New parasite inhibitors encompassing novel conformationally-locked 5′-acyl sulfamoyl adenosines

Article abstract of DOI:10.1039/c2ob25879j

We describe the design, synthesis and biological evaluation of conformationally-locked 5′-acyl sulfamoyl adenosine derivatives as new parasitic inhibitors against Trypanosoma and Leishmania. The conformationally-locked (3′-endo, North-type) nucleosides have been synthesized by covalently attaching a 4′-CH₂-O-2′ bridge (Fig. 2) across C2′-C4′ of adenosine in order to reduce the conformational flexibility of the pentose ring. This is designed to decrease the entropic penalty for complex formation with the target protein, which may improve free-energy of stabilization of the complex leading to improved potency. Conformationally-locked 5′-acyl sulfamoyl adenosine derivatives (16-22) were tested against parasitic protozoans for the first time in this work, and showed potent inhibition of Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma rhodesiense and Leishmania infantum with IC₅₀ = 0.25-0.51 μM. In particular, the potent 5′-pentanyl acyl sulfamoyl adenosine derivative 17 (IC₅₀ = 0.25 μM) against intracellular L. infantum amastigotes and Trypanosoma subspecies is interesting in view of its almost insignificant cytotoxicity in murine macrophage host cells (CC₅₀ >4 μM) and in diploid human fibroblasts MRC-5 cell lines (CC₅₀ 4 μM). This work also suggests that variable alkyl chain length of the acyl group on the acylsulfamoyl side chain at 5′ can modulate the toxicity of 5′-O-sulfamoylnucleoside analogues. This conformationally-locked sulfamoyl adenosine scaffold presents some interesting possibilities for further drug design and lead optimization.

Full text of DOI:10.1039/c2ob25879j

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PAPER
New parasite inhibitors encompassing novel conformationally-locked 5′-acyl
sulfamoyl adenosines†‡
Shailesh S. Dixit, Ram Shankar Upadhayaya and Jyoti Chattopadhyaya*
Received 8th May 2012, Accepted 23rd May 2012
DOI: 10.1039/c2ob25879j
We describe the design, synthesis and biological evaluation of conformationally-locked 5′-acyl sulfamoyl
adenosine derivatives as new parasitic inhibitors against Trypanosoma and Leishmania. The
conformationally-locked (3′-endo, North-type) nucleosides have been synthesized by covalently attaching
a 4′-CH2–O-2′ bridge (Fig. 2) across C2′–C4′ of adenosine in order to reduce the conformational
flexibility of the pentose ring. This is designed to decrease the entropic penalty for complex formation
with the target protein, which may improve free-energy of stabilization of the complex leading to
improved potency. Conformationally-locked 5′-acyl sulfamoyl adenosine derivatives (16–22) were tested
against parasitic protozoans for the first time in this work, and showed potent inhibition of Trypanosoma
cruzi, Trypanosoma brucei, Trypanosoma rhodesiense and Leishmania infantum with IC50 = 0.25–0.51 μM.
In particular, the potent 5′-pentanyl acyl sulfamoyl adenosine derivative 17 (IC50 = 0.25 μM) against
intracellular L. infantum amastigotes and Trypanosoma subspecies is interesting in view of its almost
insignificant cytotoxicity in murine macrophage host cells (CC50 >4 μM) and in diploid human fibroblasts
MRC-5 cell lines (CC50 4 μM). This work also suggests that variable alkyl chain length of the acyl group
on the acylsulfamoyl side chain at 5′ can modulate the toxicity of 5′-O-sulfamoylnucleoside analogues.
This conformationally-locked sulfamoyl adenosine scaffold presents some interesting possibilities for
further drug design and lead optimization.
administered in its initial stage. The longer the person is infected
the potency of these drugs decreases. The parasites mostly hide
1. Introduction
Chagas’ disease or American trypanosomiasis is caused by the
parasitic protozoan agent Trypanosoma cruzi (T. cruzi) and is a
serious health problem in Central and South American conti-
nents, affecting about 17 million people living in poor rural
areas.1 Depending on physiological state and age of the patient it
has a mortality rate of 8–12%.2,3 Chagas’ disease is now spread-
ing to other continents due to immigration, which was once
confined to the Latin American region.4 Trypanosoma brucei
(T.b. brucei) and Trypanosoma rhodesiense (T.b. rhod.) are cau-
sative agents for human African trypanosomiasis.5,6 It is mainly
transmitted by the infected faeces of blood-sucking triatomine
bugs, infection through blood transfusion, from infected mother
to child or organ donation.2,4 To date no vaccine is available
against Chagas’ disease.4 The disease can be controlled by
chemotherapy of benznidazole (5) or nifurtimox (6) if
in muscles of the heart and digestive system in the chronic
phase, due to which patients suffer from cardiac and digestive
disorders.4 Benznidazole or nifurtimox are not effective in
the chronic phase of infection, and are also associated with high
toxicity and side effects.2,4,7–13
Leishmaniasis is a group of diseases ranging from self-healing
cutaneous ulcers to severe visceral diseases which even may
cause death. It is caused by a protozoan parasite of species of the
genus Leishmania.14,15 These are digenetic organisms present in
a flagellated promastigote in the gut of the sandfly vectors and
an intracellular amastigote in the mammalian host.14,16 Leishma-
niasis is transmitted to humans by some species of phlebotomine
sandfly. This infection is endemic in 88 countries from tropical
and sub-tropical regions affecting 12 million people, especially
those living in poor rural areas, with 2 million new cases
annually.17–19 The standard chemotherapy treatment includes
antimony-based drugs, amphotericin-B, paromomycin, and oral
miltefosine.20–27
Program of Chemical Biology, Institute of Cell and Molecular Biology,
Biomedical Centre, Uppsala University, SE-75123 Uppsala, Sweden.
E-mail: jyoti@boc.uu.se; Fax: +46-18-554495; Tel: +46-18-4714577
†This article is part of the Organic & Biomolecular Chemistry 10th
Anniversary issue.
Although significant information on the biochemistry and
physiology of these parasites is available, current treatments are
effective only in the acute stage, having several limitations
associated with toxic side effects, prolonged treatment duration
and parasite resistance.8–10,19,28–32 This high rate of therapeutic
‡Electronic supplementary information available: Spectral data (1H,
13C NMR, DEPT, HSQC, LCMS, Mass and HPLC) for all new com-
pounds are included. See DOI: 10.1039/c2ob25879j
This journal is © The Royal Society of Chemistry 2012
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failure calls for new rational approaches to develop alternative
drugs.
In view of the antiparasitic activity displayed by 2′,3′-bis-
hydroxy sulfamoyl adenosine (1, Fig. 1) and aminoacylsul-
famoyl adenosine (ascamycin41 4, Fig. 1), we became interested
in exploring acyl-sulfamoyl derivatives of adenosines with a
modified locked-sugar backbone (C, Fig. 2) to see its effect
against Trypanosoma subsp. and Leishmania. Further retro-
synthetic analysis to synthesize sugar-locked 2′-O,4′-C-methyl-
ene-linked bicyclic ribonucleosides (C, Fig. 2) of acylsulfamoyl
adenosine (A) is given in Fig. 2. The main feature of the syn-
thesis is how the constituent pentose-sugar containing a –CH2–
Modified adenosine derivatives are known to possess anti-
protozoal activity.33–38 Adenosine derivatives display anti-
trypanosomal activity by inhibiting glyceraldehyde-3-phosphate
dehydrogenase (GAPDH),34–36,38,39 or polyamine syntheses.33,40
The strategy used so far to develop new inhibitors of these
enzymes involves base subunit modification and/or carbohydrate
modification (at 2′ and/or 5′) of adenosine. An important way of
discovering new antitrypanosomal and antileishmanial agents is
exploring new scaffolds, studying their SAR and structurally
optimizing validated lead compounds.
bridge, connecting the 2′-O
̲
of ribose with the 4′-C
̲
(C, Fig. 2)
has locked the flexible pentose-sugar to the fixed 3′-endo
Fig. 1 Known inhibitors of Trypanosoma and Leishmania: compounds 1–4 are known to possess anti-parasitic activity. Compounds 5–10 are the
standard front-line drugs: against Trypanosoma spp. (5 & 6); Leishmania (7, 8, 9 & 10).
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Fig. 2 2′-O, 4′-C-Methylene-linked bicyclic ribonucleoside bond is shown with a pink dotted line in the conformationally-constrained core B. D is
the general bicyclic structure according to IUPAC rule. General formula C represents the series of compounds synthesized in this work.
conformation. Because of the reduction in the conformational
flexibility of the pentose sugar, we expect that there will be a
decrease of the entropic penalty for the complex formation with
the target protein, which will in turn give improved free-energy
of stabilization of the complex, and hence increased potency.
Here we describe the results of studies involving our novel
antiprotozoal conformationally-locked sulfamoyl derivatives of
adenosine with new modifications at the 2′, 4′ and 5′ centers
in the sugar moiety. Most of these compounds 16–2242 are
extremely potent against Trypanosomiasis and Leishmaniasis,
with IC50 of 0.25–0.51 μM. In particular, compound 17 (in vitro
activity, IC50 = 0.25 μM), which is potent against intracellular
L. infantum amastigotes and Trypanosoma subspecies, is inter-
esting in view of its almost insignificant cytotoxicity in murine
macrophage host cells (CC50 >4 μM) and in diploid human
fibroblasts MRC-5 cell line against mammalian cells (CC50
4 μM). Growth inhibition assays were performed on compounds
16–22 (Table 1) against protozoans including epimastigote form
of T. cruzi, T. brucei subsp. brucei, T. brucei subsp. rhodesiense
and L. infantum.
Debenzylation of sulfonamide 15 using Pd(OH)2/C and
HCO2NH4 furnished alcohol 16 in good yield (87%). The target
compounds 17–22 were synthesized by treating sulfonamide 16
with the corresponding N-hydroxy succinimide aliphatic ester in
dry DMF and Cs2CO3 to furnish the synthesis of target com-
pounds 17–22 in 15–34% yield.
2.2. Antiprotozoal activity
Growth inhibition assays against protozoans including epimasti-
gote form of T. cruzi (Tulahuen 2 strain), T. brucei subsp. brucei
427, T. brucei subsp. rhodesiense STIB900 and L. infantum
(MHOM/MA (BE)/67) were performed on compounds 16–22
(Table 1). Parasites were grown in the presence of the com-
pounds and the percentage growth inhibition was determined
against control (no drug added to the medium). A cytotoxicity
assay44 involving diploid human lung fibroblasts (MRC-5) was
performed to assess the toxicity of compounds against mamma-
lian cells. The results are expressed as percent reduction in cell
viability compared to untreated control.
3. Discussion
2. Results
All compounds (16–22) reported here were found to be very
active in vitro against protozoans including epimastigote form of
T. cruzi, T. brucei, T. rhodesiense and L. infantum, having IC50s
in the range of 0.25–0.49 μM; 0.25 μM; 0.25 μM and
0.25–0.51 μM respectively. These compounds were tested for
their cytotoxicity in murine macrophage host cells (Cytotoxic
2.1. Chemistry
Locked nucleic acid (LNA), compound 1443 (Scheme 1) served
as the starting material for the synthesis of compounds 16–22.
Compound 14, on sulfamoylation reaction with sulfamoyl chlor-
ide in dry DMF and Et3N, gave sulfonamide 15 (31%).
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Table 1 In vitro activity (IC50a, μM) of compounds against T. cruzi, T. brucei brucei (T.b. brucei), T. brucei rhodesiense (T.b. rhod.) and Leishmania
infantum (L. inft.)
Compd. no.
R
T. cruzi (IC50, μM) T.b. brucei (IC50, μM) T.b. rhod. (IC50, μM) L. inft. (IC50, μM) Cytotoxicity (μM)
16
17
18
19
20
21
22
5b
—
0.25
0.25
0.25
0.25
0.25
0.25
0.49
3.66
1.80
—
0.25
0.25
0.25
0.25
0.25
0.25
0.25
—
—
—
—
—
0.25
0.25
0.25
0.25
0.25
0.25
0.25
—
—
—
—
—
0.25
0.40
0.25
0.25
0.32
0.25
0.51
—
—
8.8
5.6
>32
0.1
0.25
4
1
1
1
–(CH2)4CH3
–(CH2)8CH3
–(CH2)10CH3
–(CH2)12CH3
–(CH2)13CH3
–(CH2)16CH3
Benznidazole
Nifurtimox
1
1
NDc
NDc
>32
>32
>32
>32
6b
7b
Allopurinol
8b
Sodium stibogluconate
Pentamidine
Amphotericin B
—
—
—
9b
10b
—
—
Data are displayed as mean SD, as obtained from quadruplicate wells. Cytotoxicity in murine macrophage host cells (Cytotoxic concentration; CC50
> 4 μM) and (CC50 0.25–4 μM) in human fibroblasts MRC-5 cell line.a IC50 = compound concentration (μM) required to reduce parasite viability by
50%. b Compounds 5–10 are standard control drugs used in respective experiments, Trypanosoma spp. (5 & 6), Leishmania (7, 8, 9 & 10); c ND = not
determined.
Scheme 1 Synthesis of conformationally-locked 5′-acylsulfamoyl LNA adenosine analogs (16–22). Reagents and conditions: (i) dry DMF, dry
Et3N, H2NSO2Cl (in dry DCM), 1 h; 31% (ii) dry EtOH, 20% Pd(OH)2/C, HCO2NH4, 80 °C, 15 h; 87% (iii) dry DMF, Cs2CO3, corresponding
N-hydroxy succinimide ester, rt, 1–2 h, 17 (20%), 18 (27%), 19 (22%), 20 (25%), 21 (34%) and 22 (15%).
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concentration; CC50 >4 μM) and (CC50 0.25–4 μM) in human
fibroblasts MRC-5 cell lines.
phosphate moiety is mimicked by the isosteric sulfamate, which
readily crosses cellular membranes due to the nonionic nature of
the sulfamoyl ester group. However, the compounds (1–4,
Fig. 1) from the literature were found to be extremely toxic.52–54
Therefore, we became interested in designing a new class of
molecules in which 9-adeninyl and the 5′-sulfamate moieties
were retained as active pharmacophores. We also introduced
conformationally-locked pentose-sugar as a unique sugar moiety
that is devoid of any intrinsic sugar flexibility. We argued that
the resulting adenosine derivatives 16–22 are likely to be
biologically active against parasites without enhancing the
toxicity of the molecules, because it is known that the sugar con-
formation seems to play an important role47 in dictating the
antiparasitic activity of adenosine sulfamoyl analogues;47 the
C3′-endo sugar (North-type) conformation of ribose sugar in
ribavirin derivative 11 [1-(5′-O-sulfamoyl-β-D-ribofuranosyl)-
Compound 16 seems to be more toxic to MRC-5 cell lines
whereas other molecules (17–22) were not found to be toxic up
to 1–4 μM concentration. These cytotoxicity results further
suggest the importance of the alkyl chain (R group, Table 1),
since compound 16 which is devoid of an acyl chain was found
to be toxic at the level of its IC50 concentration (0.25 μM),
whereas compound 17 (IC50 4.0 μM) with a pentanyl chain is
non-toxic up to 4 μM concentration. Increasing the chain length
from (C-9 to C-17) in compounds 18–22 resulted in increased
cytotoxicity (CC50 of 1 μM). Compounds 16–22 are analogues
of adenosine and this class of molecules is known to possess
antitrypanosomal and antileishmanial activity.45,46 5′-O-acyl sul-
famoyl adenosine (1) derivatives have previously been reported
(IC50 = 0.5–50 μM) for their antiparasitic activity.47 The wide
activity of these compounds acting against Trypanosoma and
Leishmania can be explained based on earlier studies which
suggests that Trypanosoma (T. Cruzi) enzymes are structurally
and functionally similar to Leishmania enzymes.48 Both orga-
nisms have a high degree of sequence similarity (64%) in
ergosta-type steroids which is a main membrane steroid.48 There-
fore it is quite reasonable to suggest that the compounds that
inhibit the growth of Trypanosoma may also have an inhibitory
effect on Leishmania. Apart from this, purine analogs are metab-
olized by these parasites to nucleotides and aminated to the
analogs of adenine nucleotides.49 This halts protein synthesis
and causes the breakdown of RNA. The metabolic pathways for
purines in protozoans differ significantly from the corresponding
pathways in human beings. As an example, allopurinol (7,
Fig. 1) is nontoxic to human beings and is aminated to adenine
nucleotide analogs by parasites.49
1,2,4-triazole-3-carboxamide] gives an ED50 of 0.03 μg mL−1
,
whereas a C2′-endo sugar (South-type) conformation of ribose
sugar in compound 12 [1-(5′-O-sulfamoyl-β-D-ribofuranosyl)-
1,2,4-triazole-3-thiocarboxamide] and compound 13 [1-(5′-O-
sulfamoyl-β-D-ribofuranosyl)-1,2,4-triazole-3-carbonitrile] was
found to be relatively less active, with ED50 of 2.5 μg mL−1 and
5.0 μg mL−1, respectively (Fig. 3).
3.2. Role of the sugar conformation
The role of the pentose-sugar conformation seems to play an
important role in determining the antiparasitic activity of adeno-
sine sulfamoyl analogues. The crystal structures of compounds
11–13 (Fig. 3) clearly showed the role of the sugar conformation
in that compound 11 (ED50 = 0.03 μg mL−1), which displayed
the best activity against L. donovani, is locked in C3′-endo
(North-type) conformation.47 On the other hand, compounds 12
& 13 (Fig. 3) are locked in C3′-exo (South-type) conformation,
and found to be poorly active with ED50 = 2.5 μg mL−1 and
5.0 μg mL−1, respectively.47 Despite the conformational differ-
ences in the sugar ring in these compounds 11–13 (Fig. 3), the
5′-sulfamoyl moiety is similarly oriented in these molecules with
respect to the ribose ring, with the NH2 group positioning itself
over the sugar-ring.47 This finding supports the orientation of the
3.1. Antiparasitic structure–activity comparison amongst
adenosine analogs, 1–4 with 16–22
The structural and functional design of previously synthesized
5′-O-sulfamoyladenosine analogues (1–4, Fig. 1)41,47,50,51 and
our present compounds (16–22, Table 1) are all inspired by
naturally-occurring adenosine 5′-phosphate (AMP), wherein the
Fig. 3 Sulfamoyl derivatives of ribavirin in North and South type conformations.
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5′-sulfamoyl group being relatively less important than the sugar
conformation, and further supports the crucial role of the sugar
conformation in determining the antiparasitic activity.47 There-
fore, based on this rationale, we synthesized compounds (16–22
in Scheme 1) by covalently attaching a 4′-CH2–O-2′ bridge
(Fig. 2) across C2′–C4′ of adenosine, which conformationally-
locked (3′-endo, North-type) the nucleosides and led to excellent
anti-parasitic activity (IC50 = 0.25–0.51 μM).
analyses were performed on E-Merck 60 F 254 precoated
aluminium thin layer chromatographic plates. All air-sensitive
reactions were carried out under a nitrogen atmosphere. Melting
points were determined on a Büchi melting point B-540 instru-
ment and are uncorrected. 1H and 13C NMR spectra were
recorded on a Bruker Biospin 400 MHz spectrometer in the indi-
cated solvents (TMS as an internal standard). The values of
chemical shifts are expressed in ppm and the coupling constants
(J) in hertz (Hz). Mass spectra were recorded in API 2000
LC/MS/MS system spectrometer and the IR spectra were
recorded on Perkin Elmer FT-IR spectrometer.
4. Conclusion
In conclusion, this study provides valuable data that 3′-endo-
locked 5′-acyl sulfamoylnucleosides 16–22 exhibit interesting
antileishmanial & antitrypanosomal properties with insignificant
toxicity to mammalian cells. Their unique and potent activity
profile represents new important directions in the search for
novel antiparasitic agents as a “proof-of-concept” that bicyclic
5′-acyl sulfamoyl adenosines possess strong antitrypanosomal
and antileishmania activity. This class of molecules should be
explored further to bridge the gap of unmet need for new anti-
parasitic agents to combat neglected diseases. This work also
suggests that variable chain length of the 5′-acyl group can
modulate the toxicity of 5′-O-sulfamoylnucleoside analogues.
Additional studies with compounds 16–22 on selected parasitic
species, pharmacological profiling of the individual compound,
mechanism-of-action studies involving enzymatic assays, and dose
response toxicity evaluations are currently under investigation.
(1S,3R,4R,7S)-7-Benzyloxy-1-methanesulfonamide-3-(adenine-
7-yl)-2,5-dioxabicyclo [2.2.1]heptane (15). To a solution of sulfa-
moyl chloride (6.8 mmol) in dry DCM was added ((1S,3R,4S,
7R)-3-(6-amino-9H-purin-9-yl)-7-(benzyloxy)-2,5-dioxa-bicyclo-
[2.2.1]heptan-1-yl)methanol, 1443 (1.0 g, 2.71 mmol) in dry
DMF (25 mL) and dry Et3N (0.49 mL, 3.54 mmol) at 0 °C.
Reaction mixture was stirred at rt for 1 h. Reaction was quenched
with water (60 mL) and extracted with ethyl acetate (3 × 50 mL).
Aqueous layer was basified to pH = 8 by 2 M aq. NaOH
solution. Aqueous layer was extracted with ethyl acetate
(3 × 30 mL). Combined organic layer was washed with water
(2 × 30 mL) and then with brine (40 mL). Organic layer was
dried over anhydrous sodium sulphate, filtered and concentrated
under vacuum to give crude product. Crude product was purified
by column chromatography (silica gel 100–200 mesh, eluent:
4% MeOH in DCM) to give pure compound 15 (0.400 g, 31%).
1H NMR (400 MHz, DMSO-d6): δ 3.92 (d, J = 8.0 Hz, 1 H,
H6′′), 4.04 (d, J = 8.0 Hz, 1 H, H6′), 4.41 (d, J = 11.6 Hz, 1 H,
H5′′), 4.54 (s, 1 H, H3′), 4.57 (d, J = 11.6 Hz, 1 H, H5′),
4.67 (d, J = 2.44 Hz, 2 H, CH2Ph), 4.85 (s, 1 H, H2′), 6.02 (s, 1
H, H1′), 7.26–7.40 (m, 7 H, 2 H D2O exchangeable, NH2–SO3–,
OCH2Ph), 7.70 (s, 2 H, D2O exchangeable, C6-NH2), 8.14 (s,
1 H, H2), 8.20 (s, 1 H, H8). 13C NMR (100.6 MHz, DMSO-d6):
δ 64.9 (C5′), 71.2 (CH2Ph), 71.8 (C6′), 76.9 (C2′), 77.8 (C3′),
84.5 (C4′), 85.3 (C1′), 118.8 (C9), 127.5 (Ar–C), 127.7 (Ar–C),
128.2 (Ar–C), 137.6 (Ar–C), 138.1 (C2), 148.6 (Ar–C),
152.7 (C8), 156.0 (C6). m/z calculated for [M]+ C18H20N6O6S,
448.45; found, 448.70.
5. Implication
The uniqueness of the sugar-locked 2′-O,4′-C-methylene-linked
bicyclic ribonucleosides of acylsulfamoyl adenosine is that their
3′-endo-locked conformation has successfully inhibited the
growth of protozoan parasites with insignificant cytotoxicity in
murine macrophage host cells (CC50 >4 μM) and in diploid
human fibroblasts MRC-5 cell line against mammalian cells
(CC50 4 μM). The notable chemical feature of the 3′-endo locked
compounds is their pendant 2′-O, 4′-C-methylene fused bridge
on the pentofuranosylribose unit, which virtually restricts the
conformational flexibility of the pentose-sugar ring, hence redu-
cing entropic contribution to the binding free-energy to the
target protein. The activity of these molecules in parasite infected
primary mouse peritoneal macrophages is now followed by a
search for the target enzyme in the parasites. This would allow
us to determine the inhibition constant of the target enzyme as
well as the pharmacokinetic properties of the 3′-endo locked
compounds, which we believe would lead us to determine the
crystal structure of the complex to advance the structure–activity
relationship of these new bicyclic ribonucleosides of acylsulfa-
moyl adenosine molecules.
(1S,3R,4R,7S)-7-Hydroxy-1-methanesulfonamide-3-(adenine-
7-yl)-2,5-dioxabicyclo [2.2.1]heptane (16). To a solution of 15
(5.0 g, 13.95 mmol) in EtOH (100 mL) was added 20%
Pd(OH)2/C (3.0 g) and ammonium formate (52.79 g, 837 mmol)
and heated to reflux. The same amount of ammonium formate
and 20% Pd(OH)2/C was added after 5 h and reaction was
further refluxed for 10 h. The reaction mixture was filtered over
celite and organic phase was evaporated to give crude product.
Crude product was purified by column chromatography (silica
gel 100–200 mesh, eluent: 8–10% MeOH in DCM) to give pure
1
compound 16 (3.5 g, 87%). H NMR (400 MHz, DMSO-d6):
δ 3.87 (d, J = 7.9 Hz, 1 H, H6′′), 4.02 (d, J = 7.9 Hz, 1 H, H6′),
4.34–4.46 (m, 2 H, H5′′, H2′), 4.50 (s, 1 H, H3′), 4.54–4.64
(m, 1 H, H5′), 5.95 (s, 1 H, H1′), 6.02 (d, J = 4.0 Hz, 1 H, D2O
exchangeable, OH), 7.36 (s, 2 H, D2O exchangeable, NH2–
SO3–), 7.67 (s, 2 H, D2O exchangeable, C6–NH2–), 8.15 (s,
1 H, H2), 8.26 (s, 1 H, H8). 13C NMR (100.6 MHz, DMSO-d6):
δ 65.5 (C5′), 70.9 (C2′), 71.3 (C6′), 79.3 (C3′), 85.26 (C1′),
6. Experimental section
6.1. Chemistry – general experimental methods
All chemicals and reagents used were of reagent grade. Purifi-
cation and drying of reagents and solvents was carried out
according to literature procedure.55 Thin layer chromatographic
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85.31 (C4′), 118.9 (Ar–C), 138.1 (C8), 148.6 (Ar–C), 152.8
(C2), 156.0 (C6). m/z calculated for [M + H]+ C11H14N6O6S,
358.33; found, 359.30.
(CvO). m/z calculated for [M]+ C21H32N6O7S, 512.58; found,
513.00.
((1R,3R,4R,7S)-3-(6-Amino-9H-purin-9-yl)-7-hydroxy-2-oxabi-
cyclo[2.2.1]heptan-1-yl)methyl dodecanoylsulfamate (19). Pro-
cedure B, White solid (0.100 g, 22%), mp 131–133 °C.
1H NMR (400 MHz, DMSO-d6): δ 0.84 (app t, J = 7.0 Hz, 3 H,
CH3), 1.14–1.27 (m, 16 H, CH2), 1.38–1.54 (m, 2 H, CH2),
2.14 (t, J = 7.24 Hz, 2 H, CH2), 3.81 (d, J = 8.56 Hz,1 H, H6′′),
3.96 (d, J = 8.0 Hz, 1 H, H6′), 4.36 (s, 1 H, H2′), 4.44 (s, 1 H,
H3′), 4.48 (d, J = 12.0 Hz, 1 H, H5′′), 4.61 (d, J = 12.0 Hz, 1 H,
H5′), 5.91 (s, 1 H, H1′), 5.97 (s, 1 H, D2O exchangeable, OH),
7.34 (s, 2 H, D2O exchangeable, NH2), 8.14 (s, 1 H, H2),
8.24 (s, 1 H, H8). 13C NMR (100.6 MHz, DMSO-d6): δ 14.0
(CH3), 22.1 (CH2), 24.6 (CH2), 28.6 (CH2), 28.7 (CH2), 28.8
(CH2), 29.0 (CH2), 31.3 (CH2), 36.6 (CH2), 66.8 (C5′), 70.7
(C2′), 71.2 (C6′), 79.3 (C3′), 85.4 (C1′), 118.9 (Ar–C), 138.0
(C8), 148.5 (Ar–C), 152.7 (C2), 156.3 (C6), 173.8 (CvO). m/z
calculated for [M]+ C23H36N6O7S, 540.63; found: 540.90.
General procedure A, for N-hydroxy succinimide esters
Lauric acid (0.502 g, 2.51 mmol) was dissolved in dry DCM
(3 mL), DIC (0.38 mL, 2.51 mmol) and N-hydroxy succinimide
(0.289 g, 2.51 mmol) was added. Reaction was stirred at room
temperature under nitrogen atmosphere for 15 min, filtered
through cotton bed and filtrate was concentrated under vacuum
to give pure lauric N-hydroxy succinimide ester.
General procedure B, for compounds 17–22
The respective N-hydroxy succinimide aliphatic ester (3 eq) was
dissolved in dry DMF (3 mL), sulfonamide 16 (0.300 g,
0.83 mmol, 1 eq) and Cs2CO3 (0.409 g, 1.25 mmol, 1.5 eq)
were added to it and stirred at room temperature under nitrogen
atmosphere for 1.5 h. Reaction was quenched by water (20 mL),
extracted with ethyl acetate (3 × 25 mL), combined organic layer
was again washed with water (2 × 15 mL) and then with brine
(20 mL). Organic layer was dried over anhydrous sodium
sulfate, filtered and concentrated under vacuum to give crude
product. Crude product was purified by column chromatography
(silica gel 100–200 mesh, eluent: 10% MeOH in DCM) to give
pure compound.
((1R,3R,4R,7S)-3-(6-Amino-9H-purin-9-yl)-7-hydroxy-2,5-dioxa-
bicyclo[2.2.1]heptan-1-yl)methyl tetradecanoylsulfamate (20). Pro-
cedure B, white solid (0.120 g, 25%), mp 158–161 °C. 1H NMR
(400 MHz, DMSO-d6): δ 0.85 (app t, J = 7 Hz, 3 H, CH3),
0.97–1.34 (m, 20 H, CH2), 1.38–1.51 (m, 2 H, CH2), 2.22 (t, J =
7.3 Hz, 2 H), 3.82 (d, J = 8.0 Hz, 1 H, H6′′), 3.97 (d, J =
8.0 Hz, 1 H, H6′), 4.36 (s, 1 H, H2′), 4.44 (s, 1 H, H3′), 4.49 (d,
J = 12.0 Hz, 1 H, H5′′), 4.62 (d, J = 12.0 Hz, 1 H, H5′), 5.90 (s,
1 H, H1′), 5.96 (br-s, 1 H, D2O exchangeable, OH), 7.34 (s,
2 H, D2O exchangeable, NH2), 8.14 (s, 1 H, H2), 8.24 (s, 1 H,
H8). 13C NMR (100.6 MHz, DMSO-d6): δ 14.0 (CH3), 22.1
(CH2), 25.4 (CH2), 28.7 (CH2), 28.8 (CH2), 28.94 (CH2), 28.99
(CH2), 29.03 (CH2), 31.3 (CH2), 64.2 (C5′), 70.7 (C2′), 71.3
(C6′), 79.1 (C3′), 85.3 (C1′), 85.8 (C4′), 118.9 (Ar–C), 138.0
(C8), 148.5 (Ar–C), 152.7 (C2), 156.0 (C6), 176.7 (CvO). m/z
calculated for [M]+ C25H40N6O7S, 568.69; found, 569.10.
((1R,3R,4R,7S)-3-(6-Amino-9H-purin-9-yl)-7-hydroxy-2,5-dioxa-
bicyclo[2.2.1]heptan-1-yl)methyl hexanoylsulfamate (17). Pro-
cedure B, white solid (0.080 g, 20%), mp 121–124 °C. 1H NMR
(400 MHz, DMSO-d6): δ 0.81 (app t, J = 7.0 Hz, 3 H, CH3),
1.16–1.27 (m, 4 H, CH2), 1.39–1.48 (m, 2 H, CH2), 2.02–2.14
(m, 2 H, CH2), 3.80 (d, J = 8.0 Hz, 1 H, H6′′), 3.96 (d, J =
8.0 Hz, 1 H, H6′), 4.35 (s, 1 H, H3′), 4.40–4.55 (m, 3 H, H2′,
H5′′, H5′), 5.90 (s, 1 H, H1′), 5.93 (s, 1 H, D2O exchangeable,
OH), 7.34 (s, 2 H, D2O exchangeable, NH2), 8.14 (s, 1 H, H2),
8.24 (s, 1 H, H8). 13C NMR (100.6 MHz, DMSO-d6):
14.2 (CH3), 22.3 (CH2), 25.1 (CH2), 31.3 (CH2), 37.9, (CH2),
66.0 (C5′), 71.1 (C2′), 71.6 (C6′), 79.6 (C3′), 85.6 (C1′),
86.0 (C4′), 119.4 (Ar–C), 138.4 (C8), 148.9 (Ar–C), 153.2 (C2),
156.4 (C6), 176.0 (CvO). m/z calculated for [M]+ C17H24-
N6O7S, 456.47; found 457.00.
((1R,3R,4R,7S)-3-(6-Amino-9H-purin-9-yl)-7-hydroxy-2,5-diox-
abicyclo[2.2.1]heptan-1-yl)methyl pentadecanoylsulfamate (21).
Procedure B, white solid (0.150 g, 34%), mp decomposes at
155 °C. 1H NMR (400 MHz, DMSO-d6): δ 0.84 (app t, J =
6.8 Hz, 3 H, CH3), 1.12–1.30 (m, 22 H, CH2), 1.38–1.52 (m,
2 H, CH2), 2.19 (app t, J = 7.3 Hz, 2 H, CH2), 3.82 (d, J =
8.0 Hz, 1 H, H6′′), 3.98 (d, J = 8.0 Hz, 1 H, H6′), 4.37 (s, 1 H,
H2′), 4.46 (s, 1 H, H3′), 4.54 (d, J = 12.0 Hz, 1 H, H5′′), 4.69
(d, J = 12.0 Hz, 1 H, H5′), 5.92 (s, 1 H, H1′), 7.36 (s, 2 H, D2O
exchangeable, NH2), 8.14 (s, 1 H, H2), 8.24 (s, 1 H, H8).
13C NMR (100.6 MHz, DMSO-d6): δ 14.0 (CH3), 22.1 (CH2),
24.3 (CH2), 28.5 (CH2), 28.8 (CH2), 28.96 (CH2), 29.1 (CH2),
31.4 (CH2), 35.9 (CH2), 67.9 (C5′), 70.8 (C2′), 71.2 (C6′), 79.3
(C3′), 85.3 (C1′), 85.5 (C4′), 119.0 (Ar–C), 138.0 (C8), 148.5
(Ar–C), 152.8 (C2), 156.1 (Ar–C), 172.60 (CvO). m/z calcu-
lated for [M]+ C26H42N6O7S, 582.71; found, 583.00.
((1R,3R,4R,7S)-3-(6-Amino-9H-purin-9-yl)-7-hydroxy-2,5-dioxa-
bicyclo[2.2.1]heptan-1-yl)methyl decanoylsulfamate (18). Pro-
cedure B, white solid (0.080 g, 27%), mp 115–117 °C. 1H NMR
(400 MHz, DMSO-d6): δ 0.83 (app t, J = 7.0 Hz, 3 H, CH3),
1.11–1.32 (m, 12 H, CH2), 1.38–1.55 (m, 2 H, CH2), 2.08–2.25
(m, 2 H, CH2), 3.81 (d, J = 8.0 Hz, 1 H, H6′′), 3.96 (d, J = 8.0
Hz, 1 H, H6′), 4.36 (s, 1 H, H2′), 4.44 (s, 1 H, H3′), 4.48 (d, J =
12.0 Hz, 1 H, H5′′), 4.60 (d, J = 11.1 Hz, 1 H, H5′), 5.91 (s,
1 H, H1′), 5.96 (s, 1 H, D2O exchangeable, OH), 7.34 (s, 2 H,
D2O exchangeable, NH2), 8.14 (s, 1 H, H2), 8.24 (s, 1 H, H8),
12.2 (br-s, 1 H, NH). 13C NMR (100.6 MHz, DMSO-d6): δ
13.9 (CH3), 22.1 (CH2), 24.7 (CH2), 28.56 (CH2), 28.63 (CH2),
28.8 (CH2), 31.2 (CH2), 36.8 (CH2), 66.4 (C5′), 70.7 (C2′),
71.2 (C6′), 79.2 (C3′), 85.3 (C1′), 85.5 (C4′), 118.9 (Ar–C),
137.9 (C8), 148.4 (Ar–C), 152.7 (C2), 156.0 (C6), 174.3
((1R,3R,4R,7S)-3-(6-Amino-9H-purin-9-yl)-7-hydroxy-2,5-dioxa-
bicyclo[2.2.1]heptan-1-yl)methyl stearoylsulfamate (22). Pro-
cedure B, white solid (0.075 g, 15%), mp 218–221 °C. 1H NMR
(400 MHz, DMSO-d6): δ 0.85 (app t, J = 6.8 Hz, 3 H, CH3),
0.97–1.35 (m, 28 H, CH2), 1.38–1.52 (m, 2 H, CH2), 2.22 (t,
J = 7.3 Hz, 2 H, CH2), 3.82 (d, J = 8.0 Hz, 1 H, H6′′), 3.97 (d,
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6121–6129 | 6127

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J = 8.0 Hz, 1 H, H6′), 4.38 (s, 1 H, H2′), 4.46 (s, 1 H, H3′),
4.57 (d, J = 12.0 Hz, 1 H, H5′′), 4.74 (d, J = 12.0 Hz, 1 H, H5′),
5.92 (s, 1 H, H1′), 6.01 (s, 1 H, D2O exchangeable, OH),
7.35 (s, 2 H, D2O exchangeable, NH2), 8.13 (s, 1 H, H2), 8.24
(s, 1 H, H8). 13C NMR (100.6 MHz, DMSO-d6): δ 14.02 (CH3),
22.2 (CH2), 24.0 (CH2), 28.4 (CH2), 28.7 (CH2), 29.1 (CH2),
31.3 (CH2), 35.4 (CH2), 68.5 (C5′), 70.8 (C2′), 71.1 (C6′), 79.4
(C3′), 85.14 (C1′), 85.5 (C4′), 118.97 (Ar–C), 138.0 (C8), 148.5
(Ar–C), 152.8 (C2), 156.0 (C6), 171.7 (CvO). m/z calculated
for [M]+ C29H48N6O7S, 624.79; found, 624.90.
axenic medium (BHI-tryptose) complemented with 5% fetal calf
serum. Cells from 5-day-old culture (stationary phase) were
inoculated to 50 mL of fresh culture medium to give an initial
concentration of 1 × 106 cells per mL. Cell growth was followed
by measuring the absorbance of the culture at 600 nm every day.
Before inoculation, the medium was supplemented with the indi-
cated amount of the studied compound from a stock solution in
DMSO. The final concentration of DMSO in the culture medium
never exceeded 0.4%, and the control was run in the presence of
0.4% DMSO and in the absence of compound. No effect on
epimatigotes’ growth was observed by the presence of up to 1%
DMSO in the culture medium. To determine IC50 values, 50%
inhibitory concentrations, parasite growth was followed in the
absence (control) and presence of increasing concentrations of
the corresponding compound. At day 5, the absorbance of the
culture was measured and related to the control. The IC50 value
was taken as the concentration of compound needed to reduce
the absorbance ratio to 50%.
6.2. Biological activity-methods
6.2.1. Amastigote culture and drug sensitivity assay. The
laboratory strain of Leishmania infantum was used for in vitro
screening of synthesized compounds. The in vitro sensitivity of
compounds (16–22) to amastigotes44 was determined in primary
mouse peritoneal macrophages (MPø).56 For the drug sensitivity
assay, the 16 μM compound stock solutions were four-fold seri-
ally diluted in DMSO, followed by an additional dilution in
culture medium (RPMI 1640 supplemented with 20 mM L-gluta-
mine, 16.5 mM NaHCO3, 5% heat-inactivated fetal calf serum
[FCSi], and 2% P/S solution). The highest in-test drug concen-
tration was 64 μg mL−1. Assays were performed in quadruplet in
96-well microtiter tissue culture plates (Nunc), with each well
containing the test compound dilutions together with 3 × 104
MPø and 3 × 105 amastigotes per well. After 5 days of incu-
bation at 37 °C in 5% CO2–air, intracellular amastigote burdens
(and cytotoxicity) were microscopically assessed after Giemsa
staining. The results are expressed as percent reduction of para-
site burden compared to the level in untreated control wells, and
the 50% inhibitory concentration (IC50) and 50% cytotoxic con-
centration (CC50) on MPø was determined.
6.2.4. In vitro cytotoxicity against mammalian cells. Diploid
human lung fibroblasts (MRC-5; BioWittaker) were cultured in
minimum essential medium (MEM; Gibco), supplemented with
20 mM L-glutamine, 16.5 mM NaHCO3, 5% FCSi, and 2% P/S
solution. The assay was performed in 96-well tissue culture
plates, with each well containing the test compound dilutions
together with 5 × 103 MRC-5 cells per well. After 7 days of
incubation at 37 °C in 5% CO2–air, cell viability was assessed
after addition of Alamar Blue58 (2 μL of a 1/10 solution per
well), and fluorescence was measured (Fluostar Optima [550-nm
excitation, 590-nm emission]) after 4 h of incubation at 37 °C.
The results are expressed as percent reduction in cell viability
compared to untreated control wells, and
determined.
a CC50 was
6.2.2. In vitro activity against Trypanosoma brucei subsp.
rhodesiense and Trypanosoma brucei subsp. Brucei. T. brucei
subsp. rhodesiense STIB900 BSF trypomastigotes were main-
tained in HMI-18 medium with 15% heat-inactivated fetal calf
serum (Harlan-SeraLab, United Kingdom) at 37 °C in a 5%
CO2–95% air mixture. Trypomastigotes were washed and resus-
pended in fresh medium at a concentration of 2 × 105 mL−1. The
top concentration for the test compounds was 64 μM. Five differ-
ent concentrations of drug were tested in triplicate. Plates were
incubated for 72 h at 37 °C in a 5% CO2–95% air mixture.
At 72 h, the plates were assessed microscopically before alamar-
Blue was added. Plates were read after 5 to 6 h on a Gemini
Fluorescent plate reader (Softmax Pro. 3.1.1, Molecular Devices,
United Kingdom) at an excitation/emission of 530/585 nm, with
a filter cutoff at 550 nm. 50% inhibition concentration (IC50)
values were calculated with Msxlfit (IDBS, United Kingdom).
For studies with T. brucei subsp. brucei bloodstream forms,
trypomastigotes were maintained in HMI-9 medium with 10%
heat-inactivated fetal calf serum (Gibco) at 37 °C in a 5% CO2–
95% air mixture. The HMI-9 medium was supplemented with
1 μg mL−1 of ergosterol, which was dissolved in DMSO. Pro-
cyclic forms were grown in SDM-79 with 10% heat-inactivated
fetal calf serum at 27 °C.
Abbreviations
DCM
DIC
DMF
dichloromethane
diisopropylcarbodiimide
N,N-dimethylformamide
DMSO dimethyl sulfoxide
Et3N
EtOH
triethylamine
ethanol
MeOH methanol
MIC
mp
minimum inhibitory concentration
melting point
NMR
SAR
THF
TLC
nuclear magnetic resonance
structure–activity relationship
tetrahydrofuran
thin layer chromatography
Acknowledgements
We are thankful to Drugs for Neglected Diseases Initiative
(DNDi), Geneva, Switzerland for testing our compounds against
protozoan parasites. We also thank Rahul Salunke and Ashish
Tripathi for initial work. Generous financial support from
the European Union (Project No. 222965, Project name:
New approaches to target Tuberculosis, Call identifier:
6.2.3. In vitro activity against Trypanosoma cruzi.57 T. cruzi
epimastigotes (Tulahuen 2 strain) were grown at 28 °C in an
6128 | Org. Biomol. Chem., 2012, 10, 6121–6129
This journal is © The Royal Society of Chemistry 2012

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30 H. Cerecetto, R. Di Maio, M. Gonzalez, M. Risso, G. Sagrera,
G. Seoane, A. Denicola, G. Peluffo, C. Quijano, A. O. Stoppani,
M. Paulino, C. Olea-Azar and M. A. Basombrio, Eur. J. Med. Chem.,
2000, 35, 343–350.
FP7-Health-2007-B) and Uppsala University is also gratefully
acknowledged.
31 J. Keiser, A. Stich and C. Burri, Trends Parasitol., 2001, 17, 42–49.
32 F. A. Molfetta, A. T. Bruni, K. M. Honorio and A. B. da Silva,
Eur. J. Med. Chem., 2005, 40, 329–338.
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