Synthesis and in vitro antileishmanial activity of 5-substituted-2′- deoxyuridine derivatives

Article abstract of DOI:10.1016/j.bioorg.2005.07.001

We report herein the synthesis and the in vitro antileishmanial evaluation of 5-substituted-2′-deoxyuridine nucleosides. The most active compound against Leishmania donovani promastigotes was Thia-dU (3a) with an IC ₅₀ = 3 μM. This compound exh

Full text of DOI:10.1016/j.bioorg.2005.07.001

Bioorganic Chemistry 33 (2005) 439–447
www.elsevier.com/locate/bioorg
Synthesis and in vitro antileishmanial activity
of 5-substituted-2Ј-deoxyuridine derivatives
Corinne Peyron a, Rachid Benhida a,¤, Christian Bories b,
Philippe M. Loiseau b,¤
a Laboratoire de Chimie Bioorganique UMR-CNRS 6001, Université de Nice-Sophia Antipolis,
Parc Valrose F-06108, Nice Cédex 2, France
b Chimiothérapie Antiparasitaire, UMR-CNRS 8076, Université Paris-Sud,
92290 Châtenay Malabry F-92290, France
Received 13 June 2005
Available online 15 September 2005
Abstract
We report herein the synthesis and the in vitro antileishmanial evaluation of 5-substituted-
2Ј-deoxyuridine nucleosides. The most active compound against Leishmania donovani promas-
tigotes was Thia-dU (3a) with an IC50 D 3 ꢀM. This compound exhibited the same activity as
zidovudine (3Ј-azido-2Ј-deoxythymidine) used as nucleoside reference compound. Considering
the cytotoxicity of synthetic compounds on peritoneal murine macrophages, the most toxic
compound was MeThio-dU (3d) with a MTC at 10 ꢀM. Only Methia-dU (3b) was active
against intramacrophagic amastigotes with an IC50 D 6.5 ꢀM. This latter can now be evaluated
in vivo, for further investigations through structure-based drug design.
2005 Elsevier Inc. All rights reserved.
Keywords: Nucleosides; Antileishmanial activity
*
Corresponding authors. Fax: +33 492076151 (R. Benhida), +33 146835557 (P.M. Loiseau)
E-mail addresses: benhida@unice.fr (R. Benhida), loiseau@cep.u-psud.fr (P.M. Loiseau).
0045-2068/$ - see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2005.07.001

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C. Peyron et al. / Bioorganic Chemistry 33 (2005) 439–447
1. Introduction
The chemotherapy of visceral leishmaniasis (VL) is still based on pentavalent anti-
monials and amphotericin B in the case of resistance to antimonials which frequently
occurs in India (Bihar) and Souddan [1]. Despite the progress of amphotericin B for-
mulations able to reduce its toxicity and to enhance its antileishmanial eVect, and the
recent launch of miltefosine as the Wrst orally active antileishmanial drug, there is an
urgent need for new molecules active against Leishmania sp. because of some toxico-
logical and economical limitations for these drugs [2]. Some nucleoside derivatives
have interesting antileishmanial properties. Sinefungin (Fig. 1), a natural nucleoside
antibiotic produced by Streptomyces griseolus, is structurally related to S-adenosyl-L-
methionine and exhibits strong antifungal and antiparasitic activities both in vitro
and in vivo [3]. Various uracil analogs of sinefungin have been previously synthesized
to optimize the structural characteristics of this series [4]. This article describes the
synthesis of 5-substituted-2Ј-deoxyuridine nucleosides and their biological activity
on both Leishmania donovani promastigote and amastigote stages and their cytotox-
icity on peritoneal murine macrophages. The rationale relies on chemical modula-
tions at the 5-position that is critical for biological activity of pyrimidine nucleosides.
The choice of aryl substituents such as benzothiazole, thiazole, benzimidazole, and
thiophene systems, well known for their antiprotozoal activities was justiWed in the
aim to obtain a synergistic eVect in the biological activity by a combination of two
active moieties in one molecule. The putative mechanism of action of these com-
pounds will be discussed in this article.
2. Materials and methods
2.1. General methods
All reactions were run under an atmosphere of nitrogen. Tetrahydrofuran (THF)
was purchased from commercial sources and dried over 4 Å molecular sieves before
use. All other reagents and solvents were purchased from commercial sources and
used without puriWcation. Chromatography was made on silica gel purchased from
Merck (silica gel 60, 0.063–0.200 mm). Analytical thin-layer chromatography (TLC)
was carried out using 0.2 mm commercial silica gel plates (silica gel 60 with Xuores-
cent indicator UV254). Mass spectra were recorded on an Esquire 3000 Plus equip-
ment, in both positive or negative Electron Spray ionization (ESI) mode and are
reported as m/z. Proton nuclear magnetic resonance (1H NMR) spectra and carbon-
13 nuclear magnetic resonance (13C NMR) spectra were recorded with a Bruker
Fig. 1. Structure of sinefungin.

C. Peyron et al. / Bioorganic Chemistry 33 (2005) 439–447
441
Advance Spectrometer at 200 or 500 MHz. Chemical shifts are reported in delta (ꢀ)
units, parts per million (ppm) downWeld from used solvents, speciWed for each prod-
uct. Coupling constants are reported in Hertz (Hz).
2.1.1. Synthesis of the free nucleoside 3h: analytical and spectral data for selected
products
2.1.1.1. Indolyltin derivative 4. To a solution of Boc-protected indole (10mmol) in
THF (30mL) was slowly added one equivalent of lithium diisopropylamide (2 M
solution in hexane/THF, 5 mL) at ¡78 °C. After 30 min, a solution of Bu3SnCl
(1.1 equiv., 11 mmol) in THF (5 mL) was added dropwise. The reaction was stopped
after 3h by addition of aq. NH4Cl solution (20 mL) and the mixture was extracted
with diethyl ether (3 £ 30 mL). The organic layers were dried over MgSO4 Wltered and
the solvents were evaporated under vacuo. The obtained crude oil was used in the
next coupling step without further puriWcation. 1H NMR (CDCl3, 200 MHz) ꢀ (ppm)
0.88–1.02 (m, 15H, Bu), 1.60–1.73 (m, 12H, Bu), 1.75 (s, 9H, tBu), 6.77 (d, 1H,
J D 0.6 Hz, H-2 ind.), 7.18–7.33 (m, 3H, H-ind.), 8.01 (d, 1H, J D8.8 Hz, H-4 ind.). 13C
NMR (CDCl3, 50MHz) ꢀ (ppm) 11.69 ppm (CH2), 13.70 (CH3), 27.47 (CH2), 28.28
(CH3-Boc), 29.27 (CH2), 83.82 (C-(CH3)3), 115.34 (C-4 ind.), 118,63 (C-2 ind.), 120.09
(CH-ind.), 122.25 (CH-ind.), 125.63 (CH-ind.), 132.51 (C-Ar), 137.50 (C-Ar), and
152.26 (CO). MS (ESI+) m/z D508 (MH+).
2.1.1.2. 3Ј,5Ј-Di-O-acetyl-5-(N-tertbutoxycarbonylindolyl-2)-2Ј-deoxyuridine (1h). To
a solution of 1 (2 mmol) and indole-tin derivative 4 (4 mmol) in DMF (10 mL) was
added Pd(Ph3)2Cl2 (0.1mmol), CuI (0.2 mmol) and Et3N (2.4 mmol). The mixture was
heated at 70°C for 3 h cooled and Wltered over celite. The solvent was removed and
the residue puriWed by Xash chromatography (CH2Cl2/AcOEt: 100/0 to 85/15) to
1
yield 1h as a colorless oil (67%). H NMR (CDCl3, 500 MHz) ꢀ (ppm) 1.46 (s, 9H,
tBu), 1.79 (s, 3H, CH3), 2.00 (s, 3H, CH3), 2.19 (m, 1H, H-2Ј), 2.43 (ddd, 1H, J D1.9,
5.8 and 14.3 Hz, H-2Ј), 4.16 (m, 1H, H-4Ј), 4.22 (m, 2H, H-5Ј), 5.13 (m, 1H, H-3Ј), 6.28
(dd, 1H, J D8.2 and 5.7 Hz, H-1Ј), 6.43 (s, 1H, H-3 ind.), 7.10 (t, 1H, J D7.4 Hz, H-5 or
H-6 ind.), 7.20 (t, 1H, J D7.7 Hz, H-6 or H-5 ind.), 7.40 (d, 1H, J D7.9 Hz, H-4 or H-7
ind.), 7.54 (s, 1H, H-6), 8.02 (d, 1H, J D8.5Hz, H-7 or H-4 ind.). 13C NMR (CDCl3,
125MHz) ꢀ (ppm) 20.82 (CH3), 20.93 (CH3), 27.91 (tBu), 37.76 (C-2Ј), 62.33 (C-5Ј),
75.09 (C-3Ј), 83.92 (C-(CH3)3), 85.41 (C-4Ј), 86.03 (C-1Ј), 110.64 (C-3 ind.), 111.53 (C-
5), 115.40 (C-7 ind.), 122.62 (CH-ind.), 124.55 (CH-ind.), 128.42 (CH-ind.), 131.14 (C-
ind.), 131.93 (C-ind.), 132.01 (C-6), 149.76 (CO), 150.19 (CO), 161.82 (CO), and 170.56
(CO). MS (ESI+) m/z D450 [(MNa)+-Boc].
2.1.1.3. 3Ј,5Ј-Di-O-tolouyl-5-(N-tertbutoxycarbonylindolyl-2)-2Ј-deoxyuridine (2h). 2h
1
was obtained by using the same experimental conditions as for 1h (43% yield). H
NMR (CDCl3, 500 MHz) ꢀ (ppm) 1.56 (s, 9H, tBu), 2.19 (s, 3H, CH3), 2.39 (m, 1H, H-
2Ј), 2.44 (s, 3H, CH3), 2.78 (dd, 1H, J D5.4 and 14.5 Hz, H-2Ј), 4.56 (m, 1H, H-4Ј), 4.62
(d, 1H, J D11.0Hz, H-5Ј), 4.81 (d, 1H, J D 12.3 Hz, H-5Ј), 5.67 (br d, 1H, J D5.9Hz,
H-3Ј), 6.23 (s, 1H, H-3 ind.), 6.56 (t, 1H, J D5.5Hz, H-1Ј), 6.81 (d, 2H, J D7.5 Hz, H-
tol), 7.22 (m, 1H, H-5 or H-6 ind.), 7.28 (m, 2H, H-tol), 7.32 (m, 1H, H-6 or H-5 ind.),

442
C. Peyron et al. / Bioorganic Chemistry 33 (2005) 439–447
7.39 (d, 1H, J D7.8 Hz, H-4 or H-7 ind.), 7.62 (s, 1H, H-6), 7.64 (d, 2H, J D 7.2 Hz, H-
tol), 7.97 (d, 2H, J D7.2 Hz, H-tol), and 8.10 (d, 1H, J D 8.5Hz, H-7 or H-4 ind.). 13
C
NMR (CDCl3, 125 MHz) ꢀ (ppm) 21.45 (CH3), 21.60 (CH3), 27.83 (tBu), 38.28 (C-2Ј),
64.05 (C-5Ј), 74.90 (C-3Ј), 82.91 (C-4Ј), 83.64 (C-(CH3)3), 84.98 (C-1Ј), 110.62 (C-3
ind.), 112.20 (C-5), 115.32 (C-7 ind.), 120.54 (CH-ind.), 122.49 (CH-ind.), 124.45 (CH-
ind.), 125.86 (C-Ar), 126.11 (C-Ar), 128.42 (C-Ar), 128.86 (CH-tol), 128.97 (CH-tol),
129.17 (CH-tol), 129.70 (CH-tol), 130.26 (C-Ar), 134.78 (C-6), 136.86 (C-Ar), 144.05
(C-Ar), 144.47 (C-Ar), 149.47 (CO), 149.90 (CO), 161.48 (CO), 165.80 (CO), and
165.96 (CO). MS (ESI+) m/z D702 (MNa+).
2.1.1.4. 3Ј,5Ј-di-O-tolouyl-5-(indolyl-2)-2Ј-deoxyuridine (indole-deprotected side
product [5]). This compound was obtained as a side product in the palladium cou-
pling step. It is also an advanced intermediate in the synthesis of nucleoside 3h. 1H
NMR (CDCl3, 500 MHz) ꢀ (ppm) 2.16 (m, 1H, H-2Ј), 2.27 (s, 3H, CH3), 2.42 (s, 3H,
CH3), 2.75 (dd, 1H, J D 5.2 and 14.3 Hz, H-2Ј), 4.51 (m, 1H, H-4Ј), 4.72 (m, 2H, H-
5Ј), 5.54 (br d, 1H, J D 5.9 Hz, H-3Ј), 6.29 (br m, 1H, H-1Ј), 6.45 (s, 1H, H-3 ind.),
7.02 (t, 1H, J D 7.4 Hz, H-6 ind. or H-5 ind.), 7.09 (t, 1H, J D 7.8 Hz, H-5 ind. or H-6
ind.), 7.11 (d, 2H, J D 8.1 Hz, H-tol), 7.27 (d, 2H, J D 8.1 Hz, H-tol), 7.34 (d, 1H,
J D 7.9 Hz, H-4 ind. or H-7 ind.), 7.41 (d, 1H, J D 8.2 Hz, H-7 ind. or H-4 ind.), 7.92
(d, 2H, J D 7.9 Hz, H-tol), 7.97 (d, 2H, J D 8.2 Hz, H-tol), 8.05 (s, 1H, H-6), and 10.46
(s, 1H, NH). 13C NMR (CDCl3, 125 MHz) ꢀ (ppm) 21.55 (CH3), 21.70 (CH3), 38.36
(C-2Ј), 64.00 (C-5Ј), 74.65 (C-3Ј), 83.29 (C-4Ј), 85.90 (C-1Ј), 98.02 (C-3 ind.), 107.55
(C-5), 111.35 (CH-ind.), 119.75 (CH-ind.), 120.06 (CH-ind.), 121.81 (CH-ind.),
126.33 (2£ C-Ar), 127.75 (2£ C-Ar), 129.28 (CH-tol), 129.39 (CH-tol), 129.56 (CH-
tol), 129.80 (CH-tol), 134.28 (C-6), 136.14 (C-Ar), 144.51 (C-Ar), 144.56 (C-Ar),
149.13 (CO), 162.34 (CO), 166.02 (CO), and 166.39 (CO). MS (ESI+) m/z D 580
(MH)+, 603 (MNa)+.
2.1.1.5. 5-(N-tertbutoxycarbonylindolyl-2)-2Ј-deoxyuridine (5). To a solution of the
protected nucleoside 1h or 2h obtained above (0.5 mmol) in MeOH (5 mL) was
added K2CO3 (3 equiv.) and the reaction mixture was stirred over night at room
temperature. The mixture was Wltered and concentrated in vacuo. The residual oil
was puriWed on silica gel chromatography (CH2Cl2/MeOH: 95/5 to 90/10) to aVord
1
quantitative yield of pure product 5 as a foam. H NMR (CD3OD, 500 MHz) ꢀ
(ppm) 1.53 (s, 9H, tBu), 2.28 (m, 1H, H-2Ј), 2.31 (m, 1H, H-2Ј), 3.70 (dd, 1H, J D 3.3
and 11.9 Hz, H-5Ј), 3.78 (dd, 1H, J D 3.0 and 11.9 Hz, H-5Ј), 3.94 (m, 1H, H-4Ј), 4.41
(m, 1H, H-3Ј), 6.33 (t, 1H, J D 6.6 Hz, H-1Ј), 6.59 (s, 1H, H-3 ind.), 7.19 (t, 1H,
J D 7.5 Hz, H-5 or H-6 ind.), 7.26 (t, 1H, J D 7.5 Hz, H-6 or H-5 ind.), 7.52 (d, 1H,
J D 7.6 Hz, H-4 or H-7 ind.), 8.13 (d, 1H, J D 8.3 Hz, H-7 or H-4 ind.), 8.19 (s, 1H, H-
6). 13C NMR (CD3OD, 125 MHz) ꢀ (ppm) 28.43 (tBu), 42.05 (C-2Ј), 62.84 (C-5Ј),
72.39 (C-3Ј), 85.28 (C-(CH3)3), 87.07 (C-4Ј), 89.31 (C-1Ј), 111.86 (C-3 ind.), 112.60
(C-5), 116.44 (C-7 or C-4 ind.), 121.91 (C-4 or C-7 ind.), 124.05 (C-5 or C-6 ind.),
125.79 (C-6 or C-5 ind.), 130.43 (C-ind.), 133.20 (C-ind.), 138.81 (C-ind.), 139.17 (C-
6), 151.50 (CO), 152.34 (CO), and 164.97 (CO). MS (ESI+) m/z D 466 (MNa)+, 482
(MK)+.

C. Peyron et al. / Bioorganic Chemistry 33 (2005) 439–447
443
2.1.1.6. 5-(indolyl-2)-2Ј-deoxyuridine (3h). To a solution of 5 (133mg, 0.3mmol) in
dioxane (2mL) was added TFA (4mL) and the reaction mixture was stirred over night
at room temperature. The mixture was evaporated and the residual oil was loaded on
silica gel column and eluted with CH2Cl2/MeOH (95/5 to 85/15) to aVord 81 mg (79%)
of 3h as a foam. 1H NMR (DMSO-d6, 500MHz) ꢀ (ppm) 2.21 (m, 1H, H-2Ј), 2.30 (m,
1H, H-2Ј), 3.65 (m, 1H, H-5Ј), 3.71 (m, 1H, H-5Ј), 3.85 (m, 1H, H-4Ј), 4.34 (m, 1H, H-
3Ј), 6.26 (t, 1H, JD6.6Hz, H-1Ј), 6.81 (s, 1H, H-3 ind.), 6.97 (t, 1H, JD7.4 Hz, H-5 or
H-6 ind.), 7.05 (t, 1H, JD7.1Hz, H-6 or H-5 ind.), 7.46 (m, 2H, H-4 and H-7 ind.), 8.45
(s, 1H, H-6). 13C NMR (DMSO-d6, 125MHz) ꢀ (ppm) 38.86 (C-2Ј), 60.48 (C-5Ј), 69.54
(C-3Ј), 84.10 (C-4Ј), 86.83 (C-1Ј), 98.46 (C-3 ind.), 105.82 (C-5), 110.81 (CH-ind.),
118.61 (CH-ind.), 118.96 (CH-ind.), 120.51 (CH-ind.), 127.22 (C-ind.), 127.26 (C-ind.),
130.53 (C-ind.), 136.01 (C-6), 148.88 (CO), and 161.18 (CO). MS (ESI+) m/zD344
(MH+). UV (MeOH) ꢁmax 213nm (ꢂ 54,800), 288nm (ꢂ 31,400), 331nm (ꢂ 29,700).
2.2. Toxicity on macrophages [6]
The toxicity of the nucleosides was evaluated on mouse peritoneal macrophages
which were then used for the intramacrophagic amastigotes in vitro model. Peritoneal
macrophages were harvested from female CD1 mice (Charles River, Cléon, France) 3
days after an intraperitoneal injection of 1.5ml of sodium thioglycolate (Biomérieux)
and were dispensed into eight-well chamber slides (LabTek Ltd.) at a concentration of
5£104/well (400ꢀl/well) in RPMI 1640 medium supplemented with 10% heat-inacti-
vated fetal calf serum, 25mM Hepes, and 2mM L-glutamine (Life Technologies,
Cergy-Pontoise, France). Four hours after the macrophages were plated, they were
washed to eliminate Wbroblasts. The culture medium was renewed 48 h later and a new
culture medium containing the drug was added. The experiment was stopped at day 5,
and the viability of promastigotes was checked using the tetrazolium-dye (MTT) col-
orimetric method. The 50% inhibitory concentrations (IC50) were determined by linear
regression analysis, expressed in ꢀM§SD and the maximum tolerated concentration
(MTC) expressed in ꢀM was evaluated for each compound.
2.3. Antileishmanial activity [6]
Leishmania donovani (MHOM/IN/80/DD8) promastigotes used in this study were
cultured in Hepes (25 mM)-buVered RPMI 1640 medium enriched with 10% heat-
inactivated fetal calf serum (hi-FCS) and 50ꢀg/ml gentamycin at 27 °C in a dark
environment.
2.3.1. In vitro evaluation on promastigote forms
The antileishmanial screening was performed in Xat-bottomed 96-well plastic tis-
sue-culture trays maintained at 27°C in an atmosphere of 95% air/5% CO2. Promas-
tigote forms from a logarithmic phase culture were suspended to yield 106 cells/ml
after hemocytometer counting. Each well was Wlled with 100 ꢀl of the parasite sus-
pension, and plates were incubated at 27 °C for 1 h before drug addition. The com-
pounds to be tested were dissolved in DMSO and then added to each well to obtain

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C. Peyron et al. / Bioorganic Chemistry 33 (2005) 439–447
the Wnal concentration of 100 ꢀM and further concentrations were twice diluted. At
up to 2% (v/v), DMSO had no eVect on parasite growth. Each concentration was
screened in triplicate. The viability of promastigotes was checked using the tetrazo-
lium-dye (MTT) colorimetric method. The MTT cell proliferation assay is a colori-
metric assay system, which measures the reduction of a tetrazolium component
(MTT) into an insoluble formazan product by the mitochondria of viable cells. After
incubation of the cells with the MTT reagent, a detergent solution was added to lyse
the cells and solubilize the coloured crystals. The samples were read using an ELISA
plate reader at a wavelength of 570 nm. The amount of colour produced was directly
proportional to the number of viable cells. The results are expressed as the concentra-
tions inhibiting parasite growth by 50% (IC50) after a 3-day incubation period. Pent-
amidine and amphotericin B were used as antileishmanial reference compounds.
2.3.2. In vitro evaluation on intramacrophage amastigotes
Concerning the amastigote in vitro model, the macrophages were infected after a
24h incubation period with promastigote forms of L. donovani DD8 wild type (WT)
in a stationary phase at a ratio of 10 parasites per macrophage, to obtain 87% of
infected macrophages and 10§ 3 amastigotes per macrophage. At 18 h after the pro-
mastigotes had entered macrophages, the free promastigotes were eliminated and
intramacrophagic amastigotes were treated at various concentrations of the com-
pounds. Pentamidine and amphotericin B were used as reference compounds. Each
experiment was performed in triplicate. The culture medium was renewed 48h later
and a new culture medium containing the drug was added. The experiment was
stopped at day 5, and the percentage of infected macrophages was evaluated micro-
scopically after Giemsa staining. The 50% inhibitory concentrations (IC50) were
determined by linear regression analysis, and expressed in ꢀM § SD.
3. Results and discussion
The free nucleosides 3a–h were synthesized in two to three steps and in good over-
all yields (34–85%). The key step is based on palladium catalyzed Stille cross coupling
[7] of protected 5-iodo-2Ј-deoxyuridine 1 or 2 [8] and aryltin derivatives (Scheme 1).
A typical example is shown in Scheme 2 for the synthesis of indole nucleoside 3h.
Thus, the indole-tin derivative 4 was Wrst obtained in 91% overall yield from indole
by amine protection (Boc2O, DMAP) followed by metalation–transmetalation of the
resulting protected indole (LDA/THF then Bu3SnCl). Palladium coupling of 4 and 5-
iodo-2Ј-deoxyuridine 1 or 2 aVorded Xuorescent products 1h and 2h in 67 and 43%
yield, respectively. When the coupling step was carried out without CuI /Et3N, prod-
ucts 1h and 2h were isolated in low yields together with their Boc-cleaved compounds
[5]. 1h and 2h were then cleanly deprotected by using sequential treatment with
K2CO3/MeOH and TFA to give the free nucleoside 3h.
All compounds were evaluated for their in vitro antileishmanial activity (promas-
tigotes and intramacrophagic amastigotes) and cytotoxicity (MTC). Zidovudine, a
nucleoside compound, and the currently used drugs pentamidine and amphotericin

C. Peyron et al. / Bioorganic Chemistry 33 (2005) 439–447
445
Scheme 1. Synthesis of 5-heteroaryl-2Ј-deoxyuridine.
Scheme 2. Typical synthesis: reagents and conditions: (a) (i) Boc2O (1.2 equiv.), DMAP (10% molar),
CH3CN, (ii) LDA (1.2 equiv.), THF, ¡78 °C then Bu3SnCl (1.2 equiv.); (b) 1 or 2, Pd(PPh3)2Cl2
(0.05 equiv.), DMF, CuI (0.1 equiv.); Et3N (1.2 equiv.); (c) (i) K2CO3/MeOH (3 equiv.) to give 5 then (ii) aq.
TFA, dioxane.
B were used as references. All results are listed in Table 1. The most active compound
against promastigotes was Thia-dU (3a) (IC50 D3.1 ꢀM) and all other compounds
were at least 5–10-fold less active. Thia-dU 3a exhibited the same activity as zidovu-
dine used as nucleoside reference compound. The substituent on position 5 of the uri-
dine ring had a minor eVect on the activity against promastigotes. Thus, whatever the
heterocycle was thiazole (3a), thiophene (3c) or their substituted (3b, 3d, and 3e) or
benzannulated analogs (3f, 3g, and 3h), the activity was maintained at the same level.
In the same manner, the replacement of the methyl group on the thiophene ring by a
bromine atom (3d vs. 3e) did not signiWcantly change the activity against promasti-
gotes. When a methyl group is added on the thiazole ring at position 5 (3a vs. 3b), the
IC50 decreased 10-fold.

446
C. Peyron et al. / Bioorganic Chemistry 33 (2005) 439–447
Table 1
Antileishmanial activity of compounds 3a–h (IC50 deWned as inhibitory concentration 50%) and their tox-
icity on murine macrophage (MTC deWned as a maximum tolerated concentration)
Compound
L. donovani DD8
Cytotoxicity on
(abbreviation)
peritoneal murine
macrophages MTC (ꢀM)
Promastigotes
IC50 (ꢀM) § SD
Intramacrophagic
amastigotes IC50 (ꢀM) § SD
>100
6.5 § 0.7
>50
>10
>100
>50
>50
>50
3a (Thia-dU)
3b (Methia-dU)
3c (Thio-dU)
3d (MeThio-dU)
3e (BrThio-dU)
3f (BenzoThia-dU)
3g (BenzoThio-dU)
3h (Ind-dU)
3.1 § 0.2
26.2 § 2.8
30.3 § 3.2
25.2 § 1.9
18.3 § 1.5
32.4 § 3.3
20.2 § 1.8
19.1 § 2.1
3.1 § 0.4
100
50
50
10
100
50
50
50
>100
15
Zidovudine
Pentamidine
Amphotericin B
>100
14.6 § 1.6
0.4 § 0.1
7.2 § 0.6
0.1 § 0.1
5
The antileishmanial evaluations were performed as previously described [6].
The activity against intramacrophagic amastigotes did not reXect the activity on
promastigotes since only Methia-dU (3b) was active, with an IC50 at 6.5 ꢀM. There-
fore, some data have to be taken into consideration to explain these results. Zidovu-
dine like Thia-dU (3a), although active on promastigotes was inactive on
intramacrophagic amastigotes. Considering the low toxicity of these compounds on
uninfected macrophages, they probably cannot enter the phagolysosome containing
the amastigotes within the macrophage. It is assumed that nucleosides do not pas-
sively enter the parasite and L. donovani, like all other Kinetoplastida, is a purine
auxotroph [9]. This parasite is incapable of purine biosynthesis and must therefore
salvage purine nucleobases or nucleosides from its host. This process is initiated by
purine transporters on the parasite cell surface [10]. Furthermore, it has been demon-
strated that pyrimidine nucleosides are recognized by these transporters [11].
The diVerence of activities of compounds 3a–h against the promastigote forms
could be partially ascribed to the transporter recognition. Concerning the parasite
stage, a recent study revealed that, unlike the promastigote stage, the amastigote pos-
sesses two distinct adenosine transporters T1 and T2 both with high aYnities [12].
One of these transporters (T1) is identical to the adenosine/pyrimidine nucleoside
transporter of the promastigote previously described. The second transporter (T2) is
speciWc to the amastigote stage and only transports purine nucleosides. Therefore, we
can consider that synthetic compounds 3a–h could use the same transporter both in
promastigote and amastigote forms and, the diVerences of activity observed could be
related to the diYculty for the compound to cross over the phagolysosome mem-
brane. Whereas the substituent on the heterocycle at the C-5 position was of high
importance, the nature of the heterocycle itself (thiazole or a thiophene rings) did not
induce great changes of the biological activities. Thia-dU (3a), the most active com-
pound on promastigotes, seemed incapable to cross over the phagolysosome
membrane whereas Methia-dU (3b) had signiWcant activity on intramacrophagic
amastigotes.

C. Peyron et al. / Bioorganic Chemistry 33 (2005) 439–447
447
Furthermore, no compound from this series was active against sinefungin-resis-
tant L. donovani LRC (data not shown) indicating a cross resistance between these
nucleosides and sinefungin. A synergy was searched between Thia-dU (3a) or
Methia-dU (3b) and pentamidine or amphotericin B. Surprisingly, no synergistic
eVect was observed despite diVerent mode of action of the molecules in association.
4. Conclusion
In summary, we reported a short and eYcient synthesis of nucleoside analogs
together with their antileishmanial activity. All these compounds were active in vitro
in micromolar range. Moreover, compound 3b was active on both amastigotes and
promastigotes and has now to be studied in vivo to assess its relevance in visceral
leishmaniasis chemotherapy.
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