Amphiphilic anionic analogues of galactosylceramide: Synthesis, anti-HIV-1 activity, and gp120 binding

Article abstract of DOI:10.1021/jm0011124

We describe the synthesis together with the results of anti-HIV-1 activity and gp120-monolayer binding experiments of new galactosyl amphiphiles, analogues of galactosylceramide, an alternative receptor used by HIV to infect CD4 negative cells. These compounds consist of single- and double-chain amphiphiles containing one or two galactose residues. To favor their clustering into galactosyl-rich microdomains, their molecular structure contains also an amino group or several hydroxyls or anionic groups, such as carboxylate, sulfate, sulfonate, and phosphate. Among the 12 new galactosylated compounds reported, a specific anti-HIV activity, although moderate (IC₅₀ from 10 to 50 μM), was detected only for three of them, i.e., I-GalSer-[CO2Na][C14], II-GalSer[C14][C7SO3Na], and II-GalSer[C2SO4Na][C14], which contain an anionic group. The marked increase of surface pressure which was observed upon addition of gp120 into the aqueous subphase underneath the monolayers containing these galactolipids indicated gp120 insertion into the monolayers, suggesting that binding of these three derivatives to HIV-1 gp120 may be responsible for their anti-HIV activity.

Full text of DOI:10.1021/jm0011124

2188
J . Med. Chem. 2001, 44, 2188-2203
Am p h ip h ilic An ion ic An a logu es of Ga la ctosylcer a m id e: Syn th esis, An ti-HIV-1
Activity, a n d gp 120 Bin d in g
Barbara Faroux-Corlay,† J acques Greiner,† Raphae¨l Terreux,‡ Daniel Cabrol-Bass,‡ Anne-Marie Aubertin,§
Pierre Vierling,*,† and J acques Fantini#
Laboratoire de Chimie Bioorganique, UMR 6001 CNRS-Universite´ de Nice Sophia-Antipolis, Faculte´ des Sciences,
Parc Valrose, 06108 Nice, France, LARTIC, Universite´ de Nice Sophia-Antipolis, Faculte´ des Sciences, Parc Valrose,
06108 Nice, France, INSERM U74, Institut de Virologie, 67000 Strasbourg, France, and Institut Me´diterrane´en de Recherche
en Nutrition, UMR-INRA, Faculte´ des Sciences St-J e´rome, 13397 Marseille Ce´dex 20, France
Received November 8, 2000
We describe the synthesis together with the results of anti-HIV-1 activity and gp120-monolayer
binding experiments of new galactosyl amphiphiles, analogues of galactosylceramide, an
alternative receptor used by HIV to infect CD4 negative cells. These compounds consist of
single- and double-chain amphiphiles containing one or two galactose residues. To favor their
clustering into galactosyl-rich microdomains, their molecular structure contains also an amino
group or several hydroxyls or anionic groups, such as carboxylate, sulfate, sulfonate, and
phosphate. Among the 12 new galactosylated compounds reported, a specific anti-HIV activity,
although moderate (IC50 from 10 to 50 µM), was detected only for three of them, i.e., I-Ga lSer -
[CO2Na ][C14], II-Ga lSer [C14][C7SO3Na ], and II-Ga lSer [C2SO4Na ][C14], which contain
an anionic group. The marked increase of surface pressure which was observed upon addition
of gp120 into the aqueous subphase underneath the monolayers containing these galactolipids
indicated gp120 insertion into the monolayers, suggesting that binding of these three derivatives
to HIV-1 gp120 may be responsible for their anti-HIV activity.
In tr od u ction
was also necessary for their infection by HIV, raising
the possibility that chemokine receptors may function
as coreceptors for HIV entry into CD4(-)/GalCer(+)
cells.10 Analogues of GalCer which would strongly
interact with the V3 loop are thus potential inhibitors
of HIV uptake and infection.
Compounds intended to inhibit the human immuno-
deficiency virus (HIV) at the early stages of its replica-
tion cycle, such as its adsorption on and entry into cells,
are worthy candidates for combination therapy with the
drugs presently used in clinics. HIV infects mainly CD4-
(+) lymphoid cells. Their infection is initiated by the
binding of HIV (through its envelope glycoprotein
gp120) to CD4 and then to a cellular coreceptor of the
chemokine receptor family, such as mainly CCR5 or
CXCR4,1-6 according to the cell type infected and the
virus tropism. The interactions of the gp120/CD4 com-
plex with these coreceptors involve the V3 loop of gp120,
which, thus, plays a key role in the complex fusion
process of the HIV envelope with the cellular membrane.
HIV can also infect in vitro many CD4(-) cells, includ-
ing hepatocytes, natural killer cells, and neural and
colon epithelial cells.7 The infection of these two latter
cell types has been proposed to occur through the
binding of the V3 loop to the galactosylceramide (Gal-
Cer) receptor (see structure in Table 1) they express and
more particularly to GalCer-rich microdomains. The
nonlinear relationships found between GalCer concen-
tration in a bilayer and its binding to gp120 suggests
indeed the involvement of GalCer-rich microdomains,8,9
thus providing an attachment platform for the virus
onto the cell. It has been further established that
coexpression of CXCR4 on CD4(-) colon epithelial cells
These findings have urged the synthesis of various
galactolipids and the examination of their anti-HIV
activity.11-21 As a part of our contribution into this field,
we recently reported on GalCer analogues deriving from
â-D-galactose and â-D-galactosamine (D,L)-, D- and L-
serine (GalSer), L-cysteine (GalCys), and ethanolamine
(GalAE), and possessing fluorocarbon and/or hydrocar-
bon hydrophobic chains (see examples in Table 1).22,23
Several of the derivatives reported by others and our
group were found to exhibit a moderate in vitro anti-
HIV activity (IC50 in the 1-220 µM range11-23) on CD4-
(+) or on CD4(-)/GalCer(+) cell lines, whereas none of
the GalCys derivatives was found to be active.23 More-
over, only some of these anti-HIV active galactolipids
inhibited the binding of [3H]suramin (a polysulfonyl
compound which displays a high affinity for the V3 loop)
to SPC3, a synthetic peptide which contains the con-
served GPGRAF region of the V3 loop.15,23 These results
most likely indicated that the neutralization of the
virion through masking of this conserved V3 loop region
was not the only mechanism involved in the HIV-1
antiviral activity of these GalCer analogues which might
also affect HIV-1 infection by post-binding inhibition of
HIV-1 entry into CD4(+) cells.
* To whom correspondence should be addressed. Tel: 33 (0)4 92 07
61 43. Fax: 33 (0)4 92 07 61 51. E-mail: vierling@unice.fr.
† Laboratoire de Chimie Bioorganique, UMR 6001 CNRS-Universite´
de Nice Sophia-Antipolis.
Unfortunately, neither the 3D structure of native
gp120 nor that of the gp120/GalCer complex are known.
Therefore, it is most difficult to design new GalCer
analogues that would be more efficient anti-HIV com-
‡ LARTIC, Universite´ de Nice Sophia-Antipolis.
§ INSERM U74, Institut de Virologie.
#
Institut Me´diterrane´en de Recherche en Nutrition, UMR-INRA.
10.1021/jm0011124 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/19/2001

Anionic Analogues of Galactosylceramide
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2189
Ta ble 1. Chemical Structure and Code Name of the Galactosylceramide (GalCer) Analogues
pounds than the former generation ones. Aiming at this
goal and in order to have a better understanding of the
role played by the structural elements of the GalCer
analogues that influence their antiviral activity, we
have performed a molecular modeling and dynamic
study which included all structural analogues of GalCer
known to display an anti-HIV activity11-16,21 as well as
those known to be inactive,22,23 these molecules being
inserted or not within a model membrane.24 Most
importantly, these molecular simulations (which will be
reported elsewhere) indicated that the most active
molecules were those that exhibited a stronger tendency
to form galactosyl-rich microdomains within the mem-
brane, in agreement with the above-mentioned experi-
mental data from literature.8,9
We describe here the synthesis together with the
results of biological testing, including the anti-HIV-1
activity in various cell cultures, and of gp120 binding
experiments, of new series of amphiphilic galactosyl
derivatives. These potential HIV inhibitors displayed in
Table 1 were partly designed according to our molecular
modeling and dynamic simulations24 and to SAR data
from literature.11-16,21-23 Their molecular structure
contains, in close proximity to or at variable distances
from the galactosyl hydrophilic head, either an amino
group or several hydroxyls or anionic groups, such as
carboxylate, sulfate, sulfonate, and phosphate. One
compound (i.e. bisLa c, Table 1) with its ,bouquet.-
like structure contains two galactosyl hydrophilic heads
connected to a hydrophobic double-chain moiety. Several
of these structural features were shown by our molec-
ular modeling and dynamic study to favor the clustering
of these derivatives within a model membrane, either
through an intermolecular hydrogen bonding network
or through complexation with divalent cations (Mg2+
Ca2+).24
,
Another feature from literature that was found to
result in a substantial antiviral activity increase was
the substitution of a carboxylate group for a terminal
methyl in the alkyl chain of a GalCer analog.15 There-
fore, we have designed the highly fluorinated II-Ga lAE-
[F 8C7][C11CO2Na ] in order to increase the anti-HIV
activity of the previously prepared II-Ga lAE[C16]-
[F 8C7].23 The antiviral activity of II-Ga lAE[C16]-
[F 8C7] was attributed to the presence of the lipophobic
fluorinated chain, which favors its self-association into
galactosyl-rich patches when incorporated within con-
ventional membranes. The anomeric â-configuration of

2190 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Faroux-Corlay et al.
Sch em e 1. Synthetic Route to the Hydrocarbon Single-Chain I-GalSer and Double-Chain II-GalSer Derivativesa
a
(i) RNH2/EDC/HOBt/CH2Cl2 with R ) -(CH2)11CO2Me for 3a and R ) -(CH2)2OH for 3b; (ii) morpholine/DMF; (iii) MeOH/Et3N/H2O
(2:1:1); (iv) 0.1 N NaOH in MeOH-H2O (9:1); (v) succinic anhydride/Et3N/CH2Cl2; (vi) tetradecanoic acid N-hydroxysuccinimide ester/
CH2Cl2 or THF; (vii) SO3‚pyridine, pyridine.
galactosyl was selected for all galactosylated derivatives
reported here, as this configuration seems to be essential
for anti-HIV activity.9,25 Finally, the GalSer series
derive from L-serine because this configuration was
found to led the more anti-HIV active compounds.23
ascertained by comparison with the 1H NMR data
reported in the literature28 and with 13C NMR data for
similar derivatives.28-30 Its â-configuration is confirmed
for instance by the anomeric C-1 resonance at 101.8
ppm.
The single chain compound presenting the serine
carboxylate function, i.e., I-Ga lSer [CO2Na ][C14], was
obtained from A in three steps with 57% overall yield
(Scheme 1, path A). These steps consisted of the Fmoc-
deprotection of A by action of morpholine, then acylation
with tetradecanoic N-hydroxysuccinimide ester, and
deacetylation with a MeOH:Et3N:H2O mixture followed
by a cation-exchange.23
Resu lts a n d Discu ssion
Ch em istr y. Ga lSer Ser ies. All the galactosylated
serine lipids described here and shown in Schemes 1
and 2 were prepared from the galactosylated L-serine
synthon, A, using conventional coupling and deprotec-
tion reactions. Compounds of the I-Ga lSer series
(Scheme 1) were obtained by condensing the selected
functional group either onto its deprotected amino or
onto its acid function. Compounds of the II-Ga lSer
series (Schemes 1 and 2) were basically obtained by
condensing successively two different functional groups,
the first one onto the acid and the second one onto the
amino function of A. These strategies which rely on the
use of the glycosylated amino acid building block A
benefited from the recent development of efficient
methods for its synthesis.23,26-28 These highly versatile
strategies were further preferred to that consisting first
in preparing the serine amino acid building blocks
containing the suitable functional group(s) or chain(s)
and then in their subsequent serine O-glycosylation,
which is a most delicate step to perform in good yields.
The structure of the key compound A was established
The two first steps of the preparation of I-Ga lSer -
[C 11C O 2N a ][N H 2],
I I -G a lS e r [C 11C O 2N a ]-
[C3CO2Na ], II-Ga lSer [C2OH][C14], and II-Ga lSer -
[C2SO4Na ][C14] (Scheme 1, path B) and of the anionic
II-Ga lSer [C14]-based series (Scheme 2) were very
similar. They consisted of the condensation of A with
the appropriate amine (e.g. methyl 12-aminodode-
canoate, ethanolamine, and tetradecylamine, respec-
tively) in the presence of EDC/HOBt, followed by Fmoc-
deprotection. Derivatives 3a , 3b (Scheme 1), and 9
(Scheme 2) were thus obtained with nearly 75, 60, and
60% yield, respectively.
Compound 3a , by means of a two- and three-step
process, was then converted into I-Ga lSer [C11CO2Na ]-
[NH2] and II-Ga lSer [C11CO2Na ][C3CO2Na ], respec-
tively (Scheme 1). 5 was obtained in fairly good yield
1
by H and 13C NMR spectral assignments and further

Anionic Analogues of Galactosylceramide
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2191
Sch em e 2. Synthetic Route to the Hydrocarbon Double-Chain II-GalSer Derivatives (PGal and Gal as Defined in
Scheme 1)a
a
(i) CH3(CH2)13NH2/EDC/HOBt/CH2Cl2; (ii) morpholine/DMF; (iii) CH2dCHCH2O(O)C(CH2)10CO2H/EDC/HOBt/CH2Cl2; (iv) Pd(Ph3)4/
Et2NH, CH2Cl2; (v) MeOH/Et3N/H2O (2:1:1); (vi) succinic anhydride/Et3N/CH2Cl2; vii) a: N-hydroxysuccinimide/EDC/DMF, b: taurine/
Et3N; (viii) (PhO)2P(O)Cl, pyridine; (ix) H2/PtO2/MeOH/Et3N; (x) Et3N/H2O.
(95%) by addition of succinic anhydride on 3a in the
presence of Et3N. Deacetylation of 3a and 5, with a
MeOH:Et3N:H2O mixture, and methyl ester hydrolysis
using 0.1 N NaOH afforded I-Ga lSer [C11CO2Na ]-
[NH2] and II-Ga lSer [C11CO2Na ][C3CO2Na ], respec-
tively. Owing to the low stability of the O-glycosyl-serine
bond in basic media due to the acidity of the CH serine
bond, the ester hydrolysis has to be carried out very
carefully in order to avoid â-elimination.23 While the
hydrolysis of the methyl ester of the single-chain
compound 4 was rapid and clean (I-GalSer [C11CO2Na]-
[NH2] was obtained with 80% yield), hydrolysis of the
double-chain compound 5a needed 3 days for comple-
tion and was less selective (II-Ga lSer [C11CO2Na ]-
[C3CO2Na ] was obtained with only 65% yield). This
result confirmed that the stability toward hydrolysis of
glycosyl-serine derivatives is higher for those having an
unsubstituted amino-serine function.31
Compound 3b by coupling with tetradecanoic N-
hydroxysuccinimide ester gave 7 (70%), which, after
conventional deacetylation, afforded II-Ga lSer [C2OH]-
[C14] (35% yield from A). Alternatively, 7 when sub-
mitted to O-sulfation with the sulfur trioxide-pyridine
complex in pyridine, followed by deacetylation and
conversion into its Na+ salt gave II-Ga lSer [C2SO4Na ]-
[C14] (25% yield from A).
catalytic Pd(PPh3)4 and diethylamine as transfer agent.32
The allyl ester protection was preferred to the methyl
ester one. Indeed, all our attempts to perform the
hydrolysis in basic media of the methyl ester analogue
of the allyl ester 10 were unsuccessful as mainly
â-elimination products were formed. This further shows
the low stability of the double-chain galactoserine
derivatives under basic conditions. On the other hand,
the N-succinic derivative II-Ga lSer [C14][C3CO2Na ]
was obtained by coupling 9 with succinic anhydride in
the presence of Et3N, followed by deacetylation of 12
thus obtained and a cation exchange (44% overall yield
from A). Compound 12 was further used for the
synthesis of the taurine derivative II-Ga lSer [C14]-
[C7SO3Na ] which was obtained by in situ activation
of acid 12 with HOSu/EDC and then reaction with
taurine in basic medium, followed by conventional
acetyl-deprotection of 13 thus produced and cation
exchange (30% overall yield from A).
Where the synthesis of the N-phosphoserine deri-
vative II-Ga lSer [C14][P (OMe)(ONa )] is concerned
(Scheme 2), it was prepared from 9 using a three-step
procedure, which included (i) phosphorylation of 9 with
diphenyl chlorophosphate in pyridine yielding 14 (80%),
(ii) unexpected conversion (see below) of 14 into the
dimethyl phosphate derivative 15 (87% yield) by bub-
bling hydrogen through a methanolic solution of 14,
catalytic PtO2 and Et3N,33 and (iii) hydrolysis of one of
the dimethyl ester phosphate bonds of 15 in aqueous
Et3N. When the diphenyl phosphate deprotection of 14
was performed using the conventional procedure (ab-
sence of Et3N), we observed dephosphorylation and the
quantitative formation of the starting material 9. This
is most probably due to the low stability of the N-
phosphoric acid serine intermediate and autocatalytic
hydrolysis. To neutralize the in situ generated N-
The synthesis of the anionic II-Ga lSer [C14]-
[C11CO2Na ], II-Ga lSer [C14][C3CO2Na ], II-Ga lSer -
[C14][C7SO3Na], and II-GalSer [C14][P (OMe)(ONa)]
compounds was performed from the key amino synthon
9, as outlined in Scheme 2. Thus, the coupling of 9 with
dodecanedioic acid monoallyl ester in the presence of
EDC/HOBt gave the double-chain compound 10 in 65%
yield, which after allyl deprotection, deacetylation, and
cation exchange afforded II-Ga lSer [C14][C11CO2Na ].
The allyl cleavage was performed in 90% yield with

2192 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Faroux-Corlay et al.
Sch em e 3. Synthetic Route to the Hydrocarbon and
Fluorocarbon Double-Chain Galactosyl-Amidoethanol
II-GalAE Derivative (PGal and Gal as Defined in
Scheme 1)a
hydrolyze the second methyl ester phosphate bond
without degradation were unsuccessful.
GalAE Ser ies. The fluorinated galactolipid II-GalAE-
[F 8C7][C11CO2Na ] which is derived from ethanola-
mine (Scheme 3) was obtained by galactosylation of 16
using the Schmidt procedure,23,34 and then deacetylation
of 17 thus was produced by a MeOH:Et3N:H2O mixture
and hydrolysis of the methyl ester 18 in aqueous NaOH
(25% overall yield from 16). For the galactosylation of
16, the Schmidt method, although it requires the
preparation of 2,3,4,6-tetra-O-acetyl-R-D-galactopyra-
nosyl trichloroacetimidate as galactose donor, proved
more efficient (35% yield) than the direct galactosylation
with the commercially available 1,2,3,4,6-penta-O-acetyl-
D-galactopyranose and BF3‚Et2O as Lewis acid (25%
yield). The aglycone 16 was prepared in four steps from
2-benzyloxyethylamine23 in 50% overall yield using
conventional procedures, including acylation with 7-(per-
fluorooctyl)heptanoic acid23 in the presence of EDC/
HOBt, LiAlH4 reduction of the amide bond, then acy-
lation with dodecanedioic acid monomethyl ester in the
presence of PyBrop/Et3N, and finally benzyl-deprotec-
tion by hydrogenolysis.
a
(i) CF3(CF2)7(CH2)6CO2H/EDC/HOBt/Et3N/CH2Cl2; (ii) LiAlH4/
THF; (iii) CH3O(O)C(CH2)10CO2H/PyBrop/Et3N/CH2Cl2; (iv) H2/
Pd/C, AcOH/MeOH; (v) GalOC(dNH)CCl3/TMSOTf/CHCl3, -10
°C; (vi) 2:1:1 MeOH/Et3N/H2O; (vii) 0.3 N NaOH/1:9 MeOH/H2O.
phosphoric acid, we performed the deprotection step in
the presence of Et3N. Under these conditions, we
obtained compound N-(dimethoxyphosphoryl)serine 15
as a result of a formal transesterification of 14 by
methanol and of the expected deacetylation of the
galactose residue. The structure of 15 was confirmed
by 1H, 13C-DEPT, 31P NMR, and mass spectrometry.
More particularly, the presence of the P(OMe)2 group
is attested by a doublet (J H,P 11.2 Hz) at 3.66 ppm
integrating for six protons and by two doublets at 54.3
La c Ser ies. The synthesis of the lactobionamide
derivatives, m on oLa c and bisLa c, is presented in
Scheme 4. Condensation of 1 or 2 equiv of the com-
mercial lactobionic acid 19 with amine 20 or diamine
21 in methanol gave m on oLa c (60% yield) or bisLa c
(50% yield), respectively. This condensation step with
lactobionic acid is very advantageous for introducing a
galactosyl moiety; as in one step and without the need
of any delicate glycosylation reaction, one obtains
compounds with the required galactosyl â-configuration
(which is already present in the starting material).35
2
and 54.4 ppm with characteristic J C,P coupling con-
stants of ∼6 Hz, attributable to two methoxy groups in
1
the H and 13C-DEPT spectrum, respectively.
The hydrolysis of one of the dimethyl ester phosphate
bonds of 15 was performed in aqueous Et3N and
afforded quantitatively II-GalSer [C14][P (OMe)(ONa)],
after cation-exchange and lyophilization. These condi-
tions were expected to lead to the hydrolysis of both
phosphate esters. However, a 31P NMR monitoring
of the hydrolysis of 15 showed its total consumption
after 24 h of stirring at room temperature and its
conversion into the monoester II-GalSer [C14][P (OMe)-
(O-,Et3NH+)] and no further evolution within the next
5 days. Indeed, the signal of the starting compound at
12.8 ppm was replaced by a resonance at 8.9 ppm, which
is corresponding to that of the isolated material (as its
sodium salt). The presence of one POMe group was
unambiguously confirmed by 13C-DEPT (doublet at 51.8
Str u ctu r a l Ch a r a cter iza tion . All the new galacto-
sylated compounds shown in Table 1 were characterized
by 1H and 13C NMR (and also by 19F NMR for II-Ga lAE-
[F 8C7][C11CO2Na ]), and/or mass spectrometry, and
elemental analyses. In addition to the specific NMR
features already discussed for the different series of
1
compounds, their respective H and 13C NMR spectra
were all fully consistent with the â-configuration of the
galactosyl residue. The 13C shifts of the pyranosyl ring
carbons were indeed in accordance with those reported
in the literature, and more particularly the anomeric
carbon resonances were found in an expected 102.9-
2
ppm with J C,P ) 6 Hz, corresponding to a methoxy),
31P NMR, and mass spectrometry. Our attempts to
Sch em e 4: Synthetic Route to the Lactobionamide Derivatives (Figures Refer to C and H Atom Numbering for 1H and
13C NMR; Gal as Defined in Scheme 1)a
a
(i) MeOH, ∆.

Anionic Analogues of Galactosylceramide
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2193
Ta ble 2. Anti-HIV-1 Activity and Cytotoxicity of the GalCer Analogues
compounds
IC50 (µM) CEM-SS
IC50 (µM) MT-4
CC50 (µM) CEM-SS
CC50 (µM) MT-4
I-Ga lSer [CO2Na ][C14]
I-Ga lSer [C11CO2Na ][NH2]
II-Ga lSer [C14][C3CO2Na ]
II-Ga lSer [C14][C11CO2Na ]
II-Ga lSer [C11CO2Na ][C3CO2Na ]
II-Ga lSer [C2OH][C14]
II-Ga lSer [C2SO4Na ][C14]
II-Ga lSer [C14][C7SO3Na ]
II-Ga lSer [C14][P O(OMe)(ONa )]
II-Ga lAE[F 8C7][C11CO2Na ]a
m on oLa c
20
>100
>100
>100
>100
>100
10
>100
>100
>100
>100
>100
>100
20
>100
>100
>18
>100
33
>100
>100
>100
>100
>100
>100
>100
>100
>100
>18
>100
>100
>100
>100
>100
>100
>100
>100
>100
>18
50
>100
>18
>100
32
>100
44
>100
38
bisLa c
a
This compound was tested as PL/CH/glycolipid liposome formulation in a 2:1:0.15 molar ratio; the mean size of the liposomes with
the associated standard deviation (SD) was 75 (25) nm, as measured by light scattering. In aqueous solution, no activity was detected for
a concentration of up to 10 µM.
106.5 ppm range.30 Furthermore, the anomeric proton
signals could be seen in several cases, as a doublet in a
4.20-4.36 ppm range with a J 1,2 coupling constant of
even further away from galactose, a sulfonated group
as in II-Ga lSer [C14][C7SO3Na ]. The anti-HIV activity
in this GalSer series is decreasing on increasing the
spacer length between the anionic sulfate/sulfonate and
the serine core (II-Ga lSer [C2SO4Na ][C14] vs II-
Ga lSer [C14][C7SO3Na ]). This anti-HIV activity is lost
(i) on replacing the hydrophobic anchor by a hydrophilic
chain (I-Ga lSer [C14][NH2] vs I-Ga lSer [C11CO2Na ]-
[NH2], hence on introducing a carboxylate at the
terminus of the hydrophobic spacer, see discussion
below), (ii) on replacing the anionic sulfate by a neutral
hydroxyl (II-Ga lSer [C2SO4Na ][C14] vs II-Ga lSer -
[C2OH][C14]), or (iii) on increasing the spacer length
between the carboxylate and the serine core (I-Ga lSer -
[CO2Na ][C14] vs II-Ga lSer [C14][C3CO2Na ] or II-
Ga lSer [C14][C11CO2Na ]).
3
7.20-7.36 Hz, characteristic of trans-1,2 one.30
Where the doubling of some 1H and/or 13C signals,
which is seen for derivative II-Ga lAE[F 8C7]-
[C11CO2Na ], is concerned, it indicates rather the
presence of conformational isomers in solution due to
restricted rotation of the amide bond, as it has been
already described.23,36
Biologica l Eva lu a tion . The aim of this study was
to synthesize new GalCer analogues that were designed
according to molecular modeling and dynamic simula-
tions24 and to SAR data from literature11-16,21-23 and
expected to block HIV-1 cellular entry. The antiviral
activity against HIV-1 (LAI and IIIB) and cytotoxicity
of this second generation of GalCer analogues was
evaluated in vitro on CEM-SS and MT4 cells according
to published procedures.37-39 The data are collected in
Table 2.
Among the 12 new galactosylated compounds reported
here, a specific anti-HIV activity, although moderate
(IC50 from 10 µM to 50 µM), was detected only for three
of them on the CEM-SS cell lines, i.e., I-Ga lSer -
[CO2Na ][C14], II-Ga lSer [C2SO4Na ][C14], and II-
Ga lSer [C14][C7SO3Na ], which belong to the GalSer
series. The sulfate II-Ga lSer [C2SO4Na ][C14], which
is the more active on CEM-SS cells, is the sole compound
found to exhibit an anti-HIV activity on MT4 cells (IC50
) 20 µM). Furthermore, no cytotoxicity was detected for
these derivatives for concentrations up to 100 µM on
both cell lines.
Compounds I-Ga lSer [CO2Na ][C14] and II-Ga lSer -
[C2SO4Na ][C14] were found to display higher anti-
HIV activities on CEM-SS cells than the previously
reported first generation of GalSer derivatives (Table
1) for which IC50 of 40-60 µM only were measured.23
Furthermore, none of these first generation compounds
was active on MT4 cells by contrast to the sulfate II-
Ga lSer [C2SO4Na ][C14] which protects to some extent
these cells from HIV infection.
Concerning structure-activity relationships in the
GalSer series, the most significant fact is that the more
potent drugs were those possessing a hydrophobic
anchor and, in close proximity to the galactose polar
head, an ammonium/amine function (I-Ga lSer [C14]-
[NH2]) or an anionic carboxylate (I-Ga lSer [CO2Na ]-
[C14]), sulfate (II-Ga lSer [C2SO4Na ][C14]), or, and
Our results show also that introducing a sulfate or a
sulfonate in place of a carboxylate or a phosphate is
most beneficial for anti-HIV activity. Indeed, the sulfate
derivative II-Ga lSer [C2SO4Na ][C14] inhibits HIV
infection of both CEM-SS and MT4 cell lines by contrast
to its carboxylate analog I-Ga lSer [CO2Na ][C14] which
is active only on CEM-SS cells and at a lower level than
the sulfate. Furthermore, if an anti-HIV activity was
detected for the sulfonate II-Ga lSer [C14][C7SO3Na ],
no such activity was found for its phosphate II-Ga lSer -
[C14][P (O)OMe)(ONa )] or its carboxylate II-Ga lSer -
[C14][C3CO2Na ] homologues, while the opposite ten-
dency was expected. Indeed, with the phosphate and
carboxylate groups being located closer to the galac-
toserine core than the sulfonate, one expected a higher
anti-HIV activity for these phosphate and carboxylate
derivatives, as discussed above. These data underscore
that both the nature of the anionic group and its close
proximity to the galactose are of importance for anti-
HIV activity. They are in perfect agreement with the
recent observation that sulfatide, the natural sulfated
congener of GalCer, has a potent anti-HIV activity in
GalCer-expressing intestinal cells.10 In addition, II-
Ga lSer [C2SO4Na ][C14] inhibits HIV-1 infection of
T-cells, whereas the antiviral activity of sulfatide is
limited to GalCer-expressing CD4-negative cells. To our
knowledge, this is the first time that a synthetic sulfated
analogue of GalCer with such a broad anti-HIV activity
is described.
In the GalAE series, no anti-HIV activity was detected
for II-Ga lAE[F 8C7][C11CO2Na ] neither as an aque-
ous solution nor as a liposomal formulation and for

2194 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Faroux-Corlay et al.
concentration of up to about 20 µM (we were unable to
prepare more concentrated and stable dispersions with
this derivative). Thus, introducing a carboxylate group
at the end of one of the lipophilic chains of II-Ga lAE-
[C16][F 8C7], which was endowed with a substantial
anti-HIV activity (IC50 ) 24 µM on HT-29 cells23), has
rather a detrimental effect on or, at least, does not
improve anti-HIV activity. This is also the case, as
discussed above when going from I-Ga lSer [C14][NH2]
to I-Ga lSer [C11CO2Na ][NH2]. These results contrast
with the substantial increase of anti-HIV activity which
was noticed in the literature for such a structural
modification.14 Therefore, the presence of a carboxylate
group at the end of the hydrophobic chains of an
analogue does not improve systematically its antiviral
activity. This may be linked to the self-organizing
properties of synthetic glycolipid analogues in aqueous
phases, as recently discussed.21
ing to saturation) into the subphase of monolayers
prepared at an initial pressure of 10 mN/m and then
by recording the increase in surface pressure which is
caused by the penetration of rgp120 into the monolayer.
Among the three compounds tested, only II-Ga lSer -
[C14][C7SO3Na ] formed a stable monolayer. However,
stable monolayers could be obtained when I-Ga lSer -
[CO2Na ][C14] and II-Ga lSer [C2SO4Na ][C14] were
incorporated into a glucosylceramide (GlcCer) mono-
layer, providing their relative content did not exceed 44
and 70%, respectively. The low stability of the pure
I-GalSer [CO2Na][C14] or II-GalSer [C2SO4Na][C14]
monolayers is most probably related to their substantial
solubility in water. For these reasons, gp120 binding
was evaluated onto these mixed monolayers, since the
glycoprotein does not bind to GlcCer.43
Addition of rgp120 into the aqueous subphase
underneath the II-Ga lSer [C14][C7SO3Na ], I-Ga lSer -
[CO2Na ][C14]/GlcCer, or II-Ga lSer [C2SO4Na ][C14]/
GlcCer monolayer induced a marked increase of surface
pressure with a maximal ∆π increase of 8.5, 9.5, or 8.5
mN/m, respectively. Comparatively, a maximal ∆π
increase of 2.5 and 12 mN/m was measured for a GlcCer
(negative control) and GalCer (positive control) mono-
layer, respectively. This is a clear evidence of gp120
insertion into the galactolipid monolayers resulting from
a primary interaction with the galactose residue. These
data suggest that binding of these three derivatives to
HIV-1 gp120 may be responsible for their anti-HIV
activity in CD4-positive cells. This is in agreement with
the current hypothesis that the assembly of the HIV-1
fusion complex requires, in addition to CD4 and a
coreceptor, specific glycolipids which are present in
restricted areas (microdomains) of the plasma mem-
brane.9,40,45,46
Concerning the lactose derived compounds, neither
m on oLa c nor bisLa c displayed any specific anti-HIV
activity, bisLa c being rather cytotoxic. For m on oLa c
and bisLa c, we expected, at least, an HIV inhibition
level in the same range as that reported for closely
related N-alkyl-1-deoxylactitols13,15 (IC50 ) 1-10 µM;
CEM-SS). More particularly, we expected a substantial
HIV inhibition potency for bisLa c: its ,bouquet.-like
structure of the two galactosyl units connected to a
hydrophobic anchor was aimed at enhancing its interac-
tion with gp120, hence decreasing cellular infection.8,9,40
The cytotoxicity of bisLa c could be due to its potential
detergent properties on cell membranes. Due to its
pronounced cone-shaped geometry (polar head volume
. hydrophobic chain volume), bisLa c when dispersed
in water is expected to form rather micelles41 (long-chain
lactobionamide analogues were indeed reported to self-
assemble into micelles42). Such aggregates are known
to be more detergent on cell membranes than com-
pounds organized into lamellar phases.
Con clu sion
The results of the present study underscore the
interest of synthesizing carbohydrate-based therapeutic
agents as alternative strategies for infectious diseases
treatments.21,47 Our future efforts will focus on demon-
strating the clustering of the anti-HIV active GalSer
derivatives into galactosyl-rich microdomains when
incorporated into a membrane and the importance of
the amino and anionic group in the formation of such
microdomains, of their nature, and of their location with
respect to the galactosyl residue, all factors that have
an impact on anti-HIV activity, as evidenced in this
study.
Where a correlation between the molecular modeling
and dynamic simulations performed on some of the
galactolipids reported here and their anti-HIV activity
is concerned, it appears that clustering of these galac-
tolipids within a membrane, as evidenced from our
simulation study,24 is not a sufficient condition for anti-
HIV activity. This simulation study indicated indeed
that the galactolipids reported here that were found to
display an anti-HIV activity (i.e. I-Ga lSer [CO2Na ]-
[C14], II-Ga lSer [C2SO4Na ][C14], and II-Ga lSer -
[C14][C7SO3Na ]), should cluster into galactosyl-rich
microdomains. However, this study showed also forma-
tion of such clusters for II-Ga lSer [C14][C3CO2Na ]
which was found to be inactive.
Exp er im en ta l Section
Ch em ica l Section . Gen er a l. Unless otherwise indicated,
reactions were conducted under an anhydrous nitrogen atmo-
sphere using dry solvents and reagents. Anhydrous solvents
were prepared by standard methods. â-D-Galactose pentaac-
etate, 12-aminododecanoic acid, 1-(3-N,N-dimethylaminopro-
pyl)-3-ethylcarbodiimide (EDC), succinic anhydride, sulfur
trioxide pyridine complex (SO3‚pyridine), tetradecylamine,
dodecanedioic acid, allyl bromide, tetrakis(triphenylphosphine-
)palladium(0) [Pd(PPh3)4], diethyl dodecanedioate, and N-
hydroxysuccinimide (HOSu) were purchased from Aldrich,
boron trifluoride diethyl etherate (BF3/Et2O) and 1-hydroxy-
benzotriazole (HOBt) from Sigma, and taurine and lactobionic
acid from Fluka. Diphenyl chlorophosphate (PhO)2P(O)Cl and
platinum oxide monohydrate were purchased from Accros, N-9-
(fluorenylmethoxycarbonyl)-L-serine from Propeptide, and 1-bro-
Su r fa ce-P r essu r e Mea su r em en ts - gp 120 Bin d -
in g t o Glycolip id Mon ola yer s a t t h e Air -Wa t er
In ter fa ce. To provide further insight into the inhibition
mechanism of the anti-HIV active GalCer analogues
reported here, i.e., I-Ga lSer [CO2Na ][C14], II-Ga lSer -
[C14][C7SO3Na], and II-GalSer [C2SO4Na][C14], they
were also tested for gp120 binding. This was done by
analyzing the interaction of HIV-1 recombinant gp120
with the monolayer these lipids form when spread at
the air-water interface, using a procedure reported
elsewhere.9,43,44 These experiments were performed by
adding rgp120 (at a concentration of 10 nM correspond-

Anionic Analogues of Galactosylceramide
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2195
motris(pyrrolidino)-phosphonium hexafluorophosphate (Py-
Brop) from Novabiochem. N-Hydroxysuccinimide ester of
tetradecanoic acid was prepared by reaction of myristic acid
and N-hydroxysuccinimide in the presence of EDC. Methyl 12-
aminododecanoate was prepared by esterification of the cor-
responding acid with methanol. The 2,3,4,6-tetra-O-acetyl-R-
D-galactopyranosyl trichloroacetimidate (GalOC()NH)CCl3)
and 7-(perfluorooctyl)heptanoic acid were prepared as de-
scribed previously.23 2-Benzyloxyethylamine was prepared in
two steps (60%) from commercial N-(tert-butoxycarbonyl)-
ethanolamine.23 Dodecanedioic acid monoallyl ester was ob-
tained by reacting, in DMF, dodecanedioic acid with allyl
bromide in the presence of K2CO3 [Rf ) 0.51 (95:5 CHCl3-
MeOH). 1H NMR (CDCl3): δ 10.80 [bs, 1 H, C(O)OH], 5.85
(ddt, 1 H, J ) 17.3, 11.6 and 5.7 Hz, 1H, OCH2CH)CH2), 5.24
(dd, J ) 17.3 Hz, J ) 1.5 Hz, 1H, OCH2CH)CHaHb), 5.16 (dd,
J ) 11.6 Hz, J ) 1.5 Hz, 1H, OCH2CH)CHaHb), 4.50 (d, J )
5.7 Hz, 2H, OCH2CH)CH2), 2.30 [t, J ) 7.3 Hz, 2H, CH2C-
(O)OH], 2.26 [t, J ) 7.4 Hz, 2H, CH2C(O)OAll], 1.60-1.50 [m,
4H, CH2CH2C(O)], 1.30-1.15 [m, 12H, (CH2)6]. 13C NMR
(CDCl3): δ 180.0 [C(O)OH], 173.6 [C(O)OAll], 132.4 (CH)CH2),
118.1 (CH)CH2), 65.0 (OCH2CH)), 34.3 and 34.1 [CH2C(O)],
29.4, 29.3, and 29.1 [(CH2)6], 25.0 and 24.7 [CH2CH2C(O)]].
Dodecanedioic acid monomethyl ester was obtained from
diethyl dodecanedioate and Ba(OH)2 in MeOH as an oil [Rf )
0.48 (95:5 CHCl3-MeOH). 1H NMR (CDCl3): δ 3.61 (s, 3H,
OCH3), 2.28 [t, J ) 7.3 Hz, 2H, CH2C(O)OH], 2.25 [t, J ) 7.5
Hz, 2H, CH2C(O)OMe], 1.60-1.50 [m, 4H, CH2CH2C(O)],
1.40-1.15 [m, 12H, (CH2)6]. 13C NMR (CDCl3): δ 180.1 [C(O)-
OH], 174.5 [C(O)OMe], 51.5 (OCH3), 34.2 [CH2C(O)], 29.4, 29.3,
29.2, and 29.1 [(CH2)6], 25.0 and 24.8 [CH2CH2C(O)]]. 3-O-
(2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)-NR-9-(fluorenyl-
methoxycarbonyl)-L-serine, A, was prepared according to
published procedures and authentified by comparison of its
Gen er a l F m oc Clea va ge (P r oced u r e B). The N-Fmoc
derivatives were deprotected in a 1:2 morpholine-DMF solu-
tion at room temperature for 4 h. After evaporation, the
compounds were purified by chromatography.
Gen er a l Am in o Acid Cou p lin g (P r oced u r e C). EDC (1.1
equiv) was added to a CH2Cl2 solution of acid (1 equiv), amine
(1 equiv), and HOBt (1.1 equiv). The reaction mixture was
stirred at 0 °C for 1 h and then at room-temperature overnight.
The reaction mixture diluted with CHCl3 was successively
washed with 5% KHSO4, water, 8% NaHCO3, and then water
until neutrality. After drying with Na2SO4, filtration, evapora-
tion, and purification, the compound was obtained.
Syn th esis of I-Ga lSer [CO2Na ][C14] (Sch em e 1). 3-O-
(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la ctop yr a n osyl)-L-ser in e, 1.
The procedure B when applied to 600 mg (0.93 mmol) of A
gave, after chromatography (95:5 CHCl3-MeOH), 335 mg
1
(80%) of 1 as an oil. Rf ) 0.25 (4:1 CHCl3-MeOH). H NMR
(CDCl3): δ 5.27 (m, 1H, H-4 Gal), 5.10-4.95 (m, 2H, H-2,3
Gal), 4.40-3.60 (m, 7H, H-1â,5,6,6′ Gal and OCH2CH), 2.10-
1.90 [m, 12H, CH3C(O)], 1.80 (s, 2H, NH2). 13C NMR (CDCl3):
δ 177.5 [C(O)OH], 170.6, 170.2, and 170.0 [CH3C(O)], 101.8
(C-1â Gal), 70.8 and 70.6 (C-3,5 Gal), 69.5 (OCH2CH), 69.0
(C-2 Gal), 67.0 (C-4 Gal), 61.0 (C-6 Gal), 55.9 (OCH2CH), 20.6,
20.5, and 20.4 [CH3C(O)].
3-O-(2,3,4,6-Tet r a -O-a cet yl-â-D-ga la ct op yr a n osyl)-N-
(tetr a d eca n oyl)-L-ser in e, 2. Three hundred milligrams (0.69
mmol) of 1 and 235 mg (0.69 mmol) of N-hydroxysuccinimide
ester of tetradecanoic acid in 10 mL of THF were stirred at
room temperature for 24 h. After evaporation of the solvent,
the crude residue was dissolved in CHCl3 and washed with 1
N HCl and water until neutrality. After drying over Na2SO4
and filtration, the solvent was evaporated, and the residue was
purified by chromatography (CH2Cl2 to 9:1 CH2Cl2-MeOH)
giving 350 mg (80%) of 2 as a white solid. Rf ) 0.30 (9:1
1
NMR data with literature:28 Rf ) 0.3 (9:1 CHCl3-MeOH). H
1
CHCl3-MeOH). H NMR (CDCl3): δ 6.71 (m, 1H, NH), 5.33
NMR (CDCl3): δ 7.66 (d, J ) 8.0 Hz, 2H, Fmoc), 7.50 (d, J )
8.0 Hz, 2H, Fmoc), 7.35-7.20 (m, 4H, Fmoc), 5.90 (m, 1H, NH),
5.27 (m, 1H, H-4 Gal), 5.10-4.95 (m, 2H, H-2,3 Gal), 4.40-
3.60 [m, 10H, H-1â,5,6,6′ Gal, OCH2CH and C(O)OCH2CH],
2.10-1.90 [m, 12H, CH3C(O)]. 13C NMR (CDCl3): δ 177.5
[C(O)OH], 170.6, 170.2 and 170.0 [CH3C(O)O], 156.9 [NHC-
(O)O], 144.0, 143.8, and 141.3 (C, Fmoc), 127.8, 127.2, 125.2,
and 120.1 (CH, Fmoc), 101.8 (C-1â Gal), 70.8 and 70.6 (C-3,5
Gal), 69.5 (OCH2CH), 69.0 (C-2 Gal), 67.0 (C-4 Gal), 66.9 (C(O)-
OCH2), 61.0 (C-6 Gal), 55.4 (OCH2CH), 47.2 [C(O)OCH2CH],
20.6, 20.5 and 20.4 [CH3C(O)].
(bs, 1H, H-4 Gal), 5.03 (m, 2H, H-2,3 Gal), 4.50 (m, 2H, H-1â,5
Gal), 4.02 (m, 5H, H-6,6′ Gal and OCH2CH), 2.18 [m, 2H,
CH2C(O)], 2.08, 2.02, 1.99, and 1.91 [4s, each 3HCH3C(O)], 1.55
[m, 2H, CH2CH2C(O)], 1.19 [m, 20H, [(CH2)10], 0.81 (t, J ) 6.5
Hz, 3H, CH2CH3). 13C NMR (CDCl3): δ 174.9 [C(O)NH and
C(O)OH], 170.5, 170.2, and 169.9 [CH3C(O)], 101.4 (C-1â Gal),-
70.8 (C-3,5 Gal), 69.0 (OCH2CH and C-2 Gal), 67.1 (C-4 Gal),
61.0 (C-6 Gal), 54.1 (OCH2CH), 36.5 [CH2C(O)], 31.9 (CH2CH2-
CH3), 29.8, 29.6, and 29.4 [(CH2)8], 25.6 [CH2CH2C(O)], 22.7
(CH2CH3), 20.8, 20.7, 20.6, and 20.5 [CH3C(O)], 14.2 (CH2CH3).
3-O -(â-D-G a la c t o p y r a n o s y l)-N -(t e t r a d e c a n o y l)-L -
ser in e (Sod iu m Sa lt), I-Ga lSer [CO2Na ][C14]. The proce-
dure A when applied to 165 mg (0.25 mmol) of 2 gave, after
treatment with Amberlite IRC-50 ion-exchange (Na+), 115 mg
(90%) of I-Ga lSer [CO2Na ][C14]. Rf ) 0.30 (35:15:2 CHCl3-
MeOH-H2O). [R]D ) +8.0° (c 0.25; MeOH). 1H NMR (CDCl3-
CD3OD): δ 4.43 (sl, 1H, H-4 Gal), 4.26 (m, 2H, H-2,3 Gal),
3.85 (bs, 1H, H-1â Gal), 3.73 (m, 3H, H-5,6,6′ Gal), 3.51 (m,
3H, OCH2CH), 2.23 [t, J ) 7.4 Hz, 2H, CH2C(O)], 1.59 [m, 2H,
CH2CH2C(O)], 1.24 [m, 20H, (CH2)10], 0.85 (t, J ) 6.5 Hz, 3H,
CH3). 13C NMR (CDCl3-CD3OD): δ 176.0 [C(O)ONa], 174.2
[C(O)NH], 103.8 (C-1â Gal), 74.9 (C-5 Gal), 73.0 (C-3 Gal), 70.7
(C-2 Gal), 70.5 (OCH2CH), 68.9 (C-4 Gal), 61.1 (C-6 Gal), 54.5
(OCH2CH), 35.9 [CH2C(O)], 31.5 (CH2CH2CH3), 29.4, 29.3,
29.2, 29.1, and 28.9 [(CH2)8], 25.4 [CH2CH2C(O)], 22.2 (CH2-
CH3), 13.2 (CH3). This assignment was further confirmed by
DEPT. Anal. Calc. for C23H42NO9Na‚1/2H2O (508.58): C, 54.32;
H, 8.52; N, 2.75. Found: C, 54.21; H, 8.90; N, 2.80.
Column chromatography purifications were carried out
using Merck silica gel 60 (mesh 70-230) and/or Sephadex LH
20 gel. The purity of all new compounds was checked by thin-
layer chromatography (TLC), NMR, mass spectrometry, and/
or elemental analysis. TLC analyses were performed on
precoated silica gel F254 plates (Merck) and visualized by UV
light (λ ) 254 nm) and by charring with KMnO4, 50% MeOH-
H2SO4, or ninhydrine. Optical rotations were measured at 589
1
nm with a Perkin-Elmer 141 polarimeter (1-dm cell). H, 19F,
13C, and 31P NMR spectra were recorded on a Bruker AC 200
spectrometer at 200, 188.3, 50.3, and 81 MHz, respectively.
Chemicals shifts are given in ppm (δ) relative to the solvent
1
peak (i) CHCl3 (δ 7.27) or CH3OD (δ 3.35) for H and (ii) CDCl3
(δ 76.9) for 13C and relative to external reference CCl3F and
75% H3PO4 for 19F and 31P, respectively. Electrospray mass
analyses, positive or negative mode, were run on a TSQ 7000
Finnigan Mat spectrometer. Elemental analyses were per-
formed by the Service Central de Microanalyze of the CNRS.
Syn th esis of I-Ga lSer [C11CO2Na ][NH2] (Sch em e 1).3-
O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la ctop yr a n osyl)-L-ser in e-
11-m eth oxyca r bon ylu n d eca n a m id e, 3a . Procedure C when
applied to 63 mg (0.33 mmol) of EDC and to 2 mL of CH2Cl2
containing 0.20 g (0.30 mmol) of A, 69 mg (0.30 mmol) of
methyl 12-aminododecanoate, and 42 mg (0.31 mmol) of HOBt,
afforded after chromatographic purification (98:2 CHCl3-
MeOH) 0.35 g (95%) of 3-O-(2,3,4,6-tetra-O-acetyl-â-D-galac-
topyranosyl)-NR-9-(fluorenylmethoxycarbonyl)-L-serine-11-meth-
oxycarbonylundecanamide [Rf ) 0.76 (95:5 CHCl3-MeOH). 1H
NMR (CDCl3): δ 7.73 (d, J ) 7.2 Hz, 2H, Fmoc), 7.56 (d, J )
Gen er a l De-O-a cetyla tion (P r oced u r e A). â-D-Galacto-
pyranoside 3a , 5, 2, 7, 8, 11, 12, 13, or 17 (0.10 mmol) in 1.8
mL of a 2:1:1 MeOH-Et3N-H2O mixture was stirred at room
temperature for 2 h. After evaporation of the solvents, the
crude residues were purified by chromatography (CHCl3 to 7:3
or 3:2 CHCl3-MeOH) affording the corresponding compound
4, 6, I-Ga lSer [CO2Na ][C14], II-Ga lSer [C2OH][C14], II-
Ga lSer [C2SO4Na ][C14], II-Ga lSer [C14][C11CO2Na ], II-
Ga lSer [C14][C3CO2Na ], II-Ga lSer [C14][C7SO3Na ], and
18 as white solids, respectively.

2196 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Faroux-Corlay et al.
7.2 Hz, 2H, Fmoc), 7.40-7.25 (m, 4H, Fmoc), 6.37 (bs, 1H,
NHCH2), 5.75 [d, J ) 6.7 Hz, 1H, NHC(O)O], 5.35 (bs, 1H,
H-4 Gal), 5.12 (m, 1H, H-2 Gal), 4.99 (dd, J ) 10.4, J ) 3.2
Hz, 1H, H-3 Gal), 4.52 (d, J ) 7.5 Hz, 1H, H-1â Gal), 4.45-
3.95 [m, 9H, H-5,6,6′ Gal, OCH2CH, and C(O)OCH2CH], 3.62
[s, 3H, C(O)OCH3], 3.21 (m, 2H, NHCH2), 2.26 [t, J ) 7.4 Hz,
2H, CH2C(O)OCH3], 2.10, 1.99 and 1.95 [s,s,s, 12H, CH3C(O)],
1.65-1.45 [m, 4H, NHCH2CH2 and CH2CH2C(O)OCH3], 1.22
[m, 14H, (CH2)7]. 13C NMR (CDCl3): δ 174.4 [C(O)NH], 170.5,
170.2, 170.1, 169.6 and 169.0 [CH3C(O)O and CH3OC(O)],
156.0 [NHC(O)O], 143.9, 143.7 and 141.4 (C, Fmoc), 127.9,
127.2, 125.1 and 120.1 (CH, Fmoc), 102.1 (C-1â Gal), 71.2 (C-3
Gal), 70.7 (C-5 Gal), 70.1 (OCH2CHNH), 68.8 (C-2 Gal), 67.1
[C-4 Gal and C(O)OCH2], 61.4 (C-6 Gal), 54.0 [OCH2CHNH],
51.5 [C(O)OCH3], 47.3 [C(O)OCH2CH], 39.9 (NHCH2), 34.2
[CH2C(O)OCH3], 29.6, 29.5, 29.3, and 29.2 [(CH2)7], 26.9
[NHCH2CH2], 25.0 [CH2CH2C(O)OCH3], 20.8, 20.7, and 20.6
[CH3C(O)]]. Procedure B when applied to 130 mg (0.15 mmol)
of this methoxycarbonylundecanamide derivative afforded 80
mg (80%) of 3a as a white solid after chromatography (CHCl3
to 7:3 CHCl3-MeOH). Rf ) 0.79 (4:1 CHCl3-MeOH). 1H NMR
(CDCl3): δ 7.33 (d, J ) 6.5 Hz, 1H, NHCH2), 5.35 (bd, J ) 3.3
Hz, 1H, H-4 Gal), 5.15 (dd, J ) 7.8 Hz, J ) 10.4 Hz, 1H, H-2
Gal), 4.97 (dd, J ) 10.4 Hz, J ) 3.3 Hz, 1H, H-3 Gal), 4.48 (d,
J ) 7.8 Hz, 1H, H-1â Gal), 4.15-3.35 [m, 9H, H-5,6,6′ Gal,
OCH2CH, and C(O)OCH3], 3.18 (q, J ) 6.6 Hz, 2H, NHCH2),
2.27 [t, J ) 7.4 Hz, 2H, CH2C(O)OCH3], 2.12, 2.04, 2.02, and
1.95 [4s, each 3HCH3C(O)], 1.90 (s, 2H, NH2), 1.65-1.45 [m,
4H, NHCH2CH2 and CH2CH2C(O)OCH3], 1.23 [m, 14H, (CH2)7].
13C NMR (CDCl3): δ 174.4 [C(O)NH], 171.8, 171.2, 170.2,
170.1, and 169.6 [CH3C(O)O and CH3OC(O)], 101.4 (C-1â Gal),
72.5 (C-3 Gal), 70.9 (C-5 Gal and OCH2CHNH2), 68.9 (C-2 Gal),
67.1 (C-4 Gal), 61.3 (C-6 Gal), 55.2 (CHNH2), 51.5 [C(O)OCH3],
39.3 (NHCH2), 34.2 [CH2C(O)OCH3], 29.5, 29.4, 29.3, and 29.2
[(CH2)7], 26.9 [NHCH2CH2], 25.0 [CH2CH2C(O)OCH3], 20.8,
20.7 ,and 20.6 [CH3C(O)].
3-O-(â-D-Ga la ctop yr a n osyl)-L-ser in e-11-m eth oxyca r bo-
n ylu n d eca n a m id e, 4. The procedure A when applied to 80
mg (0.12 mmol) of 3a gave, after recrystallization in Et2O, 45
mg (76%) of 4. Rf ) 0.27 (65:36:4 CHCl3-MeOH-H2O). 1H
NMR (CDCl3-CD3OD): δ 4.25 (d, J ) 6.6 Hz, 1H, H-1â Gal),
4.00-3.45 (m, 12H, H-2s6 Gal, OCH2CH and C(O)OCH3], 3.18
(t, J ) 6.9 Hz, 2H, NHCH2), 2.30 [t, J ) 7.2 Hz, 2H, CH2C-
(O)OCH3], 1.65-1.50 [m, 4H, NHCH2CH2 and CH2CH2C(O)-
OCH3], 1.28 [m, 14H, (CH2)7]. 13C NMR (CDCl3-CD3OD): δ
175.1 [C(O)NH], 171.8 [C(O)OCH3], 103.9 (C-1â Gal), 75.8 (C-5
Gal), 73.8 (C-3 Gal), 71.4 (OCH2CHNH2), 70.9 (C-2 Gal), 69.2
(C-4 Gal), 61.6 (C-6 Gal), 54.6 (CHNH2), 51.2 [C(O)OCH3], 39.7
(NHCH2), 34.0 [CH2C(O)OCH3], 29.7, 29.6, 29.5, 29.4, and 29.2
[(CH2)7], 27.0 [NHCH2CH2], 25.0 [CH2CH2C(O)OCH3].
3-O-(â-D-Ga la ctop yr a n osyl)-L-ser in e-11-ca r boxyu n d e-
ca n a m id e (Sod iu m Sa lt ), I-Ga lSer [C11CO2Na ][NH 2].
Forty-five milligrams (0.094 mmol) of 4 were stirred with 3.3
mL of 0.1 N NaOH 9:1 MeOH-H2O, at room temperature for
3 h. The reaction mixture was purified by chromatography
(49:1 to 3:2 CHCl3-MeOH) affording 37 mg (80%) of I-Ga lSer -
[C11CO2Na ][NH2] as a white solid. Rf ) 0.18 (65:36:4
CHCl3-MeOH-H2O). [R]D ) +7.3° (c 0.28; 4:1 MeOH-H2O).
1H NMR (CD3OD): δ 4.36 (d, J ) 7.0 Hz, 1H, H-1â Gal), 4.25-
3.45 (m, 9H, H-2-6 Gal and OCH2CH), 3.21 (t, J ) 6.6 Hz, 2H,
NHCH2), 2.14 [t, J ) 7.2 Hz, 2H, CH2C(O)ONa], 1.65-1.45
[m, 4H, NHCH2CH2 and CH2CH2C(O)ONa], 1.27 [m, 14H,
(CH2)7]. 13C NMR (CD3OD): δ 181.5 [C(O)O], 172.0 [C(O)NH],
104.8 (C-1â Gal), 76.9 (C-5 Gal), 74.7 (C-3 Gal), 72.3 (OCH2-
CHNH), 71.3 (C-2 Gal), 70.2 (C-4 Gal), 62.5 (C-6 Gal), 55.5
(CHNH2), 40.6 (NHCH2), 38.0 [CH2C(O)O], 30.7, 30.6, 30.5,
30.4, and 30.3 [(CH2)7], 27.9 [NHCH2CH2], 27.2 [CH2CH2C-
(O)O]. Anal. Calc.for C21H39N2O9Na‚3/2H2O (513.56): C, 49.11;
H, 8.24; N, 5.45. Found: C, 49.12; H, 8.25; N, 5.06.
and 30 µL (0.21 mmol) of Et3N in CH2Cl2 (3 mL). After having
been stirred at room temperature for 24 h, the solution was
washed with 5% KHSO4 and then water. After having been
dried with Na2SO4 and filtration, the solvent was evaporated,
and the residue was purified by chromatography (CHCl3 to
9:1 CHCl3-MeOH) giving 80 mg (95%) of 5 as a white solid.
Rf ) 0.24 (95:5 CHCl3-MeOH). 1H NMR (CDCl3): δ 7.04 (d, J
) 7.3 Hz, 1H, CHNH), 6.71 [t, J ) 5.1 Hz, 1H, C(O)NHCH2),
5.36 (bd, J ) 3.0 Hz, 1H, H-4 Gal), 5.12 (dd, J ) 7.5, J ) 10.4
Hz, 1H, H-2 Gal), 5.01 (dd, J ) 10.4, J ) 3.2 Hz, 1H, H-3 Gal),
4.60 (d, J ) 7.4 Hz, 1H, H-1â Gal), 4.56 (m, 1H, H-5 Gal), 4.10
(m, 2H, H-6,6′ Gal), 3.93 (m, 2H, OCH2CH), 3.72 (m, 1H,
OCH2CH), 3.63 (s, 3H, OCH3), 3.18 (m, 2H, NHCH2), 2.62 [m,
2H, CH2C(O)OH], 2.50 [m, 2H, NHC(O)CH2], 2.27 [t, J ) 7.4
Hz, 2H, CH2C(O)OCH3], 2.12, 2.02, and 1.95 [s,s,s, 12H, CH3C-
(O)], 1.60-1.40 [m, 4H, NHCH2CH2 and CH2CH2C(O)OCH3],
1.40-1.20 [m, 14H, (CH2)7]. 13C NMR (CDCl3): δ 175.9 [C(O)-
OH], 174.4, 172.2, and 170.7 [C(O)NH and C(O)OCH3], 170.2,
170.1, 169.7, and 169.4 [CH3C(O)], 102.1 (C-1â Gal), 71.2, 70.8,
69.7, and 67.2 (C2-5 Gal and OCH2CH), 61.4 (C-6 Gal), 52.6
(OCH2CH), 51.5 (OCH3), 40.0 (NHCH2), 34.2 [CH2C(O)OCH3],
30.7 [NHC(O)CH2CH2C(O)OH], 29.6, 29.4, 29.3, and 29.2
[(CH2)7], 26.9 [NHCH2CH2], 25.0 [CH2CH2C(O)OCH3], 20.8 and
20.6 [CH3C(O)].
3-O-(â-D-Ga la ctop yr a n osyl)-N-(3-ca r boxyp r op ion yl)-L-
ser in e-11-m eth oxyca r bon ylu n d eca n a m id e, 6. The proce-
dure A when applied to 75 mg (0.11 mmol) of 5 gave 50 mg
1
(76%) of 6. Rf ) 0.30 (71:26:4 CHCl3-MeOH-H2O). H NMR
(CD3OD-D2O): δ 4.48 (t, J ) 4.3 Hz, 1 H, H-4 Gal), 4.30-
4.18 (m, 2H, H-1â,3 Gal), 3.83-3.70 (m, 4H, H-2,5,6,6′ Gal),
3.64 (s, 3H, OCH3), 3.60-3.50 (m, 3H, OCH2CH), 3.22-3.02
[m, 8H, +HN(CH2CH3)3 and NHCH2], 2.58-2.42 (m, 4H,
CH2CH2C(O)ONHEt3), 2.30 [t, J ) 7.3 Hz, 2H, CH2C(O)OCH3],
1.59-1.46 [m, 4H, NHCH2CH2 and CH2CH2C(O)OCH3], 1.30-
1.10 [m, 23H, (CH2)7 and +HN(CH2CH3)3]. 13C NMR (CD3-
OD): δ 180.3 [C(O)O], 177.0, 176.3, and 172.1 [C(O)NH and
C(O)OCH3], 104.6 (C-1â Gal), 76.6 (C-5 Gal), 74.4 (C-3 Gal),
72.2 (C-2 Gal), 70.1 (C-4 Gal), 70.0 (OCH2CH), 62.3 (C-6 Gal),
55.0 (OCH2CH), 52.5 (OCH3), 47.7 [+NH(CH2CH3)3], 40.7
(NHCH2), 34.8 [CH2C(O)OCH3], 33.1 and 32.9 [NHC(O)-
CH2CH2C(O)O], 30.4, 30.3, 30.1, 30.0, 29.9, and 29.8 [(CH2)7],
27.7 (NHCH2CH2), 25.9 [CH2CH2C(O)OCH3], 9.3 [+NH-
(CH2CH3)3]. This assignment was further confirmed by DEPT.
3-O-(â-D-Ga la ctop yr a n osyl)-N-(3-ca r boxyp r op ion yl)-L-
ser in e-11-car boxyu n decan am ide (Sodiu m Salt), II-GalSer -
[C11CO2Na ][C3CO2Na ]. Fifty milligrams (0.074 mmol) of
6 were stirred with 2.6 mL of 0.1 N 9:1 NaOH MeOH-H2O,
at room temperature for 3 days. The reaction mixture was
purified by gel filtration on Sephadex LH20 (MeOH) and then
silica gel chromatography (49:1 to 1:1 CHCl3-MeOH) affording
30 mg (66%) of II-Ga lSer [C11CO2Na ][C3CO2Na ] as a white
solid. Rf ) 0.50 (8:5:1 CHCl3-MeOH-H2O). [R]D ) +7.1° (c
1
0.26; H2O). MS (ESI) m/z ) 563.6 (M - 2Na + H)-. H NMR
(D2O): δ 4.46 (m, 1H, H-4 Gal), 4.31 (d, J ) 7.4 Hz, 1H, H-1â
Gal), 4.01 (dd, J ) 10.6, J ) 5.4 Hz, 1H, H-3 Gal), 3.84-3.41
(m, 7H, H-2,5,6,6′ Gal and OCH2CH), 3.13 (m, 2H, NHCH2),
2.47 [m, 4H, NHC(O)CH2CH2C(O)ONa], 2.18 [t, J ) 6.9 Hz,
2H, CH2C(O)ONa], 1.55-1.30 [m, 4H, NHCH2CH2 and CH2-
CH2C(O)ONa], 1.30-1.10 [m, 14H, (CH2)7]. 13C NMR (D2O):
δ 181.5 and 180.1 [C(O)ONa], 175.8 and 171.1 [C(O)NH], 102.9
(C-1â Gal), 75.2 (C-5 Gal), 72.7 (C-3 Gal), 70.7 (C-2 Gal), 68.7
(OCH2CH), 68.6(C-4 Gal), 60.9 (C-6 Gal), 53.9 (OCH2CH), 39.6
(NHCH2), 36.0 [CH2C(O)ONa], 32.0 and 31.6 [NHC(O)-
CH2CH2C(O)O], 29.0, 28.8, 28.6, and 28.5 [(CH2)7], 26.2
(NHCH2CH2), 25.3 [CH2CH2C(O)ONa]. Anal. Calc. for
C25H42N2O12Na2‚2H2O (644.62): C, 46.58; H, 7.19; N, 4.35.
Found: C, 46.92; H, 7.22; N, 4.50.
Syn th esis of II-Ga lSer [C2OH][C14] (Sch em e 1).3-O-
(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la ctop yr a n osyl)-L-ser in e-2-
h yd r oxyeth a n a m id e, 3b. Procedure C, when applied to 95
mg (0.49 mmol) of EDC, 320 mg (0.48 mmol) of A, 35 µL (0.57
mmol) of ethanolamine, and 70 mg (0.51 mmol) of HOBt,
afforded after chromatography (49:1 CHCl3-MeOH) 310 mg
(90%) of 3-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-NR-
Syn th esis of II-GalSer [C11CO2Na][C3CO2Na] (Sch em e
1). 3-O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la ctop yr a n osyl)-N-(3-
ca r b oxyp r op ion yl)-L-ser in e-11-m et h oxyca r b on ylu n d e-
ca n a m id e, 5. Twenty-six milligrams (0.26 mmol) of succinic
anhydride was added to a solution of 71 mg (0.12 mmol) of 3a

Anionic Analogues of Galactosylceramide
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2197
9-(fluorenylmethoxycarbonyl)-L-serine-2-hydroxyethanamide
as a white solid [Rf ) 0.62 (9:1 CHCl3-MeOH). 1H NMR
(CDCl3): δ 7.71 (d, J ) 7.2 Hz, 2H, Fmoc), 7.54 (d, J ) 7.2 Hz,
2H, Fmoc), 7.40-7.23 (m, 4H, Fmoc), 6.91 (t, J ) 6.5 Hz, 1H,
NHCH2), 5.89 [d, J ) 6.9 Hz, 1H, NHC(O)O], 5.33 (bs, 1H,
H-4 Gal), 5.11 (dd, J ) 10.3 Hz, J ) 7.7 Hz, 1H, H-2 Gal),
4.98 (dd, J ) 10.5 Hz, J ) 3.2 Hz, 1H, H-3 Gal), 4.47 (d, J )
7.6 Hz, 1H, H-1â Gal), 4.36 [m, 2H, C(O)OCH2CH], 4.15-3.80
[m, 6H, H-5,6,6′ Gal, OCH2CH and C(O)OCH2CH], 3.72 (m,
1H, OCH2CH), 3.63 (m, 2H, CH2OH), 3.37 (m, 2H, NHCH2),
2.10, 1.98, and 1.94 [s,s,s, 12H, CH3C(O)]. 13C NMR (CDCl3):
δ 170.7, 170.3, 170.1, 170.0, and 169.8 [CH3C(O)O and C(O)-
NH], 156.0 [NHC(O)O], 143.8 and 141.4 (C, Fmoc), 127.9,
127.2, 125.1, and 120.1 (CH, Fmoc), 101.9 (C-1â Gal), 71.2
(C-3 Gal), 70.6 (C-5 Gal), 70.0 (OCH2CH), 68.9 (C-2 Gal), 67.1
[C-4 Gal and C(O)OCH2], 61.4 (CH2OH and C-6 Gal), 54.4
(OCH2CH), 47.2 [C(O)OCH2CH], 42.5 (NHCH2), 20.8, 20.7, and
20.6 [CH3C(O)]]. The procedure B when applied to 300 mg
(0.43 mmol) of this hydroxyethanamide derivative gave after
chromatography (95:5 CHCl3-MeOH) 130 mg (65%) of 3b as
a white solid. Rf ) 0.20 (95:5 CHCl3-MeOH). 1H NMR
(CDCl3): δ 7.63 (t, J ) 5.6 Hz, 1H, NHCH2), 5.31 (bs, 1H, H-4
Gal), 5.10 (dd, J ) 10.5 Hz, J ) 7.7 Hz, 1H, H-2 Gal), 4.94
(dd, J ) 10.5 Hz, J ) 3.3 Hz, 1H, H-3 Gal), 4.44 (d, J ) 7.7
Hz, 1H, H-1â Gal), 4.10 (m, 2H, H-6,6′ Gal), 3.90-3.75 (m,
3H, H-5 Gal and OCH2CH), 3.60 (t, J ) 5.0 Hz, 2H, CH2OH),
3.51 (m, 1H, OCH2CH), 3.34 (m, 2H, NHCH2), 2.49 (bs, 3H,
OH and NH2), 2.10, 2.00, 1.98, and 1.91 [4s, each 3H, CH3C-
(O)]. 13C NMR (CDCl3): δ 170.6 [C(O)NHCH2], 170.2, 170.0,
and 169.8 [CH3C(O)], 102.1 (C-1â Gal), 71.4 (C-3 Gal), 70.6
(C-5 Gal), 69.9 (OCH2CH), 68.9 (C-2 Gal), 67.1 (C-4 Gal), 61.4
(CH2OH and C-6 Gal), 55.2 (OCH2CH), 42.5 (NHCH2), 20.8,
20.6, and 20.5 [CH3C(O)].
(CH2CH3), 13.1 (CH3). This assignment was further confirmed
by DEPT. Anal. Calc. for C25H48N2O9‚3/2H2O (547.69): C,
54.83; H, 9.39; N, 5.11. Found: C, 54.35; H, 8.99; N, 5.07.
Syn th esis of II-Ga lSer [C2SO4Na ][C14] (Sch em e 1).3-
O-(2,3,4,6-Tet r a -O-a cet yl-â-D-ga la ct op yr a n osyl)-N-(t et -
r adecan oyl)-L-ser in e-2-su lfoxyeth an am ide (Sodiu m Salt),
8. Fifty-five milligrams (0.081 mmol) of 7 and 40 mg (0.25
mmol) of SO3‚pyridine in 3 mL of pyridine were stirred at room
temperature for 1 h. Then, 20 mg of Na2CO3 in 5 mL of water
was added to the reaction mixture at 0 °C. After evaporation
of the solvents, the crude residue was extracted twice with
1:1 CHCl3-MeOH mixture. The organic phases were evapo-
rated and purified by chromatography (95:5 to 9:1 CHCl3-
MeOH) giving 50 mg (80%) of 8 as a white solid. Rf ) 0.34
(4:1 CHCl3-MeOH). 1H NMR (CD3OD-CDCl3): δ 5.32 (bs, 1H,
H-4 Gal), 5.00 (m, 2H, H-2,3 Gal), 4.54 (d, J ) 7.0 Hz, 1H,
H-1â Gal), 4.45 (m, 1H, H-5 Gal), 4.10-3.75 (m, 7H, OCH2CH,
CH2OSO3Na, and H-6,6′ Gal), 3.46 (m, 2H, NHCH2), 2.19 [t,
J ) 7.5 Hz, 2H, CH2C(O)], 2.08, 1.99, and 1.90 [s,s,s, 12H,
CH3C(O)], 1.53 [m, 2H, CH2CH2C(O)], 1.45-1.20 [m, 20H,
(CH2)10], 0.80 (t, J ) 6.6 Hz, 3H, CH2CH3). 13C NMR (CD3OD-
CDCl3): δ 176.4 [NHC(O)CH2], 172.2 [C(O)NHCH2], 171.8,
171.5, and 171.4 [CH3C(O)], 102.1 (C-1â Gal), 72.1 and 72.0
(C-3,5 Gal), 70.1 (OCH2CH), 69.7 (C-2 Gal), 68.4 (C-4 Gal), 67.7
(CH2OSO3Na), 62.5 (C-6 Gal), 54.4 (OCH2CH), 40.4 (NHCH2),
37.3 [CH2C(O)], 33.0 (CH2CH2CH3), 30.8, 30.7, 30.5, and 30.4
[(CH2)8], 26.7 [CH2CH2C(O)], 23.7 (CH2CH3), 21.7, 21.6, and
21.5 [CH3C(O)], 15.0 (CH2CH3).
3-O-(â-D-Galactopyr an osyl)-N-(tetr adecan oyl)-L-ser in e-
2-su lfoxyeth a n a m id e (Sod iu m Sa lt),II-Ga lSer [C2SO4Na ]-
[C14]. The procedure A when applied to 50 mg (0.063 mmol)
of 8 gave, after treatment with Amberlite IRC-50 ion exchange
(Na+), 30 mg (80%) of II-Ga lSer [C2SO4Na ][C14]. Rf ) 0.18
(35:15:2 CHCl3-MeOH-H2O). MS (ESI) m/z ) 599.5 (M -
3-O-(2,3,4,6-Tet r a -O-a cet yl-â-D-ga la ct op yr a n osyl)-N-
(tetr a d eca n oyl)-L-ser in e-2-h yd r oxyeth a n a m id e, 7. One
hundred twenty milligrams (0.25 mmol) of 3b and 93 mg (0.28
mmol) of N-hydroxysuccinimide ester of tetradecanoic acid
were stirred in 5 mL of CH2Cl2 for 3 days. The reaction mixture
diluted with CH2Cl2 was successively washed with 5% KHSO4,
water, 8% NaHCO3, and then water until neutrality. After
drying with Na2SO4 and filtration, the residue purified by
chromatography (95:5 CHCl3:MeOH) gave 120 mg (70%) of 7
as a white solid. Rf ) 0.36 (9:1 CHCl3-MeOH). 1H NMR
(CDCl3): δ 6.98 (m, 1H, NHCH2), 6.62 (m, 1H, CHNH), 5.32
(bs, 1H, H-4 Gal), 5.06 (dd, J ) 7.7 Hz, J ) 10.4 Hz, 1H, H-2
Gal), 4.96 (dd, J ) 10.4 Hz, J ) 3.3 Hz, 1H, H-3 Gal), 4.55 (m,
2H, H-1â,5 Gal), 4.25-3.85 (m, 4H, OCH2CH and H-6,6′ Gal),
3.63 (m, 3H, CH2OH and OCH2CH), 3.36 (m, 2H, NHCH2),
2.78 (s, 1H, OH), 2.18 [t, J ) 7.5 Hz, 2H, CH2C(O)], 2.10, 2.00,
and 1.91 [s,s,s, 12H, CH3C(O)], 1.53 [m, 2H, CH2CH2C(O)],
1.35-1.10 [m, 20H, (CH2)10], 0.80 (t, J ) 6.5 Hz, 3 H, CH2-
CH3). 13C NMR (CDCl3): δ 173.8 [NHC(O)CH2], 170.6 [C(O)-
NHCH2], 170.2, 170.0, and 169.8 [CH3C(O)], 102.1 (C-1â Gal),
71.4 (C-3 Gal), 70.6 (C-5 Gal), 69.9 (OCH2CH), 68.9 (C-2 Gal),
67.1 (C-4 Gal), 61.4 (CH2OH and C-6 Gal), 52.5 (OCH2CH),
42.5 (NHCH2), 36.4 [CH2C(O)], 32.0 (CH2CH2CH3), 29.6, 29.5,
29.3, 29.1, and 28.8 [(CH2)8], 25.5 [CH2CH2C(O)], 22.7 (CH2-
CH3), 20.8, 20.6, and 20.5 [CH3C(O)], 14.1 (CH2CH3).
1
Na)-. H NMR (1:1 CD3OD-CDCl3): δ 4.62 (m, 1H, H-4 Gal),
4.30 (d, J ) 7.2 Hz, 1H, H-1â Gal), 4.20 (dd, J ) 10.2 Hz, J )
4.1 Hz, 1H, H-3 Gal), 4.13 (m, 2H, H-2,5 Gal), 3.90-3.50 (m,
9H, H-6,6′ Gal, CH2CH2OSO3Na, and OCH2CH), 2.32 [t, J )
7.0 Hz, 2H, CH2C(O)], 1.65 [m, 2H, CH2CH2C(O)], 1.40-1.20
[m, 20H, (CH2)10], 0.92 (t, J ) 6.8 Hz, 3H, CH3). 13C NMR (1:1
CD3OD-CDCl3): δ 174.8 [NHC(O)CH2], 170.8 [C(O)NHCH2],
103.6 (C-1â Gal), 75.0 (C-5 Gal), 73.1 (C-3 Gal), 70.8 (C-2 Gal),
68.9 (OCH2CH and C-4 Gal), 65.8 (CH2OSO3Na), 61.0 (C-6
Gal), 53.1 (OCH2CH), 38.9 (NHCH2), 35.4 [CH2C(O)], 31.5
(CH2CH2CH3), 29.2, 29.1, 29.0, 28.9, and 28.8 [(CH2)8], 25.1
[CH2CH2C(O)], 22.1 (CH2CH3), 13.0 (CH3). This assignment
was further confirmed by DEPT. Anal. Calc. for C25H47N2O12
-
NaS‚6H2O (730.89): C, 41.09; H, 8.14; N, 3.83; S, 4.39.
Found: C, 41.13; H, 7.90; N, 3.89; S, 4.16.
Syn th esis of II-Ga lSer [C14][C11CO2Na ] (Sch em e 2).
3-O -(2,3,4,6-T e t r a -O -a c e t y l-â-D-g a la c t o p y r a n o s y l)-L -
ser in e Tetr a d eca n a m id e, 9. Procedure C, when applied to
145 mg (0.75 mmol) of EDC, 450 mg (0.70 mmol) of A, 160 mg
(0.73 mmol) of tetradecylamine, and 105 mg (0.77 mmol) of
HOBt, gave after chromatography (CHCl3) 480 mg (80%) of
3-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-NR-9-(fluore-
nylmethoxycarbonyl)-L-serine tetradecanamide as a white solid
[Rf ) 0.5 (4:1 CH2Cl2-Et2O). [R]D ) +5.7° (c 1.1; 4:1 CHCl3-
MeOH). IR (ν cm-1, KBr): 1750 (CdO ester), 1700 (CdO
carbamate), 1655 (CdO amide). 1H NMR (CDCl3-CD3OD): δ
7.68 (d, J ) 8.0 Hz, 2H, Fmoc), 7.55 (d, J ) 8.0 Hz, 2H, Fmoc),
7.35-7.20 (m, 4H, Fmoc), 5.24 (bs, J ) 3.2 Hz, 1H, H-4 Gal),
3-O-(â-D-Galactopyr an osyl)-N-(tetr adecan oyl)-L-ser in e-
2-h yd r oxyeth a n a m id e, II-Ga lSer [C2OH][C14]. The pro-
cedure A when applied to 70 mg (0.10 mmol) of 7 gave 45 mg
(85%) of II-Ga lSer [C2OH][C14]. Rf ) 0.56 (70:30:4 CHCl3-
MeOH-H2O). SM (ESI) m/z ) 543.5 (M + Na)+. 1H NMR (1:1
CDCl3-CD3OD): δ 4.56 (dd, J ) 4.5, J ) 5.6 Hz, 1H, H-4 Gal),
4.20 (d, J ) 7.2 Hz, 1H, H-1â Gal), 4.14 (dd, J ) 10.3 Hz, J )
4.4 Hz, 1H, H-3 Gal), 3.85-3.40 (m, 9H, H-2,5,6,6′ Gal, CH2-
OH, and OCH2CH), 3.30 (m, 2H, NHCH2), 2.24 [t, J ) 7.5 Hz,
2H, CH2C(O)], 1.58 [m, 2H, CH2CH2C(O)], 1.24 [m, 20H,
(CH2)10], 0.84 (t, J ) 6.1 Hz, 3H, CH3). 13C NMR (1:1 CDCl3-
CD3OD): δ 174.8 [NHC(O)CH2], 170.8 [C(O)NHCH2], 103.8 (C-
1â Gal), 75.2 (C-5 Gal), 73.2 (C-3 Gal), 70.9 (C-2 Gal), 68.9
(OCH2CH), 68.7 (C-4 Gal), 60.9 (C-6 Gal), 59.9 (CH2OH), 53.2
(OCH2CH), 41.5 (NHCH2), 35.4 [CH2C(O)], 31.4 (CH2CH2CH3),
29.1, 29.0, 28.9, and 28.8 [(CH2)8], 25.2 [CH2CH2C(O)], 22.1
3
5.00 (dd, J ) 7.4 Hz, J ) 10.4 Hz, 1H, H-2 Gal), 4.92 (dd, J
) 10.4, Hz J ) 3.2 Hz, 1H, H-3 Gal), 4.40 (d, J ) 7.4 Hz, 1H,
H-1â Gal), 4.30-3.60 [m, 9 H, H-5,6,6′ Gal, OCH2CH, and
C(O)OCH2CH], 3.15 (m, 2H, NHCH2), 2.06, 1.97, 1.94, and 1.91
[4s, each 3HCH3C(O)], 1.50-1.10 [m, 24H, (CH2)12], 0.80 (t, J
) 7.0 Hz, 3H, CH2CH3). 13C NMR (CDCl3): δ 170.8 [C(O)NH],
170.4, 170.3, 169.9, and 169.4 [CH3C(O)], 156.3 [NHC(O)O],
143.7 and 141.3 (C, Fmoc), 127.1, 127.8, 124.9, and 120.0 (CH,
Fmoc), 101.5 (C-1â Gal), 70.9 and 70.8 (C-3,5 Gal), 69.6 (OCH2-
CH), 68.7 (C-2 Gal), 67.1 (C-4 Gal), 66.9 [C(O)OCH2], 61.3 (C-6
Gal), 54.2 (OCH2CH), 47.1 [C(O)OCH2CH], 39.7 (NHCH2), 31.9
(CH2CH2CH3), 29.7, 29.6, 29.5, 29.3, 29.2, and 29.1 [(CH2)9],

2198 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Faroux-Corlay et al.
26.8 (NHCH2CH2), 22.6 (CH2CH3), 20.54, 20.45, 20.41, and
20.38 [CH3C(O)], 13.9 (CH2CH3)]]. The procedure B when
applied to 150 mg (0.17 mmol) of this tetradecanamide
derivative gave, after chromatography (CH2Cl2 to 1:1 CH2Cl2-
Et2O), 76 mg (76%) of 9 as a white solid. Rf ) 0.6 (49:1 CHCl3-
MeOH). 1H NMR (CDCl3): δ 7.34 (t, J ) 5.6 Hz, 1H, NHCH2),
5.33 (bs, J ) 3.2 Hz, 1H, H-4 Gal), 5.12 (dd, J ) 7.8 Hz, J )
10.4 Hz, 1H, H-2 Gal), 4.95 (dd, J ) 10.4 Hz, J ) 3.2 Hz, 1H,
H-3 Gal), 4.48 (d, J ) 7.8 Hz, 1H, H-1â Gal), 4.07 (m, 2H,
H-6,6′ Gal), 3.86 (m, 2H, H-5 Gal and OCHaHb), 3.77 (t, J )
8.2 Hz, 1H, OCHaHb), 3.48 (dd, J ) 8.2 Hz, J ) 4.4 Hz, 1H,
OCH2CH), 3.15 (q, J ) 6.6 Hz, 2H, NHCH2), 2.08, 2.00, 1.98,
and 1.91 [4s, each 3HCH3C(O)], 1.70 (bs, 2H, NH2), 1.50-1.10
[m, 24H, (CH2)12], 0.81 (t, J ) 6.6 Hz, 3H, CH3). 13C NMR
(CDCl3-CD3OD): δ 171.0 [C(O)NH], 170.5, 170.2, 170.1, and
169.7 [CH3C(O)], 101.3 (C-1â Gal), 71.9 (OCH2CH), 71.0 (C-3
Gal), 70.8 (C-5 Gal), 68.9 (C-2 Gal), 67.1 (C-4 Gal), 61.3 (C-6
Gal), 55.0 (OCH2CH), 39.4 (NHCH2), 32.0 (CH2CH2CH3), 29.9,
29.74, 29.70, 29.60, 29.55, 29.40, and 29.36 [(CH2)9], 27.0
(NHCH2CH2), 22.8 (CH2CH3), 20.9, 20.8, 20.7, and 20.6 [CH3C-
(O)], 14.2 (CH2CH3).
34.0 [CH2C(O)OH], 32.0 (CH2CH2CH3), 29.8, 29.6, 29.5, 29.3,
29.1, 28.9, and 28.8 [(CH2)6 and (CH2)9], 26.9 (NHCH2CH2),
25.5 [NHC(O)CH2CH2], 24.7 [CH2CH2C(O)OH], 22.7 (CH2CH3),
20.8, 20.7, and 20.4 [CH3C(O)], 14.2 (CH2CH3).
3-O-(â-D-Galactopyr an osyl)-N-(11-car boxyu n decan oyl)-
L-ser in e Tetr a d eca n a m id e (Sod iu m Sa lt), II-Ga lSer [C14]-
[C11CO2Na ]. The procedure A when applied to 65 mg (0.077
mmol) of 11 gave, after treatment with Amberlite IRC-50 ion-
exchange (Na+), 45 mg (90%) of II-Ga lSer [C14][C11CO2Na ].
Rf ) 0. 69 (71:26:4 CHCl3-MeOH-H2O). IR (ν cm-1, KBr):
1
1696 (CdO acide), 1645 (CdO amide). H NMR (CDCl3-CD3-
OD-D2O): δ 4.51 (m, 1H, H-4 Gal), 4.23 (d, J ) 7.4 Hz, 1H,
H-1â Gal), 4.08 (dd, J ) 10.4 Hz, J ) 3.3 Hz, 1H, H-3 Gal),
3.80 (m, 1H, H-2 Gal), 3.69 (m, 3H, H-5,6,6′ Gal), 3.48 (m, 3H,
OCH2CH), 3.14 (m, 2H, NHCH2), 2.24 [t, J ) 7.8 Hz, 2H,
CH2C(O)ONa], 2.14 [t, J ) 7.9 Hz, 2H, NHC(O)CH2], 1.60-
1.35 [m, 6H, NHCH2CH2, NHC(O)CH2CH2 and CH2CH2C(O)-
OH], 1.35-1.10 [m, 34H, (CH2)6 and (CH2)11CH3], 0.82 (t, J )
6.6 Hz, 3H, CH3). 13C NMR (DMSO-d6): δ 172.2 and 169.3
[C(O)NH and C(O)ONa], 104.1 (C-1â Gal), 75.2 (C-5 Gal), 73.1
(C-3 Gal), 70.4 (C-2 Gal), 68.0 (C-4 Gal), 69.4 (OCH2CH), 60.4
(C-6 Gal), 52.6 (OCH2CH), 38.5 (NHCH2), 35.1 [NHC(O)CH2],
34.6 [CH2C(O)ONa], 31.2 (CH2CH2CH3), 29.0, 28.7, and 28.6
[(CH2)6 and (CH2)9], 26.2 (NHCH2CH2), 25.0 [NHC(O)CH2CH2],
24.8 [CH2CH2C(O)OH], 22.0 (CH2CH3), 13.9 (CH3). This as-
signment was further confirmed by DEPT. Anal. Calc. for
3-O-(2,3,4,6-Tet r a -O-a cet yl-â-D-ga la ct op yr a n osyl)-N-
(11-a llyloxyca r bon yl-u n d eca n oyl)-L-ser in e Tetr a d eca n a -
m id e, 10. Procedure C when applied to 49 mg (0.26 mmol) of
EDC, 160 mg (0.25 mmol) of 9, 68 mg (0.25 mmol) of
dodecanedioic acid monoallyl ester, and 38 mg (0.28 mmol) of
HOBt afforded afetr chromatography (CHCl3) 140 mg (65%)
of 10 as a white solid. Rf ) 0.54 (49:1 CHCl3-MeOH). 1H NMR
(CDCl3): δ 6.58 [m, 2H, C(O)NH], 5.83 (m, 1H, OCH2CH)CH2),
5.34 (bd, J ) 2.5 Hz, 1H, H-4 Gal), 5.27-5.05 (m, 3H, H-2 Gal
and OCH2CHdCH2), 4.97 (dd, J ) 10.4 Hz, J ) 3.3 Hz, 1H,
H-3 Gal), 4.56 (d, J ) 7.7 Hz, 1H, H-1â Gal), 4.50 (m, 3H, H-5
Gal and OCH2CHdCH2), 4.13 (m, 2H, H-6,6′ Gal), 4.05 (m,
2H, OCH2CH), 3.66 (m, 1H, OCH2CH), 3.16 (m, 2H, NHCH2),
2.25 [t, J ) 7.3 Hz, 2H, CH2C(O)OAll], 2.15 [t, J ) 7.2 Hz,
2H, NHC(O)CH2], 2.08, 1.99, 1.98, and 1.91 [4s, each 3HCH3C-
(O)], 1.65-1.45 [m, 6H, CH2CH2C(O)O and NHCH2CH2], 1.35-
1.10 [m, 34H, (CH2)6 and (CH2)11CH3], 0.80 (t, J ) 6.6 Hz, 3H,
CH2CH3]. 13C NMR (CDCl3): δ 173.4, 173.3, and 170.5 [C(O)-
OAll and C(O)NH], 170.1, 169.9, 169.5, and 169.4 [CH3C(O)],
132.5 (OCH2CHdCH2), 118.0 (OCH2CHdCH2), 102.2 (C-1â
Gal), 71.3 (C-3 Gal), 70.8 (C-5 Gal), 69.9 (OCH2CH), 68.9 (C-2
Gal), 67.2 (C-4 Gal), 64.9 (OCH2CHdCH2), 61.4 (C-6 Gal), 52.2
[OCH2CH], 39.8 (NHCH2), 36.4 [NHC(O)CH2], 34.3 [CH2C-
(O)O], 32.0 (CH2CH2CH3), 29.7, 29.6, 29.4, and 29.1 [(CH2)6
and (CH2)9CH2CH2CH3], 26.9 (NHCH2CH2), 25.6 [CH2CH2C-
(O)NH], 25.0 [CH2CH2C(O)O], 22.7 (CH2CH3), 20.8, 20.6, and
20.5 [CH3C(O)], 14.1 (CH2CH3).
C
35H65N2O10Na‚11/2H2O (795.98): C, 52.81; H, 8.23; N, 3.52;
Na, 2.89. Found: C, 53.00; H, 8.84; N, 3.17; Na, 2.33.
Syn th esis of II-Ga lSer [C14][C3CO2Na ] (Sch em e 2).3-
O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la ctop yr a n osyl)-N-(3-ca r -
boxyp r op ion yl)-L-ser in e Tetr a d eca n a m id e, 12. Twenty
milligrams (0.20 mmol) of succinic anhydride were added to a
solution of 120 mg of 9 (0.19 mmol) and 60 µL of Et3N (0.43
mmol) in CH2Cl2 (3 mL). After stirring at room temperature
for 24 h, the solution was washed with 5% KHSO4 and then
water. After drying over Na2SO4 and filtration, the solvent was
evaporated, and the residue was purified by chromatography
(CHCl3 to 9:1 CHCl3-MeOH) giving 130 mg (95%) of 12 as a
white solid. Rf ) 0.34 (95:5 CHCl3-MeOH). 1H NMR (CDCl3):
1
δ 7.10 [d, J ) 6.9 Hz, 1H, CHNH], 6.84 [m, H, C(O)NHCH2],
1
5.32 (bd, J ) 2.8 Hz, H, H-4 Gal), 5.05 (dd, J ) 7.4 Hz, J )
1
1
10.4 Hz, H, H-2 Gal), 5.01 (dd, J ) 10.4 Hz, J ) 3.0 Hz, H,
H-3 Gal), 4.58 (d, J ) 7.3 Hz, 1H, H-1â Gal), 4.54 (m, 1H,
H-5 Gal), 4.08 (m, 2H, H-6,6¢ Gal), 3.94 (m, 2H, OCH2CH),
1
3.71 (m, H, OCH2CH), 3.15 (dt, J ) 6.6 Hz, J ) 5.8 Hz, 2H,
NHCH2), 2.53 [m, 2H, CH2C(O)OH], 2.45 [m, 2H, NHC(O)-
CH2], 2.08, 1.98, and 1.91 [s,s,s, 12H, CH3C(O)], 1.42 (m, 2H,
NHCH2CH2), 1.30-1.10 [m, 22H, (CH2)11CH3], 0.80 (t, J )
6.3 Hz, 3H, CH2CH3). 13C NMR (CDCl3): δ 176.4 [C(O)OH],
172.4 and 170.4 [C(O)NH], 169.9, 169.8, 169.5, and 169.3
[CH3C(O)], 101.6 (C-1â Gal), 70.8 (C-3 Gal), 70.5 (C-5 Gal),
69.5 (C-2 Gal and OCH2CH), 66.9 (C-4 Gal), 61.1 (C-6 Gal),
52.4 (OCH2CH), 39.7 (NHCH2), 31.7 (CH2CH2CH3), 30.9 [NHC-
(O)CH2], 30.0 [CH2C(O)OH], 29.5 and 29.1 [(CH2)9], 26.7
(NHCH2CH2), 22.5 (CH2CH3), 20.6 and 20.4 [CH3C(O)], 13.9
(CH2CH3).
3-O-(2,3,4,6-Tet r a -O-a cet yl-â-D-ga la ct op yr a n osyl)-N-
(11-ca r boxyu n d eca n oyl)-L-ser in e Tetr a d eca n a m id e, 11.
Seventy-four milligrams of 10 (0.084 mmol), 130 µL of Et2NH
(1.25 mmol), and 5.0 mg (4.4 × 10-3 mmol) of Pd[P(Ph)3]4 in
CH2Cl2 (3 mL) were stirred for 2 h at room temperature. After
evaporation of the solvent, the solution was washed with 5%
KHSO4 and then water. After drying over Na2SO4 and filtra-
tion, the solvent was evaporated, and the residue was purified
by chromatography (CHCl3 to 1:1 CHCl3-Et2O) giving 65 mg
(90%) of 11 as a white solid. Rf ) 0.36 (95:5 CHCl3-MeOH).
1H NMR (CDCl3): δ 7.02 [d, J ) 7.1 Hz, 1H, CHNH], 6.66 [t,
J ) 6.5 Hz, 1H, C(O)NHCH2], 5.36 (bd, J ) 3.1 Hz, 1H, H-4
Gal), 5.13 (dd, J ) 7.7 Hz, J ) 10.4 Hz, 1H, H-2 Gal), 5.00
(dd, J ) 10.4 Hz, J ) 3.3 Hz, 1H, H-3 Gal), 4.64 (m, 1H, H-5
Gal), 4.57 (d, J ) 7.7 Hz, 1H, H-1â Gal), 4.17 (m, 2H, H-6,6′
Gal), 3.96 (m, 2H, OCH2CH), 3.69 (m, 1H, OCH2CH), 3.20 (m,
2H, NHCH2), 2.30 [t, J ) 7.1 Hz, 2H, CH2C(O)OH], 2.23 [t, J
) 7.1 Hz, 2H, NHC(O)CH2], 2.12, 2.03, 2.01, and 1.95 [4s, each
3HCH3C(O)], 1.70-1.45 [m, 6H, NHCH2CH2, NHC(O)CH2CH2
and CH2CH2C(O)OH], 1.35-1.10 [m, 34H, (CH2)6 and (CH2)11-
CH3], 0.84 (t, J ) 6.5 Hz, 3H, CH2CH3). 13C NMR (CDCl3): δ
177.8 [C(O)OH], 173.8 and 170.6 [C(O)NH], 170.2, 170.1, 169.9,
and 169.6 [CH3C(O)], 102.4 (C-1â Gal), 71.3 (C-3 Gal), 70.8
(C-5 Gal), 70.1 (OCH2CH), 68.9 (C-2 Gal), 67.2 (C-4 Gal), 61.4
(C-6 Gal), 52.3 (OCH2CH), 40.0 (NHCH2), 36.3 [NHC(O)CH2],
3-O-(â-D-Ga la ctop yr a n osyl)-N-(3-ca r boxyp r op ion yl)-L-
ser in e Tetr a d eca n a m id e (Sod iu m Sa lt), II-Ga lSer [C14]-
[C3CO2Na ]. The procedure A when applied to 120 mg (0.16
mmol) of 12 gave, after treatment with Amberlite IRC-50 ion-
exchange (Na+), 45 mg (76%) of II-Ga lSer [C14][C3CO2Na ].
Rf ) 0.44 (71:26:4 CHCl3-MeOH-H2O). MS (ESI) m/z ) 561.4
1
(M - Na)-. H NMR (CD3OD-D2O): δ 4.46 (m, 1H, H-4 Gal),
4.28 (d, J ) 7.0 Hz, 1H, H-1â Gal), 4.17 (m, 1H, H-3 Gal), 3.80-
3.35 (m, 7H, OCH2CH and H-2,5,6,6′ Gal), 3.13 (m, 2H,
NHCH2), 2.46 [m, 4H, CH2CH2C(O)ONa], 1.43 (m, 2H,
NHCH2CH2), 1.30-1.10 [m, 22H, (CH2)11CH3], 0.80 (t, J ) 6.7
Hz, 3H, CH3). 13C NMR (D2O at 50 °C): δ 176.4 [C(O)ONa],
171.5 [C(O)NH], 104.0 (C-1â Gal), 76.0 (C-5 Gal), 73.8 (C-3
Gal), 71.7 (C-2 Gal), 69.9 (C-4 Gal), 69.6 (OCH2CH), 61.8 (C-6
Gal), 54.6 (OCH2CH), 40.5 (NHCH2), 33.1 (CH2CH2CH3), 31.3
and 31.4 [NHC(O)CH2 and CH2C(O)OH], 30.5, 30.1, and 29.7
[(CH2)9], 27.6 (NHCH2CH2), 23.3 (CH2CH3), 14.5 (CH3). This
assignment was confirmed by DEPT.

Anionic Analogues of Galactosylceramide
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2199
Syn th esis of II-Ga lSer [C14][C7SO3Na ] (Sch em e 2).
3-O-(2,3,4,6-Tet r a -O-a cet yl-â-D-ga la ct op yr a n osyl)-N-[(3-
(2-su lfoeth ylca r ba m oyl)p r op ion yl]-L-ser in e Tetr a d eca n -
a m id e, 13. Twenty milligrams (0.11 mmol) of EDC were added
at 0 °C to a solution of 80 mg (0.11 mol) of 12 and 26 mg (0.23
mmol) of N-hydroxysuccinimide in 3 mL of DMF. The reaction
mixture was stirred at 0 °C for 30 min and then at room
temperature for 24 h. Eighteen milligrams (0.14 mmol) of
taurine and 30 µL (0.22 mmol) of Et3N were then added, and
the mixture was stirred for 24 h. After evaporation of DMF
under vacuo, the residue was chromatographed on silica gel
(CHCl3 to 4:1 CHCl3-MeOH) giving 60 mg (65%) of 13 as a
white solid. Rf ) 0.37 (4:1 CHCl3-MeOH). 1H NMR (CD3OD):
δ 5.31 (bs, 1H, H-4 Gal), 5.03 (m, 2H, H-2,3 Gal), 4.56 (d, J )
6.9 Hz, 1H, H-1â Gal), 4.47 (m, 1H, H-5 Gal), 4.10 (m, 2H,
H-6,6′ Gal), 4.00-3.70 (m, 3H, OCH2CH), 3.54 (m, 2H, NHCH2-
CH2SO3), 3.15 (m, 2H, NHCH2), 2.95 (m, 2H, NHCH2CH2SO3),
2.60-2.40 [m, 4H, NHC(O)CH2CH2], 2.08, 1.99, 1.98, and 1.91
[4s, each 3H, CH3C(O)], 1.52-1.40 (m, 2H, NHCH2CH2), 1.30-
1.10 [m, 22H, (CH2)11CH3], 0.80 (t, J ) 6.3 Hz, 3H, CH2CH3).
13C NMR (CDCl3-CD3OD): δ 173.4, 173.3, and 170.8 [C(O)-
NH], 170.4, 170.3, 170.1, and 169.7 [CH3C(O)], 101.4 (C-1â
Gal), 70.9 and 70.8 (C-3,5 Gal), 69.2 (OCH2CH), 69.0 (C-2 Gal),
67.2 (C-4 Gal), 61.2 (C-6 Gal), 53.0 (OCH2CH), 46.4 (NHCH2-
CH2SO3), 40.1 (NHCH2), 35.4 (NHCH2CH2SO3), 32.0 (CH2CH2-
CH3), 30.9 and 30.8 [NHC(O)CH2CH2], 29.8, 29.4, and 29.2
[(CH2)9], 27.1 (NHCH2CH2), 22.7 (CH2CH3), 20.7 and 20.6
[CH3C(O)], 14.1 (CH2CH3).
(C-2 Gal), 67.1 (C-4 Gal), 61.3 (C-6 Gal), 54.7 (OCH2CH), 39.9
(NHCH2), 32.0 (CH2CH2CH3), 29.8, 29.6, and 29.4 [(CH2)8], 26.9
(NHCH2CH2), 22.5 (CH2CH3), 20.8 and 20.6 [CH3C(O)], 14.2
(CH3). 31P{1H} NMR (CDCl3): δ -1.52 (s).
3-O-(â-D-Ga la ctop yr a n osyl)-N-(d im eth oxyp h osp h or yl)-
L-ser in e Tetr a d eca n a m id e, 15. Hydrogen was bubbled
through a suspension of 140 mg (0.17 mmol) of 14, 40 mg of
platinum(IV) oxide monohydrate, and 100 µL of Et3N in 10
mL of MeOH at room temperature for 20 h. After filtration,
evaporation, and chromatography (95:5 to 4:1 CHCl3-MeOH),
80 mg (87%) of 15 as a white solid were obtained. Rf ) 0.55
(4:1 CHCl3-MeOH). MS (ESI) m/z ) 593.5 (M + Na)+. 1H
NMR (CDCl3-CD3OD): δ 4.18 (d, J ) 7.2 Hz, 1H, H-1â Gal),
4.08 (dd, J ) 10.0 Hz, J ) 4.1 Hz, 1H, H-3 Gal), 3.66 (d, J H,P
) 11.2 Hz, 6H, OCH3), 3.85-3.40 (m, 8H, H-2,4-6 Gal and
OCH2CH), 3.14 (m, 2H, NHCH2), 1.60-1.10 [m, 24H, (CH2)12],
0.82 (t, J ) 6.6 Hz, 3H, CH2CH3). 13C NMR (CDCl3-CD3OD):
3
δ 172.6 [d, J C,P ) 5 Hz, C(O)NH], 105.2 (C-1â Gal), 76.5 (C-5
Gal), 74.6 (C-3 Gal), 72.3 (C-2 Gal), 72.1 (d, 3J C,P ) 5 Hz, OCH2-
CHNH), 70.1 (C-4 Gal), 62.6 (C-6 Gal), 56.5 (CHNHP), 54.4
2
and 54.3 [d,d, J C,P ) 6 Hz, OCH3], 40.7 (NHCH2), 33.0 (CH2-
CH2CH3), 30.8, 30.7, 30.4, and 30.3 [(CH2)8], 27.9 (NHCH2CH2),
23.7 (CH2CH3), 14.7 (CH2CH3). 31P{1H} NMR (CDCl3-CD3-
OD): δ 9.90 (s) [12.8 (s) in D2O].
3-O-(â-D-Ga la ctop yr a n osyl)-N-(h yd r oxym eth oxyp h os-
p h or yl)-L-ser in e Tetr a d eca n a m id e (Sod iu m Sa lt), II-
Ga lSer [C14][OP (OMe)ONa ]. Forty milligrams (0.070 mmol)
of 15 and 100 µL of Et3N in 5 mL of water were stirred at
room temperature for 5 days. The crude residue was eluted
on Amberlite IRC-50 ion-exchange (Na+) column and then
lyophilized giving 38 mg (95%) of II-Ga lSer [C14][OP (OMe)-
ONa ] as a white solid. Rf ) 0.67 (71:26:4 CHCl3-MeOH-H2O).
3-O-(â-D-Galactopyr an osyl)-N-[(3-(2-su lfoeth ylcar bam -
oyl)pr opion yl]-L-ser in e Tetr adecan am ide, II-GalSer [C14]-
[C7SO3Na ]. The procedure A when applied to 57 mg (0.068
mmol) of 13 gave, after treatment with Amberlite IRC-50 ion-
exchange (Na+), 40 mg (80%) of II-Ga lSer [C14][C7SO3Na ]
as a white solid. Rf ) 0.17 (71:26:4 CHCl3-MeOH-H2O). [R]D
) -11.7° (c 0.28; MeOH). IR (ν cm-1, KBr): 1649 (CdO amide),
1
SM (ESI) m/z ) 601.5 (M + Na)+. H NMR (D2O): δ 4.33 (d,
J ) 7.0 Hz, 1H, H-1â Gal), 4.10-3.40 (m, 12H, OCH3, OCH2CH
and H-2s6 Gal), 3.12 (m, 2H, NHCH2), 1.60-1.10 [m, 24H,
(CH2)12], 0.82 (t, J ) 6.6 Hz, 3H, CH2CH3). 13C NMR (CDCl3-
1
1210 (SdO). MS (ESI) m/z ) 668.5 (M-Na)-. H NMR (CD3-
3
OD): δ 4.54 (m, 1H, H-4 Gal), 4.28 (d, J ) 7.3 Hz, 1H, H-1â
Gal), 4.23 (m, 2H, H-2,3 Gal), 3.84-3.70 (m, 3H, H-5,6,6′ Gal),
3.58 (m, 5H, OCH2CH and NHCH2CH2SO3), 3.17 (m, 2H,
NHCH2), 2.98 (t, J ) 6.8 Hz, 2H, NHCH2CH2SO3), 2.65-2.48
[m, 4H, NHC(O)CH2CH2), 1.51 (m, 2H, NHCH2CH2), 1.30-
1.10 [m, 22H, (CH2)11CH3], 0.80 (t, J ) 6.6 Hz, 3H, CH3). 13C
NMR (CD3OD): 176.8, 176.1, and 173.4 [C(O)NH], 106.5 (C-
1â Gal), 78.2 (C-5 Gal), 76.3 (C-3 Gal), 73.9 (C-2 Gal), 71.9
(C-4 Gal), 71.8 (OCH2CH), 64.1 (C-6 Gal), 56.5 (OCH2CH), 52.9
(NHCH2CH2SO3), 42.3 (NHCH2), 38.1 (NHCH2CH2SO3), 34.5
(CH2CH2CH3), 33.8 and 33.6 [NHC(O)CH2CH2], 32.3, 32.2,
31.9, and 31.8 [(CH2)9], 29.5 (NHCH2CH2), 25.2 (CH2CH3), 15.9
(CH3). This assignment was confirmed by DEPT. Anal. Calc.
for C29H54N3O12NaS‚2H2O (727.84): C, 47.86; H, 8.03; N, 5.77;
S, 3.16. Found: C, 47.85; H, 8.01; N, 5.47; S, 3.78.
CD3OD): δ 173.3 [d, J C,P ) 5 Hz, C(O)NH], 103.4 (C-1â Gal),
75.0 (C-5 Gal), 72.7 (C-3 Gal), 71.5 and 71.4 (OCH2CHNH),
70.6 (C-2 Gal), 68.5 (C-4 Gal), 60.8 (C-6 Gal), 55.6 (CHNHP),
2
51.8 (d, J C,P ) 6 Hz, OCH3), 39.5 (NHCH2), 31.9 (CH2CH2-
CH3), 29.8, 29.7, 29.4, and 28.9 [(CH2)8], 26.9 (NHCH2CH2),
22.5 (CH2CH3), 13.8 (CH2CH3). 31P {1H} NMR (D2O): δ 8.90
(s). Anal. Calc. for C24H48N2O10NaP‚5H2O (668.69): C, 43.11;
H, 8.74; N, 4.19; P, 4.63. Found: C, 42.95; H, 8.04; N, 4.17; P,
4.14.
Syn th esis of II-Ga lAE[F 8C7][C11CO2Na ] (Sch em e 3).
Meth yl N-2-Hyd r oxyeth yl-N-[7-(F-octyl)h ep tyl]-11-ca r -
boxa m id ou n d eca n oa te, 16. Procedure C when applied to
1.37 g (7.14 mmol) of EDC, 3.94 g (7.19 mmol) of 7-(perfluo-
rooctyl)heptanoic acid, 1.34 g (7.16 mmol) of 2-benzyloxyethy-
lamine hydrochloride, 1 mL of Et3N (7.2 mmol), and 0.96 g
(7.12 mmol) of HOBt afforded after chromatography (49:1
CHCl3-MeOH) 4.8 g (95%) of N-(2-benzyloxy)-7-(F-octyl)-
heptanamide as an oil [Rf ) 0.76 (4:1 CHCl3-MeOH). 1H NMR
(CDCl3): δ 7.33 (m, 5H, Ph), 4.90 (bs, 1H, NH), 4.52 (s, 2H,
OCH2Ph), 3.54 (m, 2H, CH2OBn), 3.49 (t, J ) 5.4 Hz, 2H,
Syn th esis of II-Ga lSer [C14][OP (OMe)ONa ] (Sch em e
2).3-O-(2,3,4,6-Tet r a -O-a cet yl-â-D-ga la ct op yr a n osyl)-N-
(d ip h en oxyp h osp h or yl)-L-ser in e Tetr a d eca n a m id e, 14.
One hundred sixty microliters (0.76 mmol) of diphenyl chlo-
rophosphate in 2 mL of pyridine were added to 95 mg (0.15
mmol) of 9 in 2 mL of pyridine at 0 °C. The mixture was stirred
at 0 °C for 2 h. The reaction was poured into ice water, and
the precipitate was filtered, washed twice with ice water, and
dried. The residue was purified by chromatography (CHCl3 to
49:1 CHCl3-MeOH) giving 105 mg (80%) of 14 as a white solid.
NHCH2), 2.17 [t, J ) 7.1 Hz, 2H, CH2C(O)], 2.04 (tt, J H,F
)
18.8 Hz, J ) 8.2 Hz, 2H, CH2CF2), 1.70-1.60 [m, 4H, CH2-
CH2CF2 and CH2CH2C(O)], 1.38-1.30 [m, 4H, (CH2)2]. 13C
NMR (CDCl3): δ 173.0 [C(O)NH], 138.0 (C, Ph), 128.6 and
128.0 (CH, ortho and meta Ph), 127.9 (CH, para Ph), 73.3
(OCH2Ph), 69.1 (CH2OBn), 39.4 (CH2NH), 36.6 [CH2C(O)], 30.9
1
Rf ) 0.60 (95:5 CHCl3-MeOH). H NMR (CDCl3): δ 7.25 (m,
2
10H, Ph), 6.37 (t, J ) 5.6 Hz, 1H, NHCH2), 5.34 (bs, J ) 3.3
Hz, 1H, H-4 Gal), 5.12 (dd, J ) 7.8 Hz, J ) 10.4 Hz, 1H, H-2
Gal), 4.95 (dd, J ) 10.4 Hz, J ) 3.3 Hz, 1H, H-3 Gal), 4.40 (d,
J ) 7.8 Hz, 1H, H-1â Gal), 4.25-4.00 (m, 4H, H-5,6,6′ Gal
and OCHaHb), 3.87 (m, 1H, OCHaHb), 3.60 (dd, J ) 6.8, J )
9.7 Hz, 1H, OCH2CH), 3.11 (m, 2H, NHCH2), 2.34 (m, 1H,
NHP), 2.09, 2.02, 1.96, and 1.94 [4s, each 3HCH3C(O)], 1.40-
1.10 [m, 24H, (CH2)12], 0.84 (t, J ) 6.6 Hz, 3H, CH2CH3). 13C
NMR (CDCl3): δ 170.5, 170.2, 170.1, 169.9, and 169.4 [CH3C(O)
and C(O)NH], 150.6 (d, 2J C,P ) 8.6 Hz, C Ph), 130.0 (CH, meta
Ph), 125.4 (CH, para Ph), 120.4 (d, 3J C,P ) 4.2 Hz, C ortho Ph),
101.8 (C-1â Gal), 71.1 and 70.7 (C-3,5 Gal and OCH2CH), 68.8
(t, J C,F ) 22.2 Hz, CH2CF2), 28.9 [(CH2)2], 25.5 [CH2CH2C-
(O)], 20.1 (CH2CH2CF2). 19F NMR (CDCl3): identical to that
of 16, see below]. Four portions of 1.02 g of LiAlH4 were added
over 2 h to 4.15 g (6.1 mmol) of this amide in 50 mL of
anhydrous THF; the suspension was then stirred at room
temperature for 24 h. After hydrolysis with a 5:1 THF-H2O
solution, then 15% NaOH, and water, the mixture was filtered
on Celite, and the solvents were evaporated. The crude product
diluted with CHCl3 was washed with water until neutrality
and precipitated as its HCl salt. Filtration, washing with water
and then ether, and drying afforded 3.4 g of N-[2-(benzyloxy)-
ethyl]-7-(F-octyl)heptylamine (80%) [Rf ) 0.72 (4:1 CHCl3-

2200 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Faroux-Corlay et al.
1
MeOH). H NMR (CDCl3-CD3OD): δ 7.27 (m, 5H, Ph), 4.49
Meth od E. The procedure described for the preparation of the
compound A when applied to 0.30 g (0.37 mmol) of 16, 0.19 g
(0.48 mmol) of 1,2,3,4,6-penta-O-acetyl-D-galactopyranose, and
150 µL of BF3‚Et2O (1.22 mmol) gave after chromatography
(CHCl3) 90 mg (25%) of 17 as an oily compound. Rf ) 0.54 (3:1
(s, 2H, OCH2Ph), 3.74 (t, J ) 5.2 Hz, 2H, CH2OBn), 3.09 (m,
2H, CH2CH2OBn), 2.88 (t, J ) 7.1 Hz, 2H, CH2NH2+), 1.97
(tt, J H,F ) 18.8, J ) 8.2 Hz, 2H, CH2CF2), 1.69 (m, 2H, CH2-
CH2NH2+), 1.51 (m, 2H, CH2CH2CF2), 1.28 [m, 6H, (CH2)3].
13C NMR (CDCl3-CD3OD): δ 138.0 (C, Ph), 129.5 and 129.2
(CH, ortho and meta Ph), 129.0 (CH, para Ph), 74.5 (OCH2-
Ph), 65.8 (CH2OBn), 48.7 (CH2CH2OBn), 48.0 (CH2NH2+), 31.7
(t, J C,F ) 22.2 Hz, CH2CF2), 29.8 and 29.6 [(CH2)3], 27.3 (CH2-
CH2CH2NH2+), 26.7 (CH2CH2NH2+), 21.0 (CH2CH2CF2). 19F
NMR (CDCl3): identical to that of 16, see below]. PyBrop (1.4
g, 3.0 mmol) was added at 0 °C to a solution of 20 mL of CH2-
Cl2 containing 0.73 g (3.0 mmol) of dodecanedioic acid monom-
ethyl ester, 2.1 g (3.0 mmol) of this amine, and 1.4 mL (10
mmol) of Et3N. The reaction mixture was stirred at 0 °C for 1
h and then at room temperature for 1 day. Workup, as
described in procedure C, gave after chromatography (CHCl3)
2.1 g (78%) of methyl N-[2-(benzyloxy)ethyl]-N-[7-(F-octyl)-
1
CH2Cl2-Et2O). H NMR (CDCl3): δ 5.36 (bd, J ) 2.5 Hz, 1H,
H-4 Gal), 5.13 (dd, J ) 7.7 Hz, J ) 10.4 Hz, 1H, H-2 Gal),
4.99 (dd, J ) 10.3 Hz, J ) 3.4 Hz, 1H, H-3 Gal), 4.44 (d, J )
7.7 Hz, 1H, H-1â Gal), 4.11 (m, 2H, H-6,6′ Gal), 3.91 (m, 2H,
CH2OGal), 3.70 (m, 1H, H-5 Gal), 3.63 (s, 3H, OCH3), 3.48 (m,
2H, NCH2CH2O), 3.28 (m, 2H, CH2CH2CH2N), 2.28 [m, 4H,
CH2C(O)OMe and NC(O)CH2], 2.11, 2.01, and 1.94 [s,s,s, 12H,
CH3C(O)], 1.92 (tt, J H,F ) 18.8 Hz, J ) 8.2 Hz, 2H, CH2CF2),
1.58 [m, 8H, CH2CH2CF2, NC(O)CH2CH2, CH2CH2C(O)OMe,
and CH2CH2CH2N], 1.27 [m, 18H, (CH2)3 and (CH2)6]. 13C
NMR (CDCl3): δ 174.1, 173.1, and 173.0 [C(O)OMe and
C(O)N], 170.1, 170.0, 169.9, and 169.1 [CH3C(O)], 101.5 and
101.3 (C-1â Gal), 70.9 and 70.8 (C-3,5 Gal), 69.0 and 68.6 (C-2
Gal), 68.5 and 67.7 (GalOCH2), 67.2 and 67.1 (C-4 Gal), 61.2
(C-6 Gal), 51.2 (OCH3), 49.3 and 47.1 (NCH2CH2O), 49.0 and
45.8 [CH2CH2CH2N], 34.0 [CH2C(O)OMe], 33.2 and 33.0 [NC-
heptyl]-11-carboxamidoundecanoate as a colorless oil [Rf
)
1
0.30 (CHCl3). H NMR (CDCl3): δ 7.28 (m, 5H, Ph), 4.50 and
4.48 (2s, 2H, OCH2Ph), 3.64 (s, 3H, CH3O), 3.53 [m, 4H, CH2-
OBn and CH2CH2CH2N), 3.31 (t, J ) 7.6 Hz, 2H, CH2CH2-
OBn), 2.31 [m, 4H, CH2C(O)OMe and NC(O)CH2], 1.98 (tt, J H,F
) 18.8, J ) 8.2 Hz, 2 H, CH2CF2), 1.59 [m, 8H, CH2CH2CF2,
NC(O)CH2CH2, CH2CH2C(O)OMe and CH2CH2CH2N], 1.26 [m,
18H, (CH2)3 and (CH2)6]. 13C NMR (CDCl3): δ 173.4, 173.3,
and 173.1 [C(O)OMe and C(O)N], 138.4 and 137.9 (C, Ph),
128.5 and 128.4 (CH, ortho and meta Ph), 127.8 and 127.6 (CH,
para Ph), 73.5 and 73.2 (OCH2Ph), 69.0 and 68.4 (CH2OBn),
51.4 (OCH3), 49.4 and 46.3 (CH2CH2OBn), 47.6 and 46.2 (CH2-
CH2CH2N), 34.1 [CH2C(O)OMe], 33.2 and 33.1 [NC(O)CH2],
30.9 (t, J C,F ) 22.2 Hz, CH2CF2), 29.5, 29.3, 29.2, and 29.1
[(CH2)2 and (CH2)6], 27.6 (CH2CH2CH2N), 26.8 and 26.7
(CH2CH2CH2N), 25.5 [CH2CH2C(O)OMe], 25.0 [NC(O)CH2CH2],
20.1 (CH2CH2CF2). The doubling of some signals indicates the
presence of two conformational isomers due to the amide bond.
19F NMR (CDCl3): identical to that of 16, see below]. Hydrogen
was bubbled for 5 h at room temperature through a solution
of 1.97 g (2.20 mmol) of this methyl ester derivative in MeOH
(15 mL) and AcOH (2 mL) containing 0.2 g of Pd/C (10% w/w).
The mixture was diluted with CHCl3 and filtered through
Celite. The organic phase was washed with water, dried over
Na2SO4, filtered, and evaporated to dryness under vacuo
affording 1.5 g (85%) of 16 as a white solid. Rf ) 0.30 (CHCl3).
1H NMR (CDCl3): δ 3.74 (t, J ) 4.6 Hz, 2H, CH2OH), 3.64 (s,
3H, CH3O), 3.50 (t, J ) 4.7 Hz, 2H, CH2CH2OH), 3.26 (t, J )
7.5 Hz, 2H, CH2CH2CH2N), 2.32 [m, 4H, CH2C(O)OMe and
NC(O)CH2], 2.04 (tt, J H,F ) 18.8 Hz, J ) 8.2 Hz, 2 H, CH2-
CF2), 1.59 [m, 8H, CH2CH2CF2, NC(O)CH2CH2, CH2CH2C(O)-
OMe, and CH2CH2CH2N], 1.35-1.20 [m, 18H, (CH2)3 and
(CH2)6]. 13C NMR (CDCl3): δ 175.5, 174.4, and 173.7 [C(O)-
OMe and C(O)N], 62.9 and 60.6 (CH2OH), 51.5 (OCH3), 50.2
and 46.1 (CH2CH2OH), 49.8 and 46.1 (CH2CH2CH2N), 34.2
[CH2C(O)OMe], 33.4 and 33.2 [NC(O)CH2], 30.9 (t, 2J C,F ) 22.2
Hz, CH2CF2), 29.5, 29.3, 29.2, and 29.1 [(CH2)2 and (CH2)6],
27.6 (CH2CH2CH2N), 26.8 and 26.7 (CH2CH2N), 25.4 [CH2-
CH2C(O)OMe], 25.0 [NC(O)CH2CH2], 20.1 (CH2CH2CF2). The
doubling of some signals indicates the presence of two confor-
mational isomers due to the amide bond. 19F NMR (CDCl3): δ
-81.3 (3F, CF3), -115.0 (2F, CF2CH2), -122.4 [6F, (CF2)3],
-123.3 (2F, CF2 γ CH2), -124.1 (2F, CF2 δ CH2), -126.7 (2F,
CF2CF3).
2
(O)CH2], 30.9 (t, J C,F ) 22.2 Hz, CH2CF2), 29.5, 29.4, 29.3,
29.2, 29.1, 29.0, and 28.9 [(CH2)2 and (CH2)6], 27.5 (CH2CH2-
CH2N), 26.7 and 26.6 (CH2CH2CH2N), 25.4 [CH2CH2C(O)-
OMe], 24.9 [NC(O)CH2CH2], 20.6, 20.5, 20.4, 20.3, and 20.0
[CH3C(O)], 20.1 (CH2CH2CF2). The doubling of some signals
indicates the presence of conformational isomers due to the
1
1
amide bond. Correlated H/13C COSY experience confirmed H
and 13C signal attributions. 19F NMR (CDCl3): identical to that
of 16.
Meth yl N-[2-(â-D-ga la ctop yr a n osyloxy)eth yl]-N-[7-(F-
octyl)h ep tyl]-11-ca r boxa m id ou n d eca n oa te, 18. The pro-
cedure A when applied to 200 mg (0.17 mmol) of 17 gave 140
mg (80%) of 18. Rf ) 0.32 (9:1 CHCl3-MeOH). 1H NMR
(CDCl3): δ 5.10-3.00 (m, 16H, H-1-6 Gal, CH2CH2OGal,
OCH3, and CH2CH2CH2N), 2.21 [m, 4H, CH2C(O)OMe and NC-
(O)CH2], 1.97 (tt, J H,F ) 18.8 Hz, J ) 8.2 Hz, 2H, CH2CF2),
1.51 [m, 8H, CH2CH2CF2, NC(O)CH2CH2, CH2CH2C(O)OMe,
and CH2CH2CH2N], 1.19 [m, 18H, (CH2)3 and (CH2)6]. 13C NMR
(CD3OD-CDCl3): δ 174.7 and 174.3 [C(O)OMe and C(O)N],
103.6 (C-1â Gal), 74.9 (C-5 Gal), 73.5 (C-3 Gal), 71.0 (C-2 Gal),
68.7 (C-4 Gal), 66.9 (GalOCH2), 61.0 (C-6 Gal), 50.9 (OCH3),
48.9 (NCH2CH2O), 45.7 and 45.6 (CH2CH2CH2N), 33.6 [CH2C-
(O)OMe], 33.0 and 32.7 [NC(O)CH2], 30.5 (t, J C,F ) 22.2 Hz,
CH2CF2), 29.1, 28.9, 28.8, and 28.6 [(CH2)2 and (CH2)6], 27.0
(CH2CH2CH2N), 26.4 and 26.3 (CH2CH2CH2N), 25.4 [CH2-
CH2C(O)OMe], 25.2 and 24.6 [NC(O)CH2CH2], 19.8 (CH2CH2-
CF2). The doubling of some signals indicates the presence of
two conformational isomers due to the amide bond. 19F NMR
(CDCl3): identical to that of 16.
N-[2-(â-D-Ga la ct op yr a n osyloxy)et h yl]-N-[7-(F -oct yl)-
h ep tyl]-11-ca r boxa m id ou n d eca n oa te (Sod iu m Sa lt), II-
Ga lAE [F 8C7][C11CO2Na ]. One hundred milligrams (0.10
mmol) of 18 were stirred with 1 mL of 0.3 N NaOH 1:9 MeOH-
H2O, at room temperature for 5 days. The reaction mixture
was purified by chromatography (49:1 to 4:1 CHCl3-MeOH)
affording 90 mg (90%) of II-Ga lAE[F 8C7][C11CO2Na ] as a
white solid. Rf ) 0.28 (9:1 CHCl3-MeOH). [R]D ) +2.9° (c 0.35;
1
MeOH). SM (ESI) m/z ) 950.5 (M-Na)-. H NMR (CD3OD):
δ 4.22-3.28 (m, 13 H, H-1-6 Gal, CH2CH2OGal and CH2-
3
CH2CH2N), 2.41 [t, J ) 7.2 Hz, 2H, CH2C(O)ONa], 2.24 [t, J
) 7.3 Hz, 2H, NC(O)CH2], 2.10 (tt, J H,F ) 18.8 Hz, J ) 8.2 Hz,
2H, CH2CF2), 1.56 [m, 8H, CH2CH2CF2, NC(O)CH2CH2, CH2-
CH2C(O)OMe, and CH2CH2CH2N], 1.29 [m, 18H, (CH2)3 and
(CH2)6]. 13C NMR (CD3OD): δ 177.6 [C(O)O], 176.2 and 175.8
[C(O)N], 105.3 (C-1â Gal), 76.7 and 76.6 (C-5 Gal), 75.0 and
74.9 (C-3 Gal), 72.4 and 72.3 (C-2 Gal), 70.3 and 70.2 (C-4 Gal),
68.5 (GalOCH2), 62.5 and 62.4 (C-6 Gal), 50.2 and 47,0 (NCH2-
CH2O), 48.8 (CH2CH2CH2N), 34.9 and 34.8 [CH2C(O)O], 34.2
and 33.9 [NC(O)CH2], 31.6 (t, J C,F ) 22.2 Hz, CH2CF2), 30.5,
30.4, 30.3, 30.2, 30.1, 30.0, and 29.9 [(CH2)2 et (CH2)6], 28.3
(CH2CH2CH2N), 27.7 and 27.5 (CH2CH2CH2N), 26.7 and 26.6
[CH2CH2C(O)OMe], 26.0 and 25.9 [NC(O)CH2CH2], 21.2 (CH2-
CH2CF2). This assignment was confirmed by DEPT 13C NMR.
Meth yl N-[2-O-(2,3,4,6-tetr a -O-a cetyl-â-D-ga la ctop yr a -
n osyloxy)eth yl]-N-[7-(F-octyl)h eptyl]-11-car boxam idou n -
d eca n oa te, 17. Meth od D. A solution of 50 µL (0.26 mmol)
of TMSOTf in 5 mL of CHCl3 was added dropwise at -30 °C
to a solution of 0.6 g (0.747 mmol) of 16 and 0.45 g (0.91 mmol)
of GalOC(dNH)CCl3 in CHCl3 (20 mL) and containing 4 g of
molecular sieves (4 Å). The resulting mixture was stirred at
-30 °C for 6 h, then diluted with CHCl3, filtered through
Celite, and washed with a 8% NaHCO3 and then with water
until neutrality. The organic phase was dried over Na2SO4,
filtered, and evaporated in vacuo. Purification by silica gel
chromatography (CHCl3) gave 0.24 g (35%) of 17 as an oil.

Anionic Analogues of Galactosylceramide
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2201
The doubling of some signals indicates the presence of two
conformational isomers due to the amide bond. 19F NMR
(CDCl3): identical to that of 16. Anal. Calc. for C35H49F17NO9-
Na (973.736): C, 43.10; H, 5.70; N, 1.44. Found: C, 43.16; H,
5.68; N, 1.54.
recombinant gp120 (rgp120, HIV-1-IIIB isolate) into the sub-
phase. The maximal increase in surface pressure (∆πmax) was
determined after the equilibrium was reached (60-120 min,
depending on the extent of pressure variation). All experiments
were carried out in triplicate, and each experiment was
performed with a freshly prepared film.
Syn th esis of Mon ola c a n d BisLa c (Sch em e 4). Syn th e-
sis of N-{2-[2,3-(Ditetr adecan am ido)pr opion am ido]eth yl}-
la ctobion a m id e, Mon ola c. One hundred milligrams (0.17
mmol) of 2048 and 65 mg (0.17 mmol) of lactobionic acid 19 in
6 mL of MeOH were refluxed for 24 h. After filtration, the
precipate was washed twice with MeOH, dried, and purified
by chromatography (90:10:1 to 70:30:1 CHCl3-MeOH-H2O).
One hundred milligrams (60%) of m on oLa c as a white solid
were obtained. Rf ) 0.70 (70:30:3 CHCl3-MeOH-H2O). SM
Biologica l Section
Sa m p le P r ep a r a tion . All the galactolipids were
assayed in biological tests as solution in Dubelcco
modified phosphate buffered saline (DPBS). II-Ga lAE-
[F 8C7][C11CO2Na ] was also tested formulated as PL/
CH/II-Ga lAE[F 8C7][C11CO2Na ] (2:1:0.15 molar ratio;
PL ) soya phospholipon; CH ) cholesterol) liposomes
(higher amounts of galactolipid led to liposomes that
aggregate very rapidly). These liposomes were prepared
according to the procedure described in ref 23. Average
particle sizes and size distributions were measured by
laser light scattering on a Coulter N4MD submicron
particle analyzer after preparation of the liposomes and
then periodically during storage at room temperature
(the particle sizes reported in Table 1 correspond to a
storage period of one week). The formulation was tested
within 1 week after preparation. Its phospholipid and
galactolipid concentration was determined as described
elsewhere.23 Galactose determination in the preparation
was also done by 19F NMR using CF3CH2OH as internal
standard. These determinations were consistent with
the theoritical values.
1
(ESI) m/z ) 929.8 (M + Na)+. H NMR (CD3OD-DMSO-d6): δ
4.30 (m, 2H, H-2 and H-1â Gal), 3.80-3.10 [m, 14H, H-1,5-9
and H-2-6 Gal], 3.13 (m, 4H, H-3,4), 2.10 [m, 4H, CH2(CH2)11-
CH3], 1.60-1.45 [m, 4H, CH2CH2(CH2)10CH3], 1.23 [m, 40H,
(CH2)10CH3], 0.85 (t, J ) 6.2 Hz, 6H, CH3). 13C NMR (DMSO-
d6): δ 172.9, 172.6, 172.3, and 170.0 [C(O)NH], 104.5 (C-1â
Gal), 82.8 (C-7), 75.6 (C-5 Gal), 73.2 (C-3 Gal), 71.1 (C-2 Gal),
70.3 (C-4 Gal), 72.1, 71.3, and 68.2 (C-5,6,8), 62.3 (C-9), 60.6
(C-6 Gal), 53.1 (C-2), 40.9, 38.1, and 37.7 (C-1,3,4), 35.3 [NHC-
(O)CH2(CH2)11CH3], 31.2 (CH2CH2CH3), 29.0, 28.9, 28.8, and
28.7 [(CH2)8], 25.2 and 25.0 [NHC(O)CH2CH2(CH2)10CH3], 22.0
(CH2CH3), 13.8 (CH3). Anal. Calc. for C45H86N4O14‚3/2H2O
(934.22): C, 57.86; H, 9.44; N, 6.00. Found: C, 57.83; H, 9.52;
N, 6.14.
Syn th esis of N-{[2-(La ctobion a m id o)eth yl]-N-{2-[2,3-
(d it e t r a d e ca n a m id o)p r op ion a m id o]e t h yl}la ct ob ion -
a m id e, Bisla c. Fifty milligrams (0.076 mmol) of 2148 and 60
mg (0.16 mmol) of lactobionic acid in 6 mL of MeOH were
refluxed for 24 h. After evaporation, the residue was chro-
matographed (40:25:4 CHCl3-MeOH-H2O), and the product
obtained was washed with Et2O and lyophilized giving 50 mg
(50%) of bisLa c as a white solid. Rf ) 0.70 (8:5:1 CHCl3-
MeOH-H2O). [R]D ) +7.6° (c 0.26; MeOH). SM (ESI) m/z )
1353.5 (M + Na)+. 1H NMR (CD3OD): δ 4.60 (m, 1H, H-2),
4.50 (d, J ) 7.0 Hz, 2H, H-1â Gal), 4,38 (m, 2H, H-4 Gal), 4.26
(m, 2H, H-3 Gal), 4.00-4.85 (m, 4H, H-2,5 Gal), 3.81-3.15 (m,
24 H, H-1,3,6-18 and H-5,6,6′ Gal), 2.61 (m, 6H, H-4,5,12),
2.19 [m, 4H, CH2(CH2)11CH3], 1.65-1.50 [m, 4H, CH2CH2-
(CH2)10CH3], 1.28 [m, 40 H, (CH2)10CH3], 0.89 (t, J ) 6.2 Hz,
6 H, CH3). 13C NMR (CDCl3-CD3OD): δ 177.0, 176.2, 174.9,
174.8, and 172.0 [C(O)NH], 105.5 (C-1â Gal), 83.5 and 83.4
(C-7,14), 77.0 (C-5 Gal), 74.6 (C-3 Gal), 72.9 (C-2 Gal), 72.6
(C-4 Gal), 73.9, 72.3 and 70.2 (C-8-10,15-17), 63.6 (C-11,18),
62.6 (C-6 Gal), 54.8 (C-4,5,12), 54.2 (C-2), 42.0 (C-1), 39.1 and
38.6 (C-3,6,13), 37.1 [NHC(O)CH2(CH2)11CH3], 32.9 (CH2CH2-
CH3), 30.8, 30.7, 30.6, 30.5, and 30.4 [(CH2)8], 26.9 and 26.7
[NHC(O)CH2CH2(CH2)10CH3], 23.6 (CH2CH3), 14.4 (CH3). Anal.
Calc. for C61H114N6O25‚5H2O (1421.67): C, 51.54; H, 8.79; N,
5.91. Found: C, 51.57; H, 8.76; N, 5.67.
Su r fa ce-P r essu r e Mea su r em en ts: gp 120 Bin d in g to
Glycolip id Mon ola yer s a t th e Air -Wa ter In ter fa ce. The
surface pressure was measured with the µT trough S micro-
tensiometer (Kibron Inc., Helsinki, Finland), and the data were
collected and analyzed by the FilmWare 2.3 software. The
apparatus was maintained at a temperature of 25 °C through-
out the experiments. Pure lipids were prepared at a concentra-
tion of 1 mg/mL in 11:5:4 (v:v:v) hexane:chloroform:methanol,
as previously described.49 The lipid was carefully spread onto
the surface of the trough filled with 800 µL of pure water
obtained by filtration through a milli-Q water purification
system (Millipore, Saint-Quentin, France). The amount of lipid
was calculated in order to form a stable monomolecular film
with a surface pressure of 10 mN/m, as previously determined
by compression isotherms experiments. At least 5 min were
allowed for the spreading solvent to evaporate and for the lipid
molecules to distribute themselves throughout the film at the
air-water interface. The stability of the monolayer was then
checked by recording the pressure variations ∆π for 10 min
(the monolayer can be considered as stable when ∆π is lower
than 0.2 mN/m over this period), before addition of 10 nM
Cells a n d Vir u ses. CEM-SS and MT4 cells were
cultured in RPMI medium supplemented with 10% fetal
calf serum. HIV-1 LAI and HIV IIIB were produced in
CEM-SS cells as previously reported.37
In h ib it ion of H IV-1 In fect ion . CE M-SS Cells.
CEM-SS cells were infected with HIV-1 LAI, as de-
scribed elsewhere.37 Five days later, the production of
HIV-1 was evaluated by measuring the reverse tran-
scriptase activity (RT) which expresses the presence of
the virus in the culture supernatant. The tested com-
pounds or liposomal formulation were added to the cell
cultures after viral adsorption. RT inhibition percent
was measured in comparison with the nontreated cells.
The galactoside 50% inhibitory concentration of virus
replication values (IC50) reported in Table 1 were
determined from the computer-generated median plot
of the dose-effect data. AZT was used as a positive
control (IC50 ) 4 nM). The effects of the galactosides on
CEM-SS cell proliferation and viability were measured
on noninfected cells using a colorimetric assay (MTT test
which is based on the capacity of living cells to reduce
MTT to formazan.)38 with various concentration of the
tested product as described elsewhere. The 50% cyto-
toxicity concentration (CC50) at which OD540 was re-
duced by one-half was calculated using the program
mentioned above. Three measurements were carried out
for each concentration of each compound on infected and
noninfected cells. The entire experiment was repeated
twice.
MT4 Cells. The anti-HIV-1 test based on MT4
infected cells was performed as described elsewhere.38,39
Briefly, the replication of HIV-1 (IIIB) was followed by
the cytopathogenic effect induced by the virus. MT4 cells
were infected with a virus dose allowing 5 days later
the death of 90% of the cells. The tested compounds
were added in the cell culture medium after viral

2202 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Faroux-Corlay et al.
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adsorption. Cell viability was measured by the colori-
metric MTT test. The drug IC50 values were determined
from the curves of the protection percentage ∆ against
drug concentration. The effects of the galactosides on
MT4 cell proliferation and viability were also measured
on noninfected cells using the colorimetric MTT test
after 5 days of incubation at 37 °C with various
concentrations of the tested product. All the measure-
ments were carried out in triplicate for each concentra-
tion of each compound, and the entire experiment was
repeated twice.
Ack n ow led gm en t. We wish to thank the ANRS and
SIDACTION for financial support.
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