Synthesis and antibacterial activity of new spiro[thiadiazoline-(pyrazolo[3,4-d]pyrimidine)] derivatives

Article abstract of DOI:10.1155/2015/982404

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.

Full text of DOI:10.1155/2015/982404

Hindawi Publishing Corporation
Journal of Chemistry
Volume 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
1
´ ´
´
´
Laboratoire de Chimie Organique Heterocyclique URAC 21, Faculte des Sciences, Universite Mohammed V, Rabat, Morocco
2Medicinal Chemistry Laboratory, Faculty of Medicine and Pharmacy, Mohammed V University, 10170 Rabat, Morocco
3
´
Laboratoire de Physico-Chimie des Materiaux Inorganiques et Organiques (LPCMIO), ENS, Rabat, Morocco
Laboratoire de Biochimie et Immunologie, Faculte des Sciences, Universite Mohammed V, Rabat, Morocco
4
´
´
Correspondence should be addressed to Youssef Ramli; yramli76@yahoo.fr
Received 24 February 2015; Revised 16 April 2015; Accepted 20 April 2015
Academic Editor: Marco Radi
Copyright © 2015 Mohammed El Fal et al. is 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.
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. ese compounds were characterized on the basis of
spectral and spectroscopic data (1H NMR, 13C, IR, and MS). e 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 ꢀg/mL against Staphylococcus aureus and Streptococcus fasciens.
1. Introduction
e 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 spirocy-
cloadducts, namely, spirothiadiazoles. e reaction of het-
erocyclic 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,2ꢀ-3H-1,3,4-thiadiazole] B (Figure 3).
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.
Heterocycles are widely distributed in nature and play a key
role in the metabolism of all living cells. Among the many het-
erocyclic compounds containing nitrogen, the pyrazolopy-
rimidine ring is very interesting and versatile scaffold for
the synthesis of potential drugs or molecular tools (Figure 1).
Among their many applications, pyrazolo[3,4-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].
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 method-
ologies 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.

2
Journal of Chemistry
Zaleplon
N
O
N
O
O
N
S
N
H
N
N
O
Indiplon
N
O
H
N
N
N
N
N
N
Pyrazolo[4,3-d]pyrimidine
O
N
N
N
N
N
N
N
N
N
Pyrazolo[1,5-a]pyrimidine
Pyrazolo[1,5-a]pyrimidine
N
N
S
N
N
Sildenafil
N
H
O
Pyrazolo[3,4-d]pyrimidine
OH
N
N
N
H
N
Allopurinol
Figure 1: Pyrazolopyrimidine containing drugs.
NHR2
O
N
N
N
CH3
N
S
N
Ph
N
N
N
N
N
N
X
N
R1
H3C
Ph
X: O,S
OCH3
Figure 2: Example of bioactive molecules derived from pyrazolo[3,4-d]pyrimidine and thiadiazole.
1
R
were recorded on a Perkin Elmer 577, using KBr disks. H
NMR and 13C 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).
S
Base
−HX
+ R-C(X)=NNHAr
Het
S
Het
N
N
Ar
(B)
(A)
Figure 3: Synthesis of spiroheterocycles.
2.1. ionation. 3.67 mmol of pyrazolo[3,4-d]pyridine is re-
fluxed in pyridine with 3.67 mmol of phosphorus pentasul-
fide for 4 hours. en the solvent is evaporated under reduced
pressure, and the precipitate formed is washed with hot water
to remove residual dimerized P S until there is colorless
2. Materials and Methods
Generally the melting points were taken on an electrothermal
2
5
capillary melting point apparatus. Infrared spectra (]-cm−1)
filtrate.

Journal of Chemistry
3
O
S
1H-Pyrazolo[3,4-d]pyrimidine-4(5H)-thione (2). Yield = 90%;
mp: 151∘C. IR: ꢂ = 1585 cm−1 1H NMR (DMSO-ꢁ6): ꢃppm:
8.11 (1H, s, CH); 8.22 (1H, s, CH); 13.47 (1H, s, NH); 13.92
(1H, s, NH). 13C NMR (DMSO-ꢁ6) ꢃppm: 105.05; 134.05 (Cq);
150.77 (CH); 151.20 (CH); 156.79 (Cq C=S). HRMS (ESI) [M +
H]: m/z = 153.17.
R
R
N
N
P2S5/pyridine
N
N
Δ
N
N
N
N
R
R
1: R = H
2: R = H (90%)
1,5-Diethyl-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-thione (2a).
1a: R = ethyl
2a: R = ethyl (65%)
Yield = 75%; mp: 150∘C. 1H NMR (DMSO-ꢁ6) ꢃ ppm: 1.3 (3H,
t, ꢄ = 7.2 Hz. CH ); 1.36 (3H, t, ꢄ = 7.2, CH ); 4.30 (2H, q, ꢄ =
1b: R = benzyl
2b: R = benzyl (67%)
3
3
Scheme 1: ionation of 1,5-dialkyl-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one 1a-b.
7.2 Hz, CH ); 4.51 (2H, q, ꢄ = 7.2 Hz, CH ); 8.15 (1H, s, CH);
2
2
8.72 (1H, s, CH). 13C NMR (DMSO-ꢁ6) ꢃppm: 14.56 (CH );
3
15.15 (CH ); 42.38 (CH ); 46.34 (CH ); 118.17; 137.09 (Cq);
3
2
2
145.09 (CH); 149.99 (CH); 179.11 (Cq, C=S). HRMS (ESI) [M +
IR-spectra of compounds 2a-d did not contain C=O-
group signals, the signal of C=S group (1500cm−1) was
present. e 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 C H N S compound 2, molecular ion at m/z 208.07
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-ꢁ6) ꢃ ppm:
3.25 (2H, s, CH ); 3.78 (2H, s, CH ); 6.85–7.12 (q, 10HAr); 8.21
2
2
(1H, s, CH), 8.89 (1H, s, CH). 13C NMR (DMSO-ꢁ6) ꢃppm:
5
4
4
(M+) corresponding to the molecular formula C H N S
51.83 (CH ); 54.31 (CH ); 118.24; 127.94 (Cq); 128.11–137.89
9
12
4
2
2
compound 2a and molecular ion at m /z 332.10 (M+) corre-
sponding to the molecular formula C H N S compound 2b
(CHAr); 145.54 (CH); 150.95 (CH); 179.82 (Cq, C=S). HRMS
(ESI) [M + H]: m/z = 33.10.
19 16
4
(Scheme 1, Figure 4) [23].
We investigated the reaction of 1,3-dipolar cycloaddi-
tion 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.
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) triethy-
lamine (2 mL) was added. e mixture was refluxed for 24
hours. e precipitate was collected by filtration and was
separated by silica gel chromatography (hexane/ethyl acetate:
8/2).
e structure of these compounds was confirmed on the
basis of their spectroscopic characteristics. e H NMR
2,5-Diethyl-3ꢀ,5ꢀ-diphenyl-2,5-dihydro-3ꢀH-spiro[pyrazolo[3,
4-d]pyrimidine-1,2ꢀ-[1,3,4]thiadiazole] (4a). Yield = 60%; mp:
165∘C. 1H NMR (DMSO-ꢁ6) ꢃ ppm 1.12 (3H, t, ꢄ = 7.2 Hz,
CH ); 1.28 (3H, t, ꢄ = 7.2 Hz, CH ); 3.37 (2H, q, ꢄ = 7.2 Hz.
1
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 and CH signals as triplets and
3
3
CH ); 4.11 (2H, q, ꢄ = 7.2 Hz, CH ); 6.83–7.67 (q, ꢄ = 7.7 Hz,
2
2
10HAr); 7.69 (1H, s, CH); 7.69 (1H, s, CH). 13C NMR (DMSO-
ꢁ6) ꢃppm: 15.61 (CH ); 16.03 (CH ); 41.88 (CH ); 42.28
3
3
2
(CH ); 99.70; 100.79; 117.03; 126.38 (Cq); 129.26–142.20
2
(CHAr); 143.87 (CH); 148.01 (CH). HRMS (ESI) [M + H]:
m/z = 403.16.
2
3
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). e 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 reac-
tion and formed of novel spiro[thiadiazoline-(pyrazolo[3,4-
d]pyrimidine)] (Scheme 3).
e 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
2,5-Dibenzyl-3ꢀ,5ꢀ-diphenyl-2,5-dihydro-3ꢀH-spiro[pyrazolo
[3,4-d]pyrimidine-1,2ꢀ-[1,3,4]thiadiazole] (4b). Yield = 60%;
mp: 185∘C. 1H NMR (DMSO-ꢁ6) ꢃ ppm: 3.65 (2H, s, CH );
2
4.28 (2H, s, CH ); 7.25–7.32 (q, ꢄ = 7.2 Hz, 20HAr); 8.81 (1H,
2
s, CH); 8.99 (1H, s, CH). 13C NMR (DMSO-ꢁ6) ꢃppm: 50.83
(CH ); 53.21 (CH ); 77.2; 118.24; 121.02; 125.13; 127.94; 128.04;
2
2
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 pen-
tasulfide in pyridine. e identification of the product was
determined by 1H NMR, 13C NMR, IR, and mass spectra.
1
>C=N- indicating the spirocarbon formation. Its H NMR

4
Journal of Chemistry
S1
C3
C4
N1
N2
C2
C1
C8
C9
C15
N4
C7
C14
C16
C17
C6
C10
C13
N3
C11
C5
C12
C19
C18
Figure 4: ORTEP presentation of compound 2b.
Ph
N
N
Ph
S
N
N
S
H
N
Cl
N
H3C
H3C
Et3N/THF
N
+
N
Ph
N
Δ
Ph
N
N
N
H3C
H3C
2a
3
4a
Scheme 2: 1,3-Dipolar cycloaddition of 1,5-diethyl-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-thione 2a and DPNI 3.
Ph
N
Ph
S
S
N
H
N
Cl
N
N
Et3N/THF
+
N
N
Ph
Ph
N
Δ
N
N
N
N
2b
3
4b
Scheme 3: 1,3-Dipolar cycloaddition of 1,5-dibenzyl-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-thione 2b and DPNI.
spectrum exhibited peaks at 7.25–7.32 ppm (20H) indicating
the presence of aromatic protons. e two singlets at 3.65 ppm
MIC values of the compound against bacteria are presented
in Table 1.
We synthesized new spirocompounds with a high yield by
cycloaddition reaction. ey have showed moderate antibac-
terial activity against selected bacteria. All these compounds
showed at an average concentration low antibacterial activity
against the bacteria, except that compounds1a 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.
and 4.28 ppm relating to two protons of the two CH groups
2
were also observed. 13C NMR spectra of 4b exhibited,
in particular, a spiro carbon signal at 77.2 ppm. e mass
spectrum shows a peak at m/z 527.39 corresponding to [M
+ H].
3.1. In Vitro Antibacterial Activity. We studied spiropyra-
zolo[3,4-d]pyrimidines newly synthesized for their antibac-
terial activity against Escherichia coli (ATTC-25922), Staphy-
lococcus aureus (ATCC-25923), Pseudomonas aeruginosa
(ATCC 27853), and Enterococcus faecalis (ATCC-29212)
strains of bacteria by the diffusion method disk [32]. e
4. Conclusion
In conclusion, a new class of heterocyclic spiro[thiadiazoline
pyrazolopyrimidine] compounds was synthesized and their

Journal of Chemistry
5
Table 1: Antibacterial activity of the compounds: MIC in ꢀg/mL.
[6] D. Briel, R. Aurich, U. Egerland, and K. Unverferth, “Synthese
substituierter 6-Phenylpyrazolo[3,4-d]pyrimidine mit poten-
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ziell Adenosin-A -antagonistischer Wirkung,” Die Pharmazie,
2ꢁ
Product
S. aureus
250
E. faecalis
125
E. coli
250
250
62.5
125
250
250
125
125
15
P. aeruginosa
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250
—
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1a
250
250
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1
1b
2
2a
2b
4a
4b
Chlor
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125
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62.5
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250
125
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3.75
1.25
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31.25
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2.5
5
5
MIC: minimum inhibitory concentration.
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structure was determined and also tested for their antibacte-
rial activity in vitro. is 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
[12] N. N. Gajera and M. C. Patel, “Synthesis, characterization
and biological screening of new spirochromanones,” Journal of
Chemical and Pharmaceutical Research, vol. 4, no. 7, pp. 3377–
3382, 2012.
[13] B. S. Rane, S. V. Deshmukh, M. G. Ghagare, R. V. Rote, and M.
N. Jachak, “Synthesis of spiro-pyrimido [4,5-b]quinoline and
study of their antimicrobial activities,” Journal of Chemical and
Pharmaceutical Research, vol. 4, no. 7, pp. 3562–3567, 2012.
e authors declare that there is no conflict of interests
regarding the publication of this paper.
Acknowledgment
e authors thank Pharmaceutical Laboratories PHARMA 5
for supporting this study.
[14] J. R. Yerrabelly, V. Chakravarthula, H. Yerrabelly et al.,
“Unusual tandem ring closing metathesis—Claisen rearrange-
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