Synthesis and immunobiological activity of an original series of acyclic lipid A mimics based on a pseudodipeptide backbone

Article abstract of DOI:10.1021/jm060482a

Nδ-L-Homoserinyl-D-ornithinol pseudodipeptides N-acylated with typical Escherichia coli lipid A fatty acid residues and mono-O- or bis-O-phosphorylated have been prepared and their properties investigated. The derivatives carrying two phosphate groups were found to be inducers of NO production. In addition, while they were unable to induce significantly the production of interleukin-6 (IL-6) by human PBMC cells, these compounds behaved also as potent antagonists of LPS-induced IL-6 production in the same human cells system. In conclusion, the molecules described here are the first members of an original class of immunobiologically active lipid A mimics based on an acyclic pseudodipeptide backbone carrying only the essential functionalities of the parent lipid A structure (OM-174). As the products exhibit very low endotoxicity and pyrogenicity, this class of lipid A mimics therefore opens a new generation of immunoadjuvants that possibly could reach clinical applications.

Full text of DOI:10.1021/jm060482a

6000
J. Med. Chem. 2006, 49, 6000-6014
Synthesis and Immunobiological Activity of an Original Series of Acyclic Lipid A Mimics Based
on a Pseudodipeptide Backbone
Olivier R. Martin,*,† Wei Zhou,‡ Xinfu Wu,‡ Sophie Front-Deschamps,† Ste´phane Moutel,†,§ Katharina Schindl,§
Patricia Jeandet,§ Claude Zbaeren,§ and Jacques A. Bauer§
Institut de Chimie Organique et Analytique, UniVersite´ d’Orle´ans, BP 6759, 45067 Orle´ans, France, Department of Chemistry, State UniVersity
of New York, P.O. Box 6016, Binghamton, New York 13902-6016, and OM PHARMA, 22 Rue du Bois-du-Lan, 1217 Meyrin, Switzerland
ReceiVed April 25, 2006
Nδ-L-Homoserinyl-D-ornithinol pseudodipeptides N-acylated with typical Escherichia coli lipid A fatty acid
residues and mono-O- or bis-O-phosphorylated have been prepared and their properties investigated. The
derivatives carrying two phosphate groups were found to be inducers of NO production. In addition, while
they were unable to induce significantly the production of interleukin-6 (IL-6) by human PBMC cells, these
compounds behaved also as potent antagonists of LPS-induced IL-6 production in the same human cells
system. In conclusion, the molecules described here are the first members of an original class of
immunobiologically active lipid A mimics based on an acyclic pseudodipeptide backbone carrying only the
essential functionalities of the parent lipid A structure (OM-174). As the products exhibit very low endotoxicity
and pyrogenicity, this class of lipid A mimics therefore opens a new generation of immunoadjuvants that
possibly could reach clinical applications.
Introduction
Lipopolysaccharides (LPS) are amphiphilic macromolecules
expressed at the outer membrane of Gram-negative bacteria that
possess extremely potent immunostimulating activities.1 LPS
(“endotoxins”) are responsible for the pathogenesis and mani-
festation of Gram-negative infections and, ultimately, for septic
shock.2 Several years ago it was well established that most of
the biological activities of these lipopolysaccharides arise from
their terminal lipophilic fragment, known as lipid A,3 which
serves as the LPS membrane anchor. While large structural
variations exist in the polysaccharide part of LPS, in particular
in the antigenic O-specific chain, the molecular structure of lipid
A is relatively conserved across Gram-negative bacteria: it is
generally constituted of a â-(1f6)-linked glucosamine disac-
charide backbone carrying two phosphate groups at O-1 and
O-4′ and six or more fatty acyl groups linked as esters and
amides (e.g. Escherichia coli lipid A; see Figure 1).3
Lipid A is endowed with an overwhelming spectrum of
biological activities (activation of macrophages, B cell mito-
genicity, enhancement of host resistance against bacterial or viral
infections and against tumors, etc.)3,4a and, since its discovery,
stimulated enormous interest from both industrial and academic
research laboratories. In particular, much effort has been
dedicated to modifications of the lipid A structures with the
Figure 1. Structure of E. coli lipid A and of OM-174.
goal of reducing the natural endotoxicity of the parent compound
while maintaining or improving its beneficial immunostimu-
Fendrix, containing Corixa’s MPL adjuvant. MPL adjuvant is
lating properties or of identifying nontoxic antagonists for the
now incorporated in two approved products and in multiple
treatment of septicemia. Several lipid A deriVatiVes are currently
vaccines nearing approval around the world. Two other late-
under investigation;4,5 for example, monophosphorylated lipid
stage GSK Bio vaccines that contain MPL include Simplirix, a
A (MPL)6 is considerably less toxic than the parent diphosphate
vaccine for genital herpes, and Cervarix, a cervical cancer
and has been the object of extensive studies, in particular as a
vaccine that targets the human papillomavirus. Additionnal GSK
vaccine adjuvant.7 To date, GlaxoSmithKline Biologicals (GSK
Bio vaccines containing MPL include a malaria vaccine, which
Bio) has received European approval for its hepatitis B vaccine,
recently completed a phase IIb trial, as well as vaccines for
tuberculosis, prostate cancer, and breast cancer. Also, modified
* Corresponding author. Phone: +33 2 38 49 45 81. Fax +33 2 38 41
72 81. E-mail: Olivier.Martin@univ-orleans.fr.
† Universite´ d’Orle´ans.
lipid A’s whose structure is derived from that of naturally
occurring nontoxic lipid A’s (from Rhodobacter capsulatus and
Rhodobacter sphaeorides) have been developed as potent
endotoxin antagonists.5b-d
‡ State University of New York.
§ OM PHARMA.
10.1021/jm060482a CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/09/2006

Acyclic Lipid A Mimics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6001
In the early 90s, a new lipid A derivative (OM-174, Figure
1) was isolated by OM PHARMA from partially degraded E.
coli LPS.8 This derivative has lost both sugar-O-acyl substituents
and therefore carries only the N-linked fatty acid residues of E.
coli lipid A, namely a (R)-3-hydroxytetradecanoyl group at N-2
and a (R)-3-dodecanoyloxytetradecanoyl group at N-2′, thus
leaving only three long-chain acyl groups on the structure.
Thorough pharmacological investigations of this new compound
revealed that it has potent antitumor activity in several in vivo
tumor models9 and that it is an effective immunoadjuvant with
very low toxicity. These remarkable biological activities pro-
vided evidence that the presence of six fatty acid residues is
not a necessary condition for lipid A derivatives to exhibit useful
immunostimulatory properties.10
The high interest of OM-174 and related compounds as
potential therapeutic agents, combined with the fact that such
structures are accessible only by way of lengthy syntheses,11
prompted us to design and develop simplified structures carrying
the main functionalities of the parent product. We report in this
paper the synthesis of a novel series of lipid A mimics based
on an acyclic pseudodipeptide backbone and the evaluation of
their immunostimulating activity using E. coli LPS as a
reference. These compounds constitute the first examples of a
new class of highly active nontoxic lipid A-mimicking struc-
tures.
Structural Design and Synthetic Planning. A simplification
of the lipid A structure that has been extensively studied is the
replacement of the “reducing” glucosamine residue of the
disaccharide scaffold by an acylated acyclic aglycon.4b,10,12,13
In these analogues, the aglycon is generally a glycosidically
linked hydroxy amino acid derivative N-acylated with a fatty
acid. Interesting immunostimulating activities were found for
several of the analogues investigated,10b which suggested that
the functional groups of the reducing sugar moiety of lipid A
are not necessary for biological activity. Further simplification
of the structure led to deceptively simple analogues such as the
O,N-bis-acylated derivative of 4-amino-5-hydroxypentanoic acid
SDZ 280.96114 [O-(R)-3-tetradecanoyloxytetradecanoyl N-(R)-
3-hydroxytetradecanoyl (4S)-4-amino-5-hydroxypentanoic acid],
a compound designed to mimic the reducing sugar component
of lipid A which effectively stimulates TNF-R release from
human peripheral blood mononuclear cells (PBMC). Bacterial
ornithine-containing lipids, recently shown to have macrophage-
activating properties,15 can also be considered as belonging to
this class of simple lipid A analogues. A fully acyclic analogue
of E. coli lipid A, constituted by a symmetric phospholipid dimer
connected to a non-carbohydrate backbone and carrying six fatty
acid chains (ER 112022), was recently developed;16 this
compound was found to interact directly with the LPS receptor
and thus to behave as a potent lipid A agonist.17
Figure 2. Proposed structure and synthetic planning.
Figure 3. Target lipid A mimics.
the preparation of the D-ornithinol-derived structure 1 (OM-
294-DP)18 as well as of the monophosphorylated derivative 2
(OM-294-MP),18 an analogue that is of interest because of its
relation to monophosphorylated lipid A (Figure 3). The resulting
structures were synthesized as described below and were found
to exhibit biological activities similar to those of the parent lipid
A, with extremely low endotoxicity.
We have designed novel acyclic analogues of lipid A based
on the OM-174 model and on the following considerations: (1)
replacement of the two sugar units by amino acid-derived
elements carrying anchoring functions at the appropriate loca-
tion; (2) replacement of the glycosidic linkage by an amide bond;
(3) conservation of the essential functions of the lipid A model
directly exposed to a putative receptor, namely, the phosphate
groups and the fatty acid chains; and (4) conservation of the
amphiphilic character of the parent lipid A model.
Results and Discussion
Synthetic Studies. To develop a convergent synthesis, it was
decided to perform the amide bond coupling late in the synthesis
using completely elaborated building blocks carrying both the
fatty acyl group and a protected phosphate. These building
blocks would arise in initial studies from DL-homoserine and
from D-ornithine.
Synthesis of Fatty Acids (Scheme 1). The known (R)-3-
benzyloxy- and (R)-3-dodecanoyloxytetradecanoic acid deriva-
tives 5 and 8 were prepared by modified or improved proce-
These considerations led to the design of a general structure
of type I (Figure 2). Considering appropriate disconnections,
such structure should be readily accessible from the building
blocks D/L-homoserine, D/L-ornithinol, or D/L-lysinol; substituted
fatty acids; and phosphorylating agents. We set out as goals

6002 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Martin et al.
Scheme 1a
Scheme 3a
a Reagents and conditions: (a) TMSCl, Et3N, THF; (b) PhCHO,
TMSOTf, Et3SiH, CH2Cl2; (c) LiOH, THF-water, 50 °C; (d) 10 N NaOH,
EtOH; (e) BnBr, Et3N, Bu4NI, EtOAc; (f) C11H23COCl, pyridine, CH2Cl2;
(g) Pd/C, H2, EtOAc-EtOH.
Scheme 2a
a Reagents and conditions: (a) (i)Cs2CO3, N NaOH; (ii) (Boc)2O,
dioxane; (iii) BnBr, DMF; (b) (PhO)2P(O)Cl, DMAP, pyridine, CH2Cl2;
(c) TFA; (d) acid 8, i-BuOCOCl, N-methylmorpholine, THF, -15 °C, 30
min then amine 18, Et3N, THF; (e) Pd/C, H2, EtOH.
canoic acid 5 to give 12 in 69% yield (mixed anhydride
technique). The Z group was then selectively cleaved in the
presence of the benzyl ether by catalytic hydrogenolysis in the
presence of triethylamine27 to give the amine building block
13.
The O-diphenylphosphoryl derivative of 12 was also prepared;
however, the amine obtained after cleavage of the Z group was
not sufficiently stable to be engaged in amide-bond-forming
reactions. Phosphorylation at that position was therefore post-
poned to a later stage of the synthesis.
a Reagents and conditions: (a) i-BuOCOCl, N-methylmorpholine, THF,
-15 °C; (b) NaBH4, H2O, -15 °C, 10 min; (c) TFA; (d) acid 5, i-BuOCOCl,
N-methylmorpholine, THF, -15 °C then amine 11, Et3N, THF; (e) Pd/C,
H2, EtOH-Et3N; (f) BOMCl, (i-Pr)2NEt, CH2Cl2; (g) Pd(OH)2/C, H2,
EtOH-Et3N.
dures.18 The benzylation of base labile 3-hydroxyalkanoates,
including methyl 3-hydroxytetradecanoate 3,19 has been achieved
using Ag2O-BnBr20a or benzyl trichloroacetimidate;20b,c how-
ever, the yield of these reactions does not exceed 60-80%, and
the product is difficult to purify. Compound 3 was therefore
benzylated by way of its trimethylsilyl ether under the reductive
conditions described by Nishizawa;21 these conditions provided
the desired product 420c in very high yield, which could be
engaged in the subsequent saponification without purification
For further studies, the free hydroxyl group of 12 was also
protected as a benzyloxymethyl (BOM) ether, and the amino
group of the resulting ornithinol derivative 14 was deprotected
under the same conditions as 12 to give 15. It is noteworthy
that both the benzyl and the benzyloxymethyl ether functions
were unaffected by the hydrogenolysis in the presence of a
tertiary amine.
For the synthesis of the homoserine derivative 20 (Scheme
3), the free racemic amino acid was first submitted to a three-
step sequence without isolation of the intermediates to form
derivative 17: conversion into the cesium carboxylate, formation
of the N-Boc derivative immediately followed by formation of
the benzyl ester by alkylation of the cesium carboxylate,28 and
phosphorylation of the remaining free OH group using (PhO)2P-
(O)Cl.29 By this sequence, compound 17 was obtained in 82%
overall yield and the formation of the corresponding lactone
was avoided.
24
to give pure 5 in 81% yield from 3.
Saponification of 3 afforded (R)-3-hydroxytetradecanoic acid,
which was converted into its benzyl ester 622 in essentially
quantitative yield by reaction with benzyl bromide and triethyl-
amine; the crude benzyl ester was esterified with dodecanoyl
chloride to give compound 7, which was purified and submitted
to hydrogenolysis to afford pure (R)-3-dodecanoyloxytetrade-
canoic acid23,24 8 in 86% yield from (R)-3-hydroxytetradecanoic
acid.
Synthesis from DL-Homoserine. The R-Boc-δ-Z-D-ornithine
derivative (9) was converted into R-Boc-δ-Z-D-ornithinol25 10
using the method reported by Martinez26 (Scheme 2). The N-Boc
group was then cleaved to give 11, and the R-amino group of
D-ornithinol could be acylated with (R)-3-benzyloxytetrade-
The N-Boc group was then cleaved under standard conditions
and the amino group of 18 was acylated with (R)-3-dodecanoyl-
oxytetradecanoic acid 8 to give the key intermediate 19. The
benzyl ester function of 19 was cleaved under mild hydro-
genolytic conditions just prior to the next reaction because of
the lability of the resulting free acid 20.

Acyclic Lipid A Mimics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6003
Scheme 4a
a Reagents and conditions: (a) IIDQ, CH2Cl2, 15 min then amine 13, CH2Cl2; (b) (BnO)2PNEt2, 1H-tetrazole, THF; (c) mCPBA, CH2Cl2; (d) Pd/C, H2,
EtOH; (e) PtO2, H2, EtOH.
Scheme 5a
analysis of the resulting coupling product 26 revealed however
that a considerable amount of R-epimerization (about 30%)
had taken place during the reaction. Epimerization could be
reduced to about 13% by carrying the reaction at lower
temperature (0 °C); better results, however, were obtained when
the coupling was performed using diisopropylcarbodiimide
in the presence of HOAt34 (∼2.5% epimerization at C-2b,
72% yield). The reduction of the â-carboxyl function of 26
was then investigated. The direct reduction of 26 with NaBH4
gave in modest yield a product resulting from the reductive
opening of an intermediate cyclic imide (see below). The
reduction was successfully achieved by way of the mixed
anhydride method:26 selective cleavage of the â-benzyl ester
of 26 followed by formation of the mixed anhydride with
isobutyl chloroformate and rapid treatment of this intermediate
with NaBH4 provided the expected diol 29 in 74% yield. This
compound was then bis-phosphorylated and the resulting product
30 was deprotected by hydrogenolysis to afford the product 1-
(*R) ()2b(R)).
a Reagents and conditions: (a) Pd/C, H2, EtOH-AcOH; (b) PtO2, H2,
EtOH.
The amide bond formation between fragments 13 and 20 was
investigated under a variety of conditions. The desired pseudo-
dipeptide 21 could be obtained in 56% yield using IIDQ (2-
isobutyloxy-1-isobutyloxycarbonyl-1,2-dihydroquinoline)30 as
the coupling agent (Scheme 4). Compound 21 was then
phosphorylated by way of the phosphoramidite technique,31 to
give derivative 22, which was then deprotected by two succes-
sive hydrogenolyses, the first one in the presence of Pd on
charcoal to cleave the benzyl groups, and the second one in the
presence of platinum oxide to cleave the phenyl phosphates.32
This sequence thus provided the diphosphorylated pseudodipep-
tidolipid 1 as a mixture of stereoisomers at C-2b. Similarly,
the monophosphorylated analogue 2 was prepared by the
deprotection of the amide precursor 21 (Scheme 5).
The same sequence of reaction was applied to the synthesis
of the 1(*S)-epimer starting form â-benzyl-L-aspartate, by way
of the (*S)-epimer of intermediate 26.
The monophosphorylated analogue 2(*R) was also obtained
by this strategy: compound 25 was coupled with the O-
benzyloxymethyl ornithinol derivative 15, to give compound
31 (Scheme 7), which was converted into 33 by the same
reductive process and then O-phosphorylated and deprotected
to give 2(*R).
Synthesis from D- or L-Aspartic Acid. Because of the
difficulties associated with the homoserine-derived fragments,
and the high cost of enantiomerically pure Hse, the synthetic
strategy was modified. It was envisioned that the homoserine
component of the final products could arise from an aspartic
acid residue33 and be generated by a reduction process after
the amide-forming reaction. Thus â-benzyl D-aspartate was
N-acylated with fatty acid 8 to afford compound 25 in high
yield (mixed anhydride method) (Scheme 6) and this product
was coupled to amine 13 in the presence of IIDQ. Careful
The same sequence of reaction was applied to the synthesis
of the 2(*S)-epimer starting form â-benzyl-L-aspartate, by way
of the (*S)-epimer of intermediate 31.
As indicated above, the treatment of 26 [∼3:1 mixture of
2b(R)- and 2b(S)-epimers] with NaBH4 gave, in 43% yield, an
unexpected product having a â-acyloxyacylamido group in the
fragment arising from the aspartate (compound 35, Scheme 8).
This compound was formed undoubtedly by way of a cyclic
imide, which underwent regioselective reductive opening to give

6004 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Martin et al.
Scheme 6a
a Reagents and conditions: (a) acid 8, i-BuOCOCl, N-methylmorpholine, THF, -15 °C, 30 min then H-D-Asp(OBn)-OH, CH3CN-H2O, Et3N; (b) amine
13, HOAt, N,N′-diisopropylcarbodiimide, CH2Cl2; (c) Pd/C, H2, EtOH-EtOAc, Et3N; (d) Pd/C, H2, EtOH-EtOAc; (e) i-BuOCOCl, N-methylmorpholine,
THF; (f) NaBH4, H2O, rt, 5 min; (g) (BnO)2PNEt2, 1H-tetrazole, THF; (h) mCPBA, CH2Cl2; (i) Pd/C, H2, EtOH.
Scheme 7a
a Reagents and conditions: (a) IIDQ, CH2Cl2, 15 min then amine 15, CH2Cl2; (b) Pd/C, H2, EtOH-EtOAc, Et3N; (c) i-BuOCOCl, N-methylmorpholine,
THF, 30 min; (d) NaBH4, H2O, 5 min; (e) (BnO)2PNEt2, 1H-tetrazole, THF; (f) mCPBA, CH2Cl2; (g) Pd/C, H2, EtOH-EtOAc.
the rearranged product 35. As this structure is of interest for
structure-activity studies, compound 35 was elaborated into
the bis-phosphorylated pseudodipeptide 37 by a similar sequence
of reactions.
Biological Studies. Endotoxicity. The endotoxicity of com-
pounds 1(*R/S), 1(*R), 1(*S) and 2(*R/S), 2(*R), and 2(*S)
was evaluated by the LAL (Limulus amoebocyte lysate)
chromogenic assay.35 This very sensitive assay is based on the
phenomenon that endotoxins induce coagulation of the blood
of the horseshoe crab (Limulus polyphemus). The results (Table
1) indicate that solutions of 0.1 mg/mL of the compounds of
the series 1 and 2 exhibit a low endotoxicity pattern, compounds
of the series 1 being even less endotoxic than compounds of
series 2.

Acyclic Lipid A Mimics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6005
Scheme 8a
and 1(*R), of compound 2(*R/S), and of the homogeneous
stereoisomers 2(*S) and 2(*R) to induce the production of NO
by murine macrophages was determined, and the results are
shown in Figure 4. The molecules of the series 1 (OM-294-
DP) are powerful inducers of NO production, some activity
being detected at the low dose of 0.01 µg/mL; this activity is
comparable to the effect of the “parent” biological molecule
OM-174. In contrast, only a faint activity could be detected for
the compounds of the second series (OM-294-MP) at the dose
of 0.2 µg/mL. The LPS control is also shown in Figure 4.
Interestingly, the results of Figure 4 show that stereochemistry
of the carbon from the aspartic acid moiety (C-2b) does not
seem to play a discriminating role for NO production by murine
macrophages.
Production of Interleukin-6 (IL-6) by Human PBMC. The
production of IL-6 by human peripheral blood mononuclear cells
(PBMC) is a further important in vitro test to screen the ability
of new compounds to stimulate the immune system. IL-6 is a
multifunctional cytokine that plays important roles in host
defense, acute phase reactions, and hematopoiesis.37 The ago-
nistic ability of compounds 1 and 2 per se to induce (at 20 µg/
mL) the production of IL-6 by human PBMC was determined,
and the results are shown in Figure 5. Interestingly, compounds
of the series 1 were unable to induce any production of IL-6,
and some compounds of the series 2s2(*R) and 2(*R/S)swere
slightly active per se, when compared to the positive control
LPS.
Finally, the ability of the compounds (at 0.02, 0.2, 2, and 20
µg/mL) to antagonize the LPS-induced (at 10 ng/mL) secretion
of IL-6 by human PBMC was also determined (Figure 6). Very
interestingly, compounds of the series 1 at 2 µg/mL, when added
90 min before LPS (or even concomitantly to LPS, not shown),
were always able to almost completely abolish the LPS-induced
production of IL-6, and this property was never observed with
compounds of the series 2 at 2 µg/mL. Interestingly, when LPS
was added before the compounds of the series 1 and 2, no
antagonistic effect could be detected any more. This may suggest
that the compounds of the series 1 and LPS compete for the
same receptor, probably TLR4. Additional experiments in vitro
and in vivo will be necessary to verify if any of the compounds
presented here will be worth developing clinically against
inflammatory pathologies such as septic shock. Finally, the
stereochemistry of the carbon from the aspartic acid moiety (C-
2b) did not play any significant role in this antagonistic effect
for any of the compounds tested.
a Reagents and conditions: (a) NaBH4, MeOH-H2O, -15 °C f rt; (b)
(BnO)2PNEt2, 1H-tetrazole, THF; (c) mCPBA, CH2Cl2; (d) Pd/C, H2, EtOH.
Table 1. Results of LAL Assaya
LAL activity
ng equiv LPS/
compd
EU/mL
mg product
1(*R/S) [OM-294-DP (R/S,R)]
1(*R) [OM-294-DP (R,R)]
1(*S) [OM-294-DP (S,R)]
2(*R/S) [OM-294-MP (R/S,R)]
2(*R) [OM-294-MP (R,R)]
2(*S) [OM-294-MP (S,R)]
874 ( 620
1072 ( 205
787 ( 558
965 ( 184
240 ( 196
216 ( 176
16238 ( 8974
14257 ( 10324
9320 ( 2462
14614 ( 8077
12831 ( 9295
8388 ( 2216
a Results are expressed in endotoxin units (EU) or as nanogram equivalent
LPS per milligram of product, one EU corresponding here to 0.09 ng equiv
LPS. Test compound concentration is 0.1 mg/mL. Average results from
three independent experiments.
Table 2. Results of Pyrogenicity in Rabbits
sum of increased temperature in three rabbits (°C)
1(*R/S)
2(*R/S)
doses (mg/kg)
[OM-294-DP (R/S,R)]
[OM-294-MP (R/S,R)]
control (water)
0.80 (pass)
0.60 (pass)
0.75 (pass)
1.35 (inconclusive)
0.80 (pass)
0.85 (pass)
0.95 (pass)
3.65 (fail)
0.001
0.01
0.1
Furthermore, the results of Table 1 show that the stereo-
chemistry of the carbon from the aspartic acid moiety
(C-2b) does not seem to play a crucial role for the endotoxic
potential.
Conclusion
Compounds of the series 1 (OM-294-DP) and 2 (OM-294-
MP) represent the first members of a new class of lipid A
mimics based on a pseudodipeptide backbone carrying only the
essential functionalities of the parent lipid A structure (OM-
174). These compounds possess very interesting partial im-
munological activities (either immunostimulating or immuno-
modulating), comparable to that of the parent lipid A, and are
practically devoid of endotoxicity. Further experiments both in
vitro and in vivo are now necessary to select from among the
compounds described here a lead compound for clinical
development.
Pyrogenicity. These in vitro results were confirmed by an
in vivo pyrogenicity test for compounds OM-294-DP [1(*R/
S)] and OM-294-MP [2(*R/S)] in the rabbit, since the pyroge-
nicity induced by the iv injection of OM-294-MP [2(*R/S)] was
always higher than the pyrogenicity induced by OM-294-DP
[1(*R/S)] (see Table 2). It should be noted that with doses up
to 0.01 mg/kg both compounds were far below the allowed
regulatory limit of defining whether a product is considered
pyrogenic (according to the European Pharmacopoeia for the
Assessment of Pyrogenicity in the Rabbit).
NO-Production by Murine Macrophages. A simple in
vitro test for evaluating immunostimulating activity is the
induction of NO synthase in macrophages, resulting in the
production of the highly reactive NO radical. The cytotoxic
activity of macrophages is due, in part, to the destructive action
of NO on microbial DNA and membranes.36 The ability of
compound 1(*R/S), of the homogeneous stereoisomers 1(*S)
Experimental Section
Methyl (R)-3-Benzyloxytetradecanoate (4). To a solution of
methyl (R)-3-hydroxytetradecanoate 3 (10.0 g, 38.7 mmol) in
anhydrous THF (150 mL) were added triethylamine (11.9 mL, 85.4
mmol) and chlorotrimethylsilane (9.8 mL, 77.2 mmol). The mixture
was stirred at room temperature until all the starting material had

6006 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Martin et al.
Figure 4. NO production by murine macrophages.
Figure 5. Production of IL-6 by human PBMCa by compounds of series 1 and 2. Concentration of IL-6 produced (pg/mL) ( SEM (n ) 3
independent experiments) at 20 µg/mL of the test compounds and at 0.01 µg/mL for LPS of E. coli (serotype 055:B5, Sigma, St. Louis, MO).
Figure 6. The antagonistic effect of compounds of the series 1 and 2 on LPS-induced IL-6 production by human PBMC. Concentration of IL-6
produced (pg/mL) ( SEM (n ) 3 independent experiments) at 0.02, 0.2, 2, and 20 µg/mL of the test compounds with addition of 0.01 µg/mL of
E. coli LPS (serotype 055:B5, Sigma, St. Louis, MO).
disappeared (17 h). CH2Cl2 (50 mL) was then added and the reaction
mixture was cooled in an ice bath. A saturated solution of NaHCO3
(5 mL) was added, followed by cold water (50 mL). The organic
phase was separated and washed with water (100 mL). The aqueous
phases were combined and washed with ether (2 × 100 mL); the
combined organic phases were dried over MgSO4 and concentrated,
thus affording the crude trimethylsilyl ether of 3 (12.7 g). To an
ice-cold solution of this crude silyl ether (12.7 g, 38.4 mmol) and
benzaldehyde (4.7 mL, 46.2 mmol) in anhydrous CH2Cl2 (300 mL)
was added trimethylsilyl triflate (0.7 mL, 3.87 mmol), and the
mixture was stirred for 1 h at 0 °C. Triethylsilane (6.1 mL, 38.2
mmol) was then added and the mixture was allowed to warm to
room temperature. After 14 h, the reaction mixture was diluted with
ether (150 mL) and washed with a saturated solution of NaHCO3
(200 mL) and then with water (100 mL). The organic phase was
dried over MgSO4 and the solvent was evaporated. The crude
methyl (R)-3-benzyloxytetradecanoate 420a,c thus obtained (18.2 g,
homogeneous by NMR) was used without further purification in
the next step.
(R)-3-Benzyloxytetradecanoic Acid (5). To a solution of crude
methyl (R)-3-benzyloxytetradecanoate 4 (18.2 g) in THF (200 mL)
was added 1 N aqueous LiOH (200 mL, 0.2 mol) and the mixture
was heated at 80 °C for 30 min and at 50 °C for 6 h. The mixture
was neutralized with 1 N aqueous HCl and then extracted with
ether (2 × 150 mL). The organic phases were combined, washed
with water, dried over MgSO4, and concentrated. The residue was
purified by flash column chromatography on silica gel (EtOAc/
hexane, 1:20 f 1:3) which afforded pure 5 (10.4 g, 81% from 3):

Acyclic Lipid A Mimics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6007
24
[R]D -5 (c 1.2, CHCl3) {lit. [R]D -4.4 (c 1.6, CHCl3)}. 13C
NMR (CDCl3, 90 MHz) δ 177.4, 138.2, 128.3, 127.8, 127.6, 75.8,
71.5, 39.6, 34.2, 31.9, 29.3-29.6, 25.1, 22.6, 14.1.
temperature for 2.5 h. The solvent was then evaporated and the
residue was purified by flash chromatography on silica gel (MeOH/
CH2Cl2, 10:1) to give 11 (5.45 g, 83%) as a colorless syrup: Rf )
0.23 (MeOH/CH2Cl2, 10:1); 1H NMR (250 MHz, CD3OD) δ 7.40-
7.20 (m, 5H, Ph), 5.00 (s, 2H, CH2Ph), 3.75 (dd, 1H, J ) 11.1, 3.8
Hz, H-1B), 3.55 (dd, 1H, J ) 11.1, 6.2 Hz, H-1A), 3.15 (m, 3H,
H-2, 2 H-5), 1.60 (m, 4H, 2 CH2); 13C NMR (62.9 MHz, CD3OD)
δ 162.3, 161.7, 161.2, 160.6, 157.5, 136.7, 128.0, 127.5, 127.3,
118.8, 114.1, 66.0, 60.6, 52.8, 26.0, 25.3.
Benzyl (R)-3-Dodecanoyloxytetradecanoate (7). To a suspen-
sion of (R)-3-hydroxytetradecanoic acid (17.5 g, 72 mmol) in ethyl
acetate (320 mL) were added benzyl bromide (25.6 mL, 0.22 mol),
triethylamine (30 mL, 0.21 mol), and tetrabutylammonium iodide
(13.3 g, 36 mmol). The mixture was stirred for 18 h at room
temperature. The solvent was then evaporated, and the residue was
taken up in ether (300 mL) and washed with saturated aqueous
NaHCO3 (250 mL) and then with water (2 × 250 mL). The organic
phase was separated and dried over MgSO4. The solvent was
evaporated, thus affording crude benzyl (R)-3-hydroxytetrade-
canoate22 6 (26.0 g). Crude 6 (22.8 g, 68 mmol) was acylated with
dodecanoyl chloride (16 mL, 69 mmol) in a mixture of pyridine
(17 mL) and CH2Cl2 (360 mL) at 0 °C and then at room temperature
for 18 h. The reaction mixture was poured into ice-water containing
5% aqueous NaHCO3 and the organic phase was separated. The
organic phase was washed with 1 N aqueous HCl and then with
brine (50 mL) and water (50 mL). The organic phase was separated,
dried over MgSO4, and filtered. The solvent was evaporated and
the residue was purified by flash chromatography on silica gel
(hexane/EtOAc, 20:1) to give 7 (31.3 g, 96% for two steps) as a
colorless syrup: [R]D +1.8 (c 5.20, CHCl3); νmax (thin film)/cm-1
(2R)-5-Benzyloxycarbonylamino-2-[(R)-3-benzyloxytetrade-
canoylamino]pentan-1-ol (12). To a cold solution (-15 °C) of
(R)-3-benzyloxytetradecanoic acid 5 (5.27 g, 15.78 mmol) in THF
(30 mL) were added N-methylmorpholine (1.89 mL, 15.78 mmol)
and isobutyl chloroformate (2.21 mL, 15.78 mmol). The reaction
mixture was stirred at -15 °C for 30 min. A solution of compound
11 (5.25 g, 14.34 mmol) in a mixture of THF (30 mL) and Et3N
(1.44 mL) was added to the reaction mixture. Stirring was continued
for 16 h at room temperature. Water (30 mL) and EtOAc (60 mL)
were then added, the organic phase was separated, and the aqueous
phase was extracted with EtOAc (60 mL) once more. The organic
layers were combined, washed with H2O (30 mL) and brine (30
mL), and dried over MgSO4. The solvent was evaporated and the
residue was crystallized from EtOAc-hexane to give 12 (5.82 g,
69%) as a white solid: mp 117.5-118.0 °C; [R]D +2 (c 1.30,
CHCl3); νmax (KBr)/cm-1 3298, 3090, 2921, 2851, 1690, 1642,
1539, 1264, 1027, 739, 697; 1H NMR (360 MHz, CDCl3) δ 7.35-
7.25 (m, 10H, Ph), 6.55 (br d, 1H, J ∼ 7.5 Hz, N(2)-H), 5.06 (s,
2H, CH2Ph), 4.98 (br, 1H, N(5)-H), 4.56 (d, 1H, J ) 11.1 Hz,
CH2Ph), 4.45 (d, 1H, J ) 11.1 Hz, CH2Ph), 3.83 (m, 2H, H-2,
H-3′), 3.50 (br dd, 1H, J ∼ 3.5, 11 Hz, H-1B), 3.43 (m, 1H, H-1A),
3.11 (m, 2H, 2 H-5), 2.80 (br, 1H, OH), 2.44 (dd, 1H, J ) 4, 14.6
Hz, H-2′B), 2.37 (dd, 1H, J ) 7.6, 14.7 Hz, H-2′A), 1.68-1.42
(m, 6H, 3 CH2), 1.33-1.25 (m, 18H, 9 CH2), 0.88 (t, 3H, CH3);
13C NMR (90 MHz, CDCl3) δ 172.2, 156.5, 138.2, 136.6, 128.5-
127.9, 76.8, 71.4, 66.6, 65.5, 51.5, 41.5, 40.7, 33.9, 31.9, 29.6-
29.3, 28.1, 26.5, 25.1, 22.6, 14.1; MS-IS 591.5 [M + Na]+, 569.5
[M + H]+. Anal. (C34H52N2O5) C, H, N.
(2R)-5-Amino-2-[(R)-3-benzyloxytetradecanoylamino]pentan-
1-ol (13). A solution of compound 12 (3.00 g; 5.27 mmol) in a
mixture of EtOH (300 mL) and Et3N (6 mL) was hydrogenated
for 2 h in the presence of 20% Pd on carbon (150 mg) at room
temperature under H2 (atmospheric pressure). The catalyst was
removed by filtration and the filtrate was concentrated in vacuo to
give amine 13 as a white solid that was used directly in the next
step without purification: Rf ) 0.20 (CH2Cl2/MeOH/Et3N 5:1:0.5);
mp 47-48 °C; [R]D +9.5 (c 1.05, CHCl3); νmax (KBr)/cm-1 3289,
3089, 2920, 2852, 1638, 1554, 1072, 740, 696; 1H NMR (360 MHz,
CDCl3) δ 7.33-7.24 (m, 5H, Ph), 6.77 (br, 1H, NH), 4.51 (AB,
2H, J ) 11.3 Hz, CH2Ph), 3.84 (m, 2H, H-2, H-3′), 3.50 (m, 2H,
2 H-1), 2.89 (br, 3H, 2 H-5, OH), 2.58 (br, 2H, NH2), 2.43-2.33
(ABX, 2H, 2 H-2′), 1.68-1.4 (m, 6H, 3 CH2), 1.33-1.25 (m, 18H,
9 CH2), 0.88 (t, 3H, CH3); 13C NMR (90 MHz, CDCl3) δ 172.0,
138.2, 128.4, 127.9, 127.7, 76.8, 71.5, 64.9, 51.5, 41.6, 41.3, 33.9,
31.9, 29.6, 29.3, 28.7, 28.1, 25.1, 22.6, 14.1; MS-IS 457.0 [M +
Na]+, 435.0 [M + H]+.
1
2925, 2854, 1740, 1465, 1163, 749; H NMR (250 MHz, CDCl3)
δ 7.35 (m, 5H, Ph), 5.21 (m, 1H, H-3), 5.18 (s, 2H, CH2Ph), 2.63
(dd, 1H, J ) 15.2, 7.1 Hz, H-2B), 2.55 (dd, 1H, J ) 15.2, 5.6 Hz,
H-2A), 2.20 (t, 2H, J ) 7.4 Hz, 2 H-2′), 1.70-1.45 (br, 4H), 1.40-
1.10 (br, 34H, 17 CH2), 0.87 (m, 6H, 2 CH3); 13C NMR (62.9 MHz,
CDCl3) δ 172.8, 170.0, 135.6, 128.3, 128.1, 128.0, 70.0, 66.2, 39.1,
34.2, 33.9, 31.8, 29.5, 29.4, 29.3, 29.2, 29.0, 24.9, 24.8, 22.5, 13.9;
MS-IS 539.5 [M + Na]+, 517.5 [M + H]+. Anal. (C33H56O4) C,
H.
(R)-3-Dodecanoyloxytetradecanoic Acid (8). To a solution of
benzyl ester 7 (3 g, 5.8 mmol) in a mixture of ethyl acetate (30
mL) and ethanol (30 mL) was added 10% Pd on carbon (300 mg).
The mixture was hydrogenated at room temperature under atmo-
spheric pressure of hydrogen for 3 h. The catalyst was then removed
by filtration and the filtrate was concentrated. The residue was
recrystallized from pentane (-20 °C). The solvent was decanted
and the solid washed rapidly with cold pentane. The mother liquors
were further crystallized to give pure 823,24 in two crops (2.13 g,
86%): mp 36-38 °C (lit.23,24 oil); NMR spectra identical to the
reported data.24
(2R)-5-Benzyloxycarbonylamino-2-tert-butyloxycarbonylami-
nopentan-1-ol (10). To a cold solution (-15 °C) of Boc-D-Orn-
(Z)-OH (5.45 g, 14.9 mmol) in THF (60 mL) were added
N-methylmorpholine (1.65 mL, 14.9 mmol) and isobutyl chloro-
formate (1.95 mL, 14.9 mmol). The mixture was stirred for 1 min
at -15 °C and then a solution of NaBH4 (1.70 g, 44.7 mmol) in
H2O (10 mL) was added (CAUTION: evolution of hydrogen).
Stirring was continued for 10 min at -15 °C, and then H2O (200
mL) was added to quench the reaction. The mixture was extracted
with EtOAc (2 × 100 mL), and the organic layers were combined
and washed with H2O (50 mL) and brine (60 mL). The organic
phase was separated and dried over MgSO4. The solvent was
evaporated and the residue was crystallized from EtOAc-hexane
to give D-ornithinol derivative 10 (4.94 g, 94%) as a white solid:
mp 47.5-48.0 °C; [R]D +9.5 (c 4.0, CDCl3); νmax (KBr)/cm-1 3343,
2978, 2945, 1697, 1530, 1267, 1172; 1H NMR (360 MHz, CDCl3)
δ 7.31-7.27 (m, 5H, Ph), 5.22 (br, 1H, NH), 5.06 (s, 2H, CH2Ph),
4.97 (br, 1H, NH), 3.66 (m, 2H, 2 H-1), 3.55 (m, 1H, H-2), 3.15
(m, 2H, 2 H-5), 1.54 (m, 4H, 2 CH2), 1.40 (s, 9H, 3 CH3); 13C
NMR (90 MHz, CDCl3) δ 156.6, 156.3, 136.6, 128.5, 128.1, 79.6
(C), 66.6 (CH2), 65.3 (CH2), 52.4 (CH), 40.8 (CH2), 28.7 (CH2),
28.4 (CH3), 26.6 (CH2); MS-IS 391.5 [M + K]+, 375.0 [M + Na]+,
370.5 [M + NH4]+, 353.0 [M + H]+. Anal. (C18H28N2O5) C, H,
N.
(2R)-5-(Benzyloxycarbonylamino)-2-[(R)-3-benzyloxytetrade-
canoylamino]pentan-1-ol Benzyloxymethyl Ether (14). To a
stirred solution of 12 (2.05 g, 3.60 mmol) in CH2Cl2 (40 mL) at
room temperature were added successively benzyl chloromethyl
ether (60%, 1.25 mL, 5.41 mmol) and N,N-diisopropylethylamine
(942 µL, 5.41 mmol). After having been stirred overnight at room
temperature, the mixture was concentrated and the residue was
purified by flash chromatography on silica gel (light petroleum/
EtOAc, 2:1) to give compound 14 (2.28 g, 92%) as a white solid:
Rf ) 0.70 (light petroleum/EtOAc, 1:3); mp 97-100 °C; [R]D -4
(c 1.08, CHCl3); νmax (KBr)/cm-1 3297, 2921, 2851, 1688, 1642,
1
1547, 1264, 695; H NMR (250 MHz, CDCl3) δ 7.40-7.20 (m,
5H, Ph), 6.46 (d, 1H, J ) 8.8 Hz, NH), 5.08 (s, 2H, CH2Ph), 4.95
(br, 1H, NH), 4.65-4.45 (2s and AB, 6H, 2 CH2Ph, OCH2O), 4.08
(m, 1H, H-3′), 3.81 (m, 1H, H-2), 3.56 (dd, 1H, J ) 3.8, 10 Hz,
H-1B), 3.43 (dd, 1H, J ) 4.1, 10 Hz, H-1A), 3.15 (m, 2H, 2 H-5),
(2R)-5-Benzyloxycarbonylamino-2-aminopentan-1-ol, Tri-
fluoroacetic Salt (11). Compound 10 (6.32 g, 17.95 mmol) was
dissolved in TFA (25 mL) and the solution was stirred at room

6008 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Martin et al.
2.45-2.3 (ABX, 2H, 2 H-2′), 1.68-1.42 (m, 6H, 3 CH2), 1.40-
1.15 (m, 18H, 9 CH2), 0.88 (t, 3H, CH3); 13C NMR (62.9 MHz,
CDCl3) δ 170.8, 156.3, 138.2, 137.6, 136.6, 128.4, 128.3, 128.2,
127.9, 127.6, 127.5, 94.8, 76.6, 71.2, 69.6, 69.5, 66.4, 48.3, 41.4,
40.7, 33.8, 31.8, 29.6, 29.5, 29.2, 29.0, 26.3, 25.0, 22.6, 14.0; MS-
IS 689.5 [M + H]+. Anal. (C42H61N2O6) C, H, N.
(2R)-5-Amino-2-[(R)-3-benzyloxytetradecanoylamino]pentan-
1-ol Benzyloxymethyl Ether (15). A solution of compound 14
(2.00 g; 2.90 mmol) in a mixture of EtOH (220 mL) and Et3N (4
mL) was hydrogenated for 3 h in the presence of 20% Pd(OH)2 on
carbon (200 mg) at room temperature under H2 (atmospheric
pressure). The catalyst was removed by filtration and the filtrate
was concentrated in vacuo. The white solid was dried under vacuum
to give amine 15 (1.58 g; 98%). [R]D -1 (c 1.20; CHCl3); νmax
Figure 7. Numbering scheme of 21.
1
1); mp 73.0-73.5 °C; H NMR (360 MHz, CDCl3) δ 7.30-7.25
(m, 9H, Ph), 7.23-7.05 (m, 6H, Ph), 5.10 (s, 2H, CH2Ph), 4.38
(m, 2H, 2 H-4), 4.05 (t, 1H, J ) 6.2 Hz, H-2), 2.34 (br, 2H, 2
H-3); 13C NMR (90 MHz, CDCl3) δ 168.6, 150.1 (m), 134.3, 129.9,
128.7, 128.6, 128.5, 125.6, 120.0 (2 d), 68.5, 64.5 (d, J ) 6 Hz),
49.7, 30.8 (d, J ) 6.6 Hz).
1
(KBr)/cm-1 3313, 2919, 2851, 1635, 1542, 1048, 695; H NMR
(250 MHz, CDCl3) δ 7.45-7.21 (m, 10H, Ph), 6.52 (d, 1H, J )
8.8 Hz, NH), 4.80-4.45 (m, 6H, 2 CH2Ph, OCH2O), 4.10 (m, 1H,
H-3′), 3.83 (m, 1H, H-2), 3.62 (dd, 1H, J ) 3.9, 10.0 Hz, H-1B),
3.47 (dd, 1H, J ) 4.4, 10.0 Hz, H-1A), 2.65 (t, 2H, J ) 6.4 Hz, 2
H-5), 2.48-2.32 (ABX, 2H, 2 H-2′), 1.80-1.40 (m, 8H, 3 CH2,
NH2), 1.40-1.20 (m, 18H, 9 CH2), 0.88 (t, 3H, J ) 6.3 Hz, CH3);
13C NMR (62.9 MHz, CDCl3) δ 170.8, 138.2, 137.6, 128.3, 127.6,
94.8, 76.7, 71.2, 69.6, 69.4, 48.4, 41.8, 41.4, 33.8, 31.8, 29.8, 29.5-
29.0, 25.1, 22.6, 14.0; MS-IS 555.5 [M + H]+.
N-[(R)-3-Dodecanoyloxytetradecanoyl] O-Bis(phenyloxy)-
phosphoryl DL-Homoserine Benzyl Ester (19). To a stirred
solution of (R)-3-dodecanoyloxytetradecanoic acid 8 (4.28 g, 10.07
mmol) in THF (30 mL) at -15 °C were added successively
N-methylmorpholine (1.11 mL, 10.07 mmol) and isobutyl chloro-
formate (1.31 mL, 10.07 mmol). Stirring was continued for 30 min
at -15 °C. A solution of 18 (5.73 g, 10.07 mmol) in a mixture of
THF (30 mL) and Et3N (5 mL) was then added to the reaction
mixture. After stirring overnight at room temperature, the solvent
was removed in vacuo and H2O (20 mL) was added to the residue.
The mixture was then extracted with EtOAc (2 × 30 mL), and the
organic phases were combined, washed successively with H2O (20
mL) and brine (20 mL), and dried over MgSO4. The solvent was
evaporated and the residue was purified by flash chromatography
on silica gel (hexane/EtOAc, 2:1) to give 19 (7.46 g, 87%) as a
white solid: Rf ) 0.29 (hexane/EtOAc, 2:1); mp 31.0-32.1 °C;
N-tert-Butoxycarbonyl O-Bis(phenyloxy)phosphoryl DL-Ho-
moserine Benzyl Ester (17). Commercially available DL-ho-
moserine hydrobromide (2.0 g, 16.78 mmol) was dissolved in H2O
(20 mL). To this solution were added successively 1 N aqueous
NaOH (16.8 mL) and solid cesium carbonate (3.00 g, 9.23 mmol).
After having been stirred for 5 min, the solution was cooled in an
ice-water bath. Dioxane (60 mL) and di-tert-butyl dicarbonate
(5.44 g, 25.17 mmol) were then added. The reaction mixture was
stirred at 0 °C for 1 h and then at room temperature for 5 h. The
solvent was removed in vacuo and the residue was dried by
coevaporation with DMF (20 mL). DMF (60 mL) and benzyl
bromide (4.50 mL, 20.13 mmol) were then added to the crude
cesium salt, which resulted in the formation of a white precipitate
of cesium bromide. The mixture was stirred for 16 h and the solvent
was then removed in vacuo. The residue was extracted with EtOAc
(2 × 20 mL). The organic layers were combined, washed succes-
sively with H2O (20 mL) and brine (20 mL), and dried over MgSO4.
The solvent was evaporated and the residue was dried under high
vacuum to give crude N-tert-butoxycarbonyl DL-homoserine benzyl
ester 16. To a solution of this crude material in CH2Cl2 (60 mL)
was added DMAP (4.11 g, 33.56 mmol), and the reaction mixture
was stirred for 10 min. Pyridine (12 mL) and diphenyl chlorophos-
phate (6.95 mL, 33.56 mmol) were then added and the solution
was stirred at room temperature for 18 h. The mixture was then
washed successively with 1 N aqueous HCl (20 mL), H2O (30 mL),
and brine (30 mL). The organic layer was separated and dried over
MgSO4. The solvent was evaporated and the residue was purified
by flash chromatography on silica gel (hexane/EtOAc, 4:1) followed
by crystallization to give 17 as a white solid (7.49 g, yield: 82%
from DL-homoserine hydrobromide): Rf ) 0.13 (hexane/EtOAc,
4:1); mp 63.5-64.0 °C; νmax (KBr)/cm-1 3304, 2974, 1740, 1702,
ν
max (KBr)/cm-1 3305, 3066, 2922, 2852, 1731, 1681, 1537, 1489,
1
1288, 1190, 1025, 954, 754, 689; H NMR (360 MHz, CDCl3) δ
7.27-7.36 (m, 9H, Ph), 7.16-7.20 (m, 6H, Ph), 6.62 (2d, 1H, J ∼
8 Hz, NH), 5.09-5.20 (m, 3H, H-3′, CH2Ph), 4.68 (m, 1H, H-2),
4.31 (m, 2H, 2 H-4), 2.39-2.51 (m, 2H, 2 H-2′), 2.20-2.34 (m,
4H, 2 CH2), 1.58 (m, 4H, 2 CH2), 1.25 (m, 34H, 17 CH2), 0.88 (m,
6H, 2 CH3); 13C NMR (90 MHz, CDCl3) δ 173.4, 171.2, 169.7,
150.4 (d), 135.1, 129.8, 128.3-128.8, 125.4, 120.0 (d), 71.0 (2
signals), 67.5, 65.3 (2 signals), 49.5 (2 signals), 41.5 and 41.3, 34.4,
34.2 and 34.0, 32.3, 32.2, 31.9, 29.2-29.6, 25.2 and 25.0, 22.7,
14.1; 31P NMR (121.5 MHz) δ -11.36, -11.37 (3JP, H ) 7.1 Hz);
MS-FAB 850.4 [M + H]+; HRMS-FAB calcd for C49H72NO9P [M
+ H]+ 850.5023, found 850.5024.
O-Bis(phenyloxy)phosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl] DL-Homoserine (20). 20% Pd-C (40 mg) was added to a
solution of 19 (85 mg, 0.1 mmol) in EtOH (20 mL) in a three-
necked flask. (The reaction is faster if the catalyst is activated with
hydrogen prior to the addition of the substrate.) Air was removed
under vacuum, then the flask was charged with hydrogen (atmo-
spheric pressure). The reaction mixture was stirred at room
temperature for 3 h, the catalyst was removed by filtration, and
the filtrate was concentrated to give labile acid 20 as a colorless
syrup that was used in the next step without purification: 13C NMR
(90 MHz, CDCl3) δ 173.9, 173.7, 170.7, 150.2, 129.9, 125.7, 120.0,
71.1, 65.9, 49.7, 41.2, 34.2, 34.1, 31.9, 29.1-29.6, 25.2, 25.0, 22.6,
14.0.
O-Bis(phenyloxy)phosphoryl Nr-[(R)-3-Dodecanoyloxytetra-
decanoyl] DL-Homoserine, N-{(4R)-5-Hydroxy-4-[(R)-3-benzyl-
oxytetradecanoylamino]pentyl}amide (21, Figure 7). IIDQ (36.1
mg, 0.12 mmol) was added to a solution of crude acid 20 (67 mg,
0.10 mmol) in CH2Cl2 (10 mL). After stirring for 15 min, a solution
of amine 13 (53 mg, 0.10 mmol) in CH2Cl2 (5 mL) was introduced
and the reaction mixture was stirred overnight. The solution was
concentrated and the residue was purified by flash chromatography
on silica gel (CH2Cl2/acetone, 5:2) to give amide 21 (66 mg, 56%)
1
1524, 1487, 1275, 1184, 1159, 964; H NMR (360 MHz, CDCl3)
δ 7.34-7.28 (m, 9H, Ph), 7.25-7.15 (m, 6H, Ph), 5.19 (br d, 1H,
NH), 5.13 (∼s, 2H, CH2Ph), 4.42 (m, 1H, H-2), 4.33 (∼q, 2H, 2
H-4), 2.26 (m, 1H, H-3B), 2.14 (m, 1H, H-3A), 1.42 (s, 9H, t-Bu);
13C NMR (90 MHz, CDCl3) δ 171.6, 155.2, 150.4 (d), 135.2, 129.8,
128.6, 128.4, 128.3, 125.4, 120.0 (d), 80.1, 67.3, 65.3 (d), 50.7,
32.7 (d), 28.2; 31P NMR (121.5 MHz) δ -11.42 (3JP, H ) 7.4 Hz);
MS-FAB 542.2 [M + H]+; HRMS-FAB calcd for C28H32NO8P [M
+ H]+ 542.1944, found 542.1956.
O-Bis(phenyloxy)phosphoryl DL-Homoserine Benzyl Ester,
Trifluoroacetic Salt (18). Compound 17 (7.88 g, 14.56 mmol) was
dissolved in TFA (15 mL) and the solution was stirred at room
temperature for 2.5 h. The solvent was then removed under high
vacuum. Purification of the residue by flash chromatography on
silica gel (MeOH/CH2Cl2, 10:1) followed by crystallization gave
18 (7.17 g, 89%) as a white solid: Rf ) 0.18 (MeOH/CH2Cl2, 10:
1
as a colorless syrup: Rf ) 0.23 (CH2Cl2/acetone, 5:2); H NMR
(360 MHz, CDCl3) δ 7.14-7.33 (m, 15H, Ph), 7.00 (br, 1H, NH),

Acyclic Lipid A Mimics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6009
6.90 (d, 0.5H, J ) 7.5 Hz, NH), 6.79 (d, 0.5H, J ) 7.4 Hz, NH),
6.67 (d, 1H, J ) 7.9 Hz, NH), 5.14 (m, 1H, H-3), 4.5 (m, 3H,
H-2b, CH2Ph), 4.29 (m, 2H, 2 H-4b), 3.83 (m, 2H, H-2a, H-3"),
3.41 (br m, 2H, 2 H-1a), 3.19 (m, 2H, 2 H-5a), 2.14-2.41 (3 m,
8H, 4 CH2), 1.46-1.54 (br m, 8H, 4 CH2), 1.22 (m, 54H, 27 CH2),
0.85 (m, 9H, 3 CH3); 13C NMR (90 MHz, CDCl3; two values given
when two signals are observed for diastereomeric species) δ 173.5,
172.0, 170.5/170.4, 170.0/169.9, 150.2 (d), 138.4/138.3, 129.9,
128.3, 127.8, 127.6, 125.6, 120.05(d)/119.9(d), 76.6, 71.4, 71.0,
66.2/66.1, 64.9, 51.4/51.2, 50.2/50.1, 41.6/41.5, 39.3, 34.4/34.0, 32.9
(br), 31.8, 29.1-29.5, 28.4/28.2, 24.9-25.4, 22.6, 14.0; 31P NMR
(121.5 MHz) δ -11.2, -11.3 (3JP, H ) 8.5 Hz); MS-FAB 1176.7
[M + H]+; HRMS-FAB calcd for C68H110N3O11P [M + H]+
1176.7956, found 1176.7933.
O-Bis(phenyloxy)phosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl] DL-Homoserine, N-{(4R)-5-Bis(benzyloxy)phosphoryl-
oxy-4-[(R)-3-benzyloxytetradecanoylamino]pentyl}amide (22).
To a stirred solution of amide 21 (300 mg, 0.26 mmol) and 1H-
tetrazole (64 mg, 0.82 mmol) in THF (10 mL) at room temperature
was added dibenzyl diethylphosphoramidite (85%, 230 mg, 0.62
mmol). Stirring was continued at room temperature for 30 min and
the solution was then cooled to -20 °C. A solution of mCPBA
(80-90%, 74 mg, 0.36 mmol) in CH2Cl2 (7 mL) was added and
the solution was stirred for 20 min at room temperature. Aqueous
sodium thiosulfate (10%, 6 mL) was added, the mixture was stirred
for 10 min and then diluted with ether (30 mL), and the organic
phase was separated. The organic layer was washed successively
with 10% aqueous Na2S2O3 solution (5×), saturated aqueous
NaHCO3 (2×), N HCl (1×). The organic phase was dried over
MgSO4 and the solvent removed in vacuo. Flash chromatography
of the residue on silica gel (CH2Cl2/acetone, 10:3) provided
compound 22 (242 mg, 65%) as a colorless oil: Rf ) 0.64 (CH2-
Cl2/acetone 5:2); νmax (thin film)/cm-1 3277, 2921, 2851, 1793,
1737, 1657, 1537, 1490, 1224, 1090, 1025, 916, 745, 695; 1H NMR
(360 MHz, CDCl3) δ 7.13-7.31 (m, 25H, Ph), 6.86 (br d, 1H, NH),
6.73 (d, 0.5H, J ) 7.5 Hz, NH), 6.68 (d, 0.5H, J ) 8.5 Hz, NH),
6.64 (d, 1H, J ) 8.2 Hz, NH), 5.17 (2 qt, 1H, H-3), 4.96 (m, 4H,
2 × POCH2Ph), 4.52 (m, 1H, H-2b), 4.47 (s, 2H, CH2Ph), 4.30 (br
q, 2H, 2 H-4b), 3.8-4.0 (3 m, 4H), 3.05-3.25 (m, 2H), 2.15-
2.41 (several m, 8H, 4 CH2), 1.4-1.56 (m, 8H) and 1.22 (m, 54H)
(31 CH2), 0.85 (m, 9H, 3 CH3); 13C NMR (90 MHz, CDCl3) δ
173.5/173.4, 173.1, 170.5/170.4, 169.9/169.7 (4 CdO), 150.4,
138.5, 135.7/135.6, 127.5-129.8, 125.5, 120.05 (d)/119.95 (d), 76.5,
71.2/71.0, 69.5/69.45, 68.7/68.6, 66.2 (d)/66.05 (d), 50.0/49.9, 48.5/
48.45, 41.7/41.4, 39.0, 34.0-34.4, 32.9-33.1, 31.8, 29.1-29.6,
27.7, 24.95-25.2, 22.6, 14.0; 31P NMR (121.5 MHz) δ -0.03 (3J
) 8 Hz, POBn), -11.00, -11.12 (3J ) 8 Hz, POPh); MS-IS 1436.5
[M + H]+.
O-Bis(phenyloxy)phosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl] DL-Homoserine, N-{(4R)-5-Dihydroxyphosphoryloxy-4-
[(R)-3-hydroxytetradecanoylamino]pentyl}amide (23). Pd-C
(20%, 40 mg) was added to a solution of 22 (160 mg, 0.11 mmol)
in EtOH (20 mL) in a three-necked flask. Air was removed under
vacuum and then the flask was charged with hydrogen (atmospheric
pressure). Stirring was continued until no more starting material
could be detected on TLC (CHCl3/MeOH/H2O, 6:4:0.6, new product
has Rf ) 0.63). The catalyst was removed by filtration, the filtrate
was concentrated in vacuo, and the residual product dried under
vacuum to give homogeneous 23 (100 mg; 78%): 13C NMR (90
MHz, CDCl3, after addition of Et3N) δ 173.6, 173.5, 173.1, 171.2,
170.7, 170.4, 151.6, 150.3, 150.2, 129.9, 129.5, 125.6, 124.4, 120.2,
120.2, 120.1, 120.0, 120.0, 71.1, 69.2, 67.8 (br), 66.3 (m), 66.0,
58.4 (Et3NH+), 50.1, 49.3, 43.3 (br), 41.4, 41.3, 39.0, 37.3, 34.5,
34.3, 32.7 (br), 31.9, 29.2-29.7, 27.5, 25.0-25.6, 22.7, 18.3 (Et3-
NH+), 14.1; 31P NMR (121.5 MHz) δ 0.56 (POH), -10.77, -11.70
(POPh); MS-FAB 1188.7 [M + Na] +; HRMS-FAB calcd for
C61H105N3O14P2 [M + Na]+ 1188.6969, found 1188.6959.
O-Dihydroxyphosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl] DL-Homoserine, N-{(4R)-5-Dihydroxyphosphoryloxy-4-
[(R)-3-hydroxytetradecanoylamino]pentyl}amide [1(*R/S)]. PtO2
(60 mg) was added to a solution of 23 (100 mg, 86 µmol) in EtOH
(20 mL) in a three-necked flask. Air was removed under vacuum
and then the flask was charged with hydrogen (atmospheric
pressure). The reaction mixture was stirred until no more starting
material could be detected (∼ 72 h). The catalyst was removed by
filtration, and the filtrate was concentrated to give 1(*R/S) (67 mg,
77%) as a white glass. The product was dissolved in 1:1 (v/v) H2O/
iPrOH containing 0.1% Et3N (pH 8-9); 2 M ammonium bicarbon-
ate was then added to reach a 25 mM final concentration. The
product was purified from this solution by RP HPLC [Bondapak
gel C18, 300 Å, 15-20 µm; column size 40 × 200 mm; solvent A,
1:9 (v/v) H2O/iPrOH containing ammonium bicarbonate (50 mM);
solvent B, 8:2 (v/v) H2O/iPrOH containing ammonium bicarbonate
(50 mM); 10 min 40% B; 10 min 40% f 80% B; 30 min 80% B;
tR ) 21 min; UV (210 nm) detection]: Rf ) 0.16 (CHCl3/MeOH/
H2O, 6:4:0.6); νmax (KBr)/cm-1 3297, 2955, 2921, 2851, 1731, 1651,
1549, 1469, 1172, 1062; 13C NMR (90 MHz, CDCl3, broad signals)
selected signals: δ 71.2, 69.3, 67.9, 63.5, 50.7, 49.6, 43.2, 41.4,
39.3, 37.2, 33.8-34.5, 32.8, 31.9, 29.1-29.8, 28.0, 27.5, 24.9-
25.8, 22.7, 14.0; 31P NMR (121.5 MHz) δ 0.98, 0.21. MS-FAB
1036.6 [M + Na]+; 1058.5 [M - H + 2Na]+; 1080.6 [M - 2H +
3Na]+; HRMS-FAB calcd for C49H97N3O14P2 [M + Na]+ 1036.6344,
found 1036.6347; HRMS-FAB calcd for C49H97N3O14P2 [M - H
+ 2Na]+ 1058.6163, found 1058.6158.
O-Bis(phenyloxy)phosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl] DL-Homoserine, N-{(4R)-5-Hydroxy-4-[(R)-3-hydroxy-
tetradecanoylamino]pentyl}amide (24). 20% Pd-C (40 mg) was
added to a solution of 21 (150 mg, 0.13 mmol) in a mixture of
EtOH (20 mL) and AcOH (0.6 mL) in a three-necked flask. Air
was removed under vacuum and the flask was charged with
hydrogen (atmospheric pressure). The mixture was stirred for 16 h
at room temperature. The catalyst was removed by filtration and
the filtrate was concentrated. Purification of the residue by flash
chromatography on silica gel (CH2Cl2/acetone, 5:4) gave compound
24 (104 mg, 74%) as a glassy solid: Rf ) 0.24 (CH2Cl2/acetone,
5:4); mp 67-68 °C;1H NMR (360 MHz, CDCl3) δ 7.15-7.35 (m,
10H, Ph), 7.03 (d, 0.5H, J ) 7.3 Hz, NH), 6.91 (d, 0.5H, J ) 7.1
Hz, NH), 6.79 (d, 1H, J ) 8.0 Hz, NH), 5.16 (m, 1H, H-3), 4.50
(m, 1H, H-2b), 4.29 (m, 2H, 2 H-4b), 3.89 (m, 2H, H-2a, H-3′′),
3.4-3.6 (m, 2H), 3.20 (m, 2H), 2.10-2.42 (m, 8H, 4 CH2), 1.38-
1.54 (m, 8H) and 1.22 (m, 54H) (31 CH2), 0.85 (m, 9H, 3 CH3);
13C NMR (90 MHz, CDCl3) δ 173.6, 173.2, 170.7/170.6, 170.3/
170.1, 150.3, 129.9, 125.7, 120.1/120.0, 120.0/119.9, 71.1/71.0,
68.8, 66.2/66.1, 64.7, 51.4, 50.3/50.1, 43.2/43.1, 41.7/41.6, 39.3,
37.3, 34.4, 32.8, 31.9, 29.1-29.6, 28.1, 25.0-25.6, 22.6, 14.0; 31
P
NMR (121.5 MHz) δ -11.28, -11.40 (3JP, H ) 7.8 Hz); MS-FAB
1086.8 [M + H]+; HRMS-FAB calcd for C61H104N3O11P [M +
H]+ 1086.7568, found 1086.7529.
O-Dihydroxyphosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl] DL-Homoserine, N-{(4R)-5-Hydroxy-4-[(R)-3-hydroxy-
tetradecanoylamino]pentyl}amide (2(*R/S)). PtO2 (30 mg) was
added to a solution of 24 (90 mg, 0.08 mmol) in EtOH (30 mL) in
a three-necked flask. Air was removed under vacuum and then the
flask was charged with hydrogen (atmospheric pressure). The
reaction mixture was stirred until no more starting material could
be detected by TLC (CHCl3/MeOH/H2O, 6:4:0.6, new product has
Rf ) 0.50). The catalyst was removed by filtration and the filtrate
was concentrated to give 2(*R/S) (60 mg, 78%) as a white solid.
The product was dissolved in iPrOH/H2O (1:1, v/v, ∼3 mg/mL)
and the pH was adjusted to pH 8-9 by addition of Et3N. A sample
from this solution was purified by preparative HPLC (conditions:
see preparation of 1(*R/S); tR ) 25 min): 13C NMR (90 MHz,
CDCl3 + a drop of CD3OD) δ 173.6, 173.3, 171.3, 170.4, 71.1,
68.8, 64.2, 62.6, 51.0, 50.2, 43.5, 41.5, 39.2, 37.3, 34.5, 33.7, 31.8,
29.2-29.8, 28.0, 25.0-25.8, 22.7, 14.1; 31P NMR (121.5 MHz) δ
0.78 (3JP, H ) 7.8 Hz); MS-FAB 956.5 [M + Na] +, 835.7 [M -
H3PO4]; HRMS-FAB calcd for C49H96N3O11P [M + Na] + 956.6680,
found 956.6674.
N-[(R)-3-Dodecanoyloxytetradecanoyl]-D-aspartic Acid, â-Ben-
zyl Ester (25). To a stirred solution of (R)-3-dodecanoyloxy-
tetradecanoic acid 8 (3.35 g, 7.85 mmol) in THF (25 mL) at -15
°C were added successively N-methylmorpholine (0.86 mL, 7.85

6010 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Martin et al.
added Et3N (4 mL) and the mixture was hydrogenated in the
presence of 10% Pd-C (120 mg) at room temperature under H2
(atmospheric pressure) for 2 h. The catalyst was removed by
filtration and the filtrate was concentrated in vacuo. The white solid
was taken in 1:1 i-PrOH/CH2Cl2 (100 mL) and the mixture was
stirred in the presence of Amberlite IR-120 (H+) ion-exchange resin
(5 mL) for 10 min. The resin was removed by filtration and the
filtrate was concentrated in vacuo and dried under vacuum to give
homogeneous acid 27 (2.25 g; 97%) as a white solid: mp 115-
117 °C; [R]D +8 (c 1.16; CHCl3); νmax (KBr)/cm-1 3292, 3096,
1729, 1702, 1641, 1547; 13C NMR (62.9 MHz, CDCl3) δ 173.9,
172.6, 171.0, 170.5, 138.1, 128.6, 128.1, 77.0, 71.7, 71.4, 64.6,
51.4, 49.7, 41.8, 39.3, 36.5, 34.6, 34.2, 32.0, 29.8, 29.5, 28.0, 25.4,
25.2, 22.8, 14.2; MS-IS 959.0 [M + H]+. Anal. (C56H99N3O9) C,
H, N.
Figure 8. Numbering scheme of 26.
mmol) and isobutyl chloroformate (1.02 mL, 7.85 mmol). After
30 min at -15 °C, a solution of H-D-Asp(OBn)-OH (Senn
Chemicals) (1.75 g, 7.85 mmol) in 3.5:1 (v/v) CH3CN/H2O (85
mL) and Et3N (3.7 mL) were added to the reaction mixture. After
having been stirred overnight at room temperature, the mixture was
partially concentrated and the residual aqueous phase was cooled
to 0°C and acidified with 10% aqueous citric acid (pH ) 3). The
mixture was then extracted with EtOAc (2 × 30 mL), and the
organic phases were combined and dried over MgSO4. The solvent
was evaporated and the residue was purified by flash chromatog-
raphy on silica gel (light petroleum/EtOAc, 2:1 containing 2% acetic
acid) to give 25 (4.00 g, 81%) as a white solid: Rf ) 0.42 (light
petroleum/EtOAc, 1:1 containing 2% acetic acid); mp 67-69 °C;
[R]D -19 (c 1.20, CHCl3); νmax (KBr)/cm-1 3333, 3080, 1736, 1702,
N-[(R)-3-Dodecanoyloxytetradecanoyl]-D-aspartic Acid, r-N-
{(4R)-5-Hydroxy-4-[(R)-3-hydroxytetradecanoylamino]pentyl}-
amide (28). Compound 26 (417 mg, 0.4 mmol, containing ∼25%
2b(S)-epimer) was hydrogenated for 3 h in a 1:1 mixture of MeOH
and EtOAc (36 mL) in the presence of 10% Pd-C (20 mg) at room
temperature under H2 (atmospheric pressure). The catalyst was then
removed by filtration and washed with a 4:1 mixture of CH2Cl2/
MeOH (50 mL). The filtrate was concentrated, thus providing free
acid 28 as a white solid (345 mg, quant.). Compound 28 was
purified by HPLC under the same conditions as compound 1(*R/
S): Rf ) 0.30 (CH2Cl2/MeOH 9:1 containing 0.5% AcOH); mp
135-137 °C; νmax (KBr)/cm-1 3450, 3301, 2957, 2922, 2852, 1735,
1
1649, 1548, 1170; H NMR (250 MHz, CDCl3) δ 8.90 (br, 1H,
COOH), 7.30-7.50 (m, 5H, Ph), 6.89 (d, 1H, J ) 7.8 Hz, NH),
5.10-5.20 (m and s, 3H, H-3, CH2Ph), 4.87 (dt, 1H, J ) 4.5, 4.5,
8 Hz, H-2), 3.09 (dd, 1H, J ) 4.4, 17.6 Hz, H-3B), 2.90 (dd, 1H,
J ) 4.5, 17.6 Hz, H-3A), 2.48 (∼d, 2H, J ) 6.3 Hz, 2 H-2′), 2.28
(t, 2H, J ) 7.5 Hz, 2 H-2′′), 1.60 (m, 4H) and 1.10-1.40 (m, 34H,
19 CH2), 0.87 (t, 6H, 2 CH3); 13C NMR (62.9 MHz, CDCl3) δ
174.1, 173.5, 171.0, 170.2, 135.2, 128.6, 128.4, 128.2, 125.8, 70.9,
66.9, 48.4, 41.2, 35.8, 34.4, 34.0, 31.8, 29.1-29.6, 25.2, 24.9, 22.6,
14.1; MS-IS 655.0 [M + Na]+, 633.0 [M + H]+. Anal. (C37H61-
NO7) C, H, N.
1
1637, 1458, 1377, 1235, 1176; H NMR (250 MHz, CDCl3/CD3-
OD 7:1; major epimer) δ 7.59 (d, 1H, J ) 8.5 Hz, NH), 7.47 (t,
1H, J ) 6 Hz, NH), 7.20 (d, 1H, J ) 8.5 Hz, NH), 5.17 (m, 1H,
H-3), 4.68 (m, 1H, H-2b), 3.8-4.0 (2m, 2H, H-3′′, H-2a), 3.57
(dd, 1H, J ) 4.1, 11.3 Hz, H-1aB), 3.47 (dd, 1H, J ) 5.0, 11.3 Hz,
H-1aA), 3.07-3.34 (m, 2H, 2 H-5a), 2.7-2.85 (m, 2H, 2 H-3b),
2.2-2.55 (m, 6H, 3 CH2), 1.2-1.65 (m, 62 H, 31 CH2), 0.88 (t,
9H, 3 CH3); 13C NMR (62.9 MHz, CDCl3/CD3OD 7:1; major
epimer) δ 173.9, 173.8, 173.2, 170.5, 71.1, 68.7, 64.1, 51.1, one
signal hidden by CD3OD signal, 43.3, 41.2, 39.1, 38.9, 37.1, 35.8,
34.3, 34.2, 31.7, 28.9-29.5, 27.5, 25.5, 25.4, 25.0, 24.8, 22.5, 13.9;
MS-IS: m/z 868.7 [M + H]+, 890.7 [M + Na]+, 912.7 [M - H +
2Na]+. Anal. (C49H93N3O9) C, H, N.
N-[(R)-3-Dodecanoyloxytetradecanoyl]-D-Aspartic Acid, r-N-
{(4R)-5-Hydroxy-4-[(R)-3-benzyloxytetradecanoylamino]pentyl}-
amide â-Benzyl Ester (26, Figure 8). To a stirred solution of
aspartic acid derivative 25 (363 mg, 0.57 mmol) and amine 13 (250
mg, 0.57 mmol) in CH2Cl2 (6 mL) at 0 °C were added successively
1-hydroxy-7-azabenzotriazole (HOAt) (94 mg, 0.69 mmol) and
N,N′-diisopropylcarbodiimide (109 µL, 0.69 mmol). The reaction
mixture was stirred for 1 h at 0 °C and overnight at room
temperature. The mixture was then diluted with CH2Cl2 (20 mL)
and water (20 mL). The organic phase was separated and washed
with 1 N HCl followed by saturated aqueous NaHCO3. The organic
phase was dried over MgSO4. The solvent was evaporated and the
residue was purified by flash chromatography on silica gel (CH2-
Cl2/acetone 3:1 f 2:1) to give 26 (436 mg; 72%) as a white solid
(less than 5% 2b(S)-epimer): Rf ) 0.27 (CH2Cl2/acetone 5:1); mp
106-108 °C; [R]D -7 (c 0.1; CHCl3); νmax (KBr)/cm-1 3288, 1729,
N-[(R)-3-Dodecanoyloxytetradecanoyl]-D-homoserine, N-{(4R)-
5-Hydroxy-4-[(R)-3-benzyloxytetradecanoylamino]pentyl}-
amide (29). To a stirred solution of acid 27 (1.71 g, 1.78 mmol,
less than 5% 2b(S)-epimer) in THF (12 mL) at 0 °C were added
successively N-methylmorpholine (196 µL, 1.78 mmol) and isobutyl
chloroformate (232 µL, 1.78 mmol). Stirring was continued at room
temperature for 30 min and the solution was again cooled to 0 °C.
A solution of NaBH4 (135 mg, 3.57 mmol) in H2O (4.5 mL) was
added (CAUTION: evolution of gas); the solution was diluted with
H2O (4.5 mL) and THF (5 mL) and stirred at room temperature
for 5 min. The organic solvent was then partially evaporated under
reduced pressure, CH2Cl2 (25 mL) was added, and the residual
aqueous phase was neutralized with 1 N HCl. The organic layer
was separated and dried over MgSO4. The solvent was evaporated
and the residue was purified by flash chromatography on silica gel
(CH2Cl2/MeOH, 14:1) to give homogeneous diol 29 (1.24 g, 74%)
as a white solid: Rf ) 0.27 (CH2Cl2/MeOH 12:1); mp 112-113
°C; νmax (KBr)/cm-1 3293, 1729, 1643, 1560, 1467, 1378; 1H NMR
(250 MHz, CDCl3) δ 7.28-7.40 (m, 5H, Ph), 6.91 (br d, 1H, J )
7.6 Hz, NH), 6.87 (br, 1H, NH), 6.61 (d, 1H, J ) 7.6 Hz, NH),
5.16 (quintet, 1H, H-3), 4.45-4.62 (m and AB, 3H, JAB ) 11.1
Hz, H-2b and CH2Ph), 3.87 (m, 2H, H-2a, H-3′′), 3.45-3.75 (m,
4H, 2 CH2), 3.23 (m, 2H, CH2), 2.40-2.55 (m, 4H, 2 H-2′′, 2 H-2),
2.29 (t, 2H, J ) 7.6 Hz, 2 H-2′), 1.96 (m, 1H, H-3bB), 1.75 (m,
1H, H-3bA), 1.45-1.70 (m, 10H) and 1.2-1.45 (m, 54H) (32 CH2),
0.88 (m, 9H, 3 CH3); 13C NMR (62.9 MHz, CDCl3) δ 173.7, 172.2,
171.7, 171.0, 138.3, 128.5, 128.0, 76.9, 71.6, 71.2, 64.8, 58.6, 51.4,
50.9, 41.7, 39.3, 36.0, 34.6, 34.1, 32.0, 29.3-29.8, 28.3, 25.6, 25.3,
25.1, 22.8, 14.2; MS-IS 945.0 [M + H]+. Anal. (C56H101N3O8) C,
H, N.
1
1637, 1552, 1466, 1172; H NMR (250 MHz, CDCl3) δ 7.25-
7.40 (m, 10H, Ph), 6.97 (br d, 1H, J ) 8.1 Hz, NH), 6.79 (br t,
1H, J ) 5 Hz, NH), 6.53 (br d, 1H, J ) 7.5 Hz, NH), 5.07-5.18
(m and AB, 3H, CH2Ph, H-3), 4.73 (m, 1H, H-2b), 4.54 (AB, 2H,
J ) 11.3 Hz, CH2Ph), 3.86 (m, 2H, H-2a, H-3′′), 3.4-3.6 (m, 2H,
2 H-1a), 3.19 (m, 2H, 2 H-5a), 3.01 (dd, 1H, J ) 4.7, 16.9 Hz,
H-3bB), 2.9 (br, 1H, OH), 2.67 (dd, 1H, J ) 6.0, 16.9 Hz, H-3bA),
2.43 (m, 4H, 2 H-2′′, 2 H-2), 2.28 (t, 2H, J ) 7.5 Hz, 2 H-2′),
1.4-1.7 (m) and 1.2-1.4 (m) (62 H, 31 CH2), 0.88 (m, 9H, 3 CH3);
13C NMR (62.9 MHz, CDCl3) δ 173.8, 172.3, 171.9, 170.5, 170.3,
138.4, 135.4, 128.7, 128.5, 128.3, 128.0, 76.9, 71.5, 71.3, 66.9,
65.2, 51.5, 49.5, 41.8, 39.4, 35.6, 34.5, 34.1, 32.0, 29.7, 29.4, 29.3,
28.2, 25.7, 25.3, 25.2, 25.1, 22.8, 14.2; MS-IS 1071.0 [M + Na]+,
1049.0 [M + H]+. Anal. (C63H105N3O9) C, H, N.
N-[(R)-3-Dodecanoyloxytetradecanoyl]-D-aspartic Acid, r-N-
{(4R)-5-Hydroxy-4-[(R)-3-benzyloxytetradecanoylamino]pentyl}-
amide (27). To a solution of compound 26 (2.53 g; 2.4 mmol, less
than 5% 2b(S)-epimer) in 1:1 (v/v) EtOH/EtOAc (150 mL) was

Acyclic Lipid A Mimics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6011
O-Bis(benzyloxy)phosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl]-D-homoserine, N-{(4R)-5-Bis(benzyloxy)phosphoryloxy-
4-[(R)-3-benzyloxytetradecanoylamino]pentyl}amide (30). To a
stirred solution of 29 (1.02 g, 1.08 mmol, less than 5% 2b(S)-
epimer) and 1H-tetrazole (454 mg, 6.48 mmol) in THF (46 mL) at
room temperature was added dibenzyl diethylphosphoramidite
(85%, 1.5 mL, 3.78 mmol). Stirring was continued for 30 min at
room temperature. The solution was cooled to -20 °C. A solution
of mCPBA (57-86%, 1.32 g, ∼7.7 mmol) in CH2Cl2 (30 mL) was
added and the solution was stirred for 45 min at room temperature.
Saturated aqueous sodium thiosulfate (25 mL) was then added and
the mixture was stirred for 10 min. The mixture was diluted with
ether (50 mL) and the organic phase was separated. The organic
layer was washed successively with saturated Na2S2O3 (5×),
saturated NaHCO3 (2 ×), and 1 N HCl (1 ×) and the solution was
dried over MgSO4. The solvent was removed in vacuo and the
residual product was purified by flash chromatography on silica
gel (CH2Cl2/acetone, 4:1 f 3:1), which provided homogeneous 30
(1.38 g, 87%) as a colorless oil: Rf ) 0.23 (CH2Cl2/acetone 5:1);
[R]D +7 (c 1.25, CHCl3); νmax (KBr)/cm-1 3286, 3065, 1727, 1633,
1555, 1463, 1380, 1266, 1019; 1H NMR (250 MHz, CDCl3) δ 7.2-
7.4 (m, 25H, Ph), 7.06 (t, 1H, J ) 5 Hz, NH), 6.76 (d, 1H, J ) 8
Hz, NH), 6.72 (d, 1H, J ) 8 Hz, NH) 5.15 (quintet, 1H, H-3),
4.93-5.08 (m, 8H, 4 POCH2Ph), 4.51 (br q, 1H, H-2b), 4.48 (s,
2H, OCH2Ph), 3.75-4.1 (m, 6H), 3.15 (m, 2H, 2 H-5a), 2.33-
2.47 (m, 4H, 2 H-2′′, 2 H-2), 2.25 (t, 2H, J ) 7.5 Hz, 2 H-2′), 2.04
(m, 2H, 2 H-3b), 1.40-1.65 (m, 10H) and 1.20-1.40 (m, 54H)
(32 CH2), 0.88 (m, 9H, 3 CH3); 13C NMR (62.9 MHz, CDCl3) δ
173.5, 171.3, 170.6, 169.7, 138.6, 135.7, 128.7, 128.4, 128.1, 127.8,
76.6, 71.4, 71.1, 69.6 (m), 68.9 (d), 64.7 (d), 50.0, 48.6 (d), 41.6,
39.2, 34.6, 34.4, 34.3, 33.3 (br), 32.0, 29.7, 29.4, 27.9, 25.3, 25.1,
22.8, 14.2; MS-IS 1483.0 [M + NH4]+, 1465.0 [M + H]+. Anal.
(C84H127N3O14P2) C, H, N, P.
1H, H-5aA), 3.09 (dd, 1H, J ) 4.2, 17.1 Hz, H-3bB), 2.98 (br t,
1H, OH), 2.66 (dd, 1H, J ) 5.9, 17.1 Hz, H-3bA), 2.35-2.55 (m,
4H, 2 H-2′′, 2 H-2), 2.27 (t, 2H, J ) 7.5 Hz, 2 H-2′), 1.4-1.7 (m)
and 1.2-1.4 (m) (62 H, 31 CH2), 0.88 (m, 9H, 3 CH3); 13C NMR
(62.9 MHz, CDCl3) δ 174, 172.4, 170.4, 170.2, 138.3, 135.4, 128.7,
128.6, 128.5, 128.2, 128.0, 76.9, 71.5, 71.2, 66.9, 65.7, 51.5, 49.2,
42.1, 41.8, 39.7, 35.8, 34.7, 34.5, 34.1, 32.0, 28.8-29.8, 25.4, 25.2,
25.1, 22.8, 14.2.
O-Dihydroxyphosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl]-L-homoserine, N-{(4R)-5-Dihydroxyphosphoryloxy-4-
[(R)-3-hydroxytetradecanoylamino]pentyl}amide (1(*S)). Com-
pound 26(*S) was submitted to the same sequence of reactions as
its epimer 26, namely partial hydrogenolysis (f 27(*S), ∼100%),
reduction of the carboxylic acid function by way of a mixed
anhydride (f 29(*S), 64%), bis-phosphorylation (f 30(*S), 92%),
and hydrogenolysis (95%), thus affording diphosphate 1(*S). A
sample was purified by HPLC under the same conditions as
compound 1(*R/S): 13C NMR (62.9 MHz, CDCl3/CD3OD) δ 173.8,
173.1, 171.5, 170.8, 71.1, 68.9, 67.7 (d), 63.0 (d), 50.2, 49.1 (d),
43.2, 41.0, 39.0, 37.1, 34.3, 34.2, 32.9 (d), 31.8, 29.0-29.5, 27.5,
25.4, 25.1, 24.9, 22.5, 13.8; MS-IS 1015.0 [M + H]+.
N-[(R)-3-Dodecanoyloxytetradecanoyl]-D-aspartic Acid, r-N-
{(4R)-5-(Benzyloxymethoxy)-4-[(R)-3-benzyloxytetradecanoyl-
amino]pentyl}amide â-Benzyl Ester (31). IIDQ (611 mg, 2.01
mmol) was added to a solution of acid 25 (1.06 g, 1.68 mmol) in
dry CH2Cl2 (81 mL); after stirring for 15 min at room temperature,
a solution of amine 15 (1.03 g; 1.84 mmol) in CH2Cl2 (35 mL)
was added and the reaction mixture was stirred for 3 h. The solution
was concentrated and the residue was purified by flash chroma-
tography on silica gel (light petroleum/EtOAc 2:1 f 1:1), which
afforded compound 31 (1.30 g; 66%; containing ∼25% 2b(S)-
epimer) as a white solid: Rf ) 0.40 (light petroleum/EtOAc 1:1);
mp 77-79 °C; νmax (KBr)/cm-1 3289, 2922, 2851, 1729, 1637,
1557, 1173, 731, 695; 1H NMR (250 MHz, CDCl3; major epimer)
δ 7.20-7.45 (m, 15H, Ph), 7.03 (d, 1H, J ) 8.3 Hz, NH), 6.86 (m,
1H, J ) 5.7 Hz, NH), 6.53 (d, 1H, J ) 8.5 Hz, NH), 5.05-5.25
(m and AB, 3H, CH2Ph, H-3), 4.77 (m, 1H, H-2b), 4.45-4.65 (m,
6H, 2 CH2Ph, OCH2O), 4.08 (m, 1H, H-2a), 3.84 (m, 1H, H-3′′),
3.57 (dd, 1H, J ) 3.7, 10.0 Hz, H-1aB), 3.43 (dd, 1H, J ) 4.4,
10.0 Hz, H-1aA), 3.20 (m, 2H, 2 H-5a), 3.02 (dd, 1H, J ) 4.4,
16.8 Hz, H-3bB), 2.66 (dd, 1H, J ) 6.4, 16.8 Hz, H-3bA), 2.36-
2.49 (m, 4H, 2 H-2′′, 2 H-2), 2.27 (t, 2H, J ) 7.5 Hz, 2 H-2′),
1.45-1.70 (m, 10H) and 1.20-1.40 (m, 54H) (32 CH2), 0.88 (m,
9H, 3 CH3); 13C NMR (62.9 MHz, CDCl3; major epimer) δ 173.6,
172.0, 171.2, 170.1, 138.4, 137.7, 135.5, 128.6, 128.5, 128.3, 127.8,
127.7, 94.9, 76.8, 71.4, 71.1, 69.6, 66.8, 49.3, 48.5, 41.8, 39.5, 35.9,
35.7, 34.5, 34.1, 32.0, 29.7, 29.4, 29.2, 25.2, 25.1, 22.7, 14.2; MS-
IS 1169.0 [M + H]+. Anal. (C71H114N3O10) C, H, N.
O-Dihydroxyphosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl]-D-homoserine, N-{(4R)-5-Dihydroxyphosphoryloxy-4-
[(R)-3-hydroxytetradecanoylamino]pentyl}amide (1(*R)). Com-
pound 30 (1.14 g; 0.78 mmol, less than 5% 2b(S)-epimer) in EtOH
(70 mL) was hydrogenated for 3 h in the presence of 10% Pd-C
(150 mg) at room temperature under hydrogen (atmospheric
pressure). The catalyst was removed by filtration and the filtrate
was concentrated in vacuo. The residual white solid was dried under
vacuum to give compound 1(*R) as the free acid (764 mg; 97%);
a sample was purified by HPLC under the same conditions as
compound 1(R/S): mp 155-157 °C; [R]D +6 (c 1.0, CHCl3); νmax
1
(KBr)/cm-1 3298, 1733, 1646, 1559, 1467, 1378, 1204, 1024; H
NMR (250 MHz, CDCl3/CD3OD 4:1) δ 5.11 (m, 1H, H-3), 4.39
(m, 1H, H-2b), 3.77-4.05 (m, 6H), 2.98-3.33 (m, 2H), 2.15-
2.49 (m, 6H, 3 CH2), 1.95 (m, 2H, 2 H-3b), 1.45-1.60 (m, 10H)
and 1.1-1.3 (m, 54H) (32 CH2), 0.80 (m, 9H, 3 CH3); 13C NMR
(62.9 MHz, CDCl3/CD3OD 4:1) δ 173.7, 172.8, 171.3, 170.6, 70.8,
68.5, 67.3 (d), 62.7 (d), 50.0, 48.8, 42.9, 40.8, 38.7, 36.9, 34.1,
33.9, 32.6 (d), 31.5, 29.0-29.2, 28.8, 27.3, 25.2, 25.0, 24.8, 24.6,
22.3, 13.6; MS-IS 1015.0 [M + H]+. Anal. (C49H97N3O14P2) C, H,
N, P.
N-[(R)-3-Dodecanoyloxytetradecanoyl]-L-aspartic Acid, r-N-
{(4R)-5-Hydroxy-4-[(R)-3-benzyloxytetradecanoylamino]pentyl}-
amide â-benzyl Ester (26(*S)). H-L-Asp(OBn)-OH (537 mg, 2.4
mmol) was N-acylated with (R)-3-dodecanoyloxytetradecanoic acid
8 (2.4 mmol) by way of its mixed anhydride under the same
conditions as described for 25, thus affording compound 25(*S)
(1.19 g, 78%; white solid; NMR spectra nearly identical to those
of 25). Compound 25(*S) (654 mg, 1.03 mmol) was coupled with
amine 13 (450 mg, 1.03 mmol) in the presence of EDCI and HOAt,
under the same conditions as described for the preparation of 26.
The reaction afforded compound 26(*S) (741 mg, 68%) as a white
solid (less than 10% of 2b(R)-epimer): mp 95-99 °C; [R]D -7 (c
1.05, CHCl3); 1H NMR (250 MHz, CDCl3) δ 7.25-7.45 (m, 10H,
Ph), 7.01 (br t, 1H, J ) 5.5 Hz, NH), 6.54 (br d, 1H, J ) 8 Hz,
NH), 5.22 (m, 1H, H-3), 5.10 (s, 2H, CH2Ph), 4.78 (m, 1H, H-2b),
4.54 (AB, 2H, J ) 11.0 Hz, CH2Ph), 4.05 (m, 1H) and 3.85 (quintet,
1H) (H-2a, H-3′′), 3.32-3.58 (m, 3H, 2 H-1a, H-5aB), ∼3.14 (m,
N-[(R)-3-Dodecanoyloxytetradecanoyl]-D-aspartic Acid, r-N-
{(4R)-5-(Benzyloxymethoxy)-4-[(R)-3-benzyloxytetradecanoyl-
amino]pentyl}amide (32). To a solution of compound 31 from
the preceding experiment (1.05 g; 0.90 mmol) in a 1:1 mixture of
EtOH and EtOAc (65 mL) was added Et3N (1.5 mL); the mixture
was hydrogenated for 1 h in the presence of 10% Pd-C (50 mg)
at room temperature under H2 (atmospheric pressure). The catalyst
was removed by filtration and the filtrate was concentrated in vacuo.
The white solid was taken in iPrOH/CH2Cl2 1:1 (50 mL) and stirred
for 10 min in the presence of Amberlite IR-120 (H+) ion-exchange
resin (3 mL). The resin was removed by filtration, the filtrate was
concentrated, and the residue dried under vacuum to give acid 32
(956 mg; 99%) as a white solid. Rf ) 0.50 (CH2Cl2/MeOH, 9:1);
mp 93-95 °C; νmax (KBr)/cm-1 3291, 1728, 1702, 1641, 1549,
1
1174, 695; H NMR (250 MHz, CDCl3; major epimer) δ 7.20-
7.43 (m, 10H, Ph), 7.14 (d, 1H, J ) 8.0 Hz, NH), 7.04 (br t, 1H,
J ) 5.4 Hz, NH), 6.85 (d, 1H, J ) 8.6 Hz, NH), 5.16 (m, 1H,
H-3), 4.75 (m, 1H, H-2b), 4.45-4.65 (m, 6H, 2 CH2Ph, OCH2O),
4.00 (m, 1H, H-2a), 3.84 (m, 1H, H-3′′), 3.58 (dd, 1H, J ) 3.7,
10.0 Hz, H-1aB), 3.43 (dd, 1H, J ) 3.4, 10.0 Hz, H-1aA), 3.32
(m, 1H, H-5aB), 3.08 (m, 1H, H-5aA), 2.91 (dd, 1H, J ) 4.6, 16.8
Hz, H-3bB), 2.64 (dd, 1H, J ) 7.6, 16.8 Hz, H-3bA), 2.35-2.52
(m, 4H, 2 H-2′′, 2 H-2), 2.27 (t, 2H, J ) 7.4 Hz, 2 H-2′), 1.45-

6012 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Martin et al.
1.65 (m, 10H) and 1.20-1.40 (m, 54H) (32 CH2), 0.88 (m, 9H, 3
CH3); 13C NMR (62.9 MHz, CDCl3) δ 173.6, 173.4, 171.9, 170.7,
170.2, 138.2, 137.6, 128.5, 128.4, 127.8, 94.9, 76.8, 71.4, 71.2,
69.6, 69.2, 49.6, 49.0, 41.6, 39.3, 36.6, 34.5, 34.0, 32.0, 29.7, 29.4,
29.2, 28.8, 25.7, 25.3, 25.1, 22.7, 14.1; MS-IS 1117.0 [M + K]+,
1079.0 [M + H]+. Anal. (C64H108N3O10) C, H, N.
29.7, 29.4, 25.8, 25.3, 25.1, 22.8, 14.2; MS-IS 1325.0 [M + H]+.
Anal. (C78H122N3O12P) C, H, N, P.
O-Dihydroxyphosphoryl N-[(R)-3-Dodecanoyloxytetradeca-
noyl]-D-homoserine,N-{(4R)-5-Hydroxy-4-[(R)-3-hydroxytetradecanoyl-
amino]pentyl}amide (2(*R)). Compound 34 from the preceding
experiment (288 mg; 0.22 mmol) in a 1:1 EtOH/EtOAc mixture
(30 mL) was hydrogenated for 7 h in the presence of 10% Pd-C
(60 mg) at room temperature under H2 (atmospheric pressure). The
catalyst was then removed by filtration and the filtrate was
concentrated in vacuo. The white solid was dried under vacuum to
give homogeneous compound 2 (containing ∼25% 2b(S)-epimer)
(200 mg; 98%): Rf ) 0.60 (CH2Cl2/MeOH/H2O 6:4:0.6); mp 150-
160 °C (dec); νmax (KBr)/cm-1 3396, 3297, 1728, 1647, 1559, 1467,
N-[(R)-3-Dodecanoyloxytetradecanoyl]-D-homoserine, N-
{(4R)-5-(Benzyloxymethoxy)-4-[(R)-3-benzyloxytetradecanoyl-
amino]pentyl}amide (33). To a stirred solution of acid 32 from
the preceding experiment (855 mg, 0.79 mmol) in THF (5 mL) at
0 °C were added successively N-methylmorpholine (86 µL, 0.79
mmol) and isobutyl chloroformate (103 µL, 0.79 mmol). Stirring
was continued for 30 min at room temperature, and the solution
was then cooled to 0 °C. A solution of NaBH4 (60 mg, 1.58 mmol)
in H2O (2 mL) was added (CAUTION: evolution of gas); the
reaction mixture was further diluted with H2O (2 mL) and THF
(2.5 mL) and stirred for 5 min at room temperature. The organic
solvent was then partially evaporated; CH2Cl2 (25 mL) was added
and the aqueous phase was neutralized with 1 N HCl. The organic
layer was separated and dried over MgSO4. The solvent was
evaporated and the residue was purified by flash chromatography
on silica gel (CH2Cl2/acetone, 4:1) to give 33 (387 mg, 46%) as a
white solid. Further elution (CH2Cl2/MeOH 9:1) then gave recov-
ered starting material 32 (193 mg, 23%). Compound 33: Rf ) 0.35
(CH2Cl2/acetone 4:1); mp 90-92 °C; νmax (KBr)/cm-1 3474, 3296,
1
1246, 1048, 721; H NMR (250 MHz, CDCl3/CD3OD 4:1, major
epimer) δ 5.11 (m, 1H, H-3), 4.30-4.50 (m, 1H, H-2b), 3.70-
4.05 (m, 4H), 3.35-3.55 (m, 2H), 3.00-3.25 (m, 2H), 2.15-2.45
(m, 6H, 3 CH2), 1.95 (m, 2H, 2 H-3b), 1.45-1.60 (m, 10H) and
1.10-1.40 (m, 54H) (32 CH2), 0.80 (m, 9H, 3 CH3); 13C NMR
(62.9 MHz, CDCl3/CD3OD 4:1) δ 173.7, 173.1, 171.3, 170.6, 70.9,
68.5, 63.7, 62.6, 50.8, 50.0, 43.0, 40.9, 38.9, 36.9, 34.1, 34.0, 32.7
(br), 31.6, 29.3, 29.2, 29.0, 28.8, 27.7 (br), 25.2, 24.8, 24.6, 22.3,
13.6; MS-IS 957.0 [M + Na]+, 935.0 [M + H]+. Anal.
(C49H96N3O11P + H2O) C, H, N, P.
(3R,S)-3-[(R)-3-Dodecanoyloxytetradecanoylamino]-4-hydroxy-
butanoicAcid,N-{(4R)-5-Hydroxy-4-[(R)-3-benzyloxytetradecanoyl-
amino]pentyl}amide (35). To a stirred solution of NaBH4 (7.5 mg,
0.20 mmol) in a 4:1 mixture of MeOH and H2O (0.2 mL) at -15
°C was added slowly compound 26 (52 mg, 49 µmol) in a 1:1
mixture of MeOH and THF (0.4 mL). Stirring was continued at
-15 °C for 30 min and at room temperature for 30 min. The solvent
was partially evaporated under reduced pressure, the residue was
taken up in EtOAc (5 mL), and the residual aqueous phase was
neutralized with 1 N HCl. The organic layer was separated, washed
with water, and dried over MgSO4. The solvent was evaporated
and the residue was purified by flash chromatography on silica gel
(CH2Cl2/MeOH, 12:1) to give diol 35 (20 mg, 43%, containing
∼30% 3b(S)-epimer) as a white solid: Rf ) 0.30 (CH2Cl2/MeOH
1
1728, 1640, 1613, 1054, 695; H NMR (250 MHz, CDCl3, major
epimer ) δ 7.42 (m, 1H, NH), 7.18-7.36 (m, 10H, Ph), 7.12 (m,
1H, NH), 6.75 (d, 1H, J ) 8.3 Hz, NH), 5.19 (m, 1H, H-3), 4.45-
4.65 (m, 7H, 2 CH2Ph, OCH2O, H-2b), 4.25 (m, 1H, OH), 4.05
(m, 1H, H-2a), 3.84 (m, 1H, H-3′′), 3.58 (m, 3H, 2 H-4b, H-1aB),
3.44 (dd, 1H, J ) 4.2, 9.8 Hz, H-1aA), 3.23 (m, 2H, 2 H-5a), 2.35-
2.52 (m, 4H, 2 H-2′′, 2 H-2), 2.27 (t, 2H, J ) 7.3 Hz, 2 H-2′), 1.98
(m, 1H, H-3bB), 1.70 (m, 1H, H-3bA), 1.45-1.65 (m, 10H) and
1.10-1.40 (m, 54H) (32 CH2), 0.88 (m, 9H, 3 CH3); 13C NMR
(62.9 MHz, CDCl3) δ 173.3, 171.5, 171.3, 170.7, 138.3, 137.6,
128.4, 128.3, 127.7, 127.6, 94.8, 76.6, 71.3, 70.9, 69.4, 58.3, 50.5,
48.6, 41.6, 41.4, 39.2, 36.1, 34.4, 34.2, 34.0, 31.9, 29.6, 29.3, 29.1,
25.6, 25.1, 25.0, 22.6, 14.1; MS-IS 1103.0 [M + K]+, 1065.0 [M
+ H]+. Anal. (C64H110N3O9) C, H, N.
1
12:1); H NMR (250 MHz, CDCl3, signals of major epimer) δ
7.28-7.37 (m, 5H, Ph), 7.11 (br m, 1H, NH), 7.01 (br d, 1H, J )
7.5 Hz, NH), 6.69 (br d, 1H, NH), 5.16 (m, 1H, H-3), 4.55 (AB,
2H, J ) 11.2 Hz, CH2Ph), 4.13 (m, 1H, H-3b), 3.80-3.95 (m, 2H,
H-3′′, H-2a), 3.71 (dd, 1H, J ) 3.7, 11.5 Hz, H-4bB), 3.42-3.63
(m, 3H, H-4bA, 2 H-1a), 3.22 (m, 3H, OH, 2 H-5a), 2.36-2.54
(m, 6H, 2 H-2′′, 2 H-2, 2 H-2b), 2.30 (t, 2H, J ) 7.3 Hz, 2 H-2′),
1.45-1.70 (m, 10H) and 1.15-1.40 (m, 54H) (32 CH2), 0.88 (m,
9H, 3 CH3); 13C NMR (62.9 MHz, CDCl3, major epimer) δ 173.9,
172.2, 171.9, 170.2, 138.1, 128.5, 127.9, 76.7, 71.4, 71.2, 64.6,
63.9, 51.3, 48.7, 41.9, 41.5, 39.3, 38.0, 34.5, 33.8, 31.9, 29.6, 29.5,
29.3, 29.1, 28.3, 25.2, 25.1, 25.0, 22.6, 14.1; MS-IS 967.0 [M +
Na]+, 945.0 [M + H]+.
O-Bis(benzyloxy)phosphoryl N-[(R)-3-Dodecanoyloxytetrade-
canoyl]-D-homoserine, N-{(4R)-5-(Benzyloxymethoxy)-4-[(R)-3-
benzyloxytetradecanoylamino]pentyl}amide (34). To a stirred
solution of compound 33 from the preceding experiment (313 mg,
0.29 mmol) and 1H-tetrazole (62 mg, 0.88 mmol) in THF (12 mL)
at room temperature was added dibenzyl diethylphosphoramidite
(85%, 267 µL, 0.67 mmol). Stirring was continued for 30 min at
room temperature and the solution was then cooled to -20 °C. A
solution of mCPBA (57-86%, 187 mg, ∼1.1 mmol) in CH2Cl2 (8
mL) was added and the mixture was stirred for 45 min at room
temperature. Saturated aqueous sodium thiosulfate (5 mL) was
added, the mixture was stirred for 10 min and then diluted with
ether (25 mL), and the organic phase was separated. The organic
layer was washed successively with saturated Na2S2O3 (5×),
saturated NaHCO3 (2×), and 1 N HCl solution (1×); the solution
was dried over MgSO4 and the solvent removed in vacuo. Flash
chromatography of the residual product on silica gel (CH2Cl2/
acetone, 4:1 f 3:1) provided 34 (361 mg, 93%) as a white solid:
Rf ) 0.46 (CH2Cl2/acetone 4:1); mp 68-70 °C; νmax (KBr)/cm-1
3286, 1727, 1635, 1557, 1455, 1381, 1266, 1204, 1174, 1025, 733,
695; 1H NMR (250 MHz, CDCl3; major epimer) δ 7.20-7.42 (m,
20H, Ph), 7.12 (m, 1H, NH), 6.67 (d, 1H, J ) 7.2 Hz, NH), 6.61
(d, 1H, J ) 9.1 Hz, NH), 5.16 (m, 1H, H-3), 4.95-5.12 (m, 4H, 2
POCH2Ph), 4.45-4.65 (m, 7H, 2 CH2Ph, OCH2O, H-2b), 3.95-
4.15 (m, 3H, 2 H-4b, H-2a), 3.84 (quintet, 1H, H-3′′), 3.57 (dd,
1H, J ) 3.8, 9.8 Hz, H-1aB), 3.44 (dd, 1H, J ) 4.3, 9.8 Hz, H-1aA),
3.20 (m, 2H, 2 H-5a), 2.35-2.46 (m, 4H, 2 H-2′′, 2 H-2), 2.26 (t,
2H, J ) 7.9 Hz, 2 H-2′), 2.03 (m, 2H, 2 H-3b), 1.45-1.65 (m,
(3R,S)-4-Bis(benzyloxy)phosphoryloxy-3-[(R)-3-dodecanoyl-
oxytetradecanoylamino]butanoic Acid N-{(4R)-5-Dibenzyloxy-
phosphoryloxy-4-[(R)-3-benzyloxytetradecanoylamino]pentyl}-
amide (36). To a stirred solution of diol 35 (20 mg, 21 µmol) and
1H-tetrazole (9 mg, 127 µmol) in THF (1 mL) at room temperature
was added dibenzyl diethylphosphoramidite (85%, 25 µL, ∼97
µmol). Stirring was continued for 30 min at room temperature and
the solution was then cooled to -20 °C. A solution of mCPBA
(57-86%, 27 mg, ∼160 µmol) in CH2Cl2 (0.6 mL) was added and
the solution was further stirred for 45 min at room temperature.
Saturated aqueous sodium thiosulfate (2 mL) was added, the mixture
was stirred for 10 min and then diluted with ether (10 mL), and
the organic phase separated. The organic layer was washed
successively with saturated Na2S2O3 (5×), saturated NaHCO3 (2×),
1 N HCl (1×); the organic solution was dried over MgSO4 and the
solvent removed in vacuo. Purification of the residual product by
flash chromatography on silica gel (CH2Cl2/acetone, 3:1 f 2:1)
provided 36 (18 mg, 58%) as a white solid: Rf ) 0.40 (CH2Cl2/
acetone 3:1); 13C NMR (62.9 MHz, CDCl3, major epimer) δ 173.5,
171.3, 170.5, 169.7, 138.5, 135.7, 128.8, 128.5, 128.1, 127.9, 76.6,
71.3, 71.0, 69.7, 68.9, 67.3, 48.7, 46.8, 41.6, 39.1, 35.9, 34.6, 34.4,
10H) and 1.15-1.40 (m, 54H) (32 CH2), 0.88 (m, 9H, 3 CH3); 13
C
NMR (62.9 MHz, CDCl3) δ 173.5, 171.2, 170.5, 169.7, 138.6,
137.8, 135.7, 128.7, 128.5, 128.1, 127.8, 127.7, 95.0, 76.9, 71.5,
71.1, 69.6, 64.8, 53.5, 50.1, 48.7, 41.8, 39.5, 34.5, 34.2, 33.6, 32.0,

Acyclic Lipid A Mimics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6013
34.1, 32.0, 29.3-29.8, 28.2, 25.5, 25.3, 25.2, 22.8, 14.2; MS-IS
1482.0 [M + NH4]+, 1465.0 [M + H]+.
of 20 µg/mL: medium, RPMI alone; positive control, LPS of E.
coli (serotype 055:B5, Sigma, St. Louis, MO); product 1, OM-
294-DP; product 2, OM-294-MP.
The surpernatants of the cultures were harvested after 24 h, and
the concentration of IL-6 was measured by an enzyme-linked
immunosorbent assay (ELISA) (Human IL-6 ELISA Set, BD
OptEIA, San Diego, CA), according to the manufacturer instruc-
tions. The detection limit was 10 pg/mL.
Inhibition and Measurement of IL-6 in Culture Supernatants.
The PBMC obtained as described above were incubated at 37 °C
and under 5% of CO2 atmosphere for 90 min with product 1 (OM-
294-DP) or product 2 (OM-294-MP) at the final concentrations of
0.02, 0.2, 2, and 20 µg/mL then LPS of E. coli (serotype 055:B5,
Sigma, St. Louis, MO) was added at the concentration of 0.01 µg/
mL.
After 24 h of incubation, the plates were centrifuged at 1500
rpm for 8 min, and the supernatant was collected and stored at
-20 °C. The concentration of IL-6 was measured by an enzyme-
linked immunosorbent assay (ELISA) (Human IL-6 ELISA Set,
BD OptEIA, San Diego, CA), according to the manufacturer
instructions. The detection limit was 10 pg/mL.
(3R,S)-4-Dihydroxyphosphoryloxy-3-[(R)-3-dodecanoyloxy-
tetradecanoylamino]butanoic Acid, N-{(4R)-5-Dihydroxyphos-
phoryloxy-4-[(R)-3-hydroxytetradecanoylamino]pentyl}amide (37).
Compound 36 (16 mg; 11 µmol) was hydrogenated for 4 h in EtOH
(1.5 mL) in the presence of 10% Pd-C at room temperature under
H2 (atmospheric pressure). The catalyst was then removed by
filtration and washed with CH2Cl2/MeOH 4:1, and the filtrate was
concentrated in vacuo. The white solid was dried under vacuum to
give homogeneous 37 (10 mg; 90%); a sample was purified by
HPLC under the same conditions as described for compound 1(R/
S): 1H NMR (250 MHz, CDCl3/CD3OD 4:1) δ 5.12 (m, 1H, H-3),
4.30 (m, 1H, H-3b), 3.7-4.04 (m, 6H), 3.15 (m, 2H, 2 H-5a), 2.38
(m, 4H, 2 CH2), 2.22 (m, 4H, CH2), 1.35-1.60 (m, 10H) and 1.10-
1.35 (m, 54H) (32 CH2), 0.88 (m, 9H, 3 CH3); 13C NMR (62.9
MHz, CDCl3/CD3OD 4:1, major epimer) δ 173.5, 171.3, 170.5,
169.7, 70.9, 68.6, 67.4, 65.9, 48.7, 46.6, 43.0, 40.9, 38.6, 37.0, 36.0,
34.1, 33.9, 31.6, 29.6, 29.3, 29.0, 28.8, 27.4, 25.2, 24.8, 24.7, 22.3,
13.6; MS-IS 1036.5 [M + Na]+, 1014.5 [M + H]+.
Biological Assays. Experimental Assay of Nitric Oxide
Production by Murine Macrophages. Six-week-old male C57/
BL6 mice (SPF quality, Charles Rivier, FR) were killed by CO2
inhalation. The hip, femur, and tibia from the posterior appendage
were removed. The bone marrow was extracted from the lumen
by injecting Dulbecco’s modified Eagle medium (DH) through the
bone after cutting both end portions. After washing, the stem cells
were resuspended (40 000 cells/mL) in DH medium supplemented
with 20% horse serum and 30% L929 cell supernatant. The cell
suspension was incubated for 8 d in an incubator at 37 °C under
8% CO2 and moisture-saturated atmosphere. Macrophages were
then detached with ice-cold PBS, washed, and resuspended in DH
medium supplemented with 5% fetal calf serum (FCS), amino acids,
and antibiotics (DHE medium). The cell density was adjusted to
700 000 cells/mL. Aqueous solutions of the products were serially
diluted in DHE medium directly in microtiter plates. The products
were tested in triplicate and each microtiter plate comprised a
negative control composed of medium. The final volume in each
well was 100 µL, and 100 µL of the cell suspension was added to
the diluted products, and the cells were incubated for 22 h in an
incubator at 37 °C, under 8% CO2 and a moisture-saturated
atmosphere. At the end of the incubation period, 100 µL of
supernatant was transferred to another microtiter plate, and the nitrite
concentration produced in each supernatant was determined by
running a Griess reaction. Griess reagent (100 µL, 5 mg/mL of
sulfanilamide + 0.5 mg/mL of N-(1-naphthyl)ethylenediamine
hydrochloride) in 2.5% aqueous phosphoric acid was added to each
well. The microtiter plates were read with a spectrophotometer
(SpectraMax Plus, Molecular Devices) at 562 nm against a reference
at 690 nm. The nitrite concentration is proportional to the nitric
oxide content being formed. The nitrite content is determined on
the basis of a standard curve. The results are given as mean value
( standard deviation and plotted as a dose-response curve.
Preparation of Human PBMC and Cell Culture. Peripheral
blood from healthy donors (Centre de transfusion, Hoˆpital Univer-
sitaire, Geneva) was centrifuged to get the buffy coat. The buffy
coat was mixed with Hanks’ balanced saline solution (HBSS,
Sigma, Buchs, Switzerland), layered over Ficoll Paque Plus
(Amersham Pharmacia) to 1.077 g/mL, and centrifuged (2800 rpm,
20 °C, 25 min). Cells harvested from the interphase were washed
twice in HBSS at 800 rpm for 15 min at room temperature, and
the pelleted cells were resuspended in HBSS. The cell counts were
performed using a Neubauer cell. All cell cultures were performed
in RPMI-1640 medium supplemented with penicillin (100 U/mL),
streptomycin (100 µg/mL), L-glutamine (2 mmol/L), and 10% fetal
calf serum (FCS), all obtained from Sigma. For in vitro stimulation,
the cells were cultured at a concentration of 1 × 106 viable cells/
well.
Supporting Information Available: Elemental analysis results,
general experimental procedures, and procedures for LAL assays
and for the determination of pyrogenicity in the rabbit. This material
is available free of charge via the Internet at http://pubs.acs.org.
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