The C-glycoside analogue of the immunostimulant α-galactosylceramide (KRN7000): Synthesis and striking enhancement of activity

Article abstract of DOI:10.1002/anie.200454215

Which source provides the better drug? The fully synthetic C-glycoside I (Y=CH₂) is about 1000 times more potent against malaria in mice than the corresponding O-glycoside (Y=O), which is derived from a natural product and itself known to show remarkable activity against a wide range of diseases. The results of comparative biological assays emphasize the potential of C-glycosides as therapeutic agents.

Full text of DOI:10.1002/anie.200454215

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named the agelasphins (1).[1] Structure–activity studies of
compounds available through synthesis revealed that a
slightly simpler analogue of the natural agelasphins, an a-
galactosyl ceramide named KRN7000 (2), had the best
activity of the compounds tested, and that the functional
groups and configuration of 2 were optimal. The only
acceptable variables are the lengths of the lipid chains, the
insertion of “taggants” at the terminus of the fatty amide
chain through amide linkages, substitution at C6 of the
galactose residue, the removal of the hydroxy group at C4 of
the phytosphingosine moiety, and the presence or absence of
an a hydroxy group in the fatty amide.[2]
Biologically Active Compounds
The C-Glycoside Analogue of the
Immunostimulant a-Galactosylceramide
(KRN7000): Synthesis and Striking Enhancement
of Activity**
Since the initial disclosures by the Kirin group, a multi-
faceted exploration of the biological effects of KRN7000 has
unveiled its remarkable activity against a disparate group of
diseases, such as cancer, including melanoma,[3] hepatic
metastases of pancreatic cancer,[4] hepatic metastases of
colon cancer,[5] and primary tumor formation in three differ-
ent models,[6] as well as malaria,[7] juvenile diabetes,[8]
hepatitis B,[9] and autoimmune encephalomyelitis,[10] in
murine/whole animal versions of all the diseases. Although
no activity against cell-culture versions of the diseases is ever
detected, in vitro activity can be measured by incubating the
glycolipid with antigen-presenting dendritic cells or their
isolated CD1d receptors and then challenging NKT-cell
hybridomas with the resulting complex.[11] This explosion of
research has led to that finding that the unifying mechanism
of action of KRN7000 is its remarkable ability to induce a
potent expansion of Va24+NKT cells. The sequence of
events is: 1) the galactosyl ceramide binds to the CD1d
receptor of antigen-presenting cells, then 2) the ceramide
receptor complex binds to NKT cells, which stimulate a
cascade of cytokines: signals for the processes that result in
the suppression of the disease.[12] It is not yet known why an a-
galactosyl ceramide of marine origin should be recognized by
a mammalian receptor and elicit a spectacular immune
response. It has also not been established whether an
endogenous mammalian substance exists as the “natural”
ligand for the CD1d receptor. Suffice to say that KRN7000 is
a potent lead compound for the development of immunostim-
ulant drugs.[13]
Guangli Yang, John Schmieg, Moriya Tsuji, and
Richard W. Franck*
In the early 1990s a research group at Kirin Pharmaceuticals
reported their results from screening lipophilic extracts from
the Okinawan sponge Agelas mauritianus. When their
extracts were tested in mice, but not in cell culture, they
observed potent antitumor activity by glycolipids that they
[*] Dr. G. Yang, Prof. Dr. R. W. Franck
Department of Chemistry
Hunter College of CUNY
New York, NY 10021 (USA)
Fax: (+1)212-772-5332
E-mail: rfranck@hunter.cuny.edu
J. Schmieg
Department of Medical and Molecular Parasitology
New York University Schoolof Medicine
New York, NY (USA)
Prof. Dr. M. Tsuji
Department of Medical and Molecular Parasitology
New York University Schoolof Medicine
New York and Aaron Diamond AIDS Research Center
The Rockefeller University
New York, NY (USA)
[**] The synthesis research at Hunter College was supported by grants
from the NIH (GM 60271) to R.W.F. and NIH-RCMI (RR03037) to
the college for chemistry department infrastructure. The bioassay
research at NYU MedicalSchoolwas supported by grants from the
NIH (AI 45606, AI 01682, and AI 47840) to M.T.
In our search for challenging targets that might showcase
our application of the Ramberg–Bäcklund reaction for C-
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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glycoside synthesis, we chose to prepare 3, the nearest C-
glycoside analogue of 2, on the basis of the well-known
rationale for the study of C-glycosides, namely that the O-
glycoside may be susceptible to enzymatic degradation,
whereas the corresponding C-glycoside is not. That the
lability of 2 may be a problem is conjecture by the present
authors, as little has been published about the metabolism of
KRN7000 in mammals.[14] The C-glycoside concept has
existed for many years and has been thoroughly reviewed.[15]
It is fair to conclude that comparisons of bioactivities of
synthetic C-glycosides with those of their O-glyco parents
have uncovered some examples of similar activity,[16] many of
diminished activity,[17] and none of significant improvement.
The most apposite data for our ceramide project are those of
Bertozzi et al., who compared the binding of the closely
matched, but not identical b-O- and b-C-glycosides 4 and 5 to
previous success with this method for C-glycoside synthesis.[21]
A second component of our plan was to establish the desired
anomeric configuration through an intramolecular hydride
transfer from a silyl group to an intermediate carbocation center.
The six-step synthetic route from commercially available
l-homoserine to the homophytosphingosine component 6 is
described in the Supporting Information. The sequence
mimics a synthesis of phytosphingosine reported by Naka-
nishi and co-workers in which l-serine was used as the
starting material.[22] The Cbz-substituted homophytosphingo-
sine 6 was then converted into the iodo derivative 9 in
anticipation of the ensuing S-glycoside formation
(Scheme 2).[23]
The synthesis was continued by treatment of the galactose
thioacetate 10 with hydrazinium acetate in DMF under
nitrogen to deprotect the thiol,[24] which was subsequently
treated with the electrophile 9 and
triethylamine to provide the b-d-thio-
galactoside 11 in 90% overall yield
(Scheme 3). Treatment of 11 with
NaOMe in MeOH to cleave the
remaining four acetate groups fol-
lowed by protection of the 4- and 6-
hydroxy groups with p-methoxyben-
zaldehyde dimethyl acetal and p-tolue-
a gp120 receptor. The binding constants of the two com-
pounds were similar, and no kinetic data was presented to
show that the longer lifetime of the C-glycoside had any
effect.[18] There are a few examples of C-glycoside constructs
that are not analogous to any specific O-glycoside. For
example, Schmidt and co-workers recently described a C-
glycoside mimetic of a complex proposed to be important in
galactosyl transfer.[19] Although syntheses of C-glycosyl
sphingosine substances somewhat related to 3 have been
reported by two other research groups,[20] these groups have
not reported the synthesis of the same analogue of KRN7000
prepared by us.
nesulfonic acid produced the 4,6-O-(4-methoxybenzylidene)-
b-d-1-thiogalactoside 12 in 86% yield.[25] Sodium hydride
promoted benzylation of the remaining hydroxy groups of 12,
In our first approach to 3, outlined retrosynthetically in
Scheme 1, we embarked on a route with a Ramberg–Bäck-
lund (RB) reaction to “stitch” a homophytosphingosine side
chain to galactose as the key feature, as our group had had
Scheme 2. Synthesis of an iodo derivative of homophytosphingosine:
a) (CH3)2C(OMe)2, PPTS, CH2Cl2, 95%; b) nBu4NF, AcOH, THF, 86%;
c) I2, PPh3, THF, reflux, 85%. Cbz=benzyloxycarbonyl, PPTS=pyridi-
nium p-toluenesulfonate.
followed by oxidation of the thiogalactoside with
MMPP, gave the sulfonyl galactoside 13 in good yield.
We could not avoid N-benzylation in this step.
The RB reaction of 13 in the presence of C2F4Br2,
tBuOH, and KOH–Al2O3 at reflux afforded the
product 14. The Z:E ratio of alkene isomers was
not determined because of the peak broadening in
the NMR spectra. The 1-O-methyl-2,3-O-dibenzyl-b-
galactoside 15 was prepared in one step from 14 by
using chlorotrimethylsilane in methanol. At this
stage, preliminary experiments with BF3-catalyzed
hydride transfer revealed that the isopropylidene
protecting group was not sufficiently robust. It was
therefore exchanged for a carbonate group. How-
ever, this carbonate protecting group would not be
suitable for the RB step, in which KOH is required.
Scheme 1. Retrosynthesis of a C-glycoside analogue of KRN7000.
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afforded the product 19 (combined yield of 76%),
which was identified by 1H NMR spectroscopy
(d(anomeric H) = 3.95 ppm, J12 = 4.6 Hz).
The final deprotection steps prior to the ultimate
amidation to form the target compound 3 were more
challenging than anticipated. The total debenzylation
of compound 19 failed under a variety of conditions,
including with the Pearlman catalyst (H2, 20%
Pd(OH)2, 30 psi), Birch reduction (Na/liquid NH3),
with 10% Pd/C, and with 4.4% formic acid in
methanol. We therefore elected to remove the car-
bonyl protecting groups before the hydrogenolytic
debenzylation process (Scheme 5).
Compound 19 was treated with NaOH at reflux in
a mixture of dioxane and H2O (1:1) to afford the
oxazolidinone 21, which was fully characterized. NOE
effects were observed between 3-H and 1’-Ha or b,
and between 5-H and 1’-Ha or b, which confirmed
that the configuration of compound 21 (and hence 19)
was correctly assigned (a isomer). Hydrolysis of 21 (at
reflux in KOH/EtOH) gave the N-benzylamine 22,
which was fully debenzylated by transfer hydrogenol-
Scheme 3. Formation of the C-glycoside linkage: a) 1. NH2NH2, AcOH, DMF;
2. 9, Et3N, 90%; b) NaOMe, MeOH; c) p-(MeO)C6H4CH(OMe)2, p-TsOH, DMF,
CH2Cl2, 86% (2 steps); d) NaH, BnBr, THF, 83%; e) MMPP, THF, H2O, EtOH,
608C, 93%; f) C2F4Br2, tBuOH, KOH, Al2O3, reflux, 70%; g) TMSCl, MeOH, 08C,
66%. Bn=benzyl, DMF=N,N-dimethylformamide, MMPP=magnesium
monoperoxyphthalate, TMS=trimethylsilyl, Ts=toluenesulfonyl.
Our protecting-group manipulations
began with 15, whereby esterification of
the primary hydroxy group at C6 and
cleavage of the acetonide group afforded
the corresponding diol 16 in good yield
(Scheme 4). Cyclic carbonation of the
diol in the presence of triphosgene
followed by silylation of the axial hy-
droxy group at C4 then afforded the silyl
ether 17.[26] The controlled addition with
a syringe pump of 17 as a solution in
CH2Cl2 (0.01m) to BF3·Et2O (5 equiv) in
Scheme 5. Removal of protecting groups followed by ceramidation: a) 1,4-dioxane, H2O, NaOH,
reflux, >90%; b) KOH, EtOH, reflux, 80%; c) 10% Pd/C, cyclohexene, HCl (1n), MeOH,
reflux, >90%; d) p-nitrophenylhexadecanoate, THF, DMAP, 48 h, room temperature, 60%;
e) Ac2O, DMAP, 80%. DMAP=4-dimethylaminopyridine.
CH2Cl2 afforded the a-C-galactosides 18
and 19, as well as the 1,4-anhydro com-
pound 20 (15%).[27] Treatment of the
silyl ether 18 with Bu4NF (1n) in THF
ysis (10% Pd/C, cyclohexene, reflux) to afford
crude 23 in 80% overall yield.[28] The fatty
amide chain was then introduced by using p-
nitrophenyl hexadecanoate as the acylating
agent[2a] to afford the target 3 in 60% yield.
Final purification was carried out by flash
chromatography on silica gel (eluant: CHCl3/
MeOH 4:1). The 1H and 13C NMR spectra,
optical rotation value ([a]2D5 = +40.8 (c = 0.13,
pyridine)), m.p. (175–1788C), and high resolu-
tion MS (FAB) spectrum: m/z = 856.7601
(C51H101O8N+H+ requires 856.7605) served to
fully characterize a sample of 3. The mass
1
spectrum and H NMR spectrum of fully acy-
Scheme 4. Introduction of the a-C-glycoside linkage: a) BzCl, Et3N, CH2Cl2, 88%; b) HCl(1 n),
Et2O, MeOH, 80%, c) triphosgene, pyridine, CH2Cl2, 83%; d) iPr2SiHCl, imidazole, DMF, 96%;
e) BF3·OEt2 (5 equiv), CH2Cl2, 08C; f) TBAF, THF. Bz=benzoyl, TBAF=tetrabutylammonium
fluoride.
lated 24 further confirmed the structure of 3.
Interestingly, the optical rotation value and
NMR data for the O- and C-glycosides showed
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a remarkable congruence, except for the chemical shifts of C1,
C1’, and C2’.[29]
a-galactosidases, whereas the O-glycoside is; thus, the C-
glycoside can remain active in the mouse for a longer period
of time. Another argument could be that different forms of
binding to the important receptors takes place, with the C-
glycoside analogue favoring more effective signaling cascades
in the immune system. Even though the former explanation is
most consistent with the prevalent hypothesis of the merit of
C-glycosides, we believe that our detailed bioassay data,
presented elsewhere, favor the latter.[32]
A second assay carried out was a melanoma challenge in
C57BL/6 mice. The animals were treated with either the C-
glycoside or the O-glycoside 2 days before the challenge with
B16 melanoma cells. In these mice, the melanoma attacks the
lungs. The mice were sacrificed after two weeks and their
lungs were examined visually for melanomas (black spots).
Figure 2 hardly needs an explanation. It is clear that the C-
glycoside at the 10-ng dosage level is far more effective than
the O-glycoside at the same dose. In fact, detailed dose–
response experiments showed that the C-glycoside 3 was 100
times more effective than the O-glycoside 2 in this assay.
The coupling constant of 8.8 Hz between 2-H and 3-H of
the galactose unit of 3 suggests that the 4C1 chair (as drawn) is
the principal conformer. Whereas the anomeric effect of the
O-glycoside serves to compensate for the steric repulsions of
the axial ceramide moiety, there is no such compensation in
the C-glycoside analogue, so its actual conformation can not
be assumed to be a chair. For example, Veyri›res and co-
workers reported two examples of a-C-galactosides for which
the coupling constants clearly show that the standard chair
can not be the principal conformer.[30]
A range of assays were carried out in which 3 was found to
show outstanding activity, even in comparison with the very
active O-glycoside 2. In the following we summarize the
results of these assays, which clearly demonstrate the
excellent potential of C-glycoside analogues as therapeutic
agents.
One assay that was carried out was a mouse malaria
model, whereby mice were treated with galactosyl ceramide
and then, after an interval, were challenged with malaria (P.
yoeli).[31] After a further interval, the animals were sacrificed,
and their livers were assayed for the sporozoite stage of
malaria. Both the O- and the C-glycoside 2 and 3 were very
effective at reducing sporozoite levels at a dosage level of 1 mg
per mouse relative to the control. However, the C-glycoside 3
continued to show excellent activity at the 1-ng level. Thus, 3
is approximately 1000 times more protective than the O-
glycoside (Figure 1).
Figure 2. Melanoma appearance (black spots) on mouse lung after
two weeks: untreated or treated with 10 ng of 2 or 3.
These very promising initial results leave many important
questions unanswered. More studies are required to extend
these findings, including the preparation of more analogues in
the search for compounds with greater potency, extensive
bioassays to determine the effectiveness of these materials in
other disease models, and modelling and biophysical studies
to find an explanation for the dramatic difference between C-
and O-glycosides in this series.
The experiments with animals were carried out with the
permission of the Institutional Animal Care and Use Com-
mittee at New York University School of Medicine. The
Laboratory Animal Protocol number is #4627-02 in the
context of NIH/GM60271.
Figure 1. Assay for sporozoites in mouse liver with 1) no glycolipid,
2) 1 ng of 2 or 3, and 3) 1 mg of 2 or 3. Number of P. Yoelii specific
18S rRNA molecules (determined 42 h after challenge with 10000
viable sporozites) is plotted against dosage of 2 or 3.
Received: March 8, 2004 [Z54215]
Published Online: June 30, 2004
The results of this experiment do not show explicitly that
the lifetime of the glycoside is a factor in the activity
difference observed between 2 and 3. Therefore, a “kinetics”
experiment was carried out. The interval between the
administration of the glycolipid and sporozoite challenge
was varied. These experiments showed that the C-glycoside is
effective with intervals of up to four days between drug
dosage and challenge, whereas the O-glycoside is effective
with only a one-day interval. One interpretation of these
results is that the C-glycoside is not degraded by endogenous
Keywords: biological activity · C-glycosides · drug design ·
immunostimulants · Ramberg–Bꢀcklund reaction
.
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[16] For a C-trisaccharide with lectin-binding behavior similar to that
of the parent O-trisaccharide, see: L. M. Mikkelsen, M. J.
Hernaiz, M. Martin-Pastor, T. Skrydstrup, J. Jimenez-Barbero,
J. Am. Chem. Soc. 2002, 124, 14940 – 14951.
3822
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 3818 –3822