Triazolone and their Derivatives for Anti -Tubercular Activities

 

Maste Meenaxi M.¹*, Ainapure R.², Patil P. B.², Bhat A. R.²

KLE University’s College of Pharmacy, JNMC Campus, Belgaum-590 010, Karnataka

*Corresponding Author E-mail: menai_us@yahoo.com

ABSTRACT:

The Nitro containing 5-oxo-[1,2,4,]triazole ring were synthesized by the nitration of compound (I) with nitric acid. Then acetic acid ethyl esters containing 5-oxo-[1,2,4,]triazole ring (II) were synthesized by the condensation of compound (I) with ethyl chloroacetate in basic media. The reaction of compound (II) with hydrazine hydrate led to formation of acid hydrazide (III). The treatment of compound(III) with different aromatic aldhydes resulted in the formation of arylidene hydrazide’s as target compounds. The structure of the synthesized Compounds were evaluated by IR, ¹H NMR spectra and elemental method of analysis. All the synthesized compounds were screened for their anti-tubercular activity. Anti-tubercular activity was carried out by using middle brook 7H9 broth base (M198) medium against Mycobacterium tuberculosis of H₃₇Rv Strain. Streptomycin was used as standard drug for comparison.

 

 

 

INTRODUCTION:

Tuberculosis is still a major treat to mankind. The increasing problem of Multi-Drug Resistant-tuberculosis has focused attention on developing new drugs that are not only active against drug resistant tuberculosis, but also shorten the lengthy therapy. There is urgent need and significant interest in developing new tubercular drugs. In developing new tubercular drugs, it is crucial to think about which targets in the tubercule bacillus are good drug targets. Several recent reviews on this topic are already available. This review will provide a brief update on the most recent developments in current TB drug discovery efforts.¹

 

A new dimension was added in the year 1980 due to the spread of HIV with high prevalence of tuberculosis and Mycobact avium complex infection among the patients².

 

The present first line drugs like INH, Pyrazinamide, Ethambutol, and Rifampicin are potent antitubercular agent. They act by inhibition of mycolic acid and RNA / DNA synthesis. They possess numerous adverse reactions³. To avoid these effects it seemed promising to look for more selective compounds, at other targets to suppress the activity⁴.

 

Lots of modifications have been made during last decades on Triazolone nucleus and their derivatives have been studied extensively for their biological activities. A survey of literature revealed that these Triazolone derivatives possess different types of potential biological activities that include Antioxidant⁵ antifungal,⁶ antihaemostatic⁷ antibacterial⁸.

 

Substituted aromatic aldehyde moiety with Triazolone nucleus for the first time has been associated to be designed for biological activity. As Triazolone nucleus moiety possess potent anti-tubercular activity⁸. From all the above forgoing facts it was thought and considered to be very interesting to synthesis new series of Triazolone derivatives fused with aromatic aldehyde for anti-tubercular activity.

 

MATERIALS AND METHODS:

The melting points of compounds were determined by open capillary tube method and are uncorrected (Table No 2). Synthesized compounds were subjected to ¹HNMR. IR spectra were recorded on spectrophotometer using KBr disc method. All solvents used were analytical grade.

 

Synthesis of 1,2,4,-triazolin-5-one⁹: A mixture of semicarbazide hydrochloride. (10g, 0.90 mol), trimethyl orthoformate (21.22g, 2.0 mol) and methanol (10 ml) was stirred at 20°C for 3 days. The solvent was then removed under reduced pressure and toluene  (10 ml) was added and the slurry was concentrated further to remove residual methanol, mixture was then cooled to 0°C and filtered to afford 1,2,4,-triazolin-5-one as a white solid.

3-Nitro-1,2,4-triazol-5-one (nitration)¹⁰: Nitric acid (90ml) was added to the (10g) of triazolone marinating the temperature between 0°C to 5°C. The mixture was heated to 60°C -70°C with constant stirring. The reaction was exothermic and brown fumes were evolved. After sometime mixture was chilled (0°C to5°C) in an ice-bath, filtered and washed with water to remove excess nitric acid. Pure nitrotriazolone was crystallized by water and Yield was found to be 80%.

 

General procedure for Mannich base condensation¹¹:A mixture of nitrotriazolone (0.01mol) in ethanol (15ml), aromatic amine (0.02mol) and formaldehyde (0.02mol) was added. The reaction mixture was refluxed for a period 2-6-hours, the solvent was poured into ice water. Resulting solid was filtered off dried and recrystallized using appropriate solvent.

 

N- substituted ethyl ester of 3-4-di substituted 1,2,4,-triazolin-5-one acetic acid ¹²:  Ethylchloroacetate (0.01mol) was added to a corresponding compound(I) (0.01mol) in dry acetone (20ml) to that solution potassium bicarbonate (1gm) was added and the mixture was refluxed for 10 hours, Acetone was removed after completion of  the reaction and the residue was crystallized from ethanol.

 

 

Table No.1: IUPAC names of the synthesized compounds

Compounds	Structures	IUPAC Names
A1		N-(2,4-dichlorophenyl) methylidene]-2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide.
A 2		N-(2,4- dichlorophenyl) methylidene]-2-(3-nitro-4{[(3-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide
A3		N-(4- methylphenyl) methylidene]-2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide.
A4		N-(2 chlorophenyl)- methylidene]-2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide
A5		N-(4-methoxyphenyl methylidene]-2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide
A6		N-(4-hydroxyphenyl) methylidene]-2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide
A7		N-(2,4- dimethoxyphenyl) methylidene]-2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide.
A8		N-(3,4- dichlorophenyl) methylidene]-2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide.
A9		N-(4- chlorophenyl) methylidene]-2-(3-nitro-4{[(4- nitrophenyl) amino] methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl) acetohydrazide
A10		2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-N-[phenylmethylidene] acetohydrazide
A11		N-(3,4- dichlorophenyl) methylidene]-2-(3-nitro-4{[(3- nitrophenyl) amino] methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl) acetohydrazide
A12		N-(4- hydroxyphenyl) methylidene]-2-(3-nitro-4{[(3- nitrophenyl)amino] methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohydrazide
A13		2-(3-nitro-4{[(4-nitrophenyl)amino]methyl}-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-N-[phenylmethylidene] acetohydrazide

 

 

Table No. 2:  Analytical data of the synthesized compound

Compd.	Mol. Formula	Mol. Wt.	m.p 0 C	Yield %	Elemental analyses Cal.			Rf Value .
					C	N	H
A1	C18H14Cl2N8O6	509	122	71	42.45 (42.43)	22.00 (22.0)	2.77 (2.77)	0.4
A2	C18H14Cl2N8O6	509	135	63	42.45 (42.43)	22.00 (22.0)	2.77 (2.7)	0.5
A3	C19H18N8O6	454	110	60	50.22 (50.22)	24.66 (24.6)	3.99 (3.99)	0.4
A4	C18H15ClN8O6	474	118	65	45.53 (45.57)	23.60 (23.6)	3.18 (3.18)	0.6
A5	C19H18N8O7	470	125	65	48.51 (48.51)	23.82 (23.8)	3.86 (3.86)	0.5
A6	C­18H16 N8O7	456	145	69	47.37 (47.37)	24.55 (24.5)	3.53 (3.53)	0.7
A7	C­20H20 N8O8	500	127	69	48.00 (48.00)	22.39 (22.4)	4.03 (4.0)	0.4
A8	C­18H14Cl2N8O6	509	151	61	42.45 (42.43)	22.00 (22.0)	2.77 (2.77)	0.6
A9	C­18H15ClN8O6	474	112	69	45.53 (45.57)	23.60 (23.6)	3.1 (3.1)	0.4
A10	C18 H16N8O6	440	146	72	49.09 (49.09)	25.45 (25.4)	3.66 (3.66)	0.7
A11	C18 H14Cl2N8O6	509	110	62	42.45 (42.43)	22.00 (22.0)	2.77 (2.77)	0.5
A12	C18 H16N8O7	456	134	61	47.37 (47.37)	24.55 (24.5)	3.53 (3.53)	0.6
A13	C18 H15N9O8	485	129	65	44.54 (44.53)	25.97 (25.9)	3.11 (3.11)	0.5

3-4-di substituted 2-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-N-acetohydrazide¹³: A solution of corresponding compound (II) (0.01mol) in n-butanol was refluxed with hydrazine hydrate (0.025mol.) for 4 hours, after cooling to room temperature a solid was appeared and this was recrystallized from an ethanol to  afford the desired product.

 

Synthesis of 3-4-di substituted 2-(5-oxo-4,5-dihydro- 1H-1,2,4- triazol-1-yl)-N-methylidene] acetamide ¹⁴.A solution of the corresponding compound (III) (0.01 mol) in ethanol was refluxed with appropriate aldehyde  (0.01 mol) for 3 hours. after cooling to room temperature, a white solid appeared. This crude product was recrystallized from an ethanol to afford the desired product. IUPAC names of all synthesized compounds are mentioned in Table No 1.

 

The antitubercular screening was carried out by Middle brook 7H9 agar medium against H₃₇Rv Strain. Middle brook 7H9 agar medium containing different derivatives (A₁ – A₁₃), standard drug as well as control, Middle brook 7H9 agar medium was inoculated with Mycobacterium tuberculosis of H₃₇Rv Strain. The inoculated bottles were incubated for 37°C for four weeks. After four weeks they were checked for growth.

 

SHEME:

 

RESULTS AND DISCUSSION:

The 3,4-disubstituted-1,2,4,-triazolin-5-one(I)was synthesized by reacting semicarbazide hydrochloride and trimethyl orthoformate in methanol. Treatment of these compounds (I) with ethylchloroacetate  in dry acetone and anhydrous K₂CO₃ yielded N-alkylated products, N- substituted ethyl ester of 3-4-di substituted 1,2,4,-triazolin-5- one acetic acid.(II)  Hydrazine hydrate and esters (II) in ethanol were refluxed for 5 hours to give desired 3-4-di substituted -2-(5-oxo-4,5-dihydro-1H-1,2,4- triazol-1-yl)acetohydrazide(III).3-4-di substituted - 2-(5-oxo-4,5-dihydro- 1H-1,2,4- triazol-1-yl)-N-methylidene] acetamide(A₁-A₁₃) were obtained upon by reaction of acid hydrazide (III) with  different aldehydes in butanol to give the final products.

 

The IR spectrum of the compounds (A₁-A₁₃) showed peaks at 3200-3361 cm^-1, NH stretching; 1650-1750 cm^-1, C=O stretching (Table No 3). The NMR spectrum of the compound (A₁-A₁₃) showed 8.3 (s, 1H, N=CH), 10.2 (s, 1H NH), 5.2 (s, N-CH₂) (Table No 4). Compounds (A₁, A₃, A₅, A₁₁) have shown antitubercular activity, which appeared to be promising (Table No 5). Mycobacterium tuberculosis H37 Rv strain was used as standard test organism. Streptomycin was used as standard drug for comparison. The MIC of streptomycin is 10mcg/mm.

 

Table No. 3: Infra Red spectral study of  synthesized compounds.

Compound Code	IR Frequency in cm-1	Type of vibrations
A1	753 1631 1593 3039 3359 1473	Ar-Cl  Str -C=O -NO2 Str = CH Str =NH Str = CN Str
A2	753 1631 1597 3056 3360 1478	Ar-Cl Str -C=O -NO2 Str = CH Str =NH Str = CN Str
A3	2850 1700 1505 3050 3300 1480	-CH3 Str -C=O -NO2 Str = CH Str =NH Str = CN Str
A4	650 1700 1530 3050 3300 1475	Ar-Cl  Str -C=O -NO2 Str = CH Str =NH Str = CN Str
A5	1160 1632 1505 3081 3360 1474	- OCH3 -C=O -NO2 Str = CH Str =NH Str = CN Str
A6	3360 1631 1595 2981 3360 1470	-OH (free) -C=O -NO2 Str = CH Str =NH Str = CN Str
A7	1145 1631 1593 3051 3361 1473	- OCH3 -C=O -NO2 Str = CH Str =NH Str = CN Str
A8	650 1631 1505 3081 3360 1473	-Ar-Cl Str -C=O -NO2 Str = CH Str =NH Str = CN Str
A9	628 1632 1505 3051 3360 1444	-Ar-Cl Str -C=O -NO2 Str = CH Str =NH Str = CN Str
A10	1632 1594 3217 3360 1444	-C=O -NO2 Str = CH Str =NH Str = CN Str
A11	669 1690 1505 3081 3330 1346	-Ar-Cl Str -C=O -NO2 Str = CH Str =NH Str = CN Str
A12	3360 1631 1595 2981 3320 1405	-OH (free) -C=O -NO2 Str = CH Str =NH Str = CN Str
A13	1630 1506 3181 3362 1300	-C=O -NO2 Str = CH Str =NH Str = CN Str

 

Table No. 4:¹H NMR spectral study of synthesized compound.

No. of protons compound code A3	δ  values in ppm
3H of CH3 2H Of N-CH2 2H of CH 2H of NH 8H of Ar-CH	2.5 5.3 8.4 9.8 6.6-7.93

 

Table No. 5: Antitubercular activity of the synthesized compounds

Sr. No.	Compounds	5 mcg/mL	10mcg/mL	25mcg/mL
1.	A1	S	S	S
2.	A2	R	S	S
3.	A3	S	S	S
4.	A4	R	R	R
5.	A5	S	S	S
6.	A6	R	S	S
7.	A7	R	R	R
8.	A8	R	R	R
9.	A9	R	R	S
10.	A10	R	R	R
11.	A11	S	S	S
12.	A12	R	S	S
13.	A13	R	R	S
STD.	Streptomycin	S	S	S

 

CONCLUSION:

From the above result it is evident that suitable molecular manipulation can still bring about compounds which could prove equal or better activity compared to the standard drug.

 

ACKNOWLEDGEMENT:

The authors are thankful to Prof. Dr. F.V. Manvi Principal and Prof. A.D. Taranali KLE’s college of Pharmacy, Belgaum for providing facilities and encouragement.

 

REFERENCES:

1)       World health organisation. global tuberculosis control; WHO report 2001; (b) world health organisation: Geneva, Switzerland; WHO/CDS/TB/2001.287. (c) world health organisation tuberculosis Fact Sheet, No. 104, 2000; http://www.who.int/inf-fs/en/fact104.html.

2)       N. Mooran, world health organisation issues another gloomy tuberculosis report, Nature Medicine, 2; 1996: 377.

3)       D. E. Jr Snider; M. Raviglione; A. Kochi: in tuberculosis: pathogenesis, protection and control; ed: B. R. Bloom, Am. Soc. Microbiology, Washington,DC, 1994: 2-11.

4)       Dye, C., Scheele, S., Dolin, P., Pathania, V., Raviglione, M.C., Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country WHO global surveillance and monitoring project. J. Am. Med Assoc. 282; 1999:677-686.

5)       Farmer P., Bayona J, Beccera M, Furin J, Henry C, Hiarr H, Kim JY, Mimic C, Nardell.E, Shin S, The dielemma of MDR TB in global era. Int J Tuberc Lung Dis, 2; 1998:869-876.

6)       Dony JF, Ahmad J, Khen Tiong Y, Epidemiology of tuberculosis and leprosy, Malaysia. Tuberculosis, 84(1-2); 2004: 8-18.

7)       Singh MM, Bano T, Pagare D Sharma N, Devi  R, Mehra M., knowledge and attitude towards tuberculosis in a slum community of Delhi. J Commun. Dis. 34(3); 2002:203-14.

8)       World health organization, TB a global emergency, WHO TB programme, Geneva:  Switzerland, WHO/TB/94,177; 1994:1-28

9)       Dhingra VK, Rajpal S, Taneja DK, Kalra D, Malhotra R, Health care seeking pattern of tuberculosis patients attending an urban TB clinic in Delhi. J Commun  Dis, 34(3) ;2002:185-92.

10)     Cameron J. C, Robert D. W, Brian C. B, Ian F.C,Antony J. Davies and Ulf-H. Dolling, A new synthesis of 1,2,4-triazolin-5-ones. Tetrahedron Letters, 41; 2000:8661.

11)     Mukundan T., Puranadre G.N., Nair J.K. Pansare S.M, Sinha P.K.,and Haridwar S.Explosive Nitrotriazolone Formulates, Defense Science Journal, 52(2); 2002:127- 133.

12)     Mauro M, Erika N,Mariangela M, Alessandro B, Marw F, Mauririo F, Marw T. Activitiy of Mannich base of 7-hydroxycoumarin against flaviviridae, Bioorganic and Medicinal Chemistry,16;2008:2591–2605.

13)     Ansari K.F, C.Lal. Synthesis and physicochemical properties and antimicrobial activities of some new benzimidazole derivatives. European Journal of Medicinal Chemistry, 44; 2009:4028–4033.

14)     Neslihan D, Sengül A. K, Ahmet D, Kemal S., Synthesis and antimicrobial activities of some new 1-(5-phenylamino-[1,3,4]thiadiazol-2-yl)methyl-5-oxo-[1,2,4]triazole and 1-(4-phenyl-5-thioxo-[1,2,4]triazol-3-yl)methyl-5-oxo -[1,2,4]triazole derivatives. European Journal of Medicinal Chemistry, 39; 2004:793–804.

 

 

 

Received on 15.04.2011        Modified on 02.05.2011

Accepted on 29.05.2011        © AJRC All right reserved