Synthesis of Novel Lysophosphatidylcholine Analogues Using Serine as Chiral Template

Article abstract of DOI:10.1021/jo034969s

Four novel lysophosphatidylcholine (lysoPC) analogues, (S)-N-stearoyl-O-phosphocholineserine methyl ester [(S)-1a], (R)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine [(R)-2a], (R)-N-stearoyl-O-phosphocholineserine methyl ester [(R)-1b], and (S)-1-lyso-2-stearoyl-amino-2-deoxy-sn-glycero-3-phosphatidylcholine [(S)-2b], were synthesized starting from serine as a chiral template. These synthetic compounds exhibited greatly enhanced hyphal transition inhibitory activity in Candida as compared to the natural lysoPC.

Full text of DOI:10.1021/jo034969s

The effects of antler on immunological function may be
the result of stimulation of cellular immunity or the
suppression of pathogenic activity. Of the various patho-
gens in humans, fungi are least vulnerable to medicines.
Only a few phospholipids exhibited antifungal activity
but none of them targeted on the morphogenic transition
suppression of fungi, to the best of our knowledge.10 We
successfully synthesized lysoPC as well as its two regio-
isomers in racemic form in order to establish a powerful
spectroscopic tool for the structural elucidation of lysoPCs
in nature.8f,g From our previous study, however, the
isolated lysoPC was proven to be not as effective as other
commercially available drugs such as amphotericin B,
clotrimazole, ketoconazole, and fluconazole on the sup-
pression of hyphal transition of C. albicans (data not
shown). In addition, sphingomyelin and phosphatidyl-
choline had no effect, suggesting that both phosphocho-
line moiety and a short chain length at the C1 position
of the glycerol backbone may be necessary in the hyphal
transition suppressing activity. These results led us to
design and synthesize four lysoPC analogues. In this
paper, we report the asymmetric synthesis of newly
designed lysoPC analogues to search for a novel hyphal
transition suppressor in C. albicans.
Syn th esis of Novel
Lysop h osp h a tid ylch olin e An a logu es Usin g
Ser in e a s Ch ir a l Tem p la te
Young-Ah Kim,† Hae-Mi Chung,† J in-Seon Park,†
Wonja Choi,*,‡ J uyoung Min,‡ Nok-Hyun Park,‡
Ki-Hwan Kim,† Gil-J a J hon,† and So-Yeop Han*,†
Department of Chemistry, Division of Molecular Life
Sciences and Department of Life Sciences, Ewha Womans
University, Seoul 120-750, Korea
syhan@ewha.ac.kr
Received J uly 6, 2003
Abstr a ct: Four novel lysophosphatidylcholine (lysoPC)
analogues, (S)-N-stearoyl-O-phosphocholineserine methyl
ester [(S)-1a ], (R)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-
3-phosphatidylcholine [(R)-2a ], (R)-N-stearoyl-O-phospho-
cholineserine methyl ester [(R)-1b], and (S)-1-lyso-2-stearoyl-
amino-2-deoxy-sn-glycero-3-phosphatidylcholine [(S)-2b], were
synthesized starting from serine as a chiral template. These
synthetic compounds exhibited greatly enhanced hyphal
transition inhibitory activity in Candida as compared to the
natural lysoPC.
Structures of novel lysoPC analogues are shown in
Figure 1. They are (S)-N-stearoyl-O-phosphocholineserine
methyl ester [(S)-1a ], (R)-1-lyso-2-stearoylamino-2-deoxy-
sn-glycero-3-phosphatidylcholine [(R)-2a ], (R)-N-stearoyl-
O-phosphocholineserine methyl ester [(R)-1b ], and
(S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phospha-
tidylcholine [(S)-2b].
Recently, lysophosphatidylcholine (lysoPC) has been of
crucial importance in various biochemical processes due
to its regulatory activity of signaling enzymes in a range
of lysoPC-responsive cells. LysoPC mediates the activa-
tion of p38, AP-1, and J NK kinases,1,2 as well as adenylyl
cyclase,3 and displays the inhibition of platelet aggrega-
tion4,5 while specifically binding to two G-protein-coupled
receptors, OGR1 and GPR4, with lower affinity than
sphingosylphosphorylcholine.6 In addition, the presence
of lysoPC resulted in the migration of lymphocytes
toward the site of inflammatory tissue by inducing the
secretion of chemotactic chemicals from macrophages.7
In the course of our study in glycerolipid chemistry,8
we isolated lysoPCs from deer antler extracts, guided by
the inhibitory activity of morphogenic transition of yeast
to hyphae in Candida albicans, and fully characterized
their structures.8e Despite the growing knowledge of deer
antler in immunological function, the molecular basis of
a cellular target or mechanism of action is unknown.8a,9
These four lysoPC analogues are designed in such a
way that the OH group at the C1 position of the glycerol
backbone is either preserved or transformed into the CO2-
CH3 group, whereas the OH group at the C2 position is
replaced by NHCO(CH2)16CH3. LysoPC analogues are
designed to possess the amide group instead of the ester
with an expectation of providing them with resistance
to enzymatic hydrolysis.
Synthesis of the first target molecule, (S)-N-stearoyl-
O-phosphocholineserine methyl ester [(S)-1a ], is illus-
(8) (a) Han, S.-Y.; Cho, S.-H.; Kim, S.-Y.; Seo, J .-T.; Moon, S.-J .;
J hon, G.-J . Bioorg. Med. Chem. Lett. 1999, 9, 59-64. (b) Kim, Y. H.;
Han, S.-Y.; Cho, S.-H.; Yoo, J . S.; J hon, G.-J . Rapid Commun. Mass
Spectrom. 1999, 13, 481-487. (c) Limb, J .-K.; Kim, Y. H.; Han, S.-Y.;
J hon, G.-J . J . Lipid Res. 1999, 40, 2169-2176. (d) Kim, Y. H.; So, K.-
Y.; Limb, J .-K.; J hon, G.-J .; Han, S.-Y. Rapid Commun. Mass Spectrom.
2000, 14, 2230-2237. (e) Min, J .; Lee, Y.-J .; Kim, Y.-A.; Park, H.-S.;
Han, S.-Y.; J hon, G.-J .; Choi, W. Biochim. Biophys. Acta 2001, 1531,
77-89. (f) Hong, J .; Kim, Y. H.; Gil, J . H.; Cho, K.; J ung, J . H.; Han,
S.-Y. Rapid Commun. Mass Spectrom. 2002, 16, 2089-2093. (g) Kim,
Y.-A.; Park, M.-S.; Kim, Y. H.; Han, S.-Y. Tetrahedron 2003, 59, 2921-
2928.
* Corresponding author.
† Department of Chemistry and Division of Molecular Life Sciences.
‡ Department of Life Sciences.
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10.1021/jo034969s CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/03/2003
10162
J . Org. Chem. 2003, 68, 10162-10165

SCHEME 2
between compound 3 and stearic acid was achieved by
using standard DCC/HOBt methodology11 to provide the
desired amide 4 in good yield. It is noteworthy to mention
that protection of the C3 hydroxyl group of compound 3
was not necessary prior to executing the amide bond
formation at the C2 amino group. Final installation of
the phosphocholine moiety into the C3 hydroxyl group
of compound 4 was the key step in our strategy. Attempts
to synthesize the phosphocholine moiety by using tri-
ethylammonium phosphocholine,12 2-bromoethyl dichlo-
rophosphate,13 and 2-chloro-2-oxo-1,3,2-dioxaphospholane14
were unsuccessful. Interestingly, we found that the
phosphocholine moiety was successfully introduced by
using the ethylene chlorophosphite-trimethylamine pro-
tocol, according to the procedure reported by Bittman.8g,15
Thus, compound 4 was treated with ethylene chlorophos-
phite in the presence of N,N-diisopropylethylamine in
THF at -15 °C to produce cyclic phosphite 5. Compound
5 was not purified due to its decomposing nature on silica.
After ring-opening reaction of compound 5 with bromine
and subsequent hydrolysis, the resulting compound 6 was
quaternized with 40% aqueous trimethylamine in CHCl3/
iPrOH/CH3CN. However, we could not trace the desired
product by thin-layer chromatography, in contrast with
our previous result in the synthesis of lysoPC.8g At this
point, we decided to purify the intermediate 6 by means
of recrystallization with acetone/methylene chloride be-
fore proceeding to the next step. The first target, (S)-1a ,
was successfully synthesized from purified 6 by treat-
ment with aqueous trimethylamine (12% yield from 4).
As a result, compound (S)-1a was obtained from L-serine
in six steps and 11% overall yield.
F IGURE 1. Structures of the novel lysophosphatidylcholine
(lysoPC) analogues: (S)-N-stearoyl-O-phosphocholineserine
methyl ester [(S)-1a ], (R)-1-lyso-2-stearoylamino-2-deoxy-sn-
glycero-3-phosphatidylcholine [(R)-2a ], (R)-N-stearoyl-O-phos-
phocholineserine methyl ester [(R)-1b], and (S)-1-lyso-2-stear-
oylamino-2-deoxy-sn-glycero-3-phosphatidylcholine [(S)-2b ].
SCHEME 1a
Synthesis of the second target, (R)-2a , was accom-
plished by reduction of the methyl ester group of (S)-1a
to the corresponding alcohol with lithium aluminum
hydride in THF,16 in 54% yield (Scheme 2).
Synthesis of the third and fourth target molecules, (R)-
1b and (S)-2b, followed the same synthetic protocol as
for (S)-1a and (R)-2a . Synthesis began with D-serine as
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a
Key: (a) dry HCl/MeOH, 2.5 h, 98%; (b) stearic acid, DCC,
HOBt, NMM, CH2Cl2, 0 °C to rt, 4 h, 90%; (c) ethylene chloro-
phosphite, DIEA, THF, -15 °C, 1.5 h; (d) (i) Br2, -15 °C, 15 min,
(ii) H2O, rt, 1 h; (e) aq 40% NMe3, CHCl3/iPrOH/CH3CN (3/5/5,
v/v), rt, 11 h, 12% (from 4).
trated in Scheme 1. L-Serine was chosen as the chiral
template and protected by treatment with saturated HCl
in methanol to provide methyl ester 3. Coupling reaction
J . Org. Chem, Vol. 68, No. 26, 2003 10163

the chiral template. The carboxylic group of D-serine was
protected as the methyl ester, which was then reacted
with stearic acid to afford N-stearoyl-D-serine methyl
ester in 87% yield for two steps. Sequential reactions of
the resulting amide with ethylene chlorophosphite, bro-
mine, and water followed by aqueous trimethylamine
produced the third target, (R)-1b (10% overall yield from
D-serine). For the installation of the hydroxyl function
at the C1 position, the ester group of (R)-1b was reduced
with lithium aluminum hydride to afford the fourth
target, (S)-2b (53% yield), with a rotation value of -5.3
(c 2.04, CH2Cl2/MeOH). Comparison with (R)-2a , which
has a positive rotation value of +5.3 (c 1.92, CH2Cl2/
MeOH), revealed that the target lysoPC analogues were
synthesized in enantiomerically pure forms. All struc-
tures of lysoPC analogues were characterized by spec-
troscopic analysis and compounds (S)-1a and (R)-2a were
further confirmed by fast atom bombardment mass (FAB-
MS) spectrometry.
For the antifungal activity data, Candida strains were
used as the test microorganism and grown in the Sab-
ouraud-dextrose broth at 30 °C for 16 h. Then C. krusei,
C. parapsilosis, C. tropicalis, C. guilliermondii, and C.
albicans were transferred to cornmeal agar plates supple-
mented with lysoPC analogues: at 1.9 µg/mL for (S)-1a
and (R)-1b and at 7.8 µg/mL for (R)-2a and (S)-2b. After
5 days of incubation, each strain was observed with a
photometric microscope. All of the lysoPC analogues
showed selective inhibitory activity against the hyphal
transition on various species of Candida, such as C.
albicans, C. krusei, C. guilliermondii, and C. parapsilosis,
but not C. tropicalis. The minimum inhibitory concentra-
tion (MIC) and IC50 were determined as reported
previously.8e The log-phase yeast culture of C. albicans
was transferred to RPMI liquid media and grown at 37
°C for 7 h with or without each of the lysoPC analogues
at various concentrations. The percentage of hyphae was
calculated by counting hyphae and the total number of
F IGURE 2. Effects of (a) (S)-1a and (R)-1b and (b) (R)-2a
and (S)-2b on the growth of SC5314. SC5314 was cultured in
Sabouraud’s dextrose broth supplemented with each isomer
at 30 °C, and growth was determined by measuring the
absorbance at 660 nm (OD660).
F IGURE 3. Additive effects of four lysoPC analogues with amphotericin B on the suppression of hyphal transition in Candida
albicans. Lower and less-toxic doses of amphotericin B (0.02 µg/mL) and IC50 values of four lysoPC analogues were added to the
culture for (S)-1a and (R)-1b (at 1 µg/mL), for (S)-2b (at 4 µg/mL), and for (R)-2a (at 2 µg/mL). Hyphae were counted after 7.5
h of growth.
10164 J . Org. Chem., Vol. 68, No. 26, 2003

TABLE 1. Min im u m In h ibitor y Con cen tr a tion (MIC)
a n d IC50 Va lu es for In h ibition of Hyp h a l Tr a n sition of
th e F ou r LysoP C An a logu es
compd
MIC (µM)
IC50 (µM)
(R)-2a
(S)-2b
(S)-1a
(R)-1b
9.5
15.3
3.5
3.8
7.6
1.8
1.8
7.0
cells (Table 1). The four lysoPC analogues exhibited
substantial antifungal activitysas measured by the
hyphal transition suppressing activity of the yeast.
They also inhibited the growth of yeast (Figure 2).
Compound (S)-1a is strongly effective in the suppression
of hyphal transition as well as in inhibition of yeast
growth. Their additive effects with amphotericin B are
shown in Figure 3. Combining the lysoPC analog with
amphotericin B suppressed hyphal transition more
strongly than each product alone. Compound (S)-2b
represented the strongest additive effect on the suppres-
sion of hyphal transition.
We also investigated the effect of lysoPC and the
synthetic compounds, (S)-1a and (R)-1b, on the mobili-
zation of intracellular calcium concentration in spleen
lymphocytes (Figure 4). The intracellular calcium con-
centration strongly increased when spleen lymphocytes
were stimulated with 25 g/mL (S)-1a (Figure 4E-b) and
(R)-1b (Figure 4D-b) as well as lysoPC (Figure 4C-b).
Because the intracellular calcium mobilization occurs
early in the lymphocyte activation, investigation of the
subsequent effect on signal transduction would confirm
whether lysoPC and synthesized lysoPC analogues affect
the immune system by the same mechanism.
In conclusion, (R)- and (S)-enantiomers of novel lysoPC
analogues were synthesized from commercially available
L- and D-serine as starting materials by a short and
efficient method. These newly designed and synthesized
lysoPC analogues exhibited much enhanced hyphal tran-
sition inhibitory activity against Candida species as
compared to the natural lysoPC. Compounds are obtained
as crystalline powder and are very hydrophilic and
lipophilic. Therefore, they could be safely administered
F IGURE 4. Calcium mobilization in response to (S)-1a and
(R)-1b stimulation. (A) Spleen lymphocytes (106 cells/mL) were
loaded with Fluo-3/AM (2 µg/mL). These cells were stimulated
with (B) ionomycin (2 µg/mL), (C-a) lysoPC (12.5 µg/mL), (C-
b) lysoPC (25 µg/mL), (C-c) lysoPC (50 µg/mL), (D-a) (R)-1b
(12.5 µg/mL), (D-b) (R)-1b (25 µg/mL), (D-c) (R)-1b (50 µg/
mL), (E-a) (S)-1a (12.5 µg/mL), (E-b) (S)-1a (25 µg/mL), and
(E-c) (S)-1a (50 µg/mL). Changes in [Ca2+]i were determined
as the percentage of positive cells that mobilized intracellular
calcium, as assessed by flow cytometry.
either parenterally or orally as pharmaceutical composi-
tions of various dosages.
Ack n ow led gm en t. This work was supported by
grants from KOSEF (ABRL-R14-2002-015-01002-0) and
MHW/MIC (01-PJ11-PG9-01NT00-0044). Y.A.K., H.M.C.,
J .S.P., J .M., N.H.P., and K.W.K. are recipients of
fellowships of the BK 21 program.
Su p p or tin g In for m a tion Ava ila ble: Experimental sec-
1
tion and H and 13C NMR spectra as well as FAB-MS spectra
of compounds (S)-1a and (R)-2a . This material is available
free of charge via the Internet at http://pubs.acs.org.
(16) (a) Patil, N. T.; Tilekar, J . N.; Dhavale, D. D. J . Org. Chem.
2001, 66, 1065-1074. (b) Eklund, P. C.; Riska, A. I.; Sjo¨holm, R. E. J .
Org. Chem. 2002, 67, 7544-7546.
J O034969S
J . Org. Chem, Vol. 68, No. 26, 2003 10165