Antileishmanial ring-substituted ether phospholipids

Article abstract of DOI:10.1021/jm020972c

Three series of ring-substituted ether phospholipids were synthesized carrying N,N,N-trimethylammonium, N-methylpiperidino, or N-methylmorpholino headgroups. The first series is substituted by 2-cyclohexyloxyethyl or 2-(4-alkylidenecyclohexyloxy)ethyl gro

Full text of DOI:10.1021/jm020972c

J . Med. Chem. 2003, 46, 755-767
755
An tileish m a n ia l Rin g-Su bstitu ted Eth er P h osp h olip id s
Nikos Avlonitis,† Eleni Lekka,† Anastasia Detsi,† Maria Koufaki,† Theodora Calogeropoulou,*,† Efi Scoulica,‡
Eleni Siapi,† Ioanna Kyrikou,† Thomas Mavromoustakos,† Andrew Tsotinis,§ Simona Golic Grdadolnik,| and
Alexandros Makriyannis
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vassileos Constantinou
Avenue, 11635 Athens, Greece, Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine,
School of Medicine, University of Crete, 71409, Heraklion, Greece, Department of Pharmaceutical Chemistry, School of
Pharmacy, University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece, National Institute of Chemistry,
Hajdrihova 19 P.O.B. 30 SI-1115, Ljubljana, Slovenia, and Center for Drug Discovery, Departments of Pharmaceutical
Sciences and Molecular & Cell Biology, University of Connecticut, 372 Fairfield Road, U 92, Storrs, Connecticut 06269-2092
Received J une 24, 2002
Three series of ring-substituted ether phospholipids were synthesized carrying N,N,N-
trimethylammonium, N-methylpiperidino, or N-methylmorpholino headgroups. The first series
is substituted by 2-cyclohexyloxyethyl or 2-(4-alkylidenecyclohexyloxy)ethyl groups, the second
series by cyclohexylidenealkyl or adamantylidenealkyl moieties, and the third series by
2-aryloxyethyl or 6-aryloxyhexyl groups in the alkyl portion of the molecule. The antileishmanial
activity of the new compounds was evaluated in vitro against the promastigote forms of L.
donovani and L. infantum using an MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide)-based microassay as a marker of cell viability. Analogues 12, 15, 24, 30, 32, 41, 43,
and 45 were more potent than the control compound miltefosine (hexadecylphosphocholine)
against both L. donovani and L. infantum while, derivatives 13 and 42 were equipotent to
miltefosine. Analogues 16, 17, 19, 20 were more potent than miltefosine against L. infantum
and compounds 27, 31, 44 were more active than miltefosine against L. donovani. Differential
scanning calorimetry (DSC) was used to probe the role of individual ether phospholipids on
the physicochemical properties of model membranes. The DSC scans showed that the active
compounds have a more profound effect on the thermotropic properties of model membrane
bilayers than the less active ones.
In tr od u ction
unacceptable side effects at effective doses. The recently
developed liposomal formulation of amphotericin B
(AmBisome) showed good curative rates for antimony
unresponsive cases of mucocutaneous leishmaniasis;
however, drug administration is technically difficult and
treatment costs are prohibitively expensive.4
Leishmaniasis is a protozoan parasitic disease en-
demic in 88 countries, which causes considerable mor-
bidity and mortality. At least 20 species of Leishmania
can be transmitted by sandfly bites, originating cutane-
ous, diffuse cutaneous, mucocutaneous, and visceral
leishmaniasis in humans, dogs, and various wild ver-
tebrate hosts. The estimated yearly incidence is 1-1.5
million cases of cutaneous leishmaniasis and 500 000
cases of visceral leishmaniasis. The population at risk
is estimated at 350 million people with an overall
prevalence of 12 million.1 Increasing risk factors are
making leishmaniasis a growing public health concern
for many countries around the world.2
The drugs most commonly used to treat leishmaniasis
are the pentavalent antimonials sodium stibogluconate
(Pentostam) and meglumine antimonate (Glucantime).
Antimonial chemotherapy requires high dose regimens
with long treatment courses using parenteral adminis-
tration.3 Second-line drugs, used in instances of anti-
monial-treatment failure, include amphotericin B (AMB),
paromomycin (aminosidine), and pentamidine. How-
ever, all of these drugs are far from satisfactory due to
The spreading resistance of the parasite toward the
standby antimonial drugs, the high toxicity of most
drugs in use, and the emergence of Leishmania/HIV
coinfection as a new disease entity has triggered a
continuous search for alternative therapies. Visceral
leishmaniasis caused by L. infantum has emerged as
an AIDS-associated opportunistic infection, particularly
in southern Europe.5 In recent years, alkyllysophospho-
lipid analogues (ALPs) have received considerable inter-
est due to their antineoplastic and immunomodulatory
properties.6 Their cytotoxic and cytostatic activity is
remarkably selective for cancer cells, and they stimulate
the host defense by activating macrophage cytotoxicity.7
Whereas the majority of the conventional anticancer
drugs may cause severe side effects due to bone marrow
suppression, ALPs are known to exert minimal hema-
tologic toxicity.8 Also normal resting vascular endothe-
lial cells are not affected by ALPs.9 Edelfosine (ET-
18-OCH3, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phos-
phocholine) is the prototype of antineoplastic ether
phospholipids and served as a lead drug for the syn-
thesis of BM 41.440 (Ilmofosine), SRI 62-834 and others.
Extensive structure-activity relationship studies on
a variety of ALPs showed that a long alkyl chain and a
* To whom correspondence should be addressed. Tel: +3010
7273833. Fax: +30107273818. e-mail: tcalog@eie.gr.
† National Hellenic Research Foundation.
‡ University of Crete.
§ University of Athens.
| National Institute of Chemistry.
University of Connecticut.
10.1021/jm020972c CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/04/2003

756 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Avlonitis et al.
phosphocholine moiety may represent the minimal
structural requirements for sufficient antineoplastic
effects of ether lipid analogues.10 This finding led to the
synthesis of the alkylphosphocholines (APCs). Within
the alkyl chain homologues, hexadecylphosphocholine
(HePC, miltefosine) has therapeutically useful antitu-
mor activity and was approved in 1992 as a drug in
Germany for the topical treatment of metastasized
mammary carcinoma.
Three series of analogues were synthesized carrying
N,N,N-trimethylammonium, N-methylpiperidino, or
N-methylmorpholino headgroups. The first series in-
corporates cyclohexyloxyethyl or 4-alkylidenecyclo-
hexyloxyethyl groups, while, the second series has
cyclohexylidene or adamantylidene moieties linked to
the polar headgroup by an oligomethylene bridge of 5
or 11 carbons. The third series introduces 2-aryloxyethyl
or 6-aryloxyhexyl groups in the alkyl portion of the
molecule.
Several in vitro and in vivo studies demonstrated that
alkylphosphocholines including HePC, and alkylgly-
cerophosphocholines such as edelfosine, ilmofosine, and
SRI-62,834 possess antileishmanial activity.11 Hexade-
cylphosphocholine was reported to be highly effective
in treating mice infected with visceral leishmaniasis
while oral treatment with miltefosine was 600-fold more
effective than the subcutaneous administration ïf pen-
tostam. On the basis of these promising observations,
miltefosine was evaluated in phase I and II clinical
trials as oral therapy for Indian visceral leishmaniasis
while phase III clinical trials are currently ongoing.12
Cure rates of 88% to 100% were obtained using doses
of 100-150 mg/day for 28 days. These results encour-
aged studies on the efficacy of miltefosine treatment for
cutaneous leishmaniasis in the New World and cur-
rently phase II studies are being conducted. In a phase
I study, the cure rate with miltefosine at doses of 100-
150 mg for 3 weeks was 94%.13 In the various clinical
trials, the main side effects associated with miltefosine
were gastrointestinal with the most common being
moderate vomiting and diarrhea. Transient elevation
of transaminases or urea/serum creatinine was noted
in a number of patients and decreased under continued
treatment. Although the toxicity associated with milte-
fosine sounds milder than that of some parenteral
therapies, gastrointestinal symptoms could be of more
consequence in severely ill patients, such as those who
are malnourished or dehydrated. In addition, treatment
of pregnant women is contraindicated because of milte-
fosine’s teratogenic properties in animals.14 Further-
more, miltefosine has a very long half-life and low
therapeutic ratio and a course of treatment leaves a
subtherapeutic level in the blood for several weeks.
These drug characteristics might be expected to encour-
age development of resistance.15 Additionally, miltefos-
ine was shown to be only temporarily effective in HIV
coinfected patients in Europe. Thus, more extensive
SAR studies are needed to further explore the role of
ether phospholipids as therapeutic agents for leishma-
nial infections.
The antileishmanial activity of the new compounds
was evaluated in vitro against the promastigote forms
of L. donovani and L. infantum using an MTT (3-(4,5-
dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide-
based enzymatic method as a marker of cell viability.
Ch em istr y
The synthetic strategy followed for the preparation
of the ether phospholipids 9-40 is depicted in Scheme
3. The respective alcohols were treated with phosphorus
oxychloride and triethylamine in THF to afford the
corresponding pyridinium salts after hydrolysis and
treatment with pyridine. These, in turn were coupled
with the appropriate choline, N-methylpiperidino, or
N-methylmorpholino headgroups in the presence of a
condensing agent 1-(mesitylenesulfonyl)-3-nitro-1,2,4-
triazolide (MSNT)16 or triisopropylbenzenesulfonyl chlo-
ride (TIPS-Cl).17 TIPS-Cl was employed for the synthe-
sis of the 2-cyclohexyloxyethyl-containing ether phos-
pholipids while MSNT was more suitable for the syn-
thesis of cyclohexylidene- and adamantylidene-contain-
ing ether phospholipids.
Quaternization of N-methylpiperidine or N-methyl-
morpholine to N-(2-hydroxyethyl)-N-methylpiperidini-
um bromide or N-(2-hydroxyethyl)-N-methylmorpholin-
ium bromide, respectively, was effected upon treatment
with 1-bromoethanol.18
Alcohols 4a ,b were synthesized through a Wittig
reaction between cyclohexanone (1) or adamantanone
(2), respectively, and 4-carboxybutyl triphenylphospho-
nium bromide followed by esterification of the unsatur-
ated acids 2a ,b with methanol, in the presence of a
catalytic amount of H2SO4, and subsequent reduction
using LiAlH4 (Scheme 1). Initially, reduction of acids
2a ,b to the desired alcohols 4a ,b proceeded in low yields.
However, the yields became quantitative when the
respective methyl esters 3a ,b were used. The synthesis
of alcohols 6a ,b is depicted in Scheme 1. Wittig reaction
of cyclohexanone (1) or adamantanone (2) with 10-
methoxycarbonyldecyl triphenylphosphonium bromide
afforded the unsaturated esters 5a ,b, respectively,
which were in turn reduced using LiAlH4 to give the
desired alcohols 6a ,b. The 10-methoxycarbonyldecyl
triphenylphosphonium bromide was prepared via es-
terification of 11-bromoundecanoic acid in MeOH/H+
and subsequent treatment of the corresponding methyl
ester with triphenylphosphine in dry acetonitrile at 80
°C for 24 h. Crystallization using diethyl ether/acetone
(1:2) afforded the pure phosphonium salt.
Since the alkylphosphocholine analogues studied so
far contain simple saturated or unsaturated alkyl chains
in the lipid portion, we set out to probe the influence of
cycloalkane rings on the antileishmanial activity. Specif-
ically, we explored the effect of the presence of 4-alky-
lidenecyclohexyl and cyclohexylidene or adamantylidene
groups in alkoxyethyl and alkoxy phosphodiester ether
lipids, respectively. These can be envisaged as resulting
from the deletion of the C2 or C1-C2 groups from the
ALP structure. We also introduced aryl substituents in
both categories in order to explore the role of an
aromatic moiety on the antileishmanial activity. In
addition, we evaluated how headgroup modifications
affected in vitro antileishmanial activity.
The 2-(4-alkylidenecyclohexyloxy)ethanols 8a -c were
synthesized as shown in Scheme 2. Wittig reaction of
1,4-cyclohexanedione monoethylene ketal with the ap-

Antileishmanial Ether Phospholipids
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 757
Sch em e 1a
a
Reagents and conditions: (a) [(CH3)3Si]2NK, THF; (b) MeOH, H+, 40 °C; (c) LiAlH4, THF.
Sch em e 2a
a
Reagents and conditions: (a) [(CH3)3Si]2NK, THF; (b) NaBH3CN, BF3‚Et2O, THF.
Sch em e 3a
Sch em e 4a
a
Reagents and conditions: (a) 1. P(O)Cl3, Et3N, THF; 2. H2O,
2-propanol; (b) pyridine, 40 °C; (c) pyridine, MSNT or TIPS-Cl,
HOCH2CH2N+R1R2R3, 40 °C.
propriate alkyltriphenylphosphonium bromide led to
the corresponding 4-alkylidenecyclohexyl-4-dioxolanes
7a -c. Reductive opening of the ketals 7a -c using BF3‚
Et2O and NaBH3CN in THF19 gave the respective 2-(4-
alkylidenecyclohexyloxy)ethanols 8a -c. Synthesis of the
aryloxyethanols or 6-aryloxyhexanols used for the prepa-
ration of ether phospholipids 35-40 was effected as
previously described by O-alkylation of phenol or 2-naph-
thol with 2-bromoethanol or 6-bromohexanol in DMF
in the presence of K2CO3.18
a
Reagents and conditions: (a) H2, 10% Pd/C, EtOAc, 1 atm.
lammonium analogues were more potent than the
respective N-methylpiperidino and N-methylmorpholino
congeners.
Catalytic hydrogenation of compounds 12, 15, 24, 27,
30 using 10% Pd/C in ethyl acetate afforded ether
phospholipids 41-45, respectively (Scheme 4).
A more detailed SAR is now described. In the 2-cy-
clohexyloxyethyl series introduction of an unsaturated
long alkyl chain at the 4 position of the cyclohexane
leads to ether phospholipids 12-20 that are active
against both Leishmania strains. In particular, the
tetradecylidene-substituted trimethylammonium com-
pound 15 is the most potent of the series and also more
potent than miltefosine with IC50 values 3.91 µM
against L. donovani and 5.25 µM against L. infantum.
The activity slightly decreases when the trimethylam-
monium group is substituted by N-methylpiperidino 16
(IC50 29.7 µM against L. donovani and 11.4 µM against
L. infantum). The decrease is more pronounced for the
N-methylmorpholino substituted analogue 17 (IC50 38.6
µM against L. donovani and 15.5 µM against L. infan-
tum). Replacement of the tetradecylidene group by
hexadecylidene results in reversal in order of activity
within the headgroup analogues 18-20 with the N-
methylmorpholino-bearing ether phospholipid, deriva-
Resu lts a n d Discu ssion
The new analogues 9-40 (Figure 1) and 41-45
(Scheme 4) were evaluated in vitro for antileishmanial
activity against promastigote cultures of Leishmania
donovani MON 703 and Leishmania infantum MON
235 using MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphe-
nyltetrazolium bromide as a marker of cell viability
(Table 1). In general 2-(4-alkylidenecyclohexyloxy)ethyl
analogues 12-20, 11-cyclohexylideneundecyl analogues
36-38 and 11-adamantylideneundecyl analogues 30-
32 are active against both Leishmania strains and
several of these are more potent than miltefosine.
Conversely, the 2-cyclohexyloxyethyl derivatives 9-11
and those bearing aromatic rings in the lipid portion,
i.e., compounds 34-40, are inactive, exhibiting IC50
values higher than 100 µΜ. Generally, the trimethy-

758 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Avlonitis et al.
F igu r e 1. Structures of ether phospholipids 9-40.
Ta ble 1. In Vitro Antileishmanial Activitya against the Promastigote Forms of L. infantum and L. donovani of the New Ether
Phospholipids
IC50 (µM)
L. infantum MON 235
IC50 (µM)
L. donovani MON 703
IC50 (µM)
L. infantum MON 235
IC50 (µM)
L. donovani MON 703
compd
compd
Miltefosine
9
22.56 ( 3.6
>100
23.71 ( 4.07
>100
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
>100
4.99 ( 1.50
>100
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
>100
>100
>100
46.85 ( 8.7
3.16 ( 0.63
5.41 ( 1.14
5.09 ( 1.86
>100
>100
>100
6.75 ( 2.4
22.58 ( 3.4
6.64 ( 1.2
>100
3.25 ( 0.65
23.07 ( 3.6
16.46 ( 1.8
5.25 ( 0.45
11.4 ( 2.4
15.5 ( 1.8
21.19 ( 2.6
6.5 ( 1.7
3.7 ( 0.71
>100
7.08 ( 1.2
22 ( 3.25
50.67 ( 3.6
3.91 ( 0.21
29.7 ( 3.6
38.6 ( 3.2
45.1 ( 7.2
21.96 ( 1.99
16.22 ( 2.29
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5.2 ( 1.5
47.6 ( 7.33
22.8 ( 1.7
>100
>100
5.65 ( 1.93
23.3 (3.5
8.4 ( 0.8
>100
9.49 ( 1.4
23.65 ( 4.4
10.3 ( 1.3
4.02 ( 2.3
2.88 ( 0.72
2.4 ( 0.6
8.7 ( 1
8.25 ( 0.25
5.97 ( 1.06
a
Results are expressed as mean ( SEM, n ) 3-4 (each run in duplicate).
tive 20, being the most active of the three with IC50
16.22 µM against L. donovani and 3.7 µM against L.
infantum followed by the N-methylpiperidino-substi-
tuted analogue 19 to the trimethylammonium-substi-
tuted 18. Both 19 and 20 are considerably more potent
than miltefosine against L. infantum (IC50 6.5 µM and
3.7 µM, respectively). Within the 2(4-dodecylidenecy-
clohexyloxy)ethyl series the choline derivative 12 is the
most active and more potent than miltefosine against
L. infantum (IC50 ) 3.25 µM) and against L. donovani
(IC50 ) 7.08 µM). Thus, within this series 12, 15, 16,
17, 19, and 20 are more potent than miltefosine against
L. infantum while, ether phospholipids 12, 15, and 20
are more active than miltefosine against L. donovani.
In the second series the 5-cyclohexylidenepentyl
derivatives 21-23 are inactive within the concentration
range tested (IC50 values greater than 100 µM), while
replacement of the cyclohexylidene portion in the above
compounds by an adamantylidene group (27-29, re-
spectively) establishes activity against L. donovani but
not L. infantum. In particular, ether phospholipid 27
carrying a choline headgroup is more potent than
miltefosine (IC50 ) 4.99 µM), while N-methylmorpholino
analogue 29 is less active (IC50 ) 46.85 µM) and the

Antileishmanial Ether Phospholipids
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 759
N-methylpiperidino compound 28 is inactive (IC50 > 100
µM).
The 11-cyclohexylideneundecyl analogues 24-26 are
very potent. 11-Cyclohexylideneundecyloxy ether phos-
pholipid 24 shows higher activity than miltefosine
against both L. donovani and L. infantum (IC50 ) 2.4
µM and IC50 ) 5.2 µM, respectively), while the respec-
tive compounds 25, 26 exhibit somewhat reduced activ-
ity. The decrease in activity of analogues 25, 26 is more
pronounced against L. infantum (IC50 ) 47.6 µM and
IC50 ) 22.8 µM, respectively).
Ether phospholipid 30 bearing an 11-adamantylide-
neundecyl group is also more potent than miltefosine
against both L. donovani and L. infantum (IC50 ) 3.16
µM and IC50 ) 6.75 µM, respectively). Within the
headgroup analogues of 30 the N-methylmorpholino one
is also more potent than miltefosine against both
leishmania strains, while the N-methylpiperidino com-
pound 32 exhibits decreased activity against L. infan-
tum and similar activity to 31 against L. donovani.
F igu r e 2. Percentage of live THP1 cells in the presence of
different concentrations of ether phospholipids
Lipophilic alkylphospholipids influence membrane
physiology in a pleiotrophic manner. In leishmania, this
effect is believed to involve ether lipid metabolism,
glycosylphosphatidylinositol (GPI) anchor biosynthesis,
and signal transduction.20 We used Differential scan-
ning calorimetry (DSC) to probe the role of individual
ether phospholipids on the physicochemical properties
of model membranes.21 DSC is a thermodynamic tech-
nique, which can be used to examine the effects of drug
molecules on the thermotropic properties of membranes.
For our experiments we used dipalmitoyl phosphatidyl
choline (DPPC) bilayers as model membranes. When
hydrated, phosphatidylcholines spontaneously form bi-
layers which are characterized by a mesomorphism. In
particular, DPPC bilayers exist in the gel phase (Lâ′)
at temperatures below 33 °C, and in the liquid crystal-
line phase (LR′) at temperatures above 42 °C. Between
33 and 42 °C the phospholipid bilayers exist in Pâ′ or
ripple phase. DSC scans of DPPC bilayers show a small
endothermic event (pretransition) around 35 °C and the
main phase transition around 41 °C. The main phase
transition is accompanied by several structural changes
in the lipid molecules as well as systematic alterations
in the bilayer geometry, but the most prominent feature
is the trans-gauche isomerization taking place in the
acyl chain conformation. The average number of gauche
conformers indicates the effective fluidity, which de-
pends not only on the temperature, but also on pertur-
bations due to the presence of a drug molecule interca-
lating between the lipids. Three adamantyl-substituted
ether phospholipids with different pharmacological
profile were chosen for the DSC studies. In particular,
compound 30 showed considerable activity against both
L. donovani and L. infantum, analogue 27 was only
active against L. donovani, and analogue 33 was inac-
tive against both strains. The compounds were tested
for these effects on bilayers at three concentrations (1%-
mol (x ) 0.01), 5%-mol (x ) 0.05) and 10% (x ) 0.10))
(Figure 3).
Analogues 33-40 of the third series had IC50 values
>100 µM regardless of the headgroup or the nature of
the aryl substituent (phenyl or naphthyl). Elongation
of the ethoxy group to hexyloxy (37, 38) did not result
in any increase in activity, while the 4-adamantylide-
nophenyl substituted derivative 34 was also inactive at
the doses tested. On the basis of the above data, it
cannot be deduced whether further elongation of the
alkoxy group would improve the antileishmanial activ-
ity.
The presence of the double bond on antileishmanial
activity was explored by comparing some of the most
potent ether phospholipids 12, 15, 24, 27, 30 with their
saturated congeners 41-45 obtained by catalytic hy-
drogenation. The absence of the double bond in the
cyclohexylidene-substituted compounds results in de-
crease in activity, suggesting that the more rigid cy-
cloalkylidene groups impart higher potencies. However,
in the adamantylidene-substituted derivatives the ab-
sence or presence of the double bond does not seem to
affect their potency.
The length of the alkyl chain of the most active
compounds varies from 5 to 11 carbon atoms for the
alkylphosphocholine analogues and from 12 to 14 for
the alkoxyethylphosphocholine analogues. This could be
advantageous for the solubility and/or the toxicity of the
new compounds and also for their metabolic clearance.
Thus, we proceeded to assess the cytotoxicity of four
of the most active ether phospholipids and miltefosine,
in the human monocytic cell line THP1. THP1 mono-
cytes infected with the appropriate Leishmania species
are used for the evaluation of the leishmanicidal activity
of compounds against the intracellular amastigote
stages of the parasite. Our preliminary results (Figure
2) showed a strong cytotoxic effect of miltefosine on
THP1 cells at concentrations as low as 12.5 µM, which
was not observed with two of the most active analogues
24 and 30. Conversely, compounds 12 and 15 are more
cytotoxic than miltefosine. However, further experi-
ments are required in order to evaluate the cytotoxicity
of the other active new ether phospholipids and get a
better understanding on the structural features that
impart enhanced activity and minimal cytotoxicity.
The two active compounds 30 and 27 had pronounced
perturbing effects on DPPC bilayers causing broadening
in the pre- and main transition peaks and lowering of
the pre- and main phase transition temperatures (Table
2). The inactive ether phospholipid 33 caused only
marginal effects on the phase properties of DPPC
bilayers (Table 2). Interestingly, compound 30 affects
the pretransition temperature to the same extent as 27
but causes more broadening of the main phase transi-
tion peak at the high concentrations of x ) 0.05 and x
) 0.10. These results may indicate similar perturbation

760 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Avlonitis et al.
F igu r e 3. DSC scans of (A) DPPC with either phospholipid 30, (B) DPPC with either phospholipid 27 and (C) DPPC with either
phospholipid 33.
Ta ble 2. Values of Onset Temperature (Tonset), Half-Width
between the open and closed conformers since all
methylene groups matched to one peak (Figure 5, D-J ).
The obtained results from the conformational analysis
Temperature (Tm1/2), Peak Temperature Tm, and Enthalpy
Change (∆H) of DPPC/Ether Phospholipid preparations
1/2
sample
Tonset
Tm
Tm
∆H (kcal/mol)
suggest a similar conformation for both compounds.
Thus, we can assume that the differential effects
observed in the DSC by the two molecules result from
their differences in the size of the alkyl chain. The
analogue 30 with the longer alkyl chain, is embedded
into the membrane bilayers causing significant pertur-
bation. In contrast, compound 27 has access into the
membrane core without disturbing the acyl chains of
the bilayer.
DPPC alone
DPPC+ 30 (x ) 0.01) 29.0 39.6 32.0 40.8
DPPC + 27( x ) 0.01) 31.5 40.2 33.9 41.2 3.1 1.1 4.8
DPPC + 33 (x ) 0.01) 31.8 39.8 34.3 40.7 2.5 1.0 5.7
-
1.9 4.0
43.3
42.9
43.4
40.9
41.9
43.8
40.2
37.7
45.8
DPPC + 30 (x ) 0.05)
-
38.1
-
40.2
-
-
1.9
-
DPPC + 27 ( x ) 0.05) 28.5 39.5 31.4 40.8
1.3 2.4
DPPC + 33 (x ) 0.05) 31.1 40.1 33.9 41.0 3.1 1.2 5.0
DPPC + 30 (x ) 0.10)
DPPC + 27 (x ) 0.10)
-
-
37.1
39.1
-
-
39.2
40.2
-
-
2.1
1.5
-
-
DPPC + 33 (x ) 0.10) 29.2 39.5 32.9 40.6 3.6 1.0 4.9
Con clu sion s
effects by both analogues on the polar region of the
membrane bilayers, while compound 30 interacts ef-
fectively with the hydrophobic region as well. The above
data indicate that the active ether phospholipids affect
the physicochemical properties of DPPC bilayers to a
greater extent than the inactive analogue.
To get some insight on the origin of the different
biological activity and interaction with DPPC bilayers
of the two adamantylidene derivatives 30 and 27 we
studied their conformations.
Con for m ation al An alysis. The conformational analy-
sis for ether phospholipid derivatives 27 and 30 was
carried out using a combination of NMR spectroscopy
and computational analysis. Molecular dynamics using
the most important ROE constraints and coupled with
minimization algorithms were initially carried out for
compound 27 and 30 to generate seven families of low
energy conformers. From those conformers only three
(Figure 4, A-C) for ether phospholipid 27 support the
ROE data.
This study evaluated how incorporation of cycloalkane
or aromatic rings in the alkyl residues and changes in
the choline based headgroup alters the antileishmanial
activity of alkylphosphocholines. The most active ana-
logues against both the promastigote forms L. donovani
and L. infantum are 2-(4-dodecylidene cyclohexyloxy)-
ethylphosphocholine (12), 2-(4-tetradecylidenecyclohex-
yloxy)ethylphosphocholine (15), 11-cyclohexylideneun-
decylphosphocholine (24), 11-adamantylideneundecyl-
phosphocholine (30), and the hydrogenated derivatives
41, 43, 45 which are all more potent than miltefosine.
Preliminary evaluation of the cytotoxicity in THP1
monocytes revealed that analogues 24 and 30 are less
cytotoxic than miltefosine, while compounds 12 and 15
exhibit higher cytotoxicity. Several of the new com-
pounds possess comparable activity to miltefosine against
L. donovani but are more potent than miltefosine
against L. infantum. With regard to headgroup SAR,
choline is preferred with respect to activity against both
leishmania strains. The introduction of cycloalkylidene
groups in alkylphosphocholines results in enhanced
activity and especially against the strain L. infantum
that causes the AIDS associated coinfection in Europe.
In particular, the presence of 2-(4-alkylidenecyclohexy-
loxy)ethyl moiety renders the resulting compounds more
active against L. infantum while the presence of ω-cy-
These conformers are characterized by a clustering
of the adamantane ring with the phosphocholine moiety.
The remaining four open forms are devoid of such
clustering and express the flexibility of the alkyl chain.
Concerning ether phospholipid 30, the resolution of
the 2D-ROESY experiment did not allow to differentiate

Antileishmanial Ether Phospholipids
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 761
F igu r e 4. Lowest energy conformers of compound 27 (A-C) resulting from the combined use of NMR spectroscopy and
computational chemistry. Hydrogens are not shown for clarity.
F igu r e 5. Lowest energy conformers of compound 30 (D-J ) resulting from the combined use of NMR spectroscopy and
computational chemistry. Hydrogens are not shown for clarity.
were carried out by the microanalytical section of the Institute
of Organic and Pharmaceutical Chemistry of the National
Hellenic Research Foundation.
clohexylidenealkyl or ω-adamantylidenealkyl groups
results in enhanced activity against L. donovani. Previ-
ous in vitro studies have shown variations in sensitivity
of Leishmania species to different classes of drugs.22 The
difference in activity between the various ether phos-
pholipids of this study may be due to dissimilarities in
both membrane sterol23,24 and lipid content25 of the two
Leishmania species studied. However, further investi-
gations are required to verify this hypothesis.
Since alkylphosphocholines have been found active
against other trypanosomatids26 such as Trypanosoma
cruzi and T. brucei as well as against the human
intestinal parasite Entamoeba histolytica,27 it will be
of interest to examine the effect of the new ether
phospholipids against these parasites.
5-Cycloh exylid en ep en ta n oic Acid (2a ). A solution of
4-carboxybutyltriphenylphosphonium bromide (5.43 g, 12.24
mmol) in anhydrous THF (20 mL) was treated with potassium
bis(trimethylsilyl)amide (4.88 g, 24.8 mmol), and the resulting
orange mixture was stirred at room temperature for 15 min.
A solution of cyclohexanone (1) (300 mg, 3.06 mmol) in
anhydrous THF (10 mL) was added dropwise, and the reaction
mixture was stirred at room temperature for 3 h. The reaction
was quenched with H2O, and the water layer was extracted
with ether to remove the unreacted cyclohexanone. The water
layer was acidified with 10% HCl to pH 2, and it was extracted
with ethyl acetate. The organic layer was washed with brine
and was dried with Na2SO4, and the solvent was evaporated
in vacuo to afford acid 2a 450 mg (80%) as an oil which was
used without further purification in the next step. 1Η ΝΜR
(δ): 5.02 (t, J ) 6.7 Hz, 1H, CdCH), 2.21 (t, J ) 7.9 Hz, 2H,
CH2CO), 1.94-1.84 (m, 6H), 1.53-1.32 (m, 8H).
Exp er im en ta l Section
Ch em istr y. All reactions were carried out under scrupu-
lously dry conditions. NMR spectra of all new compounds were
recorded on a Bruker AC 300 spectrometer operating at 300
MHz for 1H, 75.43 MHz for 13C, and 121.44 MHz for 31P. 1H
NMR spectra are reported in units δ with CHCl3 resonance
at 7.24 ppm used as the chemical shift resonance. 13C NMR
shifts are expressed in units relative to CDCl3 at 77.00 ppm,
5-Ad a m a n tylid en ep en ta n oic Acid (2b). Acid 2b was
prepared according to the procedure described for acid 2a in
75% yield, using 4-carboxybutyltriphenyl phosphonium bro-
mide (3.55 g, 8 mmol) in anhydrous THF (20 mL), potassium
bis(trimethylsilyl)amide (3.19 g, 16 mmol), and a solution of
2-adamantanone (2) (300 mg, 2 mmol) in anhydrous THF (10
1
31
mL) and refluxing the reaction mixture for 12 h. Η ΝΜR (δ):
while P NMR spectra are reported in units of δ relative to
5.00 (t, J ) 6.7 Hz, 1H, CHdC), 2.77 (s, 1H), 2.29 (t, J ) 7.9
Hz, 2H, CH2CO), 1.99-1.64 (m, 17H).
85% H3PO4 used as an external standard. Silica gel plates
(Merck F254) were used for thin-layer chromatography. Chro-
matographic purification was performed with silica gel (200-
400 mesh). Analyses indicated by the symbols of the elements
5-Cycloh exylid en ep en ta n oic Acid Meth yl Ester (3a ).
To a solution of acid 2a in methanol (30 mL) was added 1 mL

762 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Avlonitis et al.
concentrated H2SO4, and the mixture was stirred at 40 °C for
3 h. The solvent was evaporated in vacuo, and the residue was
partitioned between water and ethyl acetate. The organic layer
was washed with brine and dried over Na2SO4, and the solvent
was evaporated in vacuo. The residue was purified by flash
column chromatography (petroleum ether 40-60 °C/ethyl
acetate (95:5)) to afford ester 3a as an oil in 93% yield (450
mg); 1Η ΝΜR (δ): 4.91 (t, J ) 6.7 Hz, 1H, CdCH), 3.52 (s,
3H, COOCH3), 2.16 (t, J ) 7.9 Hz, 2H, CH2CO), 1.98-1.91
(m, 6H), 1.53-1.40 (m, 8H).
CH2OH), 2.77 (s, 1H, CHCdC), 2.28 (s, 1H, CHCdC), 1.94-
1.53 (m, 16H), 1.25 (broad s, 14H).
Gen er a l P r oced u r e for th e Syn th esis of 8-Alk ylid en e-
1,4-d ioxa sp ir o[4.5]d eca n es. To a solution of the correspond-
ing alkylphosphonium salt (0.01mol) in dry THF (15 mL) was
added potassium bis(trimethylsilyl) amide (2.19 g, 0.01 mmol),
and the resulting orange solution was stirred at room tem-
perature for 15 min. Subsequently, a solution of 1,4-dioxaspiro-
[4.5]decan-8-one (1.5 g, 0.01 mmol) in dry THF (10 mL) was
added dropwise, and the resulting mixture stirred at room
temperature for 12 h. The reaction was quenched by the
addition of saturated aqueous NaHCO3 solution, and the
aqueous layer was extracted with ethyl acetate. The organic
layer was washed with brine and dried with Na2SO4, and the
solvent evaporated in vacuo.
5-Ad a m a n tylid en ep en ta n oic Acid Meth yl Ester (3b).
Ester 3b was prepared according to the procedure described
for methyl ester 3a . The crude reaction mixture was purified
by flash column chromatography (petroleum ether 40-60 °C/
1
ethyl acetate (9:1)) to afford ester 3b in 84% yield; Η ΝΜR
(δ): 4.92 (t, J ) 6.7 Hz, 1H, CHdC), 3.59 (s, 3H, COOCH3),
2.70 (s, 1H, CHCdC), 2.27-2.21 (m, 3H, CH2CO, CHCd),
1.96-1.57 (m, 16H).
8-Dod ecylid en e-1,4-d ioxa sp ir o[4.5]d eca n e (7a ). Purifi-
cation of the crude residue by flash column chromatography
(petroleum ether 40-60 °C/ethyl acetate (9:1)) afforded alkene
7a as a viscous oil 2.8 g, (91% yield); 1Η ΝΜR (δ): 5.10 (t, J )
6.7 Hz, 1H, CHdC), 3.91 (s, 4H, OCH2CH2O), 2.25-2.16 (m,
4H), 2.00-1.90 (m, 2H), 1.63-1.58 (m, 4H), 1.23 (s, 18H,
(CH2)9), 0.83 (t, J ) 7.0 Hz, 3H, CH3).
8-Tetr a d ecylid en e-1,4-d ioxa sp ir o[4.5]d eca n e (7b). Pu-
rification of the crude residue by flash column chromatography
(petroleum ether 40-60 °C/ethyl acetate (9:1)) afforded alkene
7b as a viscous oil 1.6 g, (48% yield); 1Η ΝΜR (δ): 5.12 (t, J )
6.7 Hz, 1H, CHdC), 3.93 (s, 4H, OCH2CH2O), 2.27-2.18 (m,
4H), 1.99-1.93 (m, 2H), 1.67-1.60 (m, 4H), 1.25 (bs, 22H,
(CH2)11), 0.89 (t, J ) 7.0 Hz, 3H, CH3).
8-Hexa d ecylid en e-1,4-d ioxa sp ir o[4.5]d eca n e (7c). Pu-
rification of the crude residue by flash column chromatography
(petroleum ether 40-60 °C/ethyl acetate (9:1)) afforded alkene
7c as a viscous oil 3.6 g (quantitative); 1Η ΝΜR (δ): 5.05 (t, J
) 6.7 Hz, 1H, CHdC), 3.85 (s, 4H, OCH2CH2O), 2.18-2.11 (m,
4H), 2.09 (t, J ) 6.7 Hz, 2H, CH2CHdC), 1.63-1.52 (m, 4H),
1.35 (broad s, 26H, (CH2)13), 0.95 (t, J ) 7.0 Hz, 3H, CH3).
Gen er a l P r oced u r e for th e Syn th esis of 2-(4-Alk y-
lid en ecycloh exyloxy)eth a n ols (8a -c). A solution of the
corresponding ketal 7a -c (5 mmol) in dry THF (5 mL) was
sequentially treated with boron trifluoride etherate (0.76 mL,
6 mmol) and NaBH3CN (0.564 g, 9 mmol), and the resulting
mixture was stirred at room temperature for 2 h. The reaction
was quenched by the addition of saturated aqueous NaHCO3
solution, and the aqueous layer was extracted with ethyl
acetate. The combined organic extracts were washed with
brine and dried with anhydrous Na2SO4, and the solvent was
evaporated in vacuo.
5-Cycloh exylid en ep en ta n ol (4a ). To a suspension of
LiAlH4 (139.6 mg, 3.68 mmol) in anhydrous THF (10 mL) was
added dropwise at 0 °C a solution of ester 3a (360 mg, 1.84
mmol) in anhydrous THF (10 mL), and the resulting mixture
was stirred at room temperature for 2 h. Subsequently, the
reaction was quenched at 0 °C by a mixture of H2O/THF (1:1,
3 mL) followed by addition of ethyl acetate (30 mL). The
resulting mixture was dried with anhydrous Na2SO4, the solid
was filtered, and the filtrate was evaporated in vacuo to afford
alcohol 4a as a viscous oil in 96% yield (370 mg); 1Η ΝΜR (δ):
4.99 (t, J ) 6.7 Hz, 1H, CdCH), 3.54 (t, J ) 6.7 Hz, 2H, CH2-
OH), 3.15 (broad s, 1H, OH), 2.06-1.93 (m, 6H), 1.51-1.28
(m, 10H).
5-Ad a m a n tylid en ep en ta n ol (4b). Alcohol 4b was pre-
pared according to the procedure described for alcohol 4a .
Yield: 80%; 1Η ΝΜR (δ): 5.00 (t, J ) 6.7 Hz, 1H, CHdC),
3.62 (t, J ) 6.7 Hz, 2H, CH2OH), 2.77 (s, 1H, CHCdC), 2.29
(s, 1H, CHCdC), 2.02-1.23 (m, 18H).
11-Cycloh exyliden eu n decan oic Acid Meth yl Ester (5a).
Ester (5a ) was prepared according to the procedure described
for acid 2a using a solution of 10-methoxycarbonyldecyltriph-
enylphosphonium bromide (6.62 g, 12.24 mmol) in dry THF
(20 mL), potassium bis(trimethylsilyl)amide (2.44 g, 12.24
mmol), and cyclohexanone (1) (300 mg, 3.06 mmol) in anhy-
drous THF (10 mL) and stirring at room temperature for 2 h.
The reaction was quenched with H2O, and the water layer was
extracted with ethyl acetate. The organic layer was washed
with brine and dried with Na2SO4, and the solvent was
evaporated in vacuo. The crude residue was purified by flash
column chromatography (petroleum ether 40-60 °C/diethyl
ether (9:1)) to afford ester 5a as viscous oil 680 mg (79% yield);
1Η ΝΜR (δ): 5.07 (t, J ) 6.7 Hz, 1H, CHdC), 3.65 (s, 3H,
COOCH3), 2.31 (t, J ) 7.9 Hz, 2H, CH2CO), 2.11-1.93 (m, 4H),
1.67-1.50 (m, 8H), 1.25 (broad s, 14H, (CH2)7).
11-Adam an tyliden eu n decan oic Acid Meth yl Ester (5b).
Ester 5b was prepared according to the procedure described
for ester 5a using 10-methoxycarbonyldecyltriphenylphos-
phonium bromide (4.33 g, 8 mmol), potassium bis(trimethyl-
silyl)amide (1.59 g, 8 mmol), and 2-adamantanone (2) (300 mg,
2 mmol) and heating at 80 °C. Purification of the ester was
achieved by flash column chromatography (petroleum ether
40-60 °C/diethyl ether (9:1)) to afford ester 5b as viscous oil
330 mg (50% yield). 1Η ΝΜR (δ): 4.97 (t, J ) 6.7 Hz, 1H, CHd
C), 3.61 (s, 3H, COOCH3), 2.75 (s, 1H, CHCdC), 2.28 (t, J )
7.3 Hz, 2H, CH2CO), 2.22 (s, 1H, CHCdC), 1.92-1.55 (m, 16H),
1.24 (broad s, 12H).
2-(4-Dod ecylid en ecycloh exyloxy)eth a n ol (8a ). Purifica-
tion of the crude residue by flash column chromatography
(petroleum ether 40-60 °C/ethyl acetate (7:3)) afforded alcohol
1
8a as a viscous oil (0.849 g, 55% yield); Η ΝΜR (δ): 5.04 (t,
J ) 6.7 Hz, 1H, CHdC), 3.62 (broad s, 2H), 3.51-3.46 (m, 2H),
3.42-3.36 (m, 1H, CH2CHOCH2), 2.80 (broad s, 1H, OH),
2.41-2.36 (m, 1H), 2.19-2.15 (m, 1H), 1.95-1.77 (m, 6H),
1.41-1.33 (m, 2H, CH2CHdC), 1.20 (broad s, 18H, (CH2)9), 0.87
(t, J ) 7.0 Hz, 3H, CH3).
2-(4-Tetr a d ecylid en ecycloh exyloxy)eth a n ol (8b). Pu-
rification of the crude residue by flash column chromatography
(petroleum ether 40-60 °C/ethyl acetate (7:3)) afforded
1
alcohol 8b as a viscous oil (0.791 g, 47% yield); Η ΝΜR (δ):
5.12 (t, J ) 6.7 Hz, 1H, CHdC), 3.71-3.68 (m, 2H, OCH2CH2-
OH), 3.58-3.55 (m, 2H, OCH2CH2OH), 3.48-3.44 (m, 1H,
CH2CHOCH2), 2.47-2.41 (m, 1H), 2.26-2.21 (m, 1H), 2.19-
1.86 (m, 6H), 1.50-1.41 (m, 2H, CH2CHdC), 1.24 (broad s,
22H, (CH2)11), 0.89 (t, J ) 7.0 Hz, 3H, CH3).
11-Cycloh exylid en eu n d eca n ol (6a ). The procedure de-
scribed for alcohol 4a was followed using ester 5a (680 mg,
2.43 mmol) to afford 6a as a viscous oil (600 mg, 98% yield);
1Η ΝΜR (δ): 5.01 (t, J ) 6.7 Hz, 1H, CHdC), 3.55 (t, J ) 6.7
Hz, 2H, CH2OH), 3.12 (broad s, 1H, OH), 2.08-1.90 (m, 6H),
1.46 (broad s, 8H), 1.22 (broad s, 14H).
2-(4-Hexa d ecylid en ecycloh exyloxy)eth a n ol (8c). Puri-
fication of the crude residue by flash column chromatography
(petroleum ether 40-60 °C/ethyl acetate (7:3)) afforded alcohol
1
8c as a viscous oil (1.69 g, 93% yield); Η ΝΜR (δ): 5.12 (t, J
) 6.7 Hz, 1H, CHdC), 3.71-3.64 (m, 2H, OCH2CH2OH), 3.55-
3.50 (m, 2H, OCH2CH2OH), 3.48-3.44 (m, 1H, CH2CHOCH2),
2.45-2.40 (m, 1H), 2.25-2.20 (m, 1H), 2.12-1.85 (m, 6H),
1.51-1.42 (m, 2H, CH2CHdC), 1.27 (s, 26H, (CH2)13), 0.89 (t,
J ) 7.0 Hz, 3H, CH3).
11-Ad a m a n tylid en eu n d eca n ol (6b). Τhe procedure de-
scribed for alcohol 4a was followed using ester 5b (330 mg, 1
1
mmol) to afford 6b as a viscous oil (290 mg, 95%); Η ΝΜR
(δ): 4.99 (t, J ) 6.7 Hz, 1H, CHdC), 3.60 (t, J ) 6.7 Hz, 2H,

Antileishmanial Ether Phospholipids
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 763
Gen er a l P r oced u r e for t h e P r ep a r a t ion of E t h er
P h osp h olip id s. To a solution of phosphorus oxychloride (0.09
mL, 1 mmol) and triethylamine (0.25 mL, 1.8 mmol) in dry
THF (5 mL) was added dropwise at 0 °C a solution of the
corresponding alcohol (1 mmol) in dry THF (7 mL). The
resulting mixture was stirred for 2 h at room temperature and
subsequently hydrolyzed by the addition of water (3 mL). After
1 h of stirring at room temperature, the reaction mixture was
diluted with water, and the aqueous layer was extracted with
ethyl acetate and dichloromethane. The combined organic
extracts were washed with brine and dried with anhydrous
Na2SO4, and the solvent was evaporated in vacuo to afford the
corresponding phosphoric acid derivative, which was trans-
formed to the pyridinium salt by the addition of 7 mL of
anhydrous pyridine and stirring for 2 h at 40 °C. After cooling,
the solvent was evaporated in vacuo and pyridine (5 mL) was
added to the residue. To the resulting solution was added
dropwise, with cooling, a solution of 1-(mesitylen-2-sulfonyl)-
3-nitro-1H-1,2,4-triazole (MSNT) (0.593 g, 2 mmol) or 2,4,6-
triisopropylbenzenesulfonyl chloride (TIPS-Cl) (0.606 g, 2
mmol) in dry pyridine (2 mL) followed by the addition of
choline chloride (0.210 g, 1.5 mmol) or N-(2-hydroxyethyl)-N-
methylpiperidinium bromide (0.448 g, 1.5 mmol) or N-(2-
hydroxyethyl)-N-methylmorpholinium bromide (0.452 g, 1.5
mmol). The reaction mixture was stirred at 40 °C for 48-56
h. After cooling, the mixture was hydrolyzed by the addition
of H2O (2 mL) and 2-propanol (7 mL) and stirred for 1 h at
room temperature. The solvents were evaporated in vacuo, and
the resulting crude solid was purified by gravity column
chromatography using initially CH2Cl2/MeOH/25% NH4OH
(60/50/5) and subsequently MeOH/25% NH4OH (95/5), and the
solvents were evaporated in vacuo. The residue was diluted
with CHCl3 and filtered through a pore membrane (0.5 µM,
FH Millipore). After evaporation of the solvent, the desired
product was obtained.
CHO), 3.30 (s, 9H, N+(CH3)3), 2.40-1.72 (m, 8H), 1.42-1.33
(m, 2H), 1.24 (broad s, 18H, (CH2)9), 0.84 (t, J ) 7.0 Hz, 3H,
CH3); 31P ΝΜR (δ): -2.16; Anal. (C25H50NO5P‚H2O) C, H, N
1-{2-{[(4-D o d e c y li d e n e c y c lo h e x y lo x y )e t h y lo x y ]-
h yd r oxyp h osp h in yloxy}eth yl}-1-m eth ylp ip er id in iu m In -
n er Sa lt (13). The general procedure described above using
alcohol 8a , TIPS-Cl and N-(2-hydroxyethyl)-N-methylpiperi-
dinium bromide afforded compound 13 (0.350 g, 68%). 1Η ΝΜR
(δ): 5.16 (t, J ) 6.70 Hz, 1H, CdCH), 4.24 (bs, 2H, POCH2-
CH2N), 3.82-3.55 (m, 10H), 3.30 (broad s, 1H, CHO), 3.25 (s,
3H, N+CH3), 1.92-1.43 (m, 14H), 1.41-1.32 (m, 2H, CH2CHd),
1.19 (broad s, 18H, (CH2)9), 0.83 (t, J ) 7.0 Hz, 3H, CH3); 31P
ΝΜR (δ): -2.19; Anal. (C28H54NO5P‚2H2O) C, H, N.
1-{2-{[(4-D o d e c y li d e n e c y c lo h e x y lo x y )e t h y lo x y ]-
h ydr oxyph osph in yloxy}eth yl}-1-m eth ylm or ph olin iu m In -
n er Sa lt (14). The general procedure described above using
alcohol 8a , TIPS-Cl, and N-(2-hydroxyethyl)-N-methylmor-
1
pholinium bromide afforded compound 14 (0.330 g, 64%). Η
ΝΜR (δ): 5.11 (t, J ) 6.7 Hz, 1H, CdCH), 4.13 (s, 2H, POCH2-
CH2N), 3.82-3.32 (m, 15H), 3.16 (s, 3H, N+CH3), 1.92-1.47
(m, 8H), 1.42-1.34 (m, 2H, CH2CHdC), 1.27 (broad s, 18H,
(CH2)9), 0.83 (t, J ) 7.0 Hz, 3H, CH3); 31P ΝΜR (δ): -2.04;
Anal. (C27H52NO6P‚2H2O) C, H, N
1-{2-{[(4-Te t r a d e cylid e n e cycloh e xyloxy)e t h yloxy]-
h yd r oxyp h osp h in yloxy}et h yl}-N,N,N-t r im et h yla m m o-
n iu m In n er Sa lt (15). The general procedure described above
using alcohol 8b, TIPS-Cl, and choline chloride afforded
compound 15 (0.166 g, 33%). 1Η ΝΜR (δ): 5.05 (t, J ) 6.7 Hz,
1H, CHdC), 4.23 (broad s, 2H, POCH2CH2N), 3.88 (broad s,
2H), 3.75 (broad s, 2H), 3.55 (broad s, 2H, CH2N), 3.40-3.35
(m, 1H, CHO), 3.32 (s, 9H, N+(CH3)3), 2.41-2.37 (m, 1H), 2.17-
2.13 (m, 1H), 1.89-1.74 (m, 8H), 1.21 (broad s, 22H, (CH2)11),
0.89 (t, J ) 7.0 Hz, 3H, CH3); 31P ΝΜR (δ): -2.26; 13C NMR:
δ 136.9, 122.7, 74.3, 67.7, 64.7, 54.2, 33.4, 32.5, 31.9, 30.1, 29.6,
29.3, 27.4, 24.9, 22.6, 14.0; Anal. (C27H54NO5P) C, H, N.
1-{2-{[(4-Te t r a d e cylid e n e cycloh e xyloxy)e t h yloxy]-
h yd r oxyp h osp h in yloxy}eth yl}-1-m eth ylp ip er id in iu m In -
n er Sa lt (16). The general procedure described above using
alcohol 8b, TIPS-Cl, and N-(2-hydroxyethyl)-N-methylpiperi-
dinium bromide afforded compound 16 (0.201 g, 37%). 1Η ΝΜR
(δ): 5.21 (t, J ) 6.7 Hz, 1H, CHdC), 4.31 (bs, 2H, POCH2-
CH2N), 3.93-3.80 (m, 4H), 3.60-3.43 (m, 6H, CH2N(CH2)2),
3.30 (broad s, 4H, NCH3, CHO), 2.40-1.40 (m, 16H), 1.23
(broad s, 22H, (CH2)11), 0.88 (t, J ) 7.0 Hz, 3H, CH3); 31P ΝΜR
(δ): -2.42; Anal. (C30H58NO5P) C, H, N.
1-{2-[(Cycloh exyloxyeth yloxy)h yd r oxyp h osp h in yloxy]-
eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt (9). The
general procedure described above using 2-(cyclohexyloxy)-
ethanol,19 TIPS-Cl and choline chloride afforded compound 9
1
(80 mg, 26%). Η ΝΜR (δ): 4.27 (broad s, 2H, POCH2CH2N),
3.90 (broad s, 2H, CH2OP), 3.77 (broad s, 2H, OCH2CH2OP),
3.59 (broad s, 2H, CH2N+), 3.35 (s, 9H, N+ (CH3)3), 1.89 (broad
s, 1H, CHO), 1.69-1.18 (m, 10H); 31P ΝΜR (δ): -2.17; 13C ΝΜR
(δ): 67.7, 67.6, 66.1, 64.9, 59.2, 54.3, 32.4, 29.6, 25.7, 24.2; ESI-
MS m/z: 332.1 (M+ + Na), 310.1 (M+ + 1); Anal. (C13H28NO5P‚
H2O) C, H, N.
1-{2-{[(4-Te t r a d e cylid e n e cycloh e xyloxy)e t h yloxy]-
h ydr oxyph osph in yloxy}eth yl}-1-m eth ylm or ph olin iu m In -
n er Sa lt (17). The general procedure described above using
alcohol 8b, TIPS-Cl and N-(2-hydroxyethyl)-N-methylmorpho-
linium bromide afforded compound 17 (0.218 g, 40%). 1Η ΝΜR
(δ): 5.06 (t, J ) 6.7 Hz, 1H, CHdC), 4.07 (broad s, 2H, POCH2-
CH2N), 3.49-3.17 (m, 5H), 3.11 (s, 3H, N+CH3), 1.99-1.34 (m,
10H), 1.08 (broad s, 22H, (CH2)11), 0.78 (t, J ) 7.0 Hz, 3H,
CH3); 31P ΝΜR (δ): -1.99; Anal. (C29H56NO6P‚H2O) C, H, N.
1-{2-{[(4-H e xa d e cylid e n e cycloh e xyloxy)e t h yloxy]-
h yd r oxyp h osp h in yloxy}eth yl}- N,N,N-tr im eth yla m m o-
n iu m In n er Sa lt (18). The general procedure described above
using alcohol 8c, TIPS-Cl, and choline chloride afforded
compound 18 (0.196 g, 37%). 1Η ΝΜR (δ): 5.08 (t, J ) 6.7 Hz,
1H, CHdC), 4.09 (broad s, 2H, OP(O)CH2CH2N), 3.82 (broad
s, 2H, OCH2CH2OP), 3.71 (broad s, 2H, OCH2CH2OP), 3.51-
3.43 (m, 2H, CH2N), 3.04 (s, 10H, CHO, N+(CH3)3), 2.45-2.40
(m, 1H), 2.25-2.20 (m, 1H), 2.02-1.85 (m, 6H), 1.51-1.42 (m,
2H, CH2CHdC), 1.09 (broad s, 26H, (CH2)13), 0.71 (t, J ) 7.0
Hz, 3H, CH3); 31P NMR: δ -2.04; Anal. (C29H58NO5P‚H2O) C,
H, N.
1-{2-[(Cycloh exyloxyeth yloxy)h yd r oxyp h osp h in yloxy]-
eth yl}-1-m eth ylp ip er id in iu m In n er Sa lt (10). The general
procedure described above using 2-(cyclohexyloxy)ethanol,
TIPS-Cl, and N-(2-hydroxyethyl)-N-methylpiperidinium bro-
mide afforded compound 10 (70 mg, 20%).
1Η ΝΜR (δ): 4.28 (s, 2H, POCH2CH2N), 3.90 (broad s, 2H,
CH2OP), 3.88 (broad s, 2H, OCH2CH2OP), 3.65-3.52 (m, 6H,
CH2N(CH2)2), 3.29 (s, 3H, N+(CH3)), 1.85-1.10 (m, 17 H); 31
P
ΝΜR (δ): -1.85; 13C ΝΜR (δ): 67.7, 64.7, 64.6, 63.5, 61.8, 58.5,
58.4, 48.5, 32.3, 25.7, 24.0, 20.8, 20.1; ESI-MS m/z: 372.1 (M+
+ Na+), 350.1 (M+ + 1); Anal. (C16H32NO5P‚2H2O) C, H, N.
1-{2-[(Cycloh exyloxyeth yloxy)h yd r oxyp h osp h in yloxy]-
eth yl}-1-m eth ylm or p h olin iu m In n er Sa lt (11). The general
procedure described above using 2-(cyclohexyloxy)ethanol,
TIPS-Cl and N-(2-hydroxyethyl)-N-methylmorpholinium bro-
1
mide afforded compound 11 (0.122 g, 35%). Η ΝΜR (δ): 4.29
(broad s, 2H, POCH2CH2N), 4.01-3.56 (m, 14H), 3.38 (s, 3H,
N+(CH3)), 1.89-1.17 (m, 11H); 31P NMR (CDCl3) (δ): -2.32;
13C ΝΜR (δ): 67.6, 67.5, 64.8, 64.7, 64.4, 60.7, 58.5, 48.1, 32.4,
25.7, 24.2; ESI-MS m/z: 374.1 (M+ + Na+), 352.1 (M+ + 1).
Anal. (C15H30NO6P ‚3H2O) C, H, N
1-{2-{[(4-H e xa d e cylid e n e cycloh e xyloxy)e t h yloxy]-
h yd r oxyp h osp h in yloxy}eth yl}-1-m eth ylp ip er id in iu m In -
n er Sa lt (19). The general procedure described above using
alcohol 8c, TIPS-Cl, and N-(2-hydroxyethyl)-N-methylpiperi-
dinium bromide afforded compound 19 (0.211 g, 37%). 1Η ΝΜR
(δ): 5.13 (t, J ) 6.7 Hz, 1H, CHdC), 4.35 (broad s, 2H, POCH2),
3.87 (broad s, 2H), 3.78 (broad s, 2H), 3.62-3.45 (m, 6H), 3.26
(broad s, 4H), 2.27-1.63 (m, 14H), 1.52-1.41 (m, 2H, CH2CHd
1-{2-{[(4-D o d e c y li d e n e c y c lo h e x y lo x y )e t h y lo x y ]-
h yd r oxyp h osp h in yloxy}et h yl}-N,N,N-t r im et h yla m m o-
n iu m In n er Sa lt (12). The general procedure described above
using alcohol 8a , TIPS-Cl and choline chloride afforded
compound 12 (0.327 g, 69%). 1Η ΝΜR (δ): 5.06 (t, J ) 6.7 Hz,
1H, CdCH), 4.24 (broad s, 2H, POCH2CH2N), 3.89 (broad s,
2H), 3.76 (broad s, 2H), 3.57 (broad s, 2H), 3.40-3.35 (m, 1H,

764 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Avlonitis et al.
C), 1.24 (broad s, 26H, (CH2)13), 0.89 (t, J ) 7.0 Hz, 3H, CH3);
31P ΝΜR (δ): -2.01; 13C ΝΜR (δ): 138.0, 117.4, 75.1, 67.8, 67.7,
64.8, 63.3, 61.8, 58.7, 48.8, 37.2, 31.8, 30.1, 29.7, 29.6, 29.4,
29.3, 28.4, 27.7, 27.2, 22.5, 20.9, 20.1, 14.0; Anal. (C32H62NO5P‚
2H2O) C, H, N.
1-{2-{[(4-H e xa d e cylid e n e cycloh e xyloxy)e t h yloxy]-
h ydr oxyph osph in yloxy}eth yl}-1-m eth ylm or ph olin iu m In -
n er Sa lt (20). The general procedure described above alcohol
8c, using TIPS-Cl and N-(2-hydroxyethyl)-N-methylmorpho-
linium bromide afforded compound 20 (0.206 g, 36%). 1Η ΝΜR
(δ): 5.06 (t, J ) 6.70 Hz, 1H, CHdC), 4.41 (bs, 2H, POCH2),
3.99-3.39 (m, 15H), 3.35 (s, 3H, N+CH3), 2.45-2.40 (m, 1H,
CHCHOCH2), 2.25-2.20 (m, 1H, CH2CHOCH), 2.13-1.85 (m,
6H), 1.22 (broad s, 28H, (CH2)14), 0.89 (t, J ) 7.0 Hz, 3H, CH3);
31P ΝΜR (δ): -2.17; 13C ΝΜR (δ): 136.8, 122.8, 65.3, 60.7,
33.4, 33.3, 32.4, 31.8, 30.1, 29.6, 29.4, 29.3, 27.4, 24.9, 22.6,
14.0; Anal. (C31H60NO6P‚H2O) C, H, N.
MSNT, and N-(2-hydroxyethyl)-N-methylmorpholinium bro-
mide afforded compound 26 (0.117 g, 25%). Η ΝΜR (δ): 5.05
1
(t, J ) 6.7 Hz, 1H, CdCH), 4.29 (broad s, 2H, POCH2CH2N),
3.99-3.70 (m, 12H), 3.48 (s, 3H, N+CH3), 2.08-1.92 (m, 4H),
1.65-1.48 (m, 6H), 1.23 (s, 18H, (CH2)9); 31P ΝΜR (δ): -2.13;
13C ΝΜR (δ): 131.0, 124.8, 65.8, 64.3, 60.7, 58.5, 48.3, 37.1,
31.0, 30.9, 29.9, 29.7, 29.6, 29.5, 29.4, 29.3, 28.6, 28.2, 28.0,
27.8, 27.0, 25.8, 25.7, 17.6; Anal. (C24H46NO5P ‚3H2O) C, H, N
1-{2-[(5-Ad a m a n t y lid e n e p e n t y lo x y )h y d r o x y p h o s -
p h in yloxy]eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt
(27). The general procedure described above using alcohol 4b,
MSNT, and choline chloride afforded compound 27 (0.223 g,
58%). 1Η ΝΜR (δ): 4.96 (t, J ) 6.7 Hz, 1H, CdCH), 4.22 (broad
s, 2H, POCH2CH2N), 3.77-3.71 (m, 4H, CH2OPOCH2CH2N),
3.29 (s, 9H, N+(CH3)3), 2.75 (s, 1H, CHCdC), 2.27 (s, 1H,
CHCdC), 1.95-1.53 (m, 16H), 1.34-1.29 (m, 2H); 31P ΝΜR
(δ): -2.42; 13C ΝΜR (δ): 147.7, 115.9, 66.3, 65.5, 59.1, 54.3,
40.5, 39.8, 38.9, 37.2, 32.0, 30.6, 28.6, 26.6, 26.2; ESI-MS m/z:
408.1 (M+ + Na+), 386.1 (M+ + 1); Anal. (C20H36NO4P.3H2O)
C, H, N.
1-{2-[(5-C y c lo h e x y lid e n e p e n t y lo x y )h y d r o x y p h o s -
p h in yloxy]eth yl}- N,N,N-tr im eth yla m m on iu m In n er Sa lt
(21). The general procedure described above using alcohol 4a ,
MSNT, and choline chloride afforded compound 21 (0.219 g,
66%); 1Η ΝΜR (δ): 4.99 (t, J ) 6.7 Hz, 1H, CdCH), 4.21 (broad
s, 2H, POCH2CH2N), 3.74 (broad s, 4H, CH2OPOCH2CH2N),
3.34 (s, 9H, N+(CH3)3), 2.09-1.84 (m, 6H), 1.55-1.28 (m, 10H);
31P ΝΜR (δ): -2.16; 13C ΝΜR (δ): 139.8, 120.8, 66.1, 65.4, 59.1,
54.2, 37.1, 30.6, 28.6, 27.8, 26.9, 26.4, 25.6; ESI-MS m/z: 356.2
(M+ + Na+), 334.2 (M+ + 1); Anal. (C16H32NO4P) C, H, N.
1-{2-[(5-C y c lo h e x y lid e n e p e n t y lo x y )h y d r o x y p h o s -
p h in yloxy]eth yl}-1-m eth ylp ip er id in iu m In n er Sa lt (22).
The general procedure described above using alcohol 4a ,
MSNT, and N-(2-hydroxyethyl)-N-methylpiperidinium bro-
mide afforded compound 22 (0.153 g, 41%); 1Η ΝΜR (δ): 5.02
(t, J ) 6.7 Hz, 1H, CdCH), 4.28 (broad s, 2H, POCH2CH2N),
3.82-3.42 (m, 8H, CH2OPOCH2CH2N(CH2)2), 3.31 (s, 3H,
N+CH3), 2.08-1.48 (m, 16H), 1.23 (broad s, 6H, (CH2)3); 31P
1-{2-[(5-Ad a m a n t y lid e n e p e n t y lo x y )h y d r o x y p h o s -
p h in yloxy]eth yl}-1-m eth ylp ip er id in iu m In n er Sa lt (28).
The general procedure described above using alcohol 4b,
MSNT, and N-(2-hydroxyethyl)-N-methylpiperidinium bro-
1
mide afforded compound 28 (0.272 g, 64%). Η ΝΜR (δ): 4.93
(t, J ) 6.7 Hz, 1H, CdCH), 4.25 (broad s, 2H, POCH2CH2N),
3.79-3.60 (m, 8H, CH2OPOCH2CH2N(CH2)2), 3.32 (s, 3H, N+-
(CH3)3), 2.72 (s, 1H, CHCdC), 2.24 (s, 1H, CHCdC), 1.92-
1.50 (m, 22H), 1.31-1.26 (m, 2H); 31P ΝΜR (δ): -1.98; 13C
ΝΜR (δ): 147.6, 115.9, 65.4, 65.3, 63.5, 58.6, 58.5, 48.6, 40.5,
39.8, 38.9, 37.2, 32.0, 30.7, 30.6, 28.6, 26.6, 26.2, 20.9, 20.2;
ESI-MS m/z: 448.2 (M+ + Na+), 426.2 (M+ + 1); Anal. (C23H40
NO4P‚2.5H2O) C, H, N.
-
1-{2-[(5-Ad a m a n t y lid e n e p e n t y lo x y )h y d r o x y p h o s -
p h in yloxy]eth yl}-1-m eth ylm or p h olin iu m In n er Sa lt (29).
The general procedure described above using alcohol 4b,
MSNT, and N-(2-hydroxyethyl)-N-methylmorpholinium bro-
ΝΜR (δ): -2.04; ESI-MS m/z: 374.2 (M+ + 1); Anal. (C19H36
NO4P‚2H2O) C, H, N.
-
1
mide afforded compound 29 (0.239 g, 56%). Η ΝΜR (δ): 4.94
1-{2-[(5-C y c lo h e x y lid e n e p e n t y lo x y )h y d r o x y p h o s -
p h in yloxy]eth yl}-1-m eth ylm or p h olin iu m In n er Sa lt (23).
The general procedure described above using alcohol 4a ,
MSNT, and N-(2-hydroxyethyl)-N-methylmorpholinium bro-
(t, J ) 6.7 Hz, 1H, CdCH), 4.27 (broad s, 2H, POCH2CH2N),
3.99-3.69 (m, 12H), 3.43 (s, 3H, N+CH3), 2.73 (s, 1H, CHCd
C), 2.25 (s, 1H, CHCdC), 1.96-1.32 (m, 16H), 1.29-1.18 (m,
2H); 31P ΝΜR (δ): -2.16; 13C ΝΜR (δ): 147.8, 115.8, 65.6, 65.5,
64.3, 60.7, 58.5, 48.3, 40.5, 39.8, 38.9, 37.2, 32.0, 30.6, 30.5,
1
mide afforded compound 23 (0.153 g, 41%). Η ΝΜR (δ): 5.01
(t, J ) 6.7 Hz, 1H, CdCH), 4.29 (broad s, 2H, POCH2CH2N),
4.11-3.68 (m, 12H), 3.42 (s, 3H, N+CH3), 2.09-1.95 (m, 4H),
1.58-1.49 (m, 6H), 1.31 (broad s, 6H, (CH2)3); 31P ΝΜR (δ):
-2.23; ESI-MS m/z: 376.2 (M+ + 1); Anal. (C18H34NO5P‚2 H2O)
C, H, N.
28.6, 26.6, 26.5; ESI-MS m/z: 450.2 (M+ + Na+), 428.2 (M+
1); Anal. (C22H38NO5P‚2.5H2O) C, H, N.
+
1-{2-[(11-Ad a m a n t ylid en eu n d ecyloxy)h yd r oxyp h os-
p h in yloxy]eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt
(30). The general procedure described above using alcohol 6b,
MSNT, and choline chloride afforded compound 30 (0.248 g,
53%). 1Η ΝΜR (δ): 4.98 (t, J ) 6.7 Hz, 1H, CdCH), 4.21 (broad
s, 2H, POCH2CH2N), 3.75 (broad s, 4H, CH2OPOCH2CH2N),
3.32 (s, 9H, N+(CH3)3), 2.77 (s, 1H, CHCdC), 2.28 (s, 1H,
CHCdC), 1.91-1.53 (m, 16H), 1.23 (broad s, 14H); 31P ΝΜR
(δ): -2.16; 13C ΝΜR (δ): 147.2, 116.3, 66.1, 65.5, 59.2, 54.2,
40.5, 39.8, 38.9, 37.3, 32.0, 31.0, 30.9, 29.7, 29.6, 29.5, 29.2,
1-{2-[(11-Cycloh exylid en eu n d ecyloxy)h yd r oxyp h os-
p h in yloxy]eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt
(24). The general procedure described above using alcohol 6a ,
MSNT, and choline chloride afforded compound 24 (0.220 g,
52%). 1Η ΝΜR (δ): 5.05 (t, J ) 6.7 Hz, 1H, CdCH), 4.20 (broad
s, 2H, POCH2CH2N), 3.75-3.68 (m, 4H, CH2OPOCH2CH2N),
3.26 (s, 9H, N+(CH3)3), 2.11-1.92 (m, 4H), 1.65-1.48 (m, 6H),
1.23 (broad s, 18H, (CH2)9); 31P ΝΜR (δ): -2.45; 13C ΝΜR (δ):
131.0, 124.8, 66.1, 66.0, 59.1, 54.1, 31.0, 30.2, 29.9, 29.7, 29.6,
29.5, 29.3, 28.6, 28.0, 27.8, 27.0, 26.9, 25.9, 25.7; ESI-MS m/z:
440.2 (M+ + Na+), 418.2 (M+ + 1); Anal. (C22H44NO4P‚H2O)
C, H, N.
28.7, 26.5, 25.9; ESI-MS m/z: 492.2 (M+ + Na+), 470.2 (M+
1); Anal. (C26H48NO4P‚H2O) C, H, N.
+
1-{2-[(11-Ad a m a n t ylid en eu n d ecyloxy)h yd r oxyp h os-
p h in yloxy]eth yl}-1-m eth ylp ip er id in iu m In n er Sa lt (31).
The general procedure described above using alcohol 6b,
MSNT, and N-(2-hydroxyethyl)-N-methylpiperidinium bro-
1-{2-[(11-Cycloh exylid en eu n d ecyloxy)h yd r oxyp h os-
p h in yloxy]eth yl}-1-m eth ylp ip er id in iu m In n er Sa lt (25).
The general procedure described above using alcohol 6a ,
MSNT, and N-(2-hydroxyethyl)-N-methylpiperidinium bro-
1
mide afforded compound 31 (0.168 g, 33%). Η ΝΜR (δ): 4.98
(t, J ) 6.7 Hz, 1H, CdCH), 4.27 (broad s, 2H, POCH2CH2N),
3.84-3.52 (m, 8H, CH2OPOCH2CH2N(CH2)2), 3.32 (s, 3H,
NCH3), 2.76 (s, 1H, CHCdC), 2.27 (s, 1H, CHCdC), 1.92-1.53
(m, 22H), 1.23 (broad s, 14H); 31P ΝΜR (δ): -2.04; 31C ΝΜR
(δ): 147.2, 116.3, 65.1, 62.1, 57.3, 47.4, 40.5, 39.9, 38.9, 37.5,
32.0, 30.3, 29.6, 29.5, 29.4, 29.2, 28.7, 26.4, 25.8, 20.9, 20.2;
1
mide afforded compound 25 (0.315 g, 69%). Η ΝΜR (δ): 4.99
(t, J ) 6.7 Hz, 1H, CdCH), 4.23 (bs, 2H, POCH2CH2N), 3.78-
3.48 (m, 8H, CH2OPOCH2CH2N(CH2)2), 3.27 (s, 3H, N+CH3),
2.04-1.45 (m, 16H), 1.18 (broad s, 18H, (CH2)9); 31P ΝΜR (δ):
-2.04; 13C ΝΜR (δ): 130.9, 124.7, 65.3, 63.2, 58.4, 48.5, 37.0,
31.0, 30.9, 30.1, 29.8, 29.6, 29.5, 29.4, 29.2, 28.6, 27.9, 27.7,
ESI-MS m/z: 532.3 (M+ + Na+), 510.3 (M+ + 1); Anal. (C29H52
NO4P‚2H2O) C, H, N.
-
26.8, 25.8, 25.6; ESI-MS m/z: 480.3 (M+ +Na+), 458.3 (M+
1); Anal. (C25H48NO4P‚2.5H2O) C, H, N.
+
1-{2-[(11-Ad a m a n t ylid en eu n d ecyloxy)h yd r oxyp h os-
phinyloxy]ethyl}-1-methylmorpholinium Inner Salt (32).The
general procedure described above using alcohol 6b, MSNT,
and N-(2-hydroxyethyl)-N-methylmorpholinium bromide af-
1-{2-[(11-Cycloh exylid en eu n d ecyloxy)h yd r oxyp h os-
p h in yloxy]eth yl}-1-m eth ylm or p h olin iu m In n er Sa lt (26).
The general procedure described above using alcohol 6a ,

Antileishmanial Ether Phospholipids
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 765
1
forded compound 32 (0.235 mg, 46%). Η ΝΜR (δ): 4.99 (t, J
) 6.7 Hz, 1H, CdCH), 4.29 (broad s, 2H, POCH2CH2N), 4.00-
3.67 (m, 12H), 3.42 (s, 3H, N+CH3), 2.77 (s, 1H, CHCdC), 2.28
(s, 1H, CHCdC), 1.91-1.54 (m, 14H), 1.23 (s, 16H); 31P ΝΜR
(δ): -2.29; 13C ΝΜR (δ): 147.2, 116.3, 65.7, 64.9, 60.7, 58.5,
48.3, 40.5, 39.8, 38.9, 37.3, 32.0, 30.9, 30.4, 29.7, 29.6, 29.5,
29.3, 28.6, 26.5, 25.8; ESI-MS m/z: 534.2 (M+ + Na+), 512.2
(M+); Anal. (C28H50NO5P ‚3H2O) C, H, N.
4H), 4.19 (broad s, 6H), 3.37-3.15 (m, 6H), 2.95 (s, 3H), 1.67-
1.65 (m, 4H), 1.49-1.46 (m, 2H); 31P NMR (CDCl3/CD3OD)
(δ): -2.13; Anal. (C20H28NO5P‚4H2O) C, H, N.
Gen er a l P r oced u r e for th e Hyd r ogen a tion of th e
Un sa tu r a ted Eth er P h osp h olip id s (41-45). To a solution
of the desired ether phospholipid (1 mmol) in MeOH (10 mL)
was added 10% Pd/C (10% w/w), and the resulting mixture
was hydrogenated at 1 atm for 10 h. Filtration through Celite
and evaporation of the filtrate in vacuo afforded the pure
product.
1-{2-[(1-Ada m a n tylm eth yloxy)h yd r oxyp h osp h in yloxy]-
eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt (33). The
general procedure described above using 1-adamantylmetha-
nol, TIPS-Cl, and choline chloride afforded compound 33 (0.060
g, 40%); 1Η ΝΜR (CDCl3/CD3OD) (δ): 4.11 (broad s, 2H,
POCH2CH2N), 3.62-3.25 (m, 4H, CH2OPOCH2CH2N), 3.18 (s,
9H, N+(CH3)3), 1.83-1.45 (m, 15H); 31P NMR (CDCl3/CD3OD)
(δ): -1.41; Anal. (C16H30NO4P) C, H, N
1 -{2 -[4 -(1 -Ad a m a n t y l )p h e n y l o x y ]h y d r o x y p h o s -
p h in yloxy]eth yl}-1-m eth ylm or p h olin iu m In n er Sa lt (34).
The general procedure described above using 4-(1-adamantyl)-
phenol, TIPS-Cl, and N-(2-hydroxyethyl)-N-methylmorpho-
linium bromide afforded compound 34 (0.131 g, 30%). 1Η ΝΜR
(δ): 7.15 (d, J ) 7 Hz, 2H), 7.05 (d, J ) 7 Hz, 2H), 3.89-3.62
(m, 4H), 3.45-3.15 (m, 8H), 2.90 (s, 3H), 2.00-1.50 (m, 15H);
31P ΝΜR (δ): -7.19. Anal. (C23H34NO5P) C, H, N.
1-{2-{[2-(P h en yloxy)eth yloxy)]h ydr oxyph osph in yloxy}-
eth yl}- N,N,N-tr im eth yla m m on iu m In n er Sa lt (35). The
general procedure described above using 2-phenyloxyethanol,
TIPS-Cl, and choline chloride afforded compound 35 (0.055 g,
18%). 1Η NMR: δ 7.25-7.18 (m, 2H), 6.90-6.81 (m, 3H), 4.30-
4.08 (m, 6H), 3.67 (broad s, 2H), 3.21 (s, 9H); 31P ΝΜR (δ):
-2.51. Anal. (C13H22NO5P) C, H, N.
1-{2-{[2-(P h en yloxy)eth yloxy)]h ydr oxyph osph in yloxy}-
eth yl}-1-m eth ylm or p h olin iu m In n er Sa lt (36). The general
procedure described above using 2-phenyloxyethanol, TIPS-
Cl, and N-(2-hydroxyethyl)-N-methylmorpholinium bromide
afforded compound 36 (0.238 g, 69%). 1Η ΝΜR (δ): 7.26-7.21
(m, 2H), 6.92-6.84 (m, 3H), 4.31-4.10 (m, 6H), 3.95-3.45 (m,
10H), 3.36 (s, 3H); 31P ΝΜR (δ): -2.32; 13C ΝΜR (δ): 158.6,
129.7, 121.1, 114.6, 67.9, 64.3, 63.9, 60.6, 58.6, 47.9. Anal.
(C15H24NO6P‚2H2O) C, H, N.
1-{2-{[(4-Dod e cylcycloh e xyloxy)e t h yloxy]h yd r oxy-
p h osp h in yloxy}eth yl}-N,N,N-tr im eth yla m m on iu m In n er
1
Sa lt (41) Yield quantitative. Η ΝΜR (δ): 4.31 (broad s, 2H,
POCH2CH2N), 3.93 (broad s, 2H, CH2OPOCH2CH2N), 3.82
(broad s, 2H), 3.59 (broad s, 2H, POCH2CH2N), 3.37 (broad s,
10H, CHO, N+(CH3)3), 2.05-1.95 (m, 1H), 1.76-1.72 (m, 2H),
1.46-1.10 (m, 28H), 0.86 (t, J ) 7.0 Hz, 3H, CH3); Anal.
(C25H52NO5P) C, H, N.
1-{2-{[(4-T e t r a d e c y lc y c lo h e x y lo x y )e t h y lo x y ]h y -
d r oxyp h osp h in yloxy}et h yl}-N,N,N-t r im et h yla m m on i-
u m In n er Sa lt (42). Yield quantitative. 1Η ΝΜR (δ): 4.31
(broad s, 2H, POCH2CH2N), 3.93-3.82 (m, 4H), 3.59-3.15 (m,
12H), 1.96 (broad s, 1H), 1.76 (broad s, 2H), 1.42-1.09 (m, 32
H), 0.87 (t, J ) 7.0 Hz, 3H, CH3); Anal. (C27H56NO5P) C, H, N.
1 -{2 -[(1 1 -C y c l o h e x y l u n d e c y l o x y )h y d r o x y p h o s -
p h in yloxy]eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt
(43). Yield quantitative. 1Η ΝΜR (δ): 4.20 (broad s, 2H,
POCH2CH2N), 3.75-3.68 (m, 4H, CH2OPOCH2CH2N), 3.26 (s,
9H, N+(CH3)3), 2.09-1.12 (m, 13H), 1.23 (s, 18H, (CH2)9); Anal.
(C22H46NO4P) C, H, N.
1-{2-[(5-Ad a m a n tylp en tyloxy)h yd r oxyp h osp h in yloxy]-
eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt (44).Yield
1
quantitative Η ΝΜR (δ): 4.27 (bs, 2H, POCH2CH2N), 3.79-
3.09 (m, 13H), 2.02-1.25 (m, 23H); Anal. (C20H38NO4P) C, H,
N.
1 -{2 -[(1 1 -Ad a m a n t y l u n d e c y l o x y )h y d r o x y p h o s -
p h in yloxy]eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt
(45). Yield quantitative. 1Η ΝΜR (δ): 4.27 (broad s, 2H,
POCH2CH2N), 3.79-3.09 (m, 13H), 2.02-1.25 (m, 35H); Anal.-
(C26H50NO4P) C, H, N.
1-{2-{[6-(P h en yloxy)h exyloxy)]h ydr oxyph osph in yloxy}-
eth yl}-N,N,N-tr im eth yla m m on iu m In n er Sa lt (37). The
general procedure described above using 6-(phenyloxy)hexanol,
TIPS-Cl, and choline chloride afforded compound 37 (0.190 g,
53%). 1Η NMR (CDCl3/CD3OD) (δ): 7.17-7.14 (m, 2H), 6.85-
6.77 (m, 3H), 4.11 (broad s, 2H), 3.86-3.74 (m, 4H), 3.50-
3.20 (m, 2H), 3.10 (s, 9H), 1.70-1.35 (m, 8H); 31P NMR (δ):
-2.18; 31C NMR: δ 158.9, 129.3, 120.4, 114.3, 67.6, 66.4, 65.6,
58.7, 54.1, 30.5, 29.0, 25.6, 25.3; Anal. (C17H30NO5P‚2H2O) C,
H, N.
1-{2-{[6-(P h en yloxy)h exyloxy)]h ydr oxyph osph in yloxy}-
eth yl}-1-m eth ylm or p h olin iu m In n er Sa lt (38). The general
procedure described above using 6-(phenyloxy)hexanol, TIPS-
Cl, and N-(2-hydroxyethyl)-N-methylmorpholinium bromide
afforded compound 38 (0.197 g, 49%). 1Η ΝΜR (δ): 7.25-7.20
(m, 2H), 6.91-6.82 (m, 3H), 4.26 (broad s, 2H), 3.96-3.65 (m,
14H), 3.42 (s, 3H), 1.76-1.35 (m, 8H); 31P NMR (δ): -2.08;
Anal. (C19H32NO6P‚2H2O) C, H, N.
1-{2-{[2-(2-N a p h t h y lo x y )e t h y lo x y )]h y d r o x y p h o s -
ph in yloxy}eth yl} -N,N,N-tr im eth ylam m on iu m In n er Salt
(39). The general procedure described above using 2-(2-
naphthyloxy)ethanol, TIPS-Cl, and choline chloride afforded
compound 39 (0.265 g, 75%). 1Η NMR (CDCl3/CD3OD) (δ):
7.67-7.62 (m, 3H), 7.37-7.01 (m, 4H), 4.19-4.18 (m, 6H),
3.45-3.42 (m, 2H), 3.03 (s, 9H); 31P NMR (CDCl3/CD3OD) (δ):
-2.20; 31C NMR (CDCl3/CD3OD): δ 156.5, 134.4, 129.4, 128.9,
127.5, 126.6, 126.4, 123.7, 118.5, 106.9. 67.7, 67.6, 66.3, 63.9,
58.9; Anal. (C17H24NO5P‚2H2O) C, H, N.
NMR Sp ectr oscop y. The high-resolution spectra were
obtained using a Varian INOVA 600 MHz spectrometer. All
data were collected using pulse sequences and phase-cycling
routines provided in the Varian libraries of pulse programs.
Data processing including sine-bell apodization, Fourier trans-
formation, phasing, symmetrization, and plotting were per-
formed using Varian software packages. The ROESY experi-
ments were recorded using standard pulse sequence in the
phase-sensitive mode and were measured at 150 ms mixing
time. Typically, the homonuclear proton spectra were acquired
with 4096 data points in t2, 16-64 scans, 256-512 complex
points in t1, and a relaxation delay of 1-1.5 s.
Molecu la r Mod elin g. Computer calculations were per-
formed on a Silicon Graphics O2 workstation using Quanta
97 version of the Molecular Simulation Incorporated (MSI)
program. The energy of the two molecules built were first
minimized and then subjected to molecular dynamics experi-
ments to explore their lower energy conformers, with the use
of ROE constraints. The molecular dynamics calculations were
run for the molecule using simulation time 6 ps at a temper-
ature of 2000 K and a dielectric constant (ꢀ ) 1) that simulates
the CDCl3 environment. Family structures were generated
using the dihedral angle criterion. The lowest energy conform-
ers from each family were considered as the representatives
ones.
The RMSD map generated the cluster threshold of 1.138
for compound 27 and 1.038 for compound 30 and included
seven families for each molecule.
1-{2-{[2-(2-N a p h t h y lo x y )e t h y lo x y )]h y d r o x y p h o s -
p h in yloxy}eth yl}-1-m eth ylp ip er id in iu m In n er Sa lt (40).
The general procedure described above using 2-(2-naphthy-
loxy)ethanol, TIPS-Cl, and N-(2-hydroxyethyl)-N-methylpip-
eridinium bromide afforded compound 40 (0.271 g, 69%). Η
NMR (CDCl3/CD3OD) (δ): 7.66-7.61 (m, 3H), 7.36-7.02 (m,
Differ en tia l Sca n n in g Ca lor im etr y. 1,2-Dipalmitoyl-sn-
glycero-3-phosphorylcholine (DPPC, purity >99%) was ob-
tained from Avanti Polar Lipids (AL) and used without further
purification. Appropriate amounts of phospholipid with or
without compound under study were dissolved in spectroscopic
chloroform. The solvent was then evaporated by passing a
1

766 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Avlonitis et al.
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experiments, the samples were equilibrated at 4 °C for 4 h.
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mg) was sealed in a stainless steel capsule. Thermograms were
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were scanned at least twice until identical thermograms were
obtained using a scanning rate of 2.5 °C/min. The temperature
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All compounds were dissolved in DMSO to a final concen-
tration of 9.625 mM, and linear 3-fold dilutions were performed
in the culture medium. A 25 µL amount of promastigote
culture at 5 × 105 cells/mL was cultured in a 96-well flat-
bottom plate (Costar 3696) and incubated with 25 µL of
different drug concentrations at 26 °C. After 72 h, 10 µL of 5
mg/mL MTT in PBS (SIGMA M2128) was added, and incuba-
tion was continued for 3 h.28 The reaction was stopped by the
addition of 50 µL of 50% 2-propanol, 10% SDS under gentle
shaking for 30 min. Absorbance was measured at 550 nm with
reference at 620 nm in a TRITURUS microplate reader.
Calculation of IC50 values was done according to Hills et al.29,30
Assessem en t of Cytotoxicity in THP 1 Mon ocytic Cells.
Sta in in g w ith P I a n d SYBR-14. As a quantitative measure-
ment of the cell damage after incubation with different
concentrations of drugs dual staining with SYBR-14 and PI
(Molecular Probes, The Neatherlands) was used. THP1 cell
cultures were incubated at 1 × 106 cells/mL with different
concentrations of the compounds ranging from 50 to 1.56 µM.
After an incubation period of 72 h, approximately 4 × 106 cells
were suspended in labeling buffer (10 mM HEPES, 150 mM
NaCl, 10% BSA, pH 7.4) and 10 µg/mL PI and 0.1 mg/mL
SYBR-14 were added. The cultures were incubated at 37 °C
for 30 min before analysis by flow cytometry.
F low Cytom etr ic An a lysis. Cell samples were analyzed
on an Epics Elite model flow cytometer (Coulter, Miami, FL).
The green fluorescence SYBR-14 and the red fluorescence of
PI were excited at 488 nm. At least 10 000 cells were analyzed
per sample, and each staining experiment was repeated twice.
Data analysis was performed on fluorescence intensities that
excluded cell autofluorescence and cell debris.
Ack n ow led gm en t. We are grateful to Irini Fra-
giadaki and Helen Koutala for the flow cytometry
measurements. This work was supported by the Greek
General Secretariat for Research and Technology
(PENED 99ED 136), by EC COST D13/0019/01.
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Su p p or tin g In for m a tion Ava ila ble: Analytical data for
compounds 9-45 and assignment of 1H NMR chemical shifts
(ppm) of ether phospholipids 27 and 30. This material is
available free of charge via the Internet at http://pubs.acs.org.
(20) Lux, H.; Hart, D. T.; Parker, P. J .; Klenner, T. Ether lipid
metabolism, GPI anchor biosynthesis, and signal transduction
are putative targets for anti-leishmanial alkylphospholipid s.
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Thermotropic Properties of Ether Phospholipid AZT Conjugates.
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