Structure-activity relationships in human toll-like receptor 2-specific monoacyl lipopeptides

Article abstract of DOI:10.1021/jm3000533

Toll-like receptor 2-agonistic lipopeptides typified by S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM₂CS) compounds are potential vaccine adjuvants. We had previously determined that at least one acyl group of optimal length (C₁₆) and an appropriately orientated ester carbonyl group is essential for TLR2-agonistic activity. We now show that these structurally simpler analogues display agonistic activities with human, but not murine, TLR2. SAR studies on the monoacyl derivatives show that the optimal acyl chain length is C₁₆, and aryl substituents are not tolerated. A variety of alkyl and acyl substituents on the cysteine amine were examined. All N-alkyl derivatives were inactive. In contradistinction, short-chain N-acyl analogues were found to be highly active, with a clear dependence on the chain length. A cysteine N-acetyl analogue was found to be the most potent (EC₅₀: 1 nM), followed by the N-butyryl analogue. The N-acetyl analogue is human TLR2-specific, with its potency comparable to that of PAM₂CS.

Full text of DOI:10.1021/jm3000533

Article
pubs.acs.org/jmc
Structure−Activity Relationships in Human Toll-like Receptor 2-
Specific Monoacyl Lipopeptides
Deepak B. Salunke, Nikunj M. Shukla, Euna Yoo, Breanna M. Crall, Rajalakshmi Balakrishna,
Subbalakshmi S. Malladi, and Sunil A. David*
Department of Medicinal Chemistry, University of Kansas, Multidisciplinary Research Building, Room 320D, 2030 Becker Drive,
Lawrence Kansas 66047, United States
S
* Supporting Information
ABSTRACT: Toll-like receptor 2-agonistic lipopeptides
typified by S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cystein-
yl-S-serine (PAM2CS) compounds are potential vaccine
adjuvants. We had previously determined that at least one
acyl group of optimal length (C16) and an appropriately
orientated ester carbonyl group is essential for TLR2-agonistic
activity. We now show that these structurally simpler
analogues display agonistic activities with human, but not
murine, TLR2. SAR studies on the monoacyl derivatives show
that the optimal acyl chain length is C16, and aryl substituents
are not tolerated. A variety of alkyl and acyl substituents on the
cysteine amine were examined. All N-alkyl derivatives were
inactive. In contradistinction, short-chain N-acyl analogues
were found to be highly active, with a clear dependence on the chain length. A cysteine N-acetyl analogue was found to be the
most potent (EC50: 1 nM), followed by the N-butyryl analogue. The N-acetyl analogue is human TLR2-specific, with its potency
comparable to that of PAM2CS.
able from host molecules and are collectively referred to as
pathogen-associated molecular patterns (PAMPs).8,9 There are
10 TLRs in the human genome; these are transmembrane
proteins with an extracellular domain having leucine-rich
repeats (LRR) and a cytosolic domain called the Toll/IL-1
receptor (TIR) domain.9
INTRODUCTION
■
Vaccines are perhaps one of the most successful medical
interventions against infectious disease.1 An important
component in the design of effective vaccines is the
incorporation of appropriate immune potentiators (also termed
adjuvants) along with the antigen; adjuvants initiate early innate
immune responses, which lead to the induction of robust and
long-lasting adaptive immune responses.2 More than eight
decades have elapsed since the discovery of adjuvanticity of
aluminum salts (primarily phosphate and hydroxide) by Glenny
and co-workers,3 and the repertoire of investigational adjuvants
has grown to encompass a very wide range of materials;4
however, aluminum salt-based mineral salts (generically, and
incorrectly, termed “alum”) have, until the recent introduction
of 3-O-desacyl-4′-monophosphoryl lipid A (MPL),5 remained
the only adjuvants currently approved by the FDA. Aluminum
salts have enjoyed a good safety record but are weak adjuvants
for antibody induction, promoting a TH2-skewed, rather than a
TH1 response.6,7 Furthermore, not only are aluminum salts
ineffective at inducing cytotoxic T lymphocyte (CTL) or
mucosal IgA antibody responses but also have an undesirable
propensity to induce IgE isotype switching, which has been
associated with allergic reactions in some subjects.6,7
The ligands for these receptors are highly conserved
microbial molecules such as lipopolysaccharides (LPS)
(recognized by TLR4), lipopeptides (TLR2 in combination
with TLR1 or TLR6), flagellin (TLR5), single-stranded RNA
(TLR7 and TLR8), double-stranded RNA (TLR3), CpG motif-
containing DNA (recognized by TLR9), and profilin present
on uropathogenic bacteria (TLR 11).10,11 TLR1, -2, -4, -5, and
-6 respond to extracellular stimuli, while TLR3, -7, -8, and -9
respond to intracytoplasmic PAMPs, being associated with the
endolysosomal compartment.9 The activation of TLRs by their
cognate ligands leads to production of inflammatory cytokines,
and up-regulation of MHC molecules and costimulatory signals
in antigen-presenting cells as well as activating natural killer
(NK) cells (innate immune response), in addition to priming
and amplifying T- and B-cell effector functions (adaptive
immune responses).12−15 Thus, TLR stimuli serve to link
innate and adaptive immunity13 and can therefore be exploited
Toll-like receptors (TLRs) are pattern recognition receptors
present on diverse cell types. TLRs recognize specific molecular
patterns present in molecules that are broadly shared by
pathogens but are sufficiently different so as to be distinguish-
Received: January 12, 2012
Published: March 2, 2012
© 2012 American Chemical Society
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a
Scheme 1. Syntheses of Monoacyl Lipopeptide 6d and Its Analogues
a
Reagents and conditions: (i) (a) Boc2O, Et3N, H2O, (b) H-Ser(tBu)-OMe·HCl, EDCI, HOBt, Et3N, DMF; (ii) Bu3P, CH2Cl2; (iii) 2-iodoethanol,
Et3N, DMF. For compounds 5a−5g: (iv) RCOCl, Et3N, DMAP, CH2Cl2. For compounds 5h: (v) RCOOH, HBTU, Et3N, DMAP, DMF; (vi)
CF3COOH.
as powerful adjuvants in eliciting both primary and anamnestic
immune responses.
also to attempt to understand the structural correlates
determining human versus murine TLR2 specificity.
We have recently begun to explore in detail structure−
activity relationships (SAR) of several immunostimulatory
chemotypes.16−24 Our attention has focused particularly on
agonists of TLR2, exemplified by the S-[2,3-bis(palmitoyloxy)-
(2RS)-propyl]-R-cysteinyl-S-serine (PAM2CS) chemotype,17,23
which distinguishes itself from virtually all other TLR agonists
in that although the lipopeptide is devoid of any detectable pro-
inflammatory activity in ex vivo human blood models (as
defined by the production of detectable levels of TNF-α, IL-1β,
IL-6, or IL-8),25 or of local reactogenicity and pyrogenicity in
rabbit models,18 it is potently adjuvantic in murine models of
immunization,25 suggesting that this chemotype may be a safe
and effective adjuvant. Extensive SAR on the PAM2CS class of
compounds23 led to simplified second-generation monoacyl
lipopeptides in which the spacing between the ester-linked acyl
group and the thioether was found to play a crucial role in
determining activity; homologation of the ethylene-bridged
compound (6d in Scheme 1) by one methylene unit resulted in
complete abrogation of activity.17
The structurally simpler, synthetically more accessible, and
water-soluble 6d with its potent TLR2-agonistic properties in
both primary screens employing human TLR2, as well as
secondary screens in ex vivo human blood models presented an
excellent lead. Prior to commencing immunization studies in
rodent and nonrodent models, this compound was subjected to
further evaluation. We found, entirely to our surprise, that
unlike PAM2CS, 6d showed exquisite specificity in activating
human TLR2 (hTLR2), but not murine TLR2 (mTLR2),
suggesting that the binding mode of 6d to TLR2 may be
substantially different from that of PAM2CS. This conjecture
was strengthened by the observation that N-acylation of 6d
with a palmitoyl group resulted in complete loss of hTLR2-
agonistic activity, which is unexpected given the pronounced
activity of the analogous, N-palmitoylated PAM2CS compound
(PAM3CS) and crystallographic evidence for the engagement
of the TLR2/TLR1 heterodimer by PAM3CS.26,27 These
preliminary findings warranted a detailed SAR on the
monoacylated lipopeptide chemotype, our goals being not
only to identify potentially more potent analogues of 6d but
RESULTS AND DISCUSSION
■
The SAR of the monoacyl derivatives began with a careful
exploration of the nature of the ester-linked acyl group on the
mercaptoethanol fragment of the lipopeptide; specifically, we
wished to determine the optimal chain length corresponding to
maximal TLR2-stimulatory activity and whether aryl groups
could substitute for the palmitoyl group. The syntheses of these
analogues were accomplished (Scheme 1) using the strategy
previously described by us;17 as mentioned earlier, 6d, with a
palmitoyl group (Scheme 1), was the point of departure.
Examination of this series of compounds showed a clear-cut
and straightforward SAR: 6d (palmitoyl) and 6e (stearoyl)
were active in engaging hTLR2 (3.63 and 4.79 nM, respectively,
Table 1) but entirely inactive in assays using mTLR2 (Figure
1). The shorter-chain analogues 6a−6c were completely
inactive (Table 1). Aryl substituents were found to abrogate
activity as exemplified by the complete absence of activity in the
biphenyl-4-carboxylate (6f) and biphenyl-3-carboxylate (6g) as
well as the p-terphenyl-4-carboxylate (6h) derived analogues.
The latter results were not unexpected given the dimensions of
the hydrophobic tunnel in the crystal structure of TLR2;26,28
however, we were also interested in examining the possibility of
these inactive analogues behaving as antagonists of TLR2
because there is only one known report of a low-potency
lanthionine-derived antagonist.29 None of the above-mentioned
inactive compounds were antagonistic in homotypic (using
PAM2CS as stimulus) or heterotypic (using lipoteichoic
acid30−32 as stimulus) assays in mTLR2 and hTLR2 assays
(data not shown).
Our previous SAR on the lipopeptides demonstrated the
absolute requirement of the ester-linked long-chain group and
of the importance of the orientation of the carbonyl group of
the ester; in these earlier studies, we had found that
replacement of the long-chain hydrocarbon functional group
with polar polyether or polyamine moieties were not
tolerated.17 We were interested in examining a more
conservative modification. We hypothesized that the replace-
ment of the palmitoyl group by a 2-(ditetradecylamino) acetate
fragment (10a, Scheme 2) may confer to the molecule dual
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Table 1. EC50 Values ( standard Error of Means) of
Compounds in Human TLR2-Specific Reporter Gene Assay
a
Figure 1. TLR2-specific NF-κB induction by selected analogues in
human and murine TLR2 reporter gene assays. Means and standard
deviations of quadruplicate samples are shown.
hTLR2/mTLR2 activity by mimicking PAM2CS. Our assump-
tion was incorrect because both 10a and its monoalkyl
homologue, 10b, were completely inactive (Table 1).
As mentioned earlier, N-palmitoylation of 6d (11g, Scheme
3) resulted in an unanticipated loss of TLR2-agonistic activity
(Table 1). We therefore examined in detail a variety of alkyl
and acyl substituents on the cysteine amine. The N-alkyl
derivatives 11a (ethyl), 11b (octyl), and 11c (hexadecyl)
analogues (obtained by either reductive amination using
appropriate aldehydes for 11a and 11b or by direct alkylation
in the case of 11c using hexadecyl bromide) were inactive
(Table 1). In contradistinction, the short-chain N-acyl
analogues were found to be highly active, with a clear
dependence on the chain length: the N-acetyl analogue 11d
was found to be the most potent (1 nM), followed by the N-
butyryl analogue 11e and N-octanoyl compound 11f (3.8 nM).
Further homologation (11g, palmitoyl) resulted in complete
loss of activity. Two points are to be noted; first, the active
analogues (11d−11f) retained specificity toward hTLR2 and
displayed no agonistic activity in mTLR2; second, while the
EC50 of the lead compound (6d: 3.6 nM) is, at first glance,
comparable to that of the N-acetyl derivative (11d: 1 nM), the
absolute magnitude of TLR2-induced nuclear translocation of
NF-κB is far greater for 11d and approaches that of PAM2CS
(Figure 1), indicating the higher potency of 11d.
The progressive decrease in activity with increasing N-acyl
chain length strongly suggested steric issues. We wanted to
confirm this hypothesis and also evaluate replacements of the
N-acetyl group with functionalities differing in electron-
withdrawing properties. The trifluoroacetamido derivative 11h
retains weaker activity (EC50: 4.5 nM, but low magnitude of
NF-κB activation, Figure 1), while the trichloroacetamido
analogue 11i is inactive; consistent with these results are the
observations that the methanesulfonamide (11j), trifluorome-
thanesulfonamide (11k), and p-toluenesulfonamide (11l)
a
ND denotes no activity detected at 10 μM.
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a
Scheme 2. Syntheses of Analogues of 6d
a
Reagents and conditions: (i) (a) 1-bromotetradecane, Et3N, DMF, (b) Boc2O, Et3N, CH2Cl2; (ii) LiOH, H2O, THF; (iii) (a) 4, EDCI, DMAP,
NMM, CH2Cl2, (b) CF3COOH.
Scheme 3. Syntheses of N-Alkyl, N-Acyl, and N-Sulfonyl Derivatives of 6d
a
Scheme 4. Syntheses of O-Acyl Derivatives of 6d
a
Reagents and conditions: (i) (Boc)2O, Et3N, CH2Cl2. For compound 13a: (ii) (CH3CO)2O, pyridine. For compound 13b−13c: (iii) RCl, Et3N,
THF; (iv) CF3COOH.
derivatives are all inactive. Taken together, these results
highlight the simultaneous influence of electronic and steric
effects of the substituents on the amine.
Given that the cysteine N-acyl substituents showed dramatic
differences in activity as described above, it was of interest to
explore serine O-acyl substituents as well. The syntheses of
these analogues (Scheme 4) were straightforward, requiring the
protection of the cysteine amine as the t-butyl carbamate,
followed by O-acylation with either anhydride (14a) or acyl
chlorides (14b, 14c). The activity of the O-acetyl analogue 14a
is virtually indistinguishable from that of 6d; progressive loss in
activity was evident with increasing acyl chain lengths (Figure 1
and Table 1). As was observed for all of the compounds
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a
Scheme 5. Syntheses of N-Acetyl Derivative of Homologue of 6d
a
Reagents and conditions: (i) (CH3CO)2O, Et3N, CH2Cl2.
under reduced pressure using standard rotary evaporators. Flash
column chromatography was carried out using RediSep Rf “Gold” high
performance silica columns on CombiFlash Rf instrument unless
otherwise mentioned, while thin-layer chromatography was carried out
on silica gel CCM precoated aluminum sheets. Purity for all final
compounds was confirmed to be greater than 97% by LC-MS using a
Zorbax Eclipse Plus 4.6 mm × 150 mm, 5 μm analytical reverse phase
C18 column with H2O−isopropanol or H2O−CH3CN gradients and
an Agilent ESI-TOF mass spectrometer (mass accuracy of 3 ppm)
operating in the positive ion (or negative ion, as appropriate)
acquisition mode.
described above, the O-acyl analogues were also found to be
specific for hTLR2.
Given that N-acetylation appeared to specifically enhance
TLR2-agonistic potency while preserving specificity for human
TLR2, it was interesting to examine if this modification on an
inactive homologue of 6d that we had previously identified17
would also result in augmented agonistic activity. The
propylene-bridged 3-((R)-2-amino-3-((S)-3-hydroxy-1-me-
thoxy-1-oxopropan-2-ylamino)-3-oxopropylthio)propyl palmi-
tate compound 15 was N-acetylated to furnish 16 as depicted
in Scheme 5. The N-acetylated derivative was found to be
active, albeit much weaker in potency than 11d (Table 1,
Figure 1), clearly emphasizing the role of N-acetyl group in
determining TLR2-agonistic potency, although the structural
basis for this observation remains to be elucidated.
General Procedure for the Syntheses of Compounds 5a−
5g. Synthesis of Compound 5a: 2-(((R)-3-(((S)-3-(tert-butoxy)-1-
methoxy-1-oxopropan-2-yl)amino)-2-((tert-butoxycarbonyl)-
amino)-3-oxopropyl)thio)ethyl butyrate. To a solution of
compound 4 (100 mg, 0.24 mmol) in anhydrous CH2Cl2 (5
mL) were added triethylamine (49 μL, 0.18 mmol) and 4-
dimethylaminopyridine (DMAP, 3.0 mg, 0.024 mmol), and the
reaction mixture was stirred at room temperature. After 10 min,
butyryl chloride (30 μL, 0.28 mmol) was added and the
reaction mixture was stirred for further 30 min. The solvent was
then removed using vacuum, and the residue was purified using
column chromatography (5% MeOH/CH2Cl2) to obtain the
CONCLUSION
■
Our continuing SAR studies on the TLR2-agonistic lipopeptide
chemotype have led to the identification of new analogues
possessing strong TLR2-agonistic activity that is exquisitely
human TLR2-specific. It is being increasingly appreciated that
significant differences between murine and nonrodent species
exist not only in receptor specificity to TLR ligands but also in
the cellular responses to them. As has been observed with
TLR4 ligands such as taxol,33 lipid IVa, and E5531, a synthetic
lipid A analogue,34 recent evidence suggests that significant
interspecies differences exist for TLR2 also, as exemplified by
variations in specificities for lipopeptide recognition in chimeric
TLR constructs.35 Furthermore, the coupling of these pattern
recognition receptors to downstream adaptor molecules also
appear to be distinct as shown by disparities in clinical
outcomes in humans with IRAK-4 (interleukin-1 receptor-
associated kinase 4) deficiency versus the susceptibility to
pathogens in knockout mice.36 We do not yet understand the
structural basis for this specificity, which will have to await
detailed crystallographic studies.
1
compound 5a (99 mg, 85%). H NMR (500 MHz, CDCl3) δ
7.13 (d, J = 7.8 Hz, 1H), 5.44 (s, 1H), 4.65 (dt, J = 8.1, 3.0 Hz,
1H), 4.33 (d, J = 4.5 Hz, 1H), 4.24 (t, J = 6.6 Hz, 2H), 3.82
(dd, J = 9.1, 2.9 Hz, 1H), 3.74 (s, 3H), 3.57 (dd, J = 9.1, 3.2 Hz,
1H), 3.01 (dd, J = 13.9, 5.6 Hz, 1H), 2.92 (dd, J = 13.9, 6.7 Hz,
1H), 2.84 (dd, J = 12.2, 6.1 Hz, 2H), 2.30 (t, J = 7.4 Hz, 2H),
1.65 (m, 2H), 1.45 (s, 9H), 1.13 (s, 9H), 0.94 (t, J = 7.4 Hz,
3H). 13C NMR (126 MHz, CDCl3) δ 173.61, 170.59, 170.50,
155.37, 80.40, 77.42, 77.42, 77.16, 76.91, 73.65, 63.15, 61.78,
53.84, 53.32, 52.57, 36.18, 34.98, 31.19, 28.43, 27.43, 18.50,
13.82. MS (ESI) calcd for C22H40N2O8S, m/z 492.25; found
515.24 (M + Na)+.
Compounds 5b, 5c, and 5e−5g were synthesized similarly to
compound 5a. Compound 5d was synthesized as published earlier.17
2-(((R)-3-(((S)-3-(tert-Butoxy)-1-methoxy-1-oxopropan-2-yl)-
amino)-2-((tert-butoxycarbonyl) amino)-3-oxopropyl)thio)ethyl oc-
1
tanoate (5b). Yield 119 mg, 92%. H NMR (500 MHz, CDCl3) δ
7.13 (d, J = 7.9 Hz, 1H), 5.44 (s, 1H), 4.65 (dt, J = 8.1, 3.0 Hz, 1H),
4.32 (s, 1H), 4.24 (t, J = 6.6 Hz, 2H), 3.82 (dd, J = 9.1, 2.9 Hz, 1H),
3.74 (s, 3H), 3.57 (dd, J = 9.1, 3.2 Hz, 1H), 3.01 (dd, J = 13.9, 5.5 Hz,
1H), 2.92 (dd, J = 13.9, 6.8 Hz, 1H), 2.83 (dd, J = 12.6, 6.3 Hz, 2H),
2.31 (t, J = 7.6 Hz, 2H), 1.61 (dd, J = 14.7, 7.4 Hz, 2H), 1.45 (s, 9H),
1.31−1.24 (m, 8H), 1.14 (s, 9H), 0.87 (t, J = 6.9 Hz, 3H). 13C NMR
(126 MHz, CDCl3) δ 174.05, 170.84, 170.76, 155.61, 80.64, 73.91,
63.39, 62.03, 54.09, 53.57, 52.82, 35.22, 34.57, 32.04, 31.42, 29.48,
29.32, 28.69, 27.68, 25.28, 22.99, 14.47. MS (ESI) calcd for
C26H48N2O8S, m/z 548.31; found 571.31 (M + Na)+.
As discussed above, TLR2 agonists appear unique among all
other TLR agonists in that although the lipopeptides are devoid
of any detectable pro-inflammatory activity in ex vivo human
blood models,25 or of local reactogenicity and pyrogenicity in
rabbit models,18 it is potently adjuvantic in murine models of
immunization,25 suggesting that this chemotype may be a safe
and effective adjuvant. The human-specific TLR2-agonistic
properties of the monoacyl lipopeptides precludes its evaluation
in murine models, and it remains to be examined if nonrodent
animal models (including nonhuman primates) would be
suitable surrogates to evaluate the safety and efficacy of these
analogues. These studies are currently underway.
2-(((R)-3-(((S)-3-(tert-Butoxy)-1-methoxy-1-oxopropan-2-yl)-
amino)-2-((tert-butoxycarbonyl) amino)-3-oxopropyl)thio)ethyl do-
1
decanoate (5c). Yield 124 mg, 87%. H NMR (500 MHz, CDCl3) δ
7.14 (d, J = 7.9 Hz, 1H), 5.44 (s, 1H), 4.65 (dt, J = 8.1, 3.0 Hz, 1H),
4.33 (d, J = 4.2 Hz, 1H), 4.23 (t, J = 6.6 Hz, 2H), 3.82 (dd, J = 9.1, 3.0
Hz, 1H), 3.74 (s, 3H), 3.57 (dd, J = 9.1, 3.2 Hz, 1H), 3.01 (dd, J =
13.9, 5.5 Hz, 1H), 2.92 (dd, J = 13.9, 6.8 Hz, 1H), 2.83 (dd, J = 12.6,
6.3 Hz, 2H), 2.31 (t, J = 7.6 Hz, 2H), 1.60 (dd, J = 14.6, 7.3 Hz, 2H),
1.45 (s, 9H), 1.30−1.23 (m, 16H), 1.14 (s, 9H), 0.87 (t, J = 7.0 Hz,
3H). 13C NMR (126 MHz, CDCl3) δ 174.05, 170.84, 170.76, 155.61,
80.65, 73.91, 63.39, 62.03, 54.09, 53.57, 52.82, 35.22, 34.58, 32.30,
EXPERIMENTAL SECTION
■
Chemistry. All of the solvents and reagents used were obtained
commercially and used as such unless noted otherwise. Moisture- or
air-sensitive reactions were conducted under nitrogen atmosphere in
oven-dried (120 °C) glass apparatus. The solvents were removed
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31.41, 30.00, 29.86, 29.73, 29.67, 29.54, 28.69, 27.68, 25.28, 23.08,
14.52. MS (ESI) calcd for C30H56N2O8S, m/z 604.38; found 627.37
(M + Na)+.
(t, J = 7.6 Hz, 2H), 1.64−1.55 (m, 2H), 1.35−1.18 (m, 8H), 0.87 (t, J
= 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.57, 170.31,
168.25, 62.64, 61.82, 55.38, 52.96, 52.82, 50.99, 34.30, 33.05, 31.78,
30.90, 29.21, 29.04, 24.97, 22.73, 14.19. MS (ESI) calcd for
C17H32N2O6S, m/z 392.20; found 393.21 (M + H)+.
2-(((R)-2-Amino-3-(((S)-3-hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-3-oxopropyl)thio)ethyl dodecanoate (6c). 1H NMR (500
MHz, CDCl3) δ 8.49 (d, J = 7.3 Hz, 1H), 4.68 (s, 1H), 4.36 (s, 1H),
4.23 (t, J = 6.0 Hz, 2H), 3.93 (d, J = 9.5 Hz, 1H), 3.86 (dd, J = 11.1,
4.7 Hz, 1H), 3.76 (s, 3H), 3.16 (dd, J = 14.3, 5.2 Hz, 1H), 3.02 (dd, J
= 14.2, 6.9 Hz, 1H), 2.81 (t, J = 6.4 Hz, 2H), 2.31 (t, J = 7.7 Hz, 2H),
1.59 (dd, J = 14.3, 7.2 Hz, 2H), 1.31−1.22 (m, 16H), 0.87 (t, J = 7.0
Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.83, 170.54, 168.51,
62.89, 62.07, 55.65, 53.22, 53.07, 34.57, 33.29, 32.31, 31.15, 30.03,
30.02, 29.90, 29.75, 29.69, 29.55, 25.24, 23.08, 14.52. MS (ESI) calcd
for C21H40N2O6S, m/z 448.26; found 449.27 (M + H)+.
2-(((R)-3-(((S)-3-(tert-Butoxy)-1-methoxy-1-oxopropan-2-yl)-
amino)-2-((tert-butoxycarbonyl) amino)-3-oxopropyl)thio)ethyl
1
stearate (5e). Yield 142 mg, 87%. H NMR (500 MHz, CDCl3) δ
7.13 (d, J = 7.9 Hz, 1H), 5.44 (s, 1H), 4.65 (dt, J = 8.1, 3.0 Hz, 1H),
4.33 (d, J = 4.5 Hz, 1H), 4.24 (t, J = 6.6 Hz, 2H), 3.82 (dd, J = 9.1, 2.9
Hz, 1H), 3.74 (s, 3H), 3.57 (dd, J = 9.1, 3.2 Hz, 1H), 3.01 (dd, J =
13.9, 5.5 Hz, 1H), 2.92 (dd, J = 13.9, 6.8 Hz, 1H), 2.83 (dd, J = 12.7,
6.4 Hz, 2H), 2.31 (t, J = 7.6 Hz, 2H), 1.63−1.58 (m, 2H), 1.46 (s,
9H), 1.30−1.22 (m, 28H), 1.14 (s, 9H), 0.87 (t, J = 6.9 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 174.05, 170.84, 170.76, 155.59, 80.65,
73.91, 63.38, 62.03, 54.09, 53.58, 52.82, 35.22, 34.58, 32.32, 31.42,
30.10, 30.06, 30.02, 29.88, 29.76, 29.69, 29.55, 28.69, 27.69, 25.28,
23.09, 14.53. MS (ESI) calcd for C36H68N2O8S, m/z 688.47; found
711.48 (M + Na)+.
2-(((R)-3-(((S)-3-(tert-Butoxy)-1-methoxy-1-oxopropan-2-yl)-
amino)-2-((tert-butoxycarbonyl) amino)-3-oxopropyl)thio)ethyl-
[1,1′-biphenyl]-4-carboxylate (5f). Yield 87 mg, 60%. 1H NMR
(500 MHz, CDCl3) δ 8.11 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.5 Hz,
2H), 7.63 (dd, J = 5.2, 3.3 Hz, 2H), 7.47 (t, J = 7.5 Hz, 2H), 7.40 (dt, J
= 9.3, 4.3 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 5.48 (s, 1H), 4.67 (dt, J =
8.1, 3.0 Hz, 1H), 4.52 (t, J = 6.6 Hz, 2H), 4.38 (s, 1H), 3.83 (dd, J =
9.1, 2.9 Hz, 1H), 3.73 (s, 3H), 3.57 (dd, J = 9.1, 3.2 Hz, 1H), 3.09 (dd,
J = 13.9, 5.5 Hz, 1H), 3.04−2.94 (m, 3H), 1.46 (s, 9H), 1.13 (s, 9H).
13C NMR (126 MHz, CDCl3) δ 170.61, 170.52, 166.42, 155.40,
2-(((R)-2-Amino-3-(((S)-3-hydroxy-1-methoxy-1-oxopropan-2-yl)-
1
amino)-3-oxopropyl)thio)ethyl stearate (6e). H NMR (500 MHz,
CDCl3) δ 8.47 (d, J = 7.6 Hz, 1H), 4.72−4.64 (m, 1H), 4.35 (t, J = 6.3
Hz, 1H), 4.24−4.16 (m, 2H), 3.94 (d, J = 8.9 Hz, 1H), 3.86 (dd, J =
11.6, 5.1 Hz, 1H), 3.76 (s, 3H), 3.16 (dd, J = 14.4, 5.3 Hz, 1H), 3.03−
2.97 (m, 1H), 2.81 (t, J = 6.4 Hz, 2H), 2.31 (t, J = 7.7 Hz, 2H), 1.59
(dd, J = 14.3, 7.1 Hz, 2H), 1.39−1.09 (m, 28H), 0.88 (t, J = 7.0 Hz,
3H). 13C NMR (126 MHz, CDCl3) δ 174.43, 170.15, 168.12, 62.49,
61.70, 55.26, 52.83, 52.69, 34.18, 32.94, 31.94, 30.79, 29.73, 29.71,
29.68, 29.54, 29.38, 29.32, 29.18, 24.85, 22.70, 14.13. MS (ESI) calcd
for C27H52N2O6S, m/z 532.35; found 533.37 (M + H)+.
145.94, 140.13, 130.38, 129.08, 128.79, 128.32, 127.43, 127.23, 80.44,
73.67, 63.79, 61.78, 53.88, 53.34, 52.58, 35.05, 31.28, 28.44, 27.43. MS
(ESI) calcd for C31H42N2O8S, m/z 602.27; found 625.27 (M + Na)+.
2-(((R)-3-(((S)-3-(tert-Butoxy)-1-methoxy-1-oxopropan-2-yl)-
amino)-2-((tert-butoxycarbonyl) amino)-3-oxopropyl)thio)ethyl-
[1,1′-biphenyl]-3-carboxylate (5g). Yield 81 mg, 56%. 1H NMR
(500 MHz, CDCl3) δ 8.28 (t, J = 1.6 Hz, 1H), 8.02 (d, J = 7.8 Hz,
1H), 7.79 (ddd, J = 7.7, 1.8, 1.2 Hz, 1H), 7.62 (d, J = 7.1 Hz, 2H), 7.52
(d, J = 7.7 Hz, 1H), 7.46 (dd, J = 9.9, 3.5 Hz, 2H), 7.38 (t, J = 7.4 Hz,
1H), 7.17 (d, J = 7.8 Hz, 1H), 5.48 (d, J = 3.4 Hz, 1H), 4.66 (dt, J =
8.1, 3.0 Hz, 1H), 4.52 (t, J = 6.7 Hz, 2H), 4.38 (d, J = 3.6 Hz, 1H),
3.82 (dd, J = 9.1, 2.9 Hz, 1H), 3.72 (s, 3H), 3.56 (dd, J = 9.1, 3.2 Hz,
1H), 3.08 (dd, J = 13.9, 5.4 Hz, 1H), 3.03 − 2.95 (m, 3H), 1.45 (s,
9H), 1.13 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 170.60, 170.50,
166.48, 155.39, 141.65, 140.21, 131.86, 130.61, 129.04, 128.57, 127.90,
127.32, 80.41, 73.66, 63.83, 61.76, 53.89, 53.34, 52.57, 35.05, 31.23,
28.43, 27.41. MS (ESI) calcd for C31H42N2O8S, m/z 602.27; found
625.26 (M + Na)+.
2-(((R)-2-Amino-3-(((S)-3-hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-3-oxopropyl)thio)ethyl[1,1′-biphenyl]-4-carboxylate (6f).
1H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 7.4 Hz, 1H), 8.02 (d, J
= 8.4 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.55−7.52 (m, 2H), 7.40 (t, J
= 7.4 Hz, 2H), 7.36−7.31 (m, 1H), 4.72−4.67 (m, 1H), 4.50−4.42 (m,
3H), 3.94 (d, J = 9.2 Hz, 1H), 3.87 (dd, J = 11.4, 4.7 Hz, 1H), 3.65 (s,
3H), 3.25 (dd, J = 14.3, 5.1 Hz, 1H), 3.10 (dd, J = 14.3, 6.9 Hz, 1H),
2.95 (t, J = 6.4 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 170.59,
168.60, 167.20, 146.33, 140.15, 130.62, 129.31, 128.63, 128.60, 127.62,
127.45, 63.57, 62.12, 55.67, 53.18, 51.27, 33.42, 31.25. MS (ESI) calcd
for C22H26N2O6S, m/z 446.15; found 447.16 (M + H)+.
2-(((R)-2-Amino-3-(((S)-3-hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-3-oxopropyl)thio)ethyl [1,1′-biphenyl]-3-carboxylate (6g).
1H NMR (500 MHz, CDCl3) δ 8.54 (d, J = 7.5 Hz, 1H), 8.21 (s, 1H),
7.94 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 7.2 Hz,
2H), 7.42 (dt, J = 13.5, 7.8 Hz, 3H), 7.33 (t, J = 7.4 Hz, 1H), 4.71−
4.64 (m, 1H), 4.48−4.42 (m, 3H), 3.92 (d, J = 9.2 Hz, 1H), 3.85 (dd, J
= 11.5, 4.9 Hz, 1H), 3.63 (s, 3H), 3.48 (s, 2H), 3.23 (dd, J = 14.4, 5.3
Hz, 1H), 3.08 (dd, J = 14.3, 6.9 Hz, 1H), 2.93 (t, J = 6.5 Hz, 2H). 13C
NMR (126 MHz, CDCl3) δ 170.55, 168.54, 167.25, 141.87, 140.27,
132.28, 130.50, 129.33, 129.28, 128.84, 128.71, 128.18, 127.50, 63.56,
62.10, 55.67, 53.16, 51.27, 33.38, 31.21. MS (ESI) calcd for
C22H26N2O6S, m/z 446.15; found 447.16 (M + H)+.
General Procedure for Synthesis of Compounds 6a−6g.
Synthesis of Compound 6a: 2-(((R)-2-Amino-3-(((S)-3-hydroxy-1-
methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)ethyl buty-
rate. Compound 5a (49 mg, 0.1 mmol) was dissolved in 2
mL of trifluoroacetic acid and stirred for 30 min, followed by
removal of the solvent by purging nitrogen and drying under
vacuum to obtain the residue, which was further purified using
column chromatography to obtain the trifluoroacetate salt of
Synthesis of Compound 6h: 2-(((R)-2-Amino-3-(((S)-3-hydroxy-1-
methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)ethyl[1,1′-bi-
phenyl]-3-carboxylate. To a solution of p-terphenyl-4-carboxylic acid
(33 mg, 0.12 mmol) and compound 4 (100 mg, 0.24 mmol) in
anhydrous dimethylformamide (DMF) were added triethylamine (33
μL, 0.24 mmol), DMAP (2.9 mg, 0.024 mmol), and O-benzotriazole-
N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU, 55 mg,
0.144 mmol), and the reaction mixture was stirred at room
temperature for 14 h, followed by removal of the solvent under
reduced pressure. The crude was then dissolved in ethyl acetate and
washed with water. The organic fraction was dried over anhydrous
sodium sulfate, filtered, concentrated, and purified using column
chromatography (5% MeOH/CH2Cl2) to obtain the compound 5h.
The product obtained was dissolved in 2 mL of trifluoroacetic acid and
stirred for 30 min, followed by removal of the solvent by purging
nitrogen and drying under vacuum to obtain the residue, which was
further purified using column chromatography to obtain the
1
compound 6a in quantitative yield (49 mg). H NMR (500
MHz, CDCl3) δ 8.52 (d, J = 7.7 Hz, 1H), 4.71−4.62 (m, 1H),
4.35 (t, J = 6.4 Hz, 1H), 4.28−4.17 (m, 2H), 3.97−3.90 (m,
1H), 3.87 (dd, J = 11.6, 5.1 Hz, 1H), 3.76 (s, 3H), 3.17 (dd, J =
14.4, 5.4 Hz, 1H), 3.02 (dd, J = 14.4, 7.1 Hz, 1H), 2.81 (t, J =
6.4 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H), 1.69−1.55 (m, 2H), 0.94
(t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.40,
170.35, 168.39, 62.71, 61.83, 55.35, 52.91, 52.86, 36.14, 33.16,
30.90, 18.43, 13.71. MS (ESI) calcd for C13H24N2O6S, m/z
336.14; found 337.15 (M + H)+.
Compounds 6b, 6c, and d6e−6g were synthesized similarly to
compound 6a. Compound 6d was synthesized as published earlier.17
2-(((R)-2-Amino-3-(((S)-3-hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-3-oxopropyl)thio)ethyl octanoate (6b). 1H NMR (500 MHz,
CDCl3) δ 8.49 (d, J = 7.7 Hz, 1H), 4.71 − 4.64 (m, 1H), 4.35 (t, J =
6.5 Hz, 1H), 4.26−4.18 (m, 2H), 3.96−3.90 (m, 1H), 3.86 (dd, J =
11.7, 5.2 Hz, 1H), 3.75 (s, 3H), 3.48 (s, 1H), 3.16 (dd, J = 14.5, 5.6
Hz, 1H), 3.01 (dd, J = 14.6, 7.2 Hz, 1H), 2.81 (t, J = 6.5 Hz, 2H), 2.31
1
trifluoroacetate salt of compound 6h (37 mg, 59%). H NMR (500
MHz, MeOD) δ 8.14 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.5 Hz, 2H),
7.81−7.74 (m, 2H), 7.68 (dd, J = 8.3, 1.1 Hz, 1H), 7.46 (t, J = 7.7 Hz,
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1H), 7.37 (t, J = 7.4 Hz, 1H), 4.62 (t, J = 5.0 Hz 1H), 4.59−4.53 (m,
1H), 4.17 (dd, J = 8.8, 4.7 Hz, 1H), 3.97 (dd, J = 11.2, 4.8 Hz, 1H),
3.85 (dd, J = 11.2, 3.8 Hz, 1H), 3.74 (s, 3H), 3.35 (d, J = 4.6 Hz, 1H),
3.07 (td, J = 6.5, 2.5 Hz, 2H), 3.02 (dd, J = 14.7, 8.8 Hz, 1H). 13C
NMR (126 MHz, MeOD) δ 171.67, 169.09, 167.76, 146.80, 142.49,
141.63, 139.84, 131.30, 129.98, 129.89, 128.69, 128.66, 128.61, 128.01,
127.92, 64.82, 62.57, 56.41, 53.59, 53.00, 34.29, 31.68. MS (ESI) calcd
for C28H30N2O6S, m/z 522.18; found 523.19 (M + H)+.
chloride (EDCI·HCl, 58 mg, 0.379 mmol) was added, and the reaction
mixture was stirred at room temperature for 18 h. Then 50 mL of
CH2Cl2 was added to the reaction mixture and the organic layer was
washed with water, brine, dried over anhydrous sodium sulfate, and
evaporated. The crude product was further column purified to furnish
the ester intermediate N-Boc, O-tBu protected 10a. The product
obtained was dissolved in 4 mL of trifluoroacetic acid and stirred for
30 min, followed by removal of the solvent by purging nitrogen and
drying under vacuum to obtain the residue which was further purified
using column chromatography to obtain the trifluoroacetate salt of
Syntheses of Methyl 2-(Ditetradecylamino)acetate (8a) and
Methyl 2-((tert-Butoxycarbonyl)(tetradecyl)amino)acetate (8b). To
a solution of glycine methyl ester 7 (1.0 g, 8 mmol) in DMF (5 mL),
triethylamine (4.5 mL, 32 mmol) was added, followed by 1-
bromotetradecane (3.6 mL, 12 mmol), and the reaction mixture was
stirred at room temperature for 18 h. The solvent was evaporated,
water (50 mL) was added, and the product was extracted in CH2Cl2
(100 mL). The organic layer was dried over sodium sulfate and
evaporated to afford a mixture of methyl 2-(tetradecylamino)acetate
and methyl 2-(ditetradecylamino)acetate. Di-tert-butyl dicarbonate
(1.0 g) was added to the solution of this crude mixture in anhydrous
CH2Cl2 (10 mL) followed by triethylamine (0.7 mL, 5.25 mmol) was
added, and the reaction mixture was stirred at room temperature for 1
h. The solvent was removed, and the product was column purified to
furnish compound 8a (330 mg, 9%) and compound 8b (1.01 g, 33%).
Spectroscopic evidence for two conformers for 8b was observed in
1
compound 10a in quantitative yield. H NMR (500 MHz, MeOD) δ
4.60 (t, J = 4.1 Hz, 1H), 4.48 (dtd, J = 17.9, 11.7, 6.2 Hz, 2H), 4.19 (s,
2H), 4.15 (dd, J = 8.4, 4.8 Hz, 1H), 3.97 (dd, J = 11.3, 4.6 Hz, 1H),
3.84 (dd, J = 11.3, 3.7 Hz, 1H), 3.76 (s, 3H), 3.28 (dd, J = 14.8, 4.9
Hz, 1H), 3.20 (dd, J = 9.6, 7.1 Hz, 4H), 3.03−2.87 (m, 3H), 1.72 (s,
4H), 1.45−1.25 (m, 44H), 0.90 (t, J = 7.0 Hz, 6H). 13C NMR (126
MHz, MeOD) δ 171.87, 169.24, 167.79, 66.48, 62.57, 56.35, 55.98,
54.39, 53.31, 53.01, 34.00, 33.09, 30.81, 30.79, 30.78, 30.76, 30.64,
30.51, 30.49, 30.19, 27.56, 25.03, 23.75, 14.45. MS (ESI) calcd for
C39H77N3O6S, m/z 715.55; found 716.59 (M + H)+.
Compound 10b was synthesized similarly to compound 10a.
10b: (S)-Methyl 2-((R)-2-Amino-3-((2-(2-(tetradecylamino)-
acetoxy)ethyl)thio)propanamido)-3-hydroxypropanoate. 1H NMR
(400 MHz, CDCl3) δ 8.26 (d, J = 7.7 Hz, 1H), 4.63 (dt, J = 7.6, 3.8
Hz, 1H), 4.36 − 4.24 (m, 2H), 3.95 (ddd, J = 17.8, 11.2, 3.8 Hz, 2H),
3.80 (s, 3H), 3.67 − 3.58 (m, 1H), 3.44 (s, 2H), 3.05 (dd, J = 13.6, 4.3
Hz, 1H), 2.95 (dd, J = 13.6, 6.9 Hz, 1H), 2.81 (t, J = 6.6 Hz, 2H),
2.63−2.55 (m, 2H), 1.72 (bs, 4H), 1.49 (m, 2H), 1.29−1.23 (m,
22H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
173.78, 172.27, 170.90, 63.76, 63.14, 54.87, 54.12, 52.87, 50.86, 49.74,
37.99, 32.07, 31.00, 29.96, 29.84, 29.83, 29.82, 29.80, 29.77, 29.74,
29.66, 29.51, 27.35, 22.84, 14.28. MS (ESI) calcd for C25H49N3O6S,
m/z 519.33; found 520.35 (M + H)+.
Synthesis of Compound 11a: 2-(((R)-2-(Ethylamino)-3-(((S)-3-
hydroxy-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)-
ethyl palmitate. To a solution of compound 6d (50 mg, 0.081 mmol)
in anhydrous CH2Cl2 were added acetaldehyde (3.5 mg, 0.081 mmol),
4 drops of acetic acid, and macroporous polystyrene-bound
cyanoborohydride (73 mg, 0.162 mmol). The reaction mixture was
stirred for 2 h and then filtered to remove the solid resin. The filtrate
was evaporated under vacuum to obtain the residue, which was
purified using column chromatography (4% MeOH/CH2Cl2), yielding
compound 11a (8 mg, 20%). 1H NMR (500 MHz, CDCl3) δ 8.28 (d, J
= 7.7 Hz, 1H), 4.64 (dt, J = 7.7, 3.8 Hz, 1H), 4.22 (t, J = 6.7 Hz, 2H),
4.01−3.90 (m, 2H), 3.80 (s, 3H), 3.28 (dd, J = 8.2, 3.9 Hz, 1H), 3.10
(dd, J = 13.5, 3.9 Hz, 1H), 2.84−2.71 (m, 3H), 2.68 (qd, J = 7.1, 2.9
Hz, 2H), 2.32 (t, J = 7.6 Hz, 2H), 2.08 (s, 2H), 1.66−1.54 (m, 2H),
1.35−1.21 (m, 24H), 1.15 (t, J = 7.1 Hz, 3H), 0.87 (d, J = 7.1 Hz, 3H).
13C NMR (126 MHz, CDCl3) δ 173.93, 173.55, 170.77, 63.50, 63.05,
1
both H and 13C NMR.
8a: 1H NMR (500 MHz, CDCl3) δ 3.69 (s, 3H), 3.32 (s, 2H), 2.53
(dd, J = 8.6, 6.7 Hz, 4H), 1.46−1.39 (m, 4H), 1.35−1.15 (m, 44H),
0.88 (t, J = 7.0 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 172.39,
55.26, 54.71, 51.54, 32.08, 29.85, 29.84, 29.83, 29.81, 29.79, 29.73,
29.52, 27.56, 27.53, 22.85, 14.28. MS (ESI) calcd for C31H63NO2, m/z
481.49; found 482.67 (M + H)+.
8b: 1H NMR (500 MHz, CDCl3) δ 3.95 (s, 1H), 3.86 (s, 1H), 3.73
(d, J = 2.6 Hz, 3H), 3.25 (dt, J = 19.4, 7.5 Hz, 2H), 1.42−1.50 (m,
11H), 1.34−1.19 (m, 22H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 170.91, 170.81, 156.02, 155.27, 80.16, 80.13, 52.11,
52.05, 49.27, 48.60, 48.49, 48.43, 32.06, 29.83, 29.81, 29.79, 29.75,
29.54, 29.50, 29.45, 28.50, 28.44, 28.39, 28.27, 26.93, 26.86, 22.83,
14.27. MS (ESI) calcd for C22H43NO4, m/z 385.32; found 386.33 (M
+ H)+.
Synthesis of 2-(Ditetradecylamino)acetic Acid (9a). Compound
8a (275 mg, 0.57 mmol) was dissolved in tetrahydrofuran (THF, 10
mL) and LiOH (68 mg, 2.85 mmol) in 2 mL of H2O was added, and
the reaction mixture was stirred at room temperature for 18 h. The
solvent was evaporated, water was added, the pH of the aqueous layer
was rendered acidic by the addition of 1N HCl, and the product was
extracted in CH2Cl2. The organic layer was dried over anhydrous
sodium sulfate, evaporated, and column purified to afford compound
9a as white solid (250 mg, 93%). 1H NMR (500 MHz, CDCl3) δ 3.29
(s, 2H), 3.13 (s, 1H), 2.72−2.46 (m, 4H), 1.47 (m, 4H), 1.19 (m,
44H), 0.81 (t, J = 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ
174.66, 58.26, 54.51, 31.94, 29.72, 29.71, 29.68, 29.64, 29.63, 29.51,
29.39, 27.43, 25.00, 22.70, 14.09. MS (ESI) calcd for C30H61NO2, m/z
467.47; found 468.49 (M + H)+.
61.55, 54.72, 52.88, 43.30, 35.83, 34.32, 32.07, 30.93, 29.84, 29.83,
29.80, 29.76, 29.62, 29.51, 29.42, 29.28, 25.04, 22.84, 15.50, 14.28. MS
(ESI) calcd for C27H52N2O6S, m/z 532.35; found 533.37 (M + H)+.
Compound 11b was synthesized similarly as compound 11a.
Compound 9b was synthesized similarly as compound 9a.
9b: 2-((tert-Butoxycarbonyl)(tetradecyl)amino)acetic Acid. Spec-
11b: 2-(((R)-3-(((S)-3-Hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-2-(octylamino)-3-oxopropyl) thio)ethyl Palmitate. Yield 10
1
1
troscopic evidence for two conformers was observed in both H and
mg, 20%. H NMR (500 MHz, CDCl3) δ 8.26 (d, J = 7.7 Hz, 1H),
1
13C NMR. H NMR (500 MHz, CDCl3) δ 3.88 (d, J = 9.7 Hz, 2H),
4.63 (dt, J = 7.7, 3.8 Hz, 1H), 4.22 (t, J = 6.7 Hz, 2H), 4.03−3.91 (m,
2H), 3.79 (s, 3H), 3.30 (dd, J = 7.8, 3.5 Hz, 1H), 3.10 (dd, J = 13.5,
3.9 Hz, 1H), 2.86−2.71 (m, 3H), 2.63 (td, J = 7.2, 2.2 Hz, 2H), 2.32
(t, J = 7.6 Hz, 2H), 2.05 (s, 2H), 1.67−1.55 (m, 2H), 1.51 (dd, J =
14.2, 7.1 Hz, 2H), 1.37−1.19 (m, 34H), 0.92−0.82 (m, 6H). 13C NMR
(126 MHz, CDCl3) δ 173.94, 173.41, 170.67, 63.47, 63.05, 61.57,
54.78, 52.85, 48.95, 35.71, 34.32, 32.07, 31.98, 30.94, 30.19, 29.84,
29.81, 29.77, 29.62, 29.51, 29.43, 29.41, 29.29, 27.32, 25.04, 22.84,
22.81, 14.28, 14.25. MS (ESI) calcd for C33H64N2O6S, m/z 616.45;
found 617.46 (M + H)+.
3.30−3.18 (m, 2H), 1.52−1.38 (m, 11H), 1.32−1.20 (m, 22H), 0.88
(t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.85, 175.28,
156.82, 155.32, 80.83, 80.31, 49.70, 49.16, 48.82, 48.29, 32.07, 29.84,
29.82, 29.81, 29.76, 29.68, 29.64, 29.57, 29.51, 29.46, 28.50, 28.39,
28.35, 28.25, 26.95, 26.84, 22.84, 14.28. MS (ESI) calcd for
C21H41NO4, m/z 371.30; found 394.30 (M + Na)+.
Synthesis of (S)-Methyl 2-((R)-2-Amino-3-((2-(2-
(ditetradecylamino)acetoxy)ethyl) thio)propan-amido)-3-hydroxy-
propanoate (10a). To the solution of compound 4 (80 mg, 0.189
mmol) and 9a (177 mg, 0.379 mmol) in dry CH2Cl2 (5 mL), N-
methylmorpholine (41 μL, 0.379 mmol) and DMAP (9.0 mg, 0.076
mmol) were added, and the reaction mixture was stirred in an ice bath.
After 20 min, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
Synthesis of Compound 11c: 2-(((R)-2-(Hexadecylamino)-3-(((S)-
3-hydroxy-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)-
ethyl Palmitate. To a solution of compound 6d (100 mg, 0.162
mmol) in anhydrous DMF, triethylamine (56 μL, 0.405 mmol) was
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added, followed by 1-bromohexadecane (123 μL, 0.405 mmol). The
reaction mixture was stirred at room temperature for 14 h. DMF was
evaporated at 50 °C, and the crude product obtained was purified
using column chromatography to give compound 11c (18 mg, 15%) as
white solid. 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 7.7 Hz, 1H),
4.63 (dt, J = 7.6, 3.7 Hz, 1H), 4.22 (t, J = 6.7 Hz, 2H), 4.00−3.90 (m,
2H), 3.80 (s, 3H), 3.26 (dd, J = 8.1, 3.7 Hz, 1H), 3.09 (dd, J = 13.5,
3.7 Hz, 1H), 2.83−2.72 (m, 3H), 2.61 (t, J = 7.0 Hz, 2H), 2.32 (t, J =
7.6 Hz, 2H), 1.88 (bs, 2H), 1.65−1.54 (m, 2H), 1.54−1.45 (m, 2H),
1.25 (s, 50H), 0.88 (t, J = 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3)
δ 173.90, 173.60, 170.69, 63.51, 63.06, 61.66, 54.76, 52.85, 49.01,
35.82, 34.32, 32.07, 30.92, 30.30, 29.86, 29.85, 29.81, 29.78, 29.70,
29.63, 29.51, 29.43, 29.29, 27.36, 25.04, 22.84, 14.28. MS (ESI) calcd
for C41H80N2O6S, m/z 728.57; found 729.58 (M + H)+.
Synthesis of Compound 11e: 2-(((R)-2-Butyramido-3-(((S)-3-
hydroxy-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)-
ethyl Palmitate. To a solution of compound 6d (50 mg, 0.081 mmol)
in pyridine (1 mL), butyryl chloride (10.2 μL, 0.097 mmol) was added
and the reaction mixture was stirred at room temperature for 1 h. The
volatiles were removed by evaporation, and the crude product
obtained was purified using column chromatography (10% MeOH/
1
CH2Cl2) to obtain compound 11e as a white solid (35 mg, 76%). H
NMR (500 MHz, CDCl3) δ 7.42 (d, J = 7.7 Hz, 1H), 6.59 (d, J = 7.1
Hz, 1H), 4.63 (dt, J = 8.8, 5.0 Hz, 2H), 4.35−4.19 (m, 2H), 3.96 (dt, J
= 11.5, 8.0 Hz, 2H), 3.78 (s, 3H), 3.02−2.90 (m, 2H), 2.84 (t, J = 6.6
Hz, 2H), 2.32 (t, J = 7.6 Hz, 2H), 2.27−2.19 (m, 2H), 1.73−1.55 (m,
5H), 1.35−1.19 (m, 24H), 0.95 (t, J = 7.4 Hz, 3H), 0.87 (t, J = 7.0 Hz,
3H). 13C NMR (126 MHz, CDCl3) δ 174.31, 173.88, 170.62, 170.57,
62.93, 62.77, 55.19, 52.91, 52.71, 38.45, 34.54, 34.39, 32.06, 31.31,
29.83, 29.80, 29.76, 29.62, 29.50, 29.42, 29.29, 25.02, 22.83, 19.13,
14.27, 13.84. MS (ESI) calcd for C29H54N2O7S, m/z 574.37; found
575.38 (M + H)+.
Synthesis of Compound 11d: 2-(((R)-2-Acetamido-3-(((S)-3-
hydroxy-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)-
ethyl Palmitate. To a solution of compound 6d (50 mg, 0.081 mmol)
in anhydrous CH2Cl2 were added triethylamine (17 μL, 0.121 mmol)
and acetic anhydride (8 μL, 0.081 mmol). The reaction mixture was
stirred for 2 h. The solvent was removed under vacuum to obtain the
residue, which was purified using column chromatography (6%
Compounds 11f and 11g were synthesized similarly as compound
11e.
11f: 2-(((R)-3-(((S)-3-Hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-2-octanamido-3-oxopropyl) thio)ethyl Palmitate. Yield 32
1
MeOH/CH2Cl2), furnishing compound 11d (35 mg, 79%). H NMR
1
mg, 62%. H NMR (500 MHz, CDCl3) δ 7.39 (d, J = 7.6 Hz, 1H),
(400 MHz, CDCl3) δ 7.41 (d, J = 7.7 Hz, 1H), 6.63 (d, J = 7.1 Hz,
1H), 4.67−4.57 (m, 2H), 4.37−4.17 (m, 2H), 4.01−3.90 (m, 2H),
3.79 (s, 3H), 3.26 (t, J = 6.3 Hz, 1H), 2.96 (dd, J = 6.4, 3.6 Hz, 2H),
2.83 (t, J = 6.6 Hz, 2H), 2.32 (t, J = 7.6 Hz, 2H), 2.05 (s, 3H), 1.60
(dd, J = 14.4, 7.2 Hz, 2H), 1.25 (s, 24H), 0.87 (t, J = 6.8 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 174.32, 170.95, 170.58, 170.55, 62.88,
62.76, 55.18, 52.93, 52.89, 34.56, 34.39, 32.06, 31.30, 29.84, 29.80,
29.76, 29.62, 29.50, 29.42, 29.29, 25.02, 23.23, 22.84, 14.27. MS (ESI)
calcd for C27H50N2O7S, m/z 546.33; found 547.35 (M + H)+.
Compounds 11h, 11j, and 11k were synthesized similarly as
compound 11d.
6.55 (d, J = 7.0 Hz, 1H), 4.66−4.55 (m, 2H), 4.35−4.17 (m, 2H),
4.03−3.89 (m, 2H), 3.79 (s, 3H), 3.19 (s, 1H), 2.96 (qd, J = 13.9, 6.4
Hz, 2H), 2.84 (t, J = 6.6 Hz, 2H), 2.32 (t, J = 7.6 Hz, 2H), 2.26−2.22
(m, 2H), 1.77 (s, 1H), 1.61 (dd, J = 14.5, 7.5 Hz, 4H), 1.34−1.19 (m,
31H), 0.90−0.83 (m, 6H). 13C NMR (126 MHz, CDCl3) δ 174.33,
174.01, 170.60, 170.56, 62.93, 62.79, 55.20, 52.92, 52.73, 36.62, 34.54,
34.40, 32.07, 31.80, 31.34, 29.84, 29.80, 29.77, 29.63, 29.51, 29.43,
29.34, 29.30, 29.12, 25.68, 25.03, 22.84, 22.75, 14.27, 14.21. MS (ESI)
calcd for C33H62N2O7S, m/z 630.43; found 631.44 (M + H)+.
11g: 2-(((R)-3-(((S)-3-Hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-3-oxo-2-palmitamidopropyl) thio)ethyl Palmitate. Yield 41
11h: 2-(((R)-3-(((S)-3-Hydroxy-1-methoxy-1-oxopropan-2-yl)-
1
mg, 68%. H NMR (500 MHz, CDCl3) δ 7.37 (d, J = 7.6 Hz, 1H),
amino)-3-oxo-2-(2,2,2-trifluoro-acetamido)propyl)thio)ethyl Palmi-
6.55 (d, J = 7.0 Hz, 1H), 4.61 (ddd, J = 19.9, 10.3, 5.0 Hz, 2H), 4.33
(dt, J = 11.4, 6.7 Hz, 1H), 4.24 (dt, J = 11.4, 6.4 Hz, 1H), 3.97 (qd, J =
11.6, 3.5 Hz, 2H), 3.79 (s, 3H), 2.95 (ddd, J = 30.5, 14.0, 6.5 Hz, 2H),
2.85 (t, J = 6.6 Hz, 2H), 2.34−2.30 (m, 2H), 2.27−2.21 (m, 2H), 1.62
(dd, J = 14.6, 7.3 Hz, 4H), 1.29−1.23 (m, 49H), 0.87 (t, J = 7.0 Hz,
6H). 13C NMR (126 MHz, CDCl3) δ 174.38, 174.01, 170.63, 170.53,
62.94, 62.80, 55.21, 52.94, 52.76, 36.63, 34.48, 34.41, 32.07, 31.36,
29.85, 29.81, 29.79, 29.78, 29.76, 29.65, 29.64, 29.62, 29.52, 29.49,
29.43, 29.41, 29.30, 25.69, 25.03, 22.84, 14.28. MS (ESI) calcd for
C41H78N2O7S, m/z 742.55; found 743.56 (M + H)+.
1
tate. Yield 10 mg, 21%. H NMR (500 MHz, CDCl3) δ 7.59 (d, J =
7.0 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 4.72 − 4.63 (m, 2H), 4.40−4.30
(m, 1H), 4.27 (dd, J = 12.0, 5.7 Hz, 1H), 4.00 (ddd, J = 54.9, 11.5, 3.3
Hz, 2H), 3.80 (s, 3H), 2.99 (t, J = 6.2 Hz, 2H), 2.92−2.80 (m, 2H),
2.77 (s, 1H), 2.38−2.26 (m, 2H), 1.66−1.55 (m, 2H), 1.38−1.18 (m,
24H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
174.69, 170.38, 168.95, 157.74, 157.44, 157.13, 156.83, 119.13, 116.85,
114.56, 112.28, 62.81, 62.75, 55.14, 53.06, 52.77, 34.51, 34.41, 32.06,
31.38, 29.84, 29.83, 29.80, 29.76, 29.61, 29.50, 29.40, 29.27, 24.98,
22.83, 14.27. MS (ESI) calcd for C27H47F3N2O7S, m/z 600.31; found
Synthesis of Compound 11i: 2-(((R)-3-(((S)-3-Hydroxy-1-me-
t h o x y - 1 - o x o p r o p a n - 2 - y l ) a m i n o ) - 3 - o x o - 2 - ( 2 , 2 , 2 -
trichloroacetamido)propyl)thio)ethyl Palmitate. To a solution of
compound 6d (40 mg, 0.079 mmol) in anhydrous CH2Cl2 were added
triethylamine (11 μL, 0.079 mmol), trichloroacetic acid (13 mg, 0.079
mmol), EDCI·HCl (19 mg, 0.095 mmol), and a catalytic amount of 1-
hydroxybenzotriazole (HOBt). The reaction mixture was stirred for 2
h, and the solvent was then removed under vacuum. The residue was
purified using column chromatography (5% MeOH/CH2Cl2) to
+
601.31 (M + H)+ and 618.34 (M + NH4 ).
11j: 2-(((R)-3-(((S)-3-Hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-2-(methylsulfonamido)-3-oxo-propyl)thio)ethyl Palmitate.
1
Yield 7 mg, 15%. H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.6
Hz, 1H), 5.60 (d, J = 7.9 Hz, 1H), 4.71−4.60 (m, 1H), 4.24 (dt, J =
14.1, 6.4 Hz, 3H), 4.10−3.92 (m, 2H), 3.80 (s, 3H), 3.09 (s, 3H), 3.05
(t, J = 5.7 Hz, 2H), 2.82 (t, J = 6.6 Hz, 2H), 2.71 (s, 1H), 2.33 (t, J =
7.6 Hz, 2H), 1.60 (q, J = 7.3 Hz, 3H), 1.28−1.25 (m, 23H), 0.88 (t, J =
6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 174.49, 170.46, 170.05,
62.83, 62.78, 56.26, 55.17, 53.06, 41.84, 35.62, 34.40, 32.07, 31.27,
29.84, 29.81, 29.77, 29.63, 29.51, 29.41, 29.29, 25.02, 22.84, 14.27. MS
(ESI) calcd for C26H50N2O8S2, m/z 582.30; found 583.31 (M + H)+.
11k: 2-(((R)-3-(((S)-3-Hydroxy-1-methoxy-1-oxopropan-2-yl)-
amino)-3-oxo-2-(trifluoromethyl-sulfonamido)propyl)thio)ethyl
Palmitate. Yield 39 mg, 78%. 1H NMR (500 MHz, CDCl3) δ 7.60 (d,
J = 7.9 Hz, 1H), 4.75−4.64 (m, 1H), 4.36 (t, J = 6.4 Hz, 1H), 4.28−
4.20 (m, 2H), 4.05 (dd, J = 11.5, 3.5 Hz, 1H), 3.92 (dd, J = 11.5, 3.3
Hz, 1H), 3.79 (s. 3H), 3.07−2.94 (m, 2H), 2.86−2.72 (m, 2H), 2.33
(t, J = 7.6 Hz, 2H), 1.59 (dd, J = 14.5, 7.2 Hz, 2H), 1.33−1.18 (m,
26H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
174.71, 170.56, 169.23, 123.42, 120.87, 118.32, 115.77, 62.79, 62.75,
57.35, 54.99, 53.13, 35.73, 34.37, 32.06, 31.29, 29.83, 29.79, 29.76,
29.61, 29.50, 29.40, 29.26, 24.97, 22.83, 14.26. MS (ESI) calcd for
C26H47F3N2O8S2, m/z 636.27; found 637.28 (M + H)+ and 654.31 (M
1
obtain compound 11i (6 mg, 12%). H NMR (500 MHz, DMSO) δ
8.61 (d, J = 7.6 Hz, 1H), 5.16 (t, J = 5.4 Hz, 1H), 4.57 (dd, J = 10.4,
4.0 Hz, 1H), 4.38 (dt, J = 7.7, 4.7 Hz, 1H), 4.14 (tt, J = 12.9, 5.5 Hz,
2H), 3.75 (dt, J = 10.7, 4.0 Hz, 1H), 3.63 (s, 3H), 3.03 (dd, J = 13.9,
4.0 Hz, 1H), 2.94 (dd, J = 13.9, 10.5 Hz, 1H), 2.81 (qd, J = 13.9, 7.2
Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.54−1.47 (m, 2H), 1.23 (s, 24H),
0.85 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 172.80,
170.72, 169.26, 161.53, 92.55, 63.00, 61.09, 54.79, 54.46, 51.98, 33.42,
33.20, 31.32, 29.81, 29.06, 29.03, 29.00, 28.90, 28.73, 28.71, 28.45,
24.45, 22.13, 13.99. MS (ESI) calculated for C27H47Cl3N2O7S, m/z
+
648.22; found 649.22 (M + H)+ and 666.25 (M + NH4 ).
Synthesis of Compound 11l: 2-(((R)-3-(((S)-3-Hydroxy-1-me-
thoxy-1-oxopropan-2-yl)amino)-2-(4-methylphenylsulfonamido)-3-
oxopropyl)thio)ethyl Palmitate. To a solution of compound 6d (40
mg, 0.079 mmol) in anhydrous CH2Cl2 were added triethylamine (11
μL, 0.079 mmol) and 4-methylbenzene-1-sulfonyl chloride (15 mg,
0.079 mmol). The reaction mixture was stirred for 2 h, and the solvent
+
+ NH4 ).
3360
dx.doi.org/10.1021/jm3000533 | J. Med. Chem. 2012, 55, 3353−3363

Journal of Medicinal Chemistry
Article
was then removed under vacuum. The residue was purified using
column chromatography (5% MeOH/CH2Cl2) to obtain compound
11l (25 mg, 48%). 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 8.3 Hz,
2H), 7.46 (d, J = 7.3 Hz, 1H), 7.33 (d, J = 8.0 Hz, 2H), 5.82 (d, J = 7.6
Hz, 1H), 4.53 (dt, J = 7.2, 3.6 Hz, 1H), 4.13 (td, J = 6.5, 1.6 Hz, 2H),
3.99−3.90 (m, 2H), 3.90−3.81 (m, 1H), 3.78 (s, 3H), 3.02 (dd, J =
14.2, 5.5 Hz, 1H), 2.73−2.56 (m, 4H), 2.43 (s, 3H), 2.31 (t, J = 7.6
Hz, 2H), 1.65−1.54 (m, 2H), 1.27 (d, J = 16.0 Hz, 24H), 0.88 (t, J =
7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.23, 170.23, 169.91,
144.47, 136.20, 130.06, 127.60, 62.77, 62.73, 55.66, 55.32, 53.01,
34.79, 34.33, 32.07, 31.08, 29.84, 29.80, 29.77, 29.63, 29.51, 29.42,
29.29, 25.00, 22.84, 21.74, 14.28. MS (ESI) calcd for C32H54N2O8S2,
24.97, 22.84, 18.31, 14.27, 13.62. MS (ESI) calcd for C29H54N2O7S,
m/z 574.37; found 575.37 (M + H)+.
Compound 14c was synthesized similarly as compound 14b.
14c: 2-(((R)-2-Amino-3-(((S)-1-methoxy-1-oxo-3-(palmitoyloxy)-
propan-2-yl)amino)-3-oxopropyl) thio)ethyl Palmitate. Yield 44
1
mg, 63%. H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 7.6 Hz, 1H),
6.80 (s, 4H), 4.82−4.73 (m, 1H), 4.49 (dd, J = 11.6, 4.1 Hz, 1H),
4.42−4.17 (m, 4H), 3.77 (s, 3H), 3.21 (dd, J = 14.7, 5.8 Hz, 1H), 3.02
(dd, J = 14.7, 7.7 Hz, 1H), 2.81 (t, J = 6.5 Hz, 2H), 2.30 (dt, J = 19.4,
7.6 Hz, 4H), 1.68−1.50 (m, 4H), 1.25 (s, 46H), 0.88 (t, J = 6.8 Hz,
6H). 13C NMR (126 MHz, CDCl3) δ 174.85, 174.03, 169.05, 167.77,
62.88, 62.31, 53.20, 52.74, 52.48, 34.34, 33.91, 32.78, 32.08, 30.63,
29.86, 29.83, 29.82, 29.79, 29.65, 29.52, 29.42, 29.29, 29.21, 24.96,
24.85, 22.84, 14.27. MS (ESI) calcd for C41H78N2O7S, m/z 742.55;
found 743.56 (M + H)+.
+
m/z 658.33; found 659.34 (M + H)+ and 676.37 (M + NH4 ).
Synthesis of Compound 12: 2-(((2R)-2-((tert-Butoxycarbonyl)-
amino)-3-((3-hydroxy-1-methoxy-1-oxopropan-2-yl)amino)-3-
oxopropyl)thio)ethyl Palmitate. To a solution of compound 6d (180
mg, 0.29 mmol) in anhydrous CH2Cl2 were added triethylamine (67
μL, 0.49 mmol) and di-tert-butyl dicarbonate (107 mg, 0.49 mmol).
The reaction mixture was stirred for 2 h, and the solvent was then
removed under vacuum. The residue was purified using column
chromatography (5% MeOH/CH2Cl2) to obtain the compound 12
(160 mg, 91%). 1H NMR (400 MHz, CDCl3) δ 7.27 (s, 1H), 5.47 (s,
1H), 4.66 (dt, J = 7.0, 3.3 Hz, 1H), 4.28 (ddd, J = 10.3, 8.8, 4.4 Hz,
3H), 3.99 (d, J = 3.1 Hz, 2H), 3.82 (s, 3H), 3.02 (ddd, J = 32.3, 13.9,
6.1 Hz, 2H), 2.84 (t, J = 6.6 Hz, 3H), 2.34 (t, J = 7.6 Hz, 2H), 1.62
(dd, J = 14.3, 7.0 Hz, 2H), 1.48 (s, 9H), 1.27 (s, 24H), 0.90 (t, J = 6.7
Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.19, 170.83, 170.57,
155.69, 80.92, 63.09, 62.88, 55.21, 54.17, 52.96, 34.56, 34.36, 32.07,
31.31, 29.84, 29.80, 29.76, 29.62, 29.50, 29.41, 29.28, 28.40, 25.01,
22.84, 14.27. MS (ESI) calcd for C30H56N2O8S, m/z 604.38; found
605.38 (M + H)+.
Synthesis of Compound 16: 3-(((R)-2-Acetamido-3-(((S)-3-
hydroxy-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)-
propyl Palmitate. To a solution of compound 1517 (20 mg, 0.032
mmol) in anhydrous CH2Cl2 were added triethylamine (5 μL, 0.038
mmol) and acetic anhydride (3 μL, 0.032 mmol). The reaction
mixture was stirred for 2 h. The solvent was removed under vacuum to
obtain the residue, which was purified using column chromatography
1
(6% MeOH/CH2Cl2), furnishing compound 16 (10 mg, 59%). H
NMR (500 MHz, DMSO) δ 8.41 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 8.5
Hz, 1H), 5.07 (t, J = 5.6 Hz, 1H), 4.54 (td, J = 8.9, 4.9 Hz, 1H), 4.33
(dt, J = 7.7, 4.9 Hz, 1H), 4.10−4.03 (m, 2H), 3.70 (dt, J = 11.1, 5.6 Hz,
1H), 3.66−3.58 (m, 4H), 2.82 (dd, J = 13.7, 4.9 Hz, 1H), 2.57 (ddd, J
= 9.2, 5.3, 3.0 Hz, 3H), 2.28 (td, J = 7.4, 2.8 Hz, 2H), 1.85 (s, 3H),
1.84−1.76 (m, 2H), 1.57−1.43 (m, 2H), 1.23 (s, 24H), 0.85 (t, J = 6.9
Hz, 3H). 13C NMR (126 MHz, DMSO) δ 172.96, 170.78, 170.75,
169.25, 62.41, 61.10, 54.75, 51.96, 51.88, 33.66, 33.44, 31.31, 29.06,
29.05, 29.02, 28.98, 28.89, 28.72, 28.69, 28.46, 28.15, 27.69, 24.45,
22.50, 22.12, 13.98. MS (ESI) calcd for C28H52N2O7S, m/z 560.35;
found 561.35 (M + H)+.
TLR2-Specific NF-κB Induction. The induction of NF-κB in a
TLR2-specific reporter gene assay was quantified using HEK-Blue cells
as previously described by us.23 HEK293 cells stably transfected with
either human TLR2 or murine TLR2 and alkaline phosphatase (sAP)
were obtained 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 promoters is inducible by TLR2 agonists, and extracellular
sAP in the supernatant is proportional to NF-κB induction. HEK-Blue
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 then stimulated with serially
diluted aliquots of compounds for 12 h. sAP was assayed
spectrophotometrically using an alkaline phosphatase-specific chrom-
ogen (present in the HEK-detection medium as supplied by the
vendor) at 620 nm. For antagonism assays, HEK-Blue cells were
incubated at a density of ∼105 cells/mL in a volume of 80 μL/well and
stimulated with either PAM2CS (1 μg/mL) or lipoteichoic acid (1 μg/
mL) in the presence of graded concentrations of the test compounds
as described for TLR7 previously.19,37
Synthesis of Compounds 14a: 2-(((R)-3-(((S)-3-Acetoxy-1-me-
thoxy-1-oxopropan-2-yl)amino)-2-amino-3-oxopropyl)thio)ethyl
Palmitate. To a solution of compound 12 (40 mg, 0.079 mmol) in
pyridine (1 mL) was added acetic anhydride (15 μL, 0.158 mmol).
The reaction mixture was stirred at room temperature for 1 h, and the
solvent was removed to obtain the crude N-Boc intermediate 13a. The
N-Boc group was then removed by stirring compound 13a in 1 mL of
trifluoroacetic acid for 15 min, followed by removal of solvent by
purging nitrogen. The residue was dried under vacuum to obtain
1
compound 14a in quantitative yield. H NMR (500 MHz, CDCl3) δ
8.18 (d, J = 7.7 Hz, 1H), 6.55 (s, 2H), 4.80 (dd, J = 7.5, 3.8 Hz, 1H),
4.41 (ddd, J = 27.2, 11.6, 3.9 Hz, 2H), 4.35−4.16 (m, 3H), 3.77 (s,
3H), 3.11 (ddd, J = 43.7, 14.6, 6.5 Hz, 2H), 2.82 (t, J = 6.6 Hz, 2H),
2.31 (t, J = 7.6 Hz, 2H), 2.03 (s, 3H), 1.66−1.53 (m, 2H), 1.35−1.09
(m, 24H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
174.39, 171.23, 169.23, 63.19, 62.55, 53.13, 52.48, 52.39, 34.31, 33.04,
32.07, 30.89, 29.85, 29.83, 29.81, 29.79, 29.65, 29.51, 29.45, 29.31,
24.99, 22.84, 20.66, 14.27. MS (ESI) calcd for C27H50N2O7S, m/z
546.33; found 547.34 (M + H)+.
Synthesis of Compound 14b: 2-(((R)-2-Amino-3-(((S)-3-(butyr-
yloxy)-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)thio)ethyl
Palmitate. To a solution of compound 12 (50 mg, 0.083 mmol) in
anhydrous THF were added triethylamine (36 μL, 0.25 mmol) and
butyryl chloride (25 μL, 0.25 mmol). The reaction mixture was stirred
for 2 h, and the solvent was then removed under vacuum. The residue
was purified using column chromatography (15% ethylacetate/
hexanes) to obtain the N-Boc protected intermediate 13b (25 mg,
45%). The N-Boc group was then removed by stirring compound 13b
in 1 mL of trifluoroacetic acid for 15 min, followed by removal of the
acid by purging nitrogen. The residue was thoroughly dried under
vacuum to obtain compound 14b in quantitative yield. 1H NMR (400
MHz, CDCl3) δ 7.98 (d, J = 7.6 Hz, 1H), 6.17 (s, 4H), 4.79 (dt, J =
7.6, 3.7 Hz, 1H), 4.49 (dd, J = 11.6, 4.1 Hz, 1H), 4.39−4.30 (m, 2H),
4.31−4.16 (m, 2H), 3.77 (s, 3H), 3.18 (dd, J = 14.7, 6.2 Hz, 1H), 3.05
(dd, J = 14.7, 7.1 Hz, 1H), 2.82 (t, J = 6.5 Hz, 2H), 2.29 (dt, J = 19.0,
7.5 Hz, 4H), 1.60 (dd, J = 14.7, 7.4 Hz, 4H), 1.25 (s, 24H), 0.89 (dt, J
= 14.0, 7.2 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 174.61, 173.77,
169.15, 167.87, 62.94, 62.39, 53.16, 52.64, 52.47, 35.77, 34.31, 32.80,
32.07, 30.71, 29.85, 29.82, 29.81, 29.78, 29.64, 29.51, 29.42, 29.29,
ASSOCIATED CONTENT
■
S
* Supporting Information
Characterization of intermediates and final compounds (1H,
13C, mass spectra). This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 785-864-1610. Fax: 785-864-1961. E-mail: sdavid@ku.
edu.
Notes
The authors declare no competing financial interest.
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Article
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ACKNOWLEDGMENTS
■
This work was supported by NIH/NIAID contract
HHSN272200900033C.
ABBREVIATIONS USED
■
DMF, dimethylformamide; DMAP, 4-dimethylaminopyridine;
EC50, half-maximal effective concentration; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide; ESI-TOF, electrospray
ionization-time-of-flight; HBTU, O-benzotriazole-N,N,N′,N′-
tetramethyl-uronium hexafluorophosphate; HEK, human em-
bryonic kidney; HOBt, 1-hydroxybenzotriazole; IRAK-4,
interleukin-1 receptor-associated kinase 4; MPL, monophos-
phoryl lipid A; MHC, major histocompatibility complex; NF-
κB, nuclear factor-κB; PAM, palmitoyl; sAP, secreted alkaline
phosphatase; SAR, structure−activity relationship; TH1, helper
T lymphocyte, type 1; TH2, helper T lymphocyte, type 2; THF,
tetrahydrofuran; TLR, Toll-like receptor
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