CC BY-NC-ND
0Journal of Saudi Chemical Society (2011) xxx, xxx-xxx
ORIGINAL ARTICLE
Synthesis, studies and in vitro antibacterial activity of some 5-(thiophene-2-yl)-phenyl pyrazoline derivatives
Mamta Rani *, Yusuf Mohamad
Department of Chemistry, Punjabi University, Patiala, P.O. Box 47002, Punjab, India Received 11 May 2011; accepted 2 September 2011
KEYWORDS
Pyrazolines; Phenyl hydrazine; Chalcones; Antibacterial activity
Abstract In the present investigation, a novel series of pyrazolines 2a-2d were synthesized by the cyclization of various -1-[2-(alkoxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one 1a-1d with N-substi-tuted phenyl hydrazine and thiosemicarbazide in the presence of CH3COOH and NaOH in ethanol which lead to the formation of new pyrazolines. The structures of these compounds were elucidated by, IR, 1H-NMR, 13C-NMR, ESI-MS spectral data and their purities were confirmed by elemental analyes. The in vitro antibacterial activity of these compounds was evaluated against two Gram-positive and two Gram-negative bacteria Aeromonas hydrophila, Yersinia enterocolitica, Listeria monocytogenes, and Staphylococcus aureus by microdilution method and then the minimum inhibitory concentration (MIC) of these compounds was determined. The results showed that compounds 1-[2-(benzyloxy) phenyl]-5-(thiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl (2b) and 1-[2-(naphthalen-2-ylmethoxy) phenyl]-5-(thiophene-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazole-4-yl (2d) showed most promising antibacterial activity as compared to the antibiotics gentamicin and tetracycline in (Tables 1 and 2).
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1. Introduction
* Corresponding author. Tel.: +91 1675 264235; mobile: +91 9417636159; fax: +91 175 2283073. E-mail address: Drmamtaphd@gmail.com (M. Rani).
1319-6103 © 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2011.09.002
Emerging pathogenous infections such as gastroenteritis, arthritis, listeriosis and pneumonia are caused by multidrug-resistant Gram-positive and Gram-negative pathogens. These diseases are the world's most prevalent and fatal infectious diseases. Principal players among these problematic organisms are isolates of methonia resistant Aeromonas hydrophila, Yersinia enterocolitica, Listeria monocytogenes, and Staphylococcus aureus (Daskalov, 2006; Andersen, 1988; Ogston, 1984; Saginur and Suh, 2008). It is speculated that resistance to b-lactam antibiotics is due to the production of multiple inducible, chromo-somally encoded b-lactames. Resistance to the third generation cephalosporins is known to be associated with the derepression of the chromosomal enzymes (Gofii-Urriza et al., 2000). In rare cases, through direct inactivation of the antibiotic or by
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mutations in the 16S rRNA that prevent the binding of tetracycline to the ribosome. Amoxicillin, penicillin, ampicillin, norfloxacin, Ofloxacin and ciprofloxacin are the principal drugs of choice in the treatment of bacterial infection since they are effective against extraintestinal and intestinal wall infection (Ko et al., 1996), but these are associated with several side effects such as nausea, metallic taste, dizziness, hypertension, etc. as well as resistance have been reported (Bauer et al., 1996; Doyle et al., 2007). The present strategy for new drug development is directed towards identifying the essential enzyme systems in the bacterial and developing molecules to inhibit them on our ongoing medicinal chemistry research activity. The study of chalcone derivatives has become of much interest in recent years on account of their antibacterial, antiviral, anticancer, anti-fungal, anti-hermitic and insecticidal (Marc et al., 2008; Babasaheb et al., 2010; Detsi et al., 2009) activities. Cycli-zation of chalcone such as pyrazolines dramatically increases the diversity of certain biological properties such as antibacterial, antiviral and anti-amoebic activities (Solankee et al., 2010; Zitouni et al., 2005; Fathalla et al., 2003). In view of these observation and in continuation of our group work on biologically active heterocyclic compounds (Clark et al., 2008; Khan, 2008; Khan and Yusuf, 2009a,b) and their increasing importance in pharmaceutical and biological field, it was considered of interest to synthesize some new chemical entities incorporating the molecular frame work and to evaluate their biological activities. In this regard, alkloxy chalcones would be suited for preparing pyrazoline. In this paper we have to synthesis a novel series of pyrazoline derivatives from alkoxy chalcone as antibacterial agents.
2. Chemistry
The present investigations were synthesized starting from the Claisen-Schmidt reaction of acetophenone with 2-thiophene carboxaldehyde in the presence of NaOH (50%) to give 95% yield of the starting material (2E)-l-(2-hydroxyphenyl)-3-(thio-phen-2-yl) prop-2-en-1-one a. The -1-[2-(alkoxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one 1a-1d were prepared in good yields from the O-alkylation of (a) with suitable alkylating agents (like allylbromide, benzyl chloride, bromoethylacetate, 1-chloromethylnapthalene) in the presence of K2CO3/PTC/ dry acetone, under the similar condition, were prepared by the reported method. The cyclization of the latter with phenyl hydrazine in the presence of CH3COOH in ethanolic condition led to formation of new compounds such as 1-[2-(alkoxy) phe-nyl]-3-(thiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl 2a-2d, which were crystallized from CHCl3:CH3OH (1:3) to give pure crystalline solid compounds in moderate yields. All the compounds are insoluble in water but soluble in organic solvents. The structures of these compounds 1a-1d and 2a-2d were analyzed by the rigorous analysis of their IR, *H-NMR, 13C-NMR and ESI mass spectral data.
3. Pharmacology
Antibacterial activity of newly synthesized pyrazoline compounds were evaluated against various pathogenic bacterial strains (Gram-negative and Gram-positive) viz., A.hydrophila, Y. enterocolitica, L. monocytogenes and S. aureus. The antibacterial activities were evaluated by agar disc diffusion method.
The solvent, DMSO used for the preparation of compounds did not show inhibition against the tested organisms. The results of antibacterial activity and minimum inhibitory concentration (MIC) are summarized in Tables 1 and 2, respectively.
3.1. Material and method
Pure cultures of A. hydrophila, Y. enterocolitica, L. monocytog-enes and S. aureus were grown in brain heart infusion broth for sensitivity testing. Mueller Hinton agar (Hi Media) and pyraz-oline compounds 2a-2d absolutely diluted (concentration of 40, 30, 20 and 10 mcg) were applied as described by Bauer et al. (1996) and Doyle et al. (2007). The strains were tested against the following antibiotics (Hi- Midia): gentamicin 10mcg, tetracycline 30 mcg. These were enriched with BHIB for 6-8 h at 37 0C, the cultures were streaked on Mueller Hinton agar plates using a cotton swab. With an antibiotic disc dispenser, the discs were placed on the agar surface. After 30 min of pre-diffusion time, the plates were incubated at 37 0C for 18-24 h, after incubation, the diameter of the inhibition zones were measured and compared to the interpretive chart of performance standards for antimicrobial disc susceptibility tests (Hi Media) and classified as resistant, intermediate or sensitive.
3.2. In vitro antibacterial activity
In vitro antibacterial activities of pyrazoline 2a-2d derivatives were carried out using the culture of A. hydrophila, Y. enterocolitica, L. monocytogenes and S. aureus by the disc diffusion method and then minimum inhibitory concentration (MIC) of these compounds were determined. Gentamicin and tetracycline antibiotics were used as the standard drugs, where as DMSO poured disc was used as negative control. The minimum inhibitory concentration (MIC) was evaluated by the microdilution method of test compounds, previously dissolved in dimethyl sulphoxide (DMSO) which was prepared to final concentration of 40, 50, 30, 20 and 10 i/ml. The MIC, defined as the lowest concentrations of the test compounds, which inhibits the visually growth was determined after incubation for 18 h, at 37 0C. The in vitro studies result showed that the compounds 2b and 2d are more active among all the pyrazo-lines. Compounds 2b and 2d are better antibacterial agents as compared to antibiotics. The susceptibility of the bacteria to the test compounds was determined by the formation of an inhibitory zone after 48 h of incubation at 37 0C.
These compounds showed the highest activity of 2b and 2d against A. hydrophila, Y. enterocolitica, L. monocytogenes and S. aureses. The molecular structure of these active compounds showed property of the pyrazolines which might be more efficacious drugs against these bacteria and their biological effects which could be helpful in designing more potent antibacterial agents for therapeutic use.
4. Results and discussion
All the four synthesized compounds 2a-2d were screened for their potential to inhibit emerging pathogens A. hydrophila, Y. enterocolitica, L. monocytogenes and S. aureus responsible for gastrointestinal diseases. The present study describes the
Table 1 Antibacterial activity of pyrazoline derivatives, positive control (gentamicin and tetracycline) and negative control (DMSO) measured by the halo zone test (unit, mm).
Compounds Corresponding effect on micro-organisms (unit, mm)
A. hydrophila Y. enterocolitica L. monocytogenes S. aureus
2a 14.5 11.5 13.4 14.6
2b 20.5 22.4 24.3 22.8
2c 12.5 14.5 14.5 15.5
2d 21.7 18.4 21.2 19.4
Gentamicin 21 - - 17
Tetracycline 13 20 12 14
Table 2 Minimum inhibition concentration (MIC) of bis-pyrazoline derivatives and positive control (gentamicin and tetracycline)
(unit, mcg).
Compounds (MIC) (MIC) Corresponding effect on micro-organisms (unit, mcg)
A. hydrophila Y. enterocolitica L. monocytogenes S. aureus
2a 10 10 10 10
2b 30 30 30 30
2c 40 40 40 40
2d 20 20 20 20
Tetracycline 30 30 30 30
Gentamicin 10 10 10 10
synthesis and antibacterial evaluation of some 1-[2-(alkoxy) phenyl]-3-(thiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl 2a-2d pyrazoline derivatives of -1-[2-(alkoxy) phenyl]-3-(thio-phen-2-yl) prop-2-en-1-one 1a-1d and development of a series of new pyrazoline derivatives which were synthesized in satisfactory yields (62-80%) as illustrated in Fig. 1 and their structures were characterized by spectral data. Selected compounds 2a, 2b, 2c, 2d, and 2e showed similar results when tested against A. hydrophila, Y. enterocolitica, L. monocytogenes and S. aureus. The compounds 2a and 3a exhibit more activity as compared to the standard drugs Gentamicin (10mcg) and Tetracycline (30 mcg). On the basis of the above observations, modification will be done to improve antibacterial activity.
4.1. IR spectral studies
Assignment of selected characteristic IR bands provides significant indications for the formation of the cyclized pyrazoline analogues of the phenyl hydrazine 2a-2d. In started material
Figure 1 Schematic diagram indicating the ring conformation of compound nos. 2a-2d.
chalcones (C=O) and (CH=CH), functional absorbed in the expected region; (C=O) at 1597-1655 cm"1 and (CH=CH) at 1585-1598 cm"1, respectively. The IR spectra of the compounds showed u(C=N) stretching at 1586-1601 cm"1. In addition, the absorption bands at 995-1034 cm"1 were attributed to the t(C-N) stretching vibration, which also confirm the formation of desired pyrazoline compounds.
4.2. !H-NMR spectral analysis
The 1H-NMR spectra (400 MHz, CDCl3), the 1H-NMR of the starting material chalcone 1a-1d were the two broad doublets centred at d 7.71-7.82 and 6.92-7.65 which could be ascribed to H-3 and H-2 protons, respectively, and coupling value of 15.4-15.7 Hz between these hydrogens describe the trans geometry around the C-2 and C-3 double bond. The downfield resonance of the H-3 as compared to H-2 could be ascribed to the electron deficient nature of the b-carbon in the enone moiety. Other significant signals in the aromatic region appeared at d 6.7-7.9, respectively. The major features of this spectrum were the signals of the pyrazoline 2a-2d ring proton (H-x) and (H-a and b) which were found in the region at d 5.12-5.32 (1H, dd, Jxa = 6.5-6.7 Hz, Jxb = 11.6-11.8 Hz,) and 3.42-3.82 ppm (1H, dd, Jax = 6.4-6.7 Hz, Jab = 16.8-17.6 Hz), respectively. The pyrazoline proton (H-b) and -CH2 protons appeared together at d 4.01-5.49 ppm. In a double resonance experiment, irradiation of doublet of doublet at d 5.12 (H-x) converted the doublet of doublet at 3.2 (H-a) to doublet at d 4.01-5.49 ppm, which clearly describes the inter-relationship between the H-x, b and a. The proposed expression 2a-2d is due to vicinal coupling with two magnetically non-equivalent geminal proton of adjacent carbon atom C5 pyrazoline ring. The strong deshielding of the C5 (H-a and b) protons compared with the C4 (H-x) protons of the pyrazoline ring can be assumed due to its structure Fig. 2. The protons belonging to the aromatic
ring and the other cyclic groups were observed with the expected chemical shift and integral values.
Finally, 13C-NMR (400 MHz, CDCl3) spectra of all compounds were recorded in DMSO and spectral signals which are in good agreement with the probable structures. The methylene groups for 1c & 2c were resonating at d 22.454.2 (CH3) and 72.2-72.4 (OCH2); the downfield resonance of the former suggests their placement near an electronegative oxygen atom. The carbon of-CH2 in all compounds resonates at 62.5-74.2 ppm, respectively. The C4 and C5 carbon of pyr-azolines 2a-2d resonated at 61.5-64.8 and 67.6-74.2 ppm, respectively. The carbon of -CH3 in all compounds resonates at 22.4-24.5 ppm, respectively. The carbon of (C=O) and (C=C) displayed signals at 190.5-192.2 and 137.6144.6 ppm in 1a-1d compounds. All the compounds showed signal at 112.8-155.8 ppm were assigned to the aromatic carbon. The compounds 2a-2d showed two signals at 155.6156.7 ppm assigned to (C=N), respectively. The signals were due to the aromatic carbons and the carbon at 1-N-substituted aliphatic group. The other resonances were visible at their usual position in Section 5.
4.3. ESI mass analysis
Characteristic peaks were observed in the mass spectra of all compounds, which followed the similar fragmentation pattern.
The spectrum of the compound 2a showed a molecular ion peak (M+) at m/z 411. The characteristics peaks observed within the mass spectra of pyrazoline compound are given in the experimental section.
It may be concluded that this study describes the general method for the synthesis of some pyrazolines linked through the 5-aryl ring under the normal conditions. These results show that the compounds 2b & 2d show highest antibacterial activity and overall compounds exposed sufficient activity against A. hydrophila, Y. enterocolitica, L. monocytogenes and S. aureus promising antibacterial activity. Thus the accumulation of the pyrazoline derivatives will be a better antibacterial agent as compared to gentamicin and tetracycline.
5. Experimental
The entire chemicals were purchased from Aldrich Chemical Company (USA) and were used without further purification. The reactions were monitored by percolated aluminium silica gel 60F 254 thin layer plates procured from Merck (Germany). All melting points were measured with a capillary apparatus and are uncorrected. All the compounds were routinely checked by IR, 'H-NMR, 13C-NMR, mass spectrometry and elemental analyses. IR spectra were recorded in KBr on a Per-kin-Elmer model 1620 FTIR spectrophotometer. 'H-NMR and 13C-NMR spectra were recorded at an ambient tempera-
K2CO3 / PTC /
R-CH 2-X / dry acetone
O-CH2—R
1a -1d
EtOH / A
PhNHNH 2/ glacial acitic acid
,O-CH2—R
where R = -CH=CH 2, -Ph, -COOEt,
(1a,2a ) (1b, 2b) (1c, 2c) (1d. 2d)
X = -Cl, Br + -
PTC=Bu 4N I
2a -2d
Figure 2 Schematic diagram indicating the synthesis of compound nos. 2a-2d.
0 C / EtOH
ture using Brucker spectrospin DPX-400 MHz spectropho-tometer in CDCl3 and DMSO. The following abbreviations were used to indicate the peak multiplicity s - singlet, d - doublet, t - triplet, m - multiplet. The mass spectra have been scanned on the Waters Micromass Q-T of Micro (ESI) spectrometer. Anhydrous sodium sulfate was used as a drying agent for the organic phase.
5.1. General procedure for the synthesis of (2E)-l-(2-hydroxyphenyl)-3-(thiophen-2-yl) prop-2-en-1-one (1)
A suspension of O-hydroxy acetophenone (1 equiv.) and thio-phene carboxaldehyde (1 equiv.) in ethanolic solution of NaOH (30%) was stirred for 8 h at room temperature. After the completion of the reaction, the reaction mixture was poured into acidic ice water ~ pH 2 (adjusted by HCl) to produce a solid compound which was filtered under suction and washed with H2O. The solid was filtered recrystallized from CH3OH:CHCl3 (3:1) to obtain a pure chalcone 1 (Husain et al., 2008).
Yellow needles; Yield: 95%; m.p. 86 0C; Anal. calc. for C13H10O2S: C, 67.82; H, 4.34; Found: C, 67.78; H, 4.30 %; IR (KBr) tmax (cm-1): 1636 (C=O), 2952 (OH); *H-NMR (400 MHz, CDCl3): d (ppm): 12.86 (1H, s, -OH), 8.04 (1H ,d Jfcms = 15.4 Hz, H-3), 7.82 (1H, d, Jtrans = 15.4 Hz, H-2) 7.49 (1H, d{dd}, Jv^o = 1.4, 3.6, 7.1 Hz, Ar-H), 7.45 (1H, m, Ar-H), 7.43 (1H,' m, Ar-H), 7.01 (1H, dd, Jm,o = 3.6 Hz, 7.1 Hz,Ar-H), 7.34 (1H, t, Ar-H), 7.10 (1H, t, Ar-H), 6.82 (1H, t, Ar-H); 13C-NMR (400 MHz, CDCl3): d (ppm): 191.4 (c=o), 154.7, 149.6, 148.4, 143.8 (C=C), 138.7 (C=C), 133.5, 128.8, 127.5, 127.8, 125.8, 124.7, 122.6, (Ar-C); GC-MS m/z (rel. int. %): 231 (68) [M + 1] + .
5.2. General procedure for the synthesis of (2E)-1-[2-(prop-2-en-1-yloxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one (1a)
A suspension of l-(2-hydroxyphenyl)-3-(thiophen-2-yl) prop-2-en-1-one (1) (2.0 g, 0.005 mol), allyl bromide (1.5 ml, 0.0041 mol), freshly ignited K2CO3 (1.0 g) and tetrabutylamo-nium iodide as PTC (1.0 g) in dry acetone (25 ml) was heated at reflux for 1 h with stirring. In the absence of PTC, reaction occurred in 70-80 h and yields of the alkoxy chalcones were obtained. Use of PTC in these reactions, not only decreased the reaction time up to 4-5 h but also improved the yield of 1-[2-(prop-2-en-1-yloxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one derivatives up to 80-85%. The products thus obtained were purified by passing through a silica gel column (60-120 mesh) and further crystallized from methanol to afford 1a (Kumar and Yusuf, 2006).
Light brown; Yield: 92%; m.p. 82 0C; Anal. calc. for C16H14O2S: C, 71.11; H, 5.18, Found: C,71.07; H, 5.15 %; IR (KBr) tmax (cm-1): 2923, 2871 (C-H), 1651 (c=O), 1590 (C=C); 1H-NMR (400 MHz, CDCl3): d (ppm): 7.78 (1H, d, Jtrans = 15.6 Hz, H-3), 7.62 (1H, d{dd}, Jpmo = 0.8, 1.8, 7.8 Hz, Ar-H), 7.30 (1H, d, Jtrans = 15.6 Hz, H-2), 7.04 (2H, m, Ar-H), 7.44 (1H, d, J = 1.8,7.8 Hz, Ar-H), 7.36 (1H, d, J = 5.2 Hz, Ar-H), 7.28 (1H, d, J = 5.8 Hz, Ar-H), 6.96 (1H, d, J = 5.0 Hz, Ar-H), 6.05 (1H, q, J = 6.7 Hz, -CH), 5.45 (2H, td, Jvis=1.7, 17.2 Hz -CH2), 5.25 (2H, td, J vic = 6.4 Hz, -CH2); 13C-NMR (400 MHz, CDCl3): d (ppm): 192.2 (C=O), 154.5, 153.6, 144.6 (C=C), 141.5 (C=C), 132.2, 131.6, 130.2 128.6, 126.2, 125.3, 120.2 122.3 (Ar-c), 68.4 (CH2), 62.5 (CH2), 60.3 (CH); MS: m/z (M+) 271.
5.3. (2E)-1-[2-(benzyloxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one (1b)
A suspension of l-(2-hydroxyphenyl)-3-(thiophen-2-yl) prop-2-en-1-one (1) (2.0 g, 0.001 mol), benzyl chloride (1.5 g, 0.004 mol), under similar conditions was used for 1a and was further crystallized from methanol to afford 1b.
Light yellow; Yield: 95 %; m.p. 85 oC; Anal. calc. for C20H16O2S: C, 75.0; H, 5.0; Found: C, 74.7; H, 4.6%; IR (KBr) tmax (cm-1): 3074(Ar-H), 1597 (C = O), 1597 (CH = CH); 1H-NMR (400 MHz, CDCl3): 8 /ppm: 7.71 (1H, d, Jtrans = 15.7 Hz, H-3), 7.56 (4H ,m, Ar-H), 7.42 (2H, dd, J = 7.6 Hz, Ar-H), 7.05 (1H, d{dd}, Jpmo = 0.8,1.2,7.6 Hz, Ar-H), 7.32 (3H, m, Ar-H), 7.03 (1H, m, Ar-H), 6.92 (1H, d, Jtrans = 15.7 Hz, H-2), 6.8 (1H, d, J = 7.6 Hz, Ar-H), 5.18 (2H, s, -CH2); 13C-NMR (400 MHz, CDCl3): d (ppm): 191.4 (C=O), 154.7, 153.2, 149.6, 143.8 (C=C), 142.1, 138.7 (C=C), 132.5, 131.5, 130.7, 129.2, 129.2, 128.7, 128.7, 127.5, 126,2, 125.8, 124.3, 122.4 (Ar-C), 74.2 (CH2); GC-MS m/z (rel. int. %): 321 (72) [M + 1] + .
5.4. (2E)-1-[2-(ethyloxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one (1c)
A suspension of (1) (2.0 g, 0.01 mol), bromoethyl acetate (1.2 g, 0.004 mol), under similar conditions was used for 1a and was further crystallized from methanol to afford 1c.
Light brown; Yield: 86%; m.p. 89 0C; Anal. calc. for C17H16O4S: C, 64.55; H, 5.06; Found: C, 64.52; H, 5.02 %; IR (KBr) tmax (cm-1): 2980, 2933 (methylene C-H), 3075 (Ar-H), 1655, 1751 (C=O), 1598 (CH=CH); 1H-NMR (400 MHz, CDCl3): d (ppm): 7.82 (1H, d, Jtrans = 15.6 Hz, H-3), 7.65 (1H, dd, Ar-H), 7.45 (1H, d{dd}, J№o = 0.7, 1.8, 7.5 Hz, Ar-H), 7.40 (2H, d, J = 1.0,7.6, Ar-H), 7.36 (1H, d, Jtrans = 15.6 Hz, H-2), 7.07 (2H, m, Ar-H), 6.8 (1H, d, J = 1.0,7.6, Ar-H), 4.73 (2H, s, -CH2), 4.25 (2H, q, Jvic = 7.1 Hz, -OCH2), 1.25 (3H, t, Jvic = 7.2 Hz, -CH3); 13C-NMR (400 MHz, CDCl3): d (ppm): 191.2 (2C=o), 144.6 (C=C), 155.8, 154.5 148.5, 142.6 (C=C), 131.7, 130.3, 128.5, 127.4, 126.6, 125.4, 121.6 (Ar-C), 72.4 (OCH2), 68.4 (CH2), 22.4 (CH3); MS; m/z (M+) 317.
5.5. (2E)-1-[2-(naphthalen-2-ylmethoxy) phenyl]-3-(thiophen-2-yl)prop-2-en-1-one (1d)
A suspension of (1) (2.0 g, 0.01 mol), 1-chloromethylnaptha-lene (1.4 g, 0.004 mol), under similar conditions as used for 1a and was further crystallized from methanol to afford 1d.
Light brown; Yield: 88%; m.p. 94 0C; Anal. calc. for C24H18OS: C, 77.83; H,4.86; Found: C, 77.80; H, 4.82 %; IR (KBr) tmax (cm-1): 3064 (Ar-H), 1637 (C=O), 1592 (CH=CH); 1H-NMR (400 MHz, CDCl3): d (ppm): 8.02 (1H, m, Ar-H), 7.83 (1H, d, J = 8.1 Hz, Ar-H), 7.80 (1H, d, Jtrans = 15.4 Hz, H-3), 7.74 (1H, dd, J = 1.8, 7.4 Hz, Ar-H), 7.65(1H, d, Jtrans = 15.4 Hz, H-2), 7.61 (2H, d, J= 1.9, 7.6 Hz, Ar-H), 7.24 (4H, m, Ar-H), 7.42 (1H, d{dd}, Jp,m,o = 0.8, 1.9, 7.6 Hz, Ar-H), 7.09 (2H, m, Ar-H), 6.92 (2H, m, Ar-H), 5.59 (2H, s, -CH2); 13C-NMR (400 MHz, CDCl3): d (ppm): 190.5 (C=O), 155.7, 151.7, 142.7 (C=C), 137.6 (C=C), 144.4, 132.5, 131.4, 130.6, 129.5, 129.5, 128.2, 128.2, 127.5, 126.7, 125.4, 124.5, 122.5, 122.5, 121.3, 120.6, 118.9, 118.9 (Ar-C), 66.5 (CH2); m/z (M+) 371.
5.6. General procedure for the synthesis of l-[2-(allyloxy) phenyl]-5-(thiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl (2a)
A mixture of-1-[2-(prop-2-en-1-yloxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1 -one (1a) (0.5 g, 0.0015 mol) was refluxed with phenyl hydrazine (0.800 ml, 0.004 mol) in dry EtOH (30 ml) and catalytic amount of glacial acetic acid for at 80 0C for 8 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was removed under reduced pressure and the residue obtained was purified by column chromatogra-phy (20:80, diethyl ether:petroleum ether) the obtained solid was crystallized from EtOH to yield pyrazolines 2a.
Dark brown; Yield: 88 %; m.p. 134 0C; Anal. calc. for C22H20N2OS: C, 73.33; H, 5.55; Found: C, 73.30; H, 5.52 %; IR (KBr) tmax (cm"1): 3037 (Ar-H), 3319 (N-H), 1595 (C=N); 1H-NMR (400 MHz, CDCl3): d (ppm): 7.72 (3H, m, Ar-H), 7.51 (5H, m, Ar-H), 7.04 (2H, d, Ar-H), 6.80 (1H, d, J = 7.6 Hz, Ar-H), 6.32 (1H, d{dd}, Jpmo = 1.0, 1.8, 7.6 Hz, Ar-H), 5.06 (1H, dd, Jvic = 1.1Hz,7.0Hz, -CH), 4.95 (2H, t, J = 6.4 Hz, -CH2), 4.25 (2H, dd, -CH2), 5.32 (1Hx, dd, Jxa = 6.7 Hz, Jxb= 11.7 Hz), 3.51 (1Ha, dd, Jax = 6.7 Hz, Jab = 16.8 Hz), 3.22 (1Hb, dd, Jbx = 11.7 Hz, Jba = 16.8 Hz); 13C-NMR (400 MHz, CDCl3): d (ppm):
155.6 (C=N), 153.4, 152.4, 150.2, 144.2, 132.5, 130.2, 129.9, 129.9, 128.2, 128.2, 127.3, 126.3, 125.8, 123.1, 121.2, 116.6, 114.2 (Ar-C), 72.2 (pyr. ring, C-5), 71.1 (CH), 69.4 (CH2), 63.1 (pyr. ring, C-4); m/z (M+) 361.
5.7. 1-[2-(Benzyloxy) phenyl]-5-(thiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl (2b)
Pyrazoline 2b was obtained from the reaction of -1-[2-(banzyl-oxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one 1b 1a (0.5 g, 0.0015 mol) with phenyl hydrazine (0.430 g, 0.00398 mol) under similar conditions was used for 1a and was further crystallized from ethanol to afford 2b.
Blackish brown; Yield: 92%; m.p. 128 0C; Anal. calc. for C26H22N2OS: C, 76.09; H, 5.36; Found: C, 76.05; H, 5.32 %; IR (KBr) tmax (cm"1): 3037 (Ar-H), 3319 (N-H), 1595 (C=N); 1H-NMR (400 MHz, CDCl3): d (ppm): 7.91 (5H, m, Ar-H), 7.34 (2H, m, Ar-H), 7.28 (3H, m, Ar-H), 7.06 (1H, d{dd}, Jpmo = 1.0, 1.8, 7.0 Hz, Ar-H), 6.9 (5H, m, Ar-H), 6.81 (1H, m, Ar-H), 5.25 (1Hx, dd, Jxa = 6.5 Hz, Jxb = 11.8 Hz), 5.01 (2H, s, -CH2), 3.82 (1Ha, dd, Jax = 6.5 Hz, Jab = 17.2 Hz), 3.42 (1Hb, dd, Jbx = 11.8 Hz, Jba = 17.2 Hz); 13C-NMR (400 MHz, CDCl3): d (ppm):
155.7 (C=N), 153.3, 152.2, 144.2, 142.2, 132.6, 131.8, 130.4,
129.2, 129.2, 128.7, 127.5, 126.4, 126.4, 125.8, 124.3, 124.3,
122.3, 122.3, 121.5, 120.6, 118.6, 115.6 (Ar-C), 74.2 (pyr. ring, C-5), 63.6 (CH2), 64.8 (pyr. ring, C-4); m/z (M+) 411.
5.8. 1-[2-(Ethyloxy) phenyl]-5-(thiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl (2c)
Pyrazoline 2c was obtained from the reaction of -1-[2-(ethyl-oxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one 1b 1a (0.5 g, 0.0015 mol) with phenyl hydrazine (0.730 g, 0.004 mol) under similar conditions was used for 1a and was further crystallized from ethanol to afford 2c.
Reddish brown; Yield: 95%; m.p.126 0C; Anal. calc. for C23H22N2O3S: C, 67.98; H, 5.41; N, 6.89; Found: C, 67.95; H,
4.38; N, 6.85 %; IR (KBr) tmax (cm"1): 2878, 2889 (methylene C-H), 3247 (Ar-H), 3369 (N-H), 1648, 1755 (C=N); 1H-NMR (400 MHz, CDCl3): d (ppm): 7.8 (5H, m, Ar-H), 7.06 (2H, m, Ar-H), 7.35 (1H, d{dd}, Jpmo = 0.8, 1.9, 7.6 Hz, Ar-H), 7.2 (3H, m, Ar-H), 6.81 (1H, s, Ar-H), 5.12 (1Hx, dd, Jxa = 6.5 Hz, Jxb = 11.8 Hz), 3.82(1Ha, dd, Jax = 6.5 Hz, Jab = 17.6 Hz), 3.42(1Hb, dd, Jbx = 11.8 Hz, Jba = 17.6 Hz), 4.73 (2H, s, -CH2), 4.01 (2H, q, Jvic = 7.2 Hz, -OCH2), 1.25 (3H, t, Jvic = 7.1 Hz, -CH3); 13C-NMR (400 MHz, CDCl3): d (ppm): 190.5 (C=O), 155.6 (c=N), 153.4, 152.6, 150.3 144.2,
132.5, 131.9, 130.1129.8, 129.8, 128.6, 128.6, 127.3, 124.05, 125.8, 123.1, 116.7 (Ar-C), 72.2 (OCH2), 67.6 (pyr. ring, C-5), 62.7 (pyr. ring, C-4), 63.9 (CH2), 24.5 (CH3); m/z (M+) 407.
5.9. 1-[2-(Naphthalen-2-ylmethoxy) phenyl]-5-(thiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl (2d)
Pyrazoline 2d was obtained from the reaction of -1-[2-(naph-thalen-2-ylmethoxy) phenyl]-3-(thiophen-2-yl) prop-2-en-1-one 1b 1a (0.5 g, 0.0015 mol) with phenyl hydrazine (0.830 g, 0.0043 mol) under similar conditions was used for 1a and was further crystallized from ethanol to afford 2d.
Reddish brown; Yield: 91%; m.p. 155 0C; Anal. calc. for C30H24N2OS: C, 78.26; H, 5.21; N, 6.08; Found: C,78.22; H,5.18; N, 6.05%; IR (KBr) tmax (cm"1): 3257 (Ar-H), 3314 (N-H), 1586 (C=N), 1445 (N-N); 1H-NMR (400 MHz, CDCl3): d (ppm): 7.8 (2H, dd, Jmo = 1.8 Hz, 7.4 Hz, Ar-H), 7.49 (5H, m, Ar-H), 7.10 (7H, m, Ar-H), 7.02 (2H, d{dd}, Jp,m,o =0.8, 1.9, 7.6 Hz, Ar-H), 6.81 (3H, m Ar-H), 5.49 (2H, s, -CH2), 5.23 (1Hx, dd, Jxa = 6.7 Hz, Jxb = 11.6 Hz), 3.63 (1Ha, dd, Jax = 6.7 Hz, Jab = 17.2 Hz), 3.24 (1Hb, dd, Jbx = 11.6 Hz, Jba = 17.2 Hz); 13C-NMR (400 MHz, CDCl3): d (ppm): 156.7 (C=N), 154.3, 153.2, 144.2, 142.2, 133.5, 131.5, 130.7, 129.2, 129.2, 128.7, 128.7, 128.1, 127.6, 127.2, 126.2, 126.2, 125.3, 124.2, 124.2, 123.9, 123.9, 123.1, 123.1, 121.2,
120.6, 118.7 (Ar-C), 71.3 (pyr. ring, C-5), 64.3 (CH2), 61.5(pyr. ring, C-4); m/z (M+) 461.
6. Conclusions
The novel pyrazoline derivatives were synthesized by the reaction of alkoxy chalcones with thiosemicabazide/phenyl hydrazine and were studied for their antibacterial activity. This research involves the synthesis of pyrazoline derivatives 2a-2d of alkoxy chalcones 1a-1d and antibacterial activity of these pyrazoline compounds were examined using culture A. hydrophila, Y. enterocolitica, L. monocytogenes and S. aureses. The results of antibacterial screening reveal that among all the compounds screened, compounds 2a-2d and 3a-3d showed moderate antibacterial activity while compounds 2b and 2d displayed good antibacterial activity when compared with Gentamicin and Tetracycline used as the standard drugs. Particularly, compound 2a which carries the allyoxy substituent appears to exhibit the highest antibacterial activity (zone of inhibition up to 18.4-24.3 mm) against all bacteria.
Acknowledgements
Author is highly thankful to the Rajiv Gandhi fellowship JRF (UGC), N. Delhi, India, for the generous grant. The necessary
facilities and some financial assistance provided by Prof. Baldev Singh, Head, Department of Chemistry, Punjabi University, Patiala, has also been highly acknowledged.
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