Journal of Chemistry
Volume 2015 (2015), Article ID 982404, 6 pages
http://dx.doi.org/10.1155/2015/982404

Research Article

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

Mohammed El Fal,¹ Youssef Ramli,² Abdelfettah Zerzouf,³ Ahmed Talbaoui,⁴ Youssef Bakri,⁴ and El Mokhtar Essassi¹

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

Received 24 February 2015; Revised 16 April 2015; Accepted 20 April 2015

Academic Editor: Marco Radi

Copyright © 2015 Mohammed El Fal et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

New heterocyclic compounds spiroderivatives of allopurinol of biological interest were prepared from allopurinol via thionation and 1,3-dipolar cycloaddition and were produced in high to excellent yields. These compounds were characterized on the basis of spectral and spectroscopic data (¹H NMR, ¹³C, IR, and MS). The antibacterial activity of the synthesized products was studied using bacterial strains: Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Compounds having an ethyl group showed the best activity with MIC value of 31.25 µg/mL against Staphylococcus aureus and Streptococcus fasciens.

1. Introduction

Heterocycles are widely distributed in nature and play a key role in the metabolism of all living cells. Among the many heterocyclic compounds containing nitrogen, the pyrazolopyrimidine ring is very interesting and versatile scaffold for the synthesis of potential drugs or molecular tools (Figure 1). Among their many applications, pyrazolo,4-d]pyrimidines (Figure 2) were used as inhibitor kinases [1, 2] antiviral agents [3, 4], adenosine antagonists [5–7], glutamate modulators [8], antituberculosis agents [3], and as antibiotics inhibit bacterial growth [9, 10].

Figure 1: Pyrazolopyrimidine containing drugs.

Figure 2: Example of bioactive molecules derived from pyrazolo[3,4-d]pyrimidine and thiadiazole.

In light of the great importance of spirocyclic systems containing one carbon atom common to two rings [11–13], in recent years efforts have been made in developing methodologies for the synthesis of these compounds especially by cycloaddition reactions [14–22].

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.

Generally, reactions of 1,3-dipoles with true heterocyclic thiones having the thione form A proceed via 1,3-dipolar cycloaddition to the C=S double bond to form the spirocycloadducts, namely, spirothiadiazoles. The reaction of heterocyclic thiones A with nitrilimines, generated in situ by base-catalyzed dehydrohalogenation of hydrazonoyl halides, has been described for synthesis of various derivatives of spiro[heterocycle-n,2′-3H-1,3,4-thiadiazole] B (Figure 3).

Figure 3: Synthesis of spiroheterocycles.

In continuation of our work on the synthesis of the excess of allopurinol [23–28], compounds 2 and 2a-b have been synthesized by using reported methods [29–31]. Herein we report simple efficient synthesis of spiro thiadiazoline-(pyrazolo[3,4-d]pyrimidine) derivatives 4a-b by 1,3-dipolar cycloaddition of diphenyl hydrazonoyl chloride 3 with an equimolecular amount of pyrazolo[3,4-d]pyrimidin-4(5H)-thione derivatives 2a-b. All the synthesized compounds were evaluated for their antibacterial activity.

2. Materials and Methods

Generally 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. ¹H NMR and ¹³C NMR spectra were recorded on Bruker Avance 300 NMR Spectrometer in DMSO-. 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 P₂S₅ until there is colorless filtrate.

1H-Pyrazolo[3,4-d]pyrimidine-4(5H)-thione ( 2 ). Yield = 90%; mp: 151°C. IR:  cm⁻¹¹H NMR (DMSO-): δppm: 8.11 (1H, s, CH); 8.22 (1H, s, CH); 13.47 (1H, s, NH); 13.92 (1H, s, NH). ¹³C NMR (DMSO-) δppm: 105.05; 134.05 (Cq); 150.77 (CH); 151.20 (CH); 156.79 (Cq C=S). HRMS (ESI) [M + H]: m/z = 153.17.

1,5-Diethyl-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-thione ( 2a ). Yield = 75%; mp: 150°C. ¹H NMR (DMSO-) δ ppm: 1.3 (3H, t,  Hz. CH₃); 1.36 (3H, t, , CH₃); 4.30 (2H, q,  Hz, CH₂); 4.51 (2H, q,  Hz, CH₂); 8.15 (1H, s, CH); 8.72 (1H, s, CH). ¹³C NMR (DMSO-) δppm: 14.56 (CH₃); 15.15 (CH₃); 42.38 (CH₂); 46.34 (CH₂); 118.17; 137.09 (Cq); 145.09 (CH); 149.99 (CH); 179.11 (Cq, C=S). HRMS (ESI) [M + H]: m/z = 209.07.

1,5-Dibenzyl-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-thione ( 2b ). Yield = 70%; mp: 160°C. ¹H NMR (DMSO-) δ ppm: 3.25 (2H, s, CH₂); 3.78 (2H, s, CH₂); 6.85–7.12 (q, ); 8.21 (1H, s, CH), 8.89 (1H, s, CH). ¹³C NMR (DMSO-) δppm: 51.83 (CH₂); 54.31 (CH₂); 118.24; 127.94 (Cq); 128.11–137.89 (); 145.54 (CH); 150.95 (CH); 179.82 (Cq, C=S). HRMS (ESI) [M + H]: m/z = 33.10.

2.2. 1,3-Dipolar Cycloaddition

To a solution of 1,5-diethyl-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-thione (10 mmol) and diphenylnitrilimine (1.3 × 10 mmol) in THF (30 mL) triethylamine (2 mL) was added. The mixture was refluxed for 24 hours. The precipitate was collected by filtration and was separated by silica gel chromatography (hexane/ethyl acetate: 8/2).

2,5-Diethyl-3 ′,5 ′-diphenyl-2,5-dihydro-3 ′H-spiro[pyrazolo[3,4-d]pyrimidine-1,2 ′-[1,3,4]thiadiazole] ( 4a ). Yield = 60%; mp: 165°C. ¹H NMR (DMSO-) δ ppm 1.12 (3H, t,  Hz, CH₃); 1.28 (3H, t,  Hz, CH₃); 3.37 (2H, q,  Hz. CH₂); 4.11 (2H, q,  Hz, CH₂); 6.83–7.67 (q,  Hz, ); 7.69 (1H, s, CH); 7.69 (1H, s, CH). ¹³C NMR (DMSO-) δppm: 15.61 (CH₃); 16.03 (CH₃); 41.88 (CH₂); 42.28 (CH₂); 99.70; 100.79; 117.03; 126.38 (Cq); 129.26–142.20 (); 143.87 (CH); 148.01 (CH). HRMS (ESI) [M + H]: m/z = 403.16.

2,5-Dibenzyl-3 ′,5 ′-diphenyl-2,5-dihydro-3 ′H-spiro[pyrazolo[3,4-d]pyrimidine-1,2 ′-[1,3,4]thiadiazole] ( 4b ). Yield = 60%; mp: 185°C. ¹H NMR (DMSO-) δ ppm: 3.65 (2H, s, CH₂); 4.28 (2H, s, CH₂); 7.25–7.32 (q,  Hz, ); 8.81 (1H, s, CH); 8.99 (1H, s, CH). ¹³C NMR (DMSO-) δppm: 50.83 (CH₂); 53.21 (CH₂); 77.2; 118.24; 121.02; 125.13; 127.94; 128.04; 128.15; 128.46; 128.79 (Cq); 129.12–137.9 (CHAr); 145.54 (CH); 150.95 (CH). HRM (ESI) [M + H]: m/z = 527.39.

3. 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 ¹H NMR, ¹³C NMR, IR, and mass spectra.

IR-spectra of compounds 2a-d did not contain C=O-group signals, the signal of C=S group (1500cm⁻¹) was present. The spectra of ¹³C 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 C₅H₄N₄S compound 2, molecular ion at m/z 208.07 (M+) corresponding to the molecular formula C₉H₁₂N₄S compound 2a and molecular ion at m /z 332.10 (M+) corresponding to the molecular formula C₁₉H₁₆N₄S compound 2b (Scheme 1, Figure 4) [23].

Scheme 1: Thionation of 1,5-dialkyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 1a-b.

Figure 4: ORTEP presentation of compound 2b.

We investigated the reaction of 1,3-dipolar cycloaddition of diphenyl hydrazonoyl chloride 3 with equimolecular amount of 1,5-diethyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-thione 2a in dry tetrahydrofuran in presence of triethylamine (Scheme 2); one cycloadduct was obtained as a result of 1,3-dipolar cycloaddition of diphenyl nitrile imine ylide generated in situ from diphenyl hydrazonoyl chloride and triethylamine, on the dipolarophilic group C=S.

Scheme 2: 1,3-Dipolar cycloaddition of 1,5-diethyl-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-thione 2a and DPNI 3.

The structure of these compounds was confirmed on the basis of their spectroscopic characteristics. The ¹H NMR spectra of 4a (in DMSO-) 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 CH₂ and CH₃ 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].

In order to examine the N-substitution effect of alkyl group on the 1,3-dipolar cycloaddition, 1,5-dibenzyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-thione 2a was chosen to be employed to react with DPNI. So in this case it was found that C=S group underwent the 1,3-dipolar cycloaddition reaction and formed of novel spiro[thiadiazoline-(pyrazolo[3,4-d]pyrimidine)] (Scheme 3).

Scheme 3: 1,3-Dipolar cycloaddition of 1,5-dibenzyl-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-thione 2b and DPNI.

The structure of compound 4b was established by IR, ¹H NMR, ¹³C NMR, and mass spectrum. Its IR spectrum showed a characteristic absorption band at 1647 cm⁻¹ for the C=N- indicating the spirocarbon formation. Its ¹H NMR spectrum exhibited peaks at 7.25–7.32 ppm (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 CH₂ 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].

3.1. In Vitro Antibacterial Activity

We studied spiropyrazolo[3,4-d]pyrimidines newly synthesized for their antibacterial activity against Escherichia coli (ATTC-25922), Staphylococcus aureus (ATCC-25923), Pseudomonas aeruginosa (ATCC 27853), and Enterococcus faecalis (ATCC-29212) strains of bacteria by the diffusion method disk [32]. The MIC values of the compound against bacteria are presented in Table 1.

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

Product
MIC in g/L

S. aureus
E. faecalis
E. coli
P. aeruginosa

1
250
125
250
250

1a
250
250
250
—

1b
125
62.5
62.5
250

2
250
125
125
250

2a
250
125
250
250

2b
250
250
250
250

4a
125
62.5
125
250

4b
31.25
31.25
125
125

Chlor
1.875
3.75
15
7.5

Amp
2.5
1.25
5
5

MIC: minimum inhibitory concentration.

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.

4. 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.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

The authors thank Pharmaceutical Laboratories PHARMA 5 for supporting this study.

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