Synthesis, Characterization and Biological Evaluation of N [3-Chloro-2 ...

Sayyed Hussain, et al : Synthesis, Characterization and Biological Evaluation of …

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International Journal of Drug Design and Discovery Volume 2 Issue 3 July – September 2011. 527-532

Synthesis, Characterization and Biological Evaluation of N [3-Chloro-2 (aryl)-4-oxoazitidin-1-y] pyridine-4-carboxamide Sayyed Hussain1*, Shivaji Jadhav1, Megha Rai2 and Mazahar Farooqui2,3 1

Department of Chemistry, Sir Sayyed College, Aurangabad 431001 (M S).

2

Dr Rafiq Zakaria College for Women, Aurangabad.

3

Post graduate and research centre, Maulana Azad College, Aurangabad.

ABSTRACT:

Isoniazid on addition with different aromatic aldehyde gives Schiff’s bases. The Schiff base so formed on treatment with chloroacetyl chloride and triethyl amine as a base catalyst in 1-4 Dioxan gives product 2a-2n.The synthesized compounds characterized by spectral analysis like IR, NMR & Mass and elemental analysis. The compounds were screened by anticonvulsant and antimycobacterial activity.

KEY WORDS: Isoniazid; Aromatic aldehyde (Ar-CHO) Azitidinone; base catalyst anticonvulsant; antimycobacterial activities

Introduction

Experimental Section 14

Heterocyclic compounds promote the life on earth . They are widely distributed in nature and essential to life as they play role in metabolism of living cells14. Heterocyclic ring system containing N, S, and O heteroatom exhibited chemotherapeutic and other medicinal uses. Azitidinones are a group of compounds possessing a wide spectrum of biological activities. Such as anticonvulsant and antimycobacterial. Ioniazid is effective in the treatment of TB patients and azitidinones is active as antitubercular4,6. Recently 2anticonvulsants2,3, azitidinones have been assessed for antiparkinsonism7, antiinflammatory2, 4, herbicidal4. They also function as an enzyme inhibitors4 and effective on the central nervous syst4,8. The azitidinones were tested as antidepressant7, sedatives7, and are also associated with hypnotic2,8 antimicrobial2,4 antiviral and anesthetic2 activities. In the present study a novel series of azitidinones were synthesized and characterized by means of IR, H1NMR, Mass spectral analysis and elemental analysis. In the biological investigation, the compound were screened for anticonvulsant by maximal electroshock (MES) method and antimycobacterial activities against standard strain H37Rv and 2-Human strains of mycobacterium. * For correspondence: Sayyed Hussain, Tel./ Fax: 91-240-2313876;

Mob.: 9923320201

(a) General method of synthesis of Schiff bases (1a-1n): A mixture of equimolar quantities of Ar-CHO and isoniazid were refluxed for 1 hour in 50ml of ethanol with few drops glacial acetic acid. The reaction mixture was cooled at RT and pour in ice cold water. The separated product was filtered out. The Schiff bases obtained was used for final step to form 2-azitidinones. (b) General method of synthesis of 2-azitidinones (2a-2n): A mixture of Schiff base (0.002mol) and TEA (0.004mol) was dissolved in 1-4-Dioxan (50ml). To this well stirred cooled solution of acetyl chloride (0.004mol) was added drop wise within 20 min. The reaction mixture was stir for three hours and left at RT for 48 hours. The resultant mixture was concentrated, cooled, pour into ice cold water, filter, dried and recrystallized form n-Hexane, spirit which gives compounds 2a-2n. Melting points were determined in open capillary tube and were found uncorrected. The purity of test compounds were determined by TLC on precoated SiO2 gel (HF2s4 200 mesh) Aluminium plates (E-Merck). A single spot is obtained on TLC confirmed the purity of substituted azitidinones and yield is calculated (w/w). The physical data of compound (2a-2n) are given in table 1.

E-mail: [email protected]

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International Journal of Drug Design and Discovery Volume 2  Issue 3 July – September 2011

Reaction Scheme

[Ar-CHO: Aromatic aldehyde]

Table 1 Physical data of compounds (2a-2n) Sr. No.

Compound

1

2a

2

2b

3

Ar group

Mol. Formula

Mol. Wt.

Melting point

Yield (%)

2-hydroxy phenyl

C15H12CIN3O3

317.72

205

53

3-hydroxy phenyl

C15H12CIN3O3

317.72

209

63

2c

4-hydroxy phenyl

C15H12CIN3O3

317.72

195

80

4

2d

3,4-dihydroxy phenyl

C15H12CIN3O4

333.72

215

70

5

2e

4-methyl phenyl

C16H14CIN3O2

315.75

208

68

6

2f

4-methoxy phenyl

C16H14CIN3O3

331.70

183

58

7

2g

3,4,5-trimethoxy phenyl

C18H20CIN3O5

393.8

194

61

8

2h

4-hydroxy,3-methoxy phenyl

C16H14CIN3O4

347.75

196

45

9

2i

4-dimethylamino phenyl

C17H17CIN4O2

344.70

204

62

10

2j

4-Nitro phenyl

C15H11CIN4O4

346.72

205

48

11

2k

3-nitro phenyl

C15H11CIN4O4

346.72

216

55

12

2l

4-chloro phenyl

C15H11CI2N3O2

336.17

181

46

13

2m

2-chloro phenyl

C15H11CI2N3O2

336.17

188

63

14

2n

cinnamyl

C18H16CIN3O2

341.79

206

68

Above synthesis compounds were characterized by IR H1NMR mass spectral analysis. The IR spectra of the compounds were recorded on spectrophotometer (shimadzu) using KBr disc method. H1NMR was recorded

on 300m Hz – Berker DPX200. The chemical shifts are reported as ppm downfield from TMS. The mass spectra were recorded on Finnigan MAT 8230 and the spectral data of compounds 2a-2n are given in table 2.


Table 2 Spectral analysis of compounds (2a-2n) Compound

Ar-group

IR (KBr) cm–1 -1

NMR (CDC/3) 

Mass

2a

2-Hydroxy phenyl

(-lactum: C=O): 1717cm Ar-H: 3019, C–Cl: 752, C=N: 1627, Ar-OH: 3196

11.3 (S, 1H Ar-OH), 8.7 (S,1H, Co-NH) 6.9-7.4 (M, 7H, Ar-H), 3.7 (d, 1H, N-CH) 4.7 (d, 1H, CH-Cl)

+ M = 317 + B = 288

2b

3-Hydroxy phenyl

(-lactum: C=O): 1674 Ar-H: 3026, C–Cl: 748, C=N: 1625, Ar-OH: 3200

11.2 (S, 1H Ar-OH), 8.7 (S, 1H, Co-NH) 6.9-7.4 (M, 7H, Ar-H), 4.6 (d, 1H, CH-Cl) 3.2 (d, 1H, N-CH)

+ M = 317 + B = 288

2c

4-Hydroxy phenyl


11.2 (S, 1H Ar-OH), 8.6 (S, 1H, Co-NH) 6.9-7.9 (M, 7H, Ar-H), 4.5 (d, 1H, CH-Cl) 3.2 (d, 1H, N-CH)

M+ = 317 + B = 288


10.8 (S, 1H Ar-OH), 8.6 (S, 1H, Co-NH) 6.9-7.7 (M, 7H, Ar-OH), 3.9 (d, 1H, C-Cl) 2.4 (d, 1H, NCH)

M+ = 332 + B = 288

8.6 (S, 1H CONH), 7.2-7.7 (M, 7H, Ar-H), 4.6 (d, 1H, CH-Cl) 3.7 (d, 1H, N-CH), 2.4 (Ar-CH3)

2d

3,4-dihydroxyphenyl

2e

4-Methyl phenyl

(-lactum: C=O): 1681 Ar-H: 3056, C–Cl: 712, C=N: 1621, Ar-CH3: 2851, 2923

2f

4-Methoxyphenyl

(-lactum: C=O): 1734 Ar-H: 3048, C–Cl: 780, C=N: 1619, Ar-OCH3: 1251

8.6 (S, 1H CO-NH), 6.9-7.7 (M, 7H, Ar-H), 4.6 (d, 1H, CH-Cl) 3.8 (Ar-OCH3), 3.6 (d, 1H, N-CH)

2g

3,4,5-Trimethoxy phenyl

(-lactum: C=O): 1749 Ar-H: 3061, C–Cl: 764, C=N: 1622, Ar-OCH3: 1232, 1237

8.5 (S, 1H CONH), 7.0-7.2 (M, 7H, Ar-H), 4.6 (d, 1H, CH-Cl) 3.7 (d, 1H, N-CH)

2h

4-Hydroxy 3-Methoxy phenyl

(-lactum: C=O): 1778 Ar-H: 3042, C–Cl: 753, Ar-OH: 3185, Ar-OCH3: 1237

9.8 (S, 1H, Ar-OH), 8.6 (S, 1H, CO-NH) 7.1-7.6 (M, 7H, Ar-H), 4.6 (d, 1H, CH-Cl) 3.7 (d, 1H, NCH), 3.9 (Ar-OCH3)

2i

4-Dimethyl aminophenyl

(-lactum: C=O): 1772 Ar-H: 3048, C–Cl: 743, C=N:1606, N-CH3: 2851, 2920

8.5 (S, 1H, CONH) 6.7-7.7 (M, 7H, Ar-H), 4.7 (d, 1H, CH-Cl) 3.7 (d, 1H, N-CH)

2j

4-nitrophenyl

(-lactum: C=O): 1783 Ar-H: 3062, C–Cl: 745, C=N: 1628, Ar-NO2: 1346

8.5 (S, 1H, CO-NH) 6.4-7.6 (M, 7H, Ar-H), 4.6 (d, 1H, CH-Cl) 4.2 (d, 1H, N-CH)

+ M = 346 + B = 330

2k

3-nitrophenyl



M+ = 346 B+ = 330

2l

p-chlorophenyl



+ M = 336 + B = 290

2m

2-chlorophenyl

(-lactum: C=O): 1771 Ar-H: 3028, C–Cl: 747, C=N: 1621


+ M = 336 + B = 290

2n

cinnamyl

(-lactum: C=O): 1763 Ar-H: 3048, C–Cl: 748, C=N: 1628

8.3 (S, 1H, CO-NH) 6.6-7.5 (M, 7H, Ar-H), 4.2 (d, 1H, CH-Cl) 3.9 (d, 1H, N-CH)

M+ = 327 B+ = 106

+ M = 315 + B = 290

M+ = 331 + B = 65 M+ = 391 B+ = 65

M+ = 347 + B = 288

+ M = 344 + B = 290

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International Journal of Drug Design and Discovery Volume 2  Issue 3 July – September 2011

Elemental analysis of given compounds were carried out, percentage of elements C, H, N, Cl & O are recorded at Workhardt Pvt. Ltd. and given in table 3.

Biological Evaluation Anticonvulsant evaluation by Maximal Electro Shock (MES) Method: The anticonvulsant activities of the compounds were evaluated by maximal electro shock method using rats. Where the electro shock is applied through the corneal electrode producing optic stimulation cortical excitation. The MES convulsions are divided into five phases such as (a) Toxic flexion (b) Tonic extension (c) clonic convulsion (d) stupor and (e) Recovery or death. A drug is known to posses anticonvulsant properties. If it reduces or abolishes the extensor phase of MES convulsions, for the evaluation anticonvulsant activity the total 16 groups of animals were kept fasting for 10-14 hrs. The animals were divided into 16 groups each containing 6-animals. In the 14 groups were served for testing the synthesized compounds, one as control and one as standard (Phenytoin 25mg/kg of body weight) was used as a standard drug. The activities of each group were measured after the intervals of half-an-hour are compounds administering including control and standard. Results and data are given in table 4.

Antimycobacterial evaluation In vitro anti-mycobacterial screening was done for the synthesized compounds 2c, 2e, 2f, 2i. Screened against standard strain H37Rv and two human strain [Human strain-I and Human strain-II] isolated from patients

suffering from pulmonary tuberculosis in different concentration from 12.5, 25, 50, 100, 200, 400 g/ml and the isoniazid was used as standard at a cone of 50 g/ml. Material Required 1. Lowenstein-Jensen (LJ) medium slopes containing various concentration of the compound. 2. control strain H37Rv 3. Two strains of Mycobacterium tuberculosis isolated from patients suffering from pulmonary tuberculosis. Preparation of drug containing slopes The synthesized compounds were dissolved in DMSO and added to the egg fluid salt solution in such a way that to give final concentration of 12.5, 25, 50, 100, 200, 400 g of the compounds per ml of the medium. The above was inspissated at 900C for 50 minutes only once. Preparation of the bacterial suspension The bacterial culture of a control strain and 2 test strain about 2/3 loop full (3mm internal diameter) is mixed with 1ml sterile distilled water in BIJOU bottle containing 3-5, glass beads, shaken in a vertexed bottle for about 1mm to get a uniform suspension. Susceptibility test procedure 1 loop full of bacterial suspension was inoculated on L.J. medium slopes containing the test compounds. A drug free slope is also included as a control. All the slopes were incubated at 370C for 15 days. The result and data are given in table 5 and 6.

Table 3 Elemental analysis data of compounds (2a-2n) Compound

Ar group

Elemental Analysis C

H

Cl

N

O

2a

2-hydroxy phenyl

56.70

3.81

11.16

13.23

15.11

2b

3-hydroxy phenyl

56.70

3.81

11.16

13.23

15.11

2c

4-hydroxy phenyl

56.70

3.81

11.16

13.23

15.11

2d

3,4-dihydroxy phenyl

53.98

3.62

10.62

12.59

19.18

2e

4-methyl phenyl

60.86

4.47

11.23

13.31

10.13

2f

4-methoxy phenyl

57.93

4.25

10.89

12.67

14.47

2g

3,4,5-trimethoxy phenyl

55.18

4.63

9.05

10.72

20.42

2h

4-hydroxy,3-methoxy phenyl

55.26

4.06

10.19

12.08

18.40

2i

4-dimethylamino phenyl

59.22

4.97

10.28

16.25

9.28

2j

4-Nitro phenyl

51.96

3.20

10.23

16.16

18.46

2k

3-nitro phenyl

51.96

3.20

10.23

16.16

18.46

2l

4-chloro phenyl

53.59

3.30

21.09

12.50

9.52

2m

2-chloro phenyl

53.59

3.30

21.09

12.50

9.52

2n

cinnamyl

63.25

4.74

10.37

12.29

9.36


Table 4 Anticonvulsant activity of some synthesized compounds by MES method: Mean time in various phases of convulsions (seconds)  standard error mean

Compound

Recovery

MES Test

Percentage protection (% abolition of tonic extensor phase) --

Flexion

Extensor

Control

6.833  0.40

10.00  0.73



0/6

2a

3.67  0.21

1.83  0.83



3/6

50 %

2b

3.33  0.21

2.16  0.74



2/6

33.33 %

2c

4.66  0.33

2.00  0.69



2/6

33.33 %

2d

4.33  0.49

4.66  0.49



0/6

--

2e

5.33  0.61

5.00  0.57



0/6

--

2f

4.33  0.42

1.00  0.63



4/6

66.66 %

2g

6.66  0.66

7.30  0.61



0/6

--

2h

4.66  0.49

1.00  0.63



5/6

83.33 %

2i

5.66  0.33

9.16  0.74



0/6

--

2j

6.66  0.33

9.83  0.60



0/6

--

2k

6.16  0.47

8.66  0.49



0/6

--

2l

6.50  0.42

9.66  0.61



0/6

--

2m

4.66  0.22

3.66  0.33



0/6

--

2n

4.83  0.47

5.33  0.88



0/6

--

Standard

4.00  0.36

0.00  0.00



6/6

100 %

Table 5 Antimycobacterial activity of compound 2c, 2e, 2f, 2i H37Rv

Human Strain I

Human Strain II

Conc. g/ml

2c

2e

2f

2i

2c

2e

2f

2i

2c

2e

2f

2i

12.5

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

25

++

+++

+++

+++

++

+++

+++

+++

++

+++

+++

+++

50

–

++

++

+

+

++

++

+

+

++

++

++

100

–

–

–

–

–

–

+

+

+

+

+

+

200

–

–

–

–

–

–

–

–

–

–

–

–

400

–

–

–

–

–

–

–

–

–

–

–

–

–

: No growth of mycobacterium tuberculosis

+

: Growth of mycobacterium tuberculosis below 100 colonies

++

: Growth of mycobacterium tuberculosis between 100-200 colonies

+++ : Growth of mycobacterium tuberculosis above 200 colonies

Table 6 Minimum Inhibitory Concentration Compounds

Minimum Inhibitory Concentration H37Rv

Human Strain I

Human Strain II

2c

50

100

200

2e

100

100

200

2f

100

200

200

2i

100

200

200

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International Journal of Drug Design and Discovery Volume 2  Issue 3 July – September 2011 [4]

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Conclusion

[13]

Substituted azetidinone and their derivatives are in important class of heterocyclic compounds with a diverse anti convulsant and antimycobacterial activity.

B.P. Chaudhari and V.V. Mulwad, Ind. J. Hetero. Chem. 12, 197-200 (2003).

[14]

S.K. Kulkarni, Hand book of expt. Pharmacology, PP. 6872.

[15]

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Results and Discussion All the synthesized compounds exhibit significant to moderate anticonvulsant activity. Compounds 2a, 2b, 2c, 2d, 2e, 2h, 2m, 2n showed significant anticonvulsant activity in the flexion phase, other compounds showed moderate activity. In the extensor phase 2g showed most significant activity when compared with 2a, 2b, 2c, 2d, 2e, 2h, 2m, 2n. Compounds 2i, 2j, 2k, and 2l showed reduction in the duration of the flexion and extensor phase. 2e and 2g least active in the flexion and showed significant activity in extensor phase. Compounds 2c, 2e, 2f, 2i showed significant antitubercular activity. The IR spectra of synthesized compounds given significant peak of -lactum ring in between 1681cm-1 to 1770cm-1 and suitable NMR and Mass spectral data.

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