Parallel synthesis and antileishmanial activity of ether-linked phospholipids

Article abstract of DOI:10.1016/j.bmcl.2008.07.022

The synthesis and antileishmanial activity of 18 edelfosine analogues are described. Compounds were obtained in parallel combining solid phase and solution phase synthesis. The most active analogue is characterized by the octadecyl group in position 2 of the glycerol chain. Considering that this substitution determines the loss of antitumor activity, a different mechanism of antileishmanial action can be hypothesized.

Full text of DOI:10.1016/j.bmcl.2008.07.022

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4658–4660
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Parallel synthesis and antileishmanial activity of ether-linked phospholipids
Paolo Coghi a, Nadia Vaiana a, Maria G. Pezzano a, Luca Rizzi a, Marcel Kaiser b, Reto Brun b, Sergio Romeo a,
*
a Istituto di Chimica Farmaceutica e Tossicologica ‘‘Pietro Pratesi”, Università degli Studi di Milano, Via G. Mangiagalli 25, 20133 Milano, Italy
b Department of Medical Parasitology & Infection Biology, Swiss Tropical Institute, Socinstrasse 57, 4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and antileishmanial activity of 18 edelfosine analogues are described. Compounds were
obtained in parallel combining solid phase and solution phase synthesis. The most active analogue is
characterized by the octadecyl group in position 2 of the glycerol chain. Considering that this substitution
determines the loss of antitumor activity, a different mechanism of antileishmanial action can be
hypothesized.
Received 5 June 2008
Revised 1 July 2008
Accepted 3 July 2008
Available online 10 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Leishmania donovani
Ether-linked phospholipids
Edelfosine
Parallel synthesis
Antiprotozoal activity
Leishmaniasis is an infectious disease caused by kinetoplastid
protozoans of the genera Leishmania, affecting 12 million people
around the world with an annual fatality rate of approximately
50,000 people.1 The currently available drugs are Pentostam (so-
dium stibogluconate), Glucantime (meglumine antimonate), both
are antimonial salts; Amphotericin B, a macrolide antibiotic,2 and
alkylphosphocholines.3 These drugs suffer from one or more of
the following adverse effects, rather high costs and drug resistant
parasites.2 Therefore, new safe and effective drugs are needed.
Alkyl ether phospholipids (AEPs) are metabolically stable ana-
logues of 2-lysophosphatidylcholine that display antitumor and
antiparasitic activities.4 Edelfosine (ET-18-OCH3) is the most
important member of this family and it has demonstrated antipar-
asitic activities against two strains of L. donovani.5 The first site of
interaction of phospholipid analogues in both tumor cells and pro-
tozoa is the cell membrane.6 The primary site of damage in edelfo-
sine-treated L. donovani amastigotes appears to be the flagellar
pocket and the adjacent cytoplasm.7 Most of the research on para-
sites has been focused on miltefosine, an alkylphospholipid ana-
logue (Fig. 1). Miltefosine has been registered after successful
Phase IV clinical trials under the trade name ImpavidoÒ for the oral
treatment of visceral leishmaniasis.8 Miltefosine has a long half-life
(100–200 h) in humans and a low therapeutic ratio, characteristics
that could encourage development of resistance; moreover, it is
not suitable for pregnant women because it has been shown to
be teratogenic in animals9 and it did not give satisfactory results
when administered to HIV-coinfected patients.10 Moreover,
although potential antitumor activity of miltefosine and other
phospholipid analogues was demonstrated, it is still not known
how these drugs exert their antiprotozoal activity.11,12 Recently,
fluorescent analogues of miltefosine have been prepared and con-
focal microscopy showed high concentration and homogeneous
intracellular distribution of the fluorescence in L. donovani prom-
astigotes.13 Based on these observations, it is possible to suppose
a multitarget leishmanicidal mechanism of miltefosine. Structural
modifications have been introduced in the lipid portion and the
head group of miltefosine which have led to derivatives with po-
tent activity, reduced cytotoxicity, and haemolytic activity.14–16
Although several edelfosine derivatives have been synthesized
for more than 40 years,17 very few compounds have been tested
against L. donovani. We report here the parallel synthesis and the
antileshmanial activities of 18 analogues of edelfosine, obtained
by a solid phase and solution phase synthetic approach (Table 1).
O
O
O
N
Edelfosine
O
P
O
O
O
N
O
P
O
O
Miltefosine
Figure 1. Alkyl phospholipids.
* Corresponding author. Fax: +39 02 5031 9364.
E-mail address: sergio.romeo@unimi.it (S. Romeo).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.022

P. Coghi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4658–4660
4659
Table 1
Compounds synthesized
chlorides. Dialkylglycerols 21 were then cleaved from the polymeric
support using PPTS in dichloroethane/n-butanol. Alkylation of pri-
mary alcohols on solid phase was optimized in parallel (Table 2)
using several solvents (DMF, DMSO, dimethylacetamide (DMA),
tert-butanol (t-ButOH), Pyridine) and several bases (NaH, t-ButOK,
Cs2CO3), and optimal reaction conditions were achieved using
DMA as solvent and t-ButOK as base21 (Table 2).
R2
O
O
P
O
O
O
N+
R1
O-
In the second part of our synthetic scheme (Scheme 1), the
crude alcohols 22 were reacted in parallel in solution using known
procedures to give the corresponding phosphocholines (1–18).22
The final products were purified by silica gel column chroma-
tography and analyzed by 1H NMR and mass spectrometry.23 In Ta-
ble 3, the yields of the final products are reported.
The in vitro activity of these products against axenic amastigote
forms of L. donovani was determined using a previously described
assay.24 All compounds presented IC50 values higher than miltefo-
sine, the reference compound, which shows a similar IC50 to
edelfosine. Compound 17 characterized by the O-octadecyl chain
in R2 and the benzotriazolyl substituent in R1 is the most active
R1
N
N
N
O
N
R2
a
—
1
5
2
6
3
7
4
a
—
8
9
10
compound with an IC50 of 0.66 lM, a value very similar to the
one for miltefosine. Analogues (15, 16, and 18) having the C18 alkyl
11
12
16
13
17
14
18
chain in R2, but different substituents in R1, showed higher IC50 val-
ues (6.75, 1.6, and 1.3 l
M, respectively). By changing the R2 substi-
15
C18H37
tuent with the shorter butyl alkyl chain (11–14), the activity is
almost completely abolished. The introduction of the phenyl
substituent (8–10) in R2 greatly reduced the activity. The naphthyl-
methyl and the benzyl substituents showed intermediate
a
Not obtained in sufficient amount and/or purity.
In the present work, the alkylglycerol portion of edelfosine was
modified, while the phosphocholine head group was kept constant.
Substituents have been selected from the pool of commercially
available starting materials with the aim of modifying the pharma-
cokinetics of edelfosine. The octadecyl chain (R1) was substituted
with aromatic groups (benzyl, benzotriazolmethyl, 3,5-dimethylis-
oxazolmethyl) and the small allyl group; the methyl (R2) of edelfo-
sine was replaced with long or short aliphatic chains (C4H9 or
Table 2
Optimization of Williamson reaction on solid phase
R2
Base
Solvent
Yields (%)
f/fmax (%)
a
Naphthyl-1-methyl
Naphthyl-1-methyl
Naphthyl-1-methyl
Naphthyl-1-methyl
Naphthyl-1-methyl
Octadecyl
Octadecyl
Octadecyl
Octadecyl
Octadecyl
NaH
NaH
DMSO
DMA
DMA
DMF
DMA
ndb
ndb
ndb
ndb
91
82
85
15
76
48c
55c
100c
90c
77
100
89
100
80
Cs2CO3
Cs2CO3
t-ButOK
t-ButOK
t-ButOK
t-ButOK
t-ButOK
t-ButOK
C18H37) or aromatic groups (phenyl, naphthylmethyl, or benzyl).
The first part of the synthetic scheme adopted (Scheme 1) is char-
acterized by a solid phase synthesis of dialkylglycerols 22. Solid
phase has been adopted in order to increase the selectivity of the
reaction of symmetrical 2-alkyloxy-1,3-propandiols 19 to give dial-
kylglycerols.18 Thus, commercially available or easily obtainable 2-
alkyloxy-1,3-propandiols 19 have been anchored to Merriffield’s
support functionalized by Ellman’s dihydropyranyl linker using
pyridinium p-toluenesulfonate (PPTS) as catalyst.19,20 The primary
alcohols 20 were then alkylated on solid phase under Williamson’s
conditions using various commercially available alkylbromides or
DMA
t-ButOH
DMSO
DMF
Pyridine
70
65
a
f/fmax, ratio of loads; f, mmoles of bound product per g of polimeric support after
the reaction; fmax, mmol of bound product per g of polymeric support before the
reaction.
b
nd, not detectable.
Calculated based on the recovery of starting material.
c
R2
O
+
O
O
a
O
O
OH
O
HO
OH
O
R2
19
20
R2
O
O
R1
b
O
O
O
c
HO
O
R1
O
R2
21
22
R2
O
O
O
O
O
N+
P
R1
d,e
O-
1-18
Scheme 1. Reagents and conditions: (a) PPTS, DCE; (b) R1X, t-ButOK, DMA; (c) PPTS, n-ButOH, 1,2-dichloroethane; (d) 2-Chloro-1,3,2-dioxaphospholane-2-oxide,
triethylamine; (e) trimethylamine.

4660
P. Coghi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4658–4660
Table 3
References
L. donovani inhibition assay results and yields for compounds 1–18
1. Stuart, K.; Brun, R.; Croft, S.; Fairlamb, A.; Gurtler Ricardo, E.; McKerrow, J.;
Reed, S.; Tarleton, R. J. Clin. Invest. 2008, 118, 1301.
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Compound
L. donovani
M)
Inhibition IC50
(l
Yieldsa (%)
1
2
3
4
5
6
7
8
4.15
>50
>50
26.7
13.8
33.1
>50
>50
28.2
>50
42.5
>50
>50
>50
6.75
1.60
0.66
1.30
22
10
15
33
13
15
19
20
17
17
35
27
25
28
36
27
19
20
9
10
11
12
13
14
15
16
17
18
Miltefosine
0.22–0.27
a
Yields calculated as the ratio of isolated product and polymer-bound diol 19.
17. Houlihan, W. J.; Lohmeyer, M.; Workman, P.; Cheon, S. H. Med. Res. Rev. 1995,
15, 157.
activities: compounds 5 and 1, both characterized by the benzyl
group in R1, were the most active ones (IC50 of 13.8 and 4.15
respectively).
lM,
18. Crowley, J. I.; Rapoport, H. Acc. Chem. Res. 1976, 9, 135.
19. Thompson, L. A.; Ellman, J. A. Tetrahedron Lett. 1994, 35, 9333.
20. The diol (15 mmol/g of resin) was dissolved in 1,2-dichloroethane (15 ml/g of
resin), Merrifield’s peptide resin (loading 1.0–1.5 mmol/g; 1% cross-linked)
functionalized with Elmann’s dihydropyranyl linker was added and the
mixture stirred at 60 °C for one hour. After this time, PPTS (0.50 mmol/g of
polymeric support) was added and the mixture stirred at 60 °C for 12 h. The
resin was filtered, washed with CH2Cl2 (3 times), and finally dried at 60 °C
under reduced pressure.
21. The resin (loading 1.0–1.5 mmol/g) was solvated under stirring at 60 °C in
anhydrous DMA (2 ml/100–150 mg of resin). t-ButOK was then added
(20 mmol/mmol of polymer-bound diol) and the mixture stirred vigorously
at 60 °C for 10 min. The halogenide (1 equivalent) was then added and the
resin was stirred at 60 °C for 12 h. Successively the mixture was diluted with
H2O/DMF 1:1, filtered, washed with H2O (3 times), DMF (3 times), and finally
with CH2Cl2 (3 times).
22. Rampy, M. A.; Pinchuk, A. N.; Weichert, J. P.; Skinner, R. W. S.; Fisher, S. J.;
Wahl, R. L.; Gross, M. D.; Counsell, R. E. J. Med. Chem. 1995, 38, 3156.
23. Characterization of compound 17: HPLC purity: >99% C18 reverse-phase
column (4.0 Â 250 mm), detection at 206 and 254 nm (diode array detector),
eluent: MeOH–H2O 9:1 v/v plus 10 mM H3PO4; flow rate of 1.2 ml minÀ1, tR
9.6 min; 1H NMR (CDCl3): d 0.81 (t, 3H, J = 6.7 Hz), 1.19 (br s, 30H), 1.34–1.38
(m, 2H), 3.18 (s, 9H), 3.25–3.67 (m, 5H), 3.72–3.87 (m, 2H), 4.12 (m, 2H), 6.00
(m, 2H), 7.34–7.40 (m, 1H), 7.46–7.53 (m, 1H), 7.66–7.69 (m, 1H), 7.96–7.99
(m, 1H). Mass: [M+H]+: m/z 641.5, [M+Na+]: m/z 663.3.
Compound 17 was also tested in L. donovani infected macro-
phages and resulted (IC50 0.78 M), appreciably more active than
the reference drug miltefosine (IC50 1.74 M).
l
l
From the resultsobtained, it is clear thatan adequatelevel of lipo-
philicity is required in order to improve the antiparasitic activity.
The most active compound obtained is characterized by the C18
chain at R2. This is interesting since it is known that edelfosine ana-
logues which have the C18 chain in this position do not show induc-
tion of apoptosis.6 This indicates that the mechanism of antiparasitic
action of these compounds is probably different from the antitumor
mode of action which is based on induction of apoptosis. The good
intracellularactivityofcompound17supportsfurtheranaloguesyn-
thesis to identify new potential antileishmanial candidates.
Acknowledgment
This investigation received financial assistance from UNICEF/
UNDP/World Bank/WHO Special Programme for Research and
Training in Tropical Diseases (TDR).
24. Tasdemir, D.; Kaiser, M.; Brun, R.; Yardley, V.; Schmidt, T. J.; Tosun, F.; Ruedi, P.
Antimicrob. Agents Chemother. 2006, 50, 1352.