Synthesis, gp120 binding and anti-HIV activity of fatty acid esters of 1,1-linked disaccharides

Article abstract of DOI:10.1016/j.bmc.2011.06.078

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of β-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-

Full text of DOI:10.1016/j.bmc.2011.06.078

Bioorganic & Medicinal Chemistry 19 (2011) 4803–4811
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, gp120 binding and anti-HIV activity of fatty acid esters
of 1,1-linked disaccharides
Stewart Bachan a, Jacques Fantini b, , Anjali Joshi c,d, Himanshu Garg c,d, David R. Mootoo a,
⇑
⇑
a Department of Chemistry, Hunter College, 695 Park Avenue, New York, NY 10021, USA
b Universite Paul Cezanne, Laboratoire de Biochimie et Physicochimie des Membranes Biologiques, INRA-UMR 1111, Faculte des Sciences et Techniques de Saint-Jerome,
Marseille Cedex 20 13397, France
c Center of Excellence for Infectious Disease, Department of Biomedical Sciences, Texas Tech University Health Sciences Center, El Paso, TX 79905, USA
d Department of Pediatrics, Texas Tech University Health Sciences Center, El Paso, TX,79905, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of b-galactosylceramide
(GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their
binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man
disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity
Received 22 April 2011
Revised 22 June 2011
Accepted 27 June 2011
Available online 1 July 2011
in single round infection assays with the TZM-bl cell line. IC50’s were in the 50
lM range with no toxicity
to the cells at concentrations up to 200 M. These compounds appear to inhibit virus entry at early steps
l
Keywords:
Galactosylceramide
Glycolipid
Trehalose
Fatty acid
Antiviral
in viral infection since they were inactive if added post viral entry. Although these compounds were
found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their
anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with
their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined
with the easy accessibility to structurally diverse analogues make these molecules attractive leads for
new topical anti-viral agents.
HIV
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
OH
OH
NHCOR
O
While the fusion process mediated by gp41 per se remains well
studied and targeted, the events preceding gp41 mediated fusion
including gp120 binding to receptor and coreceptor present potential
new targets for drug development.1,2 Besides the well characterized
receptor and coreceptor for HIV severalcofactors are involved in facil-
itating gp120 binding to cells.3,4 Membrane glycosphingolipids (GSL)
including GalCer 1 constitute one well-known group of cofactors5,6
(Fig. 1). In this vein, GSL analogues have attracted interest as potential
inhibitors of HIV infectivity.2,7–10 We have previously synthesized
and tested the gp120 binding and antiviral activity of GalCer ana-
logues with simple ceramide substitutes, against both X4 and R5 tro-
pic virus.11 Some of the lead compounds, represented by 2, bound
gp120 similar to GalCer and showed significant activity against HIV
envelope glycoprotein (Env)-mediated fusion. The compounds act,
presumably as GalCer mimetics, at an early step in HIV Env binding
that precedes CD4 binding as determined by kinetic and temperature
arrested state experiments. These GalCer analogues also showed
inhibitory activity against vesicular stomatitis virus (VSV) pseudo-
type virus that was similar to other antiviral compounds like
cyanovirin and suramin.12,13 The further development of these
(CH2)12CH3
HO
O
OH
OH
1
R = CH(OH)(CH2)21CH3
OH
OH
R
O
(CH2)10CH3
O
HO
OH
2a R = H
2b R = CH3(CH2)13
Figure 1. GalCer and analogues.
compounds holds promise towards new therapies that can be used
both as prophylaxis and post exposure treatment as the compounds
act at an early step in the HIV cycle. Towards this end, with the aim of
identifying new GalCer mimetics with anti-HIV activity, we de-
signed a wider set of mono- and di-saccharide-derived lipids and
evaluated their binding to the V3 loop peptide of gp120 and their
HIV inhibitory activity. These results are discussed herein.
Our analog design is based on existing SAR data that suggests the
Gal and hydrophobic residues of GalCer account for a significant part
⇑
Corresponding authors. Tel.: +1 212 772 4356; fax: +1 212 772 5332 (D.R.M.).
E-mail address: dmootoo@hunter.cuny.edu (D.R. Mootoo).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.078

4804
S. Bachan et al. / Bioorg. Med. Chem. 19 (2011) 4803–4811
of the overall binding to gp120,14 and the central polar segment
that connects the two regions acts as a scaffold that controls
the spatial positioning of the sugar and hydrophobic regions.15,16
That our first generation analogues (e.g., 2), in which the cera-
mide moiety in GalCer was replaced with simple hydrocarbon
residues, showed binding properties comparable to GalCer is con-
sistent with this model. To get insight into the conformational
and spatial requirements of the ceramide residue in GalCer, for
the purpose of developing more effective GalCer antagonists, we
argued that a monosaccharide could be used as a replacement
for the polar lipid head of GalCer. Indeed, it has been suggested
that the GlcNHAc subunit in Galb1-4GlcNAcb1 is structurally
analogous to this segment of GalCer and may mimic its binding
to gp120.17 In a similar vein, we argued that b-Gal disaccharides
such as 3, 4 and 5, with one or two fatty acid esters in the man-
nose segment, may also act as GalCer mimetics (Fig. 2). While the
gp120 binding of disaccharide glycolipids have been previously
examined, these structures were conceived as mimetics of disac-
charide GSL’s, as opposed to the monosaccharide GalCer.10,16 We
opted for a 1,1-linked disaccharide template because of the
well-defined conformational bias in the intersaccharide torsions
of such frameworks,18 and a mannose subunit, because of syn-
thetic considerations relating to stereoselective glycoside bond
construction and alcohol group differentiation. The b-Glu-disac-
2. Results and discussion
2.1. Binding to V3 loop peptide of gp120
The binding of 3–8 to a synthetic V3 loop peptide, RIQRGPG-
RAFVTIGK, corresponding to the R15K peptide, from gp120 of
HIV-1 IIIb isolate was evaluated. We and others have previously
used the R15K peptide as a model for the glycolipid binding do-
main of gp120.11,19 The change in surface pressure (
Dp) at the
air-water interface of individual glycolipid monolayers of the test
compounds, on exposure to an aqueous solution of the R15K pep-
tide (10 lM) was measured. Increase in surface pressure is asso-
ciated with integration of the peptide into the glycolipid
monolayer. The critical pressure of insertion (CPI), calculated by
extrapolation for a null increase in surface pressure is often used
as a measure of binding affinity. All the compounds tested inter-
acted with the R15K V3 peptide with CPIs in the range of 18–
49 mN/m (Fig. 3). These values were comparable to that mea-
sured for GalCer (CPI = 22–25 mN/m).11 The likeness of 3–6 to,
and the similarity of their CPIs to GalCer, are consistent with
our hypothesis that these disaccharides mimic GalCer in their
binding to the V3 peptide, but in the absence of additional data
this conclusion is very speculative. It is also noteworthy that
the monosaccharides 7 and 8 appear to bind appreciably more
strongly to the V3 peptide than the disaccharides. However, give
the structural disparity between the mono- and di-saccharide
frameworks, the relative activity of the two groups of analogues
does not provide any definitive SAR insight because the two sets
of analogues may be binding to different domains on the peptide.
Nevertheless given the possibility that 3–8 interact with the Gal-
Cer domain on the V3 peptide, and earlier findings that such com-
pounds may inhibit the attachment of HIV to host cells, the next
step was to examine their anti-HIV.
charide 6, the
a-Gal-disaccharide 9, and the a-Man monosaccha-
rides 7 and 8 were designed to probe the recognition specificity
for the b-Gal motif. The Man-monosaccharide 7 was also of inter-
est because the relative orientation of the 6-O-palmitoyl ester and
the 2-OH group was similar to the positioning of the 4-OH and
the anomeric lipid chain in Gal-monosaccharide 2a, the reference
compound from the earlier study.
HO
OR3
2.2. Anti HIV activity
OR2
HO
HO
O
OH
OR1
O
The anti-HIV activity of the synthetic glycolipids was evaluated
in a virus infection assay using the TZM-bl indicator cell line infec-
tion with CXCR4 tropic HIV (NL-Lai).20 The b-Gal-disaccharide 4
with the O-3 ester on the mannose residue, the b-Glu-disaccharide
O
OH
3 R1 = C(O)(CH2)14CH3; R2 = R3 = H
4 R3 = C(O)(CH2)14CH3; R1 = R2 = H
5 R1 = R2 = C(O)(CH2)14CH3; R3 =H
HO
OH
18
OH
O
3
OH
OR
O
16
HO
HO
4
O
14
OH
5
6 R = C(O)(CH2)14CH3
12
6
HO
OH
10
8
7
8
OR2
O
OR1
6
CH3O
7 R1 = C(O)(CH2)14CH3; R2 = H
8 R1 = R2 = C(O)(CH2)14CH3
4
2
HO
OH
0
O
OH
OH
5
10
15
20
25
30
35
40
45
50
HO
Initial pressure
π
(mN/m)
HO
O
O
Figure 3. Binding of glycolipid analogues to the V3 loop peptide. Each analog was
spread on the surface of a water subphase at various values of the initial surface
pressure. A stable monomolecular film was formed after evaporation of the solvent.
The synthetic V3 peptide was then added in the subphase at a final concentration of
OH
OR
9 R = C(O)(CH2)14CH3
10 lM. The maximal surface pressure increase (DIImax) was determined after 2–3 h
of incubation, that is, when equilibrium was reached. The data were analyzed with
Figure 2. Proposed GalCer mimetics.
the Filmware 2.5 program (Kibron, Inc.)

S. Bachan et al. / Bioorg. Med. Chem. 19 (2011) 4803–4811
4805
Table 1
IC50’s of glycolipid analogues
6 with the O-6 ester and the
ter, showed appreciable activity with IC50’s of 76, 53 and 48
respectively, which was comparable to the activity of the previ-
ously assayed monosaccharide 2a (44 M, Fig. 4, Table 1). The b-
a
-Gal-disaccharide 9 with the O-6 es-
lM,
Compound
IC50 (lM)
l
2a
3
43.51 1.63
>100
Gal-disaccharide 3 with the ester at O-6 of the mannose ring, its
4
5
6
7
75.99 6.16
>100
52.51 1.93
>100
di-O-ester 5, and the monosaccharides 7 and 8 showed no signifi-
cant activity up to 100 lM. Thus the data from the binding and
anti-HIV assays indicate that V3 loop binding by itself is not a reli-
able predictor of anti-HIV activity. This situation may arise because
the binding assay measures overall binding but anti-HIV activity
requires binding to a specific domain of the peptide. Therefore,
the binding data does not necessarily correlate with anti-HIV activ-
ity. Within the context of GalCer mimicry, it may be contended
that the disaccharides interact with the GalCer binding domain
on the V3 peptide but the monosaccharides bind other domains,
so that the relative binding affinity of the mono- and di-saccharide
analogues is not connected to their anti-HIV activity. However, this
argument does not explain why the disaccharide 3 (which is very
similar in structure to 4 and 6), and to a lesser extent 5, are active
in the binding assay but not in the anti-HIV assay. One explanation
for this apparent anomaly is that glycolipid presentation in the two
assays are different,7 so that structural factors that control the
morphology of the active glycolipid species conflict with those that
affect glycolipid-peptide recognition, thereby skewing the data. Of
course, an alternative and arguably more obvious, explanation for
the contrasting behavior of individual test compounds in the two
assays is that the mechanism for anti-viral activity is not at all con-
nected to V3 loop binding. Notwithstanding the mechanism for
anti-viral activity, the data from the anti-HIV assay suggests that
a disaccharide framework (vs monosaccharide) and one ester resi-
due (vs two), are important for anti-viral activity. That the b-galac-
8
9
GML
>100
48.35 1.74
>100
IC50’s were determined from the data in Figure 4 for
compounds with IC50’s of less than 100 mM.
shown to prevent simian immunodeficiency virus (SIV) in a rhesus
macaque model.22 The low activity observed for GML in our cellu-
lar assay suggests that the activity observed for GML in the animal
model does not result from inhibition of HIV entry. Indeed the re-
sults in the animal studies have been linked to immunological ef-
fects. That the three disaccharide analogues 4, 6 and 9 were
active but the monosaccharide esters and GML were not, suggests
a specificity for a disaccharide scaffold with a single mono fatty
acid ester.
2.3. Cytotoxicity assays
Cytotoxicity was determined for test compounds against TZM-
bl cells over a 24 h incubation period using a MTS dye reduction as-
say.23 None of the compounds were toxic up to a 200
lM dose.
2.4. Pre and post infection assays
toside 4, the b-glucoside 6 and the a-galactoside 9 were active but
the b-galactoside 3 was not, also suggests that the location of the
ester is important, and that the optimal position for the ester
may vary with the structure of disaccharide framework.
We also examined the activity of glycerol monolaurate (GML, 1-
O-lauryl-glycerol) in the TZM-bl/HIV assay. GML shows low micro-
molar activity against two other envelope viruses herpes-simplex
virus 2 and bacteriophage /6.21 Since our active glycolipids and
GML are both fatty acid esters, an obvious question was whether
the glycolipids and GML inhibit HIV infectivity by a similar mech-
anism. However, GML was not active at concentrations of up to
To determine whether the compounds affected the viral entry
process or events post viral entry, simple time of addition experi-
ments were conducted.11 TZM-bl cells were exposed to the virus
in the presence of the compounds (100
were added 4 h after infection with the virus. We also used
T20,24 an inhibitor of gp41 mediated fusion and AMD 3100,25
lM) or the compounds
a
CXCR4 antagonist that inhibits HIV Env mediated fusion and viral
entry as controls in these experiments. All active compounds
inhibited virus infection if added at the same time as the virus
(Fig. 5). However, addition of the compounds 4 h post-infection
failed to show significant inhibition. The same was true for the
100 lM. Parenthetically, it should be noted that GML has been
140
Pre Infection
120
Post Infection
100
80
60
40
20
0
2a
4
6
9
AMD
T20
Figure 4. Anti HIV activity of glycolipid analogues. TZM-bl cells were infected with
X4 HIV NL-Lai in the presence of serial dilutions of the compounds. Infectivity was
determined 24 h later by measuring luciferase activity and normalized to control
wells infected with virus in the absence of drug. Pooled data from three
independent experiments was used to fit curves using Sigma Plot software.
Figure 5. Glycolipid analogues show activity pre-infection only. TZM-bl cells were
infected with NL-Lai virus. Compounds 100 M were added either prior to infection
(pre infection) or 4 h post infection. Infectivity was determined 24 h later by
measuring luciferase activity. AMD and T20 controls are used at 2 mM. Data are
mean SD of triplicate observations.
l

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S. Bachan et al. / Bioorg. Med. Chem. 19 (2011) 4803–4811
entry inhibitors T20 and AMD3100. The absence of activity when
the compounds were added 4 h post-infection also suggest that
they did not have a direct effect on cell viability or function and
hence the inhibition is at the step of initial virus cell interaction.
This mode of action, which appears to be similar to 2a11 contrasts
with the one suggested for a 3-O-fatty acid ester of trehalose
ic position of the Gal and Glu rings was deduced from vicinal JH,H val-
ues (13 /b: J1,2 (Gal) = ca. 3.5:8.0 Hz; 14 /b: J1,2(Glu) = 3.8:8.0 Hz).
The -configuration at C-1 for the Man subunit was assigned on
a
a
a
the basis of JC,H values (13a/b: J1,1 (Man) = 175:172; 14a/b: J1,1
(Man) = 175:172 Hz.34
The separated anomers from the glycosidation reactions and the
known mannoside 2135 were next transformed to the target com-
pounds 3–9 through established alcohol protecting group se-
quences (Scheme 2). These reactions were not optimized in all
cases. Representative transformations are described. Acetate
hydrolysis on 13b followed by perbenzylation of the resulting tet-
raol provided the tetra-O-benzyl ether derivative. The 4,6-O-iso-
propylidene in this product was selectively cleaved under acidic
conditions to provide diol 15. DCC mediated esterification of 15
with 1 equiv of palmitic acid provided monoester 16 as the major
product, and with excess acid, the diester 17. Individual hydrogen-
olysis of 16 and 17 afforded the mono- and di-O-acylated disaccha-
ride targets 3 and 5. For the 3-O-ester 4, 15 was first transformed to
diol 18 via standard protecting group transformations. Selective
acylation36 of the equatorial alcohol in 18 followed by hydrogenol-
(a,a-1,1-Glu-Glu) that is structurally similar to 4. The trehalose
derivative is believed to prevent influenza proliferation by inhibit-
ing viral replication.26,27
2.5. Critical micelle concentration (CMC)
The CMC’s of selected analogues were measured in order to
determine the physical state of the glycolipids at the concentrations
used in the cellular assays. Values were obtained by tensiometer
measurements of solutions of glycolipids in water, as previously de-
scribed.11 The CMC is taken as the concentration of glycolipid that
does not induce any further decrease in surface tension. This data
was obtained in triplicate: 3 (9.8, 10 and 10.5
l
M); 4 (12, 12 and
M). Thus
12.5 M); 6 (10, 10.5, and 12 M); 7 (13, 13.5 and 13.7 l
l
l
at their active concentrations analogues 4 and 6, exist as micelles,
but this does not preclude the possibility that the active species is
monomeric. The CMC’s obtained for 3, 4 and 6 were in the range ob-
served for mono-fatty acid esters of related 1,1-linked disaccha-
rides.28,29 The diester 8 was difficult to study; a regular decrease in
surface tension from 72.8 mN/m (pure water) to 0 mN/m (glycolipid
ysis of the product provided 4. The b,
1,1-Gal-Man disaccharide 9 were prepared from 14b and 13
a-1,1-Glu-Man 6 and the a,a-
a,
respectively, following the reaction sequence used for transforma-
tion of 13b to 3. For the monosaccharide esters 7 and 8, 21 was
first converted to the 2,3-O-isopropylidene-4-O-palmitoyl- or 2,3-
O-isopropylidene-4,6-di-O-palmitoyl-derivatives following proce-
dures used for the analogous disaccharides 16 and 17. Acetal
hydrolysis in these materials led to 7 and 8, respectively.
film collapse) in the concentration range of 0.25–5
lM was ob-
served. One interpretation of this result is that 8 forms precipitates
rather than micelles in water above a concentration of 0.5 lM.
3. Summary
2.6. Synthesis
1,1-Linked Gal-Man and Glu-Man disaccharides with a long
chain fatty acid ester on the mannose residue were found to inhibit
The synthesis of the disaccharide lipids 3–6 started with assem-
bly of the 1,1-linked precursors 13b and 14b (Scheme 1). Initial gly-
cosidation reactions using 2-O-acetylated Gal donors containing
different alcohol protecting and glycosyl activating groups with
Man acceptors gave low yields of disaccharide products. A solution
to this problem, albeit not a stereoselective one, was found in the
methyl triflate promoted glycosidation of thioglycoside Man donor
12 with Gal and Glu acceptors 1030 and 11,31 respectively. The phen-
ylthio glycoside 12 was prepared from the tetra-O-acetate deriva-
tive following the procedure used for the corresponding ethylthio
glycoside.32,33 The reactions of 12 with 10 and 11 afforded in each
HIV-infectivity in the 50
lM range, with no cytotoxicity at concen-
trations up to 200 M. These compounds appear to inhibit virus en-
l
try at early steps in viral infection since they were inactive if added
post viral entry. Their structural likeness to GalCer and the observa-
tion that they bind the V3 loop peptide of HIV-1 and inhibit HIV
infectivity fits with the notion their anti-HIV activity results from
their ability to bind the GalCer domain of the V3 peptide. However,
in the absence of other data this conclusion is very tentative. That
closely related disaccharide analogues showed similar binding to
the V3 peptide but did not inhibit HIV infectivity suggests that fac-
tors other than V3 loop binding may contribute to anti-HIV activity,
or may point to completely different mechanisms. In this context it
is noteworthy that the viruscidal activity of fatty acid derivatives of
related disaccharide structures and other small molecule amphi-
philic lipids has been attributed to surfactant-like, membrane-per-
turbing properties.21,37–41 Notwithstanding the mechanistic basis
for their anti-HIV activity, the preliminary structure–activity data
obtained from this investigation suggests that a disaccharide-type
framework and the position of acylation are important for activity.
Although the trehalose-type fatty acid esters reported here have
similar anti-HIV potency to other small molecule lipids, their low
cytotoxicity and other reports that trehalose-type disaccharide es-
ters have broader viruscidal activity, combined with the easy acces-
sibility to structurally diverse analogues, make them attractive leads
for drug development.42–44
case an approximately 40% yield of a 1:1 mixture of anomers,
a
:b
disaccharides 13a:b and 14 a:b. The stereochemistry at the anomer-
O
X
O
OAc
O
O
Y
O
OH
O
AcO
OAc
PhS
10 X = OAc, Y = H
11 X = H, Y = OAc
12
MeOTf, CH2Cl2
DTBMP, MS 4A
18h
O
O
O
X
O
OAc
O
O
4. Experimental
Y
AcO
O
OAc
4.1. Surface pressure measurements
The surface pressure of glycolipid monolayers was measured
13α:13β X = OAc, Y = H (41%; α:β = ca 1: 1)
14α:14β X = H, Y = OAc (41%; α:β = ca 1: 1)
with a fully automated microtensiometer (lTROUGH SX; Kibron,
Scheme 1. Glycoside synthesis.

S. Bachan et al. / Bioorg. Med. Chem. 19 (2011) 4803–4811
4807
1. NaOMe, MeOH
2. NaH, BnBr,
nBu4NI, THF
O
1. p-TsOH
or CSA
MeOH
O
O
O
DCC, DMAP
15H31COOH,
PhH
O
O
OR2
OR1
BnO
BnO
C
O
OH
OBn
O
BnO
BnO
O
3 (37%
OBn
O
3. p-TsOH, MeOH
OH
5 (28%)
13β
2. H2, Pd/C,
MeOH
OBn
48%
OBn
15
16 R1 = C(O)(CH2)14CH3; R2 = H (86%)
17 R1 = R2 = C(O)(CH2)14CH3 (70%)
1. NaH, BnBr,
nBu4NI, THF
2. CSA, MeOH
75%
HO
OC(O)(CH2)10CH3
DCC, DMAP
C15H31COOH,
PhH
HO
H2, Pd/C,
MeOH
OBn
OBn
BnO
BnO
OH
O
OBn
O
OBn
4 (79%)
BnO
O
OBn
O
O
80%
OBn
OBn
O
BnO
19
OBn
1. p-TsOH,
MeOH
2. DCC, DMAP
15H31COOH,
PhH
18
O
O
O
O
O
C
OH
7 (8%)
O
as for 15
O
as for 3
OBn
O
O
8 (70%)
6 (28%)
OH
O
14β
BnO
BnO
75%
3. p-TsOH,
MeOH
CH3O
OBn
20
21
Scheme 2. Esterification and protecting group sequences.
Inc., Helsinki, Finland). The apparatus allowed the real-time
recording of the kinetics of interaction of a soluble ligand with
the monomolecular film, using a set of specially designed Teflon
troughs. All experiments were carried out in a controlled atmo-
sphere at 20 1 °C. Monomolecular films of test compounds were
4.3. Cytotoxicity assays
Cytotoxicity of compounds was determined by a 3-(4,5-dimeth-
ylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) dye reduction assay. Briefly TZM-bl cells
were seeded in 96-well plates and incubated overnight. Subse-
spread on pure water subphases (volume of 800 lL) from hexane–
chloroform–ethanol solution as described previously.45 After
spreading of the film, 5 min was allowed for solvent evaporation.
To measure the interaction of the V3 peptide with the individual
monolayers, the peptide was injected in the subphase with a
quent day serial dilutions of compounds starting at 200 lM were
added to the cells. The cells were incubated for another 24 h before
addition of the MTS reagent (Cell titer Aqueos one solution, Prome-
ga). Reduction of the MTS dye was determined by measuring OD at
490 nM and normalized to cells incubated with media only.
10
were recorded until reaching a stable value. The experiment was
repeated at different values of the initial surface pressure ( i) of
lL Hamilton syringe, and pressure increases (Dp) produced
p
4.4. Pre and post infection assays
the monolayer. The data were analyzed with the Filmware 2.5 pro-
gram (Kibron, Inc.). The results are expressed as the variations of
To determine the step at which the compounds showed activity
a pre/post infection time of addition assay was conducted. TZM-bl
D
p as a function of pi for the test compounds. The accuracy of
the system under the experimental conditions was 0.25 mN/m
cells were infected with NL-Lai virus. The compounds (100 lM)
for surface pressure.
were either added prior to infection (pre infection) or 4 h post
infection. For the post infection the uninfected virus was removed
by washing with PBS followed by addition of fresh media contain-
ing the compounds. For controls T20 and AMD3100 were used at
4.2. Antiviral assays
Testing of antiviral activity of test compounds was done as de-
scribed previously.11 Briefly, virus stocks were prepared by trans-
fection of 293T cells with NL-Lai full length infectious clone of
HIV using Ex gen 500 transfection reagent (Fermentas, Glen Burnie,
MD. Virus supernatant was collected 48 h post transfection,
cleared of cellular components by centrifugation, aliquoted, stored
at À70 °C. Virus titers were determined in TZM-bl cells prior to use.
For determination of antiviral activity TZM-bl cells were seeded in
96-well plates at 2 Â 104 cells per well and allowed to adhere over-
night. The subsequent day cells were infected with HIV virus in the
2 lM concn.
4.5. CMC measurements
Stock solutions of glycolipids were prepared in hexane/chloro-
form/ethanol (11:5:4, v/v/v) and injected in water with a Hamilton
microsyringe (dilution 1:1000). The surface tension was continu-
ously recorded with the Kibron microtensiometer. Below the
CMC, a drop in surface tension was recorded after each glycolipid
injection. Increasing the concentration of the added glycolipid re-
sulted in a linear decrease in surface tension. The CMC was deter-
mined as the concentration of glycolipid above which there was no
further decrease in surface tension.
presence of 20 lg/mL DEAE dextran and test compounds at various
concentrations. Virus infection was determined 24 h post infection
as luciferase activity using Brite Lite Luciferase substrate (Perkin
Elmer). Percent infection was calculated based on control wells in-
fected with virus in the absence of any compound. Normalized
data was fit using Sigma Plot software and IC50 calculated based
on the curve fit. Pooled data from at least three independent exper-
iments was used for IC50 determination for each compound.
4.6. Synthesis—general
Unless otherwise stated, all reactions were carried out under a
nitrogen atmosphere in oven-dried glassware using standard

4808
S. Bachan et al. / Bioorg. Med. Chem. 19 (2011) 4803–4811
syringe and septa technique. 1H and 13C NMR spectra were obtained
on a Bruker 500 MHz spectrometer. Chemical shifts are relative to
72.6, 74.9, 75.7, 91.0 (JH1,C1 (glu) = 172 Hz), 93.9 (JH1,C1
(man) = 175 Hz), 100.0, 110.1, 169.7, 170.2, 170.4, 170.8; HRMS
(ESI) calcd for (M+Na)+ C26H38NaO15 613.2103, found 613.2095.
For 14b: (124 mg, 20%); Rf = 0.39 (40% EtOAc/petroleum ether);
1H NMR (CDCl3) d 1.37 (s, 3H), 1.43 (s, 3H), 1.52 (s, 3H), 1.57
(s, 3H), 2.03 (s, 3H), 2.05 (s, 3H), 2.08 (s, 3H), 2.12 (s, 3H), 3.74
(m, 4H), 3.89 (dd, 1H, J = 5.0, 10.1 Hz), 4.12 (d, 1H, J = 5.7 Hz),
4.19 (m, 3H), 4.74 (d, 1H, J = 8.0 Hz, H-1 (glu)), 5.01 (d, 1H,
J = 9.6 Hz), 5.05 (t, 1H, J = 9.7 Hz), 5.23 (m, 2H); 13C NMR (CDCl3)
d 18.9, 20.8 (two signals), 26.4, 28.4, 29.1, 61.6, 62.4, 62.5, 68.4,
71.6, 72.4, 72.6, 72.9, 75.0, 75.6, 99.7 (JH1,C1 (man) = 172 Hz), 99.8
(JH1,C1 (glu) = 152 Hz), 99.9, 109.9, 169.5, 169.6, 170.4, 170.9; HRMS
(ESI) calcd for (M+Na)+ C26H38NaO15 613.2103, found 613.2101.
the deuterated solvent peak and are in parts per million (ppm). 13
C
NMR peaks are listed to the nearest 0.1 ppm. When two or more
signals occur as distinct paks within 0.1 ppm, the number of these
signals are indicated in parenthesis. Assignments for selected nuclei
were determined from 1H COSY experiments. Thin layer chromatog-
raphy (TLC) was done on 0.25 mm thick precoated silica gel HF254
aluminum sheets. Chromatograms were observed under UV (short
and long wavelength) light, and were visualized by heating plates
that were dipped in a solution of ammonium (VI) molybdate tetra-
hydrate (12.5 g) and cerium (IV) sulfate tetrahydrate (5.0 g) in 10%
aqueous sulfuric acid (500 mL). Flash column chromatography
(FCC) was performed using Silica Gel 60 (230–400 mesh) and em-
ployed a stepwise solvent polarity gradient, correlated with TLC
mobility. Solvents were purified by standard procedures or used
from commercial sources as appropriate.
4.9. 2,3,4,6-Tetra-O-benzyl-b-
D-galactopyranosyl-(1?1)-2,3-O-
isopropylidene- -mannopyranoside 15
a-D
Tetra-O-acetate 13b (0.514 g, 0.87 mmol) was treated with
NaOMe (100 mg) in dry MeOH (10 mL) under N2 for 15 min. The
mixture was then neutralized with methanolic HCl and concen-
trated under reduced pressure. The crude product was dried in va-
cuo and taken up in dry THF (15 mL). TBAI (0.032 g, 0.087 mmol)
and NaH (0.348 g, 60% dispersion in mineral oil, 8.7 mmol) were
added to the solution at 0 °C, under nitrogen. After 45 min at this
temperature, BnBr was added dropwise to the mixture and stirring
continued at rt for 15 h. The reaction was then cooled to 0 °C,
quenched with MeOH, diluted with water and extracted with
ether. The organic phase was dried (Na2SO4), filtered and concen-
trated in vacuo. FCC of the residue provided the tetra-O-benzylated
derivative as a clear oil (0.498 g, 73%): Rf = 0.53 (30% EtOAc/petro-
leum ether). To a portion of this material (100 mg, 0.129 mmol), in
dry MeOH (5 mL) was added p-TsOH (12 mg, 0.064 mmol) at rt.
The mixture was stirred for 15 min, then neutralized with Et3N
and concentrated in vacuo. FCC of the residue afforded diol 15
(60 mg, 63%) as a white solid. Rf = 0.65 (70% EtOAc/petroleum
ether); 1H NMR (CDCl3) d 1.37 (s, 3H), 1.53 (s, 3H), 3.39 (m, 1H),
3.56 (dd, 1H, J = 2.9, 6.9 Hz), 3.61 (m, 3H), 3.79 (dd, 1H, J = 3.1,
8.7 Hz), 3.83 (d, 1H, J = 2.8 Hz), 3.87 (t, 1H, J = 7.9 Hz), 4.01 (m,
1H), 4.06 (d, 1H, J = 5.7 Hz), 4.14 (m, 2H), 4.39 (d, 1H, J = 11.8 Hz),
4.48 (d, 1H, J = 11.7 Hz), 4.61 (d, 1H, J = 6.5 Hz), 4.64 (d, 1H,
J = 2.6 Hz), 4.75 (d, 2H, J = 2.9 Hz), 4.80 (d, 1H, J = 11.2 Hz), 4.87
(d, 1H, J = 11.2 Hz), 4.94 (d, 1H, J = 11.7 Hz), 5.29 (s, 1H), 7.33 (m,
20 H); 13C NMR (CDCl3) d 26.2, 28.0, 62.4, 69.3, 70.1, 70.6, 73.0,
73.3, 73.5, 73.8, 74.4, 75.3, 75.4, 78.5, 79.4, 82.5, 99.0, 102.2,
109.6, 127.6 (two signals), 127.7, 127.9, 128.0, 128.1, 128.2,
128.3, 128.4 (three signals), 137.4, 138.2, 138.3, 138.5; HRMS
(ESI) calcd for (M+Na)+ C43H50NaO11 765.3245, found 765.3243.
4.7. 2,3,4,6-Tetra-O-acetyl-
2,3:4,6-di-O-isopropylidene-
a
/b-
D
-galactopyranosyl-(1?1)-
a-
D
-mannopyranoside 13 /b
a
A mixture of mannose donor 1232,33 (4.20 g, 11.9 mmol), galact-
ose acceptor 1030 (4.10 g, 11.8 mmol), freshly activated, powdered,
4 Å molecular sieves, 2,6-di-tert-butyl-4-methylpyridine (DTBMP)
(17.3 g, 83.3 mmol) and dry CH2Cl2 (50 mL) was stirred for
30 min, at rt. The mixture was then cooled to 0 °C and MeOTf
(8.1 mL, 77.4 mmol) was slowly added. After stirring for an addi-
tional 20 h the reaction was quenched with triethylamine (3 mL).
The mixture was diluted with CH2Cl2, filtered and concentrated
in vacuo. FCC of the residue afforded 13
(1.26 g, 18%) as white amorphous solids. For 13
a
(1.59 g, 23%) and 13b
: Rf = 0.32 (30%
a
EtOAc/petroleum ether); 1H NMR (CDCl3) d 1.41 (s, 3H), 1.42 (s,
3H), 1.52 (s, 3H), 1.57 (s, 3H), 2.03 (s, 3H), 2.06 (s, 3H), 2.14 (s,
3H), 2.17 (s, 3H), 3.50 (m, 1H), 3.74 (d, 1H, J = 8.1 Hz), 3.78 (m,
2H), 4.08 (m, 1H), 4.14 (m, 1H), 4.20 (d, 1H, J = 3.4 Hz), 4.29 (t,
1H, J = 6.7 Hz), 5.21 (dd, 1H, J = 3.7, 10.9 Hz), 5.30 (s, 1H), 5.36
(dd, 1H, J = 3.15, 10.9 Hz), 5.49 (apparent d, 2H, J = 3.5 Hz, H-1
(gal), H-4 (gal)); 13C NMR (CDCl3) d 19.3, 21.2 (three signals),
26.7, 28.6, 29.6, 61.9, 62.2, 62.6, 67.6, 67.8, 67.9, 68.2, 73.0, 75.2,
76.1, 91.8 (JH1,C1 (gal) = 172 Hz), 94.2 (JH1,C1 (man) = 175 Hz),
100.3, 110.4, 170.6, 170.7, 170.8, 170.9; HRMS (ESI) calcd for
(M+Na)+ C26H38NaO15 613.2103, found 613.2110. For 13b:
Rf = 0.18 (30% EtOAc/petroleum ether); 1H NMR (CDCl3) d 1.37 (s,
3H), 1.43 (s, 3H), 1.53 (s, 3H), 1.57 (s, 3H), 2.01 (s, 3H), 2.07 (s,
3H), 2.09 (s, 3H), 2.20 (s, 3H), 3.71 (t, 1H, J = 9.81 Hz), 3.76 (d,
1H, J = 7.9 Hz), 3.82 (m, 1H), 3.89 (m, 1H), 3.97 (t, 1H, J = 6.9 Hz),
4.11 (m, 2H), 4.20 (m, 1H), 4.70 (d, 1H, J = 8.0 Hz, H-1 (gal)), 5.21
(s, 1H), 5.24 (dd, 1H, J = 8.1, 10.5 Hz), 5.41 (d, 1H, J = 4.4); 13C
NMR (CDCl3) d 18.9, 20.7, 20.8, 20.9, 21.0, 26.4, 28.3, 29.2, 61.7,
62.0, 62.3, 67.2, 69.2, 71.0, 71.4, 72.5, 75.0, 75.6, 99.8 (JH1,C1
(man) = 172 Hz), 99.9 (JH1,C1 (gal) = 154 Hz), 100.4, 109.9; HRMS
(ESI) calcd for (M+Na)+ C26H38NaO15 613.2103, found 613.2097.
4.10. 2,3,4,6-Tetra-O-benzyl-b-
D-galactopyranosyl-(1?1)-2,3-O-
isopropylidene-6-O-palmitate-
a-D
-mannopyranoside 16
To a solution of 15 (118 mg, 0.160 mmol) and palmitic acid
(45 mg, 0.176 mmol), in dry benzene (4 mL) was added DCC
(36 mg, 0.176 mmol) and DMAP (5 mg, 0.04 mmol) at 0 °C. The
reaction was stirred for 18 h at rt, then diluted with ether and fil-
tered through a bed of Celite. The filtrate was concentrated in va-
cuo and the residue purified by FCC to give 16 (100 mg, 86%
brsm): Rf = 0.4 (30% EtOAc/petroleum ether); 1H NMR (CDCl3) d
0.9 (t, 3H, J = 6.8, 7.0 Hz), 1.27 (br s, 25H), 1.36 (s, 3H), 1.51 (s,
3H), 1.62 (m, 2H), 2.35 (t, 2H, J = 7.5 Hz), 3.15 (br s, 1H), 3.46 (m,
2H), 3.55 (m, 2H), 3.60 (t, 1H, J = 6.2 Hz), 3.85 (t, 1H, J = 7.9 Hz),
3.89 (d, 1H, J = 2.4 Hz), 4.00 (m, 1H), 4.06 (m, 1H), 4.15 (t, 1H,
J = 5.9 Hz), 4.38 (d, 1H, J = 11.6 Hz), 4.43 (d, 1H, J = 11.6 Hz), 4.57
(m, 2H), 4.63 (d, 1H, J = 11.7 Hz), 4.74 (s, 1H), 4.79 (d, 1H,
J = 11.2 Hz), 4.86 (d, 1H, J = 11.2 Hz), 4.95 (d, 1H, J = 11.7 Hz), 5.30
(s, 1H), 7.34 (m, 20H); 13C NMR (CDCl3) d 14.3, 22.9, 25.3, 26.4,
4.8. 2,3,4,6-Tetra-O-acetyl-
a
/b-
D-glucopyranosyl-(1?1)-2,3:4,6-
di-O-isopropylidene- -mannopyranoside 14a
a-
D
/b
Disaccharides 14
(415 mg, 1.18 mmol), and glucose acceptor 1131 (373 mg,
1.07 mmol) via the procedure described for 13 /b. For 14
a/b were prepared from mannose donor 12
a
a:
(130 mg, 21%); Rf = 0.45 (40% EtOAc/petroleum ether); 1H NMR
(CDCl3) d 1.42 (s, 3H), 1.43 (s, 3H), 1.58 (s, 6H), 2.05 (s, 3H), 2.06
(s, 3H), 3.52 (m, 1H), 3.76 (m, 3H), 4.08 (m, 1H), 4.13 (m, 2H),
4.24 (m, 2H), 4.30 (dd, 1H, J = 4.2, 4.3 Hz), 4.97 (dd, 1H, J = 3.9,
10.3 Hz), 5.13 (t, 1H, J = 9 Hz), 5.30 (s, 1H), 5.47 (d, 1H, J = 3.8 Hz,
H1 (glu)), 5.50 (t, 1H, J = 9.9 Hz); 13C NMR (CDCl3) d 18.9, 20.7,
20.8, 20.9, 26.4, 28.3, 29.3, 61.8, 61.9, 62.3, 68.3, 68.5, 70.0, 70.3,

S. Bachan et al. / Bioorg. Med. Chem. 19 (2011) 4803–4811
4809
28.3, 29.4, 29.5 (two signals), 29.6, 29.8 (two signals), 29.9, 32.1,
34.4, 62.6, 68.5, 69.3, 69.4, 73.2, 73.4, 73.7, 74.0, 74.7, 75.4, 75.7,
77.8, 79.7, 82.8, 99.6, 102.7, 109.7, 127.7 (three signals), 128.0,
128.1, 128.2, 128.4, 128.6 (three signals), 137.8, 138.4, 138.6,
138.7, 175.4.
(two signals), 127.5 9 (three signals), 127.6, 127.8 (two signals),
128.0, 128.2 (two signals), 128.3 (three signals), 128.4, 137.9,
138.3, 138.4, 138.5, 138.6, 172.6; HRMS (ESI) calcd for (M+NH4)+
C70H92NO12 1138.6614, found 1138.6606.
4.14. 2,3,4,6-Tetra-O-benzyl-b-
D-glucopyranosyl-(1?1)-2,3-O-
4.11. 2,3,4,6-Tetra-O-benzyl-b-
isopropylidene-4,6-di-O-palmitate-
D
-galactopyranosyl-(1?1)-2,3-O-
-mannopyranoside 17
isopropylidene- -mannopyranoside 20
a-D
a-D
Protected disaccharide 14b was transformed to 20 (58 mg, 75%
brsm) following the three-step procedure described for the conver-
sion of 13b to 15 (Section 4.9) For 20: white solid; Rf = 0.18 (40%
EtOAc/petroleum ether); 1H NMR (CDCl3) d 1.37 (s, 3H), 1.54 (s,
3H), 2.22 (d, 1H, J = 4.4 Hz), 2.55 (dd, 1H, J = 5.7, 7.5 Hz), 3.48 (m,
2H), 3.56 (m, 2H), 3.61–3.70 (m, 4H), 3.78–3.82 (m, 1H), 3.95–
3.99 (m, 1H), 4.04 (d, 1H, J = 5.8 Hz), 4.14 (m, 1H), 4.51 (m, 3H),
4.67 (d, 1H, J = 7.9 Hz), 4.83 (m, 4H), 4.92 (d, 1H, J = 10.9 Hz),
5.30 (s, 1H), 7.33 (m, 20H); 13C NMR (CDCl3) d 26.4, 28.2, 62.6,
69.2, 70.3, 71.1, 73.6, 74.9, 75.2, 75.4, 75.6, 75.9, 78.0, 78.7, 82.4,
85.0, 99.4, 102.5, 109.9, 127.9, 128.0, 128.1 (two signals), 128.2,
128.3, 128.6 (two signals), 128 .7, 137.7, 138.0, 138.4, 138.5; HRMS
(ESI) calcd for (M+Na)+ C43H50NaO11 765.3245, found 765.3238.
Diol 15 (60 mg, 0.081 mmol) was treated as described in Sec-
tion 4.10, with excess palmitic acid (52 mg, 0.2 mmol). The diester
17 (68 mg, 70%) was obtained as a white solid: Rf = 0.82 (25%
EtOAc/petroleum ether); 1H NMR (CDCl3) d 0.92 (s, 6H), d 1.29
(br s, 48H), d 1.60 (s, 4H), 2.30 (s, 4H), 3.56 (m, 4H), 3.86 (dd, 1H,
J = 7.8, 9.6 Hz), 3.93 (s, 1H), 4.02 (d, 2H, J = 13.7 Hz), 4.19 (d, 2H,
J = 8.4 Hz), 4.28 (d, 1H, J = 12.4 Hz), 4.40 (d, 1H, J = 11.7 Hz), 4.48
(d, 1H, J = 11.7 Hz), 4.59 (d, 1H, J = 9.5 Hz), 4.64 (d, 1H,
J = 13.4 Hz), 4.74 (s, 1H), 4.79 (d, 1H, J = 13.1 Hz), 4.88 (d, 1H,
J = 13.1 Hz), 4.94 (d, 1H, J = 13.3 Hz), 5.18 (t, 1H, J = 10.2 Hz), 5.35
(s, 1H), 7.32 (m, 20H); 13C NMR (CDCl3) d 14.3, 22.9, 25.0, 26.8,
27.9, 29.3, 29.4, 29.5 (two signals), 29.7 (two signals), 29.8, 29.9
(two signals), 32.1, 34.3, 34.4, 61.5, 67.0, 68.5, 68.8, 70.8, 73.2,
73.4, 73.7, 73.9, 74.9, 75.7 (two signals), 76.5, 79.7, 82.9, 99.3,
102.9, 110.3, 127.7, 127.9 (two signals), 128.1, 128.4, 128.6 (two
signals), 137.9, 138.4, 138.6, 138.7, 172.5, 173.8; HRMS (ESI) calcd
for (M+Na)+ C75H110NaO13 1241.7389, found 1241.7389.
4.15. b-D-Galactopyranosyl-(1?1)-6-O-palmitate-a-D-
mannopyranoside 3
A solution of 16 (100 mg, 0.1 mmol) and p-TsOH (100 mg,
0.52 mmol) in MeOH (5 mL) was stirred for 1 h. The mixture was
then quenched with saturated NaHCO3 and extracted with CH2Cl2.
The organic phase was dried (Na2SO4), filtered and concentrated in
vacuo. FCC of the residue afforded the corresponding triol (70 mg,
73%). A portion of the triol (30 mg, 0.032 mmol), 10% Pd/C (40 mg)
and HCOOH (0.1 mL) was stirred under H2 atmosphere for 20 h.
The mixture was then filtered through a bed of Celite and concen-
trated in vacuo. FCC of the residue gave 3 (9 mg, 50%): Rf = 0.36
(20% MeOH/EtOAc); 1H NMR (CD3OD) d 0.89 (t, 3H, J = 6.8 Hz),
1.28 (m, 28H), 1.61 (m, 2H), 2.36 (t, 2H, J = 7.5 Hz), 3.46 (dd, 1H,
J = 3.0, 9.7 Hz), 3.53 (m, 2H), 3.71 (m, 5H), 3.85 (d, 1H, J = 2.7 Hz),
3.95 (s, 1H), 4.05 (m, 1H), 4.25 (dd, 1H, J = 4.9, 11.95 Hz), 4.31 (d,
1H, J = 11.2 Hz), 4.41 (d, 1H, J = 7.7 Hz), 5.02 (s, 1H); 13C NMR
(CD3OD) d 14.6, 23.9, 26.2, 30.4, 30.6, 30.8, 30.9 (two signals),
33.2, 35.1, 62.2, 64.9, 68.5, 70.1, 71.6, 72.4, 72.7, 72.8, 75.1, 77.0,
103.2, 104.4, 175.9; HRMS (ESI) calcd for (M+Na)+ C28H52NaO12
603.3351, found 603.3352.
4.12. 2,3,4,6-Tetra-O-benzyl-b-D-galactopyranosyl-(1?1)-4.6-O-
benzyl- -mannopyranoside 18
a
-D
Diol 15 (61 mg, 0.082 mmol) was subjected to the standard
benzylation procedure described in Section 4.9. FCC of the crude
product yielded the di-O-benzylated derivative (71 mg,
0.08 mmol), which was taken up in dry MeOH (5 mL) and treated
with CSA (100 mg, 0.43 mmol). The mixture was stirred for
30 min, quenched with Et3N and concentrated in vacuo. FCC of
the residue afforded 18 (55 mg, 75% two steps) as a clear oil:
Rf = 0.55 (60% EtOAc/petroleum ether); 1H NMR (CDCl3) d 3.56
(m, 5H), 3.73 (dd, 1H, J = 3.15, 3.05 Hz), 3.81 (m, 2H), 3.88 (m,
2H), 3.97 (dd, 1H, J = 3.40, 3.50 Hz), 4.12 (m, 1H), 4.37 (s, 2H),
4.40 (s, 1H), 4.59 (m, 4H), 4.60 (d, 1H, J = 11.1 Hz), 4.73 (s, 2H),
4.78 (d, 1H, J = 11.1 Hz), 4.82 (d, 1H, J = 11.1 Hz), 4.94 (d, 1H,
J = 11.7 Hz), 7.24–7.39 (m, 30H); 13C NMR (CDCl3) d 68.7, 68.9,
71.1, 71.5, 71.7, 73.1, 73.6 (two signals), 73.8, 74.6, 74.8, 75.6,
75.8, 79.7, 82.7, 101.5, 103.5, 127.7, 127.8 (three signals), 127.9
(three signals), 128.0, 128.1, 128.49 (two signals), 128.5, 128.6
(three signals), 138.1, 138.4, 138.6, 138.7, 138.8.
4.16. b-D-Galactopyranosyl-(1?1)-3-O-palmitate-a-D-
mannopyranoside 4
The hexa-O-benzylether 19 (54 mg, 0.048 mmol) was subjected
to the hydrogenolysis procedure described for 3. FCC of the crude
product provided 4 (22 mg, 79%): Rf = 0.58 (20% MeOH/CH2Cl2);
1H NMR (CD3OD) d 0.90 (t, 3H, J = 6.8 Hz), 1.16 (m, 5H), 1.29 (br
s, 26H), 1.63 (m, 4H), 1.71 (m, 2H), 1.85 (m, 2H), 2.41 (t, 2H,
J = 7.45 Hz), 3.46 (m, 2H), 3.56 (m, 1H), 3.65 (m, 3H), 3.78 (m,
3H), 3.88 (d, 1H, J = 10.2 Hz), 4.10 (m, 2H), 4.44 (d, 1H,
J = 7.85 Hz), 5.04 (dd, 1H, J = 3.2, 9.8 Hz), 5.06 (s, 1H); 13C NMR
(CD3OD) d 14.6, 23.9, 26.1, 26.2, 26.9, 30.3, 30.6, 30.8, 30.9 (two sig-
nals), 33.2, 34.9, 35.2, 63.0, 63.1, 66.3, 69.7, 70.5, 72.5, 75.0, 75.3,
75.6, 77.4, 102.9, 104.7, 175.5; HRMS (ESI) calcd for C12H22NaO11
(M+Na)+ 603.3351, found 603.3351.
4.13. 2,3,4,6-Tetra-O-benzyl-b-
D-galactopyranosyl-(1?1)-4.6-O-
benzyl-3-O-palmitate- -mannopyranoside 19
a-D
To a solution of 18 (80 mg, 0.09 mmol) and palmitic acid
(26 mg, 0.099 mmol) in dry benzene was added DCC (20 mg,
0.099 mmol) and DMAP (2 mg, 0.018 mmol) at 0 °C. The reaction
was stirred for 18 h at rt, then diluted with ether, filtered through
a bed of Celite and concentrated in vacuo. FCC of the residue
yielded 19 (74 mg, 80% brsm) as a white solid. Rf = 0.30 (30%
EtOAc/petroleum ether). 1H NMR (CDCl3) d 0.90 (t, 3H, J = 6.8 Hz),
1.27 (m, 24H), 1.61 (m, 2H), 2.28 (m, 2H), 3.54 (m, 6H), 3.71 (dd,
1H, J = 3.5, 11.5 Hz), 3.89 (m, 2H), 4.03 (m, 2H), 4.23 (m, 1H),
4.35 (m, 3H), 4.47 (d, 1H, J = 11.3 Hz), 4.56 (m, 1H), 4.62 (m, 2H),
4.73 (m, 2H), 4.83 (s, 1H), 4.95 (d, 1H, J = 12.0 Hz), 5.11 (d, 1H,
J = 1.5 Hz), 5.34 (dd, 1H, J = 3.2, 9.7 Hz), 7.34 (m, 35H); 13C NMR
(CDCl3) d 14.1, 22.7, 24.8, 24.9, 25.5, 29.1, 29.2, 29.3, 29.4, 29.6
(three signals), 29.7, 31.9, 33.7, 34.4, 68.2, 68.7, 69.5, 71.7, 72.7,
72.9, 73.3, 73.6, 73.9, 74.5, 75.5, 79.3, 82.3, 101.4, 103.6, 127.4
4.17. b-D-Galactopyranosyl-(1?1)-4,6-di-O-palmitate-a-D-
mannopyranoside 5
The diester 17 (72 mg, 0.06 mmol) was dissolved in a mixture of
dry MeOH (3 mL) and dry CH2Cl2 (1 mL). CSA (25 mg, 0.11 mmol)
was then added. The reaction mixture was stirred for 1 h, then

4810
S. Bachan et al. / Bioorg. Med. Chem. 19 (2011) 4803–4811
neutralized with Et3N and evaporated under reduced pressure. FCC
of the residue gave the derived diol (32 mg, 46%): white solid.
Rf = 0.54 (35% EtOAc/petroleum ether). This material (32 mg,
0.027 mmol) was subjected to the hydrogenolysis procedure de-
scribed for 3. FCC of the product afforded 5 (13 mg, 60%): white so-
lid; Rf = 0.32 (10% MeOH/EtOAc); 1H NMR (CD3OD) d 0.90 (t, 6H,
J = 6.8 Hz), 1.31 (s, 48H), 1.61 (m, 4H), 2.35 (m, 4H), 3.48 (dd, 1H,
J = 3.3, 9.7 Hz), 3.54 (m, 2H), 3.72 (d, 2H, J = 6.3 Hz), d 3.85 (d, 1H,
J = 3.3 Hz), 3.89 (dd, 1H, J = 3.3, 9.8 Hz), 4.01 (m, 1H), 4.05 (m,
1H), 4.21 (m, 1H), 4.43 (d, 1H, J = 7.7 Hz), 5.07 (s, 1H), 5.24 (t, 1H,
J = 9.8 Hz); 13C NMR d 14.6, 23.9, 26.0, 26.1, 30.3, 30.4, 30.6, 30.7,
30.8 (two signals), 30.9, 31.0, 33.2, 35.0, 35.2, 62.4, 63.8, 70.1,
70.2, 70.6, 71.7, 72.7, 75.1, 77.1, 103.3, 104.7, 174.9, 175.5; HRMS
(ESI) calcd for (M+Na)+ C44H82NaO13 841.5641, found 841.5648.
J = 11.7 Hz), 4.99 (s, 1H), 5.03 (d, 1H, J = 3.7 Hz); 13C NMR (CD3OD)
d 14.5, 23.8, 26.1, 30.4, 30.6 (two signals), 30.7, 30.9 (two signals),
33.2, 35.1, 62.8, 64.8, 68.7, 69.7, 71.1, 71.3, 72.2, 72.5, 73.3, 95.5,
96.7, 175.7; HRMS (ESI) calcd for
589.3799 found 589.3797.
C
28H56NO12 (M+NH4)+,
Acknowledgments
This investigation was supported by grants R01 GM57865 and
SCORE S06 GM60654 from the National Institute of General Medi-
cal Sciences of the National Institutes of Health (NIH). ‘Research
Centers in Minority Institutions’ award RR-03037 from the Na-
tional Center for Research Resources of the NIH, which supports
the infrastructure and instrumentation of the Chemistry Depart-
ment at Hunter College, is also acknowledged.
4.18. b-D-Glucopyranosyl-(1?1)-6-O-palmitate-a-D-
mannopyranoside 6
Supplementary data
Diol 20 was transformed to 6 (9 mg, 28% brsm) following the
three step procedure described for the conversion of 15 to 3 (Sec-
tions 4.9, 4.10, 4.15). For 6: Rf = 0.37 (20% MeOH/EtOAc); 1H NMR
(CD3OD) d 0.92 (t, 3H, J = 6.8 Hz), 1.20 (t, 3H, J = 7.0 Hz), 1.31 (br
s, 24H), 1.64 (m, 2H), 2.23 (t, 2H, J = 7.4 Hz), 3.23 (t, 1H,
J = 8.0 Hz), 3.33 (m, 1H), 3.50 (q, 2H, J = 7.0 Hz), 3.70 (m, 3H),
3.87 (dd, 1H, J = 2.2, 11.9 Hz), 3.98 (m, 1H), 4.10 (m, 1H), 4.29
(dd, 1H, J = 5.2, 11.9 Hz), 4.36 (dd, 1H, J = 2.2, 12.0 Hz), 4.48 (d,
1H, J = 8.0 Hz), 5.03 (s, 1H); 13C NMR (CD3OD) d 12.1, 13.6, 21.9,
24.2, 28.4, 28.6, 28.8, 28.9, 31.2, 33.1, 60.7, 62.9, 65.0, 66.5, 69.3,
60.6, 70.4, 70.8, 73.3, 76.2, 76.4, 101.1, 101.7, 173.9; HRMS (ESI)
calcd for C28H52NaO12 (M+Na)+ 603.3351, found 603.3344.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.078.
References and notes
1. Meadows, D. C.; Gervay-Hague, J. ChemMedChem 2006, 1, 16.
2. McReynolds, K. D.; Gervay-Hague, J. Chem. Rev. 2007, 107, 1533.
3. Rawat, S. S.; Johnson, B. T.; Puri, A. Biosci. Rep. 2005, 25, 329.
4. Rawat, S. S.; Viard, M.; Gallo, S. A.; Blumenthal, R.; Puri, A. Glycoconjugate J.
2006, 23, 189.
5. Fantini, J.; Hammache, D.; Pieroni, G.; Yahi, N. Glycoconjugate J. 2000, 17, 199.
6. Hammache, D.; Pieroni, G.; Yahi, N.; Delezay, O.; Koch, N.; Lafont, H.; Tamalet,
C.; Fantini, J. J. Biol. Chem. 1998, 273, 7967.
7. Conboy, J. C.; McReynolds, K. D.; Gervay-Hague, J.; Saavedra, S. S. J. Am. Chem.
Soc. 2002, 124, 968.
8. Villard, R.; Hammache, D.; Delapierre, G.; Fotiadu, F.; Buono, G.; Fantini, J.
Chembiochem 2002, 3, 517.
4.19. Methyl-6-O-palmitate-a-D-mannopyranoside 7
9. Kensinger, R. D.; Yowler, B. C.; Benesi, A. J.; Schengrund, C.-L. Bioconjugate
Chem. 2004, 15, 349.
10. Lund, N.; Branch, D. R.; Mylvaganam, M.; Chark, D.; Ma, X. Z.; Sakac, D.;
Binnington, B.; Fantini, J.; Puri, A.; Blumenthal, R.; Lingwood, C. A. AIDS 2006,
20, 333.
11. Garg, H.; Francella, N.; Tony, K. A.; Augustin, L. A.; Fantini, J.; Barchi, J. J., Jr.;
Puri, A.; Mootoo, D. R.; Blumenthal, R. Antiviral Res. 2008, 80, 54.
12. Dey, B.; Lerner, D. L.; Lusso, P.; Boyd, M. R.; Elder, J. H.; Berger, E. A. J. Virol.
2000, 74, 4562.
13. Yahi, N.; Sabatier, J. M.; Nickel, P.; Mabrouk, K.; Gonzalez-Scarano, F.; Fantini, J.
J. Biol. Chem. 1994, 269, 24349.
14. Bhat, S.; Spitalnik, S. L.; Gonzalez-Scarano, F.; Silberberg, D. H. Proc. Natl. Acad.
Sci. U.S.A. 1991, 88, 7131.
15. Faroux-Corlay, B.; Clary, L.; Gadras, C.; Hammache, D.; Greiner, J.; Santaella, C.;
Aubertin, A.-M.; Vierling, P.; Fantini, J. Carbohydr. Res. 2000, 327, 223.
16. McReynolds, K. D.; Bhat, A.; Conboy, J. C.; Saavedra, S. S.; Gervay-Hague, J.
Bioorg. Med. Chem. 2002, 10, 625.
Compound 7 was prepared from 2135 following procedures
used in the conversion of 13b to 3. For 7: Rf = 0.60 (EtOAc); 1H
NMR (CDCl3) d 0.88 (t, 3H, J = 6.9 Hz), 1.26 (br s, 24H), 1.65 (m,
2H), 2.28 (br s, 1H), 2.41 (t, 2H, J = 7.4 Hz), 3.39 (s, 3H), 3.56 (t,
1H, J = 9.6 Hz), 3.69 (m, 1H), 3.83 (dd, 1H, J = 3.3, 9.3 Hz), 3.97 (s,
1H), 4.21 (dd, 1H, J = 2.0, 12.4 Hz), 4.66 (dd, 1H, J = 3.8, 12.4 Hz),
4.77 (s, 1H); 13C NMR (CDCl3) d 14.3, 22.9, 25.1, 29.3, 29.4, 29.5,
29.6, 29.8 (two signals), 29.9, 32.1, 34.4, 55.3, 63.3, 67.7, 70.4,
70.5, 71.4, 101.0, 175.5; HRMS (ESI) calcd for (M+Na)+ C23H44NaO7
455.2979, found 455.2986.
4.20. Methyl-4,6-di-O-palmitate-a-D-mannopyranoside 8
17. Székeley, Z. J. Mol. Struct. 1995, 334, 93.
18. Asensio, J. L.; Cañada, F. J.; Cheng, X.; Khan, N.; Mootoo, D. R.; Jiménez-Barbero,
J. Chem. Eur. J. 2000, 6, 1035.
19. Nehete, P. N.; Vela, E. M.; Hossain, M. M.; Sarkar, A. K.; Yahi, N.; Fantini, J.;
Sastry, K. J. Antiviral Res. 2002, 56, 233.
20. Garg, H.; Joshi, A.; Freed, E. O.; Blumenthal, R. J. Biol. Chem. 2007, 282,
16899.
21. Sands, J.; Auperin, D.; Snipes, W. Antimicrob. Agents Chemother. 1979,
15, 67.
22. Li, Q.; Estes, J. D.; Schlievert, P. M.; Duan, L.; Brosnahan, A. J.; Southern, P. J.;
Reilly, C. S.; Peterson, M. L.; Schultz-Darken, N.; Brunner, K. G.; Nephew, K. R.;
Pambuccian, S.; Lifson, J. D.; Carlis, J. V.; Haase, A. T. Nature 2009, 458,
1034.
23. Cory, A. H.; Owen, T. C.; Barltrop, J. A.; Cory, J. G. Cancer Commun. 1991, 3, 207.
24. Wild, C. T.; Shugars, D. C.; Greenwell, T. K.; McDanal, C. B.; Matthews, T. J. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 9770.
25. De Clercq, E.; Yamamoto, N.; Pauwels, R.; Balzarini, J.; Witvrouw, M.; De
Vreese, K.; Debyser, Z.; Rosenwirth, B.; Peichl, P.; Datema, R.; Thornton, D.;
Skerlj, R.; Gaul, F.; Padmanabhan, S.; Bridger, G.; Henson, G.; Abrams, M.
Antimicrob. Agents Chemother. 1994, 38, 668.
26. Boros, C.; Katz, B.; Mitchell, S.; Pearce, C.; Swinbank, K.; Taylor, D. J. Nat. Prod.
2002, 65, 108.
Compound 8 was prepared from 21 following procedures used
in the conversion of 13b to 5. For 8: Rf = 0.6 (80% EtOAc/petroleum
ether); 1H NMR d 0.90 (t, 6H, J = 6.8 Hz), 1.27 (br s, 48H), 1.64 (m,
4H), 2.36 (m, 4H), 2.72 (br s, 1H), 3.12 (br s, 1H), 3.41 (s, 3H), 3.90
(m, 2H), 3.96 (s, 1H), 4.19 (dd, 1H, J = 2.2, 12.1 Hz), 4.28 (dd, 1H,
J = 5.5, 12.1 Hz), 4.81 (s, 1H), 5.02 (t, 1H, J = 9.8 Hz). 13C NMR d
14.3, 22.9, 25.0 (two signals), 29.2, 29.3, 29.4, 29.5 (two signals),
29.6, 29.7, 29.8 (three signals), 29.9, 32.1, 34.3, 34.5, 55.3, 62.7,
68.0, 70.1, 70.6, 70.7, 100.7, 173.8, 175.0; HRMS (ESI) calcd for
C
39H74NaO8 (M+Na)+ 693.5276, found 693.5278.
4.21.
a-D-Galactopyranosyl-(1?1)-6-O-palmitate-a-D-
mannopyranoside 9
Disaccharide 13a was transformed to 9 (60 mg, 13.2% bsrm) fol-
lowing the procedures described for the conversion of 13b to 3. For
9: Rf = 0.2 (20% MeOH/EtOAc); 1H NMR (CD3OD) d 0.81 (t, 3H,
J = 6.9 Hz), 1.21 (br s, 23H), 1.52 (m, 2H), 2.26 (t, 2H, J = 7.5 Hz),
3.21 (m, 1H), 3.61 (m, 4H), 3.74 (m, 4H), 3.82 (d, 1H, J = 2.5 Hz),
3.87 (m, 1H), 4.13 (dd, 1H, J = 5.9, 11.8 Hz), 4.29 (br d, 1H,
27. Kitamoto, D.; Isoda, H.; Nakahara, T. J. Biosci. Bioeng. 2002, 94, 187.
28. Vlahov, I. R.; Vlahova, P. I.; Linhardt, R. J. J. Carbohydr. Chem. 1997, 16, 1.
29. Lang, S.; Philp, J. C.; Van Leeuw, A. J. Microbiol. 1998, 74, 59.
30. Gum, A. G.; Kappes-Roth, T.; Waldmann, H. Chem. Eur. J. 2000, 6, 3714.
31. Tiwari, P.; Misra, A. K. Tetrahedron Lett. 2006, 47, 3573.

S. Bachan et al. / Bioorg. Med. Chem. 19 (2011) 4803–4811
4811
32. Crich, D.; Sun, S. Tetrahedron 1998, 54, 8321.
33. Gómez, A. M.; Casillas, M.; Barrio, A.; Gawel, A.; López, J.-C. Eur. J. Org. Chem.
2008, 3933.
34. Bock, K.; Pedersen, C. J. Chem. Soc., Perkins. Trans. II 1974, 293.
35. Liptak, A.; Imre, J.; Nanasi, P. Carbohydr. Res. 1981, 92, 154.
36. Haines, A. B. Adv. Carbohydr. Chem. Biochem. 1976, 33, 11.
37. Shah, V.; Doncel, G. F.; Seyoum, T.; Eaton, K. M.; Zalenskaya, I.; Hagyer, R.; Azin,
A.; Gross, R. Antimicrob. Agents Chemother. 2005, 49, 4093.
38. Azim, A.; Shah, V.; Doncel, G. F.; Peterson, N.; Gao, W.; Gross, R. Bioconjugate
Chem. 2006, 17, 1523.
40. Wong, Y.-L.; Hubieki, M. P.; Curfman, C. L.; Doncel, G. F.; Dudding, T. C.; Savle, P.
S.; Gandour, R. D. Bioorg. Med. Chem. 2002, 10, 3599.
41. Macri, R. V.; Karlovská, J.; Doncel, G. F.; Du, X.; Maisuria, B. B.; Williams, A. A.;
Sugandhi, E. W.; Falkinham, J. O.; Esker, A. R.; Gandour, R. Bioorg. Med. Chem.
2009, 17, 3162.
42. Franzetti, A.; Gandolfi, I.; Bestetti, G.; Smyth, T. J. P.; Banat, I. M. Eur. J. Lipid Sci.
Technol. 2010, 112, 617.
43. Rodrigues, L.; Banat, I. M.; Teixeira, J.; Oliveira, R. J. Antimicrob. Chemother.
2006, 57, 609.
44. Kitamoto, D.; Morita, T.; Fukuoka, T.; Konoshi, M.; Imura, T. Curr. Opin. Colloid
Interface Sci. 2009, 14, 315.
45. Garmy, N.; Taieb, N.; Yahi, N.; Fantini, J. J. Lipid Res. 2005, 46, 36.
39. Katz, D. H.; Marcelletti, J. F.; Pope, L. E.; Mohammed, H. K.; Katz, L. R.;
McFadden, R. Ann. N.Y. Acad. Sci. 1994, 724, 472.