Synthesis and biological evaluation of some 6-substituted purines

Article abstract of DOI:10.1016/j.ejmech.2006.10.014

We report herein the synthesis and the in vitro antileishmanial evaluation of a series of 6-substituted purines. The most active compounds against Leishmania amazonensis promastigotes were 6-(3′-chloropropylthio)purine 2 (D.A. Benson, I. Karsch-Mizrachi, D.J. Lipman, J. Ostell, B.A. Rapp, D.L. Wheeler, Genbank. Nucleic Acids Res. 28 (2000) 15-18; E.V. Aleksandrova, P.M.I.E. Valashek, J. Med. Pharm. Chem. 35 (2001) 172-173), 6-(3′-(thioethylamine)propylthio)purine 5, 6-(α-aceticacidthio)purine 7 and 6-(6′-deoxy-1′-O-methyl-β-d-ribofuranose)purine 14 with an IC₅₀ = 50, 50, 39 and 29 μM, respectively.

Full text of DOI:10.1016/j.ejmech.2006.10.014

European Journal of Medicinal Chemistry 42 (2007) 530e537
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of some 6-substituted purines
Fernanda Gambogi Braga a, Elaine Soares Coimbra a, Magnum de Oliveira Matos a,
Arturene Maria Lino Carmo b, Marisa Damato Cancio b, Adilson David da Silva b,
*
a Departamento de Parasitologia, Microbiologia e Imunologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Juiz de Fora,
Campus Universitario, Bairro Martelos, Juiz de Fora, Minas Gerais 36036-900, Brazil
b Departamento de Quimica, Instituto de Cieˆncias Exatas, Universidade Federal de Juiz de Fora, Campus Universitario, Bairro Martelos,
Juiz de Fora, Minas Gerais 36036-900, Brazil
Received 19 May 2006; received in revised form 22 October 2006; accepted 26 October 2006
Available online 6 December 2006
Abstract
We report herein the synthesis and the in vitro antileishmanial evaluation of a series of 6-substituted purines. The most active compounds
against Leishmania amazonensis promastigotes were 6-(30-chloropropylthio)purine 2 (D.A. Benson, I. Karsch-Mizrachi, D.J. Lipman, J. Ostell,
B.A. Rapp, D.L. Wheeler, Genbank. Nucleic Acids Res. 28 (2000) 15e18; E.V. Aleksandrova, P.M.I.E. Valashek, J. Med. Pharm. Chem. 35
(2001) 172e173), 6-(30-(thioethylamine)propylthio)purine 5, 6-(a-aceticacidthio)purine 7 and 6-(60-deoxy-10-O-methyl-b-D-ribofuranose)purine
14 with an IC50 ¼ 50, 50, 39 and 29 mM, respectively.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: 6-Substituted purines; Antileishmanial activity
1. Introduction
drugs have been developed, including various colloidal and
lipid formulations and recently the oral drug miltefosine
[3,4,6]. However, the latter drug cannot be given during preg-
nancy and shows severe gastrointestinal side effects [4]. More-
over, its cost represents another limiting factor for a general
use. Other drugs such as paromomycin, allopurinol and sita-
maquine have been reported to exhibit variable cure rates
[4]. Because of these limitations, combined therapies, includ-
ing immunomodulator drugs, could be the best approach to
avoid the emergence of drug resistance.
The inability of protozoan parasites to synthesize purines
de novo is a rational therapeutical strategy for the treatment
and prevention of parasitic disease [7]. Purine and pyrimidine
antimetabolites have been highly successful against many viral
infections as well as malignancies and show great promise
against protozoal infections as well [8]. However, many ther-
apies suffer from a lack of selectivity, leading to severe side
effects. The selectivity and efficacy of purine antimetabolites
are achieved at two levels: the cell surface transporters that
mediate access to the cell, and the enzymes of the purine met-
abolic pathways that convert the prodrug to the cytotoxic
metabolite, usually a nucleotide analogue [7]. Allopurinol,
Leishmaniasis is an infection caused by multiple species of
the protozoan parasite Leishmania. This disease is endemic in
some tropical areas of the world and in underdeveloped coun-
tries, with an estimated 1.5e2 million cases per year in 88
countries [1]. Clinical manifestations occur in three major
forms in human: cutaneous, mucocutaneous and visceral,
which are fatal if untreated [2]. Recently, visceralization by
dermotropic species of this parasite has also been reported
as a complication in HIV co-infected persons [3,4].
Chemotherapeutic treatment of leishmaniasis usually relies
on the use of pentavalent antimonials such as sodium stibo-
gluconate (pentostan) and meglumine antimoniate (glucan-
time) that induce toxic side effects together with drug
resistance [3e5]. The second line of compounds used during
the treatment of unresponsive cases generally includes pent-
amidine and amphotericin B [4]. More effective and safer
* Corresponding author. Tel.: þ55 32 32293310; fax: þ55 32 32293314.
E-mail address: david.silva@ufjf.edu.br (A.D. da Silva).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.10.014

F.G. Braga et al. / European Journal of Medicinal Chemistry 42 (2007) 530e537
531
a purine nucleobase analogue, is chemically used against var-
ious manifestations of leishmaniasis [4]. In a previous study,
we have shown that 6-thiopurine derivatives have proved in-
hibitory effects in Leishmania [9,10]. So, the aim of this study
was to evaluate in vitro several 6-thiopurine derivatives against
different species of Leishmania.
bottomed 96-well plastic tissue-cultured plates maintained at
24 ꢀC. Promastigote forms from a logarithmic phase culture
were suspended to yield 2 millions of cells/mL (L. amazonen-
sis) or 3 millions of cells/mL (L. chagasi) after Newbauer hae-
mocytometer counting. Each well was filled with 100 ml of the
parasites suspension, and the plates were incubated at 24 ꢀC
for 1 h before drug addition. The compounds to be tested
were dissolved in water or DMSO and added to each well.
Up to 1% (v/v), DMSO had no effect on parasite’s growth.
Each concentration was screened in triplicate. The viability
of promastigotes of both the species was assessed by MTT
colorimetric method. The results are expressed as the concen-
trations inhibiting parasite growth by 50% (IC50) after a three-
day incubation period. Amphotericin B was used as the
reference drug and IC50 values were of 0.9 mM and 1.9 mM on
L. amazonensis and L. chagasi promastigote forms, respectively.
2. Chemistry
A series of 6-substituted purines were synthesized. As
shown in Table 1, the analogs varied with the nature of the
substituent attached to the sulfur atom. The compounds 3, 4,
5, 8, 13, 14, 18 and 22 are novel.
The compounds 2 [11,12], 6, 8 and 9 [13] were synthesized
according to the following reaction condition.
A solution of sodium 6-thiopurine, generated from the reac-
tion of 6-thiopurine with NaH in DMF at 0 ꢀC for 30 min and
at rt for over 1 h, was transferred to another flask containing
a solution of the alkylhalide in DMF at 0 ꢀC for 30 min. The
reaction mixture was stirred for an additional 48 h at 25 ꢀC.
The solvent was evaporated in vacuum until dryness. The
crude reaction product was purified by flash chromatography.
Compounds 3, 4 and 5 were obtained by the treatment of 6-(30-
chloropropylthio)purine 2 with cyclohexilamine, ethylenedi-
amine and thioethylamine, respectively, at 25 ꢀC for 48 h.
Compound 7 was obtained by treatment of 6 with KOH
(1 M) in methanol and H2O for 24 h at 25 ꢀC.
3.2. Cytotoxicity
For cytotoxicity against mammalian cells, adherent mouse
peritoneal macrophages were cultured for 48 h at 37 ꢀC with
varying concentration of the 6-substituted purines. The viabil-
ity of the macrophages was determined with the MTT assay, as
described above, and was confirmed by comparing the mor-
phology of the control group via light microscopy.
4. Biological results and discussion
The synthesis of 6-(50-deoxy-10-O-methyl-b-D-ribofuranose)
purine 14, 6-(60-deoxy-10,20,30,40-diisopropylidene-a-D-galacto-
pyranose)purine 18 and 6-(10-deoxy-20,30,40,50-diisopropyli-
dene-b-D-psicopyranose)purine 22 was achieved with a versatile
and efficient synthetic route outlined in Schemes 1e3, respec-
tively. Subsequent acid deprotection of the isopropylidene
group of 18 and 22 did not afforded the compounds hydrolyzed.
All products were purified by flash chromatography and the
reaction yields were generally between 50 and 90%. All com-
pounds were demonstrated to be of sufficient purity for use in
biological assays (>95%) by 13C nuclear magnetic resonance
(13C NMR).
In this report it was evaluated that the effect of 6-
substituted purines against two different species of Leishmania
from the New World: L. amazonensis which has been associ-
ated with all clinical forms of leishmaniasis [2] and L. chagasi
which is the causal agent of visceral disease [2], as reported in
Table 1. Based on this fact it was expected to have different
sensitivity of these parasites to the assayed drugs. Indeed, in
a general point of view, this difference was observed and the
results demonstrated that the compounds tested showed activ-
ity only against promastigotes of L. amazonensis. Among the
13 tested compounds 2, 5, 7 and 14 showed an activity against
L. amazonensis (IC50 values of 50, 50, 39 and 29 mM, respec-
tively) promastigote forms. In our assay, only those com-
pounds presenting activity against promastigotes of L.
amazonensis were further evaluated for cytotoxicity of mam-
malian cells. Interestingly, none of the compounds were found
to have significative toxicity towards mammalian cells at the
maximal concentration used (227 mM), data not shown.
The differences in sensibility between several species of
Leishmania accentuate the importance of speciation in the
treatment of leishmaniasis. Only American cutaneous leishma-
niais is associated to least 14 species of Leishmania patho-
genic to human with biochemical and molecular variation
[15].
3. Biological evaluation
3.1. In vitro antileishmanial activity
L. amazonensis (MHOM/Br/75/Josefa isolated from a pa-
tient with diffuse cutaneous leishmaniasis) and L. chagasi
(MHOM/Br/74/PP75 isolated from a patient with visceral
leishmaniasis) promastigotes were used for in vitro screening.
The antileishmanial activity was determined by colorimetric
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bro-
mide (MTT) method based on tetrazolium salt reduction by
mitochondrial dehydrogenases [14]. Briefly, promastigotes of
L. amazonensis were cultured in the Warren’s medium (brain
heart infusion plus hemin and folic acid) and promastigotes
of L. chagasi were maintained in Medium 199 (Sigma Chem-
ical Co., St Louis, MO), both supplemented with 10% fetal
bovine serum at 24 ꢀC. The screening was performed in flat-
The identification of fundamental biochemical discrep-
ancies between a parasite and its mammalian hosts offers
a promising strategy for therapeutic exploitation of parasitic
diseases. One striking metabolic disparity between protozoan
parasites and their mammalian hosts is the way that purine

532
F.G. Braga et al. / European Journal of Medicinal Chemistry 42 (2007) 530e537
Table 1
Structures and antiproliferative activities of 6-thiopurine and 6-substituted purines against L. amazonensis and L. chagasi in vitro
R
S
6
7
5
N
1
N
8
2
N
H
4
9
N
3
Compound
R
H
IC50 against L. amazonensis (mM)
>227
IC50 against L. chagasi (mM)
>227
1
>227
2'
2
3
4
50
>227
>227
1'
3'
Cl
NH
>227
>227
7'
1'
3'
2'
4'
5' 6'
NH2
2'
5'
1'
4'
3'
N
H
NH
2
2'
5'
5
6
50
>227
>227
1'
1'
3'
4'
3'
S
O
>227
2'
O
O
H
1'
7
8
39
>227
>227
O
O
O
6'
7'
5'
3'
1'
N
>227
2'
4'
2'
3'
1'
9
N
>227
>227
OCH
3
5'
O
1'
4'
13
>227
>227
2'
H
3'
H
O
O
OCH
H
3
O
14
29
>227
H
OH
HO

F.G. Braga et al. / European Journal of Medicinal Chemistry 42 (2007) 530e537
IC50 against L. amazonensis (mM)
533
Table 1 (continued)
Compound
R
IC50 against L. chagasi (mM)
O
6'
O
4'
5'
18
>227
>227
O
1'
2'
O
3'
O
O
6' 6''
2'
O
1' 1''
22
>227
>227
O
4'
5'
3'
O
O
Amphotericin B
0.9
1.9
nucleotides are synthesized [7]. Whereas mammalian cells can
synthesize the purine ring from amino acids and other small
molecules, all protozoan parasites studied to date are incapa-
ble of synthesizing purines de novo. Nucleoside transporter
has been investigated in several parasitic protozoa, for exam-
ple, Trypanosoma brucei and Leishmania species [7]. In this
parasite it was reported in promastigote forms of L. brazilien-
sis, L. major and L. donovani [7,16,17]. Recently, were re-
ported the first identification and characterization of a purine
nucleobase transport in L. mexicana amastigotes [8].
From our biological results, it is evident that 6-substituted pu-
rines exhibited potent antileishmanial activity. The overall ac-
tivity profile of compounds 2, 5, 7 and 14 demonstrated that
there is a small difference in their IC50 values on L. amazonensis.
In summary, we have demonstrated the activity of
6-substituted purines in vitro for both L. amazonensis and
L. chagasi promastigotes and the 6-(60-deoxy-10-O-methyl-b-
D-ribofuranose)purine 14 was evidently the more active
compound. Knowledge of the sensitivity of each species has
important implications for clinical treatment.
I
HO
HO
OCH3
OH
OCH3
5
O
O
O
1
4
imidazol, PPh3 , I2
HCl, CH3OH
2
3
Tolueno, reflux,
24 h, 80%
r.t. , 24 h,
86%
O
O
O
O
O
O
12
10
11
3
N
H
N
N
H
9
N
4
2
8
N
N
N
1
5
N
7
6
S
S
OCH3
5'
OCH3
1'
O
O
HCl, MeOH-H2O
24 h, 0°C, 90%
6-thiopurine, NaH,
4'
DMF, 120°C,
72 h, 85%
2'
O
3'
OH
HO
O
14
13
Scheme 1. Synthesis of the 6-(60-deoxy-10-O-methyl-b-D-ribofuranose)purine (14).

534
F.G. Braga et al. / European Journal of Medicinal Chemistry 42 (2007) 530e537
OH
OH
I
OH
O
O
6
O
O
O
O
O
acetone
5
4
imidazol, PPh3, I2
dimetoxipropano
H2SO4
O
HO
O
Tolueno, reflux
24 h, 80%
1
2
OH
3
OH
O
24 h, 92%
O
17
16
15
3
H
N
N
4
9
2
1
8
N
N
5
7
6
S
O
6-thiopurine, NaH, DMF
6'
O
O
5'
25°C, 5 days,
50%
4'
O
1'
2'
3'
18
O
Scheme 2. Synthesis of the 6-(60-deoxy-10,20,30,40-diisopropylidene-a-D-galactopyranose)purine (18).
1
5. Experimental protocols
disks). All structures were confirmed by 13C NMR and H
NMR. Spectra were recorded at 300 MHz with Bruker spec-
trometer, using tetramethylsilane (TMS) as an internal stan-
dard. Elemental analyses were carried out on a CHN-O
rapid elemental analyzer (GmbH-Germany) for C, H and N,
and the results are within ꢁ0.4% of the theoretical values.
Electron impact mass spectra were measured with an AEI
5.1. Chemistry
Melting points were determined on a Kofler hot stage appa-
ratus and are uncorrected. The IR spectra were obtained on
a Shimadzu 470 spectrophotometer (potassium bromide
O
O
OH
OH
I
OH
6
2
O
O
O
1
acetone
O
imidazol, PPh3 , I2
O
OH
4
5
3
dimetoxipropano
H2SO4
Tolueno, reflux
24 h, 50%
O
O
OH
O
O
OH
3 h, 86%
21
20
19
3
H
N
N
9
4
5
2
1
8
N
N
7
O
2'
6
6'
S
O
6-thiopurine, NaH, DMF
1'
O
5'
4'
O
90°C, 24 h,
50%
3'
O
22
Scheme 3. Synthesis of the 6-(10-deoxy-20,30,40,50-diisopropylidene-b-D-psicopyranose)purine (22).

F.G. Braga et al. / European Journal of Medicinal Chemistry 42 (2007) 530e537
535
MS 50 mass spectrometer. Merck silica gel 60 F254 plates were
used for analytical TLC; column chromatography was per-
formed on Merck silica gel (70e230 mesh).
Compounds 2 [11,12], 6 [13], 7 [13] and 9 [13] have been
described in the literature.
5.1.4. 6-(30-(Thioethylamine)propylthio)purine (5)
1-Bromo-3-chloropropane (100 mg, 0.44 mmol) was dis-
solved in DMF (4 mL) and then added to a solution of sodium
thioethylamine, generated from the reaction of thioethylamine
(150 mg, 1.3 mmol) with NaH (79 mg, 3.3 mmol) in DMF
(6 mL) at 25 ꢀC for 48 h.
The solvent was evaporated in vacuum until dryness. The
crude reaction product was purified by flash chromatography
5.1.1. 6-(30-Chloropropylthio)purine (2)
1-Bromo-3-chloropropane (1 g, 6.35 mmol) was dissolved
in EtOH at 0 ꢀC and then added to a solution of sodium 6-thi-
opurine, generated from the reaction of 6-thiopurine (1 g,
6.62 mmol) with NaH (0.15 g, 6.25 mmol) in EtOH (20 mL)
at 0 ꢀC for 30 min and at rt for over 1 h.
(eluent: CH2Cl2:MeOH 8:2) producing
5
(67 mg,
0.22 mmol) in 50% yield as a white solid, m.p. 270 ꢀC; nmax
1
(KBr): 3450 (NH2), 1646 cmꢂ1 (NH2); H NMR (300 MHz,
CDCl3): d 8.52 (s, 1H, H-2), 8.40 (s, 1H, H-8), 5.42 (s, 2H,
0
0
0
0
NH2), 4.50 (t, 2H, J5 ,4 ¼ 6.8 Hz, H-5 ), 3.30 (m, 6H, H-1 ,
H-30, H-40), 2.55 (q, 2H, H-20) (Found: C, 44.32; H, 5.68; N,
25.91. C10H15N5S2 requires C, 44.58; H, 5.61; N, 26.00).
The reaction mixture was stirred for an additional 48 h at
25 ꢀC. The solvent was evaporated in vacuum until dryness.
The crude reaction product was purified by flash chromatogra-
phy (eluent: AcOEt:hexane 4:6) producing 2 (900 mg,
3.94 mmol) in 59% yield as a white solid, m.p. 200 ꢀC; nmax
5.1.5. 6-(a-Ethylacetatethio)purine (6)
The ethyl 2-bromoacetate (1.5 g, 8.98 mmol) was dissolved
in DMF (10 mL) at 0 ꢀC and then added to a solution of so-
dium 6-thiopurine, generated from the reaction of 6-thiopurine
(1.37 g, 9 mmol) with NaH (237 mg, 9.9 mmol) in DMF
(10 mL) at 0 ꢀC for 30 min and at rt for over 1 h.
The reaction mixture was stirred for an additional 48 h at
25 ꢀC. The solvent was evaporated in vacuum until dryness.
The crude reaction product was purified by flash chromatogra-
1
(KBr): 3472 (NH), 2975 (CH), 570 cmꢂ1 (CCl); H NMR
(300 MHz, CDCl3): d 8.45 (s, 1H, H-2), 7.96 (s, 1H, H-8),
0
0
0
0
0
3.76 (t, 2H, J3 ,2 ¼ 6 Hz, H-3 ), 3.44 (t, 2H, J1 ,2 ¼ 6 Hz, H-
10), 2.17 (quint, 2H, H-20); MS (EI) m/z: 229 [M þ 1]þ; 193
[M ꢂ 35]þ; 152 þ 1 [M ꢂ 71]þ; 13C NMR (75 MHz, DMSO)
d ppm: 161 (C-2), 144 (C-8), 50 (C-30), 26 (C-10), 22 (C-20).
phy (eluent: AcOEt:hexane 7:3) producing
6 (1.37 g,
5.1.2. 6-(30-(N-Cyclohexanamine)propylthio)purine (3)
Cyclohexilamine (44 mg, 0.44 mmol) was dissolved in
DMF and then added to a solution of 6-(30-chloropropylthio)-
purine 2 (50 mg, 0.22 mmol) in DMF (3 mL).
5.7 mmol) in 64% yield as a white solid, m.p. 200 ꢀC; nmax
(KBr): 3055 (CH), 1729 (CO), 1308 (CO), 857 cmꢂ1 (CH);
1H NMR (300 MHz, CDCl3): d 8.70 (s, 1H, H-2), 8.20 (s,
0
0
0
1H, H-8), 4.32 (quart, 2H, J2 ,3 ¼ 7 Hz, H-2 ), 4.21 (s, 2H,
H-10), 1.31 (t, 3H, H-30); 13C NMR (75 MHz, CDCl3) d: 170
(C]O), 152 (C-ꢂ2), 143 (C-8), 63 (C-20), 32 (C-10), 15 (C-
30); MS TOF ES : m/z: 237 [M ꢂ 1], 192 [C7H5ON4S], 165
[C6H5N4S], 163 [C6H3N4S].
The reaction mixture was stirred for an additional 48 h at
25 ꢀC. The solvent was evaporated in vacuum until dryness.
The crude reaction product was purified by flash chromatogra-
phy (eluent: CH2Cl2:MeOH 8:2) producing 3 (36 mg,
0.12 mmol) in 56% yield as a white solid, m.p. 250 ꢀC; nmax
1
(KBr): 3450 (NH2), 1645 cmꢂ1 (NH2); H NMR (300 MHz,
5.1.6. 6-(a-Aceticacidthio)purine (7)
CDCl3): d 8.06 (s, 1H, H-2), 7.83 (s, 1H, H-8), 4.2 (s, 1H,
Compound 7 was obtained by treatment of 6 (1 g,
4.2 mmol) with KOH (10 mL, 1 M) in methanol (20 mL)
and H2O (14 mL) for 24 h at 25 ꢀC.
NH), 3.30 (m, 2H, H-10), 2.69 (t, 2H, J1 ,2 ¼ 7.2 Hz, H-1 ),
2.01 (m, 2H, H-30), 1.89e1.40 (m, 9H, H-20, H-40, H-50, H-
60, H-70) (Found: C, 57.58; H, 7.24; N, 24.10. C14H21N5S re-
quires C, 57.70; H, 7.26; N, 24.03).
0
0
0
The reaction was neutralized by 1 N HCl. The solvent was
evaporated in vacuum until dryness. The crude reaction prod-
uct was recrystallized in acetone. TLC (eluent: CH2Cl2:MeOH
8:2) producing 7 (0.8 g, 3.8 mmol) in 91% yield as a colorless
1
5.1.3. 6-(30-(N-Ethylenediamine)propylthio)purine (4)
oil; nmax (KBr): 3334 (OH), 1629 (CO), 979 cmꢂ1 (OH); H
Compound 4 was obtained by the treatment of 6-(30-chlor-
opropylthio)purine 2 (0.2 g, 0.87 mmol) with ethylenediamine
(0.26 g, 4.4 mmol) in DMF (8 mL) at 25 ꢀC for 48 h.
The solvent was evaporated in vacuum until dryness. The
crude reaction product was purified by flash chromatography
(eluent: CH2Cl2:MeOH 8:2) producing 4 (110 mg, 17 mmol)
in 50% yield as a white solid, m.p. 280 ꢀC; nmax (KBr):
3460 (NH2), 1650 cmꢂ1 (NH2); 1H NMR (300 MHz,
NMR (300 MHz, D2O): d 8.50 (s, 1H, H-2), 8.30 (s, 1H, H-
8), 4.03 (s, 2H, H-10); 13C NMR (75 MHz, D2O) d: 154 (C-
2); 146 (C-8); 37 (C-10); MS TOF MS ESꢂ: m/z: 209
[M ꢂ 1], 165 [C6H5N4S], 150 [C5H3N4S].
5.1.7. 6-(40-(Isoindoline-10,30-dione)butylthio)purine (8)
2-(4-Bromobutyl)isoindoline-1,3-dione (142 mg, 0.5 mmol)
was dissolved in EtOH and then added to a solution of sodium 6-
thiopurine, generated from the reaction of 6-thiopurine (50 mg,
0.33 mmol) with NaH (11 mg, 0.46 mmol) in EtOH (6 mL) at
0 ꢀC for 30 min and at rt for over 1 h. The reaction mixture
was stirred for an additional 48 h at 25 ꢀC. Thesolventwas evap-
orated in vacuum until dryness. The crude reaction product was
CDCl3): d 8.20 (s, 1H, H-2), 8.03 (s,01H, H-8), 7.24 (s, 1H,
0
0
0
NH), 4.58 (t, 2H, J5 ,4 ¼ 6 Hz, H-5 ), 4.04 (t, 2H, H-4 ),
3.33e3.15 (m, 4H, H-10, H-30), 2.50 (quint, 2H, H-20), 1.98
(s, 2H, NH2) (Found: C, 47.32; H, 6.38; N, 33.66.
C10H16N6S requires C, 47.60; H, 6.39; N, 33.30).

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F.G. Braga et al. / European Journal of Medicinal Chemistry 42 (2007) 530e537
purified by flash chromatography (eluent: AcOEt) producing 8
(58 mg, 0.14 mmol) in 50% yield as a white solid, m.p.
The reaction mixture was stirred for an additional 24 h at
0 ꢀC. The reaction was neutralized with NaHCO3 and the sol-
vent was evaporated in vacuum until dryness. The crude reac-
tion product was purified by flash chromatography (eluent:
CH2Cl2:MeOH 7:3) producing 14 (400 mg, 1.34 mmol) in
90% yield as a colorless oil; nmax (KBr): 3277 (OH), 2916
1
194 ꢀC; nmax (KBr): 3456 (NH2), 1641 cmꢂ1 (NH2); H NMR
(300 MHz, CDCl3): d 8.45 (s, 1H, H-2), 7.96 (s, 1H, H-8),
0
0
7.82 (s, 4H, H-60, H-70), 3.61 (t, 2H, J4 ,3 ¼ 6.6 Hz, H-4 ),
0
0
0
0
0
0
3.36 (t, 2H, J1 ,2 ¼ 6.8 Hz, H-1 ), 1.74 (m, 4H, H-2 , H-3 );
13C NMR (75 MHz, DMSO) d ppm: 167 (C]O), 151 (C-2),
134 (C-60), 131 (C-8), 123 (C-70), 36.9 (C-40), 27.3, 27.0, 26.5
(C-10, C-20, C-30) (Found: C, 57.62; H, 4.38; N, 19.76.
C17H15N5O2S requires C, 57.78; H, 4.28; N, 19.82).
1
(CH), 1094 cmꢂ1 (CeO); H NMR (300 MHz, D2O): d 8.35
(s, 1H, H-2), 8.22 (s, 1H, H-8), 4.91 (s, 1H, H-10), 04.34 (2d,
2H, H-20, H-30, J2 ,3 ¼ 5.8 Hz), 4.15 (m, 1H, H-4 ), 3.83e
0
0
3.45 (2dd, 2H, H-50, H-500, J5 ,4 ¼ 2.3 Hz, J5 ,4 ¼ 3.6 Hz,
0
0
00
0
J5 ,5 ¼ 12.3 Hz), 3.34 (s, 3H, OCH3); 13C NMR (75 MHz,
D2O) d ppm: 168 (C-5), 152 (C-2), 144 (C-8), 108 (C-10),
81 (C-30), 75 (C-20), 74 (C-40), 55 (OCH3), 32 (C-50) (Found:
C, 44.18; H, 4.69; N, 18.56. C11H14N4O4S requires C, 44.29;
H, 4.73; N, 18.78).
0
00
5.1.8. 6-((Pyridin-40-yl)methylthio)purine (9)
The 4-(bromomethyl)pyridine$HBr (132 mg, 0.152 mmol)
was dissolved in EtOH and neutralized in NaHCO3
(17.4 mg, 0.208 mmol) and then added to a solution of sodium
6-thiopurine, generated from the reaction of 6-thiopurine
(50 mg, 0.22 mmol) with NaH (5.5 mg, 0.23 mmol) in EtOH
(6 mL) at 0 ꢀC for 30 min and at rt for over 1 h. The reaction
mixture was stirred for an additional 48 h at 25 ꢀC. The sol-
vent was evaporated in vacuum until dryness. The crude reac-
tion product was purified by flash chromatography (eluent:
CH2Cl2:MeOH 10:2) producing 9 (50 mg, 0.2 mmol) in 62%
yield as a white solid, m.p. 230 ꢀC; nmax (KBr): 3450 (NH2),
5.1.11. 6-(60-Deoxy-10,20,30,40-diisopropylidene-
a-D-galactopyranose)purine (18)
6-Deoxy-6-iodo-1,2,3,4-diisopropylidene-a-D-galactopyra-
nose 17 [19] (6 g, 16.2 mmol) was dissolved in DMF (30 mL)
at 0 ꢀC and then added to a solution of sodium 6-thiopurine,
generated from the reaction of 6-thiopurine (2 g,
13.16 mmol) with NaH (0.4 g, 16.67 mmol) in DMF
(10 mL) at 0 ꢀC for 30 min and at rt for over 1 h.
The reaction mixture was stirred for an additional 5 days at
25 ꢀC. The solvent was evaporated in vacuum until dryness.
The crude reaction product was purified by flash chromatogra-
phy (eluent: AcOEt:hexane 2:1) producing 18 (2.5 g,
6.6 mmol) in 50% yield as a white solid, m.p. 136 ꢀC; nmax
(KBr): 3438 (NH), 1589 (C]C), 1113 cmꢂ1 (CeO); 1H NMR
1
1644 cmꢂ1 (NH2); H NMR (300 MHz, DMSO): d 8.71 (s,
0
0
0
1H, H-2), 8.48 (d, 2H, J2 ,3 ¼ 5.6 Hz, H-3 ), 8.46 (s, 1H, H-
8), 7.45 (d, 2H, H-20), 4.65 (s, 2H, H-10); 13C NMR
(75 MHz, DMSO) d ppm: 151 (C-2), 149 (C-30), 147 (Cq),
143 (C-8), 124 (C-20), 30.3 (C-10).
5.1.9. 6-(50-Deoxy-10-O-methyl-20,30-O-isopropylidene-
b-D-ribofuranose)purine (13)
(300 MHz, CDCl3): d 8.75 (s, 1H, H-2), 8.15 (s, 1H, H-8), 5.5
0
5-Desoxy-5-iodo-1-O-methyl-2,3-O-isopropylidene-b-D-ri-
bofuranose 12 [18] (1.2 g, 3.8 mmol) was dissolved in DMF
(10 mL) at 0 ꢀC and then added to a solution of sodium 6-thi-
opurine, generated from the reaction of 6-thiopurine (0.6 g,
3.9 mmol) with NaH (0.13 g, 5.4 mmol) in DMF (10 mL) at
0 ꢀC for 30 min and at rt for over 1 h.
The reaction mixture was stirred for an additional 72 h at
120 ꢀC. The solvent was evaporated in vacuum until dryness.
The crude reaction product was purified by flash chromatogra-
phy (eluent: AcOEt:hexane 1:1) producing 13 (1.1 g,
3.25 mmol) in 85% yield as a colorless oil; nmax (KBr):
2987 (CH), 1104 cmꢂ1 (CeO); 1H NMR (300 MHz,
(d, 1H, H-10, J1 ,2 ¼ 4.8 Hz), 4.6 (dd, 1H, H-3 , J3 ,4 ¼ 8.1 Hz,
0
0
0
0
0
0
0
0
J3 ,2 ¼ 2.4 Hz), 4.4 (dd, 1H, H-4 ), 4.29e4.25 (dd, 1H, H-2 ),
4.25e4.20 (dd, 1H, H-50, J5 ,6 ¼ J5 ,6 ¼ 6.6 Hz), 3.55e3.42
0
0
0
00
ꢂ
(m, 2H, H-60, H-600, J6 ,6 ¼ 13.4 Hz); MS TOF ES (MeOH)
m/z: 394 [M], 393 [M ꢂ 1], 165 [C6H5N4S], 151 [C5H3N4S],
MS TOF ESþ m/z: 395 [M þ 1], 279 [C12H20O5S]; 13C NMR
(75 MHz, CDCl3) d ppm: 152.5 (C-2), 132 (C-8), 129 (C-60),
110 (2Cq), 97 (C-10), 72 (C-20), 71 (C-40), 70 (C-30), 67 (C-50),
27e24 (4CH3) (Found: C, 51.61; H, 5.57; N, 14.02.
C17H22N4O5S requires C, 51.76; H, 5.62; N, 14.20).
0
00
5.1.12. 1-Deoxy-1-iodo-2,3,4,5-diisopropylidene-
b-D-psicopyranose (21)
CDCl3): d 8.85 (s, 1H, H-2), 8.40 (s, 1H, H-8), 5.14 (s, 1H,
0
H-10), 4.9 (d, 1H, H-20, J2 ,3 ¼ 5.9 Hz), 4.8 (d, 1H, H-3 ),
0
0
2,3,4,5-Diisopropylidene-b-D-psicopyranose 20 [20] (10 g,
40.3 mmol) was dissolved in toluene (200 mL) at rt and then
added iodide (13 g, 51.2 mmol), imidazole (3.5 g,
51.4 mmol) and triphenylphosphine (13.7 g, 52.2 mmol) at rt
for 30 min.
The reaction mixture was stirred for an additional 24 h at
110 ꢀC. The solvent was evaporated in vacuum until dryness.
The crude reaction product was purified by flash chromatogra-
phy (eluent: AcOEt:hexane 2:8) producing 21 (5.5 g,
15.36 mmol) in 50% yield as a colorless oil; nmax (KBr):
2988 (CeH), 1250 (CeO), 535 cmꢂ1 (CeI); 1H NMR
(300 MHz, CDCl3): d 4.48e4.54 (dd, 1H, H-4, J4,3 ¼ 2.7 Hz,
J4,5 ¼ 7.9 Hz), 4.3 (d, 1H, H-3, J3,4 ¼ 2.7 Hz), 4.23e4.19
4.65 (m, 1H, H-40), 3.8e3.6 (2dd, 2H, H-50, H-500,
0
0
00
0
0
00
J5 ,4 ¼ 2.3 Hz, J5 ,4 ¼ 3.6 Hz, J5 ,5 ¼ 12.3 Hz), 3.5 (s, 3H,
OCH3), 1.6e1.3 (2s, 6H, 2CH3); MS (IE) m/z: 229
[M þ 1]þ, 193 [M ꢂ 35]þ, 152 þ 1 [M ꢂ 71]þ; 13C NMR
(75 MHz, CDCl3) d ppm: 160 (C-5), 152 (C-2), 149 (C-8),
142 (C-6), 131 (Cq), 113 (C-10), 110 (C-30), 86 (C-20), 84
(C-40), 56 (OCH3), 32 (C-50), 27e24 (2CH3).
5.1.10. 6-(50-Deoxy-10-O-methyl-b-D-ribofuranose)purine (14)
6-(50-Deoxy-10-O-methyl-20,30-O-isopropylidene-b-D-ribo-
furanose)purine 13 (0.5 g, 1.48 mmol) was dissolved in MeOH
(20 mL) at 0 ꢀC and then added to a solution of HCl (1 N, 2 mL)
in H2O (2 mL) at 0 ꢀC for 30 min and at rt for over 12 h.

F.G. Braga et al. / European Journal of Medicinal Chemistry 42 (2007) 530e537
537
0
(dd, 1H, H-5, J5,4 ¼ 7.9 Hz, J5,6 ¼ J5,6 ¼ 10 Hz), 3.90e3.72
Amphotericin B, respectively. Jean Louis-Fourrey of the
ICSN/CNRS for his assistance with mass spectrometry.
(2dd, 2H, H-6, H-60, J6,6 ¼ 12.9 Hz, J6,5 ¼ J6 ,5 ¼ 1.8 Hz),
0
0
3.55e3.30 (2d, 2H, H-1, H-10, J1,1 ¼ 10 Hz), 1.5e1.3 (4s,
0
12H, 4CH3); 13C NMR (75 MHz, CDCl3) d ppm: 109 (C-2),
108e107 (2Cq), 100 (C-3), 72 (C-5), 70.5 (C-4), 62 (C-6),
27e24 (4CH3), 10 (C-1).
References
[1] WHO/TDR, The leishmaniasis and Leishmania/HIV co-infection, WHO
Factsheet no. 116, Geneva, 2000.
5.1.13. 6-(10-Deoxy-20,30,40,50-diisopropylidene-
b-D-psicopyranose)purine (22)
[2] G. Grimaldi Jr., R.B. Tesh, Clin. Microbiol. Rev. 6 (1993) 230e250.
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1-Deoxy-1-iodo-2,3,4,5-diisopropylidene-b-D-psicopyra-
nose 21 (4.5 g, 12.56 mmol) was dissolved in DMF (10 mL) at
0 ꢀC and then added to a solution of sodium 6-thiopurine, gen-
erated from the reaction of 6-thiopurine (1.81 g, 11.9 mmol)
with NaH (0.3 g, 12.5 mmol) in DMF (10 mL) at 0 ꢀC for
30 min and at rt for over 1 h.
A. Foroumadi, S. Emami, S. Pournourmohammadi, A. Kharazmi,
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The reaction mixture was stirred for an additional 24 h at
90 ꢀC. The solvent was evaporated in vacuum until dryness.
The crude reaction product was purified by flash chromatogra-
phy (eluent: AcOEt:hexane 7:3) producing 22 (2.4 g,
6.3 mmol) in 50% yield as a colorless oil; nmax (KBr): 3444
[8] M.I. Al-Salabi, H.P. Koning, Antimicrob. Agents Chemother. 49 (2005)
3682e3689.
ˆ
[9] A.D. Silva, A.S. Machado, C. Tempete, M. Robert-Gero, Eur. J. Med.
Chem. 29 (1994) 149e152.
(NH), 2974 (CH), 1570 (C]C), 1061 cmꢂ1 (CeO); H NMR
1
[10] P. Escobar, S. Matu, C. Marques, S.L. Croft, Acta Trop. 81 (2002) 151e157.
[11] D.A. Benson, I. Karsch-Mizrachi, D.J. Lipman, J. Ostell, B.A. Rapp,
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172e173.
(300 MHz, CDCl3) d: 8.7 (s, 1H, H-2), 8.2 (s, 1H, H-8), 4.56e
4.50 (dd, 1H, H-40, J4 ,3 ¼ 2.4 Hz, J4 ,5 ¼ 7.7 Hz), 4.34 (d,
0
0
0
0
1H, H-30), 4.22e4.16 (dd, 1H, H-50), 4.06e3.87 (2dd, 2H, H-
60, H-600, J6 ,6 ¼ 13.8 Hz), 3.91e3.70 (m, 2H, H-1 , H-1 ,
0
00
[13] P.B. Gordon, P.O. Seglen, Arch. Biochem. Biophys. 217 (1982) 282e
294;
00
0
0
00
J1 ,1 ¼ 13.1 Hz), 1.58e1.25 (4s, 12H, 4CH3); MS (MeOH)
TOF ESþ m/z: 395 [M þ 1]; 13C NMR (75 MHz, CDCl3)
d ppm: 151.5 (C-2), 141.5 (C-8), 109.5 (C-20), 109e103
(2Cq), 73 (C-50), 70.5 (C-40), 61 (C-60), 36 (C-30), 27e25
(4CH3), 24 (C-10) (Found: C, 50.08; H, 5.78; N, 14.39.
C16H22N4O5S requires C, 50.25; H, 5.80; N, 14.65).
J. Klosa, H. Kroeger, C. Meishsner, I. Winkler, M. Helsberg, E. Schrin-
ner, Eur. Pat. Appl. 1990;
L.R. Lewis, C.W. Noel, A.G. Beaman, R.K. Robins, J. Med. Pharm.
Chem. 5 (1962) 607e617.
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[15] F.T. Silveira, R. Lainson, C.E.P. Corbett, Mem. Inst. Oswaldo Cruz 99
(2004) 239e251.
Acknowledgments
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814e820.
[17] J.M. Boitz, B. Ullman, J. Biol. Chem. 281 (2006) 16084e16089.
[18] L.M. Lerner, Carbohydr. Res. 53 (1977) 177e185.
[19] J.R. Sufrin, R.J. Bernack, M.J. Morin, W. Korytnyk, J. Med. Chem. 23
(1980) 143e149.
This work was supported by FAPEMIG, CAPES and BIC/
UFJF. We are grateful to the Empresa Microbiologica Quimica
e Farmaceutica Ltda and Hospital da Universidade Federal de
Juiz de Fora for the provision of 6-mercaptopurine and
[20] S. Morgenlie, Carbohydr. Res. 107 (1982) 137e141.