INTRODUCTION:

Medical and pharmacological research provides a basis for the development of the new approaches to combat human diseases. Despite the wide availability of clinically useful anti-bacterial¹ and the anti-inflammatory^1,22 analogues a research for new anti-bacterial and anti-inflammatory agents remain indispensable. Some of the major anti-microbial and anti-inflammatory drugs have indeed considerable drawbacks in terms of limited anti-microbial spectrum or serious side effects. There is a great need for developing new anti-infective agents. The discovery of new molecules exhibiting prominent activities against infectious micro-organisms such as toxigenic staphylococci, E.coli, anaerobes, pseudomonas and others showing no cross-resistance with the existing antibiotics. In the present work we have synthesized 3,5 di-substituted pyrazoline derivatives. These nitrogen heterocycle pyrazolines represent another most active classes of compounds possessing a wide spectrum of biological activities. Most of the therapeutically useful drugs like phenylbutazone, oxyphenbutazone, antipyrine belong to pyrazoline exhibit anti-inflammatory²², analgesic²¹, anti-bacterial¹, muscle relaxant action and hypoglycemic activities²¹.

Chalcones have been extensively used to synthesize pyrazolines. In the present study, we use this strategy for the synthesis of new pyrazoline derivatives with the hope that they may possess anti-microbial activities. The chalcones were prepared from reaction of heterocyclic aldehyde with different substituted ketones in the presence of 20% sodium hydroxide in the molar ratio (1:1:1) of (aldehyde:ketone:sodiumhydroxide). This chalcones on condensation with the hydrazine hydrate gives the 3, 5 di-substituted pyrazoline derivatives.

MATERIAL AND METHODS:

The melting points of compounds were determined by open tube capillary using Thermonik precision apparatus in Celsius scale and uncorrected. IR spectra were recorded using KBr pellets on SHIMADZU PERKIN EKMER 8201 PC IR spectrophotometer, 1H NMR spectra of the final compound were recorded on BRUKER DRX 300 NMR spectrometer (300MHz). All spectra were obtained in a mixture of CDCL3 and TFA (Tri-fluro acetic acid).Mass spectra (FAB-MS) were recorded on 70V on Jeol D-300 spectrophotometer (Jeol Ltd., Tokyo, Japan).

GENERAL PROCEDURE:

In the present dissertation 3, 5 di-substituted pyrazolines have been synthesized. The synthesis consists of two steps which are as follows:

Table 2: Physical data of 3,5 di-substituted pyrazolines:

S. No.	Compound no.	Physical nature	MP ( °C )	Yield
1	7-p	Dark green crystals	125 ( °C )	85 %
2	4-p	Yellow crystals	120 °C	88 %
3	16-p	Orange crystals	115 °C	78 %
4	A	Light yellow crystals	120 °C	82 %
5	B	Yellow crystals	115 °C	87 %
6	C	Brown crystals	120 °C	80 %
7	D	Dark brown crystals	117 °C	75 %
8	E	Yellow brown crystals	124 °C	83 %
9	F	Yellow crystals	128 °C	80 %
10	G	Brown crystals	130 °C	85 %

S. No.	Compound no.	Diameter of zone of inhibition (mm)
		Bacterial organisms
		Staphylococcus aureus	Bacillus subtilis	Escherichia coli	Pseudomonas aeruginosa
1	7-p	24	13	14	12
2	4-p	17	16	17	14
3	16-p	23	18	10	15
4	A	22	15	9	11
5	B	24	12	13	11
6	C	21	13	16	12
7	D	18	11	9	14
8	E	19	14	12	12
9	F	16	12	13	11
10	Ampicillin	25	24	25	24

RESULTS AND DISCUSSION:

The physical properties are shown in table-2. All the synthesized compounds have shown anti-bacterial activities to certain extent. Sensitivity testing is carried out on all synthesized compounds as shown in the table. Some of the tested compounds such as 7p, 16p, A, B, C have shown good anti-bacterial activity and some compounds 4p, E, F, and D have shown moderate activity on tested organisms in comparison to the standard drug ampicillin.

MIC (Minimum inhibitory concentration) is also determined on all the synthesized compounds by the tube dilution technique for anti-bacterial studies. (Table-3)

CHARACTERIZATION:

The characterizations of organic compounds have been revolutionized by the progressive adoption of the wide range of spectroscopic techniques. This has been applied extensively in the preparation section to confirm the structure of the expected products. The newly synthesized compounds were subjected to the spectroscopic techniques (NMR, IR, MASS spectrometry).

The characterization requires the identification of molecular frame work, the nature of the functional groups that are present and their location with in the skeletal structure and finally establishment of any stereo chemical relationships, which might exist.

Spectral Features For Sample No: 16-p

[3(phenyl) 5-(4-chloro phenyl ) pyrazolines]

Molecular weight: 265.0

IR in cms1:

Peak at 3437 cms-1 corresponds to –NH group, peaks at 2978.52 cms-1 corresponds to –CH group, Peaks at 1591.95 cms -1corresponds to –C=N- group.

NMR: δ in PPM:

8.9 ( 1 H of –NH ), 7.7 ( 2 H of –CH=C- ), 7.4 ( 2H of –CH=CH- ), 3.7 (1H of –CH< ), 3.2 ( 2H of –CH2-), 2,9 [3H of –N (CH3) 2 ].

Mass in m/z .The molecular ion peak was observed at m/z 264.The fragment ion peak for the phenyl group is at m/z 77. Some of the important fragment ion peaks were observed at m/z 103, 146.

Sample no: 7 p:

[3(para bromo phenyl) 5(Furyl) pyrazolines].

Molecular weight: 291

IR in cms-1: Peaks at 3358.43 cms-1 corresponds to –NH- stretching.Peaks at 1582.31 cms-1 corresponds to –C=N- , Peaks at 1397.17 cms-1corresponds to –CH- .(alkenes),Peaks at 738.0 cms-1corresponds to –CH- (alkenes), Peaks at 2921.0 cms-1corresponds to –CH- stretch.

NMR: δ in PPM 7.8 (1H of -NH-), 7.2 to 7.3 (4 aromatic protons), 4.0 (1 H of –CH<), 2.9 (2H of CH2-).

MASS: .The molecular ion peak was observed at m/z 290.The fragment ion peak of phenyl group is at m/z 77.Some important fragment ion peaks were observed at 102, 181 and 183.

Sample no: 4-p.

[3(phenyl)5-(4-chloro phenyl) pyrazolines].

Molecular weight: 256.0

IR in cms-1: Peaks at 3437.49 cms-1corresponds to –NH- group stretching. Peak is at 1591.0 cms-1corresponds to –C=N group stretching. Peaks at 2978.52 cms-1corresponds to –CH- (alkanes).

NMR: δ in PPM: 7.8 (1H of –NH-), .2 to 7.3 ( 4 aromatic protons ), 4.0 (1H of –CH< ), 2.9 (2H of CH2 ).

MASS in m/z: The molecular ion peak was observed at m/z 255.The fragment ion peak of phenyl group at m/z 77.The important fragment ion peak were observed at 103.

CONCLUSION:

A Series of 3,5 pyrazoline were designed on the basis of Literature review on various pyrazoline. All the synthesized 3,5 di-substituted pyrazoline derivative remitted in product with good yield purity of all the synthesized compound were checked by their melting point. All the synthesized compounds have shown anti-bacterial activity. on tested organisms in comparison to standard drug against ampicillin. As we consider all results obtained from anti-bacterial tests together we can say that the entire compounds tested are active towards bacteria.

By analysis of spectral data of the representative compounds reveals the successful information of 3,5 di-substituted pyrazoline and also about different aromatic group at position 3 and 5.By the IR spectral analysis we can identify some of the group of 3,5 di-substituted pyrazoline derivatives such as –NH,-C=N etc. By the msss spectral analysis we can identify the compounds by parent ion peak and fragment ion peaks.

And finally by NMR spectral analysis we can identify the number of protons in our compound according to number of peaks formed.

ACKNOWLEDGEMENTS:

The authors are thankful to Indian Institute of science for their valuable help in the fast processing of spectral data and also express heartly gratitude and thankful to our esteemed guide and teacher Dr. prof. E. V. S. Subrahmanyam, principal, NGSM institute of pharmaceutical sciences, Mangalore for his guidance, valuable suggestions and constant encouragement throughout the period and providing all the facilities required for the work.

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