Synthesis of some novel amino and thiotetrazole purine derivatives and investigation of their antimicrobial activity and DNA interactions

Article abstract of DOI:10.1007/s00044-012-0140-9

A series of amino and thiotetrazole purine derivatives introduced with different alkyl groups in position 9 was synthesized. The structures of the synthesized compounds were characterized using spectroscopic methods. All the synthesized compounds were screened for their antibacterial activities against Gram-positive and Gram-negative bacteria and for their antifungal activities against yeast strains. The effect of the compounds on pBR322 plasmid DNA was studied by gel electrophoretic mobility measurements. The results of antimicrobial activity show that attachment of tetrazole group to purine bases results in disappearance of antimicrobial activity The results of the plasmid DNA interaction and the restriction studies suggest that while aminotetrazole purine derivatives cause DNA damages, thiotetrazole purine derivatives are believed to form a range of interstrand GG adducts with duplex DNA that induce global changes in the DNA conformation.

Full text of DOI:10.1007/s00044-012-0140-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0140-9
ORIGINAL RESEARCH
Synthesis of some novel amino and thiotetrazole purine
derivatives and investigation of their antimicrobial activity
and DNA interactions
•
•
•
Gulay Dilek Celik Ali Disli Yagmur Oner
Leyla Acik
Received: 28 December 2011 / Accepted: 31 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of amino and thiotetrazole purine
derivatives introduced with different alkyl groups in posi-
tion 9 was synthesized. The structures of the synthesized
compounds were characterized using spectroscopic meth-
ods. All the synthesized compounds were screened for their
antibacterial activities against Gram-positive and Gram-
negative bacteria and for their antifungal activities against
yeast strains. The effect of the compounds on pBR322
plasmid DNA was studied by gel electrophoretic mobility
measurements. The results of antimicrobial activity show
that attachment of tetrazole group to purine bases results in
disappearance of antimicrobial activity The results of the
plasmid DNA interaction and the restriction studies suggest
that while aminotetrazole purine derivatives cause DNA
damages, thiotetrazole purine derivatives are believed to
form a range of interstrand GG adducts with duplex DNA
that induce global changes in the DNA conformation.
Introduction
Bioisosteric replacement of a functional group is a widely
used technique in medicinal chemistry for the generation of
more selective and more potent analog of a lead compound
(Matta et al., 2010). Tetrazoles are of particular interest to
the medicinal chemists since they constitute the most
commonly used bioisostere of the carboxylate moiety
(Herr, 2002). Like their carboxylic acid counterparts, tet-
razoles are ionized at physiological pH and they are more
lipophilic than the corresponding carboxylates while hav-
ing similar acidity (Herr, 2002). The interchange of car-
boxylic acid for tetrazole often results in useful drugs.
Tetrazoles are screened for various biological activities
such as antiviral, antibacterial, antifungal, antiallergic,
antiulcer, anticonvulsant, anti-inflammatory, and antitu-
bercular activities (Shemyakina et al., 2011; Kumar et al.,
2011; Chermahini et al., 2008; Mohite and Bhaskar, 2011).
1,5-substituted tetrazoles are known for their pharmaceu-
tical activities as stimulants or depressants on the central
nervous system (Shemyakina et al., 2011; Mohite and
Bhaskar, 2010). Tetrazoles are currently used in cancer and
AIDS treatment (Tamura et al., 1998; Abell and Foulds,
1997).
Keywords Tetrazole ꢀ Purine derivative ꢀ
Antimetabolite ꢀ Antimicrobial activity ꢀ DNA interaction
G. Dilek Celik ꢀ A. Disli (&)
Purine analogs are of great importance to chemists as
well as to biologists as they possess antitubercular, anti-
ulcer, antimicrobial, antifungal, antineoplastic, antitumor,
antiviral, and cardiotonic properties (Hu et al., 2010;
Carmo et al., 2008; Rida et al., 2007; .Steurer et al., 2006;
Dolan and Pegg, 1997; Mitra and Kaina, 1993; Morimoto
et al., 1985). The purine antimetabolites are also used in
the treatment of autoimmune diseases (Robak et al., 2006).
Current evidence indicates that purine analogs alone or in
combination with other chemotherapeutic agents are very
effective against almost any fast-growing cells (Rida et al.,
Department of Chemistry, Gazi University,
06500 Teknikokullar, Ankara, Turkey
e-mail: adisli@gazi.edu.tr
G. Dilek Celik
e-mail: gulaydilekcelik@hotmail.com
Y. Oner ꢀ L. Acik
Department of Biology, Gazi University,
06500 Teknikokullar, Ankara, Turkey
e-mail: yagmuroner87@gmail.com
L. Acik
e-mail: leylaacik@gmail.com
123

Med Chem Res
2007). Therefore, attention has been drawn to the structural
modification of purine bases as well as introduction of
different functional groups into these bases. Looking at the
intense research activity in the tetrazole field, it was
thought the combined effects of purine and tetrazole
pharmacophores could result in interesting biological
activity. Therefore, this study describes the synthesis of
some novel amino and thiotetrazole purine derivatives, and
evaluation of their antimicrobial activities and DNA
interactions.
15.80 ppm due to NH protons of aminotetrazole. The MS
spectra also complied with the expected values.
9-Alkyl-6-(1-methyl-1H-tetrazol-5-ylthio)-9H-purines
(10–19) were synthesized by refluxing 6-chloro-9-
alkyl-9H-purines with 1-methyl-5-mercaptotetrazole in
dichloromethane (Scheme 2). The structures of compounds
1
10–19 were confirmed by FTIR, H-NMR, 13C-NMR, and
MS spectra and all spectroscopic data were in accordance
1
with the assigned structures. H-NMR spectra showed a
singlet at 4.10 ppm due to methyl protons of thiotetrazole.
All the target compounds (1–19) were reported for the
first time. The purity of the synthesized compounds was
monitored by TLC.
Result and discussion
Synthesis
Antimicrobial activity
Treatment of 6-chloropurine with some appropriate alkyl
halides in the presence of potassium carbonate generated
6-chloro-9-alkyl-9H-purines. The structures of 6-chloro-9-
alkyl-9H-purines were confirmed by comparing their
physical and spectral data with the reported ones (Okamura
et al., 2007; Lambertucci et al., 2009; Hanna et al., 1994;
Montgomery and Temple, 1961).
Results of tests with bacteria and fungi are often used as a
preliminary indicator of biological activity in the choice of
chemotherapeutic agents (Bradner and Clarke, 1958).
Therefore, all the synthesized compounds were tested for
their antimicrobial activities.
The in vitro antibacterial activities of compounds 1–19
were undertaken against Gram-positive bacteria including
Staphylococcus aureus ATCC 25923, Bacillus cereus
NRLL B-3008, Bacillus subtilis ATCC 29213, and Enter-
obacter faecalis ATCC 292112; and Gram-negative bac-
teria including Pseudomonas aeruginosa ATCC 27853,
Escherichia coli ATCC 35218, E. coli ATCC 25922, and
Proteus vulgaris ATCC 8427 by disk diffusion technique
(Clinical and Laboratory Standards Institute, 2006, 2007).
In addition, the in vitro antifungal activities of compounds
1–19 were undertaken against Candida albicans ATCC
10231 and Candida tropicalis ATCC 13803. Chloramfe-
nicol and ampicillin were used as the reference antibacte-
rial agents, and ketoconozole was used as a reference
antifungal agent under similar conditions for comparison.
9-Alkyl-N-(1H-tetrazol-5-yl)-9H-purin-6-amines (1–9)
were prepared by refluxing 6-chloro-9-alkyl-9H-purines
with 5-aminotetrazole in ethanol (Scheme 1). The products
were separated and purified using column chromatography
to provide compounds 1–9. The structures of compounds
1–9 were confirmed by FTIR, 1H-NMR, 13C-NMR, and MS
spectra. All spectroscopic data were in accordance with the
assigned structures. IR spectra of the synthesized com-
pounds exhibited a band in the region of 3,300–3,400 cm-1
which could be assigned to the characteristic NH absorption
band of tetrazole ring. NH absorption band appeared as a
broad band due to tautomeric structure of the tetrazole.
1H-NMR spectra showed two singlets at 11.90 and
N
HN
N
N
H
Cl
N
Cl
N
N
HN
N
NH2
N
N
N
N
N
N
K2CO3, RX
DMF
N
N
N
H
N
R
N
R
N
1-9
R= (1: -H; 2: -CH3; 3: -C2H5; 4: -C3H7; 5: -CH(CH3)2 6: -C6H13; 7: -CH2C6H5;
8: - CH2COOC2H5; 9: -CH2C4H7).
Scheme 1 Synthesis of aminotetrazole purine derivatives 1–9. R = (1 –H, 2 –CH3, 3 –C2H5, 4 –C3H7, 5 –CH(CH3)2, 6 –C6H13, 7 –CH2C6H5,
8 –CH2COOC2H5, 9 –CH2C4H7)
123

Med Chem Res
H3C
N
N
N
CH3
N
N
Cl
N
Cl
N
S
N
N
N
SH
N
N
N
N
N
N
N
K2CO3, RX
DMF
N
H
N
R
N
R
10-19
R= (10: -H; 11: -CH3; 12: -C2H5; 13: -C3H7; 14: -CH(CH3)2 15: -C6H13; 16: -CH2C6H5;
17: - CH2COOC2H5; 18: -CH2C4H7; 19: -CH2C6H11).
Scheme 2 Synthesis of thiotetrazole purine derivatives 10–19. R = (10 –H, 11 –CH3, 12 –C2H5, 13 –C3H7, 14 –CH(CH3)2, 15 –C6H13, 16
–CH2C6H5, 17 –CH2COOC2H5, 18 –CH2C4H7, 19 –CH2C6H11
)
Dimethyl sulfoxide (DMSO) was used as the control. The
results show that none of the synthesized compounds dis-
played antimicrobial activity against tested bacteria and
fungi.
DNA (control DNA), showing the major supercoiled (form
I) and minor nicked (form II) forms. Lanes 1–5 apply to
pBR322 plasmid DNA incubated with the compounds with
concentrations ranging from 5,000 to 312 lM.
Since a carboxylic acid group can bioisosterically be
replaced by a tetrazole, compounds 1–9 could be inter-
preted as a potential bioisostere of the corresponding pur-
ine-6-carbamic acid derivatives and compounds 10–19
could be interpreted as a potential bioisostere of the cor-
responding O-alkyl-S-purin-6-yl ester derivatives, yet the
compounds 1–19 failed to exhibit antimicrobial activity
against tested bacteria and fungi.
As pBR322 plasmid DNA was allowed to interact with
decreasing concentrations of compounds 1, 2, 3, 5, and 6,
the mobility of both form I and form II bands remained
essentially unchanged. On the other hand, the intensity of
both form I and form II bands changed at high concen-
trations. It was found that although the unreacted DNA was
not very intense, there was a pronounced increase in the
intensity of form I band at all concentrations of the com-
pounds. The increase in intensity of the band may be due to
the covalent binding of the compounds. Moreover, there
were small DNA fragments on the gel. These small DNA
pieces could be the result of DNA cleavage.
DNA and compound interactions
Small molecules that bind to DNA are useful biochemical
tools for the visualization of DNA in vitro (Palchaudhuri
and Hergenrother, 2011). Many antitumor, antineoplastic,
antimalarial, antibiotic, and antifungal agents are the small
compounds that interact with DNA (Palchaudhuri and
Hergenrother, 2007). The interaction of DNA with com-
pounds could convert covalently supercoiled form I DNA
into singly nicked circular form II, and then into linear
form III DNA (Asmafiliz et al., 2009; Huq et al., 2009).
This cleavage reaction could be monitored by gel electro-
phoresis. Therefore, interactions of compounds 1–19 with
supercoiled pBR322 plasmid DNA have been studied. The
compounds were incubated over a range of concentrations
with supercoiled pBR322 plasmid DNA in the dark at
37 °C for 24 h. The DNA cleavage by these compounds
has been examined by observing the conversion of super-
coiled DNA to the open circular form II and linear form III
DNA. Figure 1 shows the electrophoretograms for the
interaction of pBR322 plasmid DNA with compounds 1–19
at concentrations of compounds ranging from 5,000 to
312 lM. Lane P applies to the untreated pBR322 plasmid
The initial sharp increase in the intensity of the form II
band due to the presence of a small amount of compounds
points to DNA damage in which form I DNA is changed to
form II DNA. The decrease in intensity at higher concen-
trations of compounds is due to a partial damage of the
DNA caused by the covalent binding of the compounds.
When the pBR plasmid DNA interacted with decreasing
concentrations of compound 4, the mobility of form I
decreased slightly in all concentrations tested and small
DNA bands appeared. In the case of compounds 7, 8, and 9,
the mobility of form I DNA decreased with increasing con-
centrations of the compounds, and there were small DNA
fragments on the gel of compounds 8 and 9. As pBR322
plasmid DNA was allowed to interact with compound 10, the
mobility of form I gradually decreased with increasing
concentration of the compound. In the case of compounds 11
and 12, opposite effect was seen. When the pBR322 plasmid
DNA interacted with compounds 11 and 12, mobility of form
I increased with increasing concentrations of the com-
pounds. At high concentrations of compound 12, form II
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Med Chem Res
Fig. 1 Gel electrophoretic
mobility of pBR322 plasmid
DNA when incubated with
various concentrations of
compounds 1–19.
Concentrations (in lM) as
follows: lane P, untreated
pBR322 plasmid DNA; lane 1,
5000; lane 2, 2500; lane 3,
1250; lane 4, 625; lane 5, 312.
The top and the bottom bands
correspond to form II (singly-
nicked) and form I (covalently
closed circular) plasmids,
respectively
band was disappeared. The disappearance of form II band is
due to the damage of the DNA caused by the covalent
binding of the compound. In the case of compounds 13 and
14, the mobility of form I changed slightly and small DNA
fragments were observed. Furthermore, the intensity of form
II bands decreased with decreasing concentration of the
compounds. This comfirms cleavage of the two strands of the
DNA. When the pBR322 plasmid DNA interacted with
decreasing concentrations of compound 15, the mobility of
form I decreased and form II band was disappeared. In the
case of compounds 16, 17, and 18, it was observed that the
intensity of form II bands changed with the concentrations of
the compounds. Moreover, there were small DNA fragments
on the gel. The plasmid DNA interaction with these com-
pounds lead to DNA cleavage. Compound 19 also changes
the intensities of both form I and form II. While the intensity
of form I increased, the intensity of form II decreased at low
concentrations. The concentrations required to partial dam-
age of the DNA caused by covalent binding of the compound
were 312, 625, and 1250 lM. There were also small DNA
fragments on the gel.
GATCC-30 and hydrolyze the phosphodiester bond between
adjacent guanine sites (Huq et al., 2004). pBR322 contains
a single restriction site for BamHI, which converts pBR322
plasmid DNA from supercoiled form I and singly nicked
circular form II to linear form III DNA. HindIII enzymes
bind at the recognition sequence 50-A/AGCTT-30 and
cleave the sequence just after 50-adenine site and, as a result,
convert form I and form II DNA to linear form III DNA
(Gu¨mu¨s¸ et al., 2009). In order to assess whether the syn-
thesized compounds show affinity toward guanine–guanine
(GG) and/or adenine–adenine (AA) regions, we carried out
restriction endonuclease analysis of the compound–pBR322
plasmid DNA adducts digested by BamHI and HindIII
enzymes. Only one concentration for each compound was
used for the restriction analysis (1,250 lM). Figure 2 shows
the electrophoretograms applying to incubated mixtures of
pBR322 plasmid DNA and compounds 1–19 followed by
their restriction with BamHI (Fig. 2a, c) and HindIII
(Fig. 2b, d). Lane P applies to the untreated and undigested
pBR322 plasmid DNA, Lane P ? B and P ? H apply to
pBR322 plasmid DNA restricted with enzymes BamHI and
HindIII, respectively. Lanes 1–19 apply to pBR322 plasmid
DNA interacted with the compounds 1–19 followed by their
digestion with BamHI (Fig. 2a, c) and HindIII (Fig. 2b, d).
When untreated, pBR322 plasmid DNA was digested
with BamHI and HindIII, only one band corresponding to
In order to find out whether the synthesized compounds
bind or cleave DNA, restriction analyses were performed
(Huq et al., 2004; Gu¨mu¨s¸ et al., 2009). The compound–
DNA adducts were digested with BamHI and HindIII
enzymes. BamHI is known to recognize the sequence 50-G/
123

Med Chem Res
Fig. 2 Electrophoretograms applying to incubated mixtures of
pBR322 plasmid DNA and compounds 1–19 followed by their
restriction with BamHI (a, c) and HindIII (b, d). Lane P, untreated
and undigested pBR322 plasmid DNA; lane P ? B and
P ? H pBR322 plasmid DNA restricted with enzymes BamHI and
HindIII, respectively; lanes 1–19, pBR322 plasmid DNA interacted
with the compounds 1–19 followed by their digestion with BamHI (a,
c) and HindIII (b, d)
form III band was observed. In the untreated and undi-
gested pBR322 plasmid DNA, generally two bands corre-
sponding to form I and form II were observed (form I band
has the highest velocity, form II has the lowest velocity,
form III band has the intermediate velocity).
Experimental
Chemistry
All chemicals and solvents used were reagent grade (Merck
or Aldrich or Sigma) and were used without additional
purification. Reactions were monitored by thin layer
chromatography (TLC) on precoated silica gel 60 F254
plates from Merck and plates were visualized by UV light.
Column chromatography was performed using Merck Sil-
ica Gel 60 F254 (particle size: 0.63–0.200 mm; 70–230
mesh ASTM). Melting points were determined with an
Electrothermal 9100 melting point apparatus and were
uncorrected. IR spectra (4,000–400 cm-1) were recorded
using KBr disk on a Mattson 1000 FT-IR spectrometer and
In the case of incubated mixtures of pBR322 plasmid
DNA and compounds 10–19 followed by their restriction
with BamHI (Fig. 2c), two bands corresponding to form I
and form II were observed. This result shows that com-
pounds 10–19 prevent digestion with BamHI enzyme. This
may be due to the conformational change in the DNA
brought about by the covalent binding of the compounds
with the plasmid DNA. On the other hand, BamHI diges-
tion at the specific GG site is not prevented in the presence
of compounds 1–9 (Fig. 2a). All the synthesized com-
pounds also inhibit HindIII enzyme activity (Fig. 2b, d).
1
were reported in cm-1 units. H- and 13C-NMR spectra
were recorded on a Bruker spectrometer (300 MHz for 1H-
NMR and 75 MHz for 13C-NMR). Chemical shifts were
reported in d ppm units with respect to tetramethylsilane
(TMS) as an internal reference in DMSO-d6 or CDCl3
and coupling constants (J) were reported in Hz units.
Mass spectra measurements were recorded on a Waters
ACQUITY ultra performance liquid chromatography
combined with Micromass LCT PremierTM XE TOF–MS.
Conclusion
Several amino and thiotetrazole purine derivatives were
synthesized and antimicrobial activities of the synthesized
compounds were investigated. The results of antimicrobial
activity show that attachment of tetrazole group to purine
bases results in disappearance of antimicrobial activity.
Level of binding with DNA and nature of interaction with
pBR322 plasmid DNA have also been determined. The
prevention of BamHI digestion of form I and form II
pBR322 plasmid DNA with thiotetrazole purine derivatives
is believed to be due to interstrand binding that brings
about global changes in DNA conformation. On the other
hand, HindIII enzyme cuts all of the compounds interacted
with DNA.
General procedure for the preparation of 6-chloro-
9-alkyl-9H-purines
K2CO3 (1.79 g, 0.013 mol) was suspended in a stirred
solution of 6-chloropurine (2.0 g, 0.013 mol) in DMF
(30 ml). The appropriate alkyl halide (0.013 mol) was
added to the reaction mixture and the resultant was stirred
at room temperature for 24 h. The solvent was evaporated
123

Med Chem Res
under reduced pressure. The mixture was poured into water
(50 ml), and extracted with chloroform (4 9 25 ml). The
combined organic layer was dried over MgSO4 and filtered.
The solvent was evaporated in vacuo, and the residue was
purified by column chromatography on silica gel using 1:1
ethyl acetate–hexane as eluent.
3099 (Ar–H).). 1H-NMR (DMSO-d6) d: 0.78 (m, 3H,
–CH3), 1.23 (m, 6H, –CH2), 1.83 (m, 2H, –CH2), 4.27 (t,
2H, J = 7.1 Hz, –N–CH2), 8.71 (s, 1H, Ar–H), 8.74 (s, 1H,
Ar–H). 13C-NMR (DMSO-d6) d: 14.20, 22.32, 26.02,
29.40, 31.02, 44.23, 131.24, 147.92, 149.40, 151.82,
152.36. HR-MS for C11H16ClN4 ([M-H]?) Calcd:
?
239.1063; Found: 239.1057.
Synthesis of 6-chloro-9-methyl-9H-purine The compound
was prepared from methyl iodide (1.84 g, 0.013 mol) as
described above and recrystallized from diethyl ether.
Yield 45 %, mp 137–138 °C. IR (KBr) cm-1: 2922
(–C–H), 3078 (Ar–H). 1H-NMR (DMSO-d6) d: 3.83 (s, 3H,
–N–CH3), 8.64 (s, 1H, Ar–H), 8.75 (s, 1H, Ar–H).
13C-NMR (DMSO-d6) d: 30.57, 131.08, 148.49, 149.21,
Synthesis of 6-chloro-9-benzyl-9H-purine The compound
was prepared from benzyl bromide (2.21 g, 0.013 mol) as
described above and recrystallized from hexane. Yield
51 %, mp 80–82 °C. IR (KBr) cm-1: 2939 (–C–H), 3062
1
(Ar–H). H-NMR (DMSO-d6) d: 5.33 (s, 2H, –N–CH2),
7.33 (m, 5H, Ar–H), 8.79 (s, 1H, Ar–H), 8.85 (s, 1H,
Ar–H). 13C-NMR (DMSO-d6) d: 47.49, 128.13, 128.47,
129.21, 131.29, 136.47, 147.90, 149.63, 152.16, 152.26.
?
151.82, 152.65. HR-MS for C6H6ClN4 ([M-H]?) Calcd:
169.0281; Found: 169.0285.
HR-MS for C12H10ClN4 ([M-H]?) Calcd: 245.0594;
?
Synthesis of 6-chloro-9-ethyl-9H-purine The compound
was prepared from ethyl iodide (2.02 g, 0.013 mol) as
described above and recrystallized from ethyl acetate–
hexane. Yield 48 %, mp 78–80 °C. IR (KBr) cm-1: 2938
(–C–H), 3079 (Ar–H). 1H-NMR (DMSO-d6) d: 1.46 (t, 3H,
J = 7.3 Hz, –CH3), 4.32 (q, 2H, J = 7.3 Hz, –N–CH2),
8.73 (s, 1H, Ar–H) 8.76 (s, 1H, Ar–H). 13C-NMR (DMSO-
d6) d: 15.20, 39.47, 131.21, 147.40, 149.27, 151.59, 152.02.
Found: 245.0590.
Synthesis of ethyl 2-(6-chloro-9H-purin-9-yl)acetate The
compound was prepared from ethyl bromoacetate (2.16 g,
0.013 mol) as described above and recrystallized from
heptane. Yield 49 %, mp 89–90 °C. IR (KBr) cm-1: 1737
(–C=O ester), 2930 (–C–H), 3094 (Ar–H). 1H-NMR
(CDCl3) d: 1.33 (t, 3H, J = 7.1 Hz, –CH3), 4.30 (q, 2H,
J = 7.1 Hz, –O–CH2), 5.07 (s, 2H, –N–CH2), 8.21 (s, 1H,
Ar–H), 8.77 (s, 1H, Ar–H). 13C-NMR (CDCl3) d: 14.07,
44.56, 62.70, 131.22, 145.45, 151.29, 151.92, 152.26,
HR-MS for C7H8ClN4 ([M-H]?) Calcd: 183.0437;
?
Found: 183.0437.
166.44. HR-MS for C9H10ClN4O2 ([M-H]?) Calcd:
?
Synthesis of 6-chloro-9-propyl-9H-purine The compound
was prepared from 1-bromopropane (1.60 g, 0.013 mol) as
described above. Yield 63 %. IR (KBr) cm-1: 2935
(–C–H), 3073 (Ar–H). 1H-NMR (DMSO-d6) d: 0.81 (t, 3H,
J = 7.4 Hz, –CH3), 1.84 (m, 2H, –CH2), 4.22 (t, 2H,
J = 7.1 Hz, –N–CH2), 8.68 (s, 1H, Ar–H), 8.69 (s, 1H,
Ar–H). 13C-NMR (DMSO-d6) d: 11.26, 22.90, 45.79,
131.20, 147.86, 149.37; 151.73, 152.32. HR-MS for
C8H10ClN4? ([M-H]?) Calcd: 197.0594; Found: 197.0589.
241.0492; Found: 241.0485.
Synthesis of 6-chloro-9-(cyclobutylmethyl)-9H-purine The
compound was prepared from (bromomethyl)cyclobutane
(1.92 g, 0.013 mol) as described above. Yield 46 %, mp
52–55 °C. IR (KBr) cm-1: 2974 (–C–H), 3092 (Ar–H).
1H-NMR (DMSO-d6) d: 1.80 (m, 2H, –CH2), 1.95 (m, 4H,
–CH2), 2.83 (m, 1H, –C–H), 4.49 (d, 2H, J = 7.4 Hz,
–N–CH2), 8.75 (s, 1H, Ar–H), 8.85 (s, 1H, Ar–H). 13C-NMR
(DMSO-d6) d: 17.89, 25.26, 36.21, 51.41, 122.38, 142.51,
Synthesis of 6-chloro-9-isopropyl-9H-purine The com-
pound was prepared from 2-bromopropane (1.60 g,
0.013 mol) as described above and recrystallized from ethyl
acetate. Yield 32 %, mp 108–109 °C. IR (KBr) cm-1: 2933
(–C–H), 3069 (Ar–H).). 1H-NMR (DMSO-d6) d: 1.58 (d, 6H,
J = 6.8 Hz, –CH3), 4.89 (m, 1H, –C–H), 8.70 (s, 1H, Ar–H),
8.80 (s, 1H, Ar–H). 13C-NMR (DMSO-d6) d: 22.28, 48.31,
131.63, 146.19, 149.42, 151.63, 151.88. HR-MS for
C8H10ClN4? ([M-H]?) Calcd: 197.0594; Found: 197.0591.
?
151.18, 151.89, 161.95. HR-MS for C10H12ClN4
([M-H]?) Calcd: 223.0750; Found: 223.0749.
Synthesis of 6-chloro-9-(cyclohexylmethyl)-9H-purine The
compound was prepared from (bromomethyl)cyclohexane
(2.29 g, 0.013 mol) as described above. Yield 49 %, mp
70–72 °C. IR (KBr) cm-1: 2932 (–C–H), 3047 (Ar–H).
1H-NMR (CDCl3) d: 1.15(m, 2H, –CH2), 1.21(m, 4H, –CH2),
1.74 (m, 4H, –CH2), 1.90 (m, 1H, –C–H), 4.31 (d, 2H,
J = 7.2 Hz, –N–CH2), 8.22 (s, 1H, Ar–H), 8.88 (s, 1H,
Ar–H). 13C-NMR (CDCl3) d: 25.39, 25.95, 30.02, 39.50,
53.43, 122.44, 143.02, 149.48, 152.21, 161.95. HR-MS for
C12H16ClN4? ([M-H]?) Calcd: 251.1063; Found: 251.1072.
Synthesis of 6-chloro-9-hexyl-9H-purine The compound
was prepared from 1-bromohexane (2.13 g, 0.013 mol) as
described above and recrystallized from ethyl acetate.
Yield 50 %, mp 60–62 °C. IR (KBr) cm-1: 2929 (–C–H),
123

Med Chem Res
Synthesis
of
N-(1H-tetrazol-5-yl)-9H-purin-6-amine
4:1 ethyl acetate–hexane as eluent. Yield 81 %, mp
262–263 °C. IR (KBr) cm-1: 2932 (–C–H), 3074 (Ar–H),
3400-3300 (–N–H). H-NMR (DMSO-d6) d: 0.86 (t, 3H,
(1) 6-Chloropurine (0.31 g, 2.0 mmol) was refluxed with
5-aminotetrazole (0.17 g, 2.0 mmol) in ethanol (25 ml) for
5 days. The solvent was evaporated in vacuo. The product
was purified by column chromatography on silica gel using
2:1 ethyl acetate–methanol. Yield 51 %, mp [219 °C
(decomp.). IR (KBr) cm-1: 3053 (Ar–H), 3400–3300 (–N–
H). 1H-NMR (DMSO-d6) d: 8.40 (s, 1H, Ar–H), 8.60
(s, 1H, Ar–H), 11.90 (s, 1H, –N–H), 12.15 (s, 1H, –N–H),
13.50 (s, 1H, –N–H). 13C-NMR (DMSO-d6) d: 152.31,
1
J = 7.4 Hz, –CH3), 1.87 (m, 2H, –CH2), 4.22 (t, 2H,
J = 7.1 Hz, –N–CH2), 8.55 (s, 1H, Ar–H), 8.48 (s, 1H,
Ar–H), 11.91 (s, 1H, –N–H), 15.80 (s, 1H, –N–H).
13C-NMR (DMSO-d6) d: 11.38, 23.14, 45.36, 120.36,
?
144.06, 149.65, 151.42. HR-MS for C9H12N9 ([M-H]?)
Calcd: 246.1216; Found: 246.1014.
?
159.36, 158.47, 163.20, 166.96. HR-MS for C6H6N9
([M-H]?) Calcd: 204.0746; Found: 204.0686.
Synthesis of 9-isopropyl-N-(1H-tetrazol-5-yl)-9H-purin-6-
amine (5) The compound was prepared from 6-chloro-9-
isopropyl-9H-purine (0.39 g, 2.0 mmol) as described
above and purified by column chromatography on silica
gel using ethyl acetate as eluent. Yield 32 %,
mp [276 °C (decomp.). IR (KBr) cm-1: 2926 (–C–H),
General procedure for the preparation of 9-alkyl-N-
(1H-tetrazol-5-yl)-9H-purin-6-amine compounds (2–9)
1
The appropriate 6-chloro-9-alkyl-9H-purine (2.0 mmol)
was refluxed with 5-aminotetrazole (0.17 g, 2.0 mmol) in
ethanol (25 ml) for 5 days. The solvent was evaporated in
vacuo. The residue was purified by column chromatogra-
phy on silica gel to give the corresponding aminotetrazole
purine derivatives (2–9).
3076 (Ar–H), 3400–3300 (–N–H). H-NMR (DMSO-d6)
d: 1.57 (d, 6H, J = 6.8 Hz, –CH3), 4.85 (m, 1H, –C–H),
8.54 (s, 1H, Ar–H), 8.57 (s, 1H, Ar–H), 11.90 (s, 1H,
–N–H), 15.80 (s, 1H, –N–H). 13C-NMR (DMSO-d6) d:
22.51, 47.53, 120.72, 142.02, 149.79, 150.81, 151.37,
([M-H]?) Calcd:
?
151.99. HR-MS for C9H12N9
246.1216; Found: 246.1213.
Synthesis of 9-methyl-N-(1H-tetrazol-5-yl)-9H-purin-6-amine
(2) The compound was prepared from 6-chloro-9-methyl-
9H-purine (0.34 g, 2.0 mmol) as described above and
purified by column chromatography on silica gel using 2:1
ethyl acetate–hexane as eluent. Yield 35 %, mp [305 °C
(decomp.). IR (KBr) cm-1: 2979 (–C–H), 3073 (Ar–H),
Synthesis of 9-hexyl-N-(1H-tetrazol-5-yl)-9H-purin-6-
amine (6) The compound was prepared from 6-chloro-9-
hexyl-9H-purine (0.48 g, 2.0 mmol) as described above
and purified by column chromatography on silica gel using
methanol as eluent. Yield 30 %, mp 222–223 °C. IR (KBr)
cm-1: 2924 (–C–H), 3078 (Ar–H), 3400–3300 (–N–H).
1H-NMR (DMSO-d6) d: 0.82 (m, 3H, –CH3), 1.25 (m, 6H,
–CH2), 1.85 (m, 2H, –CH2), 4.24 (t, 2H, J = 7.1 Hz, –N–
CH2), 8.47 (s, 1H, Ar–H), 8.56 (s, 1H, Ar–H), 12.00 (s, 1H,
–N–H), 15.70 (s, 1H, –N–H). 13C-NMR (DMSO-d6) d:
14.27, 22.37, 26.06, 29.64, 31.05, 43.74, 120.32, 144.04,
1
3400–3300 (–N–H). H-NMR (DMSO-d6) d: 3.83 (s, 3H,
–N–CH3), 8.43 (s, 1H, Ar–H), 8.58 (s, 1H, Ar–H), 11.97 (s,
1H, –N–H), 15.78 (s, 1H, –N–H). 13C-NMR (DMSO-d6) d:
30.19, 120.18, 144.58, 149.47. HR-MS for C7H8N9
?
([M-H]?) Calcd: 218.0903; Found: 218.0904.
149.57, 151.34. HR-MS for C12H18N9 ([M-H]?) Calcd:
?
Synthesis of 9-ethyl-N-(1H-tetrazol-5-yl)-9H-purin-6-
amine (3) The compound was prepared from 6-chloro-9-
ethyl-9H-purine (0.36 g, 2.0 mmol) as described above
and purified by column chromatography on silica gel
using 2:1 ethyl acetate–hexane as eluent. Yield 34 %, mp
277–278 °C. IR (KBr) cm-1: 2977 (–C–H), 3093 (Ar–H),
288.1685; Found: 288.1682.
Synthesis of 9-benzyl-N-(1H-tetrazol-5-yl)-9H-purin-6-
amine (7)
1
3400–3300 (–N–H). H-NMR (DMSO-d6) d: 1.45 (t, 3H,
The compound was prepared from 6-chloro-9-benzyl-9H-
purine (0.49 g, 2.0 mmol) as described above and purified
by column chromatography on silica gel using 1:1 chlo-
roform–methanol as eluent. Yield 55 %, mp 271–273 °C.
IR (KBr) cm-1: 2975 (–C–H), 3010 (Ar–H), 3050
J = 7.2 Hz, –CH3), 4.30 (q, 2H, J = 7.2 Hz, –N–CH2),
8.49 (s, 1H, Ar–H), 8.56 (s, 1H, Ar–H), 12.10 (s, 1H,
–N–H), 15.80 (s, 1H, –N–H). 13C-NMR (DMSO-d6) d:
15.59, 38.98, 120.42, 143.67, 149.55, 151.16, 151.60. HR-
?
MS for C8H10N9 ([M-H]?) Calcd: 232.1059; Found:
1
(Ar–H), 3400–3300 (–N–H). H-NMR (DMSO-d6) d: 5.50
232.1062.
(s, 2H, –N–CH2), 7.40 (m, 5H, Ar–H), 8.57 (s, 1H,
Ar–H), 8.59 (s, 1H, Ar–H), 12.00 (s, 1H, –N–H), 15.80 (s,
1H, –N–H). 13C-NMR (DMSO-d6) d: 47.02, 120.33,
128.03, 128.37, 129.22, 137.09, 142.02, 149.73, 151,27.
Synthesis of 9-propyl-N-(1H-tetrazol-5-yl)-9H-purin-6-
amine (4) The compound was prepared from 6-chloro-9-
propyl-9H-purine (0.39 g, 2.0 mmol) as described above
and purified by column chromatography on silica gel using
?
HR-MS for C13H12N9 ([M-H]?) Calcd: 294.1216;
Found: 294.1224.
123

Med Chem Res
Synthesis of ethyl 2-(6-(1H-tetrazol-5-ylamino)-9H-purin-
9-yl)acetate (8)
Synthesis of 9-methyl-6-(1-methyl-1H-tetrazol-5-ylthio)-9H-
purine (11) The compound was prepared from 6-chloro-
9-methyl-9H-purine (0.34 g, 2.00 mmol) as described
above, washed with diethyl ether, and recrystallized from
The compound was prepared from ethyl 2-(6-chloro-9H-
purin-9-yl)acetate (0.48 g, 2.0 mmol) as described above
and purified by column chromatography on silica gel using
ethyl acetate as eluent. Yield 30 %, mp 241–242 °C. IR
(KBr) cm-1: 1726 (–C=O ester), 2995 (–C–H), 3078
ethyl acetate. Yield 58 %, mp 230–235 °C. IR (KBr) cm-1
:
1
2951 (–C–H), 3074 (Ar–H). H-NMR (DMSO-d6) d: 3.83
(s, 3H, –N–CH3), 4.05 (s, 3H, –N–CH3), 8.56 (s, 1H,
Ar–H), 8.65 (s, 1H, Ar–H). 13C-NMR (DMSO-d6) d: 30.37,
35.11, 130.75, 146.90, 147.70, 150.78, 152.06, 153.54.
HR-MS for C8H9N8S? ([M-H]?) Calcd: 249.0671;
Found: 249.0667.
1
(Ar–H), 3400–3300 (–N–H). H-NMR (DMSO-d6) d: 1.22
(t, 3H, J = 7.1 Hz, –CH3), 4.18 (q, 2H, J = 7.1 Hz, –O–
CH2), 5.20 (s, 2H, –N–CH2), 8.45 (s, 1H, Ar–H), 8.55 (s,
1H, Ar–H), 12.09 (s, 1H, –N–H), 15.65 (s, 1H, –N–H).
13C-NMR (DMSO-d6) d: 14.45, 44.75, 61.99, 119.93, 138.97,
Synthesis of 9-ethyl-6-(1-methyl-1H-tetrazol-5-ylthio)-9H-
purine (12) The compound was prepared from 6-chloro-
9-ethyl-9H-purine (0.36 g, 2.00 mmol) as described above,
washed with diethyl ether, and recrystallized from ethyl
acetate. Yield 75 %, mp 134–135 °C. IR (KBr) cm-1: 2974
(–C–H), 3091 (Ar–H). 1H-NMR (DMSO-d6) d: 1.44 (t, 3H,
J = 7.3 Hz, –CH3), 4.05 (s, 3H, –N–CH3), 4.29 (q, 2H,
J = 7.3 Hz, –N–CH2), 8.61 (s, 2H, Ar–H). 13C-NMR
(DMSO-d6) d: 15.34, 35.12, 39.30, 130.96, 146.80, 146.88,
150.27, 151.98, 153.69. HR-MS for C9H11N8S? ([M-H]?)
Calcd: 263.0827; Found: 263.0750.
?
144.44, 149.69, 151.97, 168.13. HR-MS for C10H12N9O2
([M-H]?) Calcd: 290.1114; Found: 290.1103.
Synthesis of 9-(cyclobutylmethyl)-N-(1H-tetrazol-5-yl)-9H-
purin-6-amine (9) The compound was prepared from
6-chloro-9-(cyclobutylmethyl)-9H-purine (0.45 g, 2.0 mmol)
as described above and purified by column chromatography
on silica gel using 2:1 ethyl acetate–hexane as eluent. Yield
30 %, mp [255 °C (decomp.). IR (KBr) cm-1: 2928 (–C–
1
H), 3099 (Ar–H), 3400–3300 (–N–H). H-NMR (DMSO-
d6) d: 1.82 (m, 2H, –CH2), 1.96 (m, 4H, –CH2), 2.85 (m,
1H, –C–H), 4.28 (d, 2H, J = 7.4 Hz, –N–CH2), 8.47 (s,
1H, Ar–H), 8.56 (s, 1H, Ar–H), 12.00 (s, 1H, –N–H), 15.80
(s, 1H, –N–H). 13C-NMR (DMSO-d6) d: 17.43, 25.11,
33.25, 48.03, 119.72, 143.46, 149.10, 150,90. HR-MS
Synthesis of 9-propyl-6-(1-methyl-1H-tetrazol-5-ylthio)-9H-
purine (13) The compound was prepared from 6-chloro-9-
propyl-9H-purine (0.39 g, 2.00 mmol) as described above,
washed with diethyl ether, and recrystallized from ethyl
acetate. Yield 68 %, mp 168–172 °C. IR (KBr) cm-1: 2937
(–C–H), 3074 (Ar–H). 1H-NMR (DMSO-d6) d: 0.90 (t, 3H,
J = 7.3 Hz, –CH3), 1.90 (m, 2H, –CH2), 4.05 (s, 3H,
–N–CH3), 4.48 (t, 2H, J = 7.2 Hz, –N–CH2), 8.60 (s, 1H,
Ar–H), 8.80 (s, 1H, Ar–H). 13C-NMR (DMSO-d6) d: 11.11,
24.79, 35.14, 49.07, 123.23, 146.72, 146.80, 150.75,
152.08, 160.26. HR-MS for C10H13N8S? ([M-H]?) Calcd:
277.0984; Found: 277.0914.
?
for C11H14N9 ([M-H]?) Calcd: 272.1372; Found:
272.1312.
Synthesis of 6-(1-methyl-1H-tetrazol-5-ylthio)-9H-purine
(10) 6-chloropurine (0.31 g, 2.00 mmol) was refluxed
with 1-methyl-5-mercaptotetrazole (0.23 g, 2.00 mmol) in
dichloromethane (25 ml) for 4 days. The solvent was
evaporated in vacuo. The product was purified by column
chromatography on silica gel using 2:1 ethyl acetate–
hexane. Yield 25 %, mp 210–215 °C. IR (KBr) cm-1
:
Synthesis of 9-isopropyl-6-(1-methyl-1H-tetrazol-5-ylthio)-
9H-purine (14) The compound was prepared from
6-chloro-9-isopropyl-9H-purine (0.39 g, 2.00 mmol) as
described above, purified by column chromatography on
silica gel using 2:1 ethyl acetate–hexane as eluent, and re-
crystallized from ethyl acetate. Yield 46 %, mp 94–98 °C.
IR (KBr) cm-1: 2934 (–C–H), 3058 (Ar–H). 1H-NMR
(DMSO-d6) d: 1.56 (d, 6H, J = 6.8 Hz, –CH3), 4.08 (s, 3H, –
N–CH3), 4.86 (m, 1H, –C–H), 8.64 (s, 1H, Ar–H), 8.71 (s,
1H, Ar–H). 13C-NMR (DMSO-d6) d: 22.56, 35.13, 48.10,
131.24, 145.31, 146.88, 149.95, 151.77, 153.77. HR-MS for
C10H13N8S? ([M-H]?) Calcd: 277.0984; Found: 277.0951.
2959 (–C–H), 3098 (Ar–H), 3428 (–N–H). 1H-NMR
(DMSO-d6) d: 4.05 (s, 3H, –N–CH3), 8.58 (s, 1H, Ar–H),
8.61 (s, 1H, Ar–H), 13.80 (s, 1H, –N–H). 13C-NMR
(DMSO-d6) d: 35.12, 145.55, 146.97, 151.79, 152.15,
152.99. HR-MS for C7H7N8S? ([M-H]?) Calcd:
235.0514; Found: 235.0470.
General procedure for the preparation of 9-alkyl-6-
(1-methyl-1H-tetrazol-5-ylthio)-9H-purine compounds
(11–19)
The appropriate 6-chloro-9-alkyl-9H-purine (2 mmol)
was refluxed with 1-methyl-5-mercaptotetrazole (0.23 g,
2.00 mmol) in dichloromethane (25 ml) for 4 days. The
solvent was evaporated in vacuo.
Synthesis of 9-hexyl-6-(1-methyl-1H-tetrazol-5-ylthio)-9H-
purine (15) The compound was prepared from 6-chloro-9-
hexyl-9H-purine (0.48 g, 2.00 mmol) as described above,
123

Med Chem Res
Synthesis of 9-(cyclohexylmethyl)-6-(1-methyl-1H-tetrazol-
5-ylthio)-9H-purine (19) The compound was prepared
from 6-chloro-9-(cyclohexylmethyl)-9H-purine (0.50 g,
2.00 mmol) as described above, washed with diethyl ether,
and recrystallized from ethyl acetate. Yield 80 %, mp
108–110 °C. IR (KBr) cm-1: 2923 (–C–H), 3067 (Ar–H).
1H-NMR (DMSO-d6) d: 1.15 (m, 2H, –CH2), 1.20 (m, 4H,
–CH2), 1.60 (m, 4H, –CH2), 1.85 (m, 1H, –C–H), 4.07
(s, 3H, –N–CH3), 4.12 (d, 2H, J = 7.2 Hz, –N–CH2), 8.59
(s, 1H, Ar–H), 8.64 (s, 1H, Ar–H).13C-NMR (DMSO-d6) d:
25.45, 26.15, 31.41, 35.14, 37.89, 49.75, 130.75, 146.87,
147.40, 150.66, 152.08, 153.80. HR-MS for C14H19N8S?
([M-H]?) Calcd: 331.1453; Found: 331.1417.
washed with diethyl ether, and recrystallized from ethyl
acetate. Yield 67 %, mp 119–122 °C. IR (KBr) cm-1: 2928
(–C–H), 3057 (Ar–H). 1H-NMR (DMSO-d6) d: 0.85 (m, 3H,
–CH3), 1.33 (m, 6H, –CH2), 1.95 (m, 2H, –CH2), 4.08 (s, 3H,
–N–CH3), 4.51 (t, 2H, J = 7.3 Hz, –N–CH2), 8.60 (s, 1H,
Ar–H), 8.80 (s, 1H, Ar–H). 13C-NMR (DMSO-d6) d: 14.27,
22.41, 25.97, 31.08, 31.65, 35.14, 47,64, 123.24, 146.67,
146.82, 150.70, 152.07, 160.28. HR-MS for C13H19N8S?
([M-H]?) Calcd: 319.1453; Found: 319.1359.
Synthesis of 9-benzyl-6-(1-methyl-1H-tetrazol-5-ylthio)-9H-
purine (16) The compound was prepared from 6-chloro-9-
benzyl-9H-purine (0.49 g, 2.00 mmol) as described above
and purified by column chromatography on silica gel using
2:1 ethyl acetate–hexane as eluent. Yield 87 %, mp
115–118 °C. IR (KBr) cm-1: 2954 (–C–H), 3020 (Ar–H),
3066 (Ar–H). 1H-NMR (DMSO-d6) d: 4.10 (s, 3H,
–N–CH3), 5.50 (s, 2H, –N–CH2), 7.34 (m, 5H, Ar–H), 8.65
(s, 1H, Ar–H), 8.80 (s, 1H, Ar–H). 13C-NMR (DMSO-d6) d:
35.14, 47.31, 128.20, 128.33, 128.49, 130.85, 136.55,
146.81, 147.01, 150.32, 152.31, 154.04. HR-MS for
C14H13N8S? ([M-H]?) Calcd: 325.0984; Found: 325.0934.
Antibacterial and antifungal testing methods
The in vitro antibacterial activity of synthesized amino
and thiotetrazole purine derivatives (compounds 1–19)
was performed against American Type Culture Collection
(ATCC) reference bacterial strain and fungi by disk dif-
fusion technique (Clinical and Laboratory Standards
Institute 2006, 2007). Gram-negative and Gram-positive
bacteria were grown in nutrient agar medium and incu-
bated at 37 °C for 24 h. The yeast strains were grown in
Sabouraud dextrose agar medium and incubated at 27 °C
for 72 h. Chloramphenicol and ampicillin were used as
the reference antibacterial agents, and ketoconazole was
used as a reference antifungal agent under similar con-
ditions for comparison. All the testings were repeated
three times.
Synthesis of ethyl 2-(6-(1-methyl-1H-tetrazol-5-ylthio)-9H-
purin-9-yl)acetate (17) The compound was prepared
from ethyl 2-(6-chloro-9H-purin-9-yl)acetate (0.48 g,
2.00 mmol) as described above, washed with diethyl ether,
and recrystallized from ethyl acetate. Yield 79 %, mp
88–92 °C. IR (KBr) cm-1: 1736 (–C=O ester), 2945 (–C–
H), 3072 (Ar–H). 1H-NMR (DMSO-d6) d: 1.20 (t, 3H,
J = 7.1 Hz, –CH3), 4.10 (s, 3H, –N–CH3), 4.18 (q, 2H,
J = 7.1 Hz, –O–CH2), 5.25 (s, 2H, –N–CH2), 8.60 (s, 1H,
Ar–H), 8.70 (s, 1H, Ar–H). 13C-NMR (DMSO-d6) d: 14.43,
35.14, 44.92, 62.12, 130.47, 146.79, 147.47, 150.57,
152.42, 154.14, 167.85. HR-MS for C11H13N8O2S? ([M-
H]?) Calcd: 321.0882; Found: 321.0867.
DNA and compound interaction
The interaction of compounds 1–19 with pBR322 plasmid
DNA was studied by agarose gel electrophoresis (Asmafiliz
et al., 2009; Huq et al., 2009). The compounds were dis-
solved in DMSO and the plasmid DNA were added to the
aliquots of decreasing concentrations of compounds rang-
ing from 5,000 to 312 lM. The mixtures were incubated in
the dark at 37 °C for 24 h and electrophoresed in 1 %
agarose gel. Electrophoresis was carried under TAE buffer
for approximately 3 h at 60 V. At the end of the electro-
phoresis, the gel was stained with ethidium bromide. The
gel was visualized under UV light using BioDoc Analyzer,
Biometra. The illuminated gel was photographed with a
video-camera and saved as a TIFF file. The experiments
were repeated three times.
Synthesis of 9-(cyclobutylmethyl)-6-(1-methyl-1H-tetrazol-
5-ylthio)-9H-purine (18) The compound was prepared
from 6-chloro-9-(cyclobutylmethyl)-9H-purine (0.45 g,
2.00 mmol) as described above, purified by column chroma-
tography on silica gel using 2:1 ethyl acetate–hexane as eluent,
and recrystallized from ethyl acetate. Yield 70 %, mp
90–94 °C. IR (KBr) cm-1: 2937 (–C–H), 3062 (Ar–H).
1H-NMR (DMSO-d6) d: 1.80 (m, 2H, –CH2), 1.95 (m, 4H,
–CH2), 2.85 (m, 1H, –C–H), 4.10 (s, 3H, –N–CH3), 4.29 (d,
2H, J = 7.4 Hz, –N–CH2), 8.61(s, 1H, Ar–H), 8.63(s, 1H, Ar–
H). 13C-NMR (DMSO-d6) d: 17.89, 25.66, 34.14, 35.13, 48.77,
130.72, 146.83, 146.90, 150.47, 152,01, 153.77. HR-MS for
C12H15N8S? ([M-H]?) Calcd: 303.1140; Found: 303.1039.
Acknowledgments The authors thank Gazi University Scientific
Research Fund (Project no. 05/2009-24) for financial support.
123

Med Chem Res
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