Synthesis and Characterization of New 3,5-Dinaphthyl Substituted 2-Pyrazolines and Study of Their Antimicrobial Activity

A number of chalcones were prepared by condensing either 1-acetylnaphthalene or substituted 1-acetylnaphthalenes with 1-naphthaldehyde or 4-dimethylamino-1-naphthaldehyde in ethanolic NaOH solutions. These chalcones were immediately reacted with hydrazine hydrochloride, phenyl hydrazine and semicarbazide hydrochloride in the presence of dry acetic acid to obtain the corresponding 2-pyrazolines. The synthesised heterocycles were characterized on the basis of their chemical properties and spectroscopic data. These compounds were tested for antimicrobial activity against a variety of test organisms: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabillis, Shigella dysentry and Salmonella typhii. The compounds containing chloro, hydroxo and dimethylamino -N(CH3)2 group as substituents on the naphthalene rings have been found to be very effective antimicrobial agents. In addition, the presence of a carboxamido -CONH2 substituent group at the N-1 position of the 2-pyrazoline rings is shown to contribute substantially to the antimicrobial activity.


Introduction
Due to the interesting activity of variously substituted pyrazolines as biological agents considerable attention has been focused on this class. The pharmaceutical importance of these compounds lies in the fact that they can be effectively utilized as antibacterial, antifungal, antiviral, antiparasitic, antitubercular and insecticidal agents [1][2][3][4][5][6]. Some of these compounds have also antiinflammatory, antidiabetic, anaesthetic and analgesic properties [7][8][9][10]. In addition, pyrazolines have played a crucial part in the development of theory in heterocyclic chemistry and also used extensively in organic synthesis [11][12][13][14][15].
Among the methods employed in synthesis of pyrazolines, condensation of a variety of substituted chalcones with hydrazine and its derivatives is commonly used [16][17][18]. 2-Pyrazolines can be conveniently synthesized by the treatment of a,ß-unsaturated carbonyl compounds with hydrazine reagents in basic and acidic media [11,16,17]. In this method, hydrazones are formed as intermediates, which can be subsequently cyclized to 2-Pyrazolines in the presence of a suitable cyclizing reagent like acetic acid [19,20].
According to a literature survey, i t was noted that very little research has been carried out regarding pyrazolines carrying naphthyl/substituted naphthyl groups as substituents on pyrazoline ring. In this area Dashi and Kadu have recently reported the synthesis and antimicrobial activity o f some new naphthyl-substituted 2-pyrazolines [21]. Therefore, as a part of our program focused on 2-pyrazolines with biological activity, and in connection with our interest in the chemistry of arylated pyrazolines, in this paper we report the synthesis and anti-microbial properties of some new 3,5-dinaphthyl substituted 2-pyrazolines. A study of the effects of certain substituent groups attached both to naphthalene and pyrazoline rings on the antimicrobial activity of these compounds was also planned.

Results and Discussion
In this paper the syntheses of twenty-four 3,5-dinaphthalene -1-yl substituted 2 -pyrazolines ( 3ag-3fk) containing certain groups as substituents both on the naphthalene and pyrazoline rings are described. These compounds were prepared by the action of hydrazine reagents 2g-2k on chalcones 1a-1f, in turn generated as intermediates by aldol condensation reactions between the corresponding 1-acetylnaphthalenes and 1-naphthaldehydes in ethanolic NaOH solution (Scheme 1).

Scheme 1
Formation of the intermediate chalcones 1a-1f was confirmed on the basis of their IR and 1 H-NMR data. They show a characteristic IR absorption peak at v 1700-1650 cm -1 indicating the presence of a conjugated carbonyl group (>C=O). As their 1 H-NMR spectra suggest, the C a -H and C ß -H protons are considerably shifted downfield to the extent that they appear in the aromatic region (6.5-8.5). As a result, these protons can hardly be distinguished from those of the aromatic rings. This is probably associated with the joint deshielding resonance and anisotropic effects of the naphthalene groups bonded to ß-carbon atom.
The chalcones 1a-1f were then reacted with hydrazines 2g-2k to give 2-pyrazoline compounds 3ag-3fk. This reaction probably takes place through mediation of an appropriate a,ß-unsaturated hydrazone, which immediately cyclizes to give a 2-pyrazoline ring in the presence of a suitable cyclizing agent like dry acetic acid under prolonged refluxing condition. Acetylation, in situ, to convert the initially obtained pyrazolines 3ag-3fg into their N-1 acetylated derivatives 3ak-3fk was observed under prolonged refluxing.
All the pyrazolines, in general, exhibited C=N stretching vibrations in the v 1687-1483 cm -1 range which can be difficult to identify due to large variations in intensity and their closeness to C=C and C=O vibrations when present. In addition, the absorption frequencies at v 3583-3485(s), 3456-3220(m), 1677-1630(s) and 834-815(s) indicating the presence of -OH, -NH 2 (usually two bands), >C=O and -Cl groups, respectively, were also typical. 1 H-NMR spectra of these compounds generally exhibit an AMX pattern for the presence of two diastereotopic protons at C-4 and one single proton at the C-5 positions. These protons appear as three doublets of doublets, respectively, in the d = 3.25-3.64, 3.92-4.67 and 5.31-6.28 ppm regions, each integrating for one proton (Table 1). It is interesting to note that, in all the 1 H-NMR spectra, a doublet integrating for one proton appears in the d = 8.92-9.56 ppm region. This probably belongs to the C' 8 -proton of the 3-naphthyl group which suffers a significant paramagnetic shift relative to other remaining aromatic protons. The large deshielding of this proton is attributed mainly to the anisotropy of C=N p-system in pyrazoline rings.

Antimicrobial activity
The compounds were tested in vitro for antimicrobial activity against the test organisms Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabillis, Shigella dysentry and Salmonella typhii at a temperature of 37 °C (±1°C). It was observed that 81% of the total samples tested show some antimicrobial activity. All the compounds showed activity against the test organisms Klebsiella pneumoniae and Staphylococcus aureus. Compounds 3cg, 3eh, 3ci, 3di and 3ei positively acted against all six organisms. Among these, 3di was found to be the most active since this compound could inhibit the microbial growth at lower concentrations. When a comparison is made between the compounds 3ag and 3bg and also between 3ah and 3bh, it a ppears that antimicrobial activity is enhanced due to the presence of an NMe 2 group as a substituent on the naphthalene ring. Furthermore, the comparison of MIC values of compounds in pairs 3bg/3cg, 3bh/3ch and also 3bh/3fh indicates that the -OH and -Cl g roups as substituents on the 3 -naphthyl ring causes a substantial increase in antimicrobial activity. This fact is further supported by comparison of the MIC values of the compounds in pairs 3cg/3dg, 3ch/3dh, 3ck/3dk and 3ci/3di. On the contrary, the comparison of MIC values between the compounds in pairs 3ch/3eh, 3ck/3ek and 3ci/3ei reveals that the presence of a methyl substituent group on the 3 -naphthyl rings contributes almost nothing to the antimicrobial activity. Finally, on the basis of the comparison of MIC values of compounds 3ag-3dg with those of compounds 3ah-3dh, it was observed that the introduction of a Ph substituent group at the N-1 position of the pyrazoline ring results in no significant change in the antimicrobial activity. While on the other hand, from such a comparison between the compounds 3ag-3dg and 3ak-3ek, and also between the compounds 3ag-3dg and 3ai-3ei, it follows that COCH 3 and CONH 2 groups bonded to the N -1 position in the pyrazoline ring have moderate and strong effects on the antimicrobial activity, respectively. Accordingly, among the heterocyclics tested, it was found that compound 3di possesses very high antimicrobial activity, which can be attributed to the simultaneous presence of -OH, -Cl, -NMe2 and -CONH2 substituent groups in this compound.
Therefore, on the basis of the observed MIC values of these compounds, it can be concluded that (i) the compounds containing -OH and -Cl groups in combination with a -NMe 2 group as substituents on the naphthalene rings can act as very effective antimicrobial agents; (ii) the dimethylamino (NMe2), group exerts distinct antimicrobial activity which is independent of the presence of other substituents; (iii) a carboxamido (CONH 2 ) group attached to the N-1 position of the 2-pyrazoline ring presents a substantial contribution in the enhancement of antimicrobial activity; (iv) the general trend for activity in decreasing order can be given as: 3di>3ci>3ei>3cg>3eh>3dk>3dg, 3eg, 3dh>3fi,3ck, 3bi>3fg, 3ek, 3fk>3fh, 3ai>3bk>3bg>3ak>3ch>3bh>3ag >3ah.

General
All melting points were determined on a Büchi 530 melting point apparatus, and are uncorrected. The 1 H-NMR spectra were recorded at ambient temperature for deuteriochlo roform solutions using tetramethylsilane as the internal standard. Jeol FX (at 90 MHz) and Brüker AM (at 200 MHz) spectrometers were used. IR spectra were recorded on a Shimadzu IR-435U-04 instrument using potassium bromide pellets. Elemental analysis was performed at the Iran Polymer Research Center, Karaj, Iran. Antibacterial tests were performed at the Biology Department, Faculty of Science, Bu-Ali Sina University, Hamedan, Iran.
General procedure for the preparation of 3,5-dinaphthyl-2-pyrazoline derivatives (3ag-3fk). Six 1,3-dinaphthyl bearing chalcones 1a-1f were prepared by stirring a mixture of the appropriate 1-acetylnaphthalene and 1 -naphthaldehyde derivative (0.1 mol of each) in ethanolic NaOH solution (80 mL) for several hours at room temperature until a yellow to orange color had developed. Then, the reaction mixture was filtered and the resulting precipitate was washed with 3% aqueous HCl. The crude material was recrystallized from ethanol (96%) to obtain a bright yellow crystalline product. The structures of these chalcones were established on the basis of their chemical properties and spectral data. Further, a solution of each of these chalcones 1a-1f (0.05 mol), in dry acetic acid (100 mL) was then reacted with an appropriate hydrazine derivative for several hours at 90-100°C until the cyclization was complete and a deep orange color developed. The reaction mixture was then evaporated in vacuo to separate the acetic acid, and the residue was recrystallized from ethanol or acetonitrile to obtain the pure crystalline 2 -pyrazolines 3ag -3fi. It was noticed that when these reactions were carried out with hydrazine itself, 2g, under prolonged refluxing conditions, acetylation, in situ, occurred to convert the initially produced 2 -pyrazolines 3ag-3fg into their acetylated derivatives 3ak-3fk (see Table 1 for melting points, yields and elemental data). The structures of these compounds were established on the basis of their chemical properties and spectral analysis ( Table 2).

Antimicrobial activity tests of the 2-pyrazolines
The above prepared 2 -pyrazolines 3ag -3fk, were assayed for their antimicrobial activities against six test organisms, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabillis, Shigella dysentry and Salmonella typhii, at a concentration of 100 mg/mL using the agar well technique [23]. Further, their M IC values against these organisms were determined by serial dilution method using DMF as a solvent and were compared with chloramphenicol as a standard antibiotic. The results obtained are given in Table 3.