SYNTHESIS OF NEW LEVOFLOXACIN DERIVATIVES AND THEIR BIOLOGICAL ACTIVITY

Headnote

ABSTRACT

Heterocyclic moieties like 2-aminobenzotriazole and 5-amino-2-mercapto-1,3,4-thiadiazole have been shown to have antibacterial, antifungal, anti-inflammatory and antitumer activities etc. Meanwhile quinolones as essential antibacterial agents are of important clinical and scientific interest in their discovery based on nalidixic acid. This attract our attention to synthesize novel compounds as important quinolone derivatives of levofloxacin linked once to 2-aminobenzotriazole and next to 5-amino-2-mercapto-1,3,4-thiadiazole with the aim of the possibility of ultering the side chain (carboxlic acid moiety) of levofloxacin to minimize the side effects of levofloxacin on gastrointestinal tract including gastric irritation and to alleviate it's side effects on joints at the same time enhancing or maintaining its antibacterial and other biological activities. The method includes the synthesis of two analogues of levofloxacin by introducing first: 2-aminobenzotiazole linked to the carboxyl moiety of levofloxacin and second: 5-amino-2-mercapto-1,3,4,thiadiazole linked at the same site of levofloxacin. For this purpose, 2-aminobenzotriazole was synthesized from benzene1,2-diamine followed by amination. Whereas 5-amino-2-mercapto-1,3,4,thiadiazole was synthesized through rearrangement of thiosemicarbazide with carbon disulphide by hot fusion. Then this step will be followed by condensation of levofloxacin via coupling reaction between the carboxylic group moiety of levofloxacin first with the amino group of 2-aminobenzotiazole and second with the amino group of 5-amino-2-mercapto-1,3,4,thiadiazole through the conventional coupling reaction using N,N-Dicyclohexylcarcodiimide (DCC) and 1-Hydroxybenzotriazole (HOBT) to obtain potent, save and higher yield products. Screening of the antimicrobial activity including antibacterial and antifungal of the two analogues was carried on using combinational agar dilution method and the inhibition zone was measured by comparism to Ofloxacin as reference drug for antibacterial activity and ketoconazol for antifungal activity of the tested compounds. The structural formula of the two analogues were consistent with the proposed structure as they were proved and characterized by different techniques i.e. melting point (m.p.), Thin layer chromatography(TLC), FTIR, Elemental microanalysis (CHNOS and F). Screening of the antimicrobial activity revealed that both analogues have significant activity both as antibacterial and antifungal. In conclusion, this work has successfully introduced new substituted amine appendages at C3 of fluoroquinolone nucleus utilizing coupling method producing new levofloxacin derivatives with enhanced antimicrobial activity.

Keywords: Heterocyclic moieties; coupling reaction; antimicrobial activity.

INTRODUCTION

Nitrogen containing heterocycles are of important roles not only for life science industry, but also in many other industrial field related to special and fine chemistry, among them 1,3,5-triazole derivatives which represent a widely used lead structure with multitude of interesting application in human field.1 Several derivatives showed antifungal, antimicrobial2, herbicidal activity and recently cytotoxic and antitumor3 activity are well elicited these derivatives as for e.g. 2-aminobenzotriazole and 5-amino-2-mercapto-1,3,4,thiadiazole.

On the other hand quinolones are of important clinical and scientific interest since their discovery based on nalidixic acid in the early 1960s.4 During the 1980s more active compounds were synthesized due to the addition of a fluorine atom at position 6 and a piperazinyl group at position 7 at the quinolone nucleus resulting in i.e. norfloxacin, an antimicrobial agent with improving activity against Gram-negative rods. Further modification at the quinolone structures resulted in ciprofloxacin and ofloxacin and based on these molecules, a cyclopropyl group at position 1 was introduced in many newer quinolones like gatifloxacin and moxifloxacin).5-11

The quinolones are now divided into four different groups due to their spectrum of different activities.12 The first group of quinolones i.e. norfloxacin and the second group, i.e. ciprofloxacin and ofloxacin have no or only little activity against obligately anaerobic bacteria.

Of the third group Levofloxacin (figure 1); is the levo- isomer and active component of the chiral molcule ofloxacin and therefore without greater change in the spectrum of activity but twice as active as ofloxacin per unit of mass.6 The fourth group include moxifloxacin and gatifloxacin.

Most of the quinolone antibacterial research has been focused on the functionality at C-7 position.13 The structure activity relationship (SAR) reveals that the C-7 substituent is the most adaptable site for chemical change and is an area that determines potency and target preference.14 During recent years a number of quinolones with substitution on piperazine ring at C-7 position of the basic structure of quinolones were synthesized.15-17

In earlier studies, the substitution of bulky residue on piperazinyl ring of levofloxacin was carried out with 2-aryl-2-oxoethyl or 2-aryl-2-oxyiminoethyl moiety18, however evidences are found in literature which reports increased antimicrobial profile of the quinolones in the form of carboxylate complexes19, which indicate that the carboxylic acid or hydrolysable group viz. ester and amide at C3 is essential for DNA gyrase binding.5

Because of lack of data in the literature concerning analogues of levofloxacin at carboxylic group, we are reporting these newly synthesized derivatives, by introducing new functionality at carboxylic group position. For this purpose the carboxylic group at C-3 was coupled by at the carbonyl carbon by various heterocyclic compounds containing amines.

The general mechanism of action employed by the fluoroquinolone class is inhibition of type II topoisomerases DNA gyrase or topoisomerase IV (topo IV) on DNA. Formation of a ternary drug-enzyme-DNA complex, which is reversed by removal of quinolone20, blocks DNA replication, RNA synthesis, and bacterial growth21.

CHEMISTRY

This involves the synthesis of two analogues of levofloxacin by introducing first: 2-aminobenzotiazole linked to the carboxyl moiety of levofloxacin and second: 5-amino-2-mercapto-1,3,4,thiadiazole linked at the same site of levofloxacin.

For this purpose, 2-aminobenzotriazole was synthesized from benzene1,2-diamine followed by amination. whereas 5-amino-2-mercapto-1,3,4,thiadiazole was synthesized through rearrangement of thiosemicarbazide with carbondisulphide by hot fusion. Then this step will be followed by condensation of levofloxacin via coupling reaction between the carboxylic group moiety of levofloxacin first with the amino group of 2-aminobenzotiazole and second with the amino group of 5-amino-2-mercapto-1,3,4,thiadiazole through the conventional coupling reaction using N,N-Dicyclohexylcarcodiimide (DCC) and 1-Hydroxybenzotriazole (HOBT) to obtain potent, save and higher yield products. Scheme 1.

RESULTS AND DISCUSSION

These were accomplished through the following steps of reactions starting from the synthesis of intermediate compounds: 2-aminobenzotriazole and 5-amino-2-mercapto-1,3,4-thiadiazole followed by coupling reaction of levofloxacin with these intermediates, using coupling reagents as in the peptide synthesis.

In the present work, the method used was the direct coupling with DCC/HOBt or DCC/ HOSu. The use of DCC is particularly convenient, since it can be simply added to the solution containing the amine and carboxyl components to be coupled. It reacts rapidly with the free carboxylic acid present to form an "active ester" intermediate (isourea), which may react with amine component directly or may proceed through an intermediate (symmetrical anhydride) as in scheme 1.

Coupling reactions mediated by DCC can be also modified by the addition of other reagents, such as HOBt and HOSu. This addition will lead to the formation of HOBt and HOSu active esters which in turn may22:

* Accelerate coupling reactions.

* Suppress byproduct formation.

* Suppress racemization.

FTIR analysis showed broad OH stretching vibration of the carboxylic group of levofloxacin extending from 3600-3100 cm-1 and strong absorption peak at 1675-1707 cm-1 due to keto carboxylic group. Our study revealed that in the spectra of all the derivatives of levofloxacin the absorption intensity of the carboxylic carbonyl group was decreased and shifted to the right near 1625 cm-1 which is indicative of the formation of amide and no peak was observed for carboxylic OH absorption and a distinct, strong and un-obscured NH stretching was observed at 3326 cm-1 indicating that carboxylic site reacted with the selected amines forming amides. Elemental microanalysis of the finally synthesized compounds (compound 5 and 6) was done in France laboratories as shown in table 2.

Biological activity

This study was designed as a comparative study of levofloxacin derivatives as shown in table 3 in relation to ofloxacin as a control. Compounds 5 and 6 were tested against a panel of microorganisms including Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Proteus spp.) using conventional agar dilution method.

The overall activity profile of compounds (5 and 6) against microorganisms revealed these derivatives are as effective as Ofloxacin in zone of inhibition values. In the terms of structure activity relationship, the antibacterial activity profile against all bacterium was altered by addition of heterocyclic intermediates containing amino group in levofloxacin molecule. It seems that this activity is due to better interaction of molecule with target enzymes or for penetration into these bacteria as shown in Table 3.

Concerning the antifungal activity, the pattern result of the antifungal activity of the tested compounds was differed from their antibacterial activity. Compound 5 showed maximum activity against Aspergillus Spp. whereas compound 6 show medium activity against this fungi and medium activity. Both compounds 5 and 6 shows lower activity against Candida species.

CONCLUSION

Development of bacterial resistance has led to the synthesis of newer and more potent quinolones. As detailed above, two carboxylic acid derivatives have been designed, synthesized, characterized and evaluated for their biological activities in vitro in order to discover potent agents against Gram-positive and Gram negative bacteria.It was observed that when amine group containing heterocyclic compound was introduced to carboxylic side, comparable antimicrobial activity against organisms were achieved from the levofloxacin nucleus.

EXPERIMENTAL

Chemistry

The parent compound (levofloxcin) was purchased from Sigma and Fluka companies (Germany). All chemicals used in preparations were of Analar quality reagents and the solvents were purified by distillation prior to use. The melting points were determined by electrothermal CIA 9300 melting point apparatus in open capillaries and were uncorrected. The ultraviolet spectra were obtained via Carrywinn U.V. Varian U.V. - visible spectrophotometer (Australia). Structures were drawn by Chemdraw Office 2008 software. IR spectra (KBr discs) were recorded on a Buck 500 FTIR scientific spectrophotometer (Iraq). Elemental microanalysis was done on a Carlo Erba elemental analyzer. This analysis was done in the micro analytical center (France). The purity of all compounds was established by single spot on the Thin-layer chromatography (TLC) plastic sheets silica gel 60 F5 precoated, 20 ×20 cm, layer thickness 0.2 mm. The spots on the chromatograms were localized using U.V. light (366 nm) (Whatmann, Merck, Germany). Iodine vapour was used as developing agent. The solvent system used employed for separation composed from (A) Methanol: Dichloromethane (10:90), (B) Strong ammonia solution: Methanol (1 % v/v), (C) Equal volumes of chloroform and solvent system B.

synthesis of levofloxacin derivatives

This includes the synthesis of benzotriazole followed by the amination with hydroxylamine-o-sulphonic acid as the following:

Synthesis of benzotriazole (compound 2)23: Benzene 1,2-diamine (0.1 mol, 10.8 gm) was dissolved in a mixture of (0.2 mol, 12 gm, 11.5 ml) of glacial acetic acid and (30 ml) water content in a (250 ml) beaker (Figure 2).

The conversion proceeds via diazotization of one of the amine groups as per figure 3.

The clear solution was cooled to (15°C) and stirred magnetically and then added a solution of (0.11 mol, 7.5 gm) of NaNO2 in (15 ml) water in one portion. The reaction mixture becomes warm within (2-3 min) and reaches a temp of about (85°C) and begins to cool while the color changes from deep red to pale brown continue stirring for (15 min), by which time the temp will have dropped to (35-40°C) and then thoroughly chilled in an ice water bath for (30 min). Then the solid compound was separated and washed with (30 ml) portion of ice cold water, the compound purity was established by TLC and its result showed that only a single spot was observed. The percentage yield, physical appearance, melting point and Rf value of this compound are listed in Table 1.

Synthesis of 2-aminobenzotriazole (compound 3)24: Hydroxylamine-O-sulphonic acid was prepared (76%) by the method of GÕSL and Meuwsen, and dried in vacuum before use.

Benzotriazole (0.1 mol, 11.9 g) was dissolved in a solution of potassium hydroxide (0.5 mol) in water (100 ml) at (60°C). Solid hydroxylamine-o-sulphonic acid (0.2 mol) was added in portions during (1 hr), the temperature being maintained at (70-75°C). The mixture was then stirred for (1 hr) at (70°C), cooled, and filtered. The alkaline solution was extracted with ether (3 x 100 ml); the precipitated potassium sulphate was washed with ether, and the combined ether extracts and washings were dried and evaporated. The resulting solid (two components by (TLC) was chromatographed on silica gel (150 g; 200-300 mesh). Petroleum (b.p. 40-60°C) ether (2:1) eluted 2-aminobenzotriazole (11%) (Figure 4), the compound purity was established by TLC and its result showed that only a single spot was observed. The percentage yield, physical appearance, melting point, and Rf value of this compound are listed in Table 1.

Synthesis of the Heterocyclic Ring 5-amino-2- mercapto-1,3,4-thiadiazole (Compound 4)25: To thiosemicarbazide (0.1mol, 10g) suspended in anhydrous ethanol (40 ml), anhydrous sodium carbonate (5.82 gm) were added and carbon disulphide (0.12 mol, 10.1 gm). The reaction mixture was heated with stirring under reflux for (1 hr), and then heated on the steam bath for 4 hrs. The solvent was largely removed by rotary evaporator and the residue was dissolved in water (44 ml), then acidified with concentrated hydrochloric acid (8.8 ml) to give the pure product (Figure 5) after recrystallization from ethanol/water. Physical appearance, melting point, and Rf values are listed in Table 1.

Synthesis of Compound 5: Conventional Solution method26 was used as a coupling method between the (2- aminobenzotriazole, 5-amino- 2-mercapto- 1,3,4- thiadiazole) and compound 3. Dicyclohexylcarbodiimide (DCC) was used as a coupling reagent in amide bond formation; While 1-hydroxybenzotriazole (HOBT) and Nhydroxysuccinamide (HOSu) were used to increase the yields of the product22 and to suppress racemization.

To a stirred solution of 2-aminobenzotriazole (1mmol, 0.134 gm) in (3 ml) N,N-Dimethylformamide (DMF), Nmethylmorpholine (NMM), (1 mmol, 0.11 ml) was added followed by stirring for 10 minutes. Solution of compound 4 (1mmol, 0.133 gm) in (3 ml) DMF was added to the reaction mixture. The mixture was then cooled to (-15 °C), followed by DCC (1mmol, 0.23 gm) addition with stirring which was continued for (72 hrs) at (0 °C) and for (48 hrs) at ambient temperature (20 °C). Ethyl acetate (10 ml) was added to the reaction mixture which is then filtered to get rid of N,N-dicyclohexylurea (DCU). The filtrate was then evaporated to dryness under vacuum and the residue was re-dissolved in ethyl acetate (10 ml), the excess DCU which was still adhesive on the amide residue was precipitated out and filtered.

The clear filtrate washed twice with (5 ml) HCL (0.1 N) solution, once with (10 ml) Distilled Water and with (10 ml) saturated NaCl solution using the separatory funnel. The ethyl acetate layer was then dried under vacuum by rotary evaporator; the remaining ethyl acetate was dried using anhydrous magnesium sulphate. The product (Figure 6) was recrystallized from (ethanol: n-pentane) mixture to get the pure product. The percentage yield, physical appearance, m.p. and Rf value are Listed in Table 1.

Synthesis of Compound 6: The same procedure was carried out as mentioned previously for compound 5 in, but starting with compound 3; (1 mmol, 0.134 gm) which was coupled with levoflxacin, (1 mmol, 0.361 gm). Figure 7.

Antimicrobial activity27

Preliminary antibacterial and antifungal activity has been carried out according towel diffusion method. According to Bauer et al 1967, the antibacterial activity of the synthesized compounds were tested in-vitro against three pathogenic bacteria, these includes (Staphylococcus aureus, Streptococcus pyogenes) as gram positive bacteria and Proteus Spp. as gram negative bacteria and against two fungi (Aspergillus Spp. and Candida Spp.). They were clinically activated and maintained on nutrient agar medium for testing antibacterial activity and Sabaroud agar medium for antifungal activity. Ofloxacin was used as a standard drug for antibacterial activity and Ketoconazol was used as a standard drug for antifungal activity.

Sensitivity Assay28

All the synthesized compounds were prepared by making a stock solution with final concentration of (120 µg/ml) and by using the equation [C1V1 * C2V2], the other concentrations (2, 5, 20, 50 µg/ml) were performed using (1% Dimethylsulphoxide). Well diffusion assay was carried out by using bacterial suspension of about (1.5 × 106 CFU/ml) obtained from McFarland turbidity standard (number 0.5).

This was used to inoculate by swabbing the surface of Mueller Hinton agar (MHA) plates. Excess liquid was air dried under a sterile hood and the impregnated discs were applied at equidistant points on top of the agar medium. In each agar plate of tested bacteria, five wells were made and (30 µml) of each concentration was added in it. The inoculated culture medium prepared as above was immediately incubated at (30 °C) for 72 hours (fungal Spp.) or (37 °C) for 24 hours (bacteria) and the antimicrobial activity was evaluated by measuring the diameter of the inhibition zone (IZ) around the disc in mm.

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