Structure-activity relationships in toll-like receptor-2 agonistic diacylthioglycerol lipopeptides

Article abstract of DOI:10.1021/jm901839g

The N-termini of bacterial lipoproteins are acylated with a (S)-(2,3-bisacyloxypropyl)cysteinyl residue. Lipopeptides derived from lipoproteins activate innate immune responses by engaging Toll-like receptor 2 (TLR2) and are highly immunostimulatory and yet without apparent toxicity in animal models. The lipopeptides may therefore be useful as potential immunotherapeutic agents. Previous structure-activity relationships in such lipopeptides have largely been obtained using murine cells, and it is now clear that significant species-specific differences exist between human and murine TLR responses. We have examined in detail the role of the highly conserved Cys residue as well as the geometry and stereochemistry of the Cys-Ser dipeptide unit. (R)-Diacylthioglycerol analogues are maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the minimal part-structure, but its stereochemistry was found not to be a critical determinant of activity. The thioether bridge between the diacyl and dipeptide units is crucial, and replacement by an oxoether bridge results in a dramatic decrease in activity.

Full text of DOI:10.1021/jm901839g

3198 J. Med. Chem. 2010, 53, 3198–3213
DOI: 10.1021/jm901839g
Structure-Activity Relationships in Toll-like Receptor-2 Agonistic Diacylthioglycerol Lipopeptides
Wenyan Wu, Rongti Li, Subbalakshmi S. Malladi, Hemamali J. Warshakoon, Matthew R. Kimbrell, Michael W. Amolins,
Rehman Ukani, Apurba Datta, and Sunil A. David*
Department of Medicinal Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047
Received December 13, 2009
The N-termini of bacterial lipoproteins are acylated with a (S)-(2,3-bisacyloxypropyl)cysteinyl residue.
Lipopeptides derived from lipoproteins activate innate immune responses by engaging Toll-like
receptor 2 (TLR2) and are highly immunostimulatory and yet without apparent toxicity in animal
models. The lipopeptides may therefore be useful as potential immunotherapeutic agents. Previous
structure-activity relationships in such lipopeptides have largely been obtained using murine cells, and
it is now clear that significant species-specific differences exist between human and murine TLR
responses. We have examined in detail the role of the highly conserved Cys residue as well as the
geometry and stereochemistry of the Cys-Ser dipeptide unit. (R)-Diacylthioglycerol analogues are
maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the
minimal part-structure, but its stereochemistry was found not to be a critical determinant of activity.
The thioether bridge between the diacyl and dipeptide units is crucial, and replacement by an oxoether
bridge results in a dramatic decrease in activity.
Introduction
peptidoglycan (PGN), a polymer of β-1f4-linked N-acetyl-
glucosamine-N-acetylmuramic acid glycan strands that are
cross-linked by short peptides.18-20 Unlike Gram-negative
bacteria that bear lipopolysaccharide on the outer leaflet of
the outer membrane, the external surface of the peptidoglycan
layer is decorated with lipoteichoic acids (LTA).21,22 LTA are
anchored in the peptidoglycan substratum via a diacylglycerol
moiety and bear a surface-exposed, polyanionic, 1-3-linked
polyglycerophosphate appendage23,24 that variesinitssubunit
composition in LTAs from various Gram-positive bacteria;
in S. aureus, the repeating subunit contains D-alanine and R-D-
N-acetylglucosamine.25 Lipoproteins are found in the bacter-
ial cytoplasmic membrane and are also common constituents
of the cell wall of both Gram-negative and Gram-positive
bacteria.25-27 The free amine of the N-terminus of lipopro-
teins is acylated with a (S)-(2,3-bis-acyloxypropyl)cysteinyl
residue which has previously been demonstrated to be
immunostimulatory,28,29 as shown by studies on synthetic
peptides containing the bis-acylthioglycerol unit,25,30,31 in
contradistinction to enterobacterial LPS which is recognized
by Toll-like receptor-4 (TLR4), PGN,32,33 LTA,34,35 lipopep-
tides,36,37 and some nonenterobacterial LPS38,39 signal via
TLR2.40,41
In comparing the various constituents of the Gram-positive
cell wall, we have found lipopeptides to be extremely potent
TLR2 agonists and yet without apparent toxicity in animal
models.42 In murine cells, PAM2CSK4 (S-[2,3-bis(palmitoyl-
oxy)-(2RS)-propyl]-R-cysteinyl-S-seryl-S-lysyl-S-lysyl-S-lysyl-
S-lysine), a commercially available, synthetic model lipopep-
tide, was equipotent in its NF-κB inducing activity relative to
LPS but elicited much lower proinflammatory cytokines while
both LPS and the lipopeptide potently induced the secretion
of a similar pattern of chemokines suggestive of the engage-
ment of downstream TRIF pathways.42 Furthermore, we
The link between immune stimulation resulting from acute
infection and the resolution of tumors has long been re-
cognized,1 and the phenomenon of infection-related “sponta-
neous regression” of cancer has been documented throughout
history, beginning as early as 2600 B.C.2,3 The foundations of
modern immunotherapy for cancer were laid in 1891 by
William B. Coley, who, in 1891, injected live streptococcal
organisms into a long-bone sarcoma lesion and observed
shrinkage of the tumor.4 More than a century has elapsed
since Coley’s empirical findings were first reported, but the
active principle(s) in Coley’s toxin remain undetermined. This
long lag period is perhaps not altogether surprising because
the recognition of innate immune system is recent,5-7 and our
understanding of the structure and function of the family of
Toll-like receptors (TLRsa)8,9 that recognize “pathogen-
associated molecular patterns (PAMPs)”10 and of the effector
mechanisms that mediate innate immune responses11-13 is yet
nascent. Nonetheless, there exists considerable potential in
harnessing innate immune responses in a wide range of disease
states, notably cancer, as evidenced by the number of TLR
agonists already in clinical trials14 and the resurgence of
interest in the immunotherapy of neoplastic states.15-17
The exoskeleton of the Gram-positive organism, similar
to that of Gram-negative bacteria, comprises underlying
*To whom correspondence should be addressed. Phone: 785-864-
1610. Fax: 785-864-1961. E-mail: sdavid@ku.edu.
a Abbreviations: EDIPA, N,N-diisopropylethylamine; IC50, concen-
tration inducing half-maximal response; IFN-γ, interferon-γ; IL-1β,
interleukin-1β; IL-12, interleukin-12; LPS, lipopolysaccharide; LTA,
lipoteichoic acid; PAM2CSK4, S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-
R-cysteinyl-S-seryl-S-lysyl-S-lysyl-S-lysyl-S-lysine; PAMP, pathogen-
associated molecular pattern; PE, phycoerythrin; PGN, peptidoglycan;
TLR, Toll-like receptor; TNF-R, tumor necrosis factor-R.
r
pubs.acs.org/jmc
Published on Web 03/19/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3199
Scheme 1a
a Reagents and conditions: (a) p-TsCl, pyr, DCM, 0 °C to room temp, 8 h; (b) N-Boc-2-aminoethanethiol, NaH, DMF, 0 °C to room temp, 8 h;
(c) 70% AcOH, room temp, 12 h; (d) C15H31COCl, pyr, DMAP, DCM, 0 °C to room temp, 10 h; (e) TFA, room temp, 30 min; (f) Boc-L-serine-OH,
EDCI, EDIPA, DMAP, DCM, 0 °C to room temp, 8 h; (g) TFA, room temp, 30 min.
have found that while LPS elicited a robust fever response in
rabbits at a dose of 100 ng/kg, a 200 μg/kg dose (2000ꢀ) of the
lipopeptide was without any discernible pyrogenic or other
apparent acute toxic effect (manuscript in preparation). These
observations suggest that the lipopeptides may be remarkably
potent, yet nontoxic immunotherapeutic agents. Indeed, in
a phase I/phase II clinical trial43 on a single, intraoperative
20-30 μg intratumoral injection of MALP-228 (a diacyl lipo-
peptide similar in its core structure to PAM2CSK4) in patients
with inoperable carcinoma of the pancreas, the lipopeptide
was well-tolerated and proved efficacious in prolonging sur-
vival.43
Considerable structure-activity relationships dictating
TLR2 binding and subsequent stimulation of innate immune
responses have been documented for a range of synthetic
lipopeptides. However, the majority of earlier SAR studies
had been performed with murine cells, many even before the
TLRs had been discovered, and it is now clear that significant
species-specific differences exist between human and murine
TLR responses36,44 as a consequence of subtle structural
differences in the ligand binding pocket within TLR2.45
The minimal structure for biological activity is the Cys-Ser
lipodipeptide46 with the lipoaminoacid Cys-OH derivative
being almost inactive;47 the remainder of the peptidic unit
appears, in large part, not to be critical for activity, since a
variety of analogues of different lengths with different amino
acid sequences were found to be equipotent.46,48-50 The (R)-
configuration at the asymmetric glyceryl carbon confers
maximum activity,30,51 and the threshold of the acyl chain
length of the two ester-bound fatty acids is eight carbons, with
shorter acyl analogues being inactive.36 Furthermore, the
recent high-resolution crystal structure of lipopeptides com-
plexed to human- and murine-derived TLR1/TLR2 hetero-
dimers45 raises additional questions, since the binding site in
TLR2 is highly unusual, being located in the convex region
formed at the border of the central and C-terminal domains
with the peptidic unit interacting with interfacial residues at
the neck region of the binding pocket.45 It was of interest to
examine whether the chirality of the highly conserved Cys
residue (L-Cys versus D-Cys) and the thioether bridge con-
necting the diacylglycerol unit to the peptide chain are critical
determinants of activity and if the thioether could be bioisos-
terically substituted by an oxoether linkage. The geometry of
the Cys-Ser dipeptide unit also appears to be crucial in that
there are key interactions with conserved Tyr316 and Pro315
residues at the neck of the binding pocket in the crystal
structure.45 We note that the carboxylic acid of Cys is in
amide linkage with the amine of serine, and we wished to test
whether inversion of the orientation of Ser, achieved by the
coupling the carboxyl group of serine to the amino group of
cysteamine, could yield analogues that were TLR2 receptor
antagonists.
Results and Discussion
Much of the previous work in the literature concerning the
structure-activity relationships in lipopeptides has utilized
murine TLR2, and in an effort to confirm the minimal part-
structure of thelipopeptidethatwas TLR-agonistic on human
TLR2, we first targeted the stereoselective syntheses of 1,2-
dipalmitoyl-(R)-3-(2-aminoethylthio)propanediol and 1,2-di-
palmitoyl-(S)-3-(2-aminoethylthio)propanediol (6a and 6b,
Scheme 1) starting from the corresponding enantiopure 1,2-
isopropylideneglycerol. The (R)-cysteamine lipoamino acid
6a was weakly active (IC50 = 1 μM, Figure 1) relative to the
commercially available reference lipopeptide PAM2CSK4
(IC50 = 67 pM), while the (S)-analogue 6b was completely
inactive. Coupling L-serine to the free amine of the cysteamine
resulted in the lipodipeptides 8a and 8b; similar to the
cysteamine analogues, only 8a was active with an IC50 of 1.2
μM; it is noted that in these latter compounds, the dipeptide
unit is inverted, since, unlike in the naturally occurring and in
synthetic PAM2CSK4, it is the carboxyl group of the serine

3200 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Wu et al.
nonbulky side chains such as Gly,46,48-50 presumably as long
as steric interactions within the narrow neck region of the
binding pocket45 are not compromised. While the PAM2CS
methyl ester compounds are indeed highly active, they are
about a fifth as potent as the reference PAM2CSK4 lipopep-
tide. We have found that, as with other amphipathic com-
pounds that we have recently characterized,52 the apparent
differences in potencies are related, at least in part, to the
substantially higher binding of the more hydrophobic
PAM2CS analogues to albumin present in cell culture media,
details of which will be published elsewhere.
It was of particular interest to examine the role of the
thioether bridge between the diacyl and dipeptide units.
O-Alkylation of (R)-1,2-isopropylideneglycerol with racemic
ethyl 2,3-ethoxypropanoate yielded the key intermediate 15,
from which the oxoether-bridged compounds were obtained
(21a and 21b, Scheme 3) in which two of the three stereo-
centers were held fixed. The activity of 21a (lipopeptide
terminating with L-Ser) was about 8 orders of magnitude
lower (IC50 = 1.2 mM) than that of PAM2CSK4, while 21b
(D-Ser) was inactive at the highest concentration tested
(10 mM, Figure 1). While these results are indicative of the
absolute requirement of the thioether linkage, we do not yet
understand why the stereochemistry of the terminal Ser
residue is manifested in differential activity only in the ether-
linked analogues (21a, 21b) but not in the thioether-linked
analogues (14a-d).
We next tested whether the ester-linked palmitoyl groups
could be replaced with potentially hydrolytically more
stable ether and amide linkages without compromising acti-
vity. Synthesis of analogue 28 with the ether-linked C16
hydrocarbon appendages could not be obtained by direct
O-alkylation of an advanced intermediate such as the depro-
tected species from series 12 under a variety of conditions.
This necessitated the installation of the ether-linked C16
groups on the glycerol backbone (23) first, followed by
transformation of the free hydroxyl to the iodo intermediate
(25) and subsequent S-alkylation with cysteine (Scheme 4).
We found that the ether analogue 28 was entirely inactive
(Figure 2).
We next investigated the bioisoteric replacement of one
(internal, secondary alcohol-derived; Scheme 5) ester as well
as both of the esters (Scheme 6) with amide-linked hydro-
carbon chains. Synthesis of the monoamide analogue 37
proceeded smoothly starting from D-Ser-OMe (29). The dia-
mide analogue, 45, however, was more problematic to synthe-
size. Acetonide deprotection of the monoamide precursor 35,
followed by attempts at converting the free hydroxyl group to
the amine via an azide intermediate, was unsuccessful. The
strategy of first N-acylating (R)-methyl 2,3-diaminopropano-
ate withpalmitoylchlorideand then convertingthe estertothe
iodo via the alcohol as described in Scheme 5 was also not
fruitful because of unexpected difficulty in the conversion of
the alcohol to the iodo group. This is probably due either to
steric bulk of the long-chain amides or to an internal H-bond
between the free alcohol and one of the amide groups. We
therefore resorted to first installing the iodo on a N,N0-di-Boc-
protected 2,3-diaminopropane-1-ol, followed by S-alkylation
with N-Troc-L-Cys-OMe (Scheme 6), affording the required
orthogonality of the protecting groups. The amines on the
diaminopropanethiol fragment of compound 41 were then
acylated with hexadecanoic acid. Next, the base-labile N-Troc
protecting group on the cysteine unit had to be converted
to the N-Boc derivative in order to carry out a subsequent
Figure 1. TLR2 agonistic activities of the lipopeptide analogues.
HEK293 cells stably transfected with a TLR2-NF-κB-SEAP repor-
ter gene was used in a 384 well format. Data points represent the
mean and SD determined on triplicate samples.
that is coupled to the lipopeptide. The L-cysteine-containing
lipoamino acid, compound 11 (Scheme 2), was also found to
beweakly TLR2-agonistic, which isincontradistinctiontothe
virtually absent activity on murine cells described earlier.47
The results emphasize differences in ligand specificity between
murine and human TLR2. The difference in activity between
the (R)- and (S)-isomers, which was consistent with pre-
vious SAR results,30,51 served to focus our subsequent SAR
in which we elected to examine only the (R)-diacylthioglycerol
analogues.
The PAM2Cys-Ser compounds of the 14seriescomprisinga
combinationof L- orD- amino acidsat eitherpositionandwith
the carboxyl terminus as either the carboxylic acid or the
methyl ester (Scheme 2) were all highly active with the IC50
values in the midpicomolar range (Figure 1), signifying that
the stereochemistry of the dipeptide unit is noncritical as long
as the orientation of the amide bond is in the correct config-
uration as discussed earlier. These results are consonant with
the crystal structure of PAM2CSK4 complexed with TLR2, in
which the carbonyl Cys-Ser amide bond forms H-bonding
with Pro315 and the NH of the amide interacts with Y326, but
the side chains of the dipeptide unit show no significant van
der Waals interactions within the binding pocket.45 The
relative unimportance of the side chains of the dipeptide core
is also in agreement with observations that in naturally
occurring lipopeptides, the second amino acid is far
less conserved and can be replaced by other residues with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3201
Scheme 2a
a Reagents and conditions: (a) PPh3, I2, imidazole, toluene, 90 °C, 2 h; (b) Boc-L- or -D-Cys-OMe, TEA, DMF, 85 °C, 4 h; (c) (i) 70% AcOH,
room temp, 24 h; (ii) C15H31COCl, pyr, DMAP, DCM, 0 °C to room temp, 8 h; (iii) TFA, room temp, 30 min; (d) (i) LiOH, THF/H2O,
room temp, 10 h; (ii) H-L-Ser(Ot-Bu)-OtBu HCl or H-L-Ser(Ot-Bu)-OMe HCl or H-D-Ser(Ot-Bu)-OMe HCl, EDCI, EDIPA, DMAP, DCM,
3
3
3
0 °C to room temp, 8 h; (e) (i) 70% AcOH, room temp,12 h; (ii) C15H31COCl, pyr, DMAP, DCM, 0 °C to room temp, 10 h; (f) TFA, room temp,
30 min.
Scheme 3a
a Reagents and conditions: (a) ethyl 2,3-epoxypropanoate, BF3 OEt2, DCM, room temp, 5 h; (b) p-TsCl, TEA, DCM, 0 °C to room temp, 8 h;
(c) NaN3, DMF, room temp, 12 h; (d) (i) PPh3, H2O, THF, reflux, 6 h; (ii) Boc2O, DCM, room temp, 8 h; (e) (i) LiOH, THF/H2O, room temp, 10 h;
3
(ii) H-L-Ser(t-Bu)-OMe HCl or H-D-Ser(t-Bu)-OMe HCl, EDCI, EDIPA, DMAP, DCM, 0 °C to room temp, 8 h; (f) (i) 70% AcOH, room temp, 12 h;
3
3
(ii) C15H31COCl, pyr, DMAP, DCM, 0 °C to room temp, 10 h; (g) TFA, room temp, 30 min.

3202 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Wu et al.
Figure 2. TLR2 agonistic activities of the ether, monoamide, and diamide lipopeptide analogues. HEK293 cells stably transfected with a
TLR2-NF-κB-SEAP reporter gene was used in a 384-well format. Data points represent the mean and SD determined on triplicate samples.
Scheme 4a
a Reagents and conditions: (a) BnBr, NaH, DMF, 0 °C to room temp, 8 h; (b) (i) 70% AcOH, room temp, 12 h; (ii) C16H33I, NaH, DMF, 0 °C to room
temp, 8 h; (c) (i) Pd/C, H2, 8 h; (ii) TsCl, pyr, DMAP, CH3CN, 70 °C, 12 h; (d) I2, KI, DMF, 80 °C, 24 h; (e) Boc-L-Cys-OMe, TEA, DMF, 85 °C, 2 h;
(f) (i) LiOH, THF/H2O, room temp, 10 h; (ii) H-L-Ser(tBu)-OMe HCl, EDCI, EDIPA, DMAP, DCM, 0 °C to room temp, 8 h; (g) TFA, room temp, 30 min.
3
base-catalyzed deesterfication step. Coupling of the terminal
Ser residue proceeded smoothly.
We were also keen to test the possibility that some of the
inactive compounds could perhaps behave as TLR2 antago-
nists, particularly with 8a and 8b, since we had designed these
compounds with inverted cysteamine-serine amide bonds in
the hope that this would be the case. In this context that TLR2-
mediated immunopathology has been implicated in a number
of inflammatory bowel diseases,53-56 and the only reported
TLR2 receptor antagonists are rather weak lipolanthionine
derivatives.57 Although we were disappointed that 6a, 6b, 21a,
b, 28, 37, and 45 were all inactive as TLR2 receptor antago-
nists, the negative results add to the SAR data in that they
demonstrate that these compounds do not engage TLR2.
The monoamide derivative 37 was found to be partially
active (IC50 = 48 nM, Figure 2) although weaker by about 3
orders of magnitude compared to the reference lipopeptide
PAM2CSK4, while the diamide derivative 45 was completely
inactive. The progressive loss of activity from the monoamide
to the diamide analogue suggests that both ester groups are
important for maximal TLR2-stimulatory activity, although
this is yet to be formally tested by synthesizing the regioisomer
of 37 with the amide group replacing the external (primary
alcohol-derived) ester group.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3203
Scheme 5a
a Reagents and conditions: (a) C15H31COCl, aqueous NaHCO3, EtOAc, room temp, 1 h; (b) 2,2-DMP, PPTS, toluene, 90 °C, 22 h; (c) LiBH4, THF,
0 °C (3 h) to room temp (6 h); (d) PPh3, I2, toluene, 90 °C, 2 h; (e) Boc-L-Cys-OMe, TEA, DMF, 85 °C, 2 h; (f) (i) Ba(OH)2, CH3CN/H2O, 60 °C, 1 h;
(ii) H-L-Ser(t-Bu)-OMe HCl, EDCI, EDIPA, DMAP, DCM, 0 °C to room temp, 8 h; (g) (i) 70% AcOH, room temp, 12 h; (ii) C15H31COCl, pyr, DMAP,
3
DCM, 0 °C to room temp, 10 h; (h) TFA, room temp, 30 min.
Scheme 6a
a Reagents and conditions: (a) (i) (Boc)2O, TEA, DCM, room temp, 2 h; (ii) MeOH, EDCI, HOBt, TEA, DMAP, DCM, 10 h; (b) NaBH4, MeOH,
THF, reflux, 4 h; (c) I2, PPh3, imidazole, DCM, 0 °C to room temp, 2 h; (d) Troc-L-Cys-OMe, TEA, DMF, 85 °C, 2 h; (e) (i) TFA, room temp, 30 min; (ii)
C15H31COOH, EDCI, HOBt, TEA, DMAP, DMF, 60 °C, 10 h; (f) (i) Zn dust, AcOH, H2O, THF, room temp, 1 h; (ii) (Boc)2O, TEA, DCM, room temp,
1 h; (g) (i) Ba(OH)2, THF/H2O, 60 °C, 1 h; (ii) H-L-Ser(tBu)-OMe HCl, EDCI, HOBt, EDIPA, DMAP, DMF, 60 °C, 10 h; (h) TFA, room temp, 30 min.
3
In addition to the lipopeptides being potentially useful as
immunotherapeutic agentsfor cancer, they alsodisplay potent
adjuvant properties. Lipopeptidesgreatly enhance MHCclass
I restricted CTL proliferation to an immunodominant influ-
enza peptide Mx58-66 ina human autologous DC/CD8þ Tcell
coculture model.58 Lipopeptide-primed dendritic cells also
stimulate the proliferation of allogeneic naive CD8þ CTLs,58
and immunization of mice with influenza-specific peptide
results in greatly enhanced numbers of interferon-γ-secreting
CD8þ CTLs when lipopeptides were used as adjuvants.59
Similarly, MALP-2 induces robust, long-lived CTL responses
against HIV-1 Tat protein.60,61 The synthetic methods that we
have developed are easily scalable, and the availability of a
free amino group in PAM2CS should allow the facile intro-
duction of electrophilic labels such as isothiocyanate for
covalent coupling to vaccine antigens. Differential protein

3204 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Wu et al.
binding should also allow considerable control of pharmaco-
kinetic properties. These are currently being investigated.
adding 70% acetic acid (AcOH/H2O = 7:3) and stirring at room
temperature for 12 h. After removal of solvent under vacuum,
the reaction residue was dissolved in EtOAc and washed with
saturated NaHCO3 solution. The aqueous layer was extracted
thrice with EtOAc. The combined EtOAc layers were washed
with brine and dried over Na2SO4. After removal of solvent
under vacuum, the residue was purified by flash column chro-
matography (hexane/EtOAc = 3:1) to afford the title com-
pounds 4a and 4b (88-91%).
Experimental Section
Chemistry. All of the solvents and reagents used were ob-
tained commercially and used as such unless noted otherwise.
Moisture- or air-sensitive reactions were conducted under argon
atmosphere in oven-dried (120 °C) glass apparatus. Solvents
were removed under reduced pressure using standard rotary
evaporators. Flash column chromatography was carried out
using silica gel 635 (60-100 mesh), while thin-layer chromato-
graphy was carried out on silica gel CCM precoated aluminum
sheets. All yields reported refer to isolated material charac-
terized by 1H, 13C NMR, and mass spectrometry. Purity for all
final compounds was confirmed to be at least 97% by LC-MS
using a Zorbax Eclipse Plus 4.6 mm ꢀ 150 mm, 5 μm analyti-
cal reverse phase C18 column with H2O-isopropanol and
H2O-CH3CN gradients and an Agilent ESI-TOF mass spectro-
meter (mass accuracy of 3 ppm) operating in the positive ion
acquisition mode.
4a: 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 8.3, 1H), 7.34
(d, J = 8.0, 1H), 4.93 (s, 1H), 3.85-3.75 (m, 1H), 3.70 (dt, J =
5.2, 10.3, 1H), 3.55 (dd, J = 5.9, 11.3, 1H), 3.31 (d, J = 5.3, 2H),
2.77-2.57 (m, 4H), 1.42 (s, 9H). 13C NMR (126 MHz, CDCl3) δ
156.20, 79.80, 70.40, 65.26, 39.85, 35.53, 32.96, 28.40. MS (ESI)
calculated for C10H21NO4S, m/z 251.12, found 252.13 (M þ H)þ
and 274.11 (M þ Na)þ.
4b: 1H NMR (400 MHz, CDCl3) δ 7.50 (s, 1H), 6.98 (s, 1H),
4.87 (s, 1H), 3.79 (ddd, J = 4.2, 6.9, 12.0, 1H), 3.75-3.68 (m,
1H), 3.55 (dt, J = 5.6, 11.3, 1H), 3.32 (d, J = 3.9, 2H), 2.78-2.55
(m, 4H), 1.43 (s, 9H). MS (ESI) calculated for C10H21NO4S, m/z
251.34, found 252.13 (M þ H)þ and 274.12 (M þ Na)þ.
Syntheses of Compounds 5a ((R)-3-(2-(tert-Butoxycarbonyla-
mino)ethylthio)propane-1,2-diyl Dipalmitate) and 5b ((S)-3-(2-
(tert-Butoxycarbonylamino)ethylthio)propane-1,2-diyl Dipalmi-
tate). O-Palmitoylation of Compounds 5a and 5b. To a solution
of compounds 4a and 4b (200 mg, 0.80 mmol) in anhydrous
DCM (8 mL) at 0 °C were added pyridine (385 μL, 4.78 mmol)
and a catalytic amount of 4-(dimethylamino)pyridine (DMAP).
Palmitoyl chloride (876 mg, 3.19 mmol) was then added drop-
wise, and the mixture was brought to room temperature, after
which the reaction solution was stirred for 10 h. The reaction
mixture was sequentially washed with water, saturated NaH-
CO3, brine and then dried over Na2SO4. After removal of
solvent under vacuum, the residue was purified by flash column
chromatography (hexane/EtOAc = 8:1) to afford the title
compounds 5a and 5b as white solids (90-92%).
Syntheses of Compounds 2a ((R)-(2,2-Dimethyl-1,3-dioxolan-
4-yl)methyl 4-Methylbenzenesulfonate) and 2b ((S)-(2,2-Dimethyl-
1,3-dioxolan-4-yl)methyl 4-Methylbenzenesulfonate). O-Tosyla-
tion of (S)-(þ)- [1a] and (R)-(-)-2,2-Dimethyl-1,3-dioxolane-
4-methanol [1b]. To enantiopure (S)-(þ)- or (R)-(-)-2,2-dimeth-
yl-1,3-dioxolane-4-methanol (compounds 1a or 1b, respectively,
1.0 g, 7.57 mmol, Sigma-Aldrich, Inc., St. Louis, MO) dissolved
in anhydrous DCM (30 mL) and cooled to 0 °C was added
pyridine (1.22 mL, 15.14 mmol), followed by p-toluenesulfonyl
chloride (p-TsCl; 2.89 g, 22.70 mmol). The reaction solution was
brought to room temperature and stirred for 8 h. After removal
of solvent under vacuum, the residue was purified by flash col-
umn chromatography (hexane/EtOAc = 49:1) to afford the
literature compounds 2a (R) and 2b (S) in 96-98% yields.62,63
Syntheses of Compounds 3a ((R)-tert-Butyl 2-((2,2-Dimethyl-
1,3-dioxolan-4-yl)methylthio)ethylcarbamate) and 3b ((S)-tert-
Butyl 2-((2,2-Dimethyl-1,3-dioxolan-4-yl)methylthio)ethylcarbamate).
To a solution of 2a or 2b (800 mg, 2.80 mmol) in anhydrous
DMF (15 mL) cooled to 0 °C was added sodium hydride (NaH;
101 mg, 4.19 mmol). After the mixture was stirred for 30 min at
0 °C, N-Boc-cysteamine (N-Boc-2-aminoethanethiol; 595 mg,
3.36 mmol) was added dropwise. The reaction solution was then
brought to room temperature and stirred for 8 h. After un-
reacted NaH was quenched with 1 M HCl, the solvent was
removed under reduced pressure, and the residue was dissolved
in EtOAc and washed with water. The water layer was extracted
thrice with EtOAc. The combined organic layers were washed
with brine and dried over Na2SO4. After removal of solvent
under reduced pressure, the residue was purified by flash column
chromatography (hexane/EtOAc = 5:1) to afford the title
compounds 3a and 3b as colorless oils (90-94%).
5a: 1H NMR (400 MHz, CDCl3) δ 5.12 (m, 1H), 4.88 (s, 1H),
4.34 (dd, J = 3.5, 11.9, 1H), 4.15 (dd, J = 6.0, 11.9, 1H), 3.30 (d,
J = 6.1, 2H), 2.68 (dd, J = 6.5, 11.5, 4H), 2.38-2.24 (m, 4H),
1.62-1.54 (m, 4H), 1.42 (s, 9H), 1.25 (d, J = 10.6, 48H), 0.86 (t,
J = 6.9, 6H). 13C NMR (126 MHz, CDCl3) δ 173.42, 173.16,
155.77, 79.50, 70.30, 63.56, 39.59, 34.31, 34.12, 43.14, 31.94,
29.72, 29.69, 29.66, 29.61, 29.52, 29.46, 29.38, 29.31, 29.26,
29.15, 29.13, 29.09, 28.39, 24.91, 24.74, 22.71, 14.15. MS (ESI)
calculated for C42H81NO6S, m/z 727.58, found 728.59 (M þ H)þ
and 750.57 (M þ Na)þ.
5b: 1H NMR (400 MHz, CDCl3) δ 5.12 (m, 1H), 4.88 (s, 1H),
4.34 (dd, J = 3.5, 11.9, 1H), 4.15 (dd, J = 6.0, 11.9, 1H), 3.30 (d,
J = 6.1, 2H), 2.68 (dd, J = 6.4, 11.5, 4H), 2.34-2.25 (m, 4H),
1.64-1.56 (m, 4H), 1.42 (s, 9H), 1.23 (s, 48H), 0.86 (t, J = 6.8,
6H). MS (ESI) calculated for C42H81NO6S, m/z 727.58, found
728.59 (M þ H)þ and 750.57 (M þ Na)þ.
3a: 1H NMR (400 MHz, CDCl3) δ 4.98 (d, J = 27.4, 1H), 4.22
(p, J = 6.2, 1H), 4.07 (dd, J = 6.1, 8.3, 1H), 3.67 (dd, J = 6.4,
8.3, 1H), 3.35 (m, 2H), 2.78-2.58 (m, 4H), 1.41 (s, 9H), 1.40 (s,
3H), 1.32 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 155.78, 109.67,
79.48, 75.58, 68.82, 39.79, 33.01, 28.40, 26.90, 25.55. MS (ESI)
calculated for C13H25NO4S, m/z 291.15, found 292.16 (M þ H)þ
and 314.14 (M þ Na)þ.
General Procedure for N-Boc Deprotection. Syntheses of
Compounds 6a ((R)-3-(2-Aminoethylthio)propane-1,2-diyl Dipal-
mitate) and 6b ((S)-3-(2-Aminoethylthio)propane-1,2-diyl Dipal-
mitate). To 5a and 5b was added excess dry trifluoroacetic acid
(TFA), and the mixture was stirred at room temperature for
30 min. TFA was removed by purging nitrogen and the residue
was thoroughly washed with diethyl ether to obtain the title
compounds as flaky white solids (98-100%).
3b: 1H NMR (400 MHz, CDCl3) δ 4.92 (s, 1H), 4.23 (p, J =
6.2, 1H), 4.12-4.04 (m, 1H), 3.68 (dd, J = 6.4, 8.3, 1H), 3.31 (m,
2H), 2.79-2.58 (m, 4H), 1.43 (s, 9H), 1.41 (d, J = 0.4, 3H), 1.34
(s, 3H). 13C NMR (126 MHz, CDCl3) δ 155.78, 109.65, 79.45,
75.57, 68.81, 39.80, 24.95, 32.98, 28.40, 26.89, 25.55. MS (ESI)
calculated for C13H25NO4S, m/z 291.15, found 292.15 (M þ H)þ
and 314.14 (M þ Naþ).
6a: 1H NMR (400 MHz, DMSO) δ 7.83 (s, 1H), 5.18-5.06 (m,
1H), 4.30 (dd, J = 2.7, 11.9, 1H), 4.10 (dd, J = 7.2, 12.0, 1H), 2.99
(t, J = 7.2, 2H), 2.90-2.65 (m, 4H), 2.32-2.14 (m, 4H),
1.58-1.42 (m, 4H), 1.23 (s, 48H), 0.84 (t, J = 6.8, 6H). 13C
NMR (126 MHz, DMSO) δ 172.47, 172.29, 69.64, 63.47, 33.62,
33.53, 33.37, 31.29, 30.87, 29.06, 29.03, 29.01, 28.93, 28.75, 28.71,
28.50, 28.42, 28.38, 24.47, 24.38, 22.08, 13.91. MS (ESI) calcu-
lated for C37H73NO4S, m/z 627.53, found 628.51 (M þ H)þ.
Syntheses of Compounds 4a ((R)-tert-Butyl 2-(2,3-Dihydroxy-
propylthio)ethylcarbamate) and 4b ((S)-tert-Butyl 2-(2,3-Di-
hydroxypropylthio)ethylcarbamate). Acetonide deprotection of
compounds 3a and 3b (900 mg, 3.09 mmol) was achieved by
1
6b: H NMR (400 MHz, DMSO) δ 7.80 (s, 1H), 5.16-5.05
(m, 1H), 4.30 (dd, J = 2.8, 11.9, 1H), 4.10 (dd, J = 7.1, 12.0,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3205
1H), 2.99 (t, J = 7.1, 2H), 2.87-2.65 (m, 4H), 2.33-2.20 (m,
4H), 1.50 (d, J = 6.2, 4H), 1.23 (s, 48H), 0.84 (t, J = 6.8, 6H).
MS (ESI) calculated for C37H73NO4S, m/z 627.53, found 628.51
(M þ H)þ.
mmol).64 The reaction mixture was stirred at 90 °C for 2 h. After
removal of toluene under reduced pressure, the residue was
dissolved in DCM, washed with saturated Na2S2O3 (to quench
the unreacted iodine), brine, and dried over Na2SO4. The
solvent was then removed under vacuum and the residue was
purified by flash column chromatography (hexane/EtOAc =
49:1) to afford the literature compound 965 as a colorless liquid
Syntheses of Compounds 7a ((6R,13R)-6-(Hydroxymethyl)-
2,2-dimethyl-4,7-dioxo-3-oxa-11-thia-5,8-diazatetradecane-13,14-
diyl Dipalmitate) and 7b ((6S,13R)-6-(Hydroxymethyl)-2,2-di-
methyl-4,7-dioxo-3-oxa-11-thia-5,8-diazatetradecane-13,14-diyl
Dipalmitate). The terminal L-serine of the dipeptide unit was
appended to 6a and 6b by conventional solution phase coupling
procedures. To 6a and 6b (200 mg, 0.27 mmol) in anhydrous
DCM (8 mL) at 0 °C were added Boc-L-Ser-OH (Bachem
Americas, Inc., Torrance, CA, 83 mg, 0.40 mmol), (3-dimethyl-
aminopropyl)-N0-ethylcarbodiimide hydrochloride (EDCI; 103
mg, 0.54 mmol), N,N-diisopropylethylamine (EDIPA; 53.4 μL,
0.32 mmol), and a catalytic amount of DMAP. The reaction
mixture was stirred at 0 °C for 30 min, then brought to room
temperature, and stirred for an additional 8 h. After removal of
solvent under vacuum, the residue was purified by flash column
chromatography (hexane/EtOAc = 3:2) to afford the title
compounds 7a or 7b as white solids (75-80%).
1
(5.44 g, 99%). H NMR (400 MHz, CDCl3) δ 4.30-4.21 (m,
1H), 4.12 (dd, J = 6.1, 8.6, 1H), 3.76 (dd, J = 5.4, 8.6, 1H), 3.23
(dd, J = 4.6, 9.8, 1H), 3.12 (dd, J = 8.5, 9.8, 1H), 1.43 (s, 1H),
1.32 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 109.47, 74.63,
68.57, 26.11, 24.57, 5.67.
Syntheses of Compounds 10a ((R)-Methyl 2-(tert-Butoxy-
carbonylamino)-3-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl-
thio)propanoate) and 10b ((S)-Methyl 2-(tert-Butoxycarbonyl-
amino)-3-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methylthio)pro-
panoate). N-Boc protected L- and D-cysteine methyl esters were
first obtained by treating H-L- and D-Cys-OMe HCl (Sigma-
3
Aldrich) with di-tert-butyl dicarbonate (Boc2O, 2.0 equiv) in
DCM, followed by slow addition of triethylamine (TEA, 1.0
equiv) at 0 °C. DCM was removed under vacuum after the
reaction was stirred at 0 °C for 2 h, and the residue was then
purified by flash column chromatography with 10% EtOAc/
hexane). S-Alkylation of Boc-L-Cys-OMe and Boc-D-Cys-OMe
with 9 was then carried out. Compound 10 was synthesized as
follows. To a solution of 9 (900 mg, 3.72 mmol) in anhydrous
DMF (8 mL) was added TEA (1.55 mL, 11.15 mmol), followed
by Boc-L- or D-Cys-OMe (673 mg, 2.86 mmol). The reaction
solution was stirred at 85 °C for 4 h. After removal of DMF
under vacuum, the residue was purified by flash column chro-
matography (hexane/EtOAc = 9:1) to afford 10a or 10b,
respectively, as viscous oils (60-63%).
7a: 1H NMR (400 MHz, CDCl3) δ 6.92 (s, 1H), 5.54 (s, 1H),
5.11 (dt, J = 5.0, 9.8, 1H), 4.36 (dd, J = 3.3, 11.9, 1H), 4.16-
4.04 (m, 3H), 3.64 (s, 1H), 3.56-3.37 (m, 2H), 2.90 (s, 1H),
2.77-2.61 (m, 4H), 2.29 (td, J = 4.5, 7.5, 4H), 1.58 (s, 4H), 1.44
(s, 9H), 1.23 (s, 48H), 0.86 (t, J = 6.8, 6H). 13C NMR (126 MHz,
CDCl3) δ 172.30, 172.13, 170.33, 154.96, 79.32, 69.11, 62.38,
61.73, 53.57, 37.04, 33.11, 32.90, 31.01, 30.72, 28.50, 28.46,
28.44, 28.29, 28.16, 28.09, 27.92, 27.10, 23.68, 21.49, 12.92.
MS (ESI) calculated for C45H86N2O8S, m/z 814.61, found
837.56 (M þ Na)þ.
7b: 1H NMR (400 MHz, CDCl3) δ 6.91 (s, 1H), 5.53 (s, 1H),
5.12 (qd, J = 3.4, 6.5, 1H), 4.35 (dd, J = 3.4, 11.9, 1H), 4.16-4.03
(m, 3H), 3.64 (s, 1H), 3.57-3.34 (m, 2H), 2.91 (s, 1H), 2.78-2.61
(m, 4H), 2.29 (td, J = 4.1, 7.6, 4H), 1.60 (s, 4H), 1.44 (s, 9H), 1.23
(s, 48H), 0.86 (t, J = 6.8, 6H). MS (ESI) calculated for
C45H86N2O8S, m/z 814.61, found 837.56 (M þ Na)þ.
10a: 1H NMR (400 MHz, CDCl3) δ 5.39 (d, J = 7.1, 1H), 4.52
(d, J = 7.3, 1H), 4.21 (p, J = 6.2, 1H), 4.06 (dd, J = 6.1, 8.3,
1H), 3.74 (s, 3H), 3.66 (dd, J = 6.4, 8.3, 1H), 3.02 (d, J = 3.2,
2H), 2.73 (dd, J = 6.0, 13.4, 1H), 2.62 (dd, J = 6.5, 13.4, 1H),
1.43 (s, 9H), 1.40 (s, 3H), 1.33 (s, 3H). 13C NMR (126 MHz,
CDCl3) δ 171.49, 155.23, 109.73, 80.18, 75.64, 68.68, 53.45,
52.58, 38.78, 35.22, 28.31, 26.84, 25.55. MS (ESI) calculated for
C15H27NO6S, m/z 349.16, found 372.10 (M þ Na)þ.
Syntheses of Compounds 8a ((R)-3-(2-((R)-2-Amino-3-hydro-
xypropanamido)ethylthio)propane-1,2-diyl Dipalmitate) and 8b
((S)-3-(2-((R)-2-Amino-3-hydroxypropanamido)ethylthio)pro-
pane-1,2-diyl Dipalmitate). 7a and 7b were deprotected with TFA
as described earlier in the general procedure for N-Boc deprotec-
tion (see syntheses of 6a, 6b). The title compounds were obtained
as flaky white solids (99-100%).
10b: 1H NMR (400 MHz, CDCl3) δ 5.43 (d, J = 7.0, 1H), 4.52
(s, 1H), 4.26-4.18 (m, 1H), 4.06 (dd, J = 6.1, 8.3, 1H), 3.76 (d,
J = 11.0, 3H), 3.66 (dd, J = 6.5, 8.3, 1H), 3.02 (ddd, J = 5.2,
13.8, 19.4, 2H), 2.75 (dd, J = 5.9, 13.5, 1H), 2.63 (dd, J = 6.3,
13.5, 1H), 1.43 (s, 9H), 1.41 (s, 3H), 1.34 (s, 3H). 13C NMR (126
MHz, CDCl3) δ 171.49, 155.23, 109.73, 80.18, 75.64, 68.68,
53.45, 52.58, 35.78, 35.22, 28.31, 26.84, 25.55. MS (ESI) calcu-
lated for C15H27NO6S, m/z 349.16, found 372.10 (M þ Na)þ.
General Procedure for Acetonide Deprotection and Palmitoy-
lation of the 1,2-Isopropylideneglycerol Unit. Synthesis of Com-
pound 11 ((R)-3-((R)-2-Amino-3-methoxy-3-oxopropylthio)pro-
pane-1,2-diyl Dipalmitate). To 10a (200 mg, 0.57 mmol) was
added 15 mL of 70% acetic acid (AcOH/H2O = 7:3), and the
mixture was stirred at room temperature for 24 h. After com-
plete removal of AcOH and water under reduced pressure,
O-palmitoylation of the diol was carried out by sequential
addition of pyridine (278 μL, 3.44 mmol), a catalytic amount
of DMAP, followed by palmitoyl chloride (709 μL, 2.29 mmol)
to the diol dissolved in anhydrous DCM (15 mL), and the
mixture was precooled to 0 °C. After being stirred at 0 °C for
30 min, the reaction solution was brought to room temperature
and stirred for 8 h. After removal of solvent under vacuum, the
residue was purified by flash column chromatography (hexane/
EtOAc = 19:1) to afford the N-Boc-protected intermediate as a
colorless oil. Finally, N-Boc deprotection was carried out as
described earlier (see syntheses of 6a, 6b) to obtain the title
compound 11 as a white glassy solid (435 mg, 95%). 1H NMR
(400 MHz, CDCl3) δ 8.01 (s, 2H), 5.13 (s, 1H), 4.41-4.06 (m,
3H), 3.81 (s, 3H), 3.20 (d, J = 41.1, 2H), 2.74 (s, 2H), 2.29 (dd,
J = 7.9, 15.5, 4H), 1.58 (d, J = 4.7, 4H), 1.23 (s, 48H), 0.86
8a: 1H NMR (400 MHz, DMSO) δ 8.51 (t, J = 5.7, 1H), 8.08
(s, 1H), 5.47 (s, 1H), 5.08 (dd, J = 4.5, 11.4, 1H), 4.31 (dd, J =
2.7, 11.9, 1H), 4.10 (dd, J = 7.1, 12.0, 1H), 3.69 (dd, J = 9.1,
33.4, 3H), 3.28 (dd, J = 6.5, 13.3, 2H), 2.74 (ddd, J = 6.6, 14.1,
21.4, 2H), 2.62 (t, J = 6.9, 2H), 2.32-2.19 (m, 4H), 1.56-1.45
(m, 4H), 1.23 (s, 48H), 0.85 (t, J = 6.8, 6H). 13C NMR (126
MHz, DMSO) δ 172.50, 172.27, 166.69, 69.88, 63.45, 60.27,
54.35, 38.52, 33.55, 33.37, 31.28, 31.04, 30.80, 29.05, 29.02,
29.00, 28.92, 28.91, 28.74, 28.71, 28.70, 28.41, 28.36, 24.47,
24.39, 22.08, 13.92. MS (ESI) calculated for C40H78N2O6S,
m/z 714.56, found 715.54 (M þ H)þ.
8b: 1H NMR (400 MHz, DMSO) δ 8.51 (t, J = 5.4, 1H), 8.08
(s, 1H), 5.48 (s, 1H), 5.09 (d, J = 5.8, 1H), 4.30 (dd, J = 2.6, 11.9,
1H), 4.10 (dd, J = 7.1, 11.9, 1H), 3.85-3.57 (m, 3H), 3.32-3.26
(m, 2H), 2.80 (dd, J = 5.8, 14.1, 1H), 2.65 (ddd, J = 7.0, 13.9,
20.3, 3H), 2.31-2.22 (m, 4H), 1.50 (d, J = 6.8, 4H), 1.23 (s,
48H), 0.84 (t, J = 6.7, 6H). 13C NMR (126 MHz, DMSO) δ
172.85, 172.63, 167.10, 70.25, 63.84, 60.65, 54.71, 38.92, 33.92,
33.75, 31.66, 31.42, 31.17, 29.44, 29.41, 29.39, 29.30, 29.13,
29.10, 29.09, 28.80, 28.75, 24.80, 22.46, 14.18. MS (ESI) calcu-
lated for C40H78N2O6S, m/z 714.56, found 715.54 (M þ H)þ.
Synthesis of Compound 9 ((R)-4-(Iodomethyl)-2,2-dimethyl-
1,3-dioxolane). To a solution of (S)-(þ)-2,2-dimethyl-1,3-diox-
olane-4-methanol (compound 1a, 3.0 g, 22.70 mmol) in toluene
(50 mL) was added triphenylphosphine (PPh3; 37.15 g, 27.38
mmol), imidazole (4.64 g, 68.10 mmol), and iodine (7.0 g, 29.51

3206 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Wu et al.
(t, J = 6.8, 6H). 13C NMR (126 MHz, CDCl3) δ 173.66, 173.63,
168.19, 69.95, 63.47, 53.73, 52.50, 34.24, 34.02, 32.91, 32.46,
31.94, 29.72, 29.70, 29.68, 29.52, 29.38, 29.30, 29.13, 24.85,
22.70, 14.13. MS (ESI) calculated for C39H75NO6S, m/z
685.53, found 686.59 (M þ H)þ.
13a ((R)-3-((R)-2-(tert-Butoxycarbonylamino)-3-((S)-1,3-di-
tert-butoxy-1-oxopropan-2-ylamino)-3-oxopropylthio)propane-
1,2-diyl dipalmitate): 1H NMR (400 MHz, CDCl3) δ 7.02 (d, J =
7.9, 1H), 5.37 (s, 1H), 5.19-5.09 (m, 1H), 4.51 (dt, J = 2.9, 8.0,
1H), 4.31 (dd, J = 3.5, 11.9, 2H), 4.14 (dd, J = 5.9, 11.9, 1H), 3.76
(dd, J = 2.9, 8.8, 1H), 3.51 (dd, J = 3.0, 8.8, 1H), 2.93 (d, J = 6.2,
2H), 2.79 (d, J =6.1, 2H), 2.30 (ddd, J = 5.8, 10.8, 12.0, 4H), 1.59
(dd, J = 6.6, 12.8, 5H), 1.43 (d, J = 2.6, 17H), 1.23 (s, 48H), 1.12
(d, J = 2.6, 9H), 0.86 (t, J = 6.8, 6H). 13C NMR (126 MHz,
CDCl3) δ 173.40, 173.13, 170.13, 168.92, 81.97, 73.17, 70.32,
63.52, 61.97, 53.51, 35.61, 34.31, 34.11, 33.71, 33.13, 31.94, 29.72,
29.68, 29.66, 29.53, 29.38, 29.33, 29.32, 29.16, 29.15, 28.30, 28.02,
27.34, 24.90, 24.74, 22.71, 14.14. MS (ESI) calculated for
C54H102N2O10S, m/z 970.73, found 993.82 (M þ Na)þ.
General Procedure for Deesterification of 10a and 10b and
Subsequent Coupling of Serine. Syntheses of Compounds 12a-d.
10a and 10b were deesterified with aqueous LiOH (165 mg, 6.87
mmol dissolved in 6 mL of water/15 mL of THF) at room
temperature for 10 h. Then 1 M HCl solution was added (to
quench unreacted LiOH and to convert the lithium salt to the
free-acid form) until a pH of 4 was reached. After removal of
THF under vacuum, the residue was extracted thrice with DCM.
The combined DCM layers were washed with brine and dried
over Na2SO4. After removal of DCM under vacuum, H-L-
Ser(tBu)-OtBu HCl or H-L-Ser(tBu)-OMe HCl or H-D-Ser-
13b ((R)-3-((R)-3-((S)-3-tert-Butoxy-1-methoxy-1-oxopro-
pan-2-ylamino)-2-(tert-butoxycarbonylamino)-3-oxopropylthio)-
3
3
1
(tBu)-OMe HCl (all from Bachem Americas, Inc., Torrance,
propane-1,2-diyl dipalmitate): H NMR (400 MHz, CDCl3) δ
3
7.06 (d, J = 8.1, 1H), 5.17 (s, 1H), 4.69-4.58 (m, 1H), 4.31 (dd,
J = 3.5, 11.9, 2H), 4.15 (dd, J = 5.9, 11.9, 1H), 3.80 (dd, J = 3.0,
9.1, 1H), 3.72 (s, 3H), 3.55 (dd, J = 3.2, 9.1, 1H), 2.94 (d, J =
6.1, 2H), 2.79 (d, J = 5.9, 2H), 2.29 (td, J = 4.9, 7.5, 4H), 1.59 (s,
4H), 1.43 (s, 9H), 1.23 (s, 48H), 1.12 (s, 9H), 0.86 (t, J = 6.8, 6H).
13C NMR (126 MHz, CDCl3) δ 173.37, 173.14, 170.44, 170.30,
155.22, 73.51, 70.29, 63.49, 61.67, 53.14, 52.41, 35.52, 34.30,
34.09, 33.20, 31.93, 29.71, 29.51, 29.37, 29.32, 29.31, 29.15,
29.14, 28.29, 27.29, 24.90, 24.88, 22.70, 14.13. MS (ESI) calcu-
lated for C51H96N2O10S, m/z 928.68, found 951.57 (M þ Na)þ.
13c ((R)-3-((R)-3-((R)-3-tert-Butoxy-1-methoxy-1-oxopro-
pan-2-ylamino)-2-(tert-butoxycarbonylamino)-3-oxopropylthio)-
CA, 437 mg, 2.06 mmol) was coupled to the deesterified inter-
mediates as described earlier for the syntheses of 7a and 7b. After
removal of solvent under vacuum, the residue was purified by
flash column chromatography (hexane/EtOAc = 4:1) to afford
the corresponding compounds 12a-d as viscous oils (32-38%).
12a ((S)-tert-Butyl 3-tert-butoxy-2-((R)-2-(tert-butoxycarbo-
nylamino)-3-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methylthio)-
propanamido)propanoate): 1H NMR (400 MHz, CDCl3) δ 7.31
(s, 1H), 5.48 (s, 1H), 4.70-4.47 (m, 2H), 4.38-4.22 (m, 2H), 4.07
(dd, J = 6.1, 8.2, 1H), 3.79-3.71 (m, 1H), 3.67 (dd, J = 6.6, 8.2,
1H), 3.59-3.45 (m, 1H), 3.10 (d, J = 6.5, 1H), 2.96 (ddd, J =
6.1, 13.9, 43.9, 1H), 2.74 (ddd, J = 6.1, 13.5, 44.1, 1H), 1.43 (s,
18H), 1.33 (s, 3H), 1.22 (s, 3H), 1.11 (d, J = 4.0, 9H). MS (ESI)
calculated for C25H46N2O8S, m/z 534.30, found 557.29 (M þ
Na)þ.
1
propane-1,2-diyl dipalmitate): H NMR (400 MHz, CDCl3) δ
7.15-7.06 (m, 1H), 5.19-5.11 (m, 1H), 4.65 (d, J = 8.4, 1H),
4.31 (dd, J = 3.6, 11.9, 2H), 4.14 (dd, J = 5.9, 11.9, 1H), 3.81
(dd, J = 2.9, 9.0, 1H), 3.72 (s, 3H), 3.53 (dd, J = 3.3, 9.0, 1H),
2.94 (dd, J = 14.0, 20.2, 2H), 2.78 (s, 2H), 2.29 (dd, J = 7.5, 13.4,
4H), 1.59 (d, J = 6.7, 4H), 1.44 (s, 9H), 1.23 (s, 47H), 1.12 (d,
J = 1.9, 9H), 0.86 (t, J = 6.9, 6H). 13C NMR (126 MHz, CDCl3)
δ 173.76, 172.63, 171.22, 170.29, 73.10, 70.42, 63.59, 55.56,
52.96, 34.36, 34.05, 31.93, 30.96, 29.73, 29.68, 29.61, 29.55,
29.38, 29.32, 29.15, 19.12, 24.84, 22.70, 14.13. MS (ESI) calcu-
lated for C51H96N2O10S, m/z 928.68, found 951.57 (M þ Na)þ.
13d ((R)-3-((S)-3-((S)-3-tert-Butoxy-1-methoxy-1-oxopro-
pan-2-ylamino)-2-(tert-butoxycarbonylamino)-3-oxopropylthio)-
12b ((S)-Methyl 3-tert-butoxy-2-((R)-2-(tert-butoxycarbo-
nylamino)-3-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methylthio)pro-
panamido)propanoate): 1H NMR (400 MHz, CDCl3) δ 7.07 (d,
J = 8.3, 1H), 5.48 (s, 1H), 4.71-4.59 (m, 1H), 4.41-4.22 (m,
2H), 4.15-4.02 (m, 1H), 3.80 (dd, J = 2.9, 9.1, 1H), 3.72 (s, 3H),
3.68 (dd, J = 6.5, 8.3, 1H), 3.55 (dd, J = 3.2, 9.1, 1H), 3.06 (ddd,
J = 5.9, 12.2, 19.8, 1H), 2.98-2.87 (m, 1H), 2.85-2.76 (m, 1H),
2.71 (dt, J = 6.8, 13.5, 1H), 1.44 (s, 9H), 1.42 (s, 3H), 1.34 (s,
3H), 1.12 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 170.47, 170.41,
155.28, 109.70, 75.70, 74.81, 73.52, 68.73, 61.69, 54.10, 53.12,
52.43, 35.61, 35.22, 28.31, 27.30, 26.87, 25.56. MS (ESI) calcu-
lated for C22H40N2O8S, m/z 492.25, found 515.18 (M þ Na)þ.
12c ((R)-Methyl 3-tert-butoxy-2-((R)-2-(tert-butoxycarbo-
nylamino)-3-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methylthio)pro-
panamido)propanoate): 1H NMR (400 MHz, CDCl3) δ 7.19 (s,
1H), 5.55 (s, 1H), 4.71 (d, J = 8.2, 1H), 4.39 (s, 1H), 4.35-4.25
(m, 1H), 4.16-4.08 (m, 1H), 3.85 (d, J = 9.0, 1H), 3.76 (s, 3H),
3.70 (t, J = 7.3, 1H), 3.57 (d, J = 9.0, 1H), 3.13 (d, J = 15.0,
1H), 2.93 (dd, J = 6.1, 13.9, 1H), 2.86-2.71 (m, 2H), 1.48 (s,
9H), 1.45 (s, 3H), 1.38 (s, 3H), 1.15 (s, 9H). 13C NMR (126 MHz,
CDCl3) δ 170.60, 170.34, 155.39, 109.70, 75.52, 74.79, 73.50,
68.68, 61.75, 53.99, 52.86, 52.41, 35.51, 30.95, 28.31, 27.30,
26.88, 25.52. MS (ESI) calculated for C22H40N2O8S m/z
492.25, found 515.18 (M þ Na)þ.
1
propane-1,2-diyl dipalmitate): H NMR (400 MHz, CDCl3) δ
7.06 (d, J = 8.1, 1H), 5.17 (s, 1H), 4.69-4.58 (m, 1H), 4.31 (dd,
J = 3.5, 11.9, 2H), 4.15 (dd, J = 5.9, 11.9, 1H), 3.80 (dd, J = 3.0,
9.1, 1H), 3.72 (s, 3H), 3.55 (dd, J = 3.2, 9.1, 1H), 2.94 (d, J =
6.1, 2H), 2.79 (d, J = 5.9, 2H), 2.29 (td, J = 4.9, 7.5, 4H), 1.59 (s,
4H), 1.43 (s, 9H), 1.23 (s, 48H), 1.12 (s, 9H), 0.86 (t, J = 6.8, 6H).
MS (ESI) calculated for C51H96N2O10S, m/z 928.68, found
951.57 (M þ Na)þ.
General Procedure for One-Step Deprotection of N-Boc and
O-tBu. Syntheses of Compounds 14a-d. 13a-d were depro-
tected with dry TFA as described earlier (general procedure
for N-Boc deprotection) which allowed for the simultaneous
deprotection of the N-Boc and O-tBu groups. Consequently, 14a
was obtained as the -Ser-OH (free acid), while 14b-d were the
-Ser-OMe esters. All title compounds (14a-d) were obtained as
white glassy solids in near-quantitative yields.
12d ((S)-Methyl 3-tert-butoxy-2-((S)-2-(tert-butoxycarbonyl-
amino)-3-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methylthio)propan-
amido)propanoate): 1H NMR (400 MHz, CDCl3) δ 7.07 (d, J =
8.3, 1H), 5.48 (s, 1H), 4.71-4.59 (m, 1H), 4.41-4.22 (m, 2H),
4.15-4.02 (m, 1H), 3.80 (dd, J = 2.9, 9.1, 1H), 3.72 (s, 3H), 3.68
(dd, J = 6.5, 8.3, 1H), 3.55 (dd, J = 3.2, 9.1, 1H), 3.06 (ddd, J =
5.9, 12.2, 19.8, 1H), 2.98-2.87 (m, 1H), 2.85-2.76 (m, 1H), 2.71
(dt, J = 6.8, 13.5, 1H), 1.44 (s, 9H), 1.42 (s, 3H), 1.34 (s, 3H),
1.12 (s, 9H). MS (ESI) calculated for C22H40N2O8S, m/z 492.25,
found 515.18 (M þ Na)þ.
14a ((S)-2-((R)-2-Amino-3-((R)-2,3-bis(palmitoyloxy)pro-
pylthio)propanamido)-3-hydroxypropanoic acid): H NMR (400
1
MHz, DMSO) δ 9.10 (d, J = 7.4, 1H), 8.82 (d, J = 7.9, 1H), 8.23
(s, 1H), 5.16 (d, J = 7.5, 1H), 4.81-4.58 (m, 1H), 4.42-4.23 (m,
2H), 4.18-4.07 (m, 1H), 3.79 (dd, J = 4.8, 11.0, 1H), 3.70-3.58
(m, 1H), 3.07(dt, J= 6.7, 13.0, 1H), 2.94-2.65 (m, 3H), 2.31-2.13
(m, 4H), 1.55-1.44 (m, 4H), 1.22 (s, 48H), 0.84 (t, J = 6.7, 6H).
13C NMR (126 MHz, DMSO) δ 179.71, 177.74, 177.51, 176.39,
74.88, 68.75, 66.29, 60.04, 56.60, 38.87, 38.78, 38.61, 38.23, 36.53,
34.30, 34.25, 34.20, 34.17, 34.13, 34.03, 33.98, 33.95, 33.76, 33.68,
33.63, 29.71, 29.62, 27.32, 19.16. MS (ESI) calculated for
C41H78N2O8S, m/z 758.55, found 759.66 (M þ H)þ.
Syntheses of Compounds 13a-d. Acetonide deprotection fol-
lowed by O-palmitoylation of 12a-d were performed as per the
general procedure outlined earlier (synthesis of compound 11) to
afford the corresponding compounds 13a-d in 90-95% yields.

Article
14b ((R)-3-((R)-2-Amino-3-((S)-3-hydroxy-1-methoxy-1-oxo-
propan-2-ylamino)-3-oxopropylthio)propane-1,2-diyl dipalmi-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3207
(ddd, J = 3.4, 6.3, 8.4, 1H), 3.54-3.38 (m, 2H), 2.42 (s, 3H), 1.35
(s, 3H), 1.31 (s, 3H), 1.19 (t, J = 7.1, 3H). 13C NMR (126 MHz,
CDCl3) δ 165.34, 143.94, 132.12, 132.09, 128.55, 126.92, 108.23,
108.20, 75.73, 73.22, 71.20, 69.59, 65.31, 60.90, 25.48, 24.18,
20.49, 12.77. MS (ESI) calculated for C18H26O8, m/z 402.13,
found 425.13 (M þ Na)þ.
1
tate): H NMR (500 MHz, CDCl3) δ 8.21 (s, 1H), 5.28 (s, 1H),
5.20-5.07 (m, 1H), 4.79-4.60 (m, 2H), 4.47-4.26 (m, 2H),
4.14-4.04 (m, 1H), 3.99-3.90 (m, 1H), 3.74 (s, 3H), 3.24-2.97
(m, 2H), 2.75 (s, 2H), 2.36-2.23 (m, 4H), 1.57 (s, 4H), 1.23 (s,
48H), 0.86 (t, J = 7.0, 6H). 13CNMR(126 MHz, CDCl3) δ174.39,
173.97, 169.90, 70.27, 70.08, 63.65, 63.52, 61.90, 52.92, 34.32, 34.05,
31.93, 29.73, 29.68, 29.55, 59.53, 29.38, 29.0, 29.14, 29.11, 24.89,
24.86, 24.82, 22.70, 14.12. MS (ESI) calculated for C42H80N2O8S,
m/z 772.56, found 773.53 (M þ H)þ.
Synthesis of Compound 17 (Ethyl 2-Azido-3-(((S)-2,2-dimethyl-
1,3-dioxolan-4-yl)methoxy)propanoate). To a solution of com-
pound 16 (380 mg, 0.94 mmol) in anhydrous DMF (10 mL) was
added sodium azide (184 mg, 2.83 mmol). The reaction mixture
was stirred at room temperature for 12 h. After removal of DMF
under vacuum, the reaction residue was dissolved in DCM,
washed with water, brine, and dried over Na2SO4. After removal
of solvent under reduced pressure, the residue was purified by
flash column chromatography (hexane/EtOAc = 9:1) to afford
14c ((R)-3-((R)-2-Amino-3-((R)-3-hydroxy-1-methoxy-1-oxo-
propan-2-ylamino)-3-oxopropylthio)propane-1,2-diyl dipalmi-
tate): 1H NMR (400 MHz, CDCl3) δ 8.86-8.37 (m, 1H),
5.40-5.22 (m, 1H), 5.21-5.01 (m, 1H), 4.72-4.42 (m, 2H),
4.42-4.16 (m, 2H), 4.08 (s, 1H), 4.01-3.88 (m, 1H), 3.73 (s, 3H),
3.39-2.98 (m, 2H), 2.73 (s, 2H), 2.29 (s, 4H), 1.56 (s, 4H), 1.23 (s,
48H), 0.85 (t, J = 6.8, 6H). 13C NMR (126 MHz, CDCl3) δ
174.22, 173.92, 70.02, 69.83, 63.66, 63.60, 53.02, 34.30, 34.05,
31.94, 29.73, 29.69, 29.56, 29.55, 29.39, 29.32, 29.16, 29.13,
24.88, 24.83, 22.70, 14.13. MS (ESI) calculated for
C42H80N2O8S, m/z 772.56, found 773.53 (M þ H)þ.
1
the title compound 17 (245 mg, 95%). H NMR (400 MHz,
CDCl3) δ 4.29-4.18 (m, 3H), 3.74 (ddd, J = 6.2, 8.4, 11.2, 1H),
3.61-3.48 (m, 2H), 1.39 (d, J = 0.9, 3H), 1.32 (d, J = 5.0, 3H),
1.29 (t, J = 7.1, 3H). 13C NMR (126 MHz, CDCl3) δ 166.54,
107.70, 107.67, 72.67, 70.55, 69.89, 64.82, 60.53, 59.86, 24.94,
23.58, 12.37. MS (ESI) calculated for C11H19N3O5, m/z 273.13,
found 296.14 (M þ Na)þ.
14d ((R)-3-((S)-2-Amino-3-((S)-3-hydroxy-1-methoxy-1-oxo-
propan-2-ylamino)-3-oxopropylthio)propane-1,2-diyl dipalmi-
tate): 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 7.87 (s, 1H),
5.32 (s, 1H), 5.13 (s, 1H), 4.46 (d, J = 80.5, 3H), 4.13 (s, 1H), 3.99
(s, 2H), 3.78 (s, 3H), 3.23 (s, 2H), 2.78 (s, 2H), 2.33 (s, 4H),
1.61 (s, 4H), 1.61 (s, 3H), 1.27 (s, 48H), 0.89 (t, J = 6.7, 6H).
13C NMR (126 MHz, CDCl3) δ 174.49, 173.76, 70.42, 63.59,
55.56, 53.70, 52.96, 48.56, 34.36, 34.05, 31.93, 29.73, 29.68,
29.55, 29.38, 29.32, 29.15, 29.12, 24.84, 22.70, 14.13. MS
(ESI) calculated for C42H80N2O8S, m/z 772.56, found 773.53
(M þ H)þ.
Synthesis of Compound 18 (Ethyl 2-(tert-Butoxycarbo-
nylamino)-3-(((S)2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)pro-
panoate). Reduction of the Azide to the Amine under Staudinger
Conditions.67 To 17 (230 mg, 0.84 mmol) dissolved in THF
(8 mL) and H2O (30.3 mg, 1.68 mmol) was added PPh3 (265 mg,
1.01 mmol). The reaction mixture was refluxed for 6 h. After
removal of solvent, the reaction residue was dissolved in anhy-
drous DCM (5 mL) and di-tert-butyl dicarbonate (Boc2O; 551
mg, 2.52 mmol). TEA (351 μL, 2.52 mmol) was added, and the
mixture was stirred at room temperature for 8 h. The solvent
was removed under vacuum and the residue was purified by
flash column chromatography (hexane/EtOAc = 17:3) to af-
ford the title compound 18 (272 mg, 93%). 1H NMR (400 MHz,
CDCl3) δ 5.37 (t, J = 8.2, 1H), 4.42-4.32 (m, 1H), 4.26-4.13
(m, 3H), 3.99 (dd, J = 6.4, 8.3, 1H), 3.94-3.86 (m, 1H),
3.75-3.63 (m, 2H), 3.55-3.40 (m, 2H), 1.43 (s, 9H), 1.38 (d,
J = 1.9, 3H), 1.33 (d, J = 0.6, 3H), 1.25 (t, J = 7.1, 3H). 13C
NMR (126 MHz, CDCl3) δ 168.21, 153.15, 107.09, 77.59, 70.07,
69.60, 69.36, 64.18, 59.17, 51.70, 25.95, 24.32, 23.02, 11.81.
MS (ESI) calculated for C16H29NO7, m/z 347.19, found 370.28
(M þ Na)þ.
Synthesis of Compound 15 (Ethyl 3-(((S)-2,2-Dimethyl-1,3-
dioxolan-4-yl)methoxy)-2-hydroxypropanoate). To a solution of
ethyl 2,3-epoxypropanoate (Sigma-Aldrich, Inc., 375 mg, 2.91
mmol) in anhydrous DCM (10 mL) was added boron trifluoride
diethyl etherate (BF3 OEt2; 83 mg, 0.58 mmol), followed by
3
(S)-(þ)-2,2-dimethyl-1,3-dioxolane-4-methanol (1a, 1.20 g, 8.72
mmol).66 The reaction mixture was stirred at room temperature
for 5 h. After removal of solvent under reduced pressure, a
mixture of ethyl 3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-
2-hydroxypropanoate (compound 15) and (S)-(þ)-2,2-dimethyl-
1,3-dioxolane-4-methanol (unreacted starting material, 1a) were
obtained. The Rf values of both the required product and the
starting material were identical, which presented difficulty in
chromatographic isolation. The mixture was therefore subjected
to selective protection of the primary hydroxyl group of the
acetonide-protected glycerol 1a with TBDMSCl (tert-butyldi-
methylsilyl chloride), which allowed the facile isolation of com-
pound 15 by flash column chromatography using 20% EtOAc/
hexane (433 mg, 60%). 1H NMR (400 MHz, CDCl3) δ 4.34-4.22
(m, 4H), 4.05 (dd, J = 6.4, 8.3, 1H), 3.88-3.80 (m, 2H), 3.75
(ddd, J = 6.3, 7.5, 8.2, 1H), 3.66-3.52 (m, 2H), 3.15 (dd, J = 6.6,
15.6, 1H), 1.44-1.27 (m, 9H). 13C NMR (126 MHz, CDCl3) δ
171.49, 171.44, 108.42, 73.59, 73.55, 72.11, 72.04, 71.56, 69.95,
69.85, 65.57, 65.54, 60.85, 28.68, 25.68, 25.67, 24.37, 24.33, 13.17.
MS (ESI) calculated for C11H10O6, m/z 248.13, found 271.12
(M þ Naþ) and 519.245 (M þ M þ Na)þ.
Syntheses of Compounds 19a ((S)-Methyl 3-tert-Butoxy-
2-(2-(tert-butoxycarbonylamino)-3-(((S)-2,2-dimethyl-1,3-dioxo-
lan-4-yl)methoxy)propanamido)propanoate) and 19b ((R)-Methyl
3-tert-Butoxy-2-(2-(tert-butoxycarbonylamino)-3-(((S)-2,2-di-
methyl-1,3-dioxolan-4-yl)methoxy)propanamido)propanoate).
Deesterification of the ethyl ester of 18 and subsequent coupling
to H-L- and -D-Ser(tBu)-OMe HCl were performed as described
3
for the syntheses of compounds 12a-d to yield 19a and 19b
(70-75%).
19a: 1H NMR (400 MHz, CDCl3) δ 7.21 (s, 1H), 5.48 (s, 1H),
4.71-4.62 (m, 1H), 4.25 (dt, J = 5.9, 11.7, 2H), 4.06-3.98 (m,
1H), 3.89 (ddd, J = 2.4, 4.0, 9.5, 1H), 3.82-3.68 (m, 5H),
3.66-3.50 (m, 4H), 1.44 (s, 9H), 1.40 (d, J = 2.4, 3H), 1.33 (d,
J = 2.2, 3H), 1.15-1.08 (m, 9H). 13C NMR (126 MHz, CDCl3)
δ 168.88, 168.87, 168.81, 108.18, 73.14, 62.15, 71.24, 71.00,
69.90, 65.30, 65.06, 60.56, 51.78, 51.03, 28.38, 26.98, 25.97,
25.43, 24.10. MS (ESI) calculated for C22H40N2O9, m/z
476.27, found 499.19 (M þ Na)þ.
Synthesis of Compound 16 (Ethyl 3-(((S)-2,2-Dimethyl-1,3-
dioxolan-4-yl)methoxy)-2-tosyloxy)propanoate). O-Tosylation
was performed by sequentially adding TEA (590 μL, 4.23 mmol)
and p-TsCl (806 mg, 4.23 mmol) to a solution of 15 (350 mg, 1.41
mmol) in anhydrous DCM (10 mL) cooled to 0 °C. The reaction
mixture was brought to room temperature and stirred for 8 h.
After removal of solvent under vacuum, the residue was purified
by flash column chromatography (hexane/EtOAc = 9:1) to
19b: 1H NMR (400 MHz, CDCl3) δ 7.21 (s, 1H), 5.49 (s, 1H),
4.66 (ddd, J = 3.2, 7.2, 11.3, 1H), 4.25 (ddd, J = 5.7, 11.6, 17.0,
2H), 4.06-3.98 (m, 1H), 3.89 (ddd, J = 2.4, 4.0, 9.5, 1H),
3.84-3.76 (m, 1H), 3.75-3.67 (m, 4H), 3.64-3.50 (m, 4H), 1.44
(s, 9H), 1.42-1.38 (m, 3H), 1.36-1.31 (m, 3H), 1.13-1.08 (m,
9H). 13C NMR (126 MHz, CDCl3) δ 167.90, 167.83, 107.19,
72.16, 71.16, 70.27, 70.03, 68.94, 64.32, 64.09, 59.58, 50.80,
50.05, 27.40, 26.00, 24.99, 24.46, 23.12. MS (ESI) calculated
for C22H40N2O9, m/z 476.27, found 499.19 (M þ Na)þ.
1
afford the title compound 16 (443 mg, 78%). H NMR (400
MHz, CDCl3) δ 7.80 (d, J = 8.4, 2H), 7.31 (d, J = 8.0, 2H),
5.02-4.96 (m, 1H), 4.18-4.06 (m, 3H), 3.96-3.77 (m, 3H), 3.62

3208 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Wu et al.
Syntheses of Compounds 20a ((S)-3-(3-((S)-3-tert-Butoxy-1-
methoxy-1-oxopropan-2-ylamino)-2-(tert-butoxycarbonylamino)-
3-oxopropoxy)propane-1,2-diyl Dipalmitate) and 20b ((S)-3-(3-
((R)-3-tert-Butoxy-1-methoxy-1-oxopropan-2-ylamino)-2-(tert-
butoxycarbonylamino)-3-oxopropoxy)propane-1,2-diyl Dipalmi-
tate). Acetonide deprotection and O-palmitoylation were per-
formed as described earlier (see syntheses of 13a-d). 20a and
20b were obtained in 80-83% yield.
adding methanol to the reaction vessel at 0 °C. After removal of
solvent, the residue was dissolved in DCM and washed with
water (1ꢀ) and the resulting water layer was extracted with
DCM (3ꢀ). The combined DCM layers were washed with brine
and dried over Na2SO4. After removal of solvent, the residue
was purified by flash column chromatography (hexane/EtOAc
= 19:1) to yield the title compound 22 (330 mg, 98%). 1H NMR
(400 MHz, CDCl3) δ 7.36-7.30 (m, 4H), 7.29-7.24 (m, 1H),
4.62-4.51 (m, 2H), 4.33-4.25 (m, 1H), 4.04 (dd, J = 6.4, 8.3,
1H), 3.73 (dd, J = 6.3, 8.3, 1H), 3.54 (dd, J = 5.7, 9.8, 1H), 3.46
(dd, J = 5.5, 9.8, 1H), 1.40 (s, 3H), 1.35 (s, 3H). 13C NMR (126
MHz, CDCl3) δ 136.87, 127.39, 126.73, 126.71, 108.39, 73.68,
72.47, 70.00, 65.81, 25.74, 24.35. MS (ESI) calculated for
C13H18O3, m/z 222.13, found 245.13 (M þ Na)þ.
20a: 1H NMR (400 MHz, CDCl3) δ 7.11 (t, J = 8.3, 1H), 5.40
(s, 1H), 5.16 (dd, J = 3.3, 5.5, 1H), 4.66 (tt, J = 3.0, 8.8, 1H),
4.38-4.22 (m, 2H), 4.18-4.06 (m, 1H), 3.96-3.76 (m, 2H), 3.71
(d, J = 1.5, 3H), 3.66-3.49 (m, 4H), 2.28 (dd, J = 7.7, 15.5, 4H),
1.58 (d, J = 5.7, 4H), 1.44 (s, 9H), 1.23 (s, 48H), 1.13-1.08 (m,
9H), 0.85 (t, J = 6.8, 6H). 13C NMR (126 MHz, CDCl3) δ
172.18, 171.88, 169.40, 168.79, 154.25, 79.11, 72.29, 69.96, 69.69,
68.70, 68.63, 68.58, 68.55, 68.48, 68.44, 68.41, 61.19, 60.94,
51.90, 51.66, 51.17, 33.06, 32.91, 30.74, 28.52, 28.48, 28.32,
28.18, 18.12, 27.94, 27.11, 26.09, 23.70, 21.51, 12.94. MS
(ESI) calculated for C51H96N2O11, m/z 912.70, found 935.54
(M þ Na)þ.
Synthesis of Compound 23 ((R)-((2,3-Bis(hexadecyloxy)pro-
poxy)methyl)benzene). Deprotection of the acetonide protecting
group of 22 was achieved by stirring the compound (180 mg,
0.81 mmol) in 70% acetic acid (AcOH/H2O = 7:3) as described
for the syntheses of 4a and 4b. After complete removal of acetic
acid and water under reduced pressure, the residue was dissolved
in DMF (5 mL) and cooled to 0 °C. The resulting diol was
O-alkylated with 1-iodohexadecane (1.14 g, 3.24 mmol) in the
presence of NaH (60% in mineral oil, 770 mg) in anhydrous
DMF (8 mL) at 0 °C. The reaction mixture was stirred for 30 min
at 0 °C, and the temperature was then raised to room tempera-
ture for 8 h. The excess NaH was quenched by slowly adding
methanol to the reaction at 0 °C. After removal of solvent, the
residue was dissolved in DCM and washed with water (1ꢀ). And
the resulting water layer was extracted with DCM (3ꢀ). The
combined DCM layers were washed with brine and dried over
Na2SO4. After removal of solvent under vacuum, the resi-
due was purified by flash column chromatography (hexane/
EtOAc = 49:1) to afford a white solid (470 mg, 92%). 1H NMR
(400 MHz, CDCl3) δ 7.31 (d, J = 4.3, 4H), 7.28-7.24 (m, 1H),
4.54 (s, 2H), 3.62-3.44 (m, 7H), 3.41 (t, J = 6.7, 2H), 1.60-1.47
(m, 4H), 1.26 (d, 52H), 0.86 (t, J = 6.8, 6H). 13C NMR (126
MHz, CDCl3) δ 138.42, 128.31, 127.58, 77.88, 73.34, 71.66,
70.79, 70.62, 70.24, 31.93, 30.09, 29.72, 29.66, 29.52, 29.38,
26.10, 22.70, 14.14. MS (ESI) calculated for C42H78O3, m/z
630.60, found 653.61 (M þ Na)þ.
20b: 1H NMR (400 MHz, CDCl3) δ 7.11 (t, J = 9.0, 1H), 5.40
(s, 1H), 5.23-5.11 (m, 1H), 4.66 (tt, J = 3.0, 8.6, 1H), 4.30 (ddd,
J = 4.3, 10.2, 15.3, 2H), 4.11 (ddt, J = 5.0, 6.0, 10.3, 1H),
3.91-3.76 (m, 2H), 3.71 (d, J = 1.3, 3H), 3.68-3.49 (m, 4H),
2.28 (dd, J = 7.8, 15.5, 4H), 1.58 (dd, J = 6.9, 12.8, 4H), 1.44 (s,
9H), 1.23 (s, 48H), 1.14-1.07 (m, 9H), 0.85 (t, J = 6.8, 6H). 13
C
NMR (126 MHz, CDCl3) δ 171.84, 171.56, 168.96, 168.46,
153.92, 78.72, 71.96, 69.62, 69.40, 68.37, 68.30, 68.25, 68.22,
68.15, 68.08, 60.86, 60.31, 51.57, 51.32, 50.84, 32.74, 32.58,
30.41, 28.19, 28.15, 27.99, 27.85, 27.79, 27.62, 26.78, 25.72,
23.38, 21.18, 12.61. MS (ESI) calculated for C51H96N2O11, m/z
912.70, found 935.54 (M þ Na)þ.
Syntheses of Compound 21a ((S)-3-(2-Amino-3-((S)-3-hydro-
xy-1-methoxy-1-oxopropan-2-ylamino)-3-oxopropoxy)propane-
1,2-diyl dipalmitate, Trifluoroacetate) and 21b ((S)-3-(2-Amino-
3-((R)-3-hydroxy-1-methoxy-1-oxopropan-2-ylamino)-3-oxo-
propoxy)propane-1,2-diyl dipalmitate, Trifluoroacetate). One-
step deprotection of the N-Boc and O-tBu groups utilizing
TFA was carried out as described earlier (syntheses of 14a-d).
Title compounds were obtained as white glassy solid 21a or 21b
(99-100%).
Synthesis of Compound 24 ((R)-2,3-Bis(hexadecyloxy)propyl
4-Methylbenzenesulfonate). O-Debenzylation followed by
O-tosylation of 23 en route to the iodo intermediate 25 was
obtained as follows: Hydrogenolysis of 23 (500 mg, 0.79
mmol) dissolved in a mixture of 20 mL MeOH/DCM (1:1)
was carried out using Pd(OH)2/C (500 mg) and H2 at 55 psi in
a Parr apparatus for 8 h. The catalyst was removed by
filtration over Celite, and the solvent was removed to afford
the O-debenzylated intermediate as a white solid (425 mg,
∼100%). After drying completely, the solid was dissolved in
anhydrous CH3CN (15 mL), followed by addition of pyridine
(320 μL, 3.96 mmol) and a catalytic amount of DMAP. Then
p-TsCl (755 mg, 3.96 mmol) was added and the reaction
mixture was heated at 70 °C for 12 h. After removal of solvent
under vacuum, the residue was purified by flash column
chromatography (hexane/EtOAc = 17:1) to afford the title
compound 24 (527 mg, 96%). 1H NMR (400 MHz, CDCl3) δ
7.77 (d, J = 8.2, 2H), 7.31 (d, J = 8.5, 2H), 4.13 (dd, J = 4.1,
10.3, 1H), 4.00 (dd, J = 5.8, 10.3, 1H), 3.62-3.54 (m, 1H),
3.47-3.41 (m, 2H), 3.38 (t, 2H), 3.33 (t, J = 6.7, 2H), 2.42 (s,
3H), 1.45 (s, 4H), 1.23 (s, 52H), 0.86 (t, J = 6.8, 6H). 13C NMR
(126 MHz, CDCl3) δ 143.66, 131.93, 128.75, 126.98, 75.16,
70.75, 69.80, 68.64, 68.30, 30.91, 28.85, 28.69, 28.65, 28.63,
28.60, 28.50, 28.46, 28.44, 28.35, 25.01, 24.94, 21.68, 20.62,
13.11. MS (ESI) calculated for C42H78O5S, m/z 694.56, found
717.52 (M þ Na)þ.
21a: 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 8.03 (d, J =
28.1, 1H), 5.28 (s, 1H), 5.15 (s, 1H), 4.71 (dd, J = 69.3, 103.2,
2H), 4.17 (ddd, J = 7.3, 17.4, 18.6, 2H), 4.06-3.81 (m, 3H), 3.76
(d, J = 14.3, 3H), 3.58 (dd, J = 10.2, 15.7, 2H), 2.28 (dd, J =
7.3, 11.9, 4H), 1.56 (s, 4H), 1.23 (s, 48H), 0.86 (t, J = 6.8, 6H).
13C NMR (126 MHz, CDCl3) δ 172.76, 172.47, 172.41, 172.34,
168.73, 68.50, 68.31, 68.22, 67.59, 61.22, 61.10, 60.49, 54.08,
53.97, 51.92, 51.63, 51.47. 32.90, 32.78, 30.63, 28.42, 28.37,
28.24, 28.08, 28.02, 27.85, 27.80, 23.55, 21.40, 12.82. MS (ESI)
calculated for C42H80N2O9, m/z 756.59, found 757.45 (M þ H)þ.
21b: 1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 8.04 (s, 1H),
5.28 (s, 1H), 5.15 (s, 1H), 4.58 (d, J = 51.7, 2H), 4.18 (d, J =
42.7, 2H), 3.90 (s, 3H), 3.76 (d, J = 14.5, 3H), 3.59 (s, 2H), 2.28
(dd, J = 7.4, 11.7, 4H), 1.56 (s, 4H), 1.23 (s, 48H), 0.86 (t, J =
6.8, 6H). 13C NMR (126 MHz, CDCl3) δ 172.11, 171.82, 171.76,
171.69, 168.06, 67.84, 67.64, 67.55, 66.89, 60.55, 60.41, 59.84,
53.42, 53.32, 51.27, 50.98, 50.82, 32.24, 32.14, 29.97, 27.76,
27.72, 27.58, 27.42, 27.35, 27.19, 27.14, 22.89, 20.74, 12.16.
MS (ESI) calculated for C42H80N2O9, m/z 756.59, found
757.45 (M þ H)þ.
Synthesis of Compound 22 ((S)-4-(Benzyloxymethyl)-2,2-di-
methyl-1,3-dioxolane). O-Benzylation of 1a was performed by
first adding sodium hydride (NaH; 60% in mineral oil, 363 mg)
to a solution of (S)-(þ)-2,2-dimethyl-1,3-dioxolane-4-methanol
(1a, 200 mg, 1.51 mmol) in anhydrous DMF (5 mL) on ice. After
the mixture was stirred for 10 min, benzyl bromide (BnBr; 360
μL, 3.03 mmol) was added slowly to the reaction mixture. The
reaction was brought to room temperature after 30 min and
stirred for an additional 8 h. NaH was quenched by slowly
Synthesis of Compound 25 ((R)-1-(1-(Hexadecyloxy)-3-iodo-
propan-2-yloxy)hexadecane). The O-tosyl derivative 24 was
converted to the iodo derivative 25. Iodine (310 mg, 12.23 mmol)
and potassium iodide (KI; 2.03 g, 12.23 mmol) were added to a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3209
solution of 25 (850 mg, 1.22 mmol) in anhydrous DMF (15 mL),
and the mixture was stirred at 80 °C for 24 h. After removal of
DMF under vacuum, the residue was diluted with DCM,
washed with water (1ꢀ), brine (1ꢀ), and dried over Na2SO4.
The solvent was removed and the residue was purified by flash
column chromatography (hexane/EtOAc = 49:1) to yield the
title compound 25 (700 mg, 88%). 1H NMR (400 MHz, CDCl3)
δ 3.51 (m, 3H), 3.45-3.39 (m, 3H), 3.37-3.24 (m, 3H),
1.60-1.50 (m, 4H), 1.23 (s, 52H), 0.86 (t, J = 6.9, 6H). 13C
NMR (126 MHz, CDCl3) δ 76.40, 70.86, 70.73, 69.25, 30.91,
28.91, 28.69, 28.66, 28.65, 28.61, 28.45, 28.43, 28.35, 25.09,
25.07, 21.68, 13.11, 6.39. MS (ESI) calculated for C35H71IO2,
m/z 650.45, found 673.42 (M þ Na)þ.
chromatography (hexane/EtOAc = 7:3) to afford the title com-
pound as a white solid (286 mg, 77%). H NMR (500 MHz,
1
CDCl3) δ 6.61 (s, 1H), 4.65-4.58 (m, 1H), 3.92 (dd, J = 3.6, 11.1,
1H), 3.83 (d, J = 11.1, 1H), 3.73 (s, 3H), 2.25-2.18 (m, 3H),
1.64-1.54 (m, 2H), 1.27-1.18 (m, 24H), 0.83 (t, J = 6.9, 3H). 13
C
NMR (126 MHz, CDCl3) δ173.98, 171.65, 63.23, 54.59, 52.68,
36.48, 31.82, 29.70, 29.67, 29.66, 29.64, 29.52, 29.37, 29.36, 29.26,
25.59, 22.69, 14.12. MS (ESI) calculated for C20H39NO4, m/z
357.29, found 358.30 (M þ H)þ and 380.28 (M þ Na)þ.
Synthesis of Compound 31 ((R)-Methyl 2,2-Dimethyl-3-palmi-
toyloxazolidine-4-carboxylate). Compound 30 was acetonide
protected with 2,2-dimethyoxypropane (2,2-DMP; 24 mL,
195.8 mmol) and pyridinium p-toluenesulfonate (PPTS; 281
mg, 1.12 mmol) by adding the above reagents to compound 30
(2.0 g, 5.59 mmol) in toluene (25 mL), and the mixture was
refluxed at 90 °C for 22 h. After the mixture was concentrated
under reduced pressure, the residue was purified by flash column
chromatography (hexane/EtOAc = 9:1) to afford the title
Synthesis of Compound 26 ((6R,10R)-Methyl 10-(Hexadecyl-
oxy)-2,2-dimethyl-4-oxo-3,12-dioxa-8-thia-5-azaoctacosane-
6-carboxylate). S-Alkylation of Boc-L-Cys-OMe with 25 was
carried out as follows: Boc-L-Cys-OMe (752 mg, 3.20 mmol)
and TEA (445 μL, 3.20 mmol) were added to a solution of 25
(260 mg, 0.40 mmol) in anhydrous DMF (8 mL). The reaction
was stirred at 85 °C for 2 h. After removal of DMF under
vacuum, the residue was purified by flash column chromato-
graphy (hexane/EtOAc = 19:1) to afford the title compound 26
(291 mg, 96%). 1H NMR (500 MHz, CDCl3) δ 5.53 (d, J = 8.0,
1H), 4.51 (s, 1H), 3.73 (s, 3H), 3.48 (m, 5H), 3.40 (m, 6.7, 2H),
3.07-2.93 (m, 2H), 2.75 (dd, J = 4.9, 13.7, 1H), 2.63 (dd, J =
6.0, 13.7, 1H), 1.57-1.49 (m, 4H), 1.42 (s, 9H), 1.31-1.20 (m,
52H), 0.85 (t, J = 7.0, 6H). 13C NMR (126 MHz, CDCl3) δ
170.63, 154.23, 28.97, 77.55, 70.68, 70.28, 69.55, 52.47, 51.47,
34.44, 33.51, 30.90, 28.97, 28.69, 28.64, 28.35, 27.28, 25.08,
25.05, 21.68, 13.13. MS (ESI) calculated for C44H87O6S, m/z
757.63, found 780.61 (M þ Na)þ.
1
compound as a white solid (2.0 g, 90%). H NMR (500 MHz,
CDCl3) δ 4.42 (dd, J = 1.3, 6.3, 1H), 4.20 (dd, J = 1.4, 9.3, 1H),
4.14 (dd, J = 6.3, 9.3, 1H), 3.78 (s, 3H), 2.18-2.03 (m, 2H), 1.67
(s, 2H), 1.63-1.57 (m, 2H), 1.54 (s, 3H), 1.28-1.20 (m, 24H),
0.85 (t, J = 7.0, 3H). 13C NMR (126 MHz, CDCl3) δ 171.15,
170.17, 96.62, 66.99, 59.49, 52.89, 35.64, 31.93, 29.70, 29.66,
29.64, 29.56, 29.48, 29.37, 29.25, 25.15, 24.57, 23.57, 22.70,
14.13. MS (ESI) calculated for C23H43NO4, m/z 397.59, found
398.33 (M þ H)þ and 420.31 (M þ Na)þ.
Synthesis of Compound 32 ((S)-1-(4-(Hydroxymethyl)-2,2-di-
methyloxazolidin-3-yl)hexadecan-1-one). The ester functional
group of 31 was reduced to the primary alcohol by first diluting
lithium borohydride (LiBH4; 2.0 M in THF, 1.89 mL, 3.77
mmol) in 5 mL of THF (5 mL) cooled to 0 °C for 30 min.
Comound 31 (500 mg, 1.26 mmol), dissolved in THF (5 mL),
was then added dropwise, after which the mixture was stirred at
0 °C for 3 h and then maintained at room temperature for an
additional 6 h. After the unreacted LiBH4 was quenched with
water, the reaction mixture was extracted with EtOAc (3ꢀ) and
the combined EtOAc layers were washed with brine and dried
over Na2SO4. The solvent was removed under vacuum and the
resulting residue was purified by flash column chromatography
(hexane/EtOAc = 4:1) to yield a white solid (489 mg, 81%). 1H
NMR (500 MHz, CDCl3) δ 4.40 (s, 1H), 4.04 (d, J = 9.0, 1H),
4.01-3.88 (m, 2H), 3.64 (d, J = 10.5, 2H), 2.39-2.26 (m, 2H),
1.62 (s, 2H), 1.59 (d, J = 17.8, 4H), 1.52 (s, 2H), 1.25 (d, J =
22.7, 24H), 0.86 (t, J = 6.9, 3H). 13C NMR (126 MHz, CDCl3) δ
170.41, 95.27, 65.43, 62.93, 58.55, 35.49, 31.94, 29.71, 29.68,
29.65, 29.54, 29.38, 26.83, 25.20, 22.93, 22.71, 14.15. MS (ESI)
calculated for C23H43NO4, m/z 369.58, found 370.33 (M þ H)þ,
392.31 (M þ Na)þ.
Synthesis of Compound 27 ((5S,8R,12R)-Methyl 8-(tert-
Butoxycarbonylamino)-12-(hexadecyloxy)-2,2-dimethyl-7-oxo-
3,14-dioxa-10-thia-6-azatriacontane-5-carboxylate). Compound
26 was obtained as a viscous oil (228 mg, 64%) via procedures
similar to those described for the syntheses of compounds 12a-d.
1H NMR (500 MHz, CDCl3) δ 5.60 (s, 1H), 4.64 (d, J = 8.1, 1H),
4.30 (s, 1H), 3.79 (dd, J = 3.0, 9.1, 1H), 3.71 (s, 3H), 3.60-3.47
(m, 7H), 3.45-3.35 (m, 2H), 2.95 (dd, J = 5.8, 13.9, 1H), 2.87(dd,
J = 6.9, 13.8, 1H), 2.78 (s, 2H), 1.58-1.49 (m, 4H), 1.43 (s, 6H),
1.30-1.20 (m, 52H), 1.11 (s, 8H), 0.85 (t, J = 7.0, 6H). 13C NMR
(126 MHz, CDCl3) δ 169.58. 169.46, 154.24, 77.23, 72.40, 70.68,
70.27, 69.54, 60.72, 52.13, 51.33, 34.57 33.34, 30.90, 28.95, 28.69,
28.64, 28.62, 28.50, 28.35, 27.28, 26.26, 25.08, 25.03, 21.68, 13.13.
MS(ESI) calculatedfor C51H100N2O8S, m/z 900.72, found923.69
(M þ Na)þ.
Synthesis of Compound 28 ((S)-Methyl 2-((R)-2-Amino-3-
((R)-2,3-bis(hexadecyloxy)propylthio)propanamido)-3-hydroxy-
propanoate, Trifluoroacetate). N-Boc deprotection with neat
TFA was performed as described for the synthesis of 6a and
6b. 1H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 4.64 (s, 1H), 4.32
(s, 1H), 4.00-3.82 (m, 1H), 3.74 (s, 3H), 3.61-3.48 (m, 3H),
3.47-3.37 (m, 3H), 3.18-3.07 (m, 1H), 3.01-2.89 (m, 1H),
2.86-2.66 (m, 1H), 1.59-1.47 (m, 4H), 1.23 (s, 48H), 0.86 (t, J =
6.8, 6H). 13C NMR (126 MHz, CDCl3) δ 170.05, 77.85, 71.80,
71.12, 70.91, 61.75, 55.32, 53.06, 52.74, 34.98, 34.32, 31.93,
29.73, 29.68, 29.55, 29.51, 29.42, 29.38, 26.06, 25.87, 22.69,
14.12. MS (ESI) calculated for C42H84N2O6S, m/z 744.61, found
645.61 (M þ H)þ.
Synthesis of Compound 33 ((R)-1-(4-(Iodomethyl)-2,2-di-
methyloxazolidin-3-yl)hexadecan-1-one). Compound 33 (151
mg, 97%) was obtained following the procedure described for
the synthesis of 9. 1H NMR (400 MHz, CDCl3) δ 4.17-4.10 (m,
2H), 4.03-3.96 (m, 1H), 3.26 (dd, J = 9.9, 11.4, 1H), 3.16 (dt,
J = 2.5, 9.9, 1H), 2.34-2.16 (m, 2H), 1.67-1.62 (m, 3H),
1.53-1.46 (m, 3H), 1.39-1.16 (m, 26H), 0.86 (t, J = 6.9, 3H).
MS (ESI) calculated for C22H42INO2 m/z 479.23, found 502.21
(M þ Na)þ.
General Procedure for S-Alkylation. Synthesis of Compound
34 (Methyl 2-(tert-Butoxycarbonylamino)-3-(((R)-2,2-dimethyl-
3-palmitoyloxazolidin-4-yl)methylthio)propanoate). To a solu-
tion of 33 (300 mg, 0.63 mmol) in anhydrous DMF (8 mL)
was added TEA (872 μL, 6.26 mmol), followed by Boc-L-Cys-
OMe (1.03 g, 4.38 mmol). The reaction solution was stirred at
85 °C for 2 h. After removal of DMF under vacuum, the residue
was purified by flash column chromatography (hexane/EtOAc
= 9:1) to afford 34 as a viscous oil (356 mg, 97%). 1H NMR (400
MHz, CDCl3) δ 5.32-5.24 (m, 1H), 4.61-4.48 (m, 1H), 4.03 (d,
J = 9.2, 1H), 3.95-3.89 (m, 1H), 3.87-3.81 (m, 1H), 3.76 (s,
3H), 3.07-2.92 (m, 2H), 2.79-2.63 (m, 2H), 2.36-2.17 (m, 2H),
Synthesis of Compound 30 ((R)-Methyl 3-Hydroxy-2-pal-
mitamidopropanoate). N-Palmitoylation of 29 was carried out
as follows: H-D-Ser-OMe HCl (29) was N-acylated by first
3
generating the free base by the addition of 10 mL of saturated
aqueous NaHCO3 solution to a solution of 29 (200 mg, 1.04
mmol, Bachem) in EtOAc (10 mL) and then adding palmitoyl
chloride (377 μL, 1.24 mmol) dropwise to the reaction. The
reaction mixture, after being stirred for 1 h, was extracted
with EtOAc (3ꢀ). The combined EtOAc layers were washed
with brine and dried over Na2SO4. After removal of
solvent under vacuum, the residue was purified by flash column

3210 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Wu et al.
1.70-1.58 (m, 6H), 1.43 (s, 9H), 1.36-1.17 (m, 27H), 0.86 (t, J =
6.9, 3H). 13C NMR (126 MHz, CDCl3) δ 171.10, 169.73, 155.02,
95.54, 80.37, 66.36, 57.42, 53.39, 52.79, 35.86, 35.49, 35.46,
31.92, 29.70, 29.69, 29.68, 29.66, 29.59, 29.37, 28.27, 26.96,
(2.33 g, 10.67 mmol) in the presence of TEA (1.49 mL, 10.67
mmol) in DCM (10 mL). After the mixture was stirred at room
temperature for 2 h, the solvent was removed under vacuum and
the residue was purified by silica flash chromatography (DCM/
MeOH = 17:3). The free carboxyl group of the intermediate was
converted to the methyl ester by dissolving it in anhydrous
DCM (10 mL), followed by sequential addition of EDCI (1.11 g,
5.81 mmol), 1-hydroxybenzotriazole hydrate (HOBt; 785 mg,
5.81 mmol), TEA (810 μL, 5.81 mmol), anhydrous MeOH (353
μL, 8.71 mmol), and a catalytic amount of DMAP. After the
mixture was stirred at room temperature for 10 h, solvent was
removed from the reaction under vacuum and the reaction
residue was dissolved in DCM, washed with water (1ꢀ), brine
(1ꢀ), and dried over Na2SO4. The residue was purified by silica
flash chromatography (hexane/EtOAc = 22: 3) to afford the
title compound 38 (702 mg, 62%). 1H NMR (400 MHz, CDCl3)
δ 5.40 (s, 1H), 4.82 (s, 1H), 4.32 (s, 1H), 3.73 (s, 3H), 3.56-3.40
(m, 2H), 1.44-1.39 (m, 18H). 13C NMR (126 MHz, CDCl3) δ
171.30, 156.13, 155.41, 80.14, 79.85, 54.17, 52.65, 42.40, 30.95,
28.29, 28.28. MS (ESI) calculated for C14H26N2O6, m/z 318.18,
found 341.17 (M þ Na)þ and 659.35 (M þ M þ Na)þ.
25.08, 22.90, 22.70, 14.15. MS (ESI) calculated for C31H158
-
N2O6S, m/z 586.40, found 609.39 (M þ Na)þ.
Synthesis of Compound 35 ((S)-Methyl 3-tert-Butoxy-2-((R)-
2-(tert-butoxycarbonylamino)-3-(((R)-2,2-dimethyl-3-palmitoyl-
oxazolidin-4-yl)methylthio)propanamido)propanoate). Compound
34 (400 mg, 0.68 mmol) in 15 mL of THF was deesterified using
procedures as described for the syntheses of 12a-d with the
exception that barium hydroxide octahydrate (645 mg, 2.04 mmol,
in 6 mL H2O at 60 °C, 1 h), and not LiOH, was used; we elected to
use the much milder Ba(OH)2 conditions in view of potential
lability of the amide bond in 34. Subsequent coupling to H-L-
Ser(tBu)-OMe HCl was carried out as described earlier for 12a-d.
3
1
Compound 35 was obtained as a viscous oil (274 mg, 55%). H
NMR (400 MHz, CDCl3) δ 7.03 (d, J = 8.2, 1H), 5.40 (s, 1H), 4.63
(d, J = 8.2, 1H), 4.35-4.24 (m, 1H), 4.11- 4.02 (m, 1H), 3.96-
3,87 (m, 2H), 3.81 (dd, J = 2.8, 9.1, 1H), 3.74-3.69 (m, 3H), 3.54
(dd, J = 3.2, 9.1, 1H), 3.05-2.94 (m, 1H), 2.93-2.79 (m, 2H),
2.77-2.67 (m, 1H), 2.38-2.23 (m, 2H), 1.61 (s, 5H), 1.53-1.48 (m,
Synthesis of Compound 39 ((R)-tert-Butyl 3-Hydroxypropane-
1,2-diyldicarbamate). Sodium borohydride (NaBH4; 285 mg,
7.54 mmol) was added to a solution of 39 (600 mg, 2.20 mmol)
for reducing the ester to the corresponding primary alcohol. We
used NaBH4 as an alternative to LiBH4 described in the synth-
esis of 32 with significantly better yields. The reaction mixture
was refluxed at 75 °C, and then anhydrous methanol (3 mL) was
added dropwise over a period of 1 h. After refluxing for an
additional 3 h, the mixture was acidified to pH 2.0 with 1 M HCl
and THF was removed under vacuum. The residue was ex-
tracted with DCM (3ꢀ), and the combined DCM layers were
washed with brine and dried over Na2SO4. The residue was
purified by silica flash chromatography (hexane/EtOAc = 3:1)
to yield 39 as a colorless oil (525 mg, 96%). 1H NMR (400 MHz,
CDCl3) δ 5.10 (s, 1H), 4.97 (s, 1H), 3.76-3.45 (m, 4H),
3.41-3.16 (m, 2H), 1.58-1.36 (m, 18H). 13C NMR (126 MHz,
CDCl3) δ 157.75, 155.74, 80.41, 79.62, 61.56, 52.27, 40.12, 30.95,
28.36, 28.29. MS (ESI) calculated for C13H16N2O5, m/z 290.18,
found 313.18 (M þ Na)þ and 603.37 (M þ M þ Na)þ.
3H), 1.31-1.20 (m, 24H), 1.12 (s, 9H), 0.85 (t, J = 6.8, 3H). 13
C
NMR (126 MHz, CDCl3) δ 173.80, 170.63, 170.51, 170.02, 95.57,
80.38, 73.69, 73.58, 66.43, 63.58, 61.68, 61.61, 57.45, 53.15, 52.50,
52.48, 51.49, 36.71, 35.40, 35.13, 34.94, 32.92, 31.93, 30.95, 29.69,
29.66, 29.65, 29.54, 29.52, 29.38, 29.37, 29.31, 28.32, 28.28, 27.30,
27.28, 26.96, 25.71, 25.16, 22.96, 22.70. MS (ESI) calculated for
C38H71N3O8S, m/z 729.50, found 752.48 (M þ Na)þ.
Synthesis of Compound 36 ((5S,8R,12R)-Methyl 8-(tert-
Butoxycarbonylamino)-2,2-dimethyl-7,14-dioxo-12-(palmitoyl-
oxymethyl)-3-oxa-10-thia-6,13-diazanonacosane-5-carboxylate).
The general procedure of acetonide deprotection and subse-
quent O-palmitoylation described earlier for the synthesis of 11
was utilized. The reaction residue was purified by flash column
chromatography (hexane/EtOAc = 3:1) to afford the inter-
mediate as a colorless oil (203 mg, 80%). 1H NMR (500 MHz,
CDCl3) δ 6.21 (d, J = 7.9, 1H), 5.56 (d, J = 6.7, 1H), 4.68-4.58
(m, 1H), 4.43-4.31 (m, 2H), 4.21 (dd, J = 5.0, 11.3, 1H), 4.08
(dd, J = 4.9, 11.3, 1H), 3.81 (dd, J = 3.0, 9.1, 1H), 3.72 (s, 3H),
3.55 (dd, J = 3.3, 9.1, 1H), 3.01 (dd, J = 5.5, 13.9, 1H), 2.91-
2.79 (m, 2H), 2.75-2.64 (m, J = 5.4, 14.0, 1H), 2.35-2.26 (m,
2H), 2.23-2.10 (m, 2H), 1.63-1.55 (br s, 4H), 1.43 (s, 6H),
1.30-1.19 (m, 52H), 1.14-1.10 (m, 8H), 0.85 (t, J = 7.0, 6H).
13C NMR (126 MHz, CDCl3) δ 173.90, 173.29, 170.59, 170.47,
155.40, 80.21, 73.57, 64.46, 61.65, 53.72, 53.14, 52.47, 48.83,
36.70, 34.12, 31.93, 29.71, 29.67, 29.55, 29.50, 29.46, 29.42,
29.37, 29.30, 29.27, 29.18, 28.31, 27.27, 25.65, 24.88, 22.70,
14.15. MS (ESI) calculated for C51H97N3O9S, m/z 927.69, found
950.68 (M þ Na)þ.
Synthesis of Compound 40 ((R)-tert-Butyl 3-Iodopropane-1,2-
diyldicarbamate). A procedure essentially identical to that de-
scribed for the synthesis of 9 was followed; however, the reaction
was carried out at 0 °C and the mixture was then warmed to
room temperature instead of at 90 °C in an effort to minimize
side reactions and improve yield. No significant improvements
in yield, unfortunately, were obtained. Compound 40 was
obtained as a white solid (489 mg, 71%). 1H NMR (400 MHz,
CDCl3) δ 5.30 (s, 1H), 4.83 (s, 1H), 3.62 (s, 1H), 3.44-3.21 (m,
4H), 1.46 (s, 18H). 13C NMR (126 MHz, CDCl3) δ 155.76,
154.44, 79.00, 50.54, 43.16 29.92, 27.31, 27.19, 7.69. MS (ESI)
calculated for C13H25N2O4, m/z 400.09, found 423.09 (M þ
Na)þ and 823.18 (M þ M þ Na)þ.
Synthesis of Compound 37 ((R)-3-((R)-2-Amino-3-((S)-3-hy-
droxy-1-methoxy-1-oxopropan-2-ylamino)-3-oxopropylthio)-2-
palmitamidopropyl Palmitate, Trifluoroacetate). The previously
described N-Boc/O-tBu deprotection procedure (see syntheses
of 14a-d) was used. The title compound was obtained as a flaky
yellow solid (99%). 1H NMR (500 MHz, CDCl3) δ 8.46 (s, 1H),
6.57 (s, 1H), 4.73-4.59 (m, 1H), 4.53-4.34 (m, 1H), 4.34-4.23
(m, 1H), 4.23-4.15 (m, 1H), 4.15-4.06 (br s, 1H), 4.03-3.84 (m,
2H), 3.79-3.67 (m, 3H), 3.21-2.93 (m, 2H), 2.90-2.63 (m, 2H),
2.37-2.10 (m, 4H), 1.64-1.49 (m, 4H), 1.32-1.16 (m, 48H),
0.85 (t, J = 6.9, 6H). 13C NMR (126 MHz, CDCl3) δ 174.32,
170.36, 170.21, 64.40, 53.13, 52.76, 52.64, 50.29, 49.20, 36.61,
34.02, 31.94, 29.75, 29.73, 29.72, 29.70, 29.68, 29.60, 29.55,
29.38, 29.34, 29.28, 29.23, 29.20, 29.12, 25.70, 24.85, 24.78,
22.70, 14.13. MS (ESI) calculated for C42H81N3O7S, m/z
771.58, found 772.59 (M þ H)þ and 794.57 (M þ Na)þ.
Synthesis of Compound 38 ((R)-Methyl 2,2,11,11-Tetra-
methyl-4,9-dioxo-3,10-dioxa-5,8-diazadodecane-6-carboxylate).
D-2,3-Diaminopropionic acid monohydrochloride (Sigma-
Aldrich, 500 mg, 3.56 mmol) was N-Boc protected by Boc2O
Synthesis of Compound 41 ((6R,10R)-Methyl 10-(tert-Butoxy-
carbonylamino)-1,1,1-trichloro-15,15-dimethyl-4,13-dioxo-3,14-
dioxa-8-thia-5,12-diazahexadecane-6-carboxylate). Orthogonal
protection of the amines of the 2,3-diaminopropionic acid
fragment and of the amine of the cysteine unit was necessary.
We elected to utilize Troc-L-Cys-OMe, which was obtained from
L-cystine dimethyl ester (Sigma-Aldrich) as follows. 2,2,2-Tri-
chloroethyl chloroformate (805 μL, 5.86 mmol) was slowly
added to a stirring solution of L-cystine dimethyl ester dihy-
drochloride (500 mg, 1.47 mmol) in anhydrous DCM (10 mL)
and pyridine (10 mL) cooled to 0 °C. The mixture was brought to
room temperature and stirred for 8 h. After removal of solvent
under vacuum, the residue was purified by flash chromato-
graphy (hexane/EtOAc = 4:1) to afford N-Troc cystine dimethyl
ester as a white solid. The disulfide bond of the N-Troc-
protected cystine ester was reduced by dissolving the solid in
THF (10 mL) and adding tributylphosphine (Bu3P; 542 μL, 2.20

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3211
mmol) and H2O (132 μL, 7.33 mmol). The mixture was stirred at
room temperature for 2 h. After removal of solvent, the residue
was purified by flash column chromatography (hexane/EtOAc
= 9:1) to afford the title compound as a oil (410 mg, 90%). The
characterization data for Troc-L-Cys-OMe (Scheme 6, step d) is
provided in the Supporting Information. S-Alkylation of Troc-
L-Cys-OMe with 40 was performed as described earlier for 26.
Compound 41 was obtained as a mixture with the oxidized
N-Troc cystine dimethyl ester as ascertained by LC-MS and 1H
NMR. The Rf values of both components were virtually iden-
tical, rendering the isolation of 41 very difficult by flash chro-
matography. Reverse-phase HPLC employing several solvent
combinations also did not yield good separations. We reasoned,
however, that N-Troc cystine dimethyl ester would be inert to
both N-Boc deprotection and N-acylation conditions that were
to follow (see synthesis of 42 below). We therefore proceeded
without further purification.
Synthesis of Compound 42 ((6R,10R)-Methyl 1,1,1-Trichloro-
4,13-dioxo-10-palmitamido-3-oxa-8-thia-5,12-diazaoctacosane-6-
carboxylate). A crude sample of 41 (200 mg, 0.34 mmol) was
dissolved in excess dry TFA and stirred at room temperature for
30 min to effect N-Boc deprotection on the 2,3-diaminopropio-
nic acid fragment. Excess TFA was removed by purging nitro-
gen. The resulting diamino intermediate was coupled with
palmitic acid (264 mg, 1.03 mmol) using EDCI (197 mg, 1.03
mmol), HOBt (139 mg, 1.03 mmol), TEA (287 mL, 2.06 mmol),
and a catalytic amount of DMAP in anhydrous DMF (8 mL).
The mixture was stirred at 60 °C for 10 h. After DMF was
removed under reduced pressure, the residue was dissolved with
DCM, washed with water (1ꢀ), brine (1ꢀ), and dried over
Na2SO4. The residue was purified by silica flash chromatogra-
phy (hexane/EtOAc = 39:11) to afford the title compound 42
(185 mg, 64%). 1H NMR (500 MHz, CDCl3) δ 6.90 (d, J = 6.5,
1H), 6.24-6.14 (m, 1H), 4.82-4.73 (m, 1H), 4.73-4.63 (m, 1H),
4.62-4.54 (m, 1H), 3.98 (br s, 1H), 3.76 (d, J = 4.3, 3H),
3.53-3.43 (m, 1H), 3.41-3.30 (m, 1H), 3.12-2.97 (m, 2H),
2.92-2.78 (m, 2H), 2.57-2.43 (m, 1H), 2.23-2.09 (m, 4H),
1.63-1.50 (m, 4H), 1.22 (s, 48H), 0.85 (t, J = 6.9, 6H). 13C
NMR (126 MHz, CDCl3) δ 173.07, 171.90, 168.27, 151.86, 92.87
72.24, 52.01, 50.44, 48.60, 34.42, 34.16, 32.08, 31.71, 29.48,
27.27, 27.24, 27.22, 27.10, 27.07, 26.92, 26.85, 23.17, 20.25,
11.69. MS (ESI) calculated for C42H78Cl3N3O6S, m/z 857.47,
found 880.45, 881.45, 882.45 (M þ Naþ with expected chlorine
isotopic mass-spectral envelope).
Synthesis of Compound 43 ((6R,10R)-Methyl 2,2-Dimethyl-
4,13-dioxo-10-palmitamido-3-oxa-8-thia-5,12-diazaoctacosane-
6-carboxylate). The base-labile N-Troc protecting group on the
cysteine unit had to be converted to the N-Boc derivative in
order to carry out the subsequent base-catalyzed deesterfication
step (see synthesis of 44 below). Accordingly, 42 (350 mg, 0.41
mmol) was dissolved in THF (3 mL), and zinc dust (266 mg, 4.07
mmol), AcOH (15 mL), and H2O (1 mL) were added to the
reaction. After the mixture was stirred at room temperature for
1 h, zinc was filtered on Celite and the filtrate was concentrated
under reduced pressure. Reprotection with Boc2O was achieved
by dissolving the completely dried intermediate in DCM, adding
Boc2O (444 mg, 2.04 mmol) in the presence of TEA (284 μL, 2.04
mmol), and stirring the reaction mixture at room temperature
for 1 h. After the solvent was removed, the residue was purified
by silica flash chromatography (hexane/EtOAc = 7:3) to yield
the desired product 43 as a white solid (217 mg, 68%). 1H NMR
(400 MHz, CDCl3) δ 6.81 (s, 1H), 5.43 (s, 1H), 4.55 (s, 1H), 4.03
(s, 1H), 3.82-3.77 (m, 3H), 3.53 (s, 1H), 3.42 (s, 1H), 3.11-2.86
(m, 3H), 2.30-2.13 (m, 4H), 1.69-1.56 (m, 4H), 1.54-1.44
(m,7H), 1.27 (s, 48H). 13C NMR (126 MHz, CDCl3) δ 173.14,
141.89, 169.32, 169.22, 78.11, 72.52, 51.39, 50.52, 48.69, 48.31,
40.02, 39.64, 34.60, 34.46, 33.77, 33.19, 32.16, 29.76, 27.55,
27.52, 27.50, 27.38, 27.36, 27.21, 27.17, 27.14, 27.12, 26.13,
23.57, 23.50, 23.48, 23.37, 20.53. MS (ESI) calculated for
C44H85N3O6S, m/z 783.62, found 806.61 (M þ Na)þ.
Synthesis of Compound 44 ((5S,8R,12R)-Methyl 8-(tert-Butoxy-
carbonylamino)-2,2-dimethyl-7,15-dioxo-12-palmitamido-3-oxa-
10-thia-6,14-diazatriacontane-5-carboxylate). Deesterification
followed by serine coupling was carried out as described for
the synthesis of 35 (75%). 1H NMR (400 MHz, CDCl3) δ 6.91 (s,
1H), 6.52 (s, 1H), 5.52 (s, 1H), 4.68-4.61 (m, 1H), 3.83-3.77 (m,
1H), 3.74-3.69 (m, 3H), 3.59-3.51 (m, 1H), 3.50-3.32 (m, 2H),
2.98-2.83 (m, 2H), 2.23-2.09 (m, 4H), 1.65-1.51 (m, 4H),
1.46-1.40 (m, 8H), 1.22 (s, 48H), 1.14-1.09 (m, 9H), 0.85 (t, J =
6.8, 6H). 13C NMR (126 MHz, CDCl3) δ 174.15, 173.17, 169.65,
169.56, 154.54, 79.12, 72.61, 60.69, 52.18, 51.40, 50.55, 41.27,
35.75, 35.61, 30.91, 29.92, 28.69, 28.66, 28.65, 28.54, 28.52,
28.39, 28.38, 28.35, 28.32, 28.30, 27.32, 27.29, 26.26, 24.76,
24.67, 21.67, 13.11. MS (ESI) calculated for C51H98N4O8S, m/
z 926.71, found 949.70 (M þ Na)þ.
Synthesis of Compound 45 ((S)-Methyl 2-((R)-2-Amino-3-
((R)-2,3-dipalmitamidopropylthio)-propanamido)-3-hydroxypro-
panoate, Trifluoroacetate). Compound 45 was deprotected with
TFA as described earlier. The title compounds were obtained as
a flaky yellow solid (99%). 1H NMR (500 MHz, CDCl3) δ
7.21-7.11 (m, 1H), 7.20-7.11 (m, 1H), 4.70-4.54 (m, 1H),
4.51-4.37 (m, 1H), 3.90 (s, 2H), 3.71 (s, 3H), 3.49-3.35 (m, 1H),
3.33-3.22 (m, 1H), 3.21-3.01 (m, 1H), 2.80-2.53 (m, 1H), 2.15
(s, 3H), 1.53 (s, 3H), 1.25 (d, J = 21.5, 49H), 0.85 (t, J = 6.9,
6H). 13C NMR (126 MHz, CDCl3) δ 132.65, 131.11, 129.02,
68.37, 52.91, 38.92, 36.93, 36.56, 36.55, 32.15, 30.56, 30.37,
29.97, 29.88, 29.83, 29.76, 29.68, 29.60, 29.56, 29.54, 29.51,
29.47, 29.46, 29.13, 25.98, 25.86, 25.77, 23.94, 23.20, 22.92,
14.41, 14.34, 14.28, 11.17. MS (ESI) calculated for C42H82-
N4O6S, m/z 770.60, found 771.60 (M þ H)þ.
NF-KB Induction. The induction of NF-κB was quantified
using HEK-Blue-2 cells as previously described by us.42 HEK-
Blue-2 cells (HEK293 cells stably transfected with TLR2, MD2,
CD14, and sAP) were from InvivoGen (San Diego, CA) and
were maintained in HEK-Blue Selection medium containing
zeocin and normocin. Stable expression of secreted alkaline
phosphatase (sAP) under control of NF-κB/AP-1 promoters is
inducible by the TLR2 agonists, and extracellular sAP in the
supernatant is proportional to NF-κB induction. HEK-Blue-2
cells were incubated at a density of ∼105 cells/mL in a volume
of 80 μL/well, in 384-well, flat-bottomed, cell culture-treated
microtiter plates until confluency was achieved and subse-
quently graded concentrations of stimuli. sAP was assayed
spectrophotometrically using an alkaline phosphatase-specific
chromogen (present in HEK-detection medium as supplied by
the vendor) at 620 nm.
Acknowledgment. This work was supported in part by
NIH/NIAID Contract HHSN272200900033C and the Uni-
versity of Kansas Start-Up Funds.
Supporting Information Available: 1H, 13C, and mass spectral
data of all intermediates and final target compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Haux, J. Infection and cancer. Lancet 2001, 358, 155–156.
(2) Bauduer, F. Medicine in Ancient Egypt, a fascinating level of
modernity. Presse Med. 2001, 30, 1236–1239.
(3) Sprunt, W. H. Imhotep. N. Engl. J. Med. 1955, 253, 778–780.
(4) Coley, W. B. A Preliminary note on the treatment of inoperable
sarcoma by the toxic product of erysipelas. Post-Graduate 1893, 8,
278–286.
(5) Medzhitov, R.; Janeway, C. A., Jr. Innate immunity: impact on the
adaptive immune response. Curr. Opin. Immunol. 1997, 9, 4–9.
(6) Medzhitov, R.; Janeway, C. A., Jr. An ancient system of host
defense. Curr. Opin. Immunol. 1998, 10, 12–15.
(7) Janeway, C. A., Jr.; Medzhitov, R. Introduction: the role of innate
immunity in the adaptive immune response. Semin. Immunol. 1998,
10, 349–350.

3212 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Wu et al.
(8) Akira, S.; Takeda, K. Functions of toll-like receptors: lessons from
KO mice. C. R. Biol. 2004, 327, 581–589.
(9) Akira, S.; Uematsu, S.; Takeuchi, O. Pathogen recognition and
whereas TLR-4 and MD-2 are not involved. J. Biol. Chem. 2003,
278, 15587–15594.
(35) Opitz, B.;Schroder, N. W.;Spreitzer, I.;Michelsen, K. S.;Kirschning,
C. J.; Hallatschek, W.; Zahringer, U.; Hartung, T.; Gobel, U. B.;
Schumann, R. R. Toll-like receptor-2 mediates Treponema glycolipid
and lipoteichoic acid-induced NF-kappaB translocation. J. Biol.
Chem. 2001, 276, 22041–22047.
innate immunity. Cell 2006, 124, 783–801.
(10) Kirschning, C. J.; Schumann, R. R. TLR2: cellular sensor for
microbial and endogenous molecular patterns. Curr. Top. Micro-
biol. Immunol. 2002, 270, 121–144.
(11) Kaisho, T.; Akira, S. Toll-like receptor function and signaling.
(36) Buwitt-Beckmann, U.; Heine, H.; Wiesmuller, K. H.; Jung, G.;
Brock, R.; Ulmer, A. J. Lipopeptide structure determines
TLR2 dependent cell activation level. FEBS J. 2005, 272, 6354–
6364.
(37) Spohn, R.; Buwitt-Beckmann, U.; Brock, R.; Jung, G.; Ulmer, A.
J.; Wiesmuller, K. H. Synthetic lipopeptide adjuvants and Toll-like
receptor 2: structure-activity relationships. Vaccine 2004, 22,
2494–2499.
J. Allergy Clin. Immunol. 2006, 117, 979–987.
(12) Kawai, T.; Akira, S. TLR signaling. Semin. Immunol. 2007, 19,
24–32.
(13) Takeuchi, O.; Akira, S. Signaling pathways activated by micro-
organisms. Curr. Opin. Cell Biol. 2007, 19, 185–191.
(14) Kanzler, H.; Barrat, F. J.; Hessel, E. M.; Coffman, R. L. Ther-
apeutic targeting of innate immunity with Toll-like receptor ago-
nists and antagonists. Nat. Med. 2007, 13, 552–559.
(38) Nahori, M. A.; Fournie-Amazouz, E.; Que-Gewirth, N. S.; Balloy,
V.; Chignard, M.; Raetz, C. R.; Saint, G., I; Werts, C. Differential
TLR recognition of leptospiral lipid A and lipopolysaccharide in
murine and human cells. J. Immunol. 2005, 175, 6022–6031.
(39) Erridge, C.; Pridmore, A.; Eley, A.; Stewart, J.; Poxton, I. R.
Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachoma-
tis and Pseudomonas aeruginosa signal via toll-like receptor 2.
J. Med. Microbiol. 2004, 53, 735–740.
(40) Farhat, K.; Riekenberg, S.; Heine, H.; Debarry, J.; Lang, R.;
Mages, J.; Buwitt-Beckmann, U.; Roschmann, K.; Jung, G.;
Wiesmuller, K. H.; Ulmer, A. J. Heterodimerization of TLR2 with
TLR1 or TLR6 expands the ligand spectrum but does not lead to
differential signaling. J. Leukocyte Biol. 2008, 83, 692–701.
(41) Heine, H.; Ulmer, A. J. Recognition of bacterial products by toll-
like receptors. Chem. Immunol. Allergy 2005, 86 (99-119), 99–119.
(42) Kimbrell, M. R.; Warshakoon, H.; Cromer, J. R.; Malladi, S.;
Hood, J. D.; Balakrishna, R.; Scholdberg, T. A.; David, S. A.
Comparison of the immunostimulatory and proinflammatory
activities of candidate Gram-positive endotoxins, lipoteichoic acid,
peptidoglycan, and lipopeptides, in murine and human cells.
Immunol. Lett. 2008, 118, 132–141.
(15) Cheever, M. A. Twelve immunotherapy drugs that could cure
cancers. Immunol. Rev. 2008, 222, 357–368.
(16) Hunder, N. N.; Wallen, H.; Cao, J.; Hendricks, D. W.; Reilly, J. Z.;
Rodmyre, R.; Jungbluth, A.; Gnjatic, S.; Thompson, J. A.; Yee, C.
Treatment of metastatic melanoma with autologous CD4þ T cells
against NY-ESO-1. N. Engl. J. Med. 2008, 358, 2698–2703.
(17) Philippines Cholera Committee. A controlled field trial of the
effectiveness of cholera and cholera El Tor vaccines in the Philip-
pines. Preliminary Report. Bull. W. H. O. 1965, 32, 603-625.
(18) Vollmer, W.; Holtje, J. V. The architecture of the murein
(peptidoglycan) in Gram-negative bacteria: vertical scaffold or
horizontal layer(s)? J. Bacteriol. 2004, 186, 5978–5987.
(19) Dmitriev, B. A.; Toukach, F. V.; Holst, O.; Rietschel, E. T.; Ehlers,
S. Tertiary structure of Staphylococcus aureus cell wall murein.
J. Bacteriol. 2004, 186, 7141–7148.
(20) Burge, R. E.; Fowler, A. G.; Reaveley, D. A. Structure of
the peptidogylcan of bacterial cell walls. I. J. Mol. Biol. 1977,
117, 927–953.
(21) Baddiley, J. Bacterial cell walls and membranes. Discovery of the
teichoic acids. BioeEssays 1989, 10, 207–210.
(22) Coley, J.; Duckworth, M.; Baddiley, J. The occurrence of lipotei-
choic acids in the membranes of Gram-positive bacteria. J. Gen.
Microbiol. 1972, 73, 587–591.
(23) Stadelmaier, A.; Morath, S.; Hartung, T.; Schmidt, R. R. Synthesis
of the first fully active lipoteichoic acid. Angew. Chem., Int. Ed.
2003, 42, 916–920.
(24) Deininger, S.; Stadelmaier, A.; von Aulock, S.; Morath, S.;
Schmidt, R. R.; Hartung, T. Definition of structural prerequisites
for lipoteichoic acid-inducible cytokine induction by synthetic
derivatives. J. Immunol. 2003, 170, 4134–4138.
(25) Fischer, W. Lipoteichoic acid and lipids in the membrane of
Staphylococcus aureus. Med. Microbiol. Immunol. 1994, 183, 61–76.
(26) Kostyal, D. A.; Butler, G. H.; Beezhold, D. H. A 48-kilodalton
Mycoplasma fermentans membrane protein induces cytokine secre-
tion by human monocytes. Infect. Immun. 1994, 62, 3793–3800.
(27) Henderson, B.; Poole, S.; Wilson, M. Bacterial modulins: a novel
class of virulence factors which cause host tissue pathology by
inducing cytokine synthesis. Microbiol. Rev. 1996, 60, 316–341.
(28) Muhlradt, P. F.; Kiess, M.; Meyer, H.; Sussmuth, R.; Jung, G.
Isolation, structure elucidation, and synthesis of a macrophage
stimulatory lipopeptide from Mycoplasma fermentans acting at
picomolar concentration. J. Exp. Med. 1997, 185, 1951–1958.
(29) Muhlradt, P. F.; Meyer, H.; Jansen, R. Identification of S-(2,3-
(43) Schmidt, J.; Welsch, T.; Jager, D.; Muhlradt, P. F.; Buchler, M. W.;
Marten, A. Intratumoural injection of the toll-like receptor-2/6
agonist “macrophage-activating lipopeptide-2” in patients with
pancreatic carcinoma: a phase I/II trial. Br. J. Cancer 2007, 97,
598–604.
(44) Grabiec, A.; Meng, G.; Fichte, S.; Bessler, W.; Wagner, H.;
Kirschning, C. J. Human but not murine toll-like receptor 2
discriminates between tri-palmitoylated and tri-lauroylated pep-
tides. J. Biol. Chem. 2004, 279, 48004–48012.
(45) Jin, M. S.; Kim, S. E.; Heo, J. Y.; Lee, M. E.; Kim, H. M.; Paik, S.
G.; Lee, H.; Lee, J. O. Crystal structure of the TLR1-TLR2
heterodimer induced by binding of a tri-acylated lipopeptide. Cell
2007, 130, 1071–1082.
(46) Prass, W.; Ringsdorf, H.; Bessler, W.; Wiesmuller, K. H.; Jung, G.
Lipopeptides of the N-terminus of Escherichia coli lipoprotein:
synthesis, mitogenicity and properties in monolayer experiments.
Biochim. Biophys. Acta 1987, 900, 116–128.
(47) Bessler, W. G.; Cox, M.; Lex, A.; Suhr, B.; Wiesmuller, K. H.;
Jung, G. Synthetic lipopeptide analogs of bacterial lipoprotein are
potent polyclonal activators for murine B lymphocytes. J. Immu-
nol. 1985, 135, 1900–1905.
(48) Metzger, J.; Wiesmuller, K. H.; Schaude, R.; Bessler, W. G.; Jung,
G. Synthesis of novel immunologically active tripalmitoyl-S-gly-
cerylcysteinyl lipopeptides as useful intermediates for immunogen
preparations. Int. J. Pept. Protein Res. 1991, 37, 46–57.
(49) Metzger, J.; Jung, G. Mycoloylpeptides and other lipopeptide
adjuvants from higher aldoketene dimers. Angew. Chem., Int. Ed.
Engl. 1987, 26, 336–338.
(50) Metzger, J.; Olma, A.; Leplawy, M. T.; Jung, G. Synthesis of a
B-lymphocyte activating R-methylserine containing lipopentapep-
tide. Z. Naturforsch. B. 1987, 42, 1197–1201.
(51) Takeuchi, O.; Kaufmann, A.; Grote, K.; Kawai, T.; Hoshino, K.;
Morr, M.; Muhlradt, P. F.; Akira, S. Cutting edge: preferentially
the R-stereoisomer of the mycoplasmal lipopeptide macrophage-
activating lipopeptide-2 activates immune cells through a toll-like
receptor 2- and MyD88-dependent signaling pathway. J. Immunol.
2000, 164, 554–557.
dihydroxypropyl)cystein in a macrophage-activating lipopep-
tide from Mycoplasma fermentans. Biochemistry 1996, 35, 7781–
7786.
(30) Metzger, J.; Jung, G.; Bessler, W. G.; Hoffmann, P.; Strecker, M.;
Lieberknecht, A.; Schmidt, U. Lipopeptides containing 2-(pal-
mitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid: synthesis,
stereospecific stimulation of B-lymphocytes and macrophages,
and adjuvanticity in vivo and in vitro. J. Med. Chem. 1991, 34,
1969–1974.
(31) Lex, A.; Wiesmuller, K. H.; Jung, G.; Bessler, W. G. A synthetic
analogue of Escherichia coli lipoprotein, tripalmitoyl pentapeptide,
constitutes a potent immune adjuvant. J. Immunol. 1986, 137,
2676–2681.
(32) Dziarski, R.; Gupta, D. The peptidoglycan recognition proteins
(PGRPs). Genome Biol. 2006, 7, 232.
(33) Dziarski, R.; Gupta, D. Staphylococcus aureus peptidoglycan is a
toll-like receptor 2 activator: a reevaluation. Infect. Immun. 2005,
73, 5212–5216.
(52) Nguyen, T. B.; Suresh Kumar, E. V.; Sil, D.; Wood, S. J.; Miller, K.
A.; Warshakoon, H. J.; Datta, A.; David, S. A. Controlling plasma
protein binding: structural correlates of interactions of hydropho-
bic polyamine endotoxin sequestrants with human serum albumin.
Mol. Pharmaceutics 2008, 5, 1131–1137.
(53) Pierik, M.; Joossens, S.; Van Steen, K.; Van Schuerbeek, N.;
Vlietinck, R.; Rutgeerts, P.; Vermeire, S. Toll-like receptor-1, -2,
and -6 polymorphisms influence disease extension in inflammatory
bowel diseases. Inflammatory Bowel. Dis. 2006, 12, 1–8.
(34) Schroder, N. W.; Morath, S.; Alexander, C.; Hamann, L.; Har-
tung, T.; Zahringer, U.; Gobel, U. B.; Weber, J. R.; Schumann, R.
R. Lipoteichoic acid (LTA) of Streptococcus pneumoniae and
Staphylococcus aureus activates immune cells via Toll-like receptor
(TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3213
(54) Canto, E.; Ricart, E.; Monfort, D.; Gonzalez-Juan, D.; Balanzo, J.;
Rodriguez-Sanchez, J. L.; Vidal, S. TNF alpha production to
TLR2 ligands in active IBD patients. Clin. Immunol. 2006, 119,
156–165.
(55) Nara, K.; Kurokawa, M. S.; Chiba, S.; Yoshikawa, H.; Tsukikawa,
S.; Matsuda, T.; Suzuki, N. Involvement of innate immunity in the
pathogenesis of intestinal Behcet’s disease. Clin. Exp. Immunol.
2008, 152, 245–251.
(56) Albert, E. J.; Marshall, J. S. Aging in the absence of TLR2 is
associated with reduced IFN-gamma responses in the large intes-
tine and increased severity of induced colitis. J. Leukocyte Biol.
2008, 83, 833–842.
(57) Seyberth, T.; Voss, S.; Brock, R.; Wiesmuller, K. H.; Jung, G.
Lipolanthionine peptides act as inhibitors of TLR2-mediated IL-8
secretion. Synthesis and structure-activity relationships. J. Med.
Chem. 2006, 49, 1754–1765.
(58) Reschner, A.; Moretta, A.; Landmann, R.; Heberer, M.; Spagnoli,
G. C.; Padovan, E. The ester-bonded palmitoyl side chains of
Pam3CysSerLys4 lipopeptide account for its powerful adjuvanti-
city to HLA class I-restricted CD8þ T lymphocytes. Eur. J.
Immunol. 2003, 33, 2044–2052.
(59) Lau, Y. F.; Deliyannis, G.; Zeng, W.; Mansell, A.; Jackson, D. C.;
Brown, L. E. Lipid-containing mimetics of natural triggers of
innate immunity as CTL-inducing influenza vaccines. Int. Immu-
nol. 2006, 18, 1801–1813.
delivery of the HIV-1 Tat protein using the synthetic lipopeptide
MALP-2 as adjuvant. Eur. J. Immunol. 2003, 33, 1548–1556.
(61) Borsutzky, S.; Ebensen, T.; Link, C.; Becker, P. D.; Fiorelli, V.;
Cafaro, A.; Ensoli, B.; Guzman, C. A. Efficient systemic and
mucosal responses against the HIV-1 Tat protein by prime/boost
vaccination using the lipopeptide MALP-2 as adjuvant. Vaccine
2006, 24, 2049–2056.
(62) Zarbin, P. H.; Arrigoni, E. B.; Reckziegel, A.; Moreira, J. A.;
Baraldi, P. T.; Vieira, P. C. Identification of male-specific chiral
compound from the sugarcane weevil Sphenophorus levis. J. Chem.
Ecol. 2003, 29, 377–386.
(63) Zhou, D.; Lagoja, I. M.; Rozenski, J.; Busson, R.; Van Aerschot,
A.; Herdewijn, P. Synthesis and properties of aminopropyl nucleic
acids. ChemBioChem 2005, 6, 2298–2304.
(64) Enders, D.; Lenzen, A.; Backes, M.; Janeck, C.; Catlin, K.;
Lannou, M. I.; Runsink, J.; Raabe, G. Asymmetric total synthesis
of the 1-epi-aglycon of the cripowellins A and B. J. Org. Chem.
2005, 70, 10538–10551.
(65) Andre, C.;Bolte,J.;Demuynck,C.Synthesesof4-deoxy-D-fructose and
enzymic affinity study. Tetrahedron: Asymmetry 1998, 9, 3737–3739.
(66) Prestat, G.; Baylon, C.; Heck, M. P.; Grasa, G. A.; Nolan, S. P.;
Mioskowski, C. New strategy for the construction of a monote-
trahydrofuran ring in Annonaceous acetogenin based on a ruthe-
nium ring-closing metathesis: application to the synthesis of
Solamin. J. Org. Chem. 2004, 69, 5770–5773.
(60) Borsutzky, S.; Fiorelli, V.; Ebensen, T.; Tripiciano, A.; Rharbaoui,
F.; Scoglio, A.; Link, C.; Nappi, F.; Morr, M.; Butto, S.; Cafaro,
A.; Muhlradt, P. F.; Ensoli, B.; Guzman, C. A. Efficient mucosal
(67) Fernandez, R.; Ros, A.; Magriz, A.; Dietrich, H.; Lassaletta, J. M.
Enantioselective synthesis of cis-R-substituted cycloalkanols and
trans-cycloalkyl amines thereof. Tetrahedron 2007, 63, 6755–6763.