due to that the successful prediction of a lead molecule and drug-like properties at the onset of drug design will pay off later in drug development.

IV. CONCLUSION

All the 10 compounds showed varied the level of activity against Gram positive and Gram negative bacteria. Overall, compounds containing methyl and halogen groups are more effective in inhibiting the selected Gram positive and Gram negative bacteria. B. Cereus and P. aeroginosa are the most susceptible and S. aureus and E. coli are the most resistant bacteria. Against the selected fungal strains, none of the ten compounds was found to be effective.

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Received: May 2, 2014.

Accepted: October 31, 2014.

Recommended by Subject Editor: Octavio Furlong.