Synthesis, Characterization and Anti-fungal activity of Some Novel Heterofused Thienotriazines

 

H R Roopa*, J. Saravanan

Department of Pharmaceutical Chemistry, PES College of Pharmacy, Bangalore-50, Karnataka, India.

*Corresponding Author E-mail: roopad24@gmail.com

 

 

ABSTRACT:

2-amino-3-N-(substituted carboxanilido)-4, 5-disubstituted thiophenes 1a – 1h were synthesized using versatile Gewald reaction. First step is preparation of substituted cyanoacetanilides (Comp. No JSR a and b) which was carried out by  Substituted anilines and Ethyl cyano acetate which was then reacted with Methylenic ketone, ammonium acetate, glacial acetic acid, benzene, sulphur to produce 2-amino-3-N-(substituted carboxanilido)-4, 5-disubstituted thiophenes JSR 1a–1h. Later the compound was subjected to diazotization to yield eight new thieno triazines. The compounds were characterized IR, 1H NMR and mass spectral data and screened for anti-fungal activity.

 

KEYWORDS: Synthesis, Thiophenes, Thienotriazines, Spectral analysis, Antifungal activity

 

 

INTRODUCTION:

In medicinal chemistry among the heterocyclic compounds sulphur containing moieties have attracted maximum attention as they have several  pharmacological  activities  as antimicrobial1-3, anticonvulasant4, antifungal5, analgesic9, antitumor10,ant- inflammatory13, antioxidant activity11 and so on. Similarly thieno triazines6-8,12 derivatives also have been reported  to possess biological activity as antifungal1. The therapeutic importance of these rings prompted us to synthesize novel benzo(b)thiophene and their thieno triazine derivatives, characterize the compound by IR, NMR and Mass spectroscopic techniques and evaluate them for their antimicrobial activity.

 

MATERIAL AND METHODS:

Chemicals:

Substituted anilines (o-anisidine and p-anisidine), ethyl  cyanoacetate,  glacial acetic acid, methylinic ketones (ethyl methyl ketone, cyclopentanone, cyclohexanone, cycloheptanone), benzene, ammonium acetate, sodium sulfate,  sulphur, diethyl amine, ethanol, sodium nitrite and Hydrocholric acid were obtained from local dealer. All other chemical used were of laboratory grade.

 

Preparation of substituted cyanoacetanilides (Comp. No JSR a and b)

A mixture of substituted anilines (0.50 M) and ethyl cyano acetate (56.5 ml; 0.50 M) were taken  in a conical flask, mixed well and heated on a heating mantle at 160-170 ºC for 5-6 hrs. Then the reaction mixture was left at room temperature for overnight. The solid obtained was collected, washed with ethanol and dried. Recrystallization  was done by acetone: water mixture (5:1)

 

Preparation of 2-cyano-2-(methylidene)-N-substituted carboxanilides

A mixture of substituted cyano acetanilide (0.04 M), appropriate methylenic ketone (4.6 ml; 0.04 M), ammonium acetate (1 g) and glacial acetic acid (2 ml) in benzene (100 ml) was refluxed with an arrangement for continuous separation of water involving dean stark apparatus. After 8 hrs the reaction mixture was cooled, diluted with 10 ml benzene and washed with sodium carbonate solution (10% w/v in water) and water successively and dried over anhydrous sodium sulphate. The solvent was removed under vacuum. The intermediate crude product obtained was immediately processed for the next step.

 

Preparation of 2- amino-3-N- substituted carboxanilido - 4,5- disubstitutedthiophenes (Comp. no. JSR 1a- 1h)

To a mixture of 2-Cyano –2- (methylidene) - N- substituted carboxanilides in alcohol (30 ml) was added sulphur (1.28 g; 0.04 M) with stirring maintaining the temperature between 40-45ºC during addition. Then to the reaction mixture, diethyl amine (4.0 ml) was added drop wise with stirring. The reaction mixture was stirred for 1 hr at 40-45ºC and chilled overnight. The solid obtained was filtered, washed with ethanol and recrystallized from isopropyl alcohol.

 

Preparation of thieno triazines (JSR 2a- 2h)

A mixture of the corresponding 2-amino-3-N-(substituted carboxanilido)-4, 5-disubstituted thiophenes 1a – 1h (0.01 M) in 10 ml of glacial acetic acid were warmed until the starting material was dissolved. The mixture was cooled to room temperature, 20 ml of concentrated HCl was added and the reaction mixture was cooled to a temperature below 5oC. To this mixture an ice cold solution of NaNO2 (0.03 M) in water (25 ml) was added drop wise with constant stirring. Temperature was maintained below 5oC. The product separated as bright yellow solid, which was filtered, dried and washed with methanol to obtain pure triazines (JSR 2a – 2h). It was obtained 40-65% yield.

 

Anti fungal studies

All the synthesized compounds were screened for their antifungal activity by agar diffusion method14,15 at a conc of 50µg/ml against Aspergillus niger and candida albicans. After 24 hours of drug addition, Zone of inhibition was measured and recorded. Miconazole Nitrate at 50µg/ml was used as standard in the experiment.

 

 

Scheme

Where:  R = p-OCH3, o-OCH3.

               R1, R2 = -CH3, - (CH2)3, - (CH2)4, - (CH2)5.

 

Table-1 Physical data of  2-Cyano-2-(methylidene) -N-substituted carboxanilides (JSR a and b), 2- amino-3-N-substituted carboxanilido-4,5-disubstituted thiophenes (JSR 1a – 1h) and New thieno-1, 2, 3-triazin – 4 – ones (JSR 2a – 2h)

 

Sr. No.

Comp.

No.

Structure

Recrystalization Solvent

M.W.

(g)

M.P.

(OC)

%

Yield

  TLC

 Solvent 

  System

Rf

 

1

 

JSR-a

 

 

acetone: water

       (5:1)

 

190

 

132

 

66.50

 

Chloroform:

Ethyl acetate(8:2)

 

0.28

 

 

2

 

 

JSR-b

 

 

 

 

acetone: water

                 (5:1)

 

 

 190

 

 

122

 

 

64.33

 

Chloroform:

Ethyl

acetate(8:2)

 

0.34

 

 

3

 

 

JSR-1a

 

 

 

    Isopropyl

      alcohol

 

 

276

 

 

130

 

 

85.18

 

Chloroform:

Ethyl acetate(9:1)

 

   0.24

 

 

4

 

 

JSR-1b

 

 

 

 

 

 

    Isopropyl

       alcohol

 

 

288

 

 

 

142

 

 

 

82.07

 

 

Chloroform:

Ethyl acetate

     (9:1)

 

 

0.78

 

 

5

 

 

 

JSR-1c

 

 

 

 

    Isopropyl

      alcohol

 

 

 

302

 

 

 

150

 

 

 

73.25

 

Chloroform:

Ethyl acetate

    (9:1)

 

 

 

0.70

 

 

6

 

 

JSR-1d

 

 

 

    Isopropyl

       alcohol

 

 

316

 

 

180

 

 

 

67.84

 

Chloroform:

Ethyl acetate

      (9:1)

 

 

0.32

 

 

7

 

 

JSR-1e

 

 

 

 

    Isopropyl

       alcohol

 

 

276

 

 

124

 

 

66.96

 

Chloroform:

Ethyl acetate(9:1)

 

 

0.35

 

8

 

JSR-1f

 

 

 

    Isopropyl

       alcohol

 

288

 

149

 

80.16

 

Chloroform:  Ethyl  

acetate(9:1)

 

0.56

 

9

 

 

JSR-1g

 

 

 

 

Isopropyl alcohol

 

 

302

 

 

131

 

 

69.84

 

Chloroform:

Ethyl acetate(9:1)

 

 

0.74

 

 

 

 

10

 

 

JSR-1h

 

 

 

 

Isopropyl alcohol

 

 

316

 

 

178

 

 

55.36

 

Chloroform:

Ethyl acetate(9:1)

 

 

0.50

 

 

11

 

 

JSR-2a

 

 

 

 

 

 

Ethanol

 

 

287

 

 

 

166

 

 

 

55.12

 

 

 

Chloroform:

Ethyl acetate

(9.5:0.5)

 

 

    0.37

 

 

 

 

12

 

 

 

JSR-2b

 

 

 

Ethanol

 

 

299

 

 

138

 

 

62.25

 

 

Chloroform:

Ethyl acetate

(9:1)

 

 

0.30

 

 

13

 

 

JSR-2c

 

 

 

Ethanol

 

 

313

 

 

160

 

 

60.69

 

Chloroform:

Ethyl acetate

(9.5:0.5)

 

 

0.23

 

14

 

 

JSR-2d

 

 

 

Ethanol

 

 

327

 

 

143

 

 

50.16

 

 

Chloroform:

Ethyl acetate

(9:1)

 

 

0.61

 

15

 

 

JSR-2e

 

 

 

Ethanol

 

 

287

 

 

110

 

 

52.75

 

 

Chloroform:

Ethyl acetate

(9:1)

 

 

0.55

 

 

16  

 

 

JSR-2f

 

 

 

Ethanol

 

 

299

 

 

178

 

 

51.24

 

 

Chloroform:

Ethyl acetate

(9:1)

 

 

0.26

 

 

17

 

 

JSR-2g

 

 

 

 

Ethanol

 

 

313

 

 

140

 

 

45.36

 

 

Chloroform:

Ethyl acetate

(9:1)

 

 

0.74

 

 

18

 

 

JSR-2h

 

 

 

 

Ethanol

 

  327

 

 

152

 

 

40.66

 

Chloroform:

Ethyl acetate

(9:1)

 

 

0.48

 

 

 

Table-2 (Spectral data) of  2-Cyano-2-(methylidene) -N-substituted carboxanilides (JSR a and b),

Comp.

No.

Structure

λmax

(nm)

IR (KBr)

 cm-1

1H NMR

 

 

 

JSR-a

 

 

 

 

279

 

3287.12 (-NH-); 3015.54(Ar-CH); 2987.78 (Ali-CH); 2235.30 (-CN); 1643.8 (C=O); 1564.32(C=C);1552.44(NH-bend); 1047.06 (C-O); 710.34(S-C).

 

_

 

 

JSR-b

 

 

 

 

272

 

3272 (-NH-); 3020.15(Ar-CH);2979.65(Ali-CH); 2250 (CN); 1697 (C=O); 1541 (NH-bend); 1543(C=C); 1123(C-O); 824(C-N).  705.86(S-C).    

 

_

 

Table-3 (Spectral data) 2- amino-3-N-substituted carboxanilido-4,5-disubstituted thiophenes (JSR 1a – 1h)

General structure

 

Comp.

No.

Structure

λmax

(nm)

IR (KBr)

 cm-1

1H NMR

(DMSO)

 

 

 

JSR-1a

 

 

 

 

342

 

3328.7 and 3222.28(-NH2); 3205.61 (-NH-); 3048.22 (Ar-CH); 2924.36(Ali-CH); 1641.48(C=O); 1539.1 (C=C); 1113.08 (C-O).

 

 

_

 

 

JSR-1b

 

 

 

 

349

 

3342.12 and 3312.08(-NH2); 3294 (-NH-); 3024 (Ar-CH); 2929 (Ali-CH); 1656 (C=O); 1521 (C=C); 1242 (C-O).

 

 

_

 

 

 

JSR-1c

 

 

 

 

353

 

3301.33 and 3296.24(-NH2); 3270 (-NH-); 3043 (Ar-CH); 2941 (Ali-CH); 1658 (C=O); 1522 (C=C); 1239 (C-O); 824(C-N).      

 

 

_

 

 

 

JSR-1d

 

 

 

 

 

354

 

3312.11 and 3293.44(-NH2); 3267 (-NH-); 3143 (Ar-CH); 2938.24 (Ali-CH); 1652 (C=O); 1527 (C=C); 1239 (C-0); 824 (C-N).        

 

8.52(s, 1H, -NH, g); 7.53 (d, 2H, Ar-H, h,k); 6.84 (d, 2H, Ar-H, i,j); 5.36 (s, 2H, NH2, f); 3.78  (s, 3H,

-OCH3, ); 2.81(t, 2H, -CH2-, e); 2.61 (t, 2H, -CH2-, a); 1.83 (s, 2H, -CH2-, d); 1.65 (t, 4H,-CH2,b,c). 

 

 

JSR-1e

 

 

 

 

  338

3332.44 and 3220.05(-NH2); 3211.41 (-NH-); 3065.8 (Ar-CH); 2932.26(Ali-CH); 1650.5(C=O); 1542.23 (C=C); 1132.80 (C-O).

8.44(d, 1H, Ar-H,h ); 8.29 (s, 1H,-NH, d); 7.02(m, 2H, Ar-H, f,g); 6.89(d, 1H, Ar-H,e)5.95 (br, 2H, NH2, c); 3.89  (s, 3H, -OCH3, ); 2.33(s, 3H, -CH3, b); 2.20 (s, 3H, -CH3, a). 

 

 

 

JSR-1f

 

 

 

 

341

 

3346.12and3302.04(-NH2); 3263.53(-NH-); 3068.05(Ar-CH); 2976.14 (Ali-CH); 1652.93 (C=O); 1591.95(C=C); 1140.82 (C-O); 824 (C-N).        

 

 

      _

 

 

 

JSR-1g

 

 

 

 

 

346

 

3315.9 and 3239.8(-NH2); 3212.68(-NH-); 3065.28 (Ar-CH); 2945.86(Ali-CH); 1655.42 (C=O); 1572.3 (C=C); 1170 (C-O); 836  (C-N).   

 

 

_

 

 

 

JSR-1h

 

 

 

 

 

349

 

3254 (-NH-); 3011 (Ar-CH); 2972 (Ali-CH); 1647 (C=O); 1540 (C=C); 1292 (C-O); 824 (C-N).        

 

 

 

_

 

Table -4 New thieno-1, 2, 3-triazin – 4 – ones (JSR 2a – 2h)

General structure

 

                                                    Comp.

No.

Structure

λmax

(nm)

IR (KBr)

 cm-1

1H NMR

(CDCl3)

 

 

 

JSR-2a

 

 

 

 

 

 

365

 

3065.45(Ar-H); 2933.26 (Ali-CH); 1694.5(C=O); 1589.4 (C=C); 1329.0 (C-O); 820.23(C-N).      

 

 

7.50 (d, 2H, Ar-H, c,f); 7.04 (d, 2H,- Ar-H, d,e); 3.87 (s, 3H, -OCH3); 2.55 (s, 6H, CH3, a,b)

 

 

 

 

 

 

JSR-2b

 

 

 

 

 

372

 

3076.44(Ar-CH); 2929 (Ali-CH);

1683 (C=O); 1523 (C=C);

1289 (C-O); 824(C-N)

 

7.52 (d, 2H, Ar-H, d,g); 7.04 (d, 2H, Ar-H, e,f);

 3.87 (s, 3H, -OCH3); 3.80(b, 2H, -CH2-, c);

3.12 (m, 2H, -CH2-, b); 2.56 (m, 2H, -CH2-, a). 

 

 

JSR-2c

 

 

 

 

 

 

374

3074.83(Ar-CH);2978.3(Ali-CH); 1687.8 (C=O); 1504.89(C=C); 1065 (C-O); 746 (C-N).709.19(C-S).

 

               _

 

 

 

JSR-2d

 

 

 

 

 

 

375

 

3134.44(Ar-CH); 2898.22 (Ali-CH); 1683 (C=O); 1524 (C=C);1213(C-0); 856 (S-C); 824 (C-N).  

 

7.49 (d, 2H, Ar-H, f,i ); 7.03 (d, 2H, Ar-H, g,h);

 3.87 (s, 3H, -OCH3); 3.37 (t, 2H, -CH2-, e); 2.99(t, 2H, -CH2-,a); 3.37 (t, 2H, -CH2-, e);

2.99 (t, 2H, -CH2-,a).1.82 (m, -CH2-, b,c,d).

 

 

JSR-2e

 

 

  358

3098.44(Ar-CH);2865.66(Ali-CH); 1694.23(C=O);1535(C=C);1198.9(C-0); 886 (S-C); 835 (C-N).  

 

               _

 

 

 

JSR-2f

 

 

 

 

 

 

363

 

3088.48(Ar-CH); 2918.48 (Ali-CH); 1700 (C=O); 1562.4(C=C); 1289 (C-O); 815(C-N).   

 

 

_

 

 

 

 

JSR-2g

 

 

 

 

 

 

 

 

367

 

 

3124.44(Ar-CH);2987(Ali-CH); 1664 (C=O); 1521 (C=C); 1225 (C-O); 824 (C-N).       .

 

 

7.49 (m, 1H, Ar-H, e); 7.37 (d, 1H,- Ar-H, h); 7.10(m, 2H, Ar-H, f,g); 3.81 (s, 3H, -OCH3, ); 3.07(d, 2H, -CH2-, d); 2.91 (t, 2H, -CH2-, a); 1.90 (m, 4H, -CH2-, b,c). 

 

 

 

JSR-2h

 

 

 

 

 

 

 

369

 

3054.82(Ar-CH);2931.91(Ali-CH); 1688.32(C=O);1562.5(C=C)1238.1222.86(C-O);818.25 (C-N); 710.08(C-S)  

 

7.07- 7.51 (m, 4H, Ar-H, f,g,h,i); 3.82 (s, 3H, -OCH3, ); 3.36 (t, 2H, -CH2-, e); 2.99 (t, 2H, -CH2-, a); 1.85 (m, 6H, -CH2-, b,c,d).

 

Table-5 Antifungal screening data of new thieno-1, 2, 3-triazine – 4 – ones (JSR 2a – 2h):

General structure

 

                                                                              JSR 2a-2h

Compound Code

R

R1and R2

    Zone of inhibition (mm)                   

Aspergillusniger

 Candida albicans

JSR 2a

p-OCH3

(CH3)2

04

15

JSR 2b

p-OCH3

(CH2)3

12

13

JSR 2c

p-OCH3

(CH2)4

09

09

JSR 2d

p-OCH3

(CH2)5

11

03

JSR 2e

o-OCH3

(CH3)2

14

12

JSR 2f

o-OCH3

(CH2)3

08

09

JSR 2g

o-OCH3

(CH2)4

13

07

JSR 2h

o-OCH3

(CH2)5

11

16

Miconazole nitrate

----------

---------

25

23

Dose concentration: 50 μg/0.1 ml;  NA : No activity;  Control  : DMF (Dimethyl formamide)

RESULTS:

From the antifungal activity results it was observed that all the compounds influenced the activity. Among the drugs tested for antifungal activities Table 5 showed that compounds JSR-2b, JSR-2e, JSR-2h exhibited potent activity by showing zone of inhibition ranging from 12mm-16mm. All these drugs showed potent activity against Candida albicans and Aspergillus niger with high zone of inhibition. All other drugs showed moderate inhibititory properties against the test organism. Miconazole nitrate exhibited potent inhibitory properties against the entire test organism.

 

DISCUSSION:

From the IR, 1H NMR, and Mass spectrum obtained, characterization of data has been done and given in table 2, 3 and 4. The formation of the new series of thienotriazines were confirmed by the shift of IR peaks between 1627 – 1640 cm -1 as seen in the starting materials to 1690 – 1700 cm -1 in the final compounds indicating the aryl cyclization due to the cyclic keto group and absence of the prominent peaks between 3217 - 3460cm-1of NH-NH2 in triazines compared to their starting thiophenes is sufficient to explain the formation of the new triazines.

 

The NMR spectra of the compounds, (JSR-2a), (JSR- 2b), (JSR- 2d), (JSR- 2g) and (JSR-2h) shows disappearance of a broad peak at δ = 5.3-5.95 of -NH2 and also a sharp singlet peak at δ = 8.52 of –NH which clearly suggest the formation of expected compounds. The compounds JSR-2c and JSR-2e were also confirmed by Mass spectrum.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Management, PES College of Pharmacy for providing necessary facilities.

 

REFERENCES:

1.       Saravanan J et al.Synthesis of some 3-substituted amino-4,5- tetramethylene  thieno[2,3-d][ 1,2,3]-triazin-4(3H)- ones as potential  antimicrobial  agents. Eur J  Med  Chem    2010;45:4365-4369.

2.       Senthil  Kumaran  et  al.  Synthesis  characterization  and antimicrobial activity of some thiophene derivatives. J of Pharm Res 2012; 5(2):1067- 69.

3.       Srivastava S  et  al.  Synthesis  and  evaluation  of  some  novel thiophenes as  potential antibacterial and   mycolytic agents. Der.Pharma Chemica 2011;3(6):103-11.

4.       Kelly, et al. 7-(2-fluorobenzyl)-4-(substituted)-7H-imidazo-[4,5-d]-1,2,3-triazines  and -7H- pyrazolo[3,4-d]-1,2,3-triazines. Synthesis and anticonvulsant activity. J  Heterocyclic Chem 1995:32(5):1417-21.

5.       Chung-KyuRyu, et al. Synthesis and antifungal activity of 5-arylamino-4,7 dioxobenzo[b] thiophenes. Bioorg Med Chem Lett 2005;15:2617-20.

6.       Jakobsen, et al. A facile one-Pot syntesis of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine and  derivatives.  Chem Abstr 1990;114:122286r:790.

7.       Jim Clark, et al. Novel tricyclic compounds containing the pyrido [5,4-b]-1,2,3-triazine system. J ChemSoc Perkin Trans 1986;711-19.

8.       Antonio Monge, et al. An unexpected reaction in the Synhesis of ethyl 1,2,3-triazino [5,4-b] indole-4- carboxylate-3-oxide. J Chem Research(S) 1988;344-45.

9.       Isloor  A  M  et  al.  Synthesis  characterization  and  biological activities of some  new benzo[b] thiophene derivatives. Eur J Med Chem.2010; 45:825-30.

10.     Asmaa A et al. New synthesis of tetra hydro benzo[4,5] thieno [2,3d] pyrimidine derivatives and schiff  bases derived from 2- aminotetrahydro  benzothiophenes  and  hetro arylcarboxyaldehydes studies on their antitumour and antimicrobial  activities.  Available  from  URL: http://idosi.org/wjc/4(2)/2009/4(2);112-117.

11.     Barot H K et al.  Synthesis of  Thiophene  Compounds  of Pharmacological    Interest.    Asian    J  Research    Chem. 2010;3(4):1065-68.

12.     Kavitha P.N et al. Microwave assisted synthesis, charaterization and antimicrobial activity of some schiff bases of 2-amino (4- chlorophenyl) thiazoles. Asian J Research Chem.2010;3(3) :751-54.

13.     Ajay D Pillai, et al. QSAR studies on some thiophene analogues as anti-inflammatory  agents: enhancement of activity by electronic parameters and its utilization for chemical lead optimization. Bioorg Med Chem 2005; 13:1275-83.

14.     Sara Tehranchian, et al., Synthesis and antibacterial activity of 1-[1,2,4-triazol-3-yl] and 1-[1,3,4- thiadiazol-2-yl]-3-methylthio-6,7-dihydro- benzo[c]thiophen-4[5H]ones. Bioorg Med Chem Lett 2005; 15:1023-25.

15.     Barry  A  L,  Antimicrobial  susceptibility  test,  principle  and  practices (Illus lea and fehinger, Philedepephia USA) 1976, pp 93-108.

 

 

 

Received on 19.03.2014         Modified on 25.03.2014

Accepted on 30.03.2014         © AJRC All right reserved

Asian J. Research Chem. 7(3):  March  2014; Page 327-334