INTRODUCTION:

A strict definition of a heterocyclic compound is one which possesses a cyclic structure with at least two different kinds of atoms in ring. One of which is carbon, and can be aliphatic or aromatic. A hetrocyclic compound usually possesses a stable ring structure which does not readily hydrolyze or depolymerize.

It is obvious that the number and variety of hetrocyclic compound with reported antimicrobial activity is extensive with respect to structure and application. Heterocyclic compounds exhibited remarkable pharmacological activities.

The hetrocyclic compounds containing pyrimidine, pyridine, and piperazine nucleus have wide range of therapeutic activity such as antitubercular, anticancer, antihelmintic, antioxidant and antimicrobial activities is also believed that the presence of N-C=S linkage is responsible for the amoebicidal, anticonvulsant, fungicidal and antiviral activities^1-4.

In this direction, the work is being pursued to investigate the antimicrobial activity of some heterocyclic compounds prepared in our laboratory. Various piperazine nucleus containing derivatives have been synthesized. All the synthesized derivatives shows better anti bacterial and anti fungal activity against various strains of bacteria and fungai viz. Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonaie. Aspergillus niger, Microsporum gypseum.

After the antimicrobial studies, it were found that hetrocyclic Compound showed excellent activity against the bacterial strain of Pseudomonas aeruginosa and compound showed very good activity against the fungal strain of Aspergillus niger.

Incorporation of oxygen, nitrogen, sulphur, or an atom of a related element into an organic ring structure in place of a carbon atom gives rise to a heterocyclic compound. Since the heterocyclic atom must form more than one bond in order to be incorporated into a ring structure, halogens do not form heterocyclic compounds although they may be substituents on a heterocyclic ring structure. Heterocyclic compounds, like polycyclic ring compounds, are usually known by non - systematic names. They may be either simple aromatic rings or non-aromatic rings. Some examples are pyridine, pyrrole, furan and thiophene.

Many heterocyclic compounds are biosynthesized by plants and animals and are biologically active. Some heterocycles are fundamental to life, such as haem derivatives in blood and the chlorophylls are essential for photosynthesis. Similarly, the paired bases found in RNA and DNA are heterocycles, as are the sugars that in combination with phosphates provide the backbones and determine the topology of these nucleic acids. The biological properties of heterocycles in general make them one of the prime interests of the pharmaceutical and biotechnology industries.

_TRIAZOLES:

In five-membered ring systems, the presence of three nitrogen atoms defines an interesting class of compounds, the triazoles. These may be of two structural types, the 1,2,3-_{Triazoles or
v-triazole (1) and 1, 2,4-triazoles or s-triazoles (2).}

The name triazole was first given to the carbon nitrogen ring system C₂H₃N₃by Bladin, who described its derivatives in 1885. Later on because of various applications, triazoles took special attention particularly by the chemical industry.

Different chemical reactions show that 1,2,4-triazole is an extremely stable nucleus, which can be regarded as aromatic in nature,_{stabilized by resonance as shown below.}

_{4,5-Disubstituted-1,2,4-triazole-3-thiones:}

Triazoles having a mercapto group at C-3 (3) constitute a special class of compounds among the substituted 1,2,4-triazoles because of the tautomerism exhibited by them. The labile hydrogen atom may be attached to either nitrogen or sulfur atom. Previous studies of IR, NMR and UV spectra have provided strong evidence that mercaptotriazoles are present predominantly _{in the thione form (4).}

Due to lability of hydrogen, both the S-substitution and N-substitution can be carried out by changing the reaction to electron deficient alkenes and alkynes, ring opening of oxiranes and acylation. At low temperatures, oxidative chlorination of mercaptotriazoles is an excellent method of obtaining the corresponding sulfonyl chloride which can be converted to sulfonamide. The mercapto group can be eliminated using reagents like Raney nickel, dilute nitric acid or hydrogen peroxide. Mercapto groups are easily converted into their methylethers with sodium hydroxide and methyliodide and these ethers have little tendency to lose methanethiol on standing. Some important reactions of thiols are shown in below.

_{5-(Substituted)-4-amino-1,2,4-triazole-3-thiones:}

Among the substituted triazoles, aminotriazoles constitute class because of their widespread biological activities eg. 3-aminotriazole, better known as amizol was the first triazole which was used as an important herbicide. Aminotriazoles may have a C-amino group or an N- amino group. C-aminotriazoles behave as normal aromatic amines and can be diazotized in aqueos mineral acid with nitrous acid, forming diazonium salts which couple with aromatic bases. The detection of 3-amino-1,2,4-triazole in plant extracts depends on this property. Both C- and N-aminotriazoles undergo other characteristic reactions of a primary amine.

The amino group of 4-amino-1,2,4-(4H)-triazole reacts with aldehydes forming the corresponding Schiff base. The Schiff bases obtained from 5-nitrofufural and 5-nitrofuryl acylic aldehyde compare favrably with furacin (5-nitrofurfural semicarbazone) in bacteriostatic properties (Eq. 1.1). As expected, ethyl chloroformate readily forms ethyl N-(1,2,4-(4H)-triazol-_{4-yl) carbamate with 4-amino-(4H)-1,2,4-triazole
(Eq. 1.2).}

_{Uses of 1,2,4-Triazoles:}

1,2,4-Triazolcs and condensed triazole systems have found considerable use in photographic industry.1,2,4-triazole derivatives e.g.N,N'-bi(5-methyl-l,2,4-triazol-3-yl) formamidines are capable of inhibiting fog formation in silver halide emulsions by incorporating in the emulsions. The addition of 5-mercaptotriazole or their soluble salts to the emulsion after development produces a greater maximum image density and prevents bronzing effect. The sodium salt of a sulfonated derivative possesses good detergent actione.g. N- Benzylated aminotriazole (14) have useful properties in inhibiting the acid fading of dyestuff. Some 4-(phenylureido)-1,2,4-triazole derivatives have been used as defoliants e.g. (15; 50.0 wt. parts) when formulated with sodium-dodecylbenzenesulfonate 3.0, sodium-ligninsulfonate 2.0, SiO: 15.0 and clay 30.0 wt. parts gives 50% wettable powder and it causes 50% defoliation of _{cotton at 0.5 kg/ha, Vs
20%with(BuS)PO.}

A new series of condensation polymers polyaminotriazoles have been prepared, which are similar to nylon. These are fibre forming and can be melt spun to give filaments which after _{drawing,
possess high strength and good affinity for dyestuffs.}

These polyaminotriazoles are condensation polymers which are produced by heating dihydrazides in presence of small amount of hydrazine. The name refers to the rearranging linkage, the 4-amino-l,2,4-triazole ring (Eq, 1.3). Some 1,2,4-triazole derivatives (16) and (17) _{have been used as stabilizers for chlorine-containing
thermoplastic polymers.}

R₁ = H,OHC_1-6 alkoxy or alkylthio CO₂H, SH

R₂ = H, OH , SH, NH, O, S, CH₂

m = 1-20, n = 1,2

(16)

R= H, NH₂ ,SH, C_1-12 alkylthio

R₁ = CO₃R₃

(17)

_OBJECTIVES:

PURPOSE OF THE WORK:

The treatment of infectious disease still remains an important and challenging problem because of emergence of new infectious disease and the increasing number of multidrug resistant microbial organisms. In spite of a large number of chemotherapeutic agents existing for medicinal use, at the same time the emergence of development of resistance to most of these in the last decades reveals a substantial need for the new classes of antimicrobial agents. There is really perceived need for the discovery of new compounds endowed with antimicrobial activity, possibly acting through mechanisms of action, which are distinct from those of well known antimicrobial agents, for which the pathogens are now resistant⁵.

Tuberculosis is believed to be present in about 1/3^rd of the world’s population⁶. Active disease following new infection, as well as reactivation of latent tuberculosis, is particularly prevalent in individuals with compromised immune systems such as those HIV positive. In addition, the emergence of drug resistance strains of Mycobacterium tuberculosis has led to the increased pressure on current chemotherapy regimens⁷. Hence there exists, urgent need for the discovery of newer molecules, which may have potential to curb the above mentioned infectious diseases.

3,5-disubstituted 1,2,4-triazole and its derivatives have been reported to posses wide spectrum of activities ranging from anti-bacterials⁸, anti-inflammatory⁹, anti-convulsant¹⁰, antineoplastic¹¹, antimalarial¹², anti-viral¹³, anti-cancer¹⁴, anti-TB¹⁵ and anti-proliferative¹⁶.

Pyridine, a heterocyclic nucleus played a vital role in the development of different medicinal agents and in the field of agrochemicals. This nucleus is present in many products such as drugs, vitamins, food, flavorings, plant dyes, adhesives and herbicides¹⁷.

It is seen from the current literature that pyridine congeners are associated with different biological properties like pesticidal^18,19, insecticides²⁰and fungicidal²¹ activity.

In view of the above mentioned facts and in continuation of our interest in the synthesis of nitrogen heterocycles to identify new candidate, that may be value in designing new, potent, selective and less toxic chemotherapeutic agent, we propose herein for the synthesis of some novel derivatives incorporating suitably substituted 1,2,4-triazole moiety with long aliphatic α,ω-dicarboxylic acid of variable lengths.

This combination suggested is an attempt to investigate the influence of such hybridization and structure variation on the anticipated biological activities, hoping the possibility that, the target derivatives might be more efficacious as antimicrobial agents.

OBJECTIVES:

The present investigation includes:

1. Preparation of N-(3-phenoxymethyl-5-mercapto-4H-1,2,4-triazol-4-yl) isonicotinamide nucleus by following literature method.

2. Preparation of dihydrazides using various α,ω- aliphatic dicarboxylic acids namely oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid by the reaction with hydrazine hydrate(99%) through their corresponding ethyl ester formation.

3. Condensing the above prepared acid dihydrazides (a-e) with 3-phenoxymethyl-5- mercapto-1,2,4-triazole to form various 5-substituted N-(3-phenoxymethyl-5-mercapto-4H-1,2,4-triazol-4-yl)isonicotinamides (6a-6e).

4. Determination of homogeneity of all the synthesized compounds by TLC technique and confirmation of the structures by determining their IR, ¹HNMR, MASS spectra.

5. Evaluation of all the synthesized compounds for their anti -tubercular profiles.

_METHODOLOGY:

INTRODUCTION:

The importance of 1,2,4-Triazole moiety has been discussed in the previous chapter. Among the many methods available for the synthesis of 1,2,4-Triazole derivatives, in the present chapter a convenient and versatile methodology has been adopted for the synthesis of 1,2,4-Triazole derivatives.

MATERIAL AND METHODS:

a) The entire chemicals used were procured from Qualingens, Himedia and Loba-chemicals. Purity of starting materials used for reaction was confirmed by checking their melting point or boiling point and by thin layer chromatography.

b) Purity of compounds was checked on “Silica Gel G” coated on laboratory micro slides prepared by dipping method or precoated plates, eluent was the mixture of different polar and non-polar solvents in varying proportions and detection was done either by observing in UV (ultra-violet) light or exposure to iodine vapours as required. The absence of TLC spots for starting materials and appearance of new TLC spot at different R_fvalue ensured the completion of reaction.

c) Melting points were determined in open capillary tube using precision melting point apparatus and uncorrected.

d) The FT-IR spectra of the synthesized compounds have been obtained from BLDE College of Pharmacy, Bijapur. The IR spectra were recorded on SHIMADZU PERKIN EKMER 8201 PC IR SPECTROMETER using a thin film on potassium bromide pellets.

e) The ¹HNMR spectra of the selected compounds have been obtained from Astra Zeneca Pharma India Ltd, Bangalore. The ¹HNMR spectra were recorded on BRUKER AVANCE II 300 NMR SPECTROMETER in a mixture of DMSO. Chemical shift (δ) values are reported as values in ppm relative to TMS (δ=0) as internal standard.

f) The Mass spectra of the selected synthesized compounds have been obtained from Oxygen Healthcare Research P. Ltd, Ahmedabad. The FAB mass spectra were recorded on JEOL SX-102/DA-6000 Mass Spectrometer.

0.1 mol of N-(5-mercapto-3-(phenoxymethyl)-4H-1,2,4-triazol-4- yl) isonicotinamide (5) and 0.1 mol of acid hydrazide (a-e) in the presence of glacial acetic acid taken in 50 ml of ethyl alcohol and refluxed in an anhydrous condition for 8hr. The reaction mixture was cooled to room temperature and filtered the product and separated. It was dried and recrystallised from absolute ethanol.

BIOLOGICAL ACTIVITIES:

Anti-Tubercular Activity:

All the synthesised 1,2,4- triazole derivatives have been evaluated for Anti-tubercular activity against Mycobacterium tuberculosis H₃₇Rv using Microplate alamar blue dye assay(MABA) .The minimum inhibitory concentration(MIC) was determined for each of the sample. The first line antitubercular drug INH was used as a refrence standard.The resulte are tabulated in table no-1 and graphically depicted in figure no-1

Anti tubercular activity using Alamar Blue Dye Method²²

The anti-mycobacterial activity of the title compounds were assessed against Mycobacterium tuberculosis H₃₇Rv using Microplate Alamar Blue Assay (MABA) method. This method is non-toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method. Briefly, 200µl of sterile deionzed water was added to all outer perimeter wells of sterile 96 wells plate to minimized evaporation of medium in the test wells during incubation. The 96 wells plate received 100 µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate. The final drug concentrations tested were 100 to 0.2 µg/ml. Plates were covered and sealed with parafilm and incubated at 37ºC for seven days. After this time, 25µl of freshly prepared 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 hrs. A blue color in the well was interpreted as no bacterial growth and pink color was scored as growth. The MIC is defined as lowest drug concentration which prevented the color change from blue to pink.

Determination of minimum inhibitory concentrations (MICs) of antimicrobial agents by agar dilution method²³:

INTRODUCTION:

Dilution methods are used to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents and are the reference methods f o r antimicrobial susceptibility testing against which other methods, such as disc diffusion are calibrated. MIC methods are Widely used in the comparative testing of new agents. In clinical laboratories, they are used to establish the susceptibility of organisms that give equivocal results in disc tests, for tests on organisms where disc tests may be unreliable and when a more accurate result is required for clinical management.

In dilution tests, microorganisms are tested for their ability to produce visible growth on a series of agar plates (agar dilution) or in microplate wells of broth (broth micro dilution) containing dilutions of the antimicrobial agent. The lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism is known as the MIC.

MEDIUM:

Although several susceptibility testing media are available in Europe, a clear choice for a reference medium remains to be determined. Mueller-Hinton (MH) agar shows no performance advantages over some other media but is probably the most widely used medium internationally, and there is a USA National Committee for clinical laboratory standards (NCCLS)d ocument which describes procedures for evaluating MH agar. MH agar which meets the requirements of the NCCLS standard is considered the reference medium.

Preparation of stock solutions:

Use an analytical balance when weighing agents. Allowance for the potency of the powder can be made by use of the following formula:

Weight of powder (mg) =

Volume of solvent (mL) X Concentration (mg/L)

Potency of powder (mg/g)

Alternatively, given a weighed amount of antimicrobial powder, the volume of diluent needed

may be calculated from the formula:

Weight of solvent (mL) =

Weight of powder ( mg) X Potency of powder (mg/g)

Concentration (mg/L)

Concentrations of stock solutions should be 1000 mg/ L or greater, although the solubility of some agents will be limiting. The actual concentrations of stock solutions will depend on the method of preparing working solutions. Sterilization of solutions is not usually necessary. If required, sterilization should be by membrane filtration, and samples before and after sterilization must be compared by assay to ensure that adsorption to the membrane has not occurred. Unless otherwise instructed by the manufacturer, store stock solutions frozen in _{aliquots at -20°C or below.
Most agents will keep at -60°C for at least 6 months. Stock solutions} must be frozen as soon as possible after preparation, used promptly on defrosting and not re - frozen.

Preparation of working solutions:

The range of concentrations tested will depend on the organisms and antimicrobial agent being tested, but a two-fold dilution series based on 1mg/L is conventionally used. Twenty- milliliter volumes of agar are commonly used in 9 cm Petri dishes for agar dilution MICs. Two alternative dilution schemes are there which involve adding 19 ml volumes of molten agar to 1ml volumes of antimicrobial solution. The more conventional method is based on diluting a 10 240 mg/ L stock solution, always measuring 1mL volumes of antimicrobial solution. The other method is based on diluting a 10000 mg/ L stock solution and involves measuring various volumes of antimicrobial solution by the use of high precision variable volume micropipettes, which are now widely available. An alternative to the method is to omit the distilled water added to make antimicrobial volumes up to 1 mL and instead to add a variable volume of molten agar to make the total volume 20 mL.

PREPARATIONOFPLATES:

Prepare agar as recommended by the manufacturer. Allow the sterilized agar to cool to 50°C in a water-bath. Prepare a dilution series of antimicrobial agents, in 25-30 mL containers. Include a drug-free control. Add 19 mL of molten agar to each container, mix thoroughly, and pour the agar into relabeled sterile p etri dishes on a level surface. Allow the plates to set at room temperature and dry the plates so that no drops of moisture remain on the surface of the agar. Do not over dry plates.

Plates should not be stored unless the agents have been shown to be stable on storage. Clavulanic acid and carbapenams are particularly unstable.

PREPARATIONOFINOCULUM:

Standardize the density of inoculums to give 10⁴ colony-forming units (CFU) per spot on the agar. Use four or five colonies of a pure culture to avoid selecting an atypical variant. The inoculums may be prepared by emulsifying overnight colonies from an agar medium or by diluting a broth culture. The broth used must not be antagonistic to the agent tested. A 0.5 McFarland standard may be used for visual comparison to adjust the suspension to a density equivalent to approximately 10⁸CFU/ mL. Alternatively, inoculate can be adjusted photo _{metrically. Dilute the
suspensions of organisms in 0.85% saline or broth to give 107CFU/mL.}

Plates must be inoculated within 30 min of standardizing the inoculums, to avoid changes in inoculum density.

INOCULATIONOFPLATES:

Mark the plates so that the orientation is obvious. Transfer diluted bacterial suspensions to the wells of an inoculums replicating apparatus. Use the apparatus to transfer the inocula to the series of agar plates, including a control plate without antimicrobial agent. Replicator pins 2.5 mm in diameter will transfer about 1 µL, i.e. an inoculum of 10⁴CFU/spot. ^{Alternatively, a micropipette or standard loop
may be used to inoculate plates. Allow the} Inoculum spots to dry at room temperature before inverting the plates for incubation.

INCUBATIONOFPLATES:

Incubate plates at 35-37°C in air for 18 h. In order to avoid uneven heating, do not stack plates more than five high. If the incubation period is extended for slow-growing organisms, the stability of the agent over the incubation period must be assessed by the inclusion of Control strains with known MICs. Avoid incubation in an atmosphere containing 5%CO₂unless necessary for growth of the organisms (e.g. Neisseria spp.).Incubate methicillin/oxacillin susceptibility tests on staphylococci at 30°C.

READING RESULTS:

The MIC is the lowest concentration of the agent that completely inhibits visible growth as judged by the naked eye, disregarding a single colony or a thin haze within the area of the inoculated spot. A trailing end point with a small number of colonies growing on concentrations several dilutions above that which inhibits most organisms should be investigated by subculture and retesting. Such trailing endpoints may indicate contamination, resistant variants, ß-lactamase producing organisms, or, if incubation is prolonged, regrowth of susceptible organisms following deterioration of the agent with sulfonamides and trimethoprim, endpoints may be seen as a reduction in growth, and a haze of growth may be seen at several dilutions above the actual MIC.

RESULTS AND DISCUSSION:

BIOLOGICAL ACTIVITIES:

ANTITUBERCULAR ACTIVITY STUDIES:

The literature survey has revealed that, the moieties containing 1,2,4- triazole nucleus have shown to posses antitubercular activity. Hence, in the present investigation all the synthesised 1,2,4-triazole derivatives (6a-6e). Have been evaluated for antitubercular activity against mycobacerium tuberculosis H37Rv following microplate alamar blue assay method. MIC was determined for each of the compound and first line antitubercular drug INH was used as the reference standard. The results of the study are tabulated in Table no-1and graphically depicted in Figure no-1

Table-1: Antitubercular activity of N-(3-(2-(3-hydrazinyl-3-oxoalkanoyl) hydrazinyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-4-yl)isonicotinamide (6a-6e) against mycobacerium tuberculosis H37Rv

Sl. No.	Samples	100	50	25	12.5	6.25	3.125	1.6	0.8	0.4	0.2
1.	6a	S	S	S	S	S	R	R	R	R	R
2.	6b	S	S	R	R	R	R	R	R	R	R
3.	6c	S	R	R	R	R	R	R	R	R	R
4.	6d	S	S	S	R	R	R	R	R	R	R
5.	6e	S	S	S	R	R	R	R	R	R	R
6.	INH	S	S	S	S	S	S	S	S	S	S

Figure-1: Antitubercular activity of N-(3-(2-(3-hydrazinyl-3-oxoalkanoyl)hydrazinyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-4-yl)isonicotinamide (6a-6e) against _{mycobacerium
tuberculosis H37 Rv}

Note *: The Mycobacterium was found to be resistant to the compounds 6d and 6e throughout the study.

DISCUSSION:

The 1,2,4-triazole derivative 6a (n=0) has shown excellent antitubercular activity by possessing

MIC of 6.25 µg/ml. The derivatives 6d (n=3) and 6e (n=4) have shown to possess the MIC of 25 µg/ml. The compound 6b (n=1) has the MIC of 50 µg/ml and that of 6c (n=2) was found to be 100 µg/ml. The potency of the compound to inhibit the growth of mycobacterium is found to be in the following order:6a > 6d and 6e > 6b > 6c

SUMMARY AND CONCLUSION:

The development of new antimicrobial therapeutic agent with enhanced potency, high selectivity and reduced toxicity is a constant process in medicinal chemistry. Exhaustive literature survey on 1,2,4-Triazoles derivatives revealed that they possess wide range of biological properties.

The purpose of the present study was to examine whether molecular modification might results in the discovery of new potential biological agents. As per the given scheme N-(5-mercapto-3- (phenoxymethyl)-4H-1,2,4-triazol-4-yl) isonicotinamide (5) was synthesised following the procedure described in methodology. The various aliphatic dicarboxylic acids differing in their length of alkyl chains were converted into their respective hydrazides and were reacted with N- (5-mercapto-3-(phenoxymethyl)-4H-1,2,4-triazol-4-yl) isonicotinamide (5) that resulted in the compounds N-(3-(2-(3-hydrazinyl-3-oxoalkanoyl)hydrazinyl)-5-(phenoxymethyl)-4H-1,2,4-triazol-4-yl)isonicotinamide (6a-6e), with good yields.

This combination suggested is an attempt to investigate the influence of such hybridization and structure variation on the anticipated biological activities hoping the possibility that the target derivatives might be more efficacious as Anti-tubercular agents.

The homogeneity of all the derivatives was established by TLC technique. The structures of the compounds were successfully established by means of IR, Proton NMR and Mass spectral studies. All the title compounds were evaluated for antitubecular activity by Microplate Alamar Blue Assay method. MIC values were determined and for the comparison of potency, the drug INH used in first line of antitubercular therapy was used as a reference standard. The compound 6a obtained as a oxalic acid derivative (n=0) has shown better potency with MIC value of 6.25µg/ml. Whereas the other compounds namely 6d and 6e have shown to possess the MIC of 25µg/ml. The remaining compounds 6b and 6c have the MIC values of 50 and 100 µg/ml.

Among the compounds evaluated for antitubercular activity 6a was found to be the most potent antitubercular agent with a least MIC value of 6.25µg/ml.

Even though, the structural activity relationship could not be arrived at, the present study is of significance in that few of the synthesised compounds shown to possess good antitubercular properties. All these results only indicate that the above type of 1,2,4-triazole moiety needs more attention and if this moiety is suitably exploited can still give better lead compounds.

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Received on 27.07.2017 Modified on 24.08.2017

Research J. Pharm. and Tech. 2018; 11(1): 153-164

DOI: 10.5958/0974-360X.2018.00029.X