Structure Activity Relationship Studies of Substituted Mannich Bases of Pyrazolo [1, 2-A][1,2,4,6]-Tetrazepine-3,7-Dione Ring System with Variable Electronegative Atoms (Urea/Thiourea/Guanidine) for Antimicrobial and Antifungal Activity

Kaumil Navnitbhai Modi*, Palakben K. Parikh, Hiren M. Marvaniya and Dhrubo Jyoti Sen

Department of Pharmaceutical Chemistry, Shri Sarvajanik Pharmacy College, Gujarat Technological University, Arvind Baug, Mehsana-384001, Gujarat, India,

*Corresponding Author E-mail: dhrubosen69@yahoo.com

ABSTRACT:

9-methyl-1,5-diphenyl-3H,7H- pyrazolo-[1,2-a][1,2,4,6]-tetrazepine-3,7-dione ring system has been synthesised by the reaction between hydrazine and ethyl acetoacetate produced pyrazolone moiety which on benzoylation produced dibenzoyl derivative which on condensation with urea produced 9-methyl-1,5-diphenyl-3H,7H- pyrazolo-[1,2-a][1,2,4,6]-tetrazepine-3,7-dione ring which on alkaline oxidation with KMnO₄/KOH produced carboxylic moiety. Treatment with thionyl chloride of produced acid chloride derivative. This on condensation with Mannich base produced by benzaldehyde with urea/thiourea/guanidine produced the desired moiety.

The synthesised compounds were characterised by N% and spectral datas and antimicrobial and antifungal screening has been performed by zone of inhibition studies and MIC calculation with standard antibiotic/antifungal drug against (gram+ve) and (gram–ve) bacterial and fungal strains on agar media after 24 hours incubation at 37°C for antimicrobial activity and 24°C for antifungal activity. Gram positive: Staphylococcus aureus ATCC 9144, Bacillus subtilis ATCC 6633, Gram negative: Escherichia coli ATCC 25922, Fungal strain: Candida albicans ATCC 10231

It has been observed that the Compound-2 (X=S) showed maximum antibacterial activity against Escherichia coli, Compound-1 (X=O) and against Bacillus subtilis and Compound-1 (X=O) and against Staphylococcus aureus. Oxygen and Sulphur has two lone pairs whereas Nitrogen has one lone pair of electrons but the electronegativity of Oxygen (X=O; 3.5), electronegativity of Sulphur (X=S; 2.4) and electronegativity of Nitrogen (X=N+H; 3.1+2.2=5.3). MIC value of all the compounds have been found as 250µg as Minimum Inhibitory Concentration by serial dilution method and MIC of Cloxacillin is 250µg and for Fluconazole is 5.5µg.

KEYWORDS: Pyrazolo-tetrazepine, Antibacterial, Antifungal, Benzoylation.

Molecular Design

X=O(Urea); X=S(Thiourea); X=NH(Guanidine)

The synthesised compounds were characterised by N% and spectral datas and antimicrobial and antifungal screening has been performed by zone of inhibition studies and MIC calculation with standard antibiotic/antifungal drug against (gram+ve) and (gram–ve) bacteria and fungal strains on agar media after 24 hours incubation at 37°C for antimicrobial activity and 24°C for antifungal activity. Gram positive: Staphylococcus aureus ATCC 9144, Bacillus subtilis ATCC 6633, Gram negative: Escherichia coli ATCC 25922, Fungal strain: Candida albicans ATCC 10231

Scheme

COMPOUND-1: C₃₂H₂₉N₇O₄COMPOUND-2: C₃₂H₂₉N₇O₃S

COMPOUND-3: C₃₂H₃₀N₈O₃

Table-1

Physicochemical Parameters

Compounds	% Yield	Melting Point °C	Molecular Formula	N% Found	N% Calcd
COMPOUND-1 (X=O)	50	104-108	C₃₂H₂₉N₇O₄	17.03	16.98
COMPOUND-2 (X-S)	20	110-114	C₃₂H₂₉N₇O₃S	16.57	16.25
COMPOUND-3 (X=NH)	25	41-45	C₃₂H₃₀N₈O₃	19.50	20.37

Table-2

UV Spectral datas (Shimadzu)

Compound-1 Compound-2 Compound-3

NMR Spectras (Bruker)

Compound-1

Antimicrobial/antifungal screening:

The microbiological assay is based upon a comparison of inhibition of growth of micro-organisms by measured concentrations of test compounds with that produced by known concentration of a standard antibiotic. Two methods generally employed are turbidometric (tube-dilution) method and cylinder plate (cup-plate) method. In the turbidometric method inhibition of growth of microbial culture in a uniform dilution of antibiotic in a fluid medium is measured. It is compared with the synthesized compounds. Here the presence or absence of growth is measured. The cylinder plate method depends upon diffusion of antibiotic from a vertical cylinder through a solidified agar layer in a Petridis or plate to an extent such that growth of added micro-organisms is prevented entirely in a zone around the cylinder containing solution of the antibiotics.The cup-plate method is simple and measurement of inhibition of microorganisms is also easy. Here we have used this method for antibacterial screening of the test compounds.^7-10

Name of Microorganism

Gram +Ve microorganisms

Staphylococcus aureus (MTCC No. 96)

Bacillus subtilis (MTCCno. 121)

Gram -Ve microorganisms

Escherichia coli (MTCC No. 521)

Preparation of medium:-

Nutrient agar 2%

Peptone 1%

Beef extract 1%

Sodium chloride 0.5%

Distilled water up to 100ml

All the ingredients were weighed and added to water. This solution was heated on water bath for about one and half-hour till it became clear. This nutrient media was sterilized by autoclave.

Apparatus:-

All the apparatus like Petridishes, Borer, pipettes, glass rods, test-tubes etc. Were properly wrapped with papers and sterilized in hot air oven.

Antimicrobial screening method

Ø All the Petri dishes were sterilized in oven at 160°C for I hour.

Ø Agar media, borer and test solutions were sterilized in autoclave at 121°C at 15psi.

Ø Molten sterile agar was poured in sterile Petri dishes asceptically.

Ø The agar was allowed to cool and the bacterial suspension was poured into the petridishes asceptically.

Ø Placing the sterile bored in the agar plate by borer and solution of the compounds was filled in the bore using pipette (0.1ml) in appropriate four quadrants of petridishes aseptically.

Incubate the Petridishes at 37°C for antimicrobial and 24ºC for antifungal for 24 hrs and observed the zone of inhibition.^11-15

ANTIMICROBIAL SCREENING BY ZONE OF INHIBITION IN MILIMETER (FILTER PAPER DISC METHOD)

Table-3

Compounds		Microorganisms
Compounds	Concⁿ (µg/ml)	Bacillus subtilis	Staphylococcus aureus	Escherichia coli
Compound-1	250	15	09	13
	500	08	18	11
	750	14	11	17
	1000	14	12	17
Compound-2	250	11	00	23
	500	11	15	09
	750	16	11	15
	1000	11	14	06
Compound-3	250	09	00	08
	500	07	00	17
	750	08	10	12
	1000	12	11	08
Cloxacillin	250	10	00	13
Control	00	07	00	11

HISTOGRAM: ANTIMICROBIAL SCREENING BY ZONE OF INHIBITION IN MILIMETER (FILTER PAPER DISC METHOD)

ANTIFUNGAL SCREENING BY ZONE OF INHIBITION IN MILIMETER (FILTER PAPER DISC METHOD)

Microorganism: Candida albicans

Compounds	Concⁿ (µg/ml)	Zone of inhibition (mm)
Compound-1	250	00
	500	00
	750	00
	1000	07
Compound-2	250	00
	500	00
	750	00
	1000	08
Compound-3	250	00
	500	00
	750	00
	1000	08
Fluconazole	250	15
Control	00	08

HISTOGRAM: ANTIFUNGAL SCREENING BY ZONE OF INHIBITION IN MILIMETER (FILTER PAPER DISC METHOD)

Result and Discussion:

Structure activity relationship studies of substituted Mannich bases of pyrazolo [1,2-a][1,2,4,6]-tetrazepine-3,7-dione ring system with variable electronegative atoms (urea/thiourea/guanidine) for antimicrobial and antifungal activity showed that the inhibition of microbial growth is as follows:

Antimicrobial activity profile of the synthesised compounds is as follows:

Compound-2(E.coli)>Compound-1(S.aureus)>Compound-1(E.coli)=Compound-3(E.coli)>Compound-2(B.subtilis)> Compound-1(B.subtilis)=Compound-2(S.aureus,E.coli)>Compound-1(B.subtilis)>Compound-1(E.coli)>Compound-1 (S.aureus)=Compound-3(B.subtilis,E.coli)>Compound-1(S.aureus,E.coli)=Compound-2(B.subtilis,S.aureus)=Compound-3 (S.aureus)>Compound-3(S.aureus)>Compound-1(S.aureus)=Compound-2(E.coli)=Compound-3(B.subtilis)>Compound-1(B.subtilis)=Compound-3(B.subtilis,E.coli)>Compound-3(B.subtilis).

Antifungal activity profile of the synthesised compounds is as follows: Compound-3(C.albicans)=Compound-2(C.albicans)>Compound-1(C.albicans).

CONCLUSION:

Acknowledgement:

The author Kaumil Navnitbhai Modi is thankful to the project guide Prof. Dr. Dhrubo Jyoti Sen and the staff members of Shri Sarvajanik Pharmacy College, Mehsana, Gujarat to fulfil the project successfully. All the authors are thankful to the Quality Assurance Department of Shri Sarvajanik Pharmacy College, Mehsana for UV and IR spectrals datas, Oxygen Healthcare, Ahmedabad for Mass spectral data and NMR spectral datas.

References:

1. Dhrubo Jyoti Sen; Investigation on newer anti-inflammatory, sedative-hypnotic and antimicrobial agents: Indian Journal of Pharmaceutical Education: 35(4), p: 178, 2001.

2. Latha, S., Selvamani, P., Sen, D.J., Gupta, J.K., Pal, T.K. and Ghosh, L.K.; Antibacterial activity of Commiphora caudata and Commiphora berryi leaves: Indian Drugs: 42(10), p: 696-698, 2005.

3. Karad, R., Vyas, R., Sen D.J. and Patel, C.N.; Antimicrobial screening of Caesalpinia crista seeds: Journal of Advances in Biological Sciences: 4, p: 87-89, 2005.

4. Dhaval M. Patel and Dr. Dhrubo Jyoti Sen; Synthesis and antimicrobial screening by SAR studies of synthesized chalcone derivatives having heterocyclic rings: International Journal of Pharmaceutical Sciences; 1(1), p: 35-41, 2009.

5. Maulik K. Prajapati and Dr. Dhrubo Jyoti Sen; Synthesis and SAR study of some new 2-Imino-3-[carboxamido p-hydroxyphenyl]-5-arylidene-4-thiazolidinone as antimicrobial agents: International Journal of Pharmaceutical Sciences; 1(1), p: 83-90, 2009.

6. Parimal M. Prajapati and Prof. Dr. Dhrubo Jyoti Sen; Synthesis and antimicrobial screening of piperidone derivative with pyrazolone substituents: The Manufacturing Pharmacist; 02(07), p: 37-44, 2010.

7. Hiren R. Patel, Parth K. Patel, Dhrubo Jyoti Sen and Amit H. Patel; Growth inhibition of microorganism by bioisosterism: International Journal of Drug Development and Research; 2(1), p: 190-196, 2010.

8. Avani Sheth, Naman Doshi, D. J. Sen, R. Badmanaban, C. N. Patel; Synthesis of picric acid and para amino phenol derivatives for anti-microbial activity: Journal of Chemical and Pharmaceutical Research; 2(2), p: 1-12, 2010.

9. Vijay K Patel, Dhrubo Jyoti Sen and C. N. Patel; Antimicrobial and antifungal screening of indanone acetic acid derivatives: Journal of Chemical and Pharmaceutical Research; 2(2), p: 50-56, 2010.

10. Parimal M. Prajapati, Dhrubo Jyoti Sen and C N Patel; Synthesis and antifungal screening of piperidone derivative with pyrazolone substituents: Journal of Chemical and Pharmaceutical Research; 2(2), p: 279-285, 2010.

11. Yatri R. Shah, Dhrubo Jyoti Sen and C.N. Patel; Schiff’s bases of piperidone derivative as microbial growth inhibitors: Journal of Chemical and Pharmaceutical Research; 2(2), p: 581-589, 2010.

12. Yatri R. Shah and Prof. Dr. Dhrubo Jyoti Sen; Schiff’s bases of piperidone derivatives as fungal growth inhibitors: The Manufacturing Pharmacist; 02(09), p: 37-44, 2010.

13. Dhrubo Jyoti Sen, Palakben K. Parikh, Julee P. Soni, Kaumil N. Modi, Hiren M. Marvaniya, Priya R. Modiya, Apexa D. Patel, Dilip R. Chavda, Deepa R. Parmar, Sanjay D. Panchal and Vidhi R. Patel; Lead identification by structure activity relationship study of schiff bases of 3-amino-2-methyl quinazolin 4-(3H)-one for antimicrobial and antifungal activity: International Journal of Pharmacology and Technology; 2(1), p: 37-43, 2010.

14. Dhrubo Jyoti Sen, Palakben K. Parikh, Julee P. Soni, Kaumil N. Modi, Hiren M. Marvaniya, Priya R. Modiya, Apexa D. Patel, Dilip R. Chavda, Deepa R. Parmar, Sanjay D. Panchal and Vidhi R. Patel; Antibacterial screening of synthesised 5-methyl-N΄-[(1E)-phenylmethylene]-pyrazine-2-carbohydrazide derivatives by zone of inhibition study and MIC: International Journal of Drug Targets; 1(1), p: 47-56, 2010.

15. Ravi Natvarlal Patel, Urviben Yashodharbhai Patel, Ripal R. Chaudhari and Prof. Dr. Dhrubo Jyoti Sen; Synthesis and antibacterial study of some new schiff’s bases of 2-hydrazinyl-1-(1H-imidazole-1-yl)-ethanone: Asian Journal of Research in Chemistry; 4(1), p: 55-57, 2011.

Received on 25.03.2011 Modified on 10.04.2011

Asian J. Research Chem. 4(5): May, 2011; Page 838-844