Synthesis of 16-mercaptohexadecylphosphocholine, a miltefosine analog with leishmanicidal activity

Article abstract of DOI:10.1016/j.bmcl.2006.07.004

The alkylphosphocholine miltefosine (n-hexadecylphosphocholine, MT) has been introduced recently as a very effective drug for the oral treatment of human leishmaniasis. However, the parasiticidal mechanism of MT at a molecular level is far from being understood. Here we report the synthesis and biological characterization of 16-mercaptohexadecylphosphocholine, a thiol analog of MT which was designed to facilitate the search of MT interacting targets within the parasite by a variety of analytical methods. This analog presents the same leishmanicidal effect as the parent drug against Leishmania donovani promastigotes and Leishmania pifanoi axenic amastigotes, and has been used to develop an affinity chromatography method to attempt the isolation of putative Leishmania proteins that bind to the phosphocholine part of the molecule.

Full text of DOI:10.1016/j.bmcl.2006.07.004

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5190–5193
Synthesis of 16-mercaptohexadecylphosphocholine, a miltefosine
analog with leishmanicidal activity
a
b
c
c
´
´
´
Valentın Hornillos, Jose Marıa Saugar, Beatriz G. de la Torre, David Andreu,
Luis Rivas,b A. Ulises Acunad,* and Francisco Amat-Guerria,*
˜
aInstituto de Quı´mica Orga´nica, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
bCentro de Investigaciones Biolo´gicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
c
`
Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra, Dr. Aiguader 80, 08003 Barcelona, Spain
dInstituto de Quı´mica Fı´sica Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
Received 30 May 2006; revised 5 July 2006; accepted 5 July 2006
Available online 25 July 2006
Abstract—The alkylphosphocholine miltefosine (n-hexadecylphosphocholine, MT) has been introduced recently as a very
effective drug for the oral treatment of human leishmaniasis. However, the parasiticidal mechanism of MT at a molecular
level is far from being understood. Here we report the synthesis and biological characterization of 16-merca-
ptohexadecylphosphocholine, a thiol analog of MT which was designed to facilitate the search of MT interacting targets
within the parasite by a variety of analytical methods. This analog presents the same leishmanicidal effect as the parent drug
against Leishmania donovani promastigotes and Leishmania pifanoi axenic amastigotes, and has been used to develop an
affinity chromatography method to attempt the isolation of putative Leishmania proteins that bind to the phosphocholine part
of the molecule.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Miltefosine (n-hexadecylphosphocholine, MT) is a syn-
thetic alkyl phospholipid with a major application in
the treatment of human leishmaniasis, a group of diseas-
es caused by the infection with Leishmania parasites.1,2
MT is the first effective oral drug against both visceral3
and cutaneous4,5 forms of the infection, preserving its
activity against antimonial-resistant parasites1,6 or on
immunodepressed patients,7 without the severe side
effects common to most of the first-line leishmanicidal
drugs.8 In contrast, the information available on the ori-
gin of the efficient parasiticidal mechanism of MT and
on the metabolism and the subcellular interactions of
the drug in Leishmania is scarce.9–11 Interestingly, it
has been known recently that MT induces apoptosis-like
death in Leishmania donovani,12,13 although the primary
targets of the drug remain to be determined. In fact, the
sole mechanism of Leishmania resistance to MT so far
described consists in a faulty uptake of the drug,14 asso-
ciated to loss-of-function of a plasma membrane
translocator.15
The current knowledge on the structure–antiparasite
activity relationship of MT analogs is also limited.1,16
It has been shown that an alkyl chain length of 16 or
18 methylene groups yields the highest in vitro activity,
while in vivo assays indicated that the C16 analog is the
most active.17,18 MT analogs with the lipophilic groups
cyclohexylideneundecyl, adamantylideneundecyl, dode-
cylidenecyclohexyloxyethyl or tetradecylidene-cyclo-
hexyloxyethyl showed more in vitro leishmanicidal
activity than MT, and among them only the two latter
compounds demonstrated higher cytotoxicity in vivo
than the parent drug. Analogs with shorter chains such
as phenoxyhexadecyl or 2-naphthyloxyethyl groups
were devoid of activity.19 Regarding the polar head
group, it has been found that compounds with the termi-
nal trimethylammonium group, as in phosphocholine,
present higher in vitro activity than the corresponding
analogs with ammonium, N-methylpiperidinium, or
N-methylmorpholinium terminal groups.17–19 In addi-
tion, a few phosphocholine derivatives have been
Keywords: Miltefosine; Hexadecylphosphocholine; Leishmania; Para-
siticide; Mercaptophosphocholine.
*
Corresponding authors. Tel.: +34 915622900x394; fax: +34
915644853; e-mail addresses: roculises@iqfr.csic.es; famat@iqog.
csic.es
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.004

V. Hornillos et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5190–5193
5191
synthesized which contain a specific reporter group
(probe). That is the case of MT analogs bearing the tet-
rafluorophenylazido photolabeling group20 or nitroxide
paramagnetic group.21 These compounds were
produced to investigate different aspects of the biologi-
cal effects of alkylphosphocholines, although the leish-
manicidal activity remains to be determined. Finally, a
fluorescent miltefosine analog has been obtained recent-
ly, in which the potent anti-Leishmania activity of the
parent drug has been preserved.22
Alcohol 3 could be also obtained, albeit with lower over-
all yield (50%), from the carboxylic acid 4, after trityl
protection of the terminal mercapto group and esterifi-
cation/reduction of the carboxylic acid group,30,31 fol-
lowing methods previously used for the incorporation
of protected mercapto groups to lipid molecules.32
Thiol 1 is stable in solid form, provided it is stored at
low temperature under an inert atmosphere, and the
freshly prepared samples are free of disulfide derivatives,
as determined by Ellman titration33 and 1H NMR spec-
troscopy. The MT-SH methanol solution is stable for
weeks at room temperature and in the presence of air.
However, 1 is slowly oxidized to disulfide in DMSO
solution, as other thiols.34
The availability of SH-substituted analogs of MT with
antiparasite properties would be of great utility in the
search of possible mechanisms of activity of the parent
drug. First, these analogs would make it possible to
develop isolation techniques of putative MT receptors
and targets in Leishmania parasites based on affinity
chromatography methods.23 In addition, self-assembled
monolayers of miltefosine may be obtained by covalent
bonding of the SH-substituted analog to a gold sur-
face.24 This derivatized gold substrate may be used as
a specific sensor of Leishmania proteins that bind to
the phosphocholine part of the molecule. Finally, the
presence of the reactive SH-group in the MT analog
would allow both the covalent attachment to the drug
of a variety of probes (as biotin) and the reversible link-
age of carrier molecules.25 With these possible applica-
tions in mind, we wanted to report the synthesis and
preliminary study of the in vitro leishmanicidal activity
of 16-mercaptohexadecylphosphocholine (1, MT-SH),
a MT analog substituted with the moderately lipophilic
mercapto group at the end of the molecule alkyl chain.
The leishmanicidal activity of MT-SH was assayed in vi-
tro following standard protocols.35,36 In this way, the
inhibition of L. donovani promastigote proliferation by
MT-SH (Fig. 1B) and, more important, of Leishmania
pifanoi axenic amastigotes (Fig. 1D)—the form respon-
sible for the pathology in vertebrates—was found to
be identical to that of the parent drug (Figs. 1A and
C). The data of Fig. 1 also show that both, the original
drug and its mercapto analog, produce a reduced effect
on the respective MT-resistant strains. Furthermore,
the concentration-dependent leishmanicidal activity
was the same for both compounds, regardless of the par-
asite stage and the MT-resistance of the isolate assayed.
In fact, the difference between the respective LD50 values
(Table 1) has no statistical significance. Note also that
the similarity of the axenic amastigotes with those ob-
tained from infected macrophages has been demonstrat-
ed by functional, morphological, antigenic, and
metabolic criteria.37
O
P
N
R
O
O
16
O
Miltefosine (MT): R = H
1
(MT-SH): R = SH
Thiol 1 was synthesized from bromoalcohol 2 in three
steps (Scheme 1): (1) reaction of 2 with trityl sulfide,
yielding the thiol-protected alcohol 3 with 90% yield;26
(2) introduction of the phosphocholine group in 3 by
reaction with 2-chloro-2-oxo-1,3,2-dioxaphospholane
and trimethylamine;27 and (3) trityl deprotection with
triethylsilane/trifluoroacetic acid.28 Thiol 1 was thus iso-
lated in pure form as a white powder in amounts in the
range of hundreds of milligrams and with fair overall
yield (36%).29
b, c
a
1
Br
OH
Ph3C
S
OH
16
16
2
3
d, e, f
HS
CO2H
15
4
Figure 1. Leishmanicidal activity of miltefosine (MT) (A and C) and
16-mercaptohexadecylphosphocholine (MT-SH) (B and D), expressed
as the percentage of proliferation inhibition relative to that of parasites
grown in the absence of drug. Both compounds were tested on
Leishmania donovani promastigotes (A and B) and on Leishmania
pifanoi axenic amastigotes (C and D). (d) MT-susceptible parasites;
(.) MT-resistant strains. For experimental conditions, see Ref. 36.
Scheme 1. Synthesis of thiol 1. Reagents and conditions: (a) Ph3CSH,
K2CO3, MeOH, Ar, 90%; (b) 2-chloro-2-oxo-1,3,2-dioxaphospholane,
Me3N, MeCN, Ar, pressure tube, À78 ꢁC, then rt, 2 h, and 70 ꢁC, 4 h,
43%; (c) triethylsilane, TFA/CH2Cl2 1:1, Ar, rt, 1 h, 83%; (d) Ph3CCl,
DMF, Ar, rt, 48 h, 60%; (e) Cl2SO, MeOH, Ar, 0 ꢁC, then rt, 1 h, 98%;
(f) DIBAL-H, MePh, Ar, rt, 1 h, 85%.

5192
V. Hornillos et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5190–5193
Table 1. Leishmanicidal (as in Fig. 1) activity of miltefosine (MT) and
16-mercaptohexadecylphosphocholine (MT-HS) in vitro
8. Croft, S. L.; Sundar, S.; Fairlamb, A. H. Clin. Microbiol.
Rev. 2006, 19, 111.
a
9. Lux, H.; Heise, N.; Klenner, T.; Hart, D.; Opperdoes, F.
R. Mol. Biochem. Parasitol. 2000, 111, 1.
Parasite
LD50 (lM)
MT
5.3 ( 0.5) 5.1 ( 0.1)
>50 >50
5.0 ( 0.2) 5.5 ( 0.1)
>50
MT-SH
10. Santa-Rita, R. M.; Henriques-Pons, A.; Barbosa, H. S.; de
Castro, S. L. J. Antimicrob. Chemother. 2004, 54, 704.
11. Rakotomanga, M.; Saint-Pierre-Chazalet, M.; Loiseau, P.
M. Antimicrob. Agents Chemother. 2005, 49, 2677.
12. Paris, C.; Loiseau, P. M.; Bories, C.; Breard, J. Antimic-
rob. Agents Chemother. 2004, 48, 852.
L. donovani promastigote
L. donovani promastigote (MT-resistant)
L. pifanoi axenic amastigote
L. pifanoi axenic amastigote (MT-resistant) >50
a LD50: drug concentration required to inhibit 50% parasite prolifer-
ation; mean values of three experiments; standard deviation given
between parentheses.
13. Verma, N. K.; Dey, C. S. Antimicrob. Agents Chemother.
2004, 48, 3010.
´
14. Perez-Victoria, F. J.; Castanys, S.; Gamarro, F. Antimic-
rob. Agents Chemother. 2003, 47, 2397.
´
15. Perez-Victoria, F. J.; Gamarro, F.; Ouellette, M.; Casta-
nys, S. J. Biol. Chem. 2003, 278, 49965.
ˆ
16. Croft, S. L.; Seifert, K.; Duchene, M. Mol. Biochem.
Parasitol. 2003, 126, 165.
17. Croft, S. L.; Neal, R. A.; Pendergast, W.; Chan, J. H.
Biochem. Pharmacol. 1987, 36, 2633.
18. Unger, C.; Maniera, T.; Kaufmann-Kolle, P.; Eibl, H.
Drugs Today 1998, 34, 133.
The use of axenic amastigotes, which proliferate in the
absence of host cells, removes interfering effects due to
the uptake and transit of MT or its analog MT-SH
through the macrophage, in order to reach the intracel-
lular amastigote.
As noted above, the reactivity of the thiol group and the
potent antiparasite effect of 1 provide the basis for sev-
eral applications to investigate the MT leishmanicidal
mechanism, as well as that of drug’s resistances that
would likely emerge in treated patients. For instance,
we have prepared an immobilized form of MT by the
facile reaction of 1 with iodoacetyl-modified agarose.38
This material is being used as an affinity column to
capture putative MT target proteins from Leishmania
parasite lysates.
19. Avlonitis, N.; Lekka, E.; Detsi, A.; Koufaki, M.; Calog-
eropoulou, T.; Scoulica, E.; Siapi, E.; Kyrikou, I.;
Mavromoustakos, T.; Tsotinis, A.; Grdadolnik, S. G.;
Makriyannis, A. J. Med. Chem. 2003, 46, 755.
20. Huang, F.; Qu, F.; Peng, Q.; Xia, Y.; Peng, L. J. Fluorine
Chem. 2005, 126, 739.
ˇ
21. Mravljak, J.; Zeisig, R.; Pecar, S. J. Med. Chem. 2005, 48,
6393.
22. Hornillos, V.: Saugar, J. M.; Luque-Ortega, J. R.; Acun˜a,
A. U.; Amat-Guerri, F., Rivas, L., in preparation.
23. Carlsson, J.; Janson, J. C.; Sparrman, M.. In Janson, J. C.,
´
Ryden, L., Eds.; Protein Purification. Principles, High-
Resolution Methods, and Applications; Wiley: New York,
1998; p 275.
Acknowledgments
24. Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164.
25. Reshetnyak, Y. K.; Andreev, O. A.; Lehnert, U.; Engel-
man, D. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 6460.
26. Compound 3: colorless powder, 90% from 2; TLC, Rf
This work was supported by projects BQU2003/4413,
SAF2002-11186E, and BIO2003-09056.CO2-O2, from
´
the Ministerio de Educacion y Ciencia (MEC) of Spain,
1
(hexane/Et2O, 1:1): 0.75; H NMR (300 MHz, CDCl3): d
and UE QLK2-CT-2001-01401, from the European
Union. VH acknowledges a FPI fellowship from
MEC. Miltefosine was kindly provided by Zentaris,
1.02–1.48 (m, 24H, H-3 to H-14), 1.37 (qn, J = 7.2 Hz, 2H,
H-15), 1.56 (qn, J = 7.3 Hz, 2H, H-2), 2.13 (t, J = 7.2 Hz,
2H, H-16), 3.64 (t, J = 7.4 Hz, 2H, CH2OH), 7.19 (t,
J = 7.1 Hz, 3H, Hp-Ph), 7.27 (t, J = 7.3 Hz, 6H, Hm-Ph),
7.41 (d, J = 7.5 Hz, 6H, Ho-Ph); 13C NMR (75 MHz,
CDCl3): d 25.7 (C-3), 28.4–29.6 (C-4 to C-15), 32.0 (C-16),
32.8 (C-2), 63.5 (C-1), 66.8 (CPh3), 126.9 (Cp), 128.2 (Cm),
130.02 (Co), 145.5 (Ci); FT IR (KBr) mmax: 3435, 2918,
2850, 1631, 1591, 1483, 1465, 1445, 743, 701 cmÀ1; MS
ES+, m/z: 539.3 [M+Na]+, 243.1 [CPh3]+; Anal. Calcd for
C29H42O2S (502.33): C, 81.22; H, 9.22; S, 6.38. Found: C,
81.25; H, 9.38; S, 6.15.
Frankfurt, Germany.
¨
References and notes
1. Sindermann, H.; Croft, S. L.; Engel, K. R.; Bommer, W.;
Eibl, H. J.; Unger, C.; Engel, J. Med. Microbiol. Immunol.
(Berl) 2004, 193, 173.
2. Berman, J. Curr. Opin. Infect. Dis. 2003, 16, 397.
3. Sundar, S.; Jha, T. K.; Thakur, C. P.; Engel, J.; Sinder-
mann, H.; Fischer, C.; Junge, K.; Bryceson, A.; Berman, J.
New Engl. J. Med. 2002, 347, 1739.
4. Soto, J.; Arana, B. A.; Toledo, J.; Rizzo, N.; Vega, J. C.;
Diaz, A.; Luz, M.; Gutierrez, P.; Arboleda, M.; Berman, J.
D.; Junge, K.; Engel, J.; Sindermann, H. Clin. Infect. Dis.
2004, 38, 1266.
5. Yardley, V.; Croft, S. L.; De Doncker, S.; Dujardin, J. C.;
Koirala, S.; Rijal, S.; Miranda, C.; Llanos-Cuentas, A.;
Chappuis, F. Am. J. Trop. Med. Hyg. 2005, 73, 272.
6. Carter, K. C.; Sundar, S.; Spickett, C.; Pereira, O. C.;
Mullen, A. B. Antimicrob. Agents Chemother. 2003, 47,
1529.
27. Quesada, E.; Delgado, J.; Gajate, C.; Mollinedo, F.; Acun˜a,
A. U.; Amat-Guerri, F. J. Med. Chem. 2004, 47, 5333.
28. Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon,
C. A. Tetrahedron Lett. 1989, 30, 2739.
29. Compound 1 (MT-SH): purified by column chromatog-
raphy (CHCl3/MeOH, 9:1, v/v and then CHCl3/MeOH/
H2O, 65:25:5) and successive precipitation from CHCl3/
acetone and CHCl3/Et2O; white powder, 32% overall yield
from 2; mp 229–231 ꢁC. Rf = 0.38 (CHCl3/MeOH/H2O,
65:25:5); 1H NMR (300 MHz, DMSO-d6): d 1.11–1.38 (m,
24H, H-3 to H-14), 1.45 (m, 2H, H-2), 1.51 (q, J = 7.0 Hz,
2H, H-15), 2.22 (t, J = 7.6 Hz, 1H, SH), 2.46 (m,
J = 7.2 Hz, 2H, H-16), 3.13 (br s, 9H, N(CH3)3), 3.48 (br
s, 2H, CH2N), 3.57 (q, J = 6.5 Hz, 2H, H-1), 3.99 (br s,
2H, OCH2CH2N); 13C NMR (75 MHz, DMSO-d6): d 23.7
(C-16), 25.5–29.1 (C-3 to C-14), 30.6 (d, J = 7.3 Hz, C-2),
7. Schraner, C.; Hasse, B.; Hasse, U.; Baumann, D.; Faeh,
A.; Burg, G.; Grimm, F.; Mathis, A.; Weber, R.;
Gunthard, H. F. Clin. Infect. Dis. 2005, 40, 120.

V. Hornillos et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5190–5193
5193
33.4 (C-15), 52.9, 53.0, 53.1 (N(CH3)3), 58.1 (d, J = 4.8 Hz,
CH2N), 63.8 (d, J = 6.0 Hz, C-1), 65.5 (br s, OCH2CH2N);
FT IR (KBr) mmax: 3028, 2917, 2849, 2551, 1483, 1471,
1243, 1079, 1060, 968 cmÀ1; ESI+ MS, m/z: 440.4 [M+H]+,
879.5 [2M+H]+, 1318.9 [3M+H]+; HR MS (L-SIMS) calcd
for (C21H46NO4PS)H+: 440.2963; found: 440.2965. Anal.
Calcd for C21H46NO4PSÆH2O (457.65): C, 55.11; H, 10.57;
N, 3.06; S, 7.29. Found: C, 55.07; H, 10.18; N, 3.22; S,
6.91.
(4· 106 parasites/mL) and transferred to a 96-microwell
plate (50 lL/well); an equal volume of MT solution in
growth medium at twice the final concentration was
added to each well. Parasites were allowed to proliferate
for 72 h at 27 ꢁC (promastigotes) or for 120 h at 32 ꢁC
(axenic amastigotes). Then, parasites were removed from
the plate and washed twice with 1 mL Hanks medium
(136 mM NaCl, 4.2 mM Na2HPO4, 4.4 mM KH2PO4,
5.4 mM KCl, and 4.1 mM NaHCO3, pH 7.2), supple-
mented with 20 mM D-glucose. Assay of inhibition of
parasite proliferation. Parasites were resuspended in
100 lL of 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) solution in Hanks
medium. MTT reduction was carried out at 26 ꢁC or
32 ꢁC for promastigotes or amastigotes, respectively, for
2 h. The resulting formazan was solubilized by addition
of an equal volume of 10% w/v sodium dodecylsulfate,
and incubated overnight at 37 ꢁC; absorbance was
30. Starck, J. P.; Nakatani, Y.; Ourisson, G. Tetrahedron
1995, 51, 2629.
31. Cao, X.-P. Tetrahedron 2002, 58, 1301.
32. Halter, M.; Nogata, Y.; Dannenberger, O.; Sasaki, T.;
Vogel, V. Langmuir 2004, 20, 2416.
33. Ellman, G. L. Archiv. Biochem. Biophys. 1959, 82, 70.
34. Capozzi, G.; Modena, G. Oxidation of Thiols. In Patai, S.,
Ed.; The Chemistry of the Thiol Group; Wiley: New York,
1974; Vol. 18, p 785.
35. Chicharro, C.; Granata, C.; Lozano, R.; Andreu, D.;
Rivas, L. Antimicrob. Agents Chemother. 2001, 45, 2441.
36. Growth conditions. Promastigotes of L. donovani
(MHOM/SD/00/1S-2D) were cultured at 26 ꢁC in
RPMI-1640 medium (Gibco), supplemented with 10%
heat-inactivated fetal calf serum (HIFCS), gentamicin,
penicillin, and 2 mM glutamine. Resistant strains were
kindly provided by Prof. S. L. Croft (London School of
Tropical Medicine and Hygiene). L. pifanoi axenic
amastigotes (MHOM/VE/60Ltrod) were grown at 32 ꢁC
in M199 medium (Gibco-BRL 31100), supplemented
with 20% HIFCS, 5% trypticase, and 50 lg/mL hemin,
pH 7.2. Its resistant strains were obtained from parasites
grown under a stepwise increase of MT concentration,
and cultured as the parental strain, except for the
addition of 40 lg/mL MT to the growth medium.
Antileishmanial activity determination in vitro. Parasites
harvested at a late growth exponential phase were
resuspended in the respective growth medium
measured at 595 nm in a 450 Bio-Rad Microplate
Reader. All assays were performed in triplicate and
the experiments were repeated at least twice. LD50 (drug
concentration required to inhibit 50% parasite prolifer-
ation) was calculated using SigmaPlot software.
37. Pan, A. A.; Duboise, S. M.; Eperon, S.; Rivas, L.;
Hodgkinson, V.; Traub-Cseko, Y.; McMahon-Pratt, D.
J. Eukaryot. Microbiol. 1993, 40, 213.
38. Linking of MT-SH to agarose. A solution of thiol 1
(2 lmol) in a mixture of methanol (0.2 mL) and pH 8.50
buffer (50 mM Tris, 5 mM EDTA) (0.8 mL) was made to
react with iodoacetyl-modified agarose (SulfoLink Cou-
pling Gel, Pierce, Rockford, IL) (1 mL). The coupling
reaction was completed after 45 min at room temperature
(first 15 min with stirring), as determined by the negative
result of Ellman test for free SH-groups. After washing
with 1 M NaCl, any remaining iodoacetyl groups in the
agarose were blocked by cysteine (50 mM, conditions as
above).