Cell permeation of a Trypanosoma brucei aldolase inhibitor: Evaluation of different enzyme-labile phosphate protecting groups

Article abstract of DOI:10.1016/j.bmcl.2006.04.010

A series of four prodrugs directed against Trypanosoma brucei aldolase bearing various transient enzyme-labile phosphate protecting groups was developed. Herein, we describe the synthesis and evaluation of cell permeation of these prodrugs. The oxymethyl

Full text of DOI:10.1016/j.bmcl.2006.04.010

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3440–3443
Cell permeation of a Trypanosoma brucei aldolase inhibitor:
Evaluation of different enzyme-labile phosphate protecting groups
a
b
a
a,
*
´
´
Laurent Azema, Christian Lherbet, Cecile Baudoin and Casimir Blonski
aLaboratoire SPCMIB, Groupe de Chimie Organique Biologique, Universite´ Paul Sabatier UMR CNRS 5068,
118 route de Narbonne, 31062 Toulouse Cedex 4, France
bDepartment of Chemistry, University of Utah, 2270 Henry Eyring Building, Salt Lake City, UT 84112, USA
Received 28 February 2006; revised 3 April 2006; accepted 3 April 2006
Available online 24 April 2006
Abstract—A series of four prodrugs directed against Trypanosoma brucei aldolase bearing various transient enzyme-labile phosphate
protecting groups was developed. Herein, we describe the synthesis and evaluation of cell permeation of these prodrugs. The
oxymethyl derivative was the most efficient prodrug with a good recovering of the free drug (IC50 = 20 lM) and without any
measurable cytotoxicity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Trypanosomiases, parasitic diseases, are endemic in
many countries of Africa and South America.1 Besides
the animal form, two human forms of the disease
occur: Trypanosoma cruzi in South America and
Trypanosoma brucei in Africa. These parasites have
developed resistance to chemotherapy and there is
an urgent need for the development of new drugs.2,3
It was found that the bloodstream form of the African
trypanosome is exclusively dependent on glycolysis for
energy production and therefore this metabolic path-
way represents a good target for the development of
new inhibitors.4–6
charged at physiological pHElsevier Ltd, did not readily
undergo passive diffusion across cellular membranes.
Accordingly, the inhibitor presented no activity on para-
sitic cultures.
This kind of problem has been widely studied during the
last twenty years, and various approaches were devel-
oped in order to enhance cell permeation of these poten-
tial drugs.12–17 Our interest focused on the use of
enzyme-labile transient phosphate protecting groups,
which mask the negative charges of the phosphate by
esterification, giving triester derivatives with increased
lipophilicity.18–20 This modification is known to facili-
tate the passive diffusion of the prodrug through the cell
membrane, followed by the delivery of the active form
of the drug by taking advantage of intracellular enzymes
such as the widely present esterases, phosphodiesterases
or reductases (Fig. 1).
In this field, our group has developed a mechanism-based
inhibitor of fructose-1,6-bisphosphate aldolase.7–9
Incubation of the glycolytic enzyme in the presence of
various dihydroxybenzaldehyde phosphate derivatives
resulted in a slow-binding inhibition of the aldolase.8,10
Interestingly, we observed that 2-hydroxybenzaldehyde-5-
phosphate inhibited the aldolase from T. brucei more
efficiently than the aldolase from mammalian origin.
The selectivity toward T. brucei was also observed for
an hydroxynaphthaldehyde phosphate inhibitor synthe-
tized in our group.11 Unfortunately, the benzaldehyde
derivative, bearing a phosphate group which is negatively
T.brucei cell
membrane
O
P
O
P
O
P
O
OR
OR
OR
OR
OH
drug-O
drug-O
drug-O
2
1
Keywords: Trypanosoma brucei; Aldolase; Phosphate; Inhibitor;
Enzyme-labile protecting group; Drug delivery.
Figure 1. Protection of the phosphate group allows for passive
diffusion through the cell membrane (arrow 1), before deprotection
by suitable cytosolic enzymes (arrow 2).
*
Corresponding author. Tel.: +33 5 6155 6486; fax: +33 5 6155
6011; e-mail: blonski@cict.fr
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.010

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L. Azema et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3440–3443
3441
Herein, we report the synthesis of selected enzyme-labile
phosphate protecting groups and their evaluation for
their ability to liberate the active drug in the presence
of suitable enzymes or parasite cell extracts. Studies on
parasite cultures are also reported. The different protect-
ing groups under study are indicated in Figure 2.
compound 5 in a good yield.30 Phosphate deprotection
with trimethylsilylbromide was followed by hydrolysis
of the pivaloic ester, thus affording compound 6 in quan-
titative yield. The silver salt of derivative 6 was prepared
by using silver nitrate. Then the phosphate derivative
was allowed to react with compounds 9–11 (Scheme 2)
to afford compounds 1, 2, and 4 in reasonable yields.
Prodrugs 1 and 2, bearing a bis(acyloxymethyl) protect-
ing group,21,22 are sensitive to esterases which are
present in the targeted parasite. Prodrug 3, which incor-
porates the S-acyl 2-thioethyl protecting group, is sensi-
tive to the same kind of enzyme, but releases an
episulfide instead of formol as a by-product.23 Finally,
prodrug 4 incorporates the S-[(2-hydroxyethyl)sulf-
idyl]-2-thioethyl group as a reductase-labile phosphate
protection.24–28
Whereas the synthesis of compound 10 was described
elsewhere,23,31–33 an original synthesis has been devel-
oped for compound 9, as shown in Scheme 2.
The first step is the mono-acetylation of dithioethanol
using acetic anhydride to afford derivative 7, which
was subsequently tosylated, leading to the new ester 8.
Nucleophilic substitution of the tosylate group by iodine
afforded precursor 9, which was used directly for the
next step (Scheme 1).
Two different synthetic pathways have been developed
to obtain compounds 1–4. Prodrugs 1, 2, and 4 were
synthesized by the reaction sequence described in
Scheme 1. Selective protection of 2,5-dihydroxybenzal-
dehyde (2,5-DHBA) on position 2 was achieved by using
pivaloyl chloride in the presence of sodium hydride.29
Subsequent phosphorylation on position 5 by using
triethylphosphite in the presence of iodine gave
Scheme 3 describes the synthesis of the S-acyl 2-thioethyl
derivative 3.
O
P
a
b
S
S
SH
HO
H
O
HO
2
O
O
12
13
O
P
S
O
O
O
c
O
2
O
R =
CH2
CH2
CH2
CH2
S
CMe3
O
O
CMe3
P
OR
OR
O
CHO
O
1
3
OH
O
O
3
SS
CHO
O
OH
Scheme 3. Synthesis of compound 3. Reagents and conditions: (a)
NaH, Et2O, 3 h, 0 ꢁC then Piv-Cl, 3 h, 78%; (b) PCl3, 1-H tetrazole,
pyridine, 3 h, then 12, pyridine, 12 h, then H2O, 30 min, 0 ꢁC, 52%; (c)
2,5-DHBA, CCl4, DIPEA, DMAP, acetonitrile, 15 min, 0 ꢁC, then 13,
10 min, 0 ꢁC, 55%.
AcO
2
4
Figure 2. Structure of the compounds with four different phosphate
protecting groups, studied herein.
O
P OEt
O
O
P O- Na+
P OR
O- Na+
O
O
OH
OH
OEt
O
OR
a, b
d, e
c
CHO
CHO
CHO
CHO
OPiv
OH
OH
1,2,4
5
6
Scheme 1. Synthesis of the potential prodrugs 1, 2, and 4. Reagents and conditions: (a) NaH, DMF, 3 h, then Piv-Cl, 12 h, 55%; (b) P(OEt)3, I2,
CH2Cl2, pyridine, 30 min, 98%; (c) Me3SiBr, 12 h, then H2O, NaOH, 10 min, 97%; (d) AgNO3, H2O, 12 h; (e) 9, 10 or 11, toluene, reflux, 12 h,
17–44%.
O
O
O
a
b
S
SS
SS
SS
O
O
HO
O
HO
OH
O
O
7
8
c
O
Cl
O
O
SS
O
Cl
O
I
O
O
10
9
11
Scheme 2. Synthesis of compound 9. Reagents and conditions: (a) Ac2O, DIPEA, CH2Cl2, 12 h, 51%; (b) TsCl, pyridine, 12 h, 75%; (c) NaI, acetone,
reflux, 12 h.

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L. Azema et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3440–3443
3442
The first step is the selective esterification of mercap-
toethanol using pivaloyl chloride and sodium hydride
to afford compound 12.24 Reaction of phosphorus tritet-
razolide (formed in situ) with the thioester 12 afforded
the H-phosphonate derivative 13. The last step was the
condensation of derivative 13 with 2,5-dihydroxybenzal-
dehyde through a Todt reaction to obtain the expected
prodrug 3, in good yield.34
phosphodiester intermediate is a very good substrate
for a phosphodiesterase.
Different behaviors occurred in the presence of the
T. brucei cell extract. Table 1 summarizes these results.
Compounds 1 and 2 released the active form of the drug,
while compound 3 was not fully enzymatically degraded
(data not shown). Indeed, we observed that the depro-
tection reaction stopped after around 20% of mono-
deprotected compound formation. This suggests that
the by-product (episulfide) may act as an inhibitor of
the esterase of the parasite. Since compound 4 was un-
able to release the free drug in the in vitro assay, it is
concluded that it was not a proper substrate of the
reductases found in the T. brucei cell extract.
The stability of the prodrugs 1–4 was assessed in 50 mM
TEA–HCl buffer, 5% DMSO at pH 7.0 and 37 ꢁC. The
degradation of these compounds (monitored by UV–vis
spectroscopy, data not shown) is ascribed to the forma-
tion of the mono-protected form of the free drug.23 The
half-lives for hydrolysis of compounds 1–4 were exam-
ined. Compound 2 is the most sensitive prodrug, with
an half-life of 28 h. The second oxymethyl type prodrug
1 is slightly more stable, with an half-life of 32 h. Even
though it is a sensitive protecting group, it is enough
to be used to deliver the drug into cell.
Finally, the prodrugs were tested in vitro, against differ-
ent parasitic systems (Table 2). Compound 2 did not
present any activity. This could stem from the nature
of the protection: poor stability in the serum used for
the assay on the one hand, and high aromaticity, leading
to a stacking effect in the membrane on the other hand.
Compound 1 was active only against T. brucei and
showed no cytotoxicity. This means that this system is
able to release the drug into the cell and that the formol
(the by-product of the deprotection reaction) is not toxic
for the human cell for tested concentrations. This result
is consistent with the fact that in this study only
T. brucei grows in a free-macrophage medium. With
the other parasites, the prodrug has to diffuse across
two cellular membranes (at the level of the macrophage
and of the parasite) to be efficient. The second step is not
made possible by the activity of the enzymes of the mac-
rophage. Compound 3 was active against both T. brucei
and the other systems studied, and was cytotoxic at the
The thioester derivative 3 appears to be much more sta-
ble, with an half-time of 49 h. Finally, compound 4 is the
most stable and only the hydrolysis of the ester group is
observed (half-time of 45 h) without formation of the
free drug.
The drug release efficiency of compounds 1–3 sensitive
to esterases (and then also to phosphodiesterases, see be-
low) was subsequently tested and monitored by HPLC.
Relative peak integrations permitted the estimation of
fully and mono-protected compounds present in the
mixture, the half-lives are summarized in Table 1. As
it is not possible to normalize these data, these experi-
ments give us an insight about the relative efficiency of
the prodrug for the release of the free drug. Compounds
1–3 were tested with a mixture of commercially available
pig liver esterase and crotale venom phosphodiesterase
II. Compounds 1 and 2 gave the same pattern, with an
accumulation of the mono-protected drug, before the
concentration of free drug increased. These profiles are
consistent with the proposed mechanism where a first
deprotection step catalyzed by an esterase is followed
by a second deprotection by phosphodiesterase or
esterase.16 Compound 2 releases the free drug at a higher
rate, probably because the cleavage site for esterase is
distant from the first created charge, allowing a second
attack by esterase. Compound 3 presents a slightly
different profile. No accumulation of the mono-
protected compound was observed, suggesting that the
Table 2. IC50 (lM) determined in the presence of different parasitic
systems with compounds 1–435
Compound
6c
1
2
3
4
T.ba
>50
>50
>50
>50
>50
20
>50
>50
>50
>50
>50
16
15
25
12
6
>50
>50
>50
>50
>50
T.ca
>50
>50
>50
>50
L.ia
P.fa
Cytotoxicityb
a Abbreviation: T.b: Trypanosoma brucei; T.c: Trypanosoma cruzi; L.i:
Leishmania infantilis; and P.f: Plasmodium falciparum.
b The cytotoxicity is determined on MRC-5 cells.
c Control.
Table 1. Half-life time of the release of the mono-protected drug (diester) and of the free drug in the presence of a mixture of standard esterase and
phosphodiesterase (mixed enzymes) or in the presence of Trypanosoma brucei (cell extract)a
Compound:
1
2
3
4
Mixed enzymes
Cell extract
Diester
Free drug
35 min
8 h 10 min
10 min
1 h 30 min
4 h 40 min
5 h
ndb
ndb
Diester
Free drug
8 h 20 min
>25 h
2 h 30 min
20 h
>25 h
>25 h
Stable
Stable
a [1–4] = 500 lM, in 50 mM TEA–HCl, pH 7, at 37 ꢁC; PLE: 80 UI; crotale venom phosphodiesterase II: 0.5 UI; T. brucei cells: 0.2 · 1019 per assay.
b nd, not determined.

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L. Azema et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3440–3443
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In this paper, we report the first study concerning the
cell permeation of an inhibitor directed against the
aldolase of T. brucei using four different enzyme-labile
phosphate protecting groups. We show that the pivaloyl
oxymethyl protection (compound 1) is especially suit-
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