Synthesis and in vivo evaluation of 4-deoxy-4,4-difluoro-KRN7000

Article abstract of DOI:10.1021/ol801663m

(Chemical Equation) The synthesis of 4-deoxy-4,4-difluoro-KRN7000 starting from phytosphingosine is described. Key steps include a regioselective benzylation of azidophytosphingosine and a deoxofluor-mediated fluorination of the corresponding 4-ketone. Th

Full text of DOI:10.1021/ol801663m

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4433-4436
Synthesis and In Vivo Evaluation of
4-Deoxy-4,4-difluoro-KRN7000
Leo Leung,† Cyrille Tomassi,† Katrien Van Beneden,‡ Tine Decruy,‡
Dirk Elewaut,‡ Tim Elliott,§ Aymen Al-Shamkhani,§ Christian Ottensmeier,§
Serge Van Calenbergh,| Joern Werner, Tony Williams,§ and Bruno Linclau*,†
School of Chemistry, UniVersity of Southampton, Highfield, Southampton SO17 1BJ,
U.K., Laboratory for Molecular Immunology and Inflammation, Department of
Rheumatology, Ghent UniVersity Hospital, Ghent UniVersity, De Pintelaan 185,
9000 Ghent, Belgium, Cancer Sciences DiVision, School of Medicine, UniVersity of
Southampton, Somers Cancer Research Building, MP824, Tremona Road, Southampton
SO16 6YD, U.K., Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical
Sciences, Ghent UniVersity, Harelbekestraat 72, 9000 Ghent, Belgium, and School of
Biological Sciences, UniVersity of Southampton, Bassett Crescent East, Southampton
SO16 7PX, U.K.
bruno.linclau@soton.ac.uk
Received July 22, 2008
ABSTRACT
The synthesis of 4-deoxy-4,4-difluoro-KRN7000 starting from phytosphingosine is described. Key steps include a regioselective benzylation of
azidophytosphingosine and a deoxofluor-mediated fluorination of the corresponding 4-ketone. This fluorination failed completely when the
adjacent 3-OH was protected as benzyl ether but proceeded well when a benzoyl group was used. The biological evaluation reveals a bias
toward Th1 cytokine induction upon Natural Killer T cell activation.
The discovery of KRN7000 1 (Figure 1)1 as potent stimulator
of Natural Killer T-cells (NKT-cells) has stimulated intense
research toward the immunomodulating properties of this
class of exogenous (R-linked) glycolipids.
It has been shown that KRN7000 binds to the CD1d
protein to form a complex that is subsequently recognized
by NKT-cells, leading to a rapid release of cytokines.
Unfortunately, both proinflammatory Th1 (eg IFN-γ and IL-
2) and immunomodulatory Th2 (e.g., IL-4 and IL-10)
Figure 1. Structures of KRN7000 (1), R-C-GalCer (2), and OCH
† School of Chemistry, University of Southampton.
‡ Department of Rheumatology, Ghent University.
§ Cancer Sciences Division, University of Southampton.
| Laboratory for Medicinal Chemistry, Ghent University.
School of Biological Sciences, University of Southampton.
(1) Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa,
E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med. Chem.
1995, 38, 2176–2187.
(3).
cytokines are released, which antagonize each other’s effects.
The development of KRN7000 analogues that lead to
polarization of cytokine release, and hence may have
10.1021/ol801663m CCC: $40.75
Published on Web 09/18/2008
2008 American Chemical Society

Scheme 1. Retrosynthetic Analysis
therapeutic potential, is currently an active area of research.2
Examples include the C-glycoside R-C-GalCer 2, which was
found to induce a markedly Th1 skewed response,3 while
truncation of one of the lipid chains, such as in OCH 3,4
caused a Th2 bias in NKT cell response.
ability to accept a hydrogen bond,10 and to investigate the
effect of this modification on the immunostimulating proper-
ties.
An R-galactosylceramide construction strategy was envi-
sioned in which the modified sphingosine 6 (Scheme 1)
would be glycosylated with the galactosyl donor 7,11
followed by azide reduction, amide bond formation with 5,11b
and global deprotection. The sphingosine derivative 6 would
be obtained by fluorination of ketone 8, which would be
accessible from the commercially available phytosphingosine
9. Hence, the project started by investigating the conversion
of 9 to 8 aiming for minimal protection/deprotection opera-
tions.
In attempting to differentiate the 4-OH from the other
alcohol groups, direct 1,3-protection, for example as silylene
acetal,12 proved not of use for our purposes. A high-yielding
selective alcohol differentiation was developed from 1-O-
trityl phytosphingosine 11 (Scheme 2). Masking the amino
group of phytosphingosine 9 as an azide13,14 was followed
by regioselective tritylation of 10.15 Subsequent azide
reduction led to 11, upon which treatment with Boc2O
surprisingly led to the 3-O carbonate 12 as the only product
in 80% yield. The origin for the selectivity is unclear. Given
that there is no additional stabilizing group present on the
nitrogen, prior amine protection followed by NfO migration
of the Boc group under steric pressure from the trityl group
is not expected under the employed reaction conditions.16
The functionalization at the 3-position was easily proven by
NMR spectroscopy. This remarkably high-yielding selective
3-OH protection step allowed us to easily obtain the desired
ketone 13 by subsequent protection of the amino group with
At present the relationship between glycolipid structure
and cytokine polarization is not completely understood, but
could relate to the stability of the glycolipid complex with
CD1d,5 or to a difference in glycolipid presenting cells.6
It has been established that the sphingosine 3-OH is much
more important than the 4-OH for antigenic activity.1,20
Following crystallographic studies of CD1d complexed with
KRN70006a,7 and related GalCers,8 it was shown that the
3-OH forms a H-bond with the CD1d Asp80. Furthermore,
the crystal structure of the human NKT TCR in complex
with CD1d bound to 1 reveals a H-bond between the 3-OH
and Arg 95 of the CDR3R-loop.9 Given the apparent pivotal
role of the 3-OH in the interaction with both proteins, it was
decided to synthesize the 4-deoxy-4,4-difluoro KRN7000
analogue 4, in which the 3-OH would display a better
hydrogen bond donating capacity, but a significantly reduced
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4434
Org. Lett., Vol. 10, No. 20, 2008

Scheme 2. First Synthesis of a 4-Deoxy-4,4-difluorophytosphingosine Derivative
trifluoroacetic anhydride, followed by oxidation of the 4-OH.
Disappointingly, fluorination of 13 proceeded very slowly,
with only 20% of the desired product 14 obtained after 3
days at 40 °C. In addition, the corresponding elimination
product 16 was isolated in 10% yield, along with remaining
starting material (27%) and deprotected 15 (<5%). The
fluoroalkene 16 was obtained as a single geometric isomer
as judged by 19F NMR. The alkene was assigned as the
Z-isomer by determination of the coupling constant between
the vinylic fluorine and proton (1H NMR, 3JF-H ) 36.6 Hz).
The desired derivative 14 was obtained in seven steps from
9, and the removal of the three different protecting groups
would need another two or three steps. Hence, it was decided
to reconsider our approach by investigating another protect-
ing strategy. Relying on the electron-withdrawing azido
group to direct functionalization of the adjacent hydroxyl
groups, a short, direct protection of the 1,3-diol moiety was
achieved by benzylation of 10 under basic conditions
(Scheme 3). By using an excess of base, but only 2 equiv of
BnBr, an acceptable 49% yield was obtained of the desired
1,3-dibenzylated azidophytosphingosine 17. Minor amounts
of 1-O-benzylated (16%) and the tribenzylated product17
(10%) were formed as well but were easily separated by
chromatography, resulting in an attractive direct selective
protection protocol. The position of the benzylation was
assigned by NMR. Oxidation of 17 gave the ketone 18 in
good yield. However, all attempts to effect fluorination of
18 completely failed.
Unfortunately, direct benzoylation of 10 under nucleophilic
conditions (BzCl, pyridine) or basic conditions (NaH, BzCl)
only led to 1,3-protection in low yield and selectivity, with
the isomers not separable by chromatography (not shown),
but a direct benzyl to benzoyl oxidation of 18 to 20 (Scheme
3) proved successful despite the moderate yield obtained.
The ketone 20 is now easily accessible in four steps from
phytosphingosine 9. As predicted, the fluorination now did
proceed, if still very slowly, with a yield of 42% for the
desired 21. The corresponding elimination product 22 was
formed in an approximate 1:1 ratio with 21, and the products
were separable by preparative HPLC. The Z-fluoroalkene 22
was again obtained as a single geometric isomer (3JF-H
)
37.0 Hz).
The final stages for the synthesis of 4 are shown in Scheme
4. Deprotection of 21 proceeded in excellent yield, and
TMSOTf-induced galactosidation with galactosyl donor 718
occurred selectively at the primary alcohol of 23. No
ꢀ-isomer was detected. Finally, azide reduction allowed
introduction of the amide chain11b which was followed by
global deprotection.
To investigate whether 4 could elicit CD1d-dependent
NKT cell activation in vivo, wild-type C57Bl/6 mice,
CD1d-/- as well as JR18-/- mice were injected with 5 µg
of 4. Injection into wild-type C57B1/6 mice resulted in
significantly reduced serum levels of IL-4 (Figure 2), while
similar levels of IFN-γ production were detected compared
to KRN7000. No cytokine production was observed when
compound 4 was injected in either JR18-/- or CD1d-/- mice,
indicating that this glycolipid induces CD1d-dependent TCR
activation of NKT cells.
With the partially successful fluorination of 13 in mind, it
was thought that an electron-withdrawing protecting group,
such as a benzoate, would result in a more reactive ketone.
Scheme 3. Direct Benzylation Approach
Org. Lett., Vol. 10, No. 20, 2008
4435

Scheme 4. Final Assembly to 4-Deoxy-4,4-difluoro KRN7000
This result confirms that the 4-OH is not required for
activity and that replacement with a gem-difluoro group at
that position is allowed. The fluorination will result in an
increased hydrogen bond donating ability for the 3-OH,10
which is expected to strengthen binding between 4 and CD1d
through the interaction with Asp-80. Conversely, the fluo-
rination will drastically reduce the hydrogen bond acceptor
ability of the 3-OH group,10 which would significantly
weaken the binding to Arg 95 of the NKT TCR. Hence, a
possible interpretation of the biological data is that a tighter
binding between the glycolipid with CD1d is beneficial for
cytokine polarization,5 and given the minimal loss in activity
compared to KRN7000 1, the interaction involving Arg 95
of the NKT TCR is of less importance.
phytosphingosine,19 featuring a selective protection reaction
of the azidosphingosine 1- and 3-OH. Fluorination of the
C4-ketone was promoted by an electron-withdrawing pro-
tecting group at the 3-OH. R-Galactosylation of 23, followed
by introduction of the ceramide side chain, yielded 4-deoxy-
4,4-difluoro-KRN7000 4 in a further four steps. Biological
evaluation showed that 4 induced CD1d-dependent TCR
activation of NKT cells and that the modification modulates
NKT cell responses resulting in the induction of a different
cytokine pattern compared to KRN7000, with a bias toward
Th1 cytokine release.
Acknowledgment. We thank Cancer Research Technol-
ogy and the Fund for Scientific Research Flanders (FWO-
Vlaanderen), for funding. K.V.B. is a FWO postdoctoral
fellow.
Supporting Information Available: Experimental details,
characterization, and copies of 1H, 13C, and (when relevant)
19F spectra for 17, 18, 20-24, and 4.This material is available
free of charge via the Internet at http://pubs.acs.org.
OL801663M
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Figure 2. In vivo cytokine release by KRN7000 and 4-deoxy-4,4-
difluoro-KRN7000 4.
(20) (a) Sidobre, S.; Hammond, K. J. L.; Be´nazet-Sidobre, L.; Maltsev,
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In summary, the synthesis of a 4-deoxy-4,4-difluorosph-
ingosine derivative 23 was achieved in five steps from
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Org. Lett., Vol. 10, No. 20, 2008