Ether phospholipid-AZT conjugates possessing anti-HIV and antitumor cell activity. Synthesis, conformational analysis, and study of their thermal effects on membrane bilayers

Article abstract of DOI:10.1021/jm001121c

The 1-O-hexadecyl-2-O-methyl-sn-glyceryl phosphodiester AZT 4 and hexadecyl-phosphodiester AZT 5 derivatives were synthesized and found to be active against HIV-1, HIV-2, and tumor cell proliferation. Compared to AZT, compound 4 possessed ca. 10-fold lowe

Full text of DOI:10.1021/jm001121c

1702
J . Med. Chem. 2001, 44, 1702-1709
Eth er P h osp h olip id -AZT Con ju ga tes P ossessin g An ti-HIV a n d An titu m or Cell
Activity. Syn th esis, Con for m a tion a l An a lysis, a n d Stu d y of Th eir Th er m a l
Effects on Mem br a n e Bila yer s
Thomas Mavromoustakos,*,† Theodora Calogeropoulou,† Maria Koufaki,† Antonios Kolocouris,† Ioanna Daliani,†
Costas Demetzos,‡ Zhaoxing Meng,§ Alexandros Makriyannis,§ J an Balzarini,# and Erik De Clercq#
National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, Vas. Constantinou 48, 116 35,
Athens, Greece, School of Pharmacy, Department of Pharmacognosy, University of Athens, Panepistimioupoli-Zografou
GR-15771 Athens, Greece, School of Pharmacy, Department of Molecular and Cell Biology and Institute of Materials Science,
University of Connecticut, Storrs, Connecticut 06269, and Rega Institute for Medical Research, Katholieke Universiteit Leuven,
B-3000 Leuven, Belgium
Received November 24, 2000
The 1-O-hexadecyl-2-O-methyl-sn-glyceryl phosphodiester AZT 4 and hexadecyl-phosphodiester
AZT 5 derivatives were synthesized and found to be active against HIV-1, HIV-2, and tumor
cell proliferation. Compared to AZT, compound 4 possessed ca. 10-fold lower anti-HIV activity
and ca. 10-fold higher anti-tumor cell activity. Compound 5 was 10-fold less potent than
compound 4 in both biological tests. In an attempt to correlate biological activity of compounds
4 and 5 with structure, their conformational and thermal effects on membrane bilayers were
compared using a combination of NMR spectroscopy, computational analysis, and Differential
Scanning Calorimetry. The obtained results showed that compound 4 adopts a compact
conformation in which the alkyl chain, the 2-methoxyglyceryl functionality, and the methyl
group of thymine are in spatial proximity, while analogue 5 possesses a less compact
conformation of the nucleoside base and the alkyl chain. The presence of the 2-methoxyglyceryl
group in compound 4 may augment its potency by inducing a turn of the alkyl chain stabilized
by hydrophobic interactions. The DSC scans show that conjugate 4 affects less effectively the
thermotropic properties of model membrane bilayers than compound 5. This may be attributed
to the fact that compound 4 is incorporated in a compact conformation and does not perturb
significantly the trans:gauche isomerization of the membrane phospholipids. In contrast,
conjugate 5 may enter with a less compact conformation and perturb more the membrane
bilayers.
In tr od u ction
branes.11,12 As a continuation of our efforts to explore
the stereoelectronic requirements for optimal effective-
ness of the lipid component of ether lipid-AZT conju-
gates, we report the synthesis of 3′-azido-3′-deoxythy-
midine 5′-[3-(1-O-hexadecyl-2-O-methyl-sn-glyceryl) phos-
phate] (4) and the evaluation of its antiviral and anti-
tumor cell activity. In an attempt to correlate biological
activity with structure, the conformational properties
and the effects on membrane bilayers of the alkylglyc-
eryl phosphate AZT conjugate 4 and the less potent
alkyl phosphate AZT conjugate 5 which is devoid of the
glycerol backbone were compared (Figure 1).
The conformational properties of 4 and 5 were ex-
plored using computational analysis coupled with ex-
perimental NOE data and their thermal effects on
phospholipid bilayers were studied using Differential
Scanning Calorimetry.
3′-Azido-3′-deoxythymidine (AZT), initially tested as
an anticancer agent, is an inhibitor of HIV-1 reverse
transcriptase and the first drug clinically used for the
treatment of AIDS.1,2 In an attempt to improve the
biological profile of AZT, various derivatives of this drug
have been synthesized.3-5 Alkyl and alkoxy,6 aryloxy,6,7
and alkyl rac-glycero and thioglycero8,9 phosphoAZT
conjugates have attracted special interest since they can
penetrate the membranes of virally infected cells and
may be subjected to in situ hydrolysis to the corre-
sponding 5′-nucleotide.6 Little is known about the
cytotoxic properties of the above-mentioned compounds,
while various synthesized alkyl phosphate derivatives
of other nucleosides exhibit cytotoxic activity against
L1210 cell cultures.10
Our laboratory has been involved in the design and
synthesis of 5′-lipophilic AZT phosphates6,7 as well as
in the study of their biophysical properties i.e., their
conformational properties and interactions with mem-
Resu lts a n d Discu ssion
Ch em istr y. 3′-Azido-3′-deoxythymidine 5′-[3-(1-O-
hexadecyl-2-O-methyl-sn-glyceryl) phosphate] (4) was
synthesized according to Scheme 1. 1-Hexadecyl-2-
methyl-sn-glycerol (1) was prepared from the 2,3-
isopropylidene-L-glyceric acid methyl ester according to
the procedure described by Bhatia and Hajdu14 with
modifications in the last two steps. Specifically, the
alkylation at position sn-1 was accomplished by treat-
* To whom correspondence should be addressed. Tel: +30-1-
7273869. Fax: +30-1-7273872, +30-1-7273831. E-mail: tmavro@eie.gr.
† National Hellenic Research Foundation, Institute of Organic and
Pharmaceutical Chemistry.
‡ University of Athens
§ University of Connecticut.
#
Katholieke Universiteit Leuven.
10.1021/jm001121c CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/01/2001

Ether Phospholipid-AZT Conjugate
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1703
(AZT-MP) into the cells but instead are converted to
AZT. To further assess this hypothesis, the hydrolytic
behavior of the phosphodiesters 4 and 5 were examined.
Conjugates 4 and 5 (1 mM concentrations) were incu-
bated in the presence of RPMI-1640 and RPMI-1640
containing 10% fetal calf serum (FCS) at 37 °C, and the
solutions were analyzed by HPLC and reverse phase
thin-layer chromatography. The obtained results show
that compounds 4 and 5 were very slowly hydrolyzed
only in serum-containing medium to afford AZT, prob-
ably through the action of phosphodiesterases and/or
phosphatases present in the FCS. On this basis how-
ever, we cannot differentiate between the formation of
AZT-MP and its rapid conversion to AZT or the direct
release of free AZT from the parent compound.
Thus, incubation of conjugate 4 in RPMI-1640 con-
taining 10% FCS resulted to 3.3% and 4.7% hydrolysis
after 21 and 45 h, respectively. Similarly, after 45 h
incubation 5.9% of compound 5 was hydrolyzed to AZT.
AZT-MP was not detected during the experiments.
Compounds 4 and 5 were also evaluated for their
inhibitory effects on the proliferation of murine leuke-
mia cells L1210/0 and L1210/TK-, murine mammary
carcinoma cells FM3A/0, and human T-lymphocyte cells
Molt4/C8 and CEM/0 (Table 2). Their inhibitory effect
on cell proliferation was expressed as a reduction of the
total number of viable tumor cells by 50% upon a 2-day
(murine leukemia L1210 and murine mammary carci-
noma FM3A) or 3-day (human T-lymphoblast Molt4/C8
and CEM) incubation of the cells at 37 °C.15,16
F igu r e 1. Chemical structures and critical torsion angles for
phospholipid AZT conjugates 4 and 5. In the same figure are
shown the most important NOEs observed for compounds 4
and 5 at 298 K.
As compared to AZT and analogue 5, compound 4 was
proved to be approximately 10-fold more potent an
inhibitor of tumor cell proliferation. Derivatives 4 and
5 were less active against TK- than L1210/0 cell growth.
However, they still retain reasonable activity in contrast
to AZT. This is in accordance with the very slow release
of AZT that was observed in the hydrolysis experiments
described above.
ment of the alcohol with NaH and 1-hexadecylbromide
in toluene at 80-100 °C. Deprotection of the trityl group
was performed using HCOOH in ether to yield alcohol
1. Thus, 1-hexadecyl-2-methyl-sn-glycerol (1) reacted
with excess o-chlorophenyl phosphodi-1,2,4-triazolide in
pyridine/acetonitrile to afford the corresponding triethy-
lammonium salt 2 after hydrolysis. This, in turn, was
coupled with AZT in the presence of the condensing
agent 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT)
to yield the phosphotriester 3 in good yield. Removal of
the o-chlorophenyl group with tetra-n-butylammonium
fluoride followed by treatment with Dowex H+ yielded
the target compound 4. Compound 5 was synthesized
according to previously reported procedures.6,7
Biologica l Assa ys. Conjugates 4 and 5 were evalu-
ated against HIV-1 (IIIB) and HIV-2 (ROD), and their
inhibitory effects were based on suppression of HIV-
induced cytopathicity (gland cell formation) in virus-
infected human T-lymphocyte CEM cell cultures.15,16
The anti-HIV activity of compound 4 was about 10-fold
lower than that of AZT and 10-fold higher than that of
analogue 5 (Table 1), although its cytotoxicity was the
highest.
High -Resolu tion NMR Sp ectr oscop y in Solu tion .
1
The H NMR spectra of compounds 4 and 5 are shown
1
in Figure 2, and the assignment of H chemical shifts
are shown in Table 3. Identification of individual peaks
was obtained by peak integration, by analogy with the
chemical shift data of 3′-azido-3′-deoxythymidine 5′-
phosphate,17 and with the aid of 2D NMR COSY and
NOESY experiments.
In particular, for compound 4, the COSY spectrum
did not allow the differentiation between the closely
resonating protons 2′ and 2′′, and thus, the assignment
of these peaks was achieved by the 2D NOESY spectra.
The 2′ proton was assigned to resonate at 2.24 ppm
because of its spatial proximity to 6 proton. The 2’’
proton was assigned to resonate at 2.17 ppm since it
correlates through space with 1′ proton. The most
informative observed NOE connectivities were found
between the protons: 1′-6 (m ) medium), 2′-6 (m), 3′-6
(m), 5′-6 (m), 5CH3-9 (s ) strong), 5CH3-13 (s), and 9-13
(s) (Figure 1). The 5CH3-13 proton correlation points to
a spatial proximity between the thymine moiety and the
alkyl chain. The NOEs found between 5CH3-9 and 5′-6
reveal a spatial relationship between the glycerol back-
bone and thymine and give valuable information about
the thymine orientation. The NOE observed between 9
To obtain more detailed information on their mech-
anism of action, compounds 4 and 5 were examined in
thymidine kinase deficient cells in order to evaluate
their ability to deliver intracellularly the 5′-nucleotide.
To this end, CEM/TK- cells were used which are
incapable of phosphorylation and, thus, activation of
AZT. As shown in Table 1, compounds 4 and 5 lost their
activity against HIV in CEM/TK- cells, suggesting that
they do not efficiently deliver AZT monophosphate

1704 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Mavromoustakos et al.
Sch em e 1a
a
Reagents and conditions: (a) C16H33Br, NaH, toluene, 100 °C; (b) HCOOH, ether; (c) o-chlorophenyl phosphodi-1,2,4-triazolide, pyridine
then, Et3N/H2O, rt; (d) MSNT, AZT, pyridine, rt; (e) n-Bu4N+F-, THF/pyridine/H2O (8:1:1, v/v/v) then, Dowex H+.
Ta ble 1. Anti-HIV-1 and Anti-HIV-2 Activity and Cytotoxicity
of the Compounds 4 and 5 and AZT in Human T-Lymphocyte
(CEM Cells)
a
EC50 (µM)
CEM/0
CEM/TK-
HIV-2
CC50 (µM)
b
compound
HIV-1
HIV-2
CEM/0
4
5
AZT
0.065
0.58
0.0064
0.08
1.0
0.006
>10
>50
>100
34.3 ( 3.0
161 ( 7
>500
a
50% Effective concentration or concentration required to
b
protect CEM cells against the cytopathicity of HIV by 50%. 50%
Cytotoxic concentration or concentration required to reduce CEM
cell viability by 50%. All data represent average values for at least
two separate experiments.
Ta ble 2. Inhibitory Effects of the Compounds 4 and 5 and AZT
on the Proliferation of Murine Leukemia Cells (L1210/0,
L1210/TK-), Murine Mammary Carcinoma Cells (FM3A/0), and
Human T-Lymphocyte Cells (Molt4/C8)
a
IC50 (µM)
compound
L1210/0
L1210/TK-
33.6 ( 1.2 68.4 ( 7.8 21.4 ( 5.6
165 ( 3 133 ( 9 144 ( 8
6.18 ( 3.55 >500 203 ( 173 62.8 ( 23.5
FM3A/0
Molt4/C8
4
5
AZT
0.98 ( 0.65
12.5 ( 3.4
a
50% Inhibitory concentration or concentration required to
inhibit cell growth by 50%. All data represent average values for
at least two separate experiments.
and 13 protons indicates an alkyl chain-glycerol back-
bone spatial relationship.
The same strategy was applied for the assignment of
the proton resonances for compound 5 (Figure 1, Table
3). The most important NOE connectivities that deter-
mine its conformation were found between protons 1′-6
(m), 2′-6 (m), 3′-6 (m), 4′-9 (s), 5CH3-9 (s), and 6-9 (m)
(Figure 1). These NOEs suggest different conformational
properties for compound 5.
Con for m ation al An alysis. The conformational analy-
sis for AZT derivatives 4 and 5 was carried out using a
combination of NMR spectroscopy and computational
analysis.
Molecular dynamics using the most important NOE
constraints and coupled with minimization algorithms
were initially carried out for compounds 4 and 5 to
generate a set of 12 families of low energy conformers.13
From those conformers only three for compound 4
(Figure 3A-C) and four for compound 5 (Figure 3D-
F igu r e 2. 1H NMR spectra of AZT derivatives 4 and 5 in
CDCl3 at 298 K.
G) were consistent with NOE data. The conformational
characteristics for these conformers in terms of nucleo-
side moiety, glycerol backbone, and alkyl chain confor-
mation are briefly discussed.
Con for m a tion of Nu cleosid e Moiety. The confor-

Ether Phospholipid-AZT Conjugate
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1705
Ta ble 3. 1H NMR Chemical Shifts (ppm) of Phospholipid AZT
Conjugates 4 and 5 Obtained in CDCl3 Solution at 298 K Using
TMS as Internal Reference
or (+)gauche-trans for low energy conformers of com-
pounds 4 and 5.
Con for m a tion of th e Glycer ol Ba ck bon e a n d
Alk yl Ch a in . The conformation of the glycerol backbone
for compound 4 was determined by the torsion angles
τ1 and τ2. Six rotamers with staggered conformations
about the two carbon-carbon glycerol bonds are pos-
sible. Torsion angles τ1 and τ2 were found to be (+)-
gauche-trans, trans-(-)gauche, (+)gauche-(-)gauche,
(+)gauche-trans, or trans-(-)gauche, (+)gauche-(-)-
gauche.
The combination of dynamics simulation and NOE
spectroscopy revealed an isomerization of an extended
all-trans alkyl chain conformer to several low energy
conformers that include trans-gauche orientations
around carbon-carbon bonds. The isomerization of the
alkyl chain creates an energetically favorable turn of
the chain and establishes a spatial proximity between
the alkyl chain, the glycerol backbone, and the methyl
group of AZT thymine as shown in low energy conform-
ers of Figure 3A-C. This stabilizing effect can explain
the observed compact anti orientation of thymine base
in compound 4 compared with derivative 5 which adopts
a more random conformation of nucleoside and alkyl
chain groups (Figure 3D-G).
compound 4
chemical shift
compound 5
chemical shift
proton
proton
14
13
12
5-CH3
2′
2′′
11
9
0.70
1.08
1.38
1.76
2.20
2.24
3.26
3.28
3.33
3.37
3.73
3.78
3.86
3.90
4.01
4.29
6.04
7.48
10
9
8
0.75
1.11
1.56
1.78
2.25
2.31
3.37
3.30
4.20
4.28
4.35
6.17
7.38
5-CH3
2′
2′′
7
4
5′
a
10
8
7
a
5′′
3′
1′
6
7′
4′
5′
5′′
a
a
3′
1′
6
a
The chemical shifts of these protons can be interchanged.
Ta ble 4. Values of Phase Pretransition and Transition
Temperatures (Tm), Half-Width Temperature (Tm1/2), and
Enthalpy Changes (∆H) of DPPC with or without Compound 4
or 5
In conclusion, the obtained results showed that the
nucleoside and alkyl chain moieties adopt a more
compact conformation in compound 4 than that of
compound 5. This higher flexibility of compound 5 does
not allow a unique family of low energy conformers that
accounts for all observed NOEs.
Differ en tia l Sca n n in g Ca lor im etr y (DSC). To
examine if the observed conformational differences
between compounds 4 and 5 may consequently induce
any differences in their physicochemical interactions
with lipid bilayers, DSC was applied. The DSC traces
of DPPC bilayers without or with either compounds 4
or 5 are shown in Figure 4.
Fully hydrated DPPC phospholipids spontaneously
form bilayers whose dynamic and thermotropic proper-
ties have been extensively studied by various biophysi-
cal methods.21,22 These methods showed that these
bilayers exist in the gel phase at temperatures lower
than 35 °C and in the liquid crystalline phase at
temperatures higher than 42 °C. The transition is
accompanied by several structural changes in the lipid
molecules as well as alterations in the bilayer geometry.
The most prominent feature during the transition 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 an additive intercalating
between the phospholipids.
The addition of x ) 0.01 (1%-molar ratio) of the more
active analogue 4 does not cause any significant change
in the main phase transition but broadens the pretran-
sition temperature. In contrast, the presence of the less
active analogue 5 causes lowering of the main phase
transition temperature by 0.6 K relative to compound
4. At x ) 0.05, the presence of either compound in DPPC
bilayers abolishes the pretransition temperature, but
the presence of compound 5 still causes more lowering
of the main phase transition temperature by 0.6 K in
samples
DPPC alone
Tm1/2
Tm
∆H
1.0
1.3
1.7
2.0
2.8
1.2
1.5
2.3
2.4
35.9, 42.3
30.9, 40.4
40.2
39.3
36.7
34.2, 41.0
40.8
40.4
5.6, 42.8
43.4
DPPC + 5 (x ) 0.01)
DPPC + 5 (x ) 0.05)
DPPC + 5 (x ) 0.1)
DPPC + 5 (x ) 0.2)
DPPC + 4 (x ) 0.01)
DPPC + 4 (x ) 0.05)
DPPC + 4 (x ) 0.10)
DPPC + 4 (x ) 0.20)
43.9
42.7
45.9
53.4
43.8
46.9
50.4
39.5
mation of the nucleoside moiety for molecules 4 and 5
was determined by studying the puckering of the sugar
ring, the geometry of the glycosidic linkage (defined by
the ø dihedral angle C2-N1-C1′-O4′), and the rotation
about the exocyclic C4′-C5′ bond (defined by the γ
dihedral angle C3′-C4′-C5′-O5′).
H1′ resonances of the ether phospholipid AZT conju-
gates 4 and 5 appeared as triplets (J ) 6.4 Hz for
compound 4 and 6.0 Hz for compound 5). A similar
spectral feature was observed for the free AZT nucleo-
side and indicates a predominant C2′-endo solution
conformation of the sugar ring. The absence of a NOE
between H2′′-H4′ also confirms a C2′-endo deoxyribose
conformation which is further supported by the H6-
H2′ NOE correlation.18-20
The heterocyclic base of compound 4 was found to
adopt an anti orientation around the glycosidic bond.
An anti orientation is characterized by ø torsion angle
values varying between -170° and -90°.20 The NOEs
between protons 5CH3-9 and 5′-6 support this orienta-
tion. For compound 5 no dipolar correlation was ob-
served between protons 5′-6. This suggests a more
flexible rotation about C1′-N1 bond which allows the
thymine base to move easily between an anti and a syn
orientation (torsion angle values varying between 50°
and 90°). The results of the dynamics calculations reflect
this flexibility as shown in the represented low energy
conformers (Figure 3D-G). The conformation around
torsion angle γ was found to be (+)gauche-(-)gauche

1706 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Mavromoustakos et al.
F igu r e 3. Low energy conformations of alkylglycerophospholipid AZT derivative 4 (A-C) and alkylphospholipid AZT derivative
5 (D-G) resulting from the combined use of NMR spectroscopy and computational chemistry. Hydrogens are omitted for better
representation.
comparison to compound 4. At high concentrations of x
) 0.10 and x ) 0.20 both compounds broaden the phase
transition and cause a lowering of the phase transition
temperature, but the effects of compound 5 are clearly
far more pronounced than those of compound 4 as it is
depicted from the values of diagnostic parameters Tm1/2
and Tm. Higher ∆H values were observed for derivative
4 as it is expected, because it probably fits better in the
membrane bilayers and it does not perturb the cooper-
ativity of the lipid chains.
The obtained results suggest that the less active
analogue 5 perturbs more membrane bilayers probably
because of its adopted more flexible conformation. The
more active compound 4 which adopts a compact
conformation may fit better into the membrane bilayer
and causes less significant effects.
moiety and alkyl chain. Thus, it seems that 2-methoxy-
glycerol backbone stabilizes the compact conformation
observed for compound 4. Such a compact conformation
may be related to its way of entering the degree of
penetration in the membrane bilayers and the less
efficient perturbation compared to 5. It remains to be
seen whether novel phospholipid AZT analogues that
adopt a compact conformation will possess improved
anti-cancer and anti-HIV properties.
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 the intermediate
compounds were recorded on a Bruker AC 300 spectrometer
1
operating at 300 MHz for H and 121.44 MHz for 31P. 1H NMR
spectra are reported in units of δ with CHCl3 resonance at
7.24 ppm used as the chemical shift resonance. 31P NMR
spectra are reported in units of δ relative to 85% H3PO4 used
as an external standard. Silica gel plates (Merck F254) were
used for thin-layer chromatography. Chromatographic puri-
fication was performed with silica gel (200-400 mesh). The
microanalysis of compound 4 was carried out by the Service
Central de Microanalyze (CNRS), France. HPLC data were
recorded using a Waters quaternary system.
It appears that the use of DSC may increase our
understanding of a possible relationship between the
conformational properties of the phospholipid AZT
conjugates and their membrane interactions.
Con clu sion
The synthetic AZT derivative 4 was found to possess
both anti-HIV and anti-tumor cell activities. Interest-
ingly, conjugate 5 which is devoid of glycerol backbone
possesses 10-fold less activity as anticancer and HIV
agent than compound 4. The two molecules differ not
only in their biological profile but also in their confor-
mational properties and thermal effects on membrane
bilayers. Compound 4 prefers a compact conformation
in which the alkyl chain, the 2-methoxyglyceryl group,
and the 5-CH3 are in close contact so as to favor
hydrophobic interactions, while compound 5 adopts a
less defined conformation with respect to the nucleoside
1-H e xa d e cyl-2-m e t h yl-3-(t r ip h e n ylm e t h yl)-sn -glyc-
er ol. To a slurry of NaH (0.132 g, 5.5 mmol, washed twice
with anhydrous hexane) in toluene (4 mL) was added at 0 °C
a solution of 2-methyl-3-(triphenylmethyl) glycerol (0.950 g,
2.75 mmol) in toluene (4 mL). The resulting mixture was
stirred at 0 °C for 0.5 h, and then 1-bromohexadecane (1.049
g, 3.44 mmol) and KI (0.057 g, 0.344 mmol) were added. The
resulting mixture was stirred at 100 °C for 12 h. The reaction
was quenched at 0 °C, by addition of MeOH (1 mL). The
mixture was diluted with ether and washed with H2O and
brine. The organic layer was dried (Na2SO4), and the solvent
was evaporated in vacuo to afford the desired product in 82%
yield after purification of the residue by flash column chro-

Ether Phospholipid-AZT Conjugate
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1707
F igu r e 4. DSC scans of DPPC bilayers alone or with incorporation of phospholipid AZT derivative 4 or 5.
matography using a mixture of petroleum ether (40-60 °C)/
ethyl acetate (95:5) as eluting solvent. Spectral data and
optical rotation were in accordance with those reported in ref
14.
was followed by extraction with dichloromethane. The organic
phase was dried (Na2SO4) and evaporated in vacuo. The
resulting (o-chlorophenyl)phosphatidic acid triethylammonium
salt 2 was used without further purification for the next step.
1-H exa d ecyl-2-m et h yl-sn -glycer ol (1). A solution of
1-hexadecyl-2-methyl-3-(triphenylmethyl)-sn-glycerol (1.2 g,
2.2 mmol) in a mixture of formic acid (90%) (4.4 mL) and
diethyl ether (4.4 mL) was stirred at room temperature for 3
h. Addition of ether was followed by extraction with saturated
aqueous NaHCO3, H2O, and brine. The solvent was evaporated
in vacuo, the resulting solid was dissolved in methanol (25
mL), and K2CO3 (6.6 mmol) was added. The reaction mixture
was stirred until TLC showed the formate esters were com-
pletely hydrolyzed. The K2CO3 was filtered off; the filtrate was
diluted with ether and washed with saturated aqueous
NH4Cl, H2O, and brine. The organic layer was dried (Na2SO4),
and the solvent was evaporated in vacuo. After purification of
the residue by flash column chromatography using petroleum
ether (40-60 °C)/ethyl acetate (75:25) as eluting solvent, the
desired product 1 was afforded in 92% yield. Spectral data and
optical rotation were in accordance with those reported in ref
14.
2-Ch lor oph en yl 5′-(3′-azido-3′-deoxyth ym idin yl) 1-Hexa-
d ecyl-2-m eth oxy-sn -glycer yl P h osp h a te (3). To an ice-
cooled solution of o-chlorophenyl phosphodichloridate (0.17 mL,
1 mmol) in acetonitrile (4 mL) were sequentially added 1,2,4-
triazole (0.155 g, 2.2 mmol) and triethylamine (0.3 mL), and
the mixture was stirred at room temperature for 30 min.
1-Hexadecyl-2-methyl-sn-glycerol (1) (0.152 g, 0.5 mmol) in
pyridine (4 mL) and after a period of 45 min triethylamine
(0.35 mL) and water (0.1 mL) were added. The mixture was
stirred for 10 min, and addition of saturated aqueous NaHCO3
Compound 2 was dissolved in pyridine (2.5 mL) and
1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazole (MSNT) (0.180 g,
0.67 mmol) and AZT (0.150 g, 0.56 mmol) was added. After
stirring at room temperature for 12 h, aqueous NaHCO3 was
added, and the mixture was extracted with dichloromethane.
The organic phase was washed with water and was saturated
with aqueous NaCl and dried over Na2SO4. The residue was
purified by column chromatography using a gradient of dichlo-
romethane/acetone (90:10-85:15) to afford 0.270 g (71.8%) of
the desired product 3. 1H NMR (300 MHz) δ 8.35 (bs, 1H, NH),
7.50-7.05 (m, 5H, ArH, H6), 6.22 (t, J ) 6.5 Hz, 1H), 4.51-
4.07 (m, 6H), 3.55-3.34 (m, 8H), 2.49-2.21 (m, 2H), 1.88 (s,
3H), 1.7-1.45 (m, 2H), 1.30-1.15 (m, 26 H), 0.88 (t, J ) 6.8
Hz, 3H); 31P NMR (121 MHz) δ -8.22, -8.29.
3′-Azid o-3′-d eoxyth ym id in e 5′-[3-(1-O-Hexa d ecyl-2-O-
m eth yl-sn -glycer yl) P h osp h a te] (4). Tetra-n-butylammo-
nium fluoride (1 mmol 1M in THF) was added to a solution of
2-chlorophenyl 5′-(3′azido-2′-deoxythymidinyl) 1-hexadecyl-2-
methoxy-glyceryl phosphate (3) (0.250 g, 0.33 mmol) in 3.5 mL
of a THF-pyridine-water (8:1:1 v/v/v) mixture. The mixture
was then stirred at room temperature for 5 h, worked up by
addition of saturated aqueous NaHCO3, and extracted with
dichloromethane. The organic phase was evaporated, and the
residue was purified by column chromatography using a
dichloromethane/methanol gradient (95:5-50:50 v/v). The
appropriate fractions were concentrated and treated with

1708 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Mavromoustakos et al.
DOWEX 50WX8 (H+) in methanol to afford the final phos-
phodiester 4 (0.147 g,68%).31P NMR δ-1.85.Anal.(C30H54N5O8P‚
H2O) C, H.
stainless steel capsules obtained from Perkin-Elmer. Thermo-
grams were obtained on a Perkin-Elmer DSC-7 calorimeter.
Prior to scanning the samples were held above their phase
transition temperature for 1-2 min to ensure equilibration.
All samples were scanned at least twice until identical
thermograms were obtained using a scanning rate of 2.5 °C/
min. The temperature scale of the calorimeter was calibrated
using indium (Tm ) 156.6 °C) as a standard sample.
Sta bility of Com p ou n d s 4 a n d 5. Conjugates 4 and 5 (1
mM concentrations) were incubated in the presence of RPMI-
1640 culture medium and RPMI-1640 culture medium con-
taining 10% fetal calf serum. The solutions were monitored
by HPLC, using an ODS10 column, an eluent of water/
acetonitrile, and a linear gradient from 100% water to 20%
water at 30 min, with a flow rate of 2 mL min-1 and detection
by UV at 265 nm. Reverse-phase thin-layer chromatography
(RP-18 TLC plates and eluent system of water/acetonitrile 1:1)
was also used for monitoring the solutions. For identification
of the possible hydrolysis products AZT-MP, hexadecyl mono-
phosphate, and 1-O-hexadecyl-2-O-methyl-sn-glycerol mono-
phosphate were synthesized and used as standards.
Ack n ow led gm en t. This work was supported by
grants from the European Commission and from the
Belgian Government (95/5). The excellent technical help
of Mrs. Ann Absillis and Lizette van Berchelaen was
highly appreciated.
Refer en ces
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Bruker 300, 400, and 500 MHz instruments. COSY and
NOESY 2D-NMR experiments were performed using pulse
sequences and phase-cycling routines provided in the Bruker
library of pulse programs. Data processing were performed
using Bruker software packages.
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the following acquisition parameters: pulse width (PW) 6.0
µs, (SW) 6023 Hz, data size (TD) 16K, recycling delay (RD)
1.0 s, number of transients (NS) 16, and digital resolution 0.37
1
Hz/pt. The acquisition parameters used for the H-1H correla-
tion COSY spectra were as follows: recycling delay (D1) 2.0
s, D0 increment 3 µs, and spectral width in F2 2512.56 Hz
and in F1 1256.28 Hz. The data sizes were as follows: 1024
and 128 data points in F1 and F2, respectively; the data were
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transformation giving digital resolution 9.81 Hz/pt. The spec-
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1
2D H-1H nuclear Overhauser enhancement (NOESY) spec-
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data sizes were as follows: 1024 and 512 data points in F1
and F2, respectively; the data were zero-filled in F1 to 1024
data points prior to 2D Fourier transformation giving digital
resolution 2.64 Hz/pt. The optimum mixing time (D9) was
found to be 0.5 s. The spectrum was processed as in the COSY
experiment.
Molecu la r Mod elin g. Computer calculations were per-
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package. Molecular mechanics calculations were carried out
using the CHARMm force field. The conformational energy of
lipid-AZT conjugate 4 and 5 were first minimized with
conjugate gradient and then with Newton-Raphson algo-
rithms, using an energy gradient tolerance of 0.01 kcal mol-1
A-1 to reach a local minimum. These structures were subjected
to an unrestrained molecular dynamics simulation at 1000 K
for 300 ps (ꢀ ) 1). Conformations were sampled every 1 ps
during the simulation, resulting in 300 randomized structures
which were subjected to restrained energy minimization using
1000 steps of conjugate gradient algorithm. The upper bound
distances used were 3.0 and 3.5 Å for strong and medium NOE
correlations correspondingly. After cluster analysis using a
torsion angle threshold of 83°, 12 family structures were
selected. The obtained low energy structures of each cluster
were further minimized to reach local minima. Three conform-
ers assigned as A-C for compound 5 (Figure 3A-C) support
the observed NOE between 9CH3-13, and four conformers
(Figure 3D-G) for compound 4 represent low energy structures
consistent with the NMR data.
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Ether Phospholipid-AZT Conjugate
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