Study of Mesoionic Compounds : Synthesis and Pharmacological Evaluation of Several 2-[ { ( 4-Substituted-1-sulphonyl ) Sydnon-3-yl } ]-1 , 3 , 4-thiadiazino ( 6 , 5b ) indoles as Antimicrobial , Insecticidal and Antihelmintic Agents

In the present study several novel 2-[{(4-Substituted-1-sulphonyl)sydnon-3-yl}]-1,3,4thiadiazino(6,5-b) indoles [4a-f] synthesised from 2-amino-1,3,4-thiadiazino (6,5-b) indole. Structures of the synthesized substituted indoles were characterized by IR, 1H-NMR, Mass and elemental analysis (C, H, N). Synthesised indole derivatives were screened for antimicrobial, insecticidal and anthelmintic activities. Compound 4a displayed significant biological spectrum.


INTRODUCTION
An enor mous amount of research on synthetic and pharmacological studies of sydnones has been reported during the last few decades 1 .Mesoionic compounds are non-benzenoid heterocycles containing different combination of hetero atoms.Sydnones being mesoionic compounds, are 1, 2, 3-oxadiazolium-5-olates and their chemistry has been widely studied by the scientific workers [2][3] due to their electronic and versatile biological profile.Literature is evident to reveal that sydnone derivatives are most important indoles 4a-f starting from 2-amino-1, 3, 4-thiadiazino (6, 5-b) indole (Scheme-1).Hence all these observation prompted us to synthesis some novel derivatives of indoles bearing syndnone moiety.It was interesting to study the biological behaviours of various substituted sydnones.

RESULTS AND DISCUSSION
Synthetic strategy has been outlined in Scheme-1.Compound 1 and 2 were synthesised by our earlier reported method 29 .The condensation reaction of compound 2 and chloro sulphonic acid in presence of catalytic amount of phosphorous pentoxide yielded the compound 3.The formation of compound 3 was evidenced by the disappearance of singlet in 1 H-NMR spectra and IR spectral band at d 4.69 ppm and 3106 cm -1 respectively.It was further evidenced by the appearance of IR spectral band at 1180 cm -1 due to the SO 2 group.The desired derivatives, 4a-f were obtained by the reaction of compound 3 and secondary amines.In 1 H-NMR spectra compounds 4, the peaks were observed at d 2.02-2.51due to hydrogens of morpholine ring.Presence of absorption band at 1460, 1120 for C-O-C of morpholine ring cleared the synthesis of derivatives 4a-f.Scheme-2 explored the general mass fragmentation of 2-[{(4-morpholino-1-sulphonyl) sydnon-3-yl}]-1,3,4-thiadiazino(6,5-b) indoles 4a.In the mass spectra of compound 4a, the molecular ion peak 420 [M + ] (15%) also confirmed the formation of the sydnonyl substituted indoles.The molecular, base peak and other peak with their relative intensities were summarised in Table 1

Antimicrobial screening
All the newly synthesized compounds 3 and 4a-f were screened for their antibacterial and antifungal activity.All the bacterial as well as fungal strains were clinical isolates, identified with conventional morphological and biochemical methods.The microorganisms employed antibacterial studies were Staphylococcus aureus, E. coli and Klabsiella pneumoniae.Disk diffusion method 30- 31 was used for determination of the preliminary antibacterial activity.Disks measuring 6.25 mm in diameter were punched from Whatman no. 1 filter paper.batches of 100 disks were dispensed to each screw-capped bottle and sterilized by dry heat at 140 °C for an hour.The test compounds were prepared with different concentrations using DMF.One milliliter containing 100 times the amount of chemical in each disk was added to each bottle, which contained 100 disks.Disks of each concentration were for placed in triplicate in nutrient agar medium seeded with fresh bacteria separately.The incubation was carried out at 37 °C for 24 h.Ampicillin trihydrate and fluconazole were used as standard drugs.Solvent and growth controls were kept and zones of inhibition were noted.The bacterial inhibition values (mm) of the tested compounds against the tested bacterial strains are recorded in Table 2. On the other hand, the newly prepared compounds were screened for their in vitro antifungal activity against Aspergillus fumigatus (plant isolate), Candida albicans and Candida glabrata, in DMF by the serial plate dilution method [32][33] .Sabouraud's agar media were prepared by dissolving peptone (1 g), D-glucose (4 g), and agar (2 g) in distilled water (100 ml) and adjusting the pH to 5.7.Normal saline was used to make a suspension of the spore of fungal strain for lawning.A loopful of particular fungal strain was transferred to 3 ml saline to get a suspension of the corresponding species.Agar media (20 ml) was poured into each petri dish.Excess suspension was decanted and the plates were dried by placing in an incubator at 37 °C for 1 h.Using an agar punch wells were made into each well labelled.A control was also prepared in triplicate and maintained at 37 °C for 3-4 days.Antifungal activity was determined by measuring the diameter of the inhibition zone.The fungicidal inhibitory (mm) values of the tested compounds against the tested fungal species are recorded in Table-2.

Insecticidal activity
Periplaneta americana was taken for insecticidal study and 1 and 2% solutions of the derivatives 3 and 4a-f were injected in the abdominal of P. americana with the help of micro syringe.The time of death had been noted as KD (Knock promising to moderate activity) value.Cypermethrin was used as standard drug.At the time of death the antennae of P. americana became motionless, the appendages shrunk and folded towards the ventral side and cockroach lay dorsally .

Anthelmintic activity
Indian adult earthworms (Pheretima posthuma) were collected from moist soil and washed with normal saline to remove all faecal matter and used for the anthelmintic activity.All the synthesized derivatives 3 and 4a-f were dissolved in minimum amount of DMF and the volume adjusted to 10 ml with saline water.All solutions of synthesized derivatives and drugs solutions were freshly prepared.Groups of six earthworms were released into desired formulations and the paralytic and lethal time noted.Albendazole was used as standard drug.Observations were made for the time taken for paralysis and death of individual worms.Paralysis was said to occur when the worms did not receive even in normal saline.Death was concluded when the worms lost their motility followed by fading away of their body colour [35][36][37][38].

EXPERIMENTAL
All the chemicals used for the preparation of desired derivatives, were obtained from Sisco Research Laboratories (SRL), Mumbai, India; Qualigen Fine Chemicals, Mumbai, India; E. Merck Ltd., New Delhi, India.The reference drugs ampicillin trihydrate, fluconazole, cypermethrin and albendazole were procured from Ind-Swift, Pharmaceutical, Panjab; Savorite Pharmaceuticals, Gujarat; Royal Crop Science, Panipat, India.The melting points of the compounds were determined in open glass capillaries with the help of thermonic melting points apparatus (Campbell Electronics, Mumbai, India) and are uncorrected.The homogeneity of all the newly synthesized compounds were routinely checked by TLC on silica gel G plates and spots were located by using iodine chamber.Elemental analysis was performed in Heraeus CHN rapid analyser.The results were found within the ±0.4% of theoretical values.Infrared spectra were recorded on Kbr pellets on a Perkin Elmer system 2000 FTIR spectrometer and 1 H-NMR spectra on bruker DPX 200 using TMS as internal standard.
. The mass spectral study of sydnonyl substituted indoles publicized that parent compound 4a cleaved and released gradually fragment radicals and neutrals by following two routes viz route A and route b.Route A produced two daughter ions [a] + and [b] + while route b also produced two daughter ions [n] + and [o] + .Ion [a] + releases N 2 radical to give ion [c] + which further expel out S radical to yield quinolinium ion [d] + .Quinolinium ion 28 [d] + splits to give ion [e] + .In ion [b] + , sydnonyl ring ruptures similarly to that of other pattern of sydnone derivatives 29 to yield two ions, ion [f] + and [j] + .Ion [f] + further cleaved into ions [g] + and [s] + .Ion [g] + transformed into ions [h] + and [i] + .Ion [j] + breaks down to give ions [k] + and [l] + and SO 2 release converted ion [l] + into ion [m] + .Ions [p] + and [q] + were obtained by the splitting of ion [n] + .Expulsion of NO radical from ion [q] + furnish ion [r] + (Table-1).