Synthesis and Microbiological Evaluation of Novel Azetidin-2–Ones Encompassing Benzimidazole

 

Kiran M. Patel* and Dr. Dhrubo Jyoti Sen

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

*Corresponding Author E-mail: kiru_patel71@yahoo.com

 

The major drawback of current treatment of infectious diseases are challenging due to resistance to antimicrobial agents and their side effects. Benzimidazole and (β-lactam) Azetidinone are the heterocyclic compounds with considerable therapeutic and pharmacological properties. In this view, the series of Azetidinone encompassing Benzimidazole with different substitution were synthesized and evaluated for antibacterial and antitubercular activities. Series of 6 compounds having Benzimidazole and Azetidinone derivatives have been synthesized. These compounds were evaluated for antibacterial and antitubercular activities. In-vitro antibacterial activity of synthesized compound was tested against Gram positive and Gram negative microorganisms (Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC 121 and Escherichia coli MTCC 521) respectively by filter paper disk method and the antitubercular activity against INH resistant Mycobacterium tuberculosis H37Rv, using the L.J. slope method. All the compounds were characterized by UV, IR, Mass and NMR. All Compounds possess a molecular ion M+2 peak due to presence of chlorine. In the ¹H NMR δ value obtained in the range of 7.2 to 8.2 signifies the presence of aromatic ring and δ value at about 3.0 to 4.0 corresponds to the presence of methylene protons. In antibacterial screening all compounds found to have more activity against gram negative bacteria E.coli. Compounds 6a, 6d and 6e found to have better antibacterial activity against gram negative bacteria E.coli. Compound 5f have better antibacterial activity against gram+ve bacteria S.aureus and B.subtilis as compared to other synthesized compounds. Among the ring substituted azetidinone derivatives (6a to 6f) were tested for their antitubercular activity in vitro against INH resistant Mycobacterium tuberculosis H37Rv, using the L.J. slope method. Compounds 6a and 6e were found to be most potent among the series having MIC 100 μg/ml, others synthesized compounds were found to be active (having MIC 250μg/ml: 6b, 500μg/ml: 6d & 6f, 750 μg/ml: 6c). None of the compound was found to be equipotent with standard isoniazid.

 

 

 

INTRODUCTION:

In recent scenario heterocycles plays a major role in drug synthesis. In that respect azetidinone plays a significant role among other heterocyclic compounds. From the literature survey, in recent years 4 Aryl-3-chloro-1-(benzimidazole-2yl-benzamido)-2-azetidinones have attracted considerable interest because of their therapeutic and pharmacological properties. 2-substitutedbenzimidazole derivatives are known to possess varied biological activities. 2-Azetidinone derivatives have been reported to possess anti-inflammatory, anticonvulsant, antielastase, anti-HCMV, fungicidal, antiviral, antibacterial, antibiotic, antimicrobial, antitumor, anti-tubercular activities and pharmacological interest.

 

The electronic nature of the substituent groups at 2 positions in Benzimidazole nucleus, 4 in azetidinone led to significant variation in antibacterial activity. Among the series compounds substituted by electron-withdrawing (-NO₂ and -Cl) and electron-donating (-OCH₃-OH and –CH₃) groups are enhanced biological activity. So it was planned to synthesize a novel series of 2-azetidinone derivatives and to check their activity as Antibacterial and Antitubercular activity. The incorporation of a 2-oxoazetidine moiety in to 2-substitutedbenzimidazole scaffold enhances its activity. The treatment of many infectious diseases are challenging due to resistance to antimicrobial agents. The emergence of resistance among bacteria to a wide variety of structurally unrelated antibacterial agents such as β-lactams, macrolides, tetracyclines and fluoroquinolones as well as selected dyes and disinfectants has become a serious public health concern so makes it necessary to continue the search for new antibacterial agents.^1,2

 

6 (a-f)

R =

(a)		(d)
(b)		(e)
(c)		(f)

 

EXPERIMENTAL SECTION:

All the chemicals use for the synthesis of title compounds were produced from S.D. Fine Chem., Finar Chem. Ltd and Loba chemicals. The chemicals were used without further purification. All the melting points were determined in open capillaries and uncorrected. Thin layer chromatography was performed on microscopic slides (2×7.5cm) coated with silica-Gel-Gf 254 and spots were visualized under UV light by exposure to iodine vapours. IR spectra of all compounds were recorded in FTIR-8400 Shimadzu. Spectrophotometer using KBr. Mass spectra were obtained using 2010EV LCMS Shimadzu instrument. The ¹H-NMR was recorded on Bruker advanced-II NMR-400 MHz instruments using DMSO-d₆ as solvent and TMS (tetra methyl silane) as internal standard, chemical shifts were expressed as δ values (ppm).

 

 

Scheme of Synthesis

 

Reagents: (a)    = 4N HCl (b)    = NH2-NH2.2H2O/C2H5OH (c)    = Substituted aldehydes (d)    = (I) ClCH2COCl    (II) 1,4 Dioxane                    (III) Triethylamine	R
	(a)	(b)	(c)	(d)	(e)	(f)

Synthetic Procedure

Preparation of 2-chloro methyl 1-H benzimidazole: (3)

The ortho-phenylenediamine (0.01 mol.) was dissolved in 4N HCl and chloro acetic acid (0.01 mol.) was added. The mixture was refluxed for 4hrs, cooled and on neutralization with sodium bicarbonate, the product was precipitated out. It was filtered, washed with water, dried and recrystallized from aqueous ethanol.

 

                                                                                                                      (3)                                                  

 

Preparation of 2-Hydrazinyl methyl 1-H benzimidazole: (4)

To the ethanolic solution of 2-chloromethyl 1-H benzimidazoles (0.02 mol.) Hydrazine hydrates were added and it was refluxed for 4-5 hrs. Hot mixture was poured in crushed ice with constant stirring. The separated solid was dissolved in mixture of HCl (10 ml) & water (40 ml) than solution was extracted with ethyl acetate, charcoal was added & the mixture was filtered. Finally compound was reprecipiteted by addition of NaHCO₃ Solution. pH was adjusted to 12. The solid material was filtered, washed and recrystallized from ethanol.

(4)

 

General method of synthesis of Schiff bases^5-8                                                        

Preparation of (Z)-1-((1-H-benzimidazole-2-yl)-methyl-2-(substituted-benzylidene)-hydrazine: 5(a-f)

Hydrazinyl methyl 1-H-benzimidazole (0.01 mol.) was dissolve in 30 ml ethanol containing few drops of glacial acetic acid. Then add substituted benzaldehyde (a-f) in equimolar quantity and reaction mixture was refluxed on a water bath for 6-8 hrs. Completion of reaction was checked by TLC. After completion of reaction the reaction mixture was cooled and poured into crushed ice with stirring solid precipitated which was filtered and recrystallized from ethanol to yield product.

5(a-f)

 

General method of synthesis of Azetidinones^5-8

1-((1-H-benzo[d]imidazole-2-yl)methylamino)-3-chloro-4-(substituted-phenyl)-azetidin-2-one: 6(a-f)  

A mixture of Schiff bases [5(a-f)] (0.001 mol.) and triethylamine (0.003 mol.) was dissolved in 1,4–dioxane (25 ml), to this well stirred cooled solution of chloro acetyl chloride (0.0012 mol) was added drop wise at 5-10⁰C. The reaction mixture was stirred for 5-6 hrs. The reaction mixture was reflux for 2 hrs and kept for two days at room temperature. After that the resulting solid was separated out, dried and recrystallized with mixture of DMF-Methanol.

 

6(a-f)

 

Physical parameters:

Table: 1. Physical Characteristics of Synthesized Compounds

Compound code	R	Molecular formula	Molecular weight	%Yield	Melting point ( oC)	Log P
5-a	4-Cl	C15H13N4Cl	282.74	82-85	182-184	3.47
5-b	4-NO2	C15H13N5O2	295.29	70-72	180-182	3.44
5-c	2-Napthyl	C19H16N4	300.14	80-82	162-164	3.91
5-d	4-OH	C15H14N4O	266.12	75-80	158-160	2.53
5-e	2-Cl	C15H13N4Cl	282.74	78-80	166-168	3.47
5-f	4-OCH3	C16H16N4O	280.32	72-75	169-170	2.79
6-a	4-Cl	C17H14Cl2N4O	361.23	35-40	230-232	3.14
6-b	4-NO2	C17H14ClN5O3	371.08	40-45	237-238	2.53
6-c	2-Napthyl	C21H17ClN4O	376.84	42-45	248-250	3.57
6-d	4-OH	C17H15ClN4O2	342.78	45-50	235-238	2.19
6-e	2-Cl	C17H14Cl2N4O	361.23	35-38	232-234	3.14
6-f	4-OCH3	C18H17ClN4O2	356.81	37-40	245-248	2.45

Spectral characteristics:

Table: 2. Spectral Characteristics of Synthesized Compounds

Compd code	R	IR (υ, cm-1)	Mass (m/e)	1H NMR (d ppm)
6-a	4-Cl	2952.72 (CH2 str.),     1752.67 (C=O str. of azetidinone), 1633 (-CONH),     1100.09 (Ar-Cl str.)	360.3[M+], 362.4[M+2],  364.0[M+4]	8.2(s, 1H, -NH benzimidazole), 6.72-7.65 (m, 8H, Ar-H), 5.0 (s, 1H, -NH), 4.1 (s, 1H, >CH-Cl), 3.91 (s, 2H, -CH2), 3.17 (d, 1H, >CH-Ar)
6-b	4-NO2	1732.36 (C=O str. of Azetidinone), 1641.65 (-CONH), 1596.95 (C=C str.), 1519.80 (C-NO2 str.), 1454.23 (CH2 band.), 1342.36 (Ar-NO2 str.)	371.0 [M+], 373.1 [M+2]	8.5(s, 1H, -NH benzimidazole), 6.72-7.70 (m, 8H, Ar-H),  5.05 (s, 1H, -NH), 4.3 (s, 1H, >CH-Cl), 3.91 (s, 2H, -CH2), 3.27 (d, 1H, > CH-Ar)
6-c	2-Napthyl	1738.51 (C=O str. of Azetidinone), 1630.33 (-CONH), 600.33 (C=C str.), 1454.23 (CH2 band.)	376.8 [M+], 378.6 [M+2]	-------
6-d	4-OH	3498.63 (Ar-OH str.), 1730.79 (C=O str. of azetidinone), 1654.10 (-CONH), 1612.93 (C=C str.)	342.7[M+], 344.6 [M+2]	------
6-e	2-Cl	3470.23 (-NH str.), 2952.72 (CH2 str.), 1752.67 (C=O str. of Azetidinone), 1633 (-CONH), 1100.09 (Ar-Cl str.)	-------	-------
6-f	4-OCH3	1738.81(C=O str. of Azetidinone), 1643.46 (-CONH), 1195.78 (Ar- OH)	356.75[M+], 358.71[M+2]	8.4 (s, 1H,-NH benzimidazole), 6.88-7.95 (m, 8H, Ar-H),      5.0(s, 1H,  -NH),      4.5(s, 1H, >CH-Cl), 3.91 (s,2H,-CH2), 3.30 (d,1H, > CH-Ar)

 

 

SCREENING OF ANTIMICROBIAL ACTIVITY

A. ANTI-BACTERIAL ACTIVITY^9-10

Table: 3. Screening of Antibacterial activities

Compound Code	Concentration  (μg/ml)	Zone of Inhibition (mm)
		Gram +ve		Gram –ve
		S.aureus	B.subtilis	E.coli
Control	100	-	-	-
	200	-	-	-
	300	-	-	-
Ofloxacin	100	14	15	24
	200	14	16	26
	300	15	18	29
6a	100	03	04	18
	200	04	06	19
	300	06	08	21
6b	100	04	00	16
	200	05	02	17
	300	07	04	18
6c	100	03	02	12
	200	03	00	13
	300	05	02	17
6d	100	03	03	16
	200	05	03	17
	300	07	03	19
6e	100	03	04	18
	200	04	06	20
	300	06	08	22
6f	100	05	06	16
	200	06	06	17
	300	07	08	18

 

Figure: 1. Histogram of Antibacterial activity

 

 

 

B. ANTITUBERCULAR ACTIVITY^11,12

Determination of Minimal Inhibition Concentrations by L.J. (Lowenstein Jensen) method (Proportion method)

Here L-J medium without potato starch with drug incorporation before inspissation’s is used. Screw-capped tubes 17 mm in diameter, containing 7ml of medium are inspissated at 85°C for 40-45 minutes.  The drug susceptibility test is carried out from a primary isolation or a sub-culture on L-J medium. A representative portion of the culture is obtained by sampling as many colonies as possible within 1 or 2 weeks after appearance of growth.  The sample is homogenized in a sterile screw capped bottle (e.g. 14ml McCartney bottle or 5ml Bijoux bottle) containing 1 ml of sterile distil water and 10 glass beads 3mm in diameter.  The mixture is homogenized in a vortex mixture for a minute and if necessary the opacity is adjusted by adding sterile distils water, down to that of a standard suspension of 1 mg/ml of BCG.  The suspension is left to settle for about 30 minutes.

 

Methods used for Primary and Secondary Screening

Each synthesized drug was diluted obtaining 2000 microgram /ml concentration, as a stock solution.

 

Primary screen

In primary screening 500 micro/ml, 250 micro/ml, and 125 micro/ml concentrations of the synthesized drugs were taken. The active synthesized drugs found in this primary screening were further tested in a second set of dilution against all microorganisms.

 

Secondary screen

The drugs found active in primary screening were similarly diluted to obtain 100 micro/ml, 50 micro/ml, 25 micro/ml, 12.5 micro/ml, 6.250 micro/ml, 3.125 micro/ml and 1.5625 micro/ml concentrations.

Reading Result

The highest dilution showing at least 99 % inhibition is taken as MIC. The result of this is much affected by the size of the inoculum. The test mixture should contain 10⁸ organism/ml.

 

The Standard Drugs

The Standard strain M.tuberculosis, H₃₇Rv is tested with each new batch of medium. The recommended drug concentrations are 4 mg/l for Streptomycin, 0.2 mg/l for Isoniazid, 40 mg/l for Rifampicin and 2 mg/ l for Ethambutol.

 

Table: 4. Minimum Inhibitory Concentration of synthesized compounds for Anti tubercular activity

METHOD	L.J.MEDIUM  [CONVENTIONAL METHOD]
BACTERIA	H37RV
CONCENTRATION	1000µg/ml, 500µg/ml, 250µg/ml, 100µg/ml, 62.5µg/ml,
	50µg/ml, 25µg/ml, 12.5µg/ml, 6.25µg/ml,3.25µg/ml,
STANDARD DRUG	ISONIAZID
SR.NO	CODE NO	MIC µg/ml	REMARKS
1	6-a	100	ISONIAZID  = 0.20 µg/ml
2	6-b	250	99 % inhibition
3	6-c	750
4	6-d	500
5	6-e	100
6	6-f	500

 

 

Figure: 2.   Histogram of Antitubercular activity

 

 

RESULTS AND DISCUSSION:

All the synthesized compounds were screened for antibacterial activity and anti-tubercular activity. Ofloxacin and Isoniazid were used as standard reference drug for antibacterial screening and antitubercular activity respectively. In antibacterial activity, Compound 6f found to have better antibacterial activity against gram +ve bacteria S.aureus and B.subtilis as compared to other synthesized compounds. Compounds 6a, 6e and 6d found to have better antibacterial activity against gram –ve bacteria (E.coli).  In antitubercular activity, Compounds 6a and 6e were found to be most potent among the series having MIC 100 µg/ml. Others synthesized compounds were found to be active (having MIC 250 µg/ml; 6b, 500 µg/ml: 6d & 6f, 750 µg/ml: 6c). All synthesized compounds were found to be less potent as compared to standard drugs. Table: 1 represents the physical characteristics of the synthesized compounds and Table: 2 for spectral datas of the same. Antibacterial screening report has been mentioned in Table: 3 and histogram in Figure: 1. Table: 4 represents the Minimum Inhibitory Concentration of synthesized compounds for Anti tubercular activity and Figure: 2 for histogram of the same. Log P of 6c has been found as 3.57 which is the maximum value and the antitubercular potency was also found the maximum (Figure: 2).

 

ACKNOWLEDGEMENT

The author Kiran M. Patel is thankful to the Department of Quality Assurance of Shri Sarvajanik Pharmacy College, Mehsana for UV and IR Datas, also special thankful to the Department of Pharmaceutical Chemistry of Shri Sarvajanik Pharmacy College, Mehsana, Gujarat, India to perform the research work successfully with the expertise of project guide Dr. Dhrubo Jyoti Sen. The author is also thankful to Microcare Laboratory, Surat for checking out anti tubercular activity of the all synthesized compounds.

 

REFERENCES

1.     Patrick GL, An introduction to Medicinal Chemistry; 1^st edition; Oxford university press, 1995, 158.

2.     Hessen MT and Kaye D. Principles of use of antibacterial agents; 18^th edition; Infectious Disease Clinics, North America, 2004, 435–450.

3.     Liu P, Muller M, Derendorf H, “Rational dosing of antibiotics: the use of plasma concentrations versus tissue concentrations.” Int J Antimicrob Agents. 2002, 19, 285–290.

4.     Rothschild B, Martin L, “Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present.” Clin Infect Dis. 2001, 33, 305– 11.

5.     Desai KG, Desai KR, “Green route for heterocyclization of 2- mercaptobenzimidazole into β- lactum segment derivatives containing –CONH- bridge with benzimidazole screening in vitro antimicrobial activity with various microorganisms.” Bioorganic and medicinal chemistry. 2006, 14, 8271-8279.

6.     Panneer S, Radhika PP, “Synthesis of novel 2-substituted benzimidazole derivatives.” Research in Biotechnology. 2011, 2(3), 50-57.

7.     Mahavir C, Shrivastav AK, Atika J and Anil K, “Synthesis and biological activity of new 3-chloro-4-(3-substituted phenyl)-1-(5-((2-methyl-1H-benzo[d]imidazol-1-yl) methyl)-1, 3, 4-thiadiazol-2-yl) azetidin-2-one.” International Journal of ChemTech Research. 2011, 03, 1556-1562.

8.     Dhakad AK, Chaturvedi SC and Smita S, “Biological evaluation studies on some substituted 3-chloro-1-[5-(5-chloro-2-phenyl-benzimidazole-1-ylmethyl)-[1, 3, 4] thiadiazole-2-yl]-azetidin-2-one.” Digest Journal of Nanomaterials and Biostructures. 2009, 04, 275-284.

9.     Ashutosh Kar, Pharmaceutical Microbiology; 1^st edition; New Age International Limited Publishers, New Delhi, 2008, 268-278.

10.   Chakraborty P,  A Text Book of Microbiology; 5^th edition; New Central Book Agency (P) Limited, Kolkata, 2005, 623.

11.   Ali MA and Yar MS, “Substituted pyrazolyl-2-toluidinomethanethione having anti-tubercular activity.” Acta Poloniae Pharmaceutica Drug Research. 2007, 64(2), 139-146.

12.   Gutierrez MC and Brisse S, “Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis.” Comp. Immunol. Microbial. Infect. 2002, 1(1), 5.

 

 

 

Received on 01.06.2012        Modified on 08.06.2012

Accepted on 20.06.2012        © AJRC All right reserved