Synthesis and antibacterial activity of new spiro [ thiadiazoline-quinoxaline ] derivatives

The 1,3-dipolar cycloaddition of 3-methylquinoxaline-2-thione and their N-alkylated derivatives to diphenylnitrile imine is presented. Using this method, spiro[thiadiazoline-quinoxaline] derivatives as biologically interesting compounds were produced in high to excellent yields. These compounds have been characterized on the basis of their spectroscopic et spectrometric data (H and C NMR, IR, MS and X-ray). Antibacterial activity of the synthesized products has been studied by employing five bacterial strains. The compounds with ethyl group showed the best activity with MIC value of 32 μg/mL against Streptococcus fasciens.


Introduction
The chemistry of quinoxalines has attracted considerable attention in the last decade for their chemical reactivity 1,2 biological properties, 3,4 and materials applications. 5,6They exhibit a wide spectrum of biological activities such as antibacterial, 7 antifungal, 8 and anticancer. 9Moreover, quinoxaline ring is a part of various antibiotics, such as Echinomycin, Levomycin and Actinoleutin that are known to inhibit growth of gram positive bacteria and to be active against various tumors.Also, thiadiazoline moieties are present in the structure of various bioactive molecules found to act as anti-inflammatory, 10 analgesic 11 and allosteric modulator. 12he 1,3-dipolar cycloaddition has been the subject of intense research over the last decade, due to its great synthetic value.The cycloaddition is a process of synthesis of five-membered heterocycles, difficult to be prepared with other ways.[15] Figure 1.Examples of bioactive molecules derived from quinoxaline and thiadiazole.
In continuation of our work on the synthesis of quinoxaline derivatives, 16,17 we report the preparation and investigation of novel spiro[thiadiazoline-quinoxaline] as potential antibacterial compounds.

Results and Discussion
Initially, we have shown that the thionation of N-alkylquinoxaline 1a-b with phosphorus pentasulfide in refluxing pyridine results in rapid formation of the corresponding alkyquinoxaline derivatives 1a-b.The products were easily obtained by evaporation of pyridine and addition of warm water to the reaction mixture and the results were excellent in terms of yields and purity.

Scheme 1. Thionation of N-alkyl quinoxalin-2-one 1a-b.
The structure of compounds 2a-b was unambiguously characterized on the basis of their IR, 1 H NMR, 13 C NMR, and mass spectra.The 1 H NMR spectra of these compounds showed in particular the presence of protons corresponding to the alkyl groups.After these, we investigated the reaction of 1,3-dipolar cycloaddition of diphenyl hydrazonoyl chloride with an equimolecular amount of 1-ethyl-3-methylquinoxaline-2-thione in dry tetrahydrofuran in the presence of the triethylamine (Scheme 2), one cycloadduct was obtained as a result of the 1,3-dipolar cycloaddition of diphenylnitrile imine ylide generated in situ from diphenyl hydrazonoyl chloride and triethylamine, on the dipolarophilic group C=S.The structural assignment of product 4a was straight forward and relied upon the elemental analysis and structural data including IR, 1 H NMR and 13 C NMR spectra and single X-ray diffraction.The 1 H NMR spectrum of compound 4a demonstrated a singlet at 2.16 ppm due to methyl protons at C3, the multiplets at 0.86 and 2.21 ppm were assigned to the ethyl protons appeared as ABX3 patterns due to the presence of the chiral center at C2.The aromatic protons appeared as a multiplet at 4.89-7.49ppm.The 13 C NMR spectrum exhibited the following signals : methyl carbons at 12.0 and 22.1 ppm, methylene carbon at 39.7 ppm, the spiro carbon at 102.6 ppm, the aromatic carbons at 113.4-132.6, the imine carbon at 156.6 and 142.7 ppm for (S-C=N).The mass spectrum shows a peak at m/z 399.1633 corresponding to [M+H].An X-ray crystallographic study of a single crystal of 4a (Figure 2, table 1) confirmed the structure deduced from NMR spectroscopic studies.In order to examine the N-substitution effect of alkyl group on the 1,3-dipolar cycloaddition, N-allyl-3-methylquinoxaline-2-thione was chosen to be employed to react with DPNI.So in this case it was found that both the double bond C=S and C=C of allyl group underwent the 1,3dipolar cycloaddition reaction and formed regioisomeric mixture of novel spiro[thiadiazolinequinoxaline] in excellent yield (Scheme 3).The two products obtained were separated by silica gel column chromatography.The structure of these compounds was confirmed on the basis of their spectroscopic characteristics.In the 1 H NMR spectrum of 4b, the allylic methylene group consists of two diastereotopic protons resonate as dd at 3.88 ppm.The other three consecutive signals correspond to the three vinylic protons resonate at 4.95, 5.11 and 5.51 ppm respectively.The aromatic protons resonate at 6.86-7.69ppm.On the other hand the spiro carbon appeared at 102.4 ppm in the 13 C NMR, the allylic carbon showed resonance at 47.6, 118.4,133.1 ppm.The 1 H NMR spectrum of 4b' shows a multiplet in the region δ 2.70-3.00for the CH2 protons of pyrazoline ring.The N-CH2 protons of the methylene group appeared as a multiplet in the region δ 3.47-3.69while the CH proton of the pyrazoline ring appears as a multiplet at δ 4.39.The aromatic protons appeared at the region 6.56-7.77ppm.The 13 C NMR shows signals at δ 36.1, 58.1 and 102.6 for CH, CH2 pyrazolinic and spiro carbon, respectively, which confirms the structure of the products.
The structure of 4b' was unequivocally evidenced by a single crystal X-ray data (Figure3; Table 1).

In vitro antibacterial activity
The compounds were evaluated for their in vitro antibacterial activity against Escherichia coli ATCC 4157, Pseudomonas aeroginosa ATCC 27853, Proteus vulgaris ATCC 13315, Staphylococcus aureus ATCC 25923 and Streptococcus fasciens ATCC 29212 using an Agar dilution method. 18The microorganisms used were procured from the Department of Microbiology Medicinal, National Institute of health, Rabat, Morocco.All bacteria were grown on Mueller-Hinton Agar (Hi-media) plates (37 °C, 24 h).The Minimum Inhibitory Concentration (MIC) was considered to be the lowest concentration that completely inhibited the growth on agar plates, disregarding a single colony or faint haze caused by the inoculum.The results of the screening are shown in Table 2.In antibacterial activity studies, an introduction of ethyl group reflected better activity 64 µg/mL against Staphylococcus aureus and 256 µg/mL against Streptococcus fasciens followed in decreasing order by 1-ethyl-spiro[thaidiazoline-quinoxaline] with 128 µg/mL against S. aureus.In contrast, surprisingly the other molecules were found to be totally inactive against all test bacteria species.By comparison of the inhibiting effects of the alkyl groups, we can deduce that the presence of ethyl group conferred the highest antibacterial action. 19,20When compared to Clotrimazole and Tetracycline, compound 2a was less active and compound 2 was more active against Streptococcus fasciens than both references.

Conclusions
In conclusion, new spiro[thiadiazoline-quinoxaline] were synthesized and their structure were determined and also assayed for their in vitro antibacterial activity.This study should extend on tests anti-inflammatory drug, antifungal and anti-cancer because the literature gives results enormously interesting on these subjects.Also of other bacteria should be also selected to widen the investigation.

Experimental Section
General.The melting points were taken on an Electrothermal capillary melting point apparatus.Infrared spectra (ν-cm -1 ) were recorded on a Perkin Elmer 577, using KBr disks. 1 H-NMR and 13 C-NMR spectra were recorded on Bruker Avance 300 NMR Spectrometer in DMSO-d6.Spectra were internally referenced to TMS.Peaks are reported in ppm downfield of TMS.Mass spectra recorded in a SYNAPT G2 HDMS (Waters) spectrometer in Electrospray Ionization (ESI).

Table 1 .
Crystallographic data of compounds 4a and 4b'

Table 2 .
Antibacterial activity of the compounds: MIC's in mg/mL