Alkyl and alkoxyethyl antineoplastic phospholipids

Article abstract of DOI:10.1021/jm9509152

Two series of phosphodiester ether lipid analogs with (N- methylmorpholino)ethyl or (N-methylpiperidino)ethyl polar head groups and long aliphatic or alkoxyethyl chains in the nonpolar portion of the molecule were synthesized as potential antineoplastic agents. The cytotoxic activity of these compounds (9-19) was evaluated in vitro against a panel of six human tumor xenografts and in two biochemical, mechanism-based screens (cdc2 kinase and cdc25 phosphatase). Analogs 13, 14, 17, and 19 showed activity in the in vitro tests. Specifically, 14 and 17 were more active than the reference compound hexadecylphosphocholine (Miltefosine, He-PC) while 13 and 19 possessed activity similar to that of the control. Of the analogs tested the one with the highest potency and least toxicity (17) has an N- methylpiperidino head group and a C₁₆ alkyl chain. In the mechanism-based tests 11 showed weak inhibitory activity in the cdc25 phosphatase screen.

Full text of DOI:10.1021/jm9509152

J . Med. Chem. 1996, 39, 2609-2614
2609
Notes
Alk yl a n d Alk oxyeth yl An tin eop la stic P h osp h olip id s
Maria Koufaki,† Vanessa Polychroniou,† Theodora Calogeropoulou,† Andrew Tsotinis,‡ Markus Drees,§
Heiner H. Fiebig,§ Sophie LeClerc, Hans R. Hendriks,| and Alexandros Makriyannis*,∇
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave.,
GR-11635 Athens, Greece, Department of Pharmacy, Division of Pharmaceutical Chemistry, University of Athens,
Panepistimiopoli Zografou, GR-15771 Athens, Greece, Department of Internal Medicine, University of Freiburg,
Freiburg, Germany, CNRS, Station Biologique, 29682 Roscoff Cedex, France, EORTC-NDDO, Amsterdam, The Netherlands,
School of Pharmacy and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269
Received December 13, 1995X
Two series of phosphodiester ether lipid analogs with (N-methylmorpholino)ethyl or (N-
methylpiperidino)ethyl polar head groups and long aliphatic or alkoxyethyl chains in the
nonpolar portion of the molecule were synthesized as potential antineoplastic agents. The
cytotoxic activity of these compounds (9-19) was evaluated in vitro against a panel of six human
tumor xenografts and in two biochemical, mechanism-based screens (cdc2 kinase and cdc25
phosphatase). Analogs 13, 14, 17, and 19 showed activity in the in vitro tests. Specifically,
14 and 17 were more active than the reference compound hexadecylphosphocholine (Miltefosine,
He-PC) while 13 and 19 possessed activity similar to that of the control. Of the analogs tested
the one with the highest potency and least toxicity (17) has an N-methylpiperidino head group
and a C16 alkyl chain. In the mechanism-based tests 11 showed weak inhibitory activity in
the cdc25 phosphatase screen.
In tr od u ction
that a long alkyl chain and a phosphocholine moiety
may represent the minimal structural requirements for
sufficient antineoplastic effects of ether lipid analogs.7,8
This finding led to the synthesis of a new group of
analogs, the alkylphosphocholines.
Ether lipid (EL) and alkyllysophospholipid (ALP)
analogs have received much interest in recent years
because of their cytotoxic effects on malignant cells in
vitro and in vivo.1 A number of these ether lipids
including rac-2-O-methyl-1-O-octadecylglycero-3-phos-
phocholine (rac-ET-18-OCH3, rac-Edelfosine),2 hexade-
cylphosphocholine (Miltefosine, He-PC),3 and others4
have been studied in some detail. ELs structurally
resemble the natural membrane components and are
completely different from the classical antitumor drugs
as their primary target appears to be the plasma
membrane. Several mechanisms of action have been
postulated to account for these biological effects, includ-
ing enhancement of the cytotoxic properties of macro-
phages, alteration of phospholipid metabolism, and
inhibition of PKC.4d,5
With the first-generation ALPs, and in particular ET-
18-OCH3 as a reference structure, many laboratories
have embarked on the chemical synthesis of a variety
of structurally related compounds and screening for
antineoplastic activity. In addition, the use of synthetic
ELs in the autologous bone marrow transplant tech-
nique is of considerable interest due to their nonhema-
totoxic effects.6
Within the alkyl chain homologs, He-PC has thera-
peutically useful antitumor activity.9 Alkylphospho-
cholines with longer alkyl chains such as octadecyl are
antitumor effective but at the same time far too toxic
to be used as potential drugs.9a In vitro studies on He-
PC revealed antineoplastic activity on HL60, U937, Raji,
and K562 leukemia cell lines. In addition, He-PC,
administered orally, was superior for the treatment of
dimethylbenzanthracene-induced rat mammary carci-
nomas when compared to intravenously administered
cyclophosphamide.7 In a clinical pilot study on breast
cancer patients with widespread skin involvement,
topically applied He-PC showed skin tumor regressions
without local or systemic side effects.7 On the basis of
these promising observations, regular phase I and II
trial studies (oral and topical applications) were per-
formed.10,11 In the phase II trials, He-PC was admin-
istered orally to patients with advanced non small cell
lung carcinoma. The treatment lasted an average of 8
weeks, but the results, as with ET-18-OCH3, were
unsatisfactory. Furthermore, there were gastrointes-
tinal side effects (WHO > 2) such as abdominal pain,
anorexia, nausea, vomiting, and diarrhea.1h Since there
seems to be a discrepancy between the biological activity
of ALPs in vitro and in clinical trials, more extensive
SAR studies are needed to further explore the role of
these molecules as therapeutic agents.
Structure-activity relationship (SAR) studies on a
variety of synthetic alkyllysophosphocholines showed
* To whom correspondence should be addressed.
† Institute of Organic and Pharmaceutical Chemistry, National
Hellenic Research Foundation.
‡ Department of Pharmacy, Division of Pharmaceutical Chemistry,
University of Athens.
§ Department of Internal Medicine, University of Freiburg.
CNRS, Station Biologique, Roscoff.
As an ongoing effort aimed at exploring the structural
and stereochemical requirements of amphipathic ether
lipids for optimal cytotoxicity and/or immunomodula-
tion,12 we studied the effect of different head groups and
| EORTC-NDDO, Amsterdam.
∇ School of Pharmacy, University of Connecticut.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(95)00915-0 CCC: $12.00 © 1996 American Chemical Society

2610 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Notes
Ta ble 1. Antimitotic and Cytotoxic Activity of Alkylphospholipids
inhibitory activity on cell cycle control enzymes, (IC50 µM)
in vitro cytotoxicity in six human tumor xenografts
Cdc2
kinase
Cdc25
phosphatase
xenograftsa
active/total
mean IC70
µg/mL
alkylphospholipid
9
10
>1000
>1000
500
290
0/6
1/6
(RXF)
1/6
(RXF)
1/6
(LXFS)
2/6
(LXFS, RXF)
>10
>10
11
12
13
14
15
>1000
>1000
>1000
>1000
>1000
40
610
>10
>10
8.6
380
1000
>1000
3/6
7.8
(LXFS, MEXF, RXF)
1/6
(LXFS)
>10
16
17
>1000
>1000
490
460
0/6
4/6
>10
5.6
(LXFL, LXFS, MEXF, OVXF)
18
>1000
>1000
>1000
300
100
25
1/6
(LXFS)
2/6
(LXFL, LXFS)
2/6
(LXFL, RXF)
>10
7.9
19
He-PC
8.0
a
Nonsmall cell lung carcinoma line LXFL 529, small cell lung carcinoma cell line LXFS 538, ovarian adenocarcinoma OVXF 1023,
melanoma MEXF 989, colorectal adenocarcinoma CXF 1103, and renal cancer RXF 423. All compounds were tested at the 10 µg/mL dose
level.
Sch em e 1. Synthetic Scheme for Alkylphospholipids
9-19
F igu r e 1. Structures of alkylphospholipids.
alkyl chains on the in vitro antineoplastic activity of
ALPs. Specifically, we explored the effect of deleting
the C2 or C1-C2 groups from the ALP structure to
obtain the corresponding alkoxyethyl and the alkoxy
phosphodiester ether lipids, respectively. We also evalu-
ated how head group modifications affected in vitro
phosphorus oxychloride in the presence of triethylamine
followed by the addition of salts 1 and 2 gave the desired
alkylphospholipids 9-19. Alkoxyethanols 6-8 were
prepared according to our previously reported method.16
cytotoxicity.
Two series of analogs were synthesized carrying
N-methylmorpholino or N-methylpiperidino head groups
and long aliphatic or alkoxyethyl chains in the alkyl
portion of the molecule (Figure 1). The cytotoxic activity
of these new compounds was then evaluated in vitro
against a panel of human tumor xenografts13 and in two
biochemical, mechanism-based screens (cdc2 kinase and
cdc25 phosphatase).14,15 The tests showed that 13, 14,
17, and 19 were active in the in vitro tests while 11 had
a weak inhibitory activity in the cdc25 phosphatase
screen.
Resu lts a n d Discu ssion
The antimitotic activity of alkylphospholipids 9-19
(Table 1) was evaluated using purified cell cycle regula-
tors as molecular targets. The first screening test uses
the p34cdc2/cyclin Bcdc13 protein-kinase, affinity-purified
on p9CKShs sepharose beads. The enzyme activity was
assayed in the presence of potential inihibitors using
Histone H1 and 32P-labeled ATP.15 The second screen-
ing test uses a highly purified human recombinant
glutathione-S-transferase/cdc25 fusion protein assayed
colorimetrically for p-nitrophenyl phosphate phos-
phatase activity in microtitration plates.14 The use of
these major cell cycle control proteins provides highly
specific mechanism-based screens for antimitotic drug
discovery. The level of activity for the cdc2 kinase and
cdc25 phosphatase screens is defined at an IC50 value
Ch em istr y
Alkylphospholipids 9-19 were synthesized according
to the general procedure depicted in Scheme 1. Quater-
nization of N-methylmorpholine or N-methylpiperidine
to N-(2-hydroxyethyl)-N-methylmorpholinium bromide
(1) or N-(2-hydroxyethyl)-N-methylpiperidinium bro-
mide (2), respectively, was effected upon treatment with
1-bromoethanol. Phosphorylation of alcohols 3-8 using

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2611
Ta ble 2. In Vitro Cytotoxicity in HTXs (IC70, µg/mL) of
of <50 µM. The calculated IC50 values for 9-19
reported in Table 1 show that only 11 exhibits weak
inhibitory activity in cdc25 (IC50 ) 40 µM). However,
this analog was inactive in cdc2 (IC50 >100 µM). All
other compounds were inactive in both screens. Simi-
larly, hexadecylphosphocholine is active in cdc25 with
an IC50 value of 25 µM but lacks inhibitory activity in
cdc2. The cytotoxicity of alkylphospholipids 9-19 was
tested in a panel of six human tumor xenografts
(HTXs)13 (Table 1). The panel consists of two sensitive
lines, nonsmall cell lung carcinoma LXFL 529 and small
cell lung carcinoma LXFS 538; two intermediately
sensitive lines, ovarian adenocarcinoma OVXF 1023 and
melanoma MEXF 989; and two resistant lines, colorectal
adenocarcinoma CXF 1103 and renal cancer RXF 423.
Alkylphospholipids
tumor
13
14
17
19
He-PC (control)
CXF
1103
LXFL
529
LXFS
538
>10.000 >10.000 >10.000 >10.000
>10.000
10.000
5.849
10.378
5.115
8.756
4.328
1.895
5.379
4.540
9.574
>10.000
>10.000
>10.000
2.754
MEXF >10.000
989
OVXF >10.000 >10.000
1023
RXF
423
mean
4.641 >10.000
7.796 >10.000
6.738
8.562
4.757
7.776
10.000
5.564
10.000
7.906
8.007
In this in vitro assay, tumor cells taken from human
tumor xenografts which were serially transplanted in
nude mice are incubated continuously (for at least 1
week) with three concentrations of the drug, 0.1, 1, and
10 µg/mL, in cell culture medium. This is done for six
HTXs, and colony formation is scored for each tumor.
Drug effects are expressed as percentage survival
obtained by comparing the mean number of colonies in
treated plates with the mean number of colonies of
control plates (test/control, % ) T/C, %). A compound
is considered to be cytotoxic if it reduces colony forma-
tion to less than 30% of the control value (T/C < 30%).
The new analogs 9-19 were compared with He-PC
which was active in two out of six human tumor
xenografts at 10 µg/mL.
Our data show that the compounds tested were
inactive at the dose levels of 0.1 and 1 µg/mL. As shown
in Table 1, at the dose level of 10 µg/mL compounds 13,
14, 17, and 19 were active with alkylphospholipids 14
and 17 being cytotoxic in a larger number of HTXs than
He-PC. Ether lipids 10, 11, 12, 15, and 18 were less
active, all having mean IC70 values greater than 10 µg/
mL while 9 and 16 showed no activity. The most active
analog 17, which contains a C16 aliphatic chain in the
alkyl portion of the molecule and a N-methylpiperidino
component in the polar head group, had a mean IC70
value equal to 5.6 µg/mL and showed overall cytotoxicity
1.5-fold higher than that of the control He-PC (IC70 ) 8
µg/mL).
The above data indicate that, in general, analogs with
C16 alkyl residues are more cytotoxic than those with
shorter aliphatic chains. Also, replacement of the long
aliphatic chains of analogs 9-11 with alkoxyethyl
groups leads to more potent compounds. With regard
to head group SAR, the N-methylpiperidino analogs
were more potent than the respective N-methylmor-
pholino and trimethylammonium congeners.
Con clu sion
This study evaluated how changes in the choline head
group and alkyl residues of alkylphosphocholines al-
tered the in vitro cytotoxic efficacy of this class of
compounds in six human tumor xenografts. The most
active ether lipid and least toxic of all the compounds
tested was 17 which has a N-methylpiperidino head
group and a C16 alkyl chain. Thus, N-methylpiperidino
may be an attractive head group modification when
designing novel alkoxyphospholipids. In contrast, in the
alkoxyethyl series the N-methylmorpholino head group
is preferred not only with respect to activity but also in
that the most promising analogs 13 and 14 are cytotoxic
against resistant tumor cell line RXF.
Earlier studies12b have shown that ether lipid analogs
exhibit selective toxicities toward cancer cells when
compared to normal cells. Even though there is still
no clearly accepted mechanism of action which could
explain this selectivity, an attractive explanation may
be based on the ability of ELs to preferentially ac-
cumulate at higher concentrations in cancer cell mem-
branes, thus resulting in the destruction of these cells.
This selective accumulation may, in turn, be due to
differences in membrane organization and/or composi-
tion between cancer and normal cells.
In more detail, 10, 11, 13, and 14 bearing the
N-methylmorpholino head group showed activity against
the resistant RXF HTX. In contrast, analogs with a
N-methylpiperidino head group (15-19) are inactive in
the RXF HTX but show cytotoxicity in the sensitive lines
LXFL or LXFS.
Exp er im en ta l Section
Among the alkoxyethyl phospholipids, 13, 14, and 19
were found to be cytotoxic (Table 2). Of these the
N-methylmorpholino analogs were less active in the
RXF HTX than He-PC. In the LXFL HTX these analogs
showed comparable activity to that of He-PC while in
the LXFS HTX they were more potent. The N-meth-
ylpiperidino alkoxyethyl phospholipid 19 was substan-
tially less active than He-PC in the RXF HTX, while it
showed increased activity in the LXFL and LXFS HTXs
compared to the control.
An tim itotic Activity. The antimitotic activity of the new
compounds was evaluated according to the procedures by
Rialet and Meijer14 and Baratte et al.15
(a ) P 34cd c2/Cyclin B Assa y. Starfish oocytes were induced
to enter into M phase with 10 µM 1MeAde, frozen in liquid
nitrogen, and stored at -80 °C. When required, the oocytes
were homogenized in homogenization buffer (1 g of oocytes/2
mL of buffer) and centrifuged for 45 min at 24000g.15,17 The
supernatant was loaded on p9CKShs-sepharose beads at a ratio
of 40 mL of supernatant/mL p9CKShs-sepharose beads. The
beads were prepared from recombinant human p9CKShs as
described in Azzi et al.18 After 30 min at 4 °C, under constant
rotation, the beads were washed extensively with bead buffer.
Active p34cdc2/cyclin B kinase was eluted with free p9CKShs (3
mg/mL). The eluted kinase was assayed using Histone H1 as
a substrate according to Arion et al.17 and Meijer et al.19 with
The most potent analog 17 showed significantly
higher activity than He-PC in all HTXs with the
exception of RXF. In particular, analog 17 was excep-
tionally potent in the LXFS HTX with an IC70 value of
1.9 µg/mL (Table 2).

2612 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Notes
a few minor modifications. Briefly, 10 µL of p34cdc2/cyclin B
kinase, 5 µL of Histone H1 (5 mg/mL), 7 µL of buffer C, and 3
µL of inhibitor were mixed in a Rhesus tube. The kinase assay
was started by addition of 5 µL of [32P]ATP (90 µM) and run
for 10 min at 30 °C. The assay was terminated by transferring
25 µL on 2.5 × 3 cm pieces of P81-phosphocellulose paper.
After 20 s, these papers were washed six times with 10 mL of
phosphoric acid/L for at least 5 min/wash. The filters were
then dried and counted in a scintillation counter. Background
activity (the kinase being replaced by buffer) was substracted
from all data.
A compound is considered to be active in the panel if it
shows cytotoxic activity in g20% of the number of tumors
tested at the dose of e10 µg/mL.
Ma ter ia ls a n d Exp er im en ta l P r oced u r es
All reactions were carried out under scrupulously dry
conditions with magnetic stirring. Organic phases were dried
over Na2SO4, and the solvent was evaporated under vacuum.
Silica gel grade 60 (200-400 mesh, E. Merck, Germany) and
ASTM (150-230 mesh, E. Merck, Germany) was used for flash
and gravity column chromatography, respectively. All com-
pounds were demonstrated to be homogeneous by analytical
TLC on precoated silica gel TLC plates (grade 60, F254, E.
Merck, Germany), and chromatograms were visualized by
phosphomolybdic acid staining. All NMR spectra were re-
corded on a Bruker AC300 spectrometer operating at 300 MHz
(b ) P 80cd c25 P h osp h a t a se Assa y. An Escherichia coli
strain containing a plasmid encoding the genes fusion con-
struct of glutathione-S-transferase (GST) and human cdc25A
under the control of IPTG was used. E. coli were first grown
in 100 µg of ampicillin/mL of LB medium. When the optical
density at 600 nm had reached a value between 0.8 and 1.00,
IPTG was added and the culture incubated for 7 h. Cells were
then harvested by a 3000g centrifugation for l5 min at 4 °C.
Pellets were kept frozen at -80 °C until extraction. The
bacterial pellet was disrupted by sonication in lysis buffer at
4 °C. The homogenate was centrifuged for 30 min at 4 °C at
100000g. The supernatant was recentrifuged under similar
conditions; the final supernatant was then immediately mixed
and rotated with glutathione-agarose beads (equilibrated with
lysis buffer) for 30 min at 4 °C. The glutathione-agarose
beads were washed three times with 10 volumes of lysis buffer,
followed by four washes with 10 volumes of Tris buffer A.
Elution of the fusion protein was induced by three or four
successive washes with 10 mM glutathione in Tris buffer A.
The efficiency of the elution was monitored by the following
phosphatase assay. Twenty microliters of GST-cdc25A protein
(diluted to an activity of ∂ OD 405 nm ) 0.2-0.3/60 min) was
mixed with 20 µL of 100 mM DTT (in Tris buffer A) and 140
µL of Tris buffer A, in 96-wells microplates. The plates were
preincubated at 37 °C for 15 min in a Denley Wellwarm 1
microplate incubator. The assays were initiated by addition
of 20 µL of 500 mM p-NPP (in Tris buffer A). After a 60 min
incubation at 37 °C, absorbance at 405 nm was measured in
a BioRad microplate reader. Blank values (no GST-cdc25A
protein added) were automatically substracted.
In Vitr o An titu m or Activity. A modification of the
double-layer soft-agar assay as described by Hamburger and
Salmon20 was used as described recently.13 Briefly, solid
human tumor xenografts were mechanically disaggregated and
subsequently incubated with an enzyme cocktail consisting of
collagenase 0.05%, DNAse 0.07%, and hyaluronidase 0.1% in
RPMI 1640 at 37 °C for 30 min. The cells were washed twice
and passed through sieves of 200 and 50 µm each size. The
percentage viable cells was determined in a hemocytometer
using trypan blue exclusion. The tumor cell suspension was
plated into 24-multiwell plates over a bottom layer consisting
of 0.2 mL of Iscoves’ modified Dulbecco’s medium with 20%
fetal calf serum and 0.7% agar. Next, 20 000 to 200 000 cells
were added to 0.2 mL of the same culture medium and 0.4%
agar and plated onto the base layer. Cytostatic drugs were
applied by continuous exposure (drug overlay) in 0.2 mL of
medium. In each assay, six control plates received the vehicle
only; drug-treated groups were plated in triplicate in three
concentrations: 0.1, 1, and 10 µg/mL. Drug effects are
expressed as percentage survival obtained by comparing the
mean number of colonies in treated plates with the mean
number of colonies of control plates (test/control, %, T/C, %).
Cytotoxicity is considered to be significant if the compound
reduced colony formation to less than 30% of the control value
(T/C < 30%).
1
for H and 121.44 MHz for 31P. 1H NMR spectra are reported
in units of δ relative to internal CHCl3 at 7.24 ppm. 31P NMR
spectra were proton noise decoupled and are reported in units
of δ relative to 85% phosphoric acid as external standard;
positive shifts are downfield. Analyses indicated by the
symbols of the elements were carried out by the microana-
lytical section of the Institute of Organic and Pharmaceutical
Chemistry of the National Hellenic Research Foundation and
the microanalytical section of the Chemistry Department of
the University College London.
Gen er a l P r oced u r e for th e Qu a ter n iza tion of N-Me-
th ylm or p h olin e a n d N-Meth ylp ip er id in e. To a stirred
solution of amine (49.5 mmol) in EtOH (10 mL) was added
dropwise at room temperature 2-bromoethanol (9.2 g, 74
mmol). The resulting mixture was refluxed for 2 h. After
cooling, the excess 2-bromoethanol and EtOH were evaporated
in vacuo. Trituration of the crude mixture with ether afforded
the desired quaternary salts.
N-(2-Hyd r oxyeth yl)-N-m eth ylm or p h olin iu m br om id e
1
(1): yield 99%; TLC (CH2Cl2/MeOH (85:15)) Rf 0.20; H NMR
(DMSO-d6) δ 5.40 (t, 1H, J ) 4.8 Hz), 4.10-3.92 (m, 6H), 3.72-
3.52 (m, 6H), 3.32 (s, 3H).
N-(2-H yd r oxyet h yl)-N-m et h ylp ip er id in iu m b r om id e
1
(2): yield 99%; TLC (CH2Cl2/MeOH (85:15)) Rf 0.20; H NMR
(DMSO-d6) δ 3.92-3.81 (m, 2H), 3.78-3.48 (m, 6H), 3.30 (s,
3H), 1.95-1.78 (m, 4H), 1.72-1.61 (m, 2H).
Gen er a l P r oced u r e for th e P r ep a r a tion of Alk ylp h os-
p h olip id s a n d (Alk oxyeth yl)p h osp h olip id s. To a stirred
solution of phosphorus oxychloride (0.307 g, 2 mmol) and
triethylamine (0.36 g, 3.6 mmol) in tetrahydrofuran (2.5 mL)
was added dropwise at 0 °C a solution of the corresponding
alcohol or alkoxyethanol (2 mmol) in tetrahydrofuran (2.5 mL).
The resulting mixture was stirred for 10 min at 0 °C and
subsequently hydrolyzed by the addition of H2O (0.5 mL). After
1 h of stirring at room temperature, the reaction mixture was
diluted with ether and the organic layer was washed with H2O,
brine and dried (Na2SO4). The solvent was evaporated in
vacuo to afford the crude phosphoric acid derivative which was
transformed to the pyridinium salt by the addition of pyridine
(14 mL) and stirring for 2 h at 50 °C. After cooling the solvent
was evaporated at high vacuum and pyridine (6 mL) was
added to the residue. To the resulting solution was added
dropwise at 0 °C a solution of triisopropylbenzenesulfonyl
chloride (1.2 g, 3.96 mmol) in pyridine (23 mL) followed by
the addition of N-(2-hydroxyethyl)-N-methylmorpholinium
bromide or N-(2-hydroxyethyl)-N-methylpiperidinium bromide
(3 mmol). The resulting mixture was stirred at 35-40 °C for
5 h. After cooling, the mixture was hydrolyzed by the addition
of H2O (1.5 mL) and 2-propanol (6 mL) and stirred for 10 min
at room temperature, and the solvents were evaporated in
vacuo. The resulting crude solid was purified by gravity
column chromatography (CH2Cl2/MeOH/NH4OH (60:50:5) fol-
lowed by MeOH/NH4OH (95:5)), and the solvents were evapo-
rated in vacuo. The residue was diluted with CHCl3 (5 mL)
and filtered through a pore membrane (0.5 µm, FH Millipore)
and the solvent removed under vacuum to give the desired
product.
The composition of the panel is based more on the sensitivity
of the lines to standard anticancer agents than on ensuring
that frequently occurring human tumor types are represented.
Currently, the panel consists of two sensitive lines, nonsmall
cell lung carcinoma line LXFL 529 and small cell lung
carcinoma LXFS 538; two intermediately sensitive lines,
ovarian adenocarcinoma OVXF 1023 and melanoma MEXF
989; and two resistant lines, colorectal adenocarcinoma CXF
1103 and renal cancer RXF 423. Most of the standard
anticancer agents are active at a dose of e1 µg/mL.
(N-Meth ylm or p h olin o)eth a n ol d od ecyl p h osp h on a te
(9): yield 25%; TLC (CH2Cl2/MeOH/NH4OH (60:50:5)) Rf 0.25;

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2613
1H NMR δ 4.38-4.15 (m, 2H), 4.05-3.85 (m, 6H), 3.70-3.58
(m, 6H), 3.44 (bs, 3H), 1.55-1.42 (m, 2H), 1.32-1.06 (m, 18H),
0.82 (t, 3H, J ) 6.3 Hz); 31P NMR δ -2.0. Anal. (C19H40NO5P‚-
2H2O) C, H, N.
(N-Met h ylm or p h olin o)et h a n ol t et r a d ecyl p h osp h o-
n a te (10): yield 21%; TLC (CH2Cl2/MeOH/NH4OH (60:50:5))
Rf 0.25; 1H NMR δ 4.32-4.20 (m, 2H), 3.95-3.85 (m, 6H),
3.75-3.58 (m, 6H), 3.45 (s, 3H), 1.55-1.42 (m, 2H), 1.32-1.12
(m, 22H), 0.82 (t, 3H, J ) 6.7 Hz); 31P NMR δ -2.0. Anal.
(C21H44NO5P‚1.45H2O) C, H, N.
(N-Met h ylm or p h olin o)et h a n ol h exa d ecyl p h osp h o-
n a te (11): yield 22%; TLC (CH2Cl2/MeOH/NH4OH (60:50:5))
Rf 0.25; 1H NMR δ 4.32-4.25 (m, 2H), 4.05 -3.90 (m, 6H),
3.80-3.55 (m, 6 H), 3.47 (s, 3H), 1.60-1.48 (m, 2H), 1.35-
1.15 (m, 26H), 0.86 (t, 3H, J ) 6.5 Hz); 31P NMR δ -2.0. Anal.
(C23H48NO5P‚0.5H2O) C, H, N.
Refer en ces
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(N-Met h ylm or p h olin o)et h a n ol 2-(d od ecyloxy)et h yl
p h osp h on a te (12): yield 40%; TLC (CH2Cl2/MeOH/NH4OH
(60:50:5)) Rf 0.20; 1H NMR δ 4.40-4.22 (m, 4H), 4.05-3.82
(m, 6H), 3.80-3.58 (m, 4H), 3.52-3.48 (m, 2H), 3.42 (s, 3H),
3.35 (t, 2H, J ) 6.9 Hz), 1.55-1.38 (m, 2H), 1.30-1.11 (m,
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Hexadecylphosphocholine in the Topical Treatment of Skin
18H), 0.82 (t, 3H, J ) 6.6 Hz); 31P NMR δ -2.0. Anal. (C21H44
NO6P‚1.5H2O) C, H, N.
-
(N-Meth ylm or p h olin o)eth a n ol 2-(tetr a d ecyloxy)eth yl
p h osp h on a te (13): yield 31%; TLC (CH2Cl2/MeOH/NH4OH
(60:50:5)) Rf 0.20; 1H NMR δ 4.35-4.18 (m, 4H), 4.00-3.85
(m, 6H), 3.74-3.59 (m, 4H), 3.52-3.49 (m, 2H), 3.41 (s, 3H),
3.36 (t, 2H, J ) 6.9 Hz), 1.58-1.42 (m, 2H), 1.30-1.18 (m,
Metastases: A Phase
I trial. In New Drugs in Oncology;
Queisser, W., Fiebig, H. H., Eds.; Karger: New York, 1989; pp
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hauser, G.; Busch, R.; Vogler, W. R.; Rastetter, J .; Berdel, W. E.
Cytotoxic Effects of Hexadecylphosphocholine in Neoplastic Cell
Lines Including Drug-Resistant Sublines in Vitro. J . Lipid
Mediators 1990, 2, 271-280.
22H), 0.83 (t, 3H, J ) 6.6 Hz); 31P NMR δ -2.0. Anal. (C23H48
NO6P‚2H2O) C, H, N.
-
(N-Meth ylm or p h olin o)eth a n ol 2-(h exa d ecyloxy)eth yl
p h osp h on a te (14): yield 48.6%; TLC (CH2Cl2/MeOH/NH4OH
(60:50:5)) Rf 0.20; 1H NMR δ 4.35-4.22 (m, 4H), 4.01-3.80
(m, 6H), 3.72-3.56 (m, 4H), 3.55-3.48 (m, 2H), 3.40 (s, 3H),
3.33 (t, 2H, J ) 6.9 Hz), 1.50-1.38 (m, 2H), 1.30-1.12 (m,
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Elevation of Leukemic Cell Intracellular Calcium by the Ether
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K.; Shoji, M.; Eibl, H.; Berdel, W. E.; Hajdu, J .; Vogler, W. R.;
Kuo, J . F. Inhibition of Protein Kinase C, (Sodium Plus Potas-
sium)-Activated Adenosine Triphosphatase, and Sodium Pump
by Synthetic Phospholipid Analogues. Cancer Res. 1990, 50,
3025-3031. (e) Bonjouklian, R.; Phillips, M. L.; Kuhler, K. M.;
Grindey, G. B.; Poore, G. A.; Schultz, R. M.; Altom, M. G. Studies
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kenyl)phosphocholines. J . Med. Chem. 1986, 29, 2472-2477. (f)
Houlihan, W. J .; Lee, M. L.; Munder, P. G.; Winslow, C. M.;
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phospholipids. Synthesis and Neoplastic Cell Growth Inhibitory
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Natschke, S. L.; Gumus, F.; Marasco, C. J ., J r.; Meyer, K. L.;
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(5) (a) Modolell, M.; Andreesen, R.; Pahlke, W.; Brugger, U.;
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the Selective Destruction of Tumor Cells Induced by Alkyl-
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Protein Kinase C by Hexadecylphosphocholine and 1-O-Octa-
decyl-2-O-methyl-rac-glycero-3-phosphocholine do not Correlate
With Inhibition of Proliferation of HL60 and K562 Cell Lines.
Eur. J . Cancer 1994, 30A, 509-515.
26H), 0.80 (t, 3H, J ) 6.6 Hz); 31P NMR δ -2.0. Anal. (C25H52
NO6P‚0.5H2O) C, H, N.
-
(N-Met h ylp ip er id in o)et h a n ol d od ecyl p h osp h on a t e
(15): yield 16%; TLC (CH2Cl2/MeOH/NH4OH (60:50:5)) Rf 0.24;
1H NMR δ 4.32-4.18 (m, 2H), 3.92-3.81 (m, 2H), 3.80-3.48
(m, 6H), 3.32 (s, 3H), 1.95-1.78 (m, 4H), 1.72-1.61 (m, 2H),
1.60-1.48 (m, 2H), 1.35-1.15 (m, 18H), 0.83 (t, 3H, J ) 6.3
Hz); 31P NMR δ -1.5. Anal. (C20H42NO4P‚2H2O) C, H, N.
(N-Meth ylp ip er id in o)eth a n ol tetr a d ecyl p h osp h on a te
(16): yield 14%; TLC (CH2Cl2/MeOH/NH4OH (60:50:5)) Rf 0.27;
1H NMR δ 4.30-4.18 (m, 2H), 3.90-3.42 (m, 8H), 3.23 (s, 3H),
1.92-1.85 (m, 4H), 1.70-1.58 (m, 2H), 1.57-1.42 (m, 2H),
1.32-1.10 (m, 22H), 0.81 (t, 3H, J ) 6.5 Hz); 31P NMR δ -1.5.
Anal. (C22H46NO4P‚H2O) C, H, N.
(N-Meth ylp ip er id in o)eth a n ol h exa d ecyl p h osp h on a te
(17): yield 16%; TLC (CH2Cl2/MeOH/NH4OH (60:50:5)) Rf 0.25;
1H NMR δ 4.15-4.06 (m, 2H), 3.70-3.32 (m, 8H), 3.15 (s, 3H),
1.78-1.62 (m, 4H), 1.58-1.30 (m, 4H), 1.22 -0.95 (m, 26H),
0.66 (t, 3H, J ) 6.5 Hz); 31P NMR δ -1.5. Anal. (C24H50
NO4P‚1.5H2O) C, H, N.
-
(N-Meth ylp ip er id in o)eth a n ol 2-(tetr a d ecyloxy)eth yl
p h osp h on a te (18): yield 37.6%; TLC (CH2Cl2/MeOH/NH4OH
(60:50:5)) Rf 0.32; 1H NMR δ 4.32-4.20 (m, 2H), 3.98-3.78
(m, 4H), 3.70-3.48 (m, 6H), 3.37 (t, 2H, J ) 6.8 Hz), 3.29 (s,
3H), 1.90-1.80 (m, 4H), 1.70-1.60 (m, 2H), 1.54-1.42 (m, 2H),
1.32-1.14 (m, 22H), 0.84 (t, 3H, J ) 6.5 Hz); 31P NMR δ -2.2.
Anal. (C24H50NO5P‚2H2O) C, H, N.
(N-Meth ylp ip er id in o)eth a n ol 2-(h exa d ecyloxy)eth yl
p h osp h on a te (19): yield 30%; TLC (CH2Cl2/MeOH/NH4OH
(60:50:5)) Rf 0.32; 1H NMR δ 4.40-4.25 (m, 2H), 4.07-3.73
(m, 4H), 3.70-3.48 (m, 6H), 3.37 (t, 2H, J ) 6.8 Hz), 3.29 (s,
3H), 1.95-1.75 (m, 4H), 1.75-1.60 (m, 2H), 1.55-1.45 (m, 2H),
1.35-1.15 (m, 28H), 0.84 (t, 3H, J ) 6.5 Hz); 31P NMR δ -2.1.
Anal. (C26H54NO5P‚1.5H2O) C, H, N.
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by KESY (Central Health Council of Greece) and by the
National Drug Organization of Greece. We would like
to express our gratitude to Dr. E. Kalatzis for perfoming
the analytical work.

2614 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Notes
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