Synthesis and antimicrobial evaluation of some new substituted purine derivatives

Article abstract of DOI:10.1016/j.bmc.2008.12.050

A series of 8,9-disubstituted adenines (4, 5, 8), 6-substituted aminopurines (10-13) and 9-(p-fluorobenzyl/cyclopentyl)-6-substituted aminopurines (16, 17, 19-30) have been prepared and the antimicrobial activities of these compounds against Staphylococcu

Full text of DOI:10.1016/j.bmc.2008.12.050

Bioorganic & Medicinal Chemistry 17 (2009) 1693–1700
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antimicrobial evaluation of some new substituted
purine derivatives
a
b
c
c
Meral Tunçbilek a, , Zeynep Atesß-Alagöz , Nurten Altanlar , Arzu Karayel , Süheyla Özbey
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandog˘an, Ankara, Turkey
b Department of Microbiology, Faculty of Pharmacy, Ankara University, 06100 Tandog˘an, Ankara, Turkey
c Department of Physics Engineering, Hacettepe University, 06800 Beytepe, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 8,9-disubstituted adenines (4, 5, 8), 6-substituted aminopurines (10–13) and 9-(p-fluoroben-
zyl/cyclopentyl)-6-substituted aminopurines (16, 17, 19–30) have been prepared and the antimicrobial
activities of these compounds against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA,
standard and clinical isolate), Bacillus subtilis, Escherichia coli and Candida albicans were evaluated. 6-
[(N-phenylaminoethyl)amino]-9H-purine (12) which has no substitution at N-9 position and 9-cyclopen-
tyl-6-[(4-fluorobenzyl)amino]-9H-purine (24) exhibited excellent activity against C. albicans with MIC
Received 5 September 2008
Revised 16 December 2008
Accepted 22 December 2008
Available online 29 December 2008
Keywords:
Purine derivatives
Antibacterial
Antifungal
3.12 lg/mL. These compounds displayed better antifungal activity than that of standard oxiconazole. Fur-
thermore, compound 22 carrying 4-chlorobenzylamino group at the 6-position of the purine moiety
exhibited comparable antibacterial activity with that of the standard ciprofloxacin against both of the
drug-resistant bacteria (MRSA, standard and clinical isolate).
Methicillin-resistant Staphylococcus aureus
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Nosocomial bloodstream infections are important causes of
morbidity and mortality. Staphylococcus aureus remains a signifi-
cant reason of nosocomial bloodstream infection.1 Methicillin-
resistant S. aureus (MRSA) isolates came into existence soon after
the introduction of methicillin and MRSA is now a major pathogen
worldwide. MRSA isolates have been associated with nosocomial
infections and rapidly developed resistance to multiple drug
classes.2
Candida infections have increased dramatically over the past
three decades. Candida spp. are the fourth most common cause
of nosocomial bloodstream infections in many hospitals and repre-
sent 10% of all bloodstream infections.3,4
Purine, purines nucleosides and their analogs have been exten-
sively investigated due to their potential activity as enzyme
inhibitors,5–8 cytotoxic,9–12 antiviral,13–16 antihyperglycemic,17
immunostimulator,18 antifungal and antibacterial agents.19–25 On
the basis of these observations, in this study, we designed and syn-
thesized novel substituted purine derivatives (4–6, 8, 10–13, 16, 17,
19–30) and evaluated their antibacterial, antifungal activities.
NH2
NH2
NH2
N
N
N
N
N
a
b
N
N
N
Br
N
H
N
N
N
1
2
3
c
d
NH2
N
NH2
NH2
N
N
H
N
N
N
N
N
e
N
N
R1
Br
N
N
N
6
7
R1=
R1=
4
5
f
NH2
N
N
H
N
N
N
8
* Corresponding author. Tel.: +90 312 2033071; fax: +90 312 2131081.
E-mail address: tuncbile@pharmacy.ankara.edu.tr (M. Tunçbilek).
Scheme 1. (a) Iodoethane, K2CO3, DMF; (b) NBS, DMF; (c) the appropriate amine;
(d) cyclopentyl bromide, K2CO3, DMFß(e) Br2, H2O; (e) ethylamine (70% in H2O).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.050

1694
M. Tunçbilek et al. / Bioorg. Med. Chem. 17 (2009) 1693–1700
thesized by nucleophilic substitution of chlorine of commercially
2. Result and discussions
2.1. Chemistry
available 6-chloropurine (9) with the appropriate amines. Com-
pound 9 was alkylated with p-fluorobenzyl chloride to give mix-
ture of 7- and 9-alkylated purines.31 Isomers were separated by
column chromatography to obtain the 9-isomers (14) in 55% and
7-isomers (15) in 21% yield. Treatment of 9-p-fluorobenzyl-6-chlo-
ropurine (14) with substituted ethylenediamines afforded the 6-
(substituted ethylenediamino)-9-(p-fluorobenzyl)purines (16, 17).
Finally, compounds 19–30 were synthesized via N-9 alkylation of
6-chloropurine 9 with cyclopentylbromide,32 and by amination of
6-chloro-9-cyclopentylpurine 18 with substituted amines. The
alkylation reaction occurred only at the N-9 atom. X-ray analysis
also confirmed the structure of compound 18 (Fig. 1).
The synthesis of the 8,9-disubstituted adenine derivatives 4, 5, 8
was carried out starting from commercially available adenine (1)
(Scheme 1). For preparation of 9-ethyl-8-(alkylamino)adenines
(4, 5), 1 was alkylated with iodoethane to give intermediate 9-eth-
yladenine (226). The reaction of 2 N-bromosuccinimide gave the
8-bromo-9-ethyladenine (326). The final products (4, 5) were
obtained by nucleophilic substitution of compound 3 with cyclo-
pentylamine or isobutylamine. The 9-cyclopentyl analoge 6 was
previously prepared from 5-amino-1-cyclopentylimidazole-4-car-
boxamidine,27 or from 6-chloro-9-cyclopentyl purine,28 or from
t-butoxycarbonyl protected adenine derivative and cyclopentanol
with Mitsunobu reaction.29 Here, 9-cyclopentyl adenine (6) was
obtained from 1 with potassium carbonate and cyclopentylbro-
mide. Reaction of 6 with bromine30 followed by ethylamine alkyl-
ation furnished 9-cyclopentyl-8-ethyladenine (8) in good yield.
6-Substituted aminopurines (10–13) and 9-(p-fluorobenzyl/
cyclopentyl)-6-substituted aminopurines (16, 17, 19–30) were
synthesized as shown in Scheme 2. Compounds 10–13 were syn-
2.2. Microbiology
The newly obtained purine derivatives (4–6, 8, 10–13, 16, 17, 19–
30) were evaluated for their in vitro antibacterial activity against
Gram positive S. aureus, methicillin-resistant S. aureus (MRSA, stan-
dart and clinical isolate), Bacillus subtilis, Gram negative Escherichia
coli and antifungal activity against Candida albicans by tube dilution
technique.33,34 Sultamicillin, ampicillin and ciprofloxacin were used
R2
HN
R2=
R2=
10
11
H
N
N
12 R2=
N
N
N
N
N
H
N
R3
N
13 R3=
N
a
N
H
N
a
R3
b
HN
Cl
N
N
N
N
N
N
N
N
Cl
N
N
N
14
c
F
N
H
F
+
N
R3=
F
16
9
H
N
Cl
N
17 R3=
N
N
N
d
15
R4
Cl
HN
N
N
N
N
N
a
N
N
N
F
18
N
25 R4=
R4=
19
F
H
N
b
20 R4=
26 R4=
H
N
OCH3
OCH3
R4=
R4=
R5
21
22
N
N
N
27 R4=
28 R4=
OCH3
N
Cl
Cl
Cl
23 R4=
N
N
R5=
30
N
R4=
R4=
24
29
F
Scheme 2. (a) The appropriate amine, EtOH; (b) 4-methylpiperidine; (c) 4-fluorobenzyl chloride, NaH, DMF; (d) cyclopentyl bromide, K2CO3, DMF.

M. Tunçbilek et al. / Bioorg. Med. Chem. 17 (2009) 1693–1700
4. Experimental
1695
4.1. Chemistry
Melting points were recorded with a capillary melting point
apparatus (Electrothermal 9100) and are uncorrected. 1H and
13C NMR spectra were recorded on VARIAN Mercury 400 FT-
NMR spectrometer (operated at 400) and are referenced to inter-
nal tetramethylsilane (TMS) at 0.0 ppm. The spin multiplicities
are indicated by the symbols s (singlet), d (doublet), dd (doublet
of doublets), t (triplet), q (quartet), m (multiplet), and br (broad).
Mass spectra were taken on Waters Micromass ZQ by using
(ESI+) method. Elemental analyses (C, H, N) were determined
on a Leco CHNS 932 instrument and gave values within 0.4%
of the theoretical values. All instrumental analysis was performed
at Ankara University, Faculty of Pharmacy. Column chromatogra-
phy was accomplished on silica gel 60 (40–63 mm particle size).
The chemical reagents used in synthesis were purchased from E.
Merck, Fluka, Sigma, Acros and Aldrich.
Figure 1. Two molecules in the asymmetric unit of 18, showing the atom
numbering scheme; displacement ellipsoids are drawn at the 40% probability level.
as antibacterial standard drugs, while miconazole and oxiconazole
were used as antifungal standard drugs whose minimum inhibitory
concentration (MIC) values are provided (Table 1).
As it indicated in Table 1 most of the compounds showed good
activity against C. albicans. 6-[(N-phenylaminoethyl)amino]-9H-
purine (12) which have no substitution at N-9 position and 9-
cyclopentyl-6-[(4-fluorobenzyl)amino]-9H-purine (24) were the
most active compounds, they were two times as active as oxicon-
4.1.1. 6-Amino-9-ethyl-8-(cyclopentylamino)-9H-purine (9-
ethyl-8-(cyclopentylamino)-9H-adenine, 4)
A solution of 3 (0.18 g, 0.74 mmol) in excess cyclopentylamine
(2 mL) was heated at 120 °C for 14 h. The reaction mixture was
concentrated by evaporation and the residue was purified by col-
umn chromatography eluting with CHCl3–MeOH (10:1) to give 4
(0.12 g, 65%) as a cream-colored solid: mp 221 °C. 1H NMR
(DMSO-d6) d 1.18 (t, 3H), 1.53–1.71 (m, 6H), 1.95–1.99 (m, 2H),
4.00 (q, 2H), 4.20–4.25 (m, 1H), 6.36 (s, 2H), 6.51 (d, 1H), 7.90
(s, 1H). MS (ESI+) m/z: 247 (100%) (M+H). Anal. Calcd for
C12H18N6: C, 58.51; H, 7.37; N, 34.12. Found: C, 58.22; H, 7.15;
N, 33.85.
azole against C. albicans with MIC 3.12 lg/mL. When compared
with miconazole, compounds 12 and 24 showed similar activity.
All the 6-(substitutedbenzylamino)-9-cyclopentylpurines (22–
27) possess good antifungal activity, with MIC values against C.
albicans in the range of 3.12–25 lg/mL. From these derivatives
compound 22, with 4-chlorobenzylamino substituted at C-6,
showed also comparable activity against MRSA standard with the
standard ciprofloxacin. Again, this compound possessed a better
activity against MRSA clinical isolate than the standard ciprofloxa-
4.1.2. 6-Amino-9-ethyl-8-(isobutylamino)-9H-purine (9-ethyl-
8-(isobutylamino)-9H-adenine, 5)
cin with MIC 6.25
22, 24 exhibited a better activity against both of the drug-resistant
bacteria than the standard ampicillin with MIC 6.25–25 g/mL.
lg/mL. 6-Substitutedbenzylamino compounds
A solution of 3 (0.08 g, 0.33 mmol) in excess isobutylamine
(1.5 mL) was refluxed with stirring for 48 h. The reaction mixture
was concentrated by evaporation and the residue was purified by
column chromatography eluting with CHCl3–isopropanol (10:2)
to give 5 (0.04 g, 52%) as a cream-colored solid: mp 188–190 °C.
1H NMR (DMSO-d6) d 0.90–0.94 (3, 9H), 1.76–1.88 (m, 1H), 3.22
(t, 2H), 4.10 (q, 2H), 7.60–7.84 (br s, 3H), 8.21 (s, 1H). MS (ESI+)
m/z: 235 (100%) (M+H). Anal. Calcd for C11H18N6: C, 56.39; H,
7.74; N, 35.87. Found: C, 56.54; H, 7.80; N, 35.73.
l
From this screening, the results reported in Table 1 show that
most of the compounds were effective against B. Subtilis; 4, 6, 10,
11, 13, 16, 19–24, 27 and 28 showed better activity than the stan-
dard ampicillin with MIC 25 lg/mL.
The synthesized compounds were also tested against Gram-
negative bacteria E. coli and10–12, 27, 29 displayed similar activity
with the standard sultamicillin with MIC 25 lg/mL. These deriva-
tives possessed also a better activity against E. Coli than the stan-
dard ampicillin.
4.1.3. 6-Amino-9-cyclopentyl-9H-purine (9-cyclopentyl-9H-
adenine, 6)
3. Conclusion
To a suspension of 1 (0.25 g, 1.85 mmol) in 4 mL of DMF was
added K2CO3 (0.29 g, 2.12 mmol). The reaction mixture was stirred
at 60 °C for 30 min. A solution of cyclopentyl bromide (0.3 mL,
2.77 mmol) in 3 mL DMF was added to the reaction mixture and
the mixture was stirred at 65 °C for 72 h. The reaction mixture
was concentrated by evaporation and the residue was purified by
column chromatography eluting with EtOAc–MeOH (10:1) to give
6 (0.18 g, 48%) as a white solid: mp 154 °C. 1H NMR (DMSO-d6) d
1.67–2.16 (m, 8H), 4.81–4.87 (m, 1H), 7.23 (s, 2H), 8.15 (s, 1H),
8.22 (s, 1H). 13C NMR (CDCl3) d 23.8, 32.8, 56.0, 119.9, 138.5,
150.1, 152.7, 155.8. MS (ESI+) m/z: 204 (100%) (M+H). Anal. Calcd
for C10H13N5: C, 59.10; H, 6.45; N, 34.46. Found: C, 58.72; H,
6.51; N, 34.53.
In this work, we have synthesized novel 8,9-disubstituted ade-
nine derivatives (4, 5, 8), 6-substituted aminopurines (10–13) and
9-(p-fluorobenzyl/cyclopentyl)-6-substituted aminopurine analogs
(16, 17, 19–30) and screened for their antimicrobial activities. All
the new compounds except 19, demonstrated potent antifungal
activity against C. albicans. Particularly, 6-substituted aminopurine
analogs 12 having no substituents on the 9-position of the purine
structure and 24 which is among 9-cyclopentyl-6-substituted ami-
nopurine derivatives displayed better antifungal efficacy than that
of the standard drug oxiconazole. Also these compounds showed
same activity as miconazole against C. albicans. Against both of
the drug-resistant bacteria, compound 22 carrying 4-chloro-
benzylamino group at the 6-position of the purine exhibited com-
parable antibacterial activity with that of the clinically used drug
ciprofloxacin. Therefore, we need to do studies on in vivo and
mode of action mechanisms of the new purines to better deter-
mine the potential of their antifungal and antibacterial activity.
4.1.4. 6-Amino-8-bromo-9-cyclopentyl-9H-purine (8-bromo-9-
cyclopentyl-9H-adenine, 7)
A solution of bromine (0.2 mL, 3.9 mmol) in water (15 mL) was
added to 6 (0.1 g, 0.49 mmol). The reaction mixture was stirred at
room temperature for 17 h. The reaction mixture was concentrated

1696
M. Tunçbilek et al. / Bioorg. Med. Chem. 17 (2009) 1693–1700
Table 1
Antibacterial and antifungal activity of compounds 4–6, 8, 10–13, 15, 16, 18–29 (MIC minimum inhibitory concentration
lg/mL)
R
N
N
X
N
N
Y
Compound
R
X
Y
S. aureus (25923)a MRSAb (431300) MRSAc E. coli (25922) B. subtilis (6633) C. albicans (10145)
H
N
4
–NH2
–C2H5
50
50
50
50
50
50
50
50
25
50
12.5
25
H
N
5
–NH2
–C2H5
6
8
–NH2
–NH2
–H
50
25
50
50
50
50
50
50
25
50
12.5
6.25
H
N
10
11
12
13
16
–H
–H
–H
–H
–H
50
50
50
25
50
50
50
50
50
50
50
50
50
50
50
25
25
25
50
50
25
25
50
25
25
6.25
12.5
3.12
25
N
H
–H
–H
–H
–H
N
H
N
N CH3
H
N
N
H
N
CH3
N
25
N
H
F
F
H
N
17
19
20
21
22
23
24
25
26
27
28
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
50
50
50
50
50
6.25
25
25
50
50
50
50
50
50
50
50
6.25
50
25
50
25
50
50
50
50
50
50
50
50
50
50
50
25
50
50
25
25
25
25
25
25
50
50
25
25
25
N
H
N
50
50
N
H
H
N
25
25
N
H
H
N
50
12.5
12.5
6.25
3.12
25
N
H
N
H
12.5
12.5
25
Cl
Cl
HN
Cl
F
N
H
F
N
H
50
F
N
H
25
12.5
12.5
25
OCH3
OCH3
N
H
50
OCH3
50
N
H
N
N CH3

M. Tunçbilek et al. / Bioorg. Med. Chem. 17 (2009) 1693–1700
1697
Table 1 (continued)
Compound
R
X
Y
S. aureus (25923)a
MRSAb (431300)
25
MRSAc
25
E. coli (25922)
B. subtilis (6633)
C. albicans (10145)
29
–H
12.5
25
50
12.5
N
H
30
–H
50
50
50
50
50
12.5
N
CH3
Sultamicillin
Ampicillin
Ciprofloxacin
Miconazole
Oxiconazole
1.56
0.78
0.78
25
50
6.25
25
50
12.5
25
50
0.39
0.78
50
0.19
3.12
6.25
a
ATCC number.
MRSA: methicillin-resistant S. aureus (standart).
MRSA: methicillin-resistant S. aureus (clinical isolate).
b
c
by evaporation and the residue was purified by column chroma-
tography eluting with hexane–EtOAc (1:1) to give 7 (0.11 g, 79%)
as a cream-colored solid: mp 193 °C. 1H NMR (DMSO-d6) d 1.66–
2.34 (m, 8H), 4.84–4.92 (m, 1H), 7.36 (s, 2H), 8.11 (s, 1H). MS
(ESI+) m/z: 282 (98%) (M+H), 284 (100%) (M+H+2). Anal. Calcd for
C10H12BrN5: C, 42.57; H, 4.29; N, 24.82. Found: C, 42.95; H, 4.56;
N, 24.79.
centrated by evaporation and the product was crystallized from
DMF–MeOH to give 13 (0.19 g, 77%) as a white solid: mp 253 °C.
1H NMR (DMSO-d6) d 3.31 (q, 2H), 3.71 (br s, 2H), 5.84 (t, 1H),
6.57 (t, 1H, J = 7.2 Hz), 6.68 (d, 2H, J = 8.4 Hz), 7.13 (t, 2H,
J = 7.6 Hz), 7.79 (br s, 1H), 8.16 (s, 1H), 8.28 (br s, 1H), 13.00 (br
s, 1H). MS (ESI+) m/z: 255 (100%) (M+H). Anal. Calcd for
C13H14N6: C, 61.40; H, 5.55; N, 33.05. Found: C, 61.04; H, 5.46; N,
32.95.
4.1.5. 6-Amino-8-(ethylamino)-9-cyclopentyl-9H-purine (8-
(ethylamino)-9-cyclopentyl-9H-adenine, 8)
4.1.9. 6-(4-Methylpiperidin-1-yl)-9H-purine (13)
A solution of 7 (0.07 g, 0.25 mmol) in excess ethylamine (70% in
water, 2 mL) was stirred at 30 °C for 48 h. The reaction mixture was
concentrated by evaporation and the residue was purified by col-
umn chromatography eluting with CHCl3–MeOH (10:1) to give 8
(0.046 g, 75%) as a cream-colored solid: mp 188 °C. 1H NMR
(DMSO-d6) d 1.17 (t, 3H), 1.56–2.26 (m, 8H), 3.35 (m, 2H), 4.56
(m, 1H), 6.29 (s, 2H), 6.54 (t, 1H), 7.84 (s, 1H). MS (ESI+) m/z: 247
(100%) (M+H). Anal. Calcd for C12H18N6Á0.1CHCl3Á0.1MeOH: C,
56.04; H, 7.13; N, 32.14. Found: C, 56.40; H, 6.79; N, 32.02.
To a solution of 9 (0.15 g, 0.97 mmol) in absolute EtOH (5 mL)
was added 4-methylpiperidine (0.23 mL, 1.94 mmol) and this mix-
ture was refluxed for 16 h. The reaction mixture was concentrated
by evaporation and the product was crystallized from MeOH–
EtOAc to give 12 (0.15 g, 71%) as a white solid: mp 265 °C. 1H
NMR (DMSO-d6) d 0.92 (d, 3H), 1.04–1.16 (m, 1H), 1.72 (m, 4H),
3.03 (m, 4H), 8.09 (s, 1H), 8.17 (s, 1H), 13.00 (br s, 1H). 13C NMR
(DMSO-d6) d 22.5, 31.3, 34.6, 45.5, 119.4, 138.4, 152.0, 152.5,
153.8. MS (ESI+) m/z: 218 (100%) (M+H). Anal. Calcd for
C11H15N5: C, 60.81; H, 6.96; N, 32.23. Found: C, 60.47; H, 6.58; N,
32.30.
4.1.6. 6-(2-Cyclohexenylethyl)amino-9H-purine (10)
To a solution of 9 (0.15 g, 0.97 mmol) in absolute EtOH (5 mL)
was added 2-(1-cyclohexenyl)ethylamine (0.27 mL, 1.94 mmol)
and this mixture was refluxed for 40 h. The reaction mixture was
concentrated by evaporation and the residue was purified by col-
umn chromatography eluting with EtOAc–MeOH (10:1) to give
10 (0.16 g, 68%) as a white solid: mp 199 °C. 1H NMR (DMSO-d6)
d 1.51 (dd, 4H), 1.94 (d, 4H), 2.22 (t, 2H), 3.55 (br s, 2H), 5.41 (s,
1H), 7.52 (br s, 1H), 8.07 (s, 1H), 8.17 (br s, 1H), 12.88 (br s, 1H).
MS (ESI+) m/z: 244 (100%) (M+H). Anal. Calcd for C13H17N5: C,
64.17; H, 7.04; N, 28.78. Found: C, 63.85; H, 6.65; N, 28.39.
4.1.10. 6-Chloro-9-(4-fluorobenzyl)-9H-purine (14) and 6-
chloro-7-(4-fluorobenzyl)-9H-purine (15)
A suspension of 9 (0.25 g, 1.62 mmol) in DMF (8 mL) was trea-
ted with NaH (0.07 g, 3.23 mmol) and stirred at room temperature
for 20 min. 4-Fluorobenzyl chloride (0.38 mL, 3.23 mmol) was
added and the mixture stirred for 45 h at room temperature. H2O
was added and then extracted with EtOAc. The extract was dried
over Na2SO4, the solvent was evaporated in vacuo, and the residue
was purified by column chromatography eluting with EtOAc–hex-
ane (7:3) to give first 14 (0.23 g, 55%) and afterwards 15 (0.09 g,
21%). Compound 14 mp 132 °C. 1H NMR (CDCl3) d 5.44 (s, 2H),
7.07 (t, 2H, J = 8.4 Hz), 7.31–7.35 (m, 2H), 8.11 (s, 1H), 8.79 (s,
1H). MS (ESI+) m/z: 263 (100%) (M+H), 265 (32%) (M+H+2). Anal.
Calcd for C12H8ClFN4: C, 54.87; H, 3.07; N, 21.33. Found: C,
54.52; H, 2.69; N, 21.26. Compound 15 mp 168 °C. 1H NMR (CDCl3)
d 5.67 (s, 2H), 7.09 (t, 2H, J = 8.8 Hz), 7.17–7.21 (m, 2H), 8.24 (s,
1H), 8.91 (s, 1H). MS (ESI+) m/z: 263 (100%) (M+H), 265 (32%)
(M+H+2). Anal. Calcd for C12H8ClFN4: C, 54.87; H, 3.07; N, 21.33.
Found: C, 54.49; H, 2.72; N, 21.30.
4.1.7. 6-(4-Methylpiperazin-1-yl)amino-9H-purine (11)
To a solution of 9 (0.13 g, 0.84 mmol) in absolute EtOH (5 mL)
was added 1-amino-4-methylpiperazine (0.2 mL, 1.68 mmol) and
this mixture was refluxed for 4 h. The reaction mixture was con-
centrated by evaporation. The product was washed with CHCl3
and crystallized from MeOH–EtOAc to give 11 (0.14 g, 71%) as a
cream-colored solid: mp 299 °C. 1H NMR (DMSO-d6) d 2.51 (s,
3H), 2.77 (s, 4H), 3.15 (br s, 4H), 8.23 (s, 1H), 8.31 (br s, 1H), 9.04
(br s, 1H), 12.20 (br s, 1H). MS (ESI+) m/z: 234 (100%) (M+H). Anal.
Calcd for C10H15N7: C, 51.49; H, 6.48; N, 42.03. Found: C, 51.57; H,
6.36; N, 41.92.
4.1.11. 6-[2-(N,N-Diethylamino)ethyl]amino-9-(4-fluorobenzyl)-
9H-purine (16)
4.1.8. 6-[2-(Phenylamino)ethyl]amino-9H-purine (12)
To a solution of 14 (0.10 g, 0.38 mmol) in absolute EtOH (4 mL)
was added N,N-diethylethylenediamine (0.21 mL, 1.52 mmol) and
this mixture was refluxed for 5 h. The reaction mixture was con-
centrated by evaporation and the residue was purified by column
To a solution of 9 (0.15 g, 0.97 mmol) in absolute EtOH (5 mL)
was added N-phenylethylenediamine (0.25 mL, 1.94 mmol) and
this mixture was refluxed for 15 h. The reaction mixture was con-

1698
M. Tunçbilek et al. / Bioorg. Med. Chem. 17 (2009) 1693–1700
chromatography eluting with EtOAc–MeOH–NH3 (10:2:0.2) to give
16 (0.09 g, 69%) as a cream-colored solid: mp 94 °C. 1H NMR
(CDCl3) d 1.11 (t, 6H), 2.70 (q, 4H), 2.84 (t, 2H), 3.78 (br s, 2H),
5.35 (s, 2H), 6.75 (br s, 1H), 7.02 (t, 2H), 7.27–7.32 (m, 2H), 7.84
(s, 1H), 8.41 (s, 1H). 13C NMR (CDCl3) d 11.3, 38.0, 46.6, 47.0,
51.6, 116.0, 116.2, 129.8 (2), 131.9 (2), 139.9, 153.6, 155.1, 161.5,
164.0. MS (ESI+) m/z: 343 (100%) (M+H). Anal. Calcd for
C18H23FN6Á0.6H2OÁ0.2CH3OH: C, 60.78; H, 7.00; N, 23.37. Found:
C, 60.44; H, 6.65; N, 23.08.
4.1.14.3. 6-[2-(Phenylamino)ethyl]amino-9-cyclopentyl-9H-pur-
ine (21). The compound was prepared from 18 and N-phenyle-
thylenediamine according to general procedure and was purified
by column chromatography (EtOAc/MeOH 10:1) to give 21
(0.08 g, 50%) as a cream-colored solid: mp 123 °C. 1H NMR
(DMSO-d6) d 1.66–2.18 (m, 8H), 3.24 (q, 2H), 3.65 (br s, 2H),
4.80–4.88 (m, 1H), 5.77 (t, 1H), 6.51 (t, 1H, J = 7.2 Hz), 6.62 (d,
2H, J = 8.0 Hz), 7.07 (t, 2H, J = 7.6 Hz), 7.81 (br s, 1H), 8.22 (s, 1H),
8.25 (br s, 1H). 13C NMR (DMSO-d6) d 24.1, 33.0, 40.5, 44.5, 56.2,
113.0, 117.7, 129.5, 138.3, 148.2, 152.9, 155.4. MS (ESI+) m/z:
323 (100%) (M+H). Anal. Calcd for C18H22N6.0.1CH3COOC2H5: C,
66.72; H, 6.94; N, 25.37. Found: C, 66.83; H, 6.55; N, 25.08.
4.1.12. 9-(4-Fluorobenzyl)-6-[2-(N-isopropylamino)ethyl]amino-
9H-purine (17)
This compound was prepared from 14 (0.09 g, 0.34 mmol) and
N-isopropylethylenediamine (0.1 mL, 1.36 mmol) according to
the procedure described in for 16 as a cream-colored solid 17
(0.07 g, 62%): mp 90 °C. 1H NMR (DMSO-d6) d 0.81 (d, 6H), 2.54–
2.60 (m, 3H), 3.40 (br s, 2H), 5.21 (s, 2H), 7.02 (t, 2H), 7.23–7.26
(m, 2H), 7.54 (br s, 1H), 8.08 (br s, 1H), 8.12 (s, 1H). MS (ESI+) m/
z: 329 (100%) (M+H). Anal. Calcd for C17H21FN6Á0.05H2OÁ0.1CH3-
COOC2H5: C, 61.81; H, 6.53; N, 24.86. Found: C, 61.94; H, 6.17; N,
24.55.
4.1.14.4. 6-(4-Chlorobenzyl)amino-9-cyclopentyl-9H-purine
(22). The compound was prepared from 18 and 4-chloroben-
zylamine according to general procedure and was purified by col-
umn chromatography (EtOAc/hexane 2:1) to give 22 (0.13 g, 71%)
as a cream-colored solid: mp 153.5 °C. 1H NMR (DMSO-d6) d 1.65–
2.09 (m, 8H), 4.64 (br s, 2H), 4.74–4.87 (m, 1H), 7.32 (s, 4H), 8.16
(s, 1H), 8.20 (s, 1H), 8.32 (br s, 1H). MS (ESI+) m/z: 328 (100%)
(M+H). Anal. Calcd for C17H18ClN5Á0.1CH3COOC2H5: C, 62.08; H,
5.62; N, 20.80. Found: C, 62.33; H, 5.25; N, 20.58.
4.1.13. 6-Chloro-9-cyclopentyl-9H-purine (18)
To a suspension of 9 (0.4 g, 2.58 mmol) in 5 mL of DMSO was
added K2CO3 (0.53 g, 3.88 mmol). The reaction mixture was stirred
at 40 °C for 20 min. A solution of cyclopentyl bromide (0.4 mL,
3.88 mmol) in 4 mL DMF was added to the reaction mixture and
the mixture was stirred at 40 °C for 48 h. The reaction mixture
was treated with H2O and extracted with EtOAc. The extract was
dried over Na2SO4, the solvent was evaporated in vacuo, and the
residue was purified by column chromatography eluting with hex-
anes–EtOAc (10:1) to give 18 (0.32 g, 55%) as a cream-colored crys-
tal: mp 97 °C. 1H NMR (CDCl3) d 1.76–2.14 (m, 6H), 2.26–2.44 (m,
2H), 4.95–5.06 (m, 1H), 8.17 (s, 1H), 8.75 (s, 1H). MS (ESI+) m/z: 223
(100%) (M+H), 225 (34%) (M+H+2). Anal. Calcd for C10H11ClN4: C,
53.94; H, 4.98; N, 25.16. Found: C, 53.78; H, 4.70; N, 25.03.
4.1.14.5. 6-(2,4-Dichlorobenzyl)amino-9-cyclopentyl-9H-pur-
ine (23). The compound was prepared from 18 and 2,4-dichlo-
robenzylamine according to general procedure and was purified
by column chromatography (EtOAc/hexane 2:1) to give 23
(0.12 g, 74%) as a cream-colored solid: mp 135 °C. 1H NMR
(DMSO-d6) d 1.66–2.10 (m, 8H), 4.68 (br s, 2H), 4.78–4.86 (m,
1H), 7.24–7.33 (m, 2H), 7.57 (s, 1H), 8.15 (s, 1H), 8.24 (s, 1H),
8.33 (br s, 1H). MS (ESI+) m/z: 362 (100%) (M+H). Anal. Calcd
for C17H17Cl2N5: C, 56.36; H, 4.73; N, 19.33. Found: C, 56.67; H,
4.80; N, 18.95.
4.1.14.6. 6-(4-Fluorobenzyl)amino-9-cyclopentyl-9H-purine
(24). The compound was prepared from 18 and 4-fluoroben-
zylamine according to general procedure and was purified by
column chromatography (EtOAc/hexane 2:1) to give 24
(0.10 g, 71%) as a cream-colored solid: mp 143 °C. 1H NMR
(DMSO-d6) d 1.64–2.13 (m, 8H), 4.64 (br s, 2H), 4.77–4.85 (m,
1H), 7.08 (t, 2H), 7.35 (t, 2H), 8.17 (s, 1H), 8.20 (s, 1H), 8.30
(br s, 1H). 13C NMR (DMSO-d6) d 24.2, 32.6, 42.9, 56.1, 115.5,
115.7, 120.2, 129.8 (2), 137.1, 140.0, 149.5, 152.8, 154.9,
160.6, 162.9. MS (ESI+) m/z: 312 (100%) (M+H). Anal. Calcd
for C17H18FN5.0.15H2O: C, 65.01; H, 5.87; N, 22.30. Found: C,
65.34; H, 5.98; N, 21.94.
4.1.14. General procedure for the synthesis of compounds 19–
30
To
a suspension of 6-chloro-9-cyclopentyl-9H-purine (18)
(0.5 mmol) in absolute EtOH (5 mL) was added the appropriate
amine (excess) and the mixture was refluxed for 8–13 h. The reac-
tion mixture was concentrated in vacuo and the residue was puri-
fied by column chromatography.
4.1.14.1. 6-[2-(N,N-Diethylamino)ethyl]amino-9-cyclopentyl-
9H-purine (19). The compound was prepared from 18 and N,N-
diethylethylenediamine according to general procedure and was
purified by column chromatography (EtOAc/MeOH/NH3 8:2:0.2)
to give 19 (0.13 g, 95%) as a cream-colored solid: mp 155 °C. 1H
NMR (DMSO-d6) d 1.26 (t, 6H), 1.68–2.15 (m, 8H), 3.18 (q, 4H),
3.19 (t, 2H), 3.87 (br s, 2H), 4.82–4.90 (m, 1H), 7.96 (br s, 1H),
8.28 (s, 1H), 10.84 (br s, 1H). MS (ESI+) m/z: 303 (100%) (M+H).
Anal. Calcd for C16H26N6: C, 63.54; H, 8.67; N, 27.79. Found: C,
63.67; H, 8.55; N, 27.68.
4.1.14.7. 6-(2,4-Difluorobenzyl)amino-9-cyclopentyl-9H-purine
(25). The compound was prepared from 18 and 2,4-difluorob-
enzylamine according to general procedure and was purified by
column chromatography (EtOAc/hexane 3:1) to give 25 (0.11 g,
74%) as a cream-colored solid: mp 127 °C. 1H NMR (DMSO-d6)
d 1.65–2.12 (m, 8H), 4.67 (br s, 2H), 4.77–4.88 (m, 1H), 6.97 (t,
1H), 7.17 (t, 1H), 7.35 (q, 1H), 8.17 (s, 1H), 8.22 (s, 1H), 8.27
(br s, 1H). MS (ESI+) m/z: 330 (100%) (M+H). Anal. Calcd for
C17H17F2N5Á0.1H2O: C, 61.66; H, 5.23; N, 21.15. Found: C,
62.03; H, 5.13; N, 20.78.
4.1.14.2. 6-[2-(N-isopropylamino)ethyl]amino-9-cyclopentyl-
9H-purine HCl (20). The compound was prepared from 18 and
N-isopropylethylenediamine according to general procedure. The
residue was purified by column chromatography (EtOAc/MeOH/
NH3 5:2:0.4) and HCl salt was prepared with conc. HCl to give 20
(0.15 g, 93%) as a white solid: mp 233 °C. 1H NMR (DMSO-d6) d
1.13 (d, 6H), 1.50–2.12 (m, 8H), 3.09 (m, 1H), 3.86 (t, 2H), 4.21–
4.82 (m, 3H), 8.35–8.67 (m, 2H), 9.21–9.43 (m, 2H). MS (ESI+) m/
z: 289 (100%) (M+H). Anal. Calcd for C15H24N6Á3HCl: C, 45.29; H,
6.84; N, 21.13. Found: C, 45.05; H, 6.71; N, 21.01.
4.1.14.8. 6-(4-Methoxybenzyl)amino-9-cyclopentyl-9H-purine
(26). The compound was prepared from 18 and 4-methoxyben-
zylamine according to general procedure and was purified by col-
umn chromatography (EtOAc/hexane 3:1) to give 26 (0.12 g, 82%)
as a cream-colored solid: mp 138 °C. 1H NMR (CDCl3) d 1.77–1.99
(m, 6H), 2.24–2.31 (m, 2H), 3.79 (s, 3H), 4.81 (br s, 2H), 4.87–4.93
(m, 1H), 6.44 (br s, 1H), 6.85 (d, 2H, Jo = 8.4 Hz), 7.31 (d, 2H,
Jo = 8.0 Hz), 7.66 (br s, 1H), 8.41 (br s, 1H). 13C NMR (CDCl3) d

M. Tunçbilek et al. / Bioorg. Med. Chem. 17 (2009) 1693–1700
1699
23.8, 32.7, 44.0, 52.3, 55.8, 114.0, 120.0, 129.0, 130.8, 137.9,
149.4, 152.9, 154.8, 159.0. MS (ESI+) m/z: 324 (100%) (M+H). Anal.
Calcd for C18H21N5O: C, 66.85; H, 6.55; N, 21.66. Found: C, 66.59;
H, 6.78; N, 21.67.
4.2.2. Culture of microorganisms
Bacteria and fungal species used were obtained from Microbiol-
ogy Department of Ankara University Faculty of Pharmacy and Re-
fik Saydam Health Institution of Health Ministry, Ankara, Turkey,
namely S. aureus (ATCC 25923), MRSA (standart, ATCC 431300),
MRSA (clinical isolate), E. coli (ATCC 25922), B. Subtilis (ATCC
6633), C. albicans (ATCC 10145). The bacterial strains were main-
tained on MHA (Mueller–Hinton Agar) medium for 24 h at 37 °C
and fungi were maintained on SDA (Sabouraud Dextrose Agar)
for 2–5 days at 25 1 °C. The bacteria and fungi inocula were pre-
pared by suspension in 9 mL of sterile water for colonies from cul-
ture on MHA and SDA medium.
4.1.14.9. 6-(2,4-Dimethoxybenzyl)amino-9-cyclopentyl-9H-pur-
ine (27). The compound was prepared from 18 and 2,4-dime-
thoxybenzylamine according to general procedure and was
purified by column chromatography (EtOAc/hexane 3:1) to give
27 (0.07 g, 44%) as a white solid: mp 142 °C. 1H NMR (CDCl3) d
1.77–2.04 (m, 6H), 2.22–2.30 (m, 2H), 3.78 (s, 3H), 3.84 (s, 3H),
4.76 (br s, 2H), 4.87–4.92 (m, 1H), 6.19 (br s, 1H), 6.41 (d, 1H,
Jo = 8 Hz), 6.46 (s, 1H), 7.30 (d, 1H, Jo = 7.6 Hz), 7.75 (br s, 1H),
8.41 (br s, 1H). MS (ESI+) m/z: 354 (100%) (M+H). Anal. Calcd
for C19H23N5O2: C, 64.57; H, 6.56; N, 19.82. Found: C, 64.25; H,
6.31; N, 19.55.
4.2.3. Tube dilution technique
The in vitro antibacterial and antifungal activity of com-
pounds were tested by the tube dilution technique.33,34 The
tube dilution technique was followed to determine the mini-
mum inhibitory concentration (MIC) of all the synthesized com-
pounds. MHB (Mueller–Hinton Broth) was used for bacteria,
SDB (Sabouraud Dextrose Broth) was used for C. albicans. The
cell density of each inoculum was adjusted in sterile water of
0.5 Mc Farland standard. A final concentration of approximately
105 CFU/mL and 104 CFU/mL for the bacteria and fungi, respec-
tively. Microbial inocula were added to the two-fold diluted
samples. After incubation for bacteria 18–24 h at 37 1 °C and
for fungi 2–5 days 25 1 °C, the last tube with no growth of
microorganism was recorded to represent MIC value expressed
4.1.14.10. 6-(4-Methylpiperazin-1-yl)amino-9-cyclopentyl-9H-pur-
ine (28). The compound was prepared from 18 and 1-amino-4-
methylpiperazine according to general procedure and was purified
by column chromatography (CHCl3/MeOH 10:2) to give 28
(0.07 g, 52%) as
a
cream-colored solid: mp 91 °C. 1H NMR
(DMSO-d6) d 1.65–2.00 (m, 6H), 2.06–2.16 (m, 5H), 2.39 (t, 4H),
2.84 (t, 4H), 4.77–4.85 (m, 1H), 8.17 (s, 1H), 8.20 (s, 1H), 8.66 (s,
1H). 13C NMR (CDCl3) d 24.0, 32.9, 45.9, 51.0, 54.4 (2), 56.2 (2),
119.2, 136.9, 138.6, 149.9, 153.5 (2). MS (ESI+) m/z: 302 (100%)
(M+H). Anal. Calcd for C15H23N7: C, 59.78; H, 7.69; N, 32.53. Found:
C, 59.55; H, 7.32; N, 32.47.
in lg/mL.
4.1.14.11. 6-(2-Cyclohexenylethyl)amino-9-cyclopentyl-9H-purine
(29). The compound was prepared from 18 and 2-(1-cyclohexe-
nyl)ethylamine according to general procedure and was purified by
column chromatography (EtOAc/hexane 1:1) to give 29 (0.10 g,
71%) as a cream-colored solid: mp 88 °C. 1H NMR (DMSO-d6) d
1.43–2.20 (m, 18H), 3.51 (br s, 2H), 4.76–4.84 (m, 1H), 5.38 (s, 1H),
7.56 (br s, 1H), 8.16 (s, 2H). 13C NMR (DMSO-d6) d 22.6, 23.1, 24.2,
25.4, 28.5, 32.6, 38.1, 39.0, 56.0, 120.2, 122.4, 135.9, 139.6, 149.2,
152.8, 155.1. MS (ESI+) m/z: 312 (100%) (M+H). Anal. Calcd for
C18H25N5Á0.1CH3COOC2H5: C, 68.62; H, 8.14; N, 21.75. Found: C,
68.78; H, 7.81; N, 21.40.
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idine according to general procedure and was purified by column
chromatography (CHCl3/MeOH 10:1) to give 30 (0.09 g, 70%) as a
cream-colored solid: mp 79 °C. 1H NMR (DMSO-d6) d 0.87 (d,
3H), 0.98–1.12 (m, 1H), 1.60–2.16 (m, 12H), 2.99 (m, 4H), 4.78–
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and 0.19 lg/mL.

1700
M. Tunçbilek et al. / Bioorg. Med. Chem. 17 (2009) 1693–1700
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