Synthesis and Antibacterial Activity of New Spiro[thiadiazoline-(pyrazolo[3,4-d]pyrimidine)] Derivatives

1Laboratoire de Chimie Organique Hétérocyclique URAC 21, Faculté des Sciences, Université Mohammed V, Rabat, Morocco 2Medicinal Chemistry Laboratory, Faculty of Medicine and Pharmacy, Mohammed V University, 10170 Rabat, Morocco 3Laboratoire de Physico-Chimie des Matériaux Inorganiques et Organiques (LPCMIO), ENS, Rabat, Morocco 4Laboratoire de Biochimie et Immunologie, Faculté des Sciences, Université Mohammed V, Rabat, Morocco

1,3-Dipolar cycloaddition is a subject of intense research during the last decade due to its great synthetic value.
The cycloaddition is a method of synthesis of five membered heterocycles, which are difficult to prepare by other means.

Materials and Methods
Generally the melting points were taken on an electrothermal capillary melting point apparatus.Infrared spectra (]-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- 6 .Spectra were internally referenced to TMS.Peaks are reported in ppm downfield of TMS.Mass spectra are recorded in a SYNAPT G2 HDMS (Waters) Spectrometer in electrospray ionization (ESI).
2.1.Thionation.3.67 mmol of pyrazolo [3,4-d]pyridine is refluxed in pyridine with 3.67 mmol of phosphorus pentasulfide for 4 hours.Then the solvent is evaporated under reduced pressure, and the precipitate formed is washed with hot water to remove residual dimerized P 2 S 5 until there is colorless filtrate.

Results and Discussion
We first prepared 1,5-diethyl-1H-pyrazolo [3,4-d]pyrimidine-4(5H)-thiones 2a-b from 1a-b by refluxing phosphorus pentasulfide in pyridine.The identification of the product was determined by 1 H NMR, 13 C NMR, IR, and mass spectra.IR-spectra of compounds 2a-d did not contain C=Ogroup signals, the signal of C=S group (1500cm −1 ) was present.The spectra of 13 C NMR corroborated the results of IR.In addition, its mass spectrum shows a molecular ion peak at m/z 152 (M+) corresponding to the molecular formula C 5 H 4 N 4 S compound 2, molecular ion at m/z 208.07 (M+) corresponding to the molecular formula C 9 H 12 N 4 S compound 2a and molecular ion at m /z 332.10 (M+) corresponding to the molecular formula C 19 H 16 N 4 S compound 2b (Scheme 1, Figure 4) [23].
The structure of these compounds was confirmed on the basis of their spectroscopic characteristics.The 1 H NMR spectra of 4a (in DMSO- 6 ) showed an aromatic multiplet in the region of 6.83 to 7.67 ppm corresponding to the aromatic protons.Two downfield singlets were observed in the region of 7691-7968 ppm representing the protons for CH in pyrimidine ring and CH in pyrazole ring because of the high incidence of the aromatic ring system deshielding protons CH pyrimidine CH pyrazole.1H NMR spectra of 4a also showed the CH 2 and CH 3 signals as triplets and multiplets and between 1.12 and 1.27 ppm and between 3.37 and 4.11 ppm, respectively. 13C NMR spectra of 4a exhibit in signal spirocarbon to 99.70 ppm, aromatic carbons 100.79 to 129.38 ppm, and the imine carbon to 142.2 and 139.93 ppm (HC=N).The mass spectrum shows a peak at m/z 403.16 corresponding to [M + H].
The structure of compound 4b was established by IR, 1 H NMR, 13 C NMR, and mass spectrum.Its IR spectrum showed a characteristic absorption band at 1647 cm −1 for the >C=Nindicating the spirocarbon formation.Its spectrum exhibited peaks at 7.25-7.32ppm (20H) indicating the presence of aromatic protons.The two singlets at 3.65 ppm and 4.28 ppm relating to two protons of the two CH 2 groups were also observed. 13C NMR spectra of 4b exhibited, in particular, a spiro carbon signal at 77.2 ppm.The mass spectrum shows a peak at m/z 527.39 corresponding to [M + H].
We synthesized new spirocompounds with a high yield by cycloaddition reaction.They have showed moderate antibacterial activity against selected bacteria.All these compounds showed at an average concentration low antibacterial activity against the bacteria, except that compounds 1a and 4a showed higher activity, even at higher concentrations.Compound 4b showed higher activity against S. aureus and E. faecalis compared to bacteria E. coli and P. aeruginosa.

Conclusion
In conclusion, a new class of heterocyclic spiro[thiadiazoline pyrazolopyrimidine] compounds was synthesized and their structure was determined and also tested for their antibacterial activity in vitro.This study is expected to take the tests of anti-inflammatory drugs, antifungal, and anticancer activity because the literature gives some very interesting results on these topics.

Table 1 :
Antibacterial activity of the compounds: MIC in g/mL.