Synthesis and antibacterial activity of 9-cyclopropyl-4-fluoro-6-oxo-6 , 9-dihydro-[ 1 , 2 , 5 ] thiadiazolo [ 3 , 4-h ] quinoline-7-carboxylic acid and its ethyl ester

We report on the synthesis and the antimicrobial activity of 9-cyclopropyl-4-fluoro-6oxo[1,2,5]thiadiazolo[3,4-h]quinoline-5-carboxylic acid (16), representative of a new class of antibacterial agents structurally related to the clinically successful fluoroquinolones. The novel 6-fluoroquinolone (16) is herein prepared, in good yield, by thermal cyclocondensation of ethyl 7,8-diamino-9-cyclopropyl-4-fluoro-6-oxoquinoxaline-7-carboxylate with thionyl chloride, followed by acid-catalysed hydrolysis of the ester intermediate (15). Their structures were characterized by IR, MS, Hand C NMR spectra and X-ray crystal structure of 16 is presented. The antimicrobial evaluation against a broad panel of wild and resistant Grampositive and Gram-negative bacteria and fungal species demonstrated the high antibacterial activity of 16, even at the concentration of 0.015 μg mL.


Introduction
Resistance to currently available antibacterial drugs is bringing alarming threat to public health and causing growing concern among people across the globe.At the same time as the old antibiotics are losing their effectiveness, the supply of new drugs is drying up.Our best weapon against this threat is to continually develop new antibiotics and new synthetic antibacterials against which bacteria have not yet developed resistance.
Scheme 1. Synthesis of thiadiazolo [3,4-h] The IR, MS, NMR spectral data for the new compounds 15 and 16 are in accordance with the assigned structures; details are given in the experimental part.Thus, the mass spectra display the correct molecular ion peaks for which the measured high resolution (HRMS) data are in good agreement with the calculated values.Based on 1 H NMR, proton-decoupled 13 C NMR spectra, DEPT 135 and DEPT 90 experiments, the ester derivative 15 displayed 1CH 3 , 2CH 2 , 3CH and 8C-quaternary carbons, whereas the parent carboxylic acid 16 showed, as expected, 1CH 2 in addition to 3CH and 8C-quaternary carbons.2D (COSY, HMQC, HMBC) experiments showed correlations that helped in the 1 H-and 13 C-signal assignments to the different carbons and the neighboring hydrogens.In HMBC experiments, distinct 'three-bond' ( 1 H, 13 C) correlations are observed between H-5 and each of C-3a, C-9a and C-6, and between H-8 and each of C-9a, C-1', C-6 and CO 2 Et/CO 2 H.

Crystallography
The structure of 16 was confirmed by means of the X-ray diffractive analysis of single-crystal.A summary of data collection and refinement parameters is given in Table 1.Selected bond angles are listed in Table 2.The molecular structure of 16, based on crystallographic data, is displayed in Figure 3.

Antimicrobial assay
The new fluoroquinolone 16 and its ester parent compound 15 were tested in vitro against a wide spectrum of Gram-positive (Table 3) and Gram-negative (Table 4) bacteria, yeasts and moulds.The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC), both expressed in µg mL -1 , were determined and compared to those of ciprofloxacin 7 as reference drug (Tables 3 and 4).Since the development of new antimicrobial agents is increasingly important due to the continual emergence of microbial strains that demonstrate multidrug resistance, both methicillin-and ciprofloxacin-resistant bacteria were assayed simultaneously.
Compound 16 shows a very strong activity against Gram-positive bacilli and staphylococci (MICs 0.015-1.5 µg mL -1 ), including methicillin-resistant Staphylococcus aureus, and against most of the Gram-negative bacteria tested (MICs 0.07-3 µg mL -1 ).A limited effect is detected for the ester parent compound 15 against the same microorganisms (MICs 3-200 µg mL -1 for Grampositive bacteria and 12-400 µg mL -1 for Gram-negative ones), confirming that the carboxylic group on 7-position is an optimal requirement for the antibacterial potency of these compounds. 27The spectrum of activity and the degree of efficacy of compound 16 against the different strains of bacteria is similar to that of ciprofloxacin 7, with MIC values identical or higher than those of the reference compound.The only exception is Staphylococcus haemolyticus that is more sensitive to compound 16 than ciprofloxacin.Both 15 and 16 act as bacteriostatic agents, being MBC values always higher than the corresponding MICs.As expected, compound 16, bearing a fluorine atom and a cyclopropyl group at C(4)-and C(9)-positions, respectively, showed increased antibacterial activity when compared with compound 5 (Tables 3 and 4). 28

Conclusions
The 9-cyclopropyl-4-fluoro-6-oxothiadiazolo[3,4-h]quinoline-7-carboxylic acid 16 described herein represents a potential antibacterial agent for treatment of serious Gram-positive and Gram-negative infections.However, this compound still lacks effectiveness against methicillinand quinolone-resistant strains.These results encourage further modifications of the fluorothiadiazoloquinoline scaffold to provide novel compounds more active than the existing quinolones.

Experimental Section
General.Pure grade thionyl chloride, benzene, methanol and dichloromethane were purchased from Acros Organics (Geel, Belgium).Ciprofloxacin used as standard quinolone was obtained from Sigma (Milano, Italy).Melting points (uncorrected) were determined on a Gallenkamp electrothermal melting-temperature apparatus. 1H-and 13 C NMR spectra were measured on a Bruker DPX-300 instrument.Chemical shifts are expressed in ppm with reference to TMS as internal standard.Electron impact mass spectra (EIMS) were obtained using a Finnigan MAT TSQ-70 spectrometer at 70 eV and at an ion source temperature of 200ºC.High-resolution mass spectra (HRMS) were measured in positive ion mode using electrospray ion trap (ESI) technique by collision-induced dissociation on a Bruker APEX-4 (7 Tesla) instrument.The samples were dissolved in acetonitrile, diluted in spray solution (methanol/water 1:1 v/v + 0.1% formic acid) and infused using a syringe pump with a flow rate of 2 µL/min.External calibration was conducted using Arginine cluster in a mass range m/z 175-871.IR spectra were recorded as KBr discs on a Nicolet Impact-400 FT-IR spectrophotometer (abbreviations: vs = very strong, s = strong, m = medium, w = weak, br = broad).Elemental analyses (C, H, N, S) were performed at the Microanalytical Laboratory of the Hashemite University, Zarqa-Jordan, and the results were found to be in good agreement (± 0.4%) with the calculated values.

X-ray diffraction
Yellow block crystals, suitable for X-ray crystallography, were grown slowly from a solution of 16 in DMF.Crystal size (mm 3 ): 0.34 x 0.26 x 0.22.Crystal data collection was made with a Siemens SMART three axis goniometer with APEX II area detector system.The data were reduced with Bruker AXS APEX 2 Vers.2.0-2 2006, and the structure was solved by the direct method using AXS SHELXTL programs Vers.2008 /4/(c) 2008.

Figure 3 .
Figure 3. ORTEP plot of the molecular structure of 16.Displacement ellipsoids are drawn at the 50 % probability level.

Table 1 .
Crystal data and structure refinement parameters for compound 16