Asymmetric synthesis of the α-D-galactosyl ceramide KRN7000 via an organocatalytic aldol reaction as key step

Article abstract of DOI:10.1055/s-0029-1218844

The asymmetric synthesis of the antitumor and immunostimulatory α-D-galactosyl ceramide KRN7000 using a (R)-proline-catalyzed enantioselective aldol reaction as key step is described. The title compound is synthesized in thirteen linear steps with excelle

Full text of DOI:10.1055/s-0029-1218844

PAPER
2979
Asymmetric Synthesis of the a-D-Galactosyl Ceramide KRN7000 via an
Organocatalytic Aldol Reaction as Key Step
A
symmetric Synth
i
esis of
e
K
RN7000ter Enders,* Violeta Terteryan, Jiří Paleček
Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, 52074 Aachen, Germany
Fax +49(241)8092127; E-mail: enders@rwth-aachen.de
Received 19 May 2010
the option of introducing flexible modifications into the
structure to access analogues.
Abstract: The asymmetric synthesis of the antitumor and immuno-
stimulatory a-D-galactosyl ceramide KRN7000 using a (R)-proline-
catalyzed enantioselective aldol reaction as key step is described.
The title compound is synthesized in thirteen linear steps with ex-
cellent stereoselectivity (de >98%, ee = 95%) employing the com-
mercially available substrates 1-pentadecanal, 2,2-dimethyl-1,3-
dioxan-5-one, hexacosanoic acid, and D-galactose.
Retrosynthetically, we envisaged the preparation of the a-
galactosyl ceramide (1) using four building blocks, name-
ly, the relatively inexpensive, commercially available D-
galactose (A), the hexacosanoic acid (B), along with the
easily prepared 1-pentadecanal (C) and the dioxanone D
(Scheme 1).
Key words: a-galactosyl ceramide, asymmetric synthesis, aldol re-
action, organocatalysis, proline
acylation
OH
HO
O
C23H47
OH
O
Through the cooperation between Japan’s Kirin Brewery
and the University of the Ryukyus, several compounds
with related chemical structures called agelasphins
(AGLs) were isolated from the naturally occurring
Okinawan marine sponge Agelas mauritianus in 1993.1
After screening their biological activities using several assay
systems, KRN7000 (also called AGL-582 or a-GalCer)
displayed the most potent immunostimulatory and antitu-
mor activities.2 This a-galactosylceramide is able to acti-
vate NKT-cells (natural killer cells) via interaction with
CD1d, a glycoprotein expressed on the surface of the an-
tigen-presenting cells.3 After activation, the NKT cells
produce large numbers of cytokines, which are responsi-
ble for the in vitro and in vivo antitumor activities.4 Addi-
tionally, the cytokines provide protection against various
autoimmune diseases5 such as type I diabetes,6 and are
efficient against hepatitis B,7 bacterial infections such as
tuberculosis8 as well as malaria.9
HO
HN
HO
O
C13H27
organocatalytic
aldol
glycosylation
OH
reductive
amination
KRN7000 (1)
O
OH
HO
HO
C25H51
O
B
O
H
HO
O
C13H27
HO
OH
A
C
O
O
D
The structure of KRN7000 (1) consists of a ceramide unit
with a non-polar aliphatic ‘tail’, a polar amidoalcohol
‘head’ and galactose, which are linked via an a-glycosidic
bond. The substituents of the ceramide unit, the length of
its aliphatic chains as well as the sugar type and its config-
uration, all play an important role in the antitumor activi-
ty.10
Scheme 1 Retrosynthetic analysis of KRN7000 (1)
The route to KRN7000 consists of an organocatalytic al-
dol reaction followed by reductive amination of the keto
group to generate three of the required stereocenters; N-
acylation and stereoselective glycosylation complete the
synthesis. As the first stereogenic step, we employed a
highly diastereo- and enantioselective proline-catalyzed
aldol reaction developed in our research group as a key
step (Scheme 2).21 The precursor aldol adduct 2 could be
obtained on a multigram scale in 59% yield with virtually
complete anti-diastereoselectivity (de >98%) and an
enantiomeric excess of 95%.
With the aim of understanding the relationship between
the chemical structure and bioactivity in order to create
more effective drugs, a variety of synthetic approaches to
KRN700011 and its analogues12–20 have been reported.
Accordingly, we were interested in developing a straight-
forward and efficient asymmetric synthesis of this impor-
tant class of immunostimulatory compounds that allows
To commence the synthesis of the KRN7000 target, the
TBS-protected syn-1,3-azido alcohol 3 was prepared from
the aldol product 2 — obtained from dioxanone D22 and 1-
pentadecanal (C)23 using the diastereo- and enantioselec-
SYNTHESIS 2010, No. 17, pp 2979–2984
x
x.
x
x
.
2
0
1
0
Advanced online publication: 29.06.2010
DOI: 10.1055/s-0029-1218844; Art ID: Z13010SS
© Georg Thieme Verlag Stuttgart · New York

2980
D. Enders et al.
PAPER
amide and, as a result, virtually quantitative conversion
into the triple silyl protected azide 5. Removal of the pri-
mary silyl protecting group was accomplished in a regio-
selective manner by employing a known literature
procedure.11b,17b The trichloroacetimidate 10 was generat-
ed from the commercially available 2,3,4,6-tetra-O-ben-
O
O
OH
(R)-proline (30 mol%)
O
CHCl3, r.t., 4 d
C14H29
C13H27
+
H
O
O
59%
O
O
C
D
2
de > 99%, ee = 95%
zyl-D-galactopyranose according to
a
procedure
previously described by Schmidt et al.26 Stereoselective
glycosylation of 6 with the galactosyl donor 10 was then
achieved in the presence of boron trifluoride etherate at
–20°C, giving the a-galactoside 7 as the major anomer (1H
NMR) in good yield (65%). The conversion of azide 7
into the corresponding primary amine was effected by a
Staudinger reduction27 using trimethylphosphine (1 M so-
lution in THF). The amine was used directly without
purification in the 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (EDCI) and hydroxybenzotria-
zole hydrate (HOBt)-mediated acylation reaction,
employing the commercially available cerotinic acid (B).
Following a procedure reported by Kim et al.,11e the re-
sulting fully protected a-GalCer 8 was treated with TBAF
in THF to give benzyl-protected KRN7000 9 in 96%
yield. To complete the synthesis, compound 9 was sub-
jected to a debenzylation reaction by catalytic hydrogena-
tion [H2/Pd(OH)2/C]. Purification of the crude product
could not be accomplished by conventional chromato-
graphic methods; however, we were able to purify the
crude solid by employing reversed-phase C18 silica gel
(MeOH–THF–CHCl3, 18:1:1). After purification,
KRN7000 (1) could be obtained with a yield of 89%.
Scheme 2 Organocatalytic aldol reaction as a key step
tive (R)-proline-catalyzed aldol reaction24 — in four steps
in an overall yield of 67%.25 The four steps involved TBS-
protection of the alcohol, diastereoselective reduction
with L-Selectride, conversion of the resulting alcohol into
a mesylate, and an SN2 displacement with sodium azide in
the presence of 18-crown-6, which took place with virtu-
ally complete inversion of configuration (de >98% by
NMR and GC analyses).
At this point we decided to attach the sugar moiety before
attempting the N-acylation, because the solubility of the
corresponding amide, which bears a polar head and two
long aliphatic chains, turned out to be low in organic sol-
vents and the resulting poor yields meant that such a se-
quence of steps in the synthesis of KRN7000 could not be
completed in a satisfactory manner.
After simultaneous deprotection of the three hydroxy
groups using Dowex exchange resin, the triol azide 4 was
protected once again with TBSOTf. The strategic advan-
tage of this route (Scheme 3) is the increased solubility of
azido-phytosphingosine 4 compared to the corresponding
O
OH
N3
OTBS
C14H29
N3
OH
TBSOTf, 2,6-lutidine
CH2Cl2, THF, 0 °C
DOWEX 50WX2
C14H29 4 steps (ref. 25)
67%
C14H29
MeOH, THF, 60 °C, 2 h
O
O
OH OH
O
O
98%
91%
4
2 (de > 98%, ee = 95%)
3 (de > 98%)
OBn
BnO
BnO
O
N3
OTBS
C14H29
N3
OTBS
10% CF3CO2H aq, THF
–15 °C to 0 °C, 15 h
10, BF3•OEt2, 4 Å MS
THF, Et2O, –20 °C, 4 h
TBSO
N3
OTBS
C14H29
HO
BnO
C14H29
O
85%
65%
OTBS
OTBS
OTBS
5
6
7
OBn
OBn
BnO
BnO
BnO
O
O
C25H51
OTBS
C14H29
C25H51
OH
O
1) Me3P (1 M soln in THF), 1 M NaOH
THF, 0 °C to r.t., 3 h
2) B, EDCI, HOBt, i-Pr2NEt
CH2Cl2, DMF, r.t., 24 h
O
BnO
HN
HN
TBAF, THF
r.t., 3 h
BnO
BnO
O
O
C14H29
96%
49%
OTBS
OH
O
9
8
OBn
OH
BnO
HO
O
C25H51
OH
O
BnO
HO
H2, Pd(OH)2/C
EtOH, CHCl3, r.t., 24 h
HN
BnO
HO
O
CCl3
O
C14H29
89%
10
NH
OH
KRN7000 (1) (de > 98%, ee = 95%)
Scheme 3 Asymmetric synthesis of KRN7000 (1)
Synthesis 2010, No. 17, 2979–2984 © Thieme Stuttgart · New York

PAPER
Asymmetric Synthesis of KRN7000
2981
(2S,3S,4R)-2-Azido-1,3,4-tri-tert-buthyldimethylsilyloxy-1,3,4-
octadecanetriol (5)
In summary, we have developed an efficient asymmetric
synthesis of the a-D-galactosyl ceramide KRN7000 by us-
ing an organocatalytic aldol reaction as key step in excel-
lent diastereo- and enantiomeric excesses (de >98%,
ee = 95%). The title compound was obtained in thirteen
steps starting from four, readily available building blocks.
This method allows access to both enantiomers of
KRN7000, depending on whether (R)- or (S)-proline is
used as catalyst. Furthermore, the prochiral ketone func-
tion of 2 can be easily converted into the epimeric amino
group.21 Theoretically, by using derivates of A, B and C,
access to distinct analogues of KRN7000 for pharmaceu-
tical studies via this route should be possible.
Compound 4 (0.50 g, 2.12 mmol) was dissolved in CH2Cl2 (30 mL)
and THF (6 mL) and cooled to 0 °C. Under argon, sequentially, 2,6-
lutidine (2.9 mL, 25.4 mmol) and TBSOTf (3.9 mL, 17.0 mmol)
were added dropwise via syringe. After 2 h, the reaction mixture
was warmed to r.t. and stirred for an additional 14 h then quenched
with sat. NaHCO3 (20 mL). The aqueous phase was extracted with
CH2Cl2 (3 × 40 mL), the combined organic layers were dried over
MgSO4, and the solvent was removed under reduced pressure. Pu-
rification by flash chromatography on silica gel (Et2O–pentane, 3:1)
provided the TBS-protected azido triol 5.
Yield: 1.43 g (98%); colorless oil; de >98% (NMR); [a]D22 +4.3 (c
2.3, CHCl3).
IR (CHCl3): 2954, 2856, 2099, 1468, 1255, 1072, 836, 778, 670
cm–1.
All solvents were dried by conventional methods. Starting materials
and reagents were purchased from commercial suppliers and used
without further purification. THF and Et2O were freshly distilled
from SOLVONA® (a sodium doped material) under argon. CH2Cl2
was freshly distilled from CaH2 under argon. Preparative column
chromatography was performed using silica gel 60, particle size
0.040–0.063 mm (230–240 mesh, flash) or reversed-phase octade-
cylsilicate (POLYGOPREP 60–50 mesh, C18-RP). Analytical TLC
was carried out by employing silica gel 60 F254 plates from Merck,
Darmstadt. Visualization of the developed chromatograms was
achieved by staining with phosphomolybdic acid solution in EtOH
and anisaldehyde. Optical rotation values were measured with a
Perkin–Elmer P241 polarimeter. Microanalyses were obtained with
a Vario EL element analyzer. Mass spectra were acquired with a
Finnigan SSQ7000 spectrometer (CI 100 eV; EI 70 eV). HRMS
data were recorded with a Finnigan MAT95 spectrometer (EI) or a
Thermo Fisher Scientific OrbitrapXL (ESI) instrument. IR spectra
were measured with a Perkin–Elmer FT-IR 1760 or a Spectrum 100
1H NMR (300 MHz, CDCl3): d = 0.01, 0.02, 0.03, 0.07, 0.08, 0.09
(6 × s, 18 H, 6 × CH3Si), 0.87 (t, J = 7.1 Hz, 3 H, H-18), 0.87–0.89
(m, 27 H, 3 × t-BuSi), 1.27 (m, 24 H, 12 × CH2, H6–17), 1.40–1.56
(m, 2 H, CH2, H-5), 3.59 (m, 2 H, H-3, H-4), 3.59–3.75 (m, 2 H, H-
1a, H-2), 3.99 (dd, J = 11.8, 1.9 Hz, 1 H, H-1b).
13C NMR (75 MHz, CDCl3): d = –5.4, –4.7, –4.4, –4.1, –3.9, –2.9
(6 × CH3Si), 14.1 (C-18), 18.1, 18.2, 18.3 (3 × C-Si), 22.7, 25.3,
25.7, 25.8, 26.02, 26.06, 29.4, 29.6, 29.7, 29.9, 31.9, 32.8, 64.1 (C-
1), 65.3 (C-2), 74.6, 75.8 (C-3, C-4).
ESI-MS: m/z (%) = 658 (37) [M+ – N2], 687 (100) [M + H]+, 709
(45) [M + Na]+.
HRMS (ESI): m/z [M + H]+ calcd for C36H80N3O3Si3: 686.5502;
found: 686.5503.
(2S,3S,4R)-2-Azido-3,4-bis-tert-butyldimethylsilyloxy-1-octa-
decaneol (6)
1
spectrophotometer. H and 13C NMR spectra were recorded with
To a stirred solution of 5 (1.20 g, 1.75 mmol) in THF (40 mL) aq
TFA (10%, 13 mL) was added dropwise at –20 °C. The reaction
temperature was gradually raised to 0°C with stirring over 4 h. The
reaction was quenched with aq NaOH (15%, 15 mL) and the reac-
tion mixture was extracted with Et2O (10 mL). The combined or-
ganic layers were washed with H2O (10 mL), sat. aq NaHCO3 (10
mL) and brine (10 mL), dried with MgSO4 and concentrated in vac-
uo. The residue was purified by column chromatography on silica
gel (pentane–EtOAc, 9:1) to give 6.
Varian Mercury 300, Gemini 300 or Inova 400 spectrometers with
TMS as the internal standard.
(2S,3S,4R)-2-Azido-1,3,4-octadecanetriol (4)
DOWEX 50WX2 (2 g) was added to a solution of 3 (1.00 g,
2.01 mmol) in MeOH (15 mL) and THF (2 mL) and the mixture was
heated at reflux for 3 h. After filtration of the resin, the solvent was
removed under reduced pressure. Purification by flash chromatog-
raphy (silica gel; CH2Cl2–MeOH, 3:1) afforded the azido triol 4.
Yield: 0.85 g (85%); colorless oil; de >98% (NMR); [a]D21 –3.6 (c
1.5, CHCl3).
Yield: 0.63 g (91%); colorless solid; mp 92 °C; de >98% (NMR
analysis); [a]D20 +10.9 (c 0.5, MeOH).
IR (CHCl3): 3346, 2991, 2891, 2167, 1235, 1054, 797, 774, 658
cm–1.
IR (CHCl3): 3293, 2916, 2848, 2097, 1463, 1253, 1066, 1011, 880,
720, 669 cm–1.
1H NMR (400 MHz, CD3OD): d = 0.87 (t, J = 6.9 Hz, 3 H, CH3, H-
18), 1.26 (m, 24 H, 12 × CH2, H6–17), 1.51–1.67 (m, 2 H, CH2, H-
5), 3.50 (m, 2 H, H-3, H-4), 3.57 (ddd, J1b–2 = 3.6 Hz, J2–3 = 7.8 Hz,
1H NMR (400 MHz, CDCl3): d = 0.09 (s, 6 H, 2 × CH3Si), 0.12 (s,
6 H, 2 × CH3Si), 0.87 (t, J = 6.6 Hz, 3 H, H-18), 0.91 (s, 18 H, 2 ×
t-BuSi), 1.26 (m, 24 H, 12 × CH2), 1.50–1.63 (m, 2 H, H-5), 2.60
(br s, 1 H, OH), 3.70 (m, 2 H, H-3, H-4), 3.75 (m, 1 H, H-2), 3.90
(m, 2 H, H-1a, H-1b).
J1a–2 = 11.2 Hz, 1 H, H-2), 3.73 (dd, J1a–1b = 14.9 Hz, J1a–2
=
13C NMR (100 MHz, CDCl3: d = –4.8, –4.5, –4.1, –4.0 (4 × CH3Si),
14.1 (C-18), 18.1, 18.2 (2 × C-Si), 22.7, 25.5, 26.0 (6 × C-CH3),
29.4, 29.6, 29.7, 29.8, 31.9, 33.7 (13 × CH2), 62.1 (C-1), 64.8 (C-2),
75.4 (C-4), 76.0 (C-3).
11.5 Hz, 1 H, H-1a), 3.89 (dd, J1a–1b = 14.9 Hz, J1b–2 = 3.3 Hz, 1 H,
H-1b).
13C NMR (100 MHz, CD3OD): d = 13.1 (CH3), 22.4, 25.4, 29.1,
29.4, 31.7, 32.5 (13 × CH2), 61.1 (C-1), 65.3 (C-2), 71.4, 74.6 (C-3/
C-4).
ESI-MS: m/z = 344 [M + H]+, 366 [M + Na]+, 709 [2M + Na]+.
HRMS (ESI): m/z [M + Na]+ calcd for C18H37N3O3Na: 366.2727;
ESI-MS: m/z (%) = 572 (100) [M + H]+, 594 (66) [M +Na]+, 610
(10) [M+ +K].
HRMS (ESI): m/z [M + H]+ calcd for C30H66N3O3Si2: 572.4637;
found: 572.4634.
found: 366.2726.
(2S,3S,4R)-2-Azido-3,4-bis-tert-butyldimethylsilyloxy-1-
(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)octadecane (7)
Compound 10 (1.00 g, 1.46 mmol), which was prepared as de-
scribed previously,11e and azidoshingosine 6 (417 mg, 0.73 mmol)
Synthesis 2010, No. 17, 2979–2984 © Thieme Stuttgart · New York

2982
D. Enders et al.
PAPER
were dissolved in Et2O (13 mL) and THF (3 mL). Freshly dried 4Å
molecular sieves (400 mg) were added and the reaction mixture was
cooled to –20 °C under an argon atmosphere. BF3·OEt2 (140 mL,
1.5 equiv) was added and the reaction mixture was stirred at –20 °C.
After 18 h, the reaction mixture was diluted with EtOAc (35 mL)
followed by filtration. The organic solution was washed with aq
NaHCO3 (15 mL) and brine (10 mL), dried with MgSO4 and con-
centrated under reduced pressure. The crude product was purified
by flash chromatography (silica gel; pentane–EtOAc, 9:1).
The NMR data are in good agreement with those reported in litera-
ture.11b,e
1H NMR (400 MHz, CDCl3): d = 0.02, 0.04, 0.06, 0.09 (4 × s, 12 H,
CH3Si), 0.85–0.90 (m, 6 H, CH3, H-18, 26¢), 0.87 (s, 9 H, t-BuSi),
0.89 (s, 9 H, t-BuSi), 1.26 (m, 66 H, 33 × CH2), 1.44–1.68 (m, 6 H,
H-5, 6, 3¢), 1.94 (t, J = 7.9 Hz, 2 H, H-2¢), 3.53 (m, 2 H, CH2, H-6¢¢),
3.61 (m, 1 H, CH, H-4), 3.77–3.98 (m, 6 H, H-1a, 3, 2¢¢, 3¢¢, 4¢¢, 5¢¢),
4.03 (dd, J = 10.3, 3.6 Hz, 1 H, H-1b), 4.10 (m, 1 H, H-2), 4.36 (d,
J = 12.1 Hz, 1 H, PhCH2), 4.55 (d, J = 11.3 Hz, 1 H, PhCH2), 4.58
(d, J = 11.8 Hz, 1 H, PhCH2), 4.65 (d, J = 12.1 Hz, 1 H, PhCH2),
4.68 (d, J = 3.3 Hz, 1 H, H-1¢¢), 4.74 (d, J = 11.3 Hz, 1 H, PhCH2),
4.80 (d, J = 11.8 Hz, 1 H, PhCH2), 4.91 (d, J = 11.5 Hz, 1 H,
PhCH2), 4.97 (d, J = 11.5 Hz, 1 H, PhCH2), 5.92 (d, J = 7.1 Hz,
1 H, N-H), 7.23–7.38 (m, 20 H, Ar-H).
Yield: 519 mg (65%); colorless oil; de >98% (NMR), [a]D21 +20.8
(c 1.3, CHCl3).
IR (CHCl3): 2921, 2877, 2109, 1644, 1541, 1464, 1365, 1239, 1109,
777 cm–1.
1H NMR (400 MHz, CDCl3): d = 0.06, 0.08, 0.09, 0.10 (4 × s, 12 H,
CH3Si), 0.88 (t, J = 5.8 Hz, 3 H, H-18), 0.87 (s, 9 H, t-BuSi), 0.89
(s, 9 H, t-BuSi), 1.26 (m, 24 H, 12 × CH2), 1.42–1.62 (m, 2 H, H-5),
3.54 (m, 2 H, CH2, H-6¢), 3.62 (m, 1 H, CH, H-4), 3.72–3.93 (m,
6 H, H-1a, 3, 2¢, 3¢, 4¢, 5¢), 3.98–4.14 (m, 2 H, H-1b, H-2), 4.4 (d,
J = 10.7 Hz, 1 H, PhCH2), 4.52 (d, J = 11.3 Hz, 1 H, PhCH2), 4.61
(d, J = 11.8 Hz, 1 H, PhCH2), 4.72 (d, J = 4.1 Hz, 1 H, H-1¢), 4.76
(d, J = 10.9 Hz, 1 H, PhCH2), 4.90 (d, J = 11.3 Hz, 1 H, PhCH2),
4.91 (d, J = 11.5 Hz, 1 H, PhCH2), 4.97 (d, J = 11.5 Hz, 2 H,
PhCH2), 7.24–7.39 (m, 20 H, Ar-H).
13C NMR (100 MHz, CDCl3): d = –5.14, –4.70, –3.96, –3.81
(4 × CH3Si), 14.3 (C-18, C-26¢), 18.3, 18.5 (2 × C-Si), 22.8, 24.0,
25.7, 26.1, 26.3, 29.40, 29.49, 29.56, 29.61, 29.69, 29.74, 29.83
(alkyl CH2), 30.1, 32.0, 33.1, 36.9, 51.1, 68.31, 69.01, 69.75, 72.8,
72.9, 73.49, 74.55, 74.86, 75.0, 75.3, 75.6, 78.7, 79.6, 82.1, 85.1,
103.9 (CH, H-1¢¢), 127.3–128.4 (CH, Ar), 137.7–138.8 (C, Ar),
172.8 (NH-CO).
ESI-MS: m/z (%) = 1447 (100) [M]+, 1469 (19) [M +Na]+.
HRMS (ESI): m/z [M + H]+ calcd for C90H152NO9Si2: 1447.1000;
13C NMR (100 MHz, CDCl3): d = –4.79 to –4.04 (6 × CH3Si), 14.1
(C-18), 18.1, 18.2 (2 × C-Si), 22.6, 25.3, 25.98, 25.99, 29.3, 29.53,
29.58, 29.6, 29.7 (alkyl CH2), 31.8 (CH2, C-5), 33.0 (CH2, C-16),
60.3 (CH, C-2), 63.4 (CH2, C-6¢), 68.3, 68.8, 69.2 (3 × CH, C-3¢, 4¢,
5¢), 69.5 (CH2, C-1), 70.4, 72.61, 72.64, 72.8, 73.2, 73.3, 73.4, 74.3,
74.6, 74.8, 75.1, 75.7, 75.8 (PhCH2), 78.5 (CH, C-3), 79.3 (CH, C-
4), 81.9 (CH, C-2¢), 103.6 (CH, H-1¢), 127.2–128.2 (CH, Ar),
137.6–138.5 (C, Ar).
found: 1447.1012.
(2S,3S,4R)-2-Hexacosanoylamino-1-(2,3,4,6-tetra-O-benzyl-a-
D-galactopyranosyl)octadecane-3,4-diol (9)
Following the procedure of Kim et al.,11e a solution of 8 (150 mg,
0.103 mmol) in THF (10 mL) was cooled to 0 °C and TBAF (4.41
mL, 1.0 M solution in THF, 4 equiv) was added dropwise. After the
mixture was stirred at r.t. for 3 h, H2O (30 mL) was added and an
the mixture was extracted with Et2O (3 × 30 mL). The combined or-
ganic layers were washed with brine (20 mL) and dried with
MgSO4. Evaporation of the solvent followed by column chromatog-
raphy (silica gel; hexane–EtOAc, 3:1) of the crude product afforded
9.
ESI-MS: m/z (%) = 1111 (100) [M+ + 17], 1116 (39) [M+ + Na].
HRMS (ESI): m/z [M + Na]+ calcd for C64H99N3O8Si2Na:
1116.6862; found: 1116.6842.
(2S,3S,4R)-3,4-Bis-tert-butyldimethylsilyloxy-2-hexacosanoyl-
amino-1-(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)octade-
cane (8)
Yield: 121 mg (96%); colorless solid; mp 79 °C (Lit.11b,e 70.5–
71.5 °C); de >98% (NMR); [a]D +30.8 (c 1.5, CHCl3) {Lit.11b
21
[a]D25 +33.3 (c 1.0, CHCl3); Lit.11e [a]D16 +27.6 (c 2.1, CHCl3)}.
Azide 7 (300 mg, 0.274 mmol) was dissolved in THF (6 mL) and
cooled to 0 °C. Me3P (1.37 mL, 1.0 M in THF, 1.37 mmol) was add-
ed to the solution and the reaction was stirred for 45 min at 0 °C and
for 3 h at r.t.. After complete conversion of the starting material (re-
action monitored by TLC), aq NaOH (1 M, 2.5 mL) was added and
the mixture was stirred for 1.5 h. EtOAc (10 mL) was then added to
the solution and the organic layers were washed with H2O (3 × 5
mL), brine (3 mL) and dried over MgSO4. Evaporation of the sol-
vent afforded the crude amine as a yellow oil, which was used for
the next step without purification.
IR (KBr): 3457, 3350, 2955, 2849, 1648, 1631, 1532, 1489, 1174,
1048, 716, 698, 659 cm–1.
The NMR data are in good agreement with those reported in litera-
ture.11b,e
1H NMR (300 MHz, CDCl3): d = 0.88 (m, 6 H, H-18, 26¢), 1.26 (m,
66 H, 33 × CH2), 1.38–1.68 (m, 6 H, H-3¢, 5, 6), 2.22 (s, 1 H, OH),
2.35 (s, 1 H, OH), 2.38 (t, J = 7.42 Hz, 2 H, H-2¢), 3.44–3.58 (m,
3 H, H-4, 6¢¢), 3.98–4.06 (m, 6 H, H-1a, 3, 2¢¢, 3¢¢, 4¢¢, 5¢¢), 4.02 (dd,
J = 3.8, 10.9 Hz, 1 H, H-1b), 4.14 (m, 1 H, H-2), 4.28–4.95 (m, 9 H,
H-1¢¢, PhCH2), 6.40 (d, J = 8.1 Hz, 1 H, NH), 7.20–7.39 (m, 20 H,
Ar-H).
13C NMR (75 MHz, CDCl3): d = 14.0 (C-18, C-26¢), 22.6, 25.6,
25.9, 29.1, 29.3, 29.6, 31.8, 32.6, 36.4, 50.8, 68.8, 70.5, 72.8, 72.9,
73.3, 73.5, 74.6, 74.7, 75.3, 76.6, 76.9, 77.4, 79.1, 99.1 (CH, H-1¢¢),
127.5–128.3 (CH, Ar), 137.2–138.1 (C, Ar), 173.9 (NH-CO).
ESI-MS: m/z (%) = 1219 (3) [M + H]+, 1069 (100), 867 (10), 467
(13), 120 (20).
HRMS (ESI): m/z [M + H]+ calcd for C78H124NO9: 1218.9270;
found: 1218.9273.
To a suspension of hexacosanoic acid B (326 mg, 0.822 mmol) in
DMF (20 mL) and CH2Cl2 (55 mL) was added EDCI (185 mg, 0.82
mmol) and HOBt (130 mg, 0.82 mmol) at 0 °C. After the mixture
was stirred 45 min at r.t., a solution of the crude amine prepared as
described above and DIPEA (0.35 mL) in CH2Cl2 (30 mL) were
added. The mixture was stirred at r.t. for 24 h, then diluted with
EtOAc–Et2O (4:1, 30 mL) and washed with sat. aq NaHCO3 (35
mL), aq HCl (1 M, 35 mL) and brine (25 mL). The organic layers
were dried over MgSO4 and the solvent was removed under reduced
pressure. Purification by flash chromatography (silica gel; hexane–
EtOAc, 4:1) afforded the desired product 8.
Yield: 194 mg (49% over 2 steps); colorless oil; de >98% (NMR);
20
24
[a]D +11.2 (c 1.0, CHCl3) {Lit.11b [a]D +15.4 (c 1.0, CHCl3);
KRN7000 [(2S,3S,4R)-1-(a-D-Galactopyranosyl)-2-hexa-
cosanoylamminooctadecane-3,4-diol; 1]
Lit.11e [a]D17 +9.5 (c 0.6, CHCl3)}.
To a solution of 9 (80 mg, 0.065 mmol) in CHCl3 (1 mL) and EtOH
(4 mL) were added Pd(OH)2/C (20% wt, 320 mg). The reaction
IR (CHCl3): 3437, 2922, 2852, 1671, 1463, 1253, 1101, 1028, 835,
757 cm–1.
Synthesis 2010, No. 17, 2979–2984 © Thieme Stuttgart · New York

PAPER
Asymmetric Synthesis of KRN7000
2983
mixture was stirred for 24 h under an atmosphere of H2 at r.t., then
filtered through a short pad of Celite and washed with CHCl3–
MeOH (1:1, 25 mL). The combined filtrate and washings were con-
centrated in vacuo and the residual solid was triturated with hex-
ane–EtOAc (1:1, 4 mL). Purification by flash chromatography
using reversed-phase C-18 silica gel (MeOH–THF–CHCl3, 18:1:1)
afforded the title compound 1.
Scherer, D. C.; Wei, J.; Kronenberg, M.; Koezuka, Y.;
Van Kaer, L. Nature Med. 2001, 7, 1052. (c) Falcone, M.;
Facciotti, F.; Ghidoli, N.; Monti, P.; Olivieri, S.;
Zaccagnino, L.; Bonifacio, E.; Casorati, G.; Sanvito, F.;
Sarvetnick, N. J. Immunol. 2004, 172, 5908.
(7) (a) Kakimi, K.; Guidotti, L. G.; Koezuka, Y.; Chisari, F. V.
J. Exp. Med. 2000, 192, 921. (b) Baron, J. L.; Gardiner, L.;
Nishimura, S.; Shinkai, K.; Locksley, R.; Ganem, D.
Immunity 2002, 16, 583.
(8) Chackerian, A.; Alt, J.; Perera, V.; Behar, S. M. Infect.
Immun. 2002, 70, 6302.
(9) (a) Hansen, D. S.; Siomos, M.; Koning-Ward, T.;
Buckingham, L.; Crabb, B. S.; Schofield, L. Eur. J.
Immunol. 2003, 33, 2588. (b) Gonzalez-Aseguinolaza, G.;
Van Kaer, L.; Bergmann, C. C.; Wilson, J. M.; Schmieg, J.;
Kronenberg, M.; Nakayama, T.; Taniguchi, M.; Koezuka,
Y.; Tsuji, M. J. Exp. Med. 2002, 195, 617.
(10) Kobayashi, E.; Motoki, K.; Yamaguchi, Y.; Uchida, T.;
Fukushima, H.; Koezuka, Y. Bioorg. Med. Chem. Lett. 1996,
4, 615.
Yield: 49 mg (89%); colorless solid; mp 185 °C (Lit.11b 189.5–
20
190.0 °C); de >98% (NMR); ee = 95%; [a]D +39.8 (c 0.2, pyri-
dine) {Lit.11b [a]D23 +42.2 (c 0.54, pyridine); Lit.11a [a]D23 +43.6 (c
1.0, pyridine)}.
IR (KBr): 3425, 2922, 2851, 1631, 1605, 1532, 1483, 1163, 1247,
1058, 748 cm–1.
1H NMR (400 MHz, pyridine-d5): d = 0.88 (t, J = 6.6 Hz, 6 H, H-18,
26¢), 1.20–1.46 (m, 66 H, 33 × CH2), 1.63–1.69 (m, 1 H, H-6a),
1.78–1.86 (m, 2 H, H-3¢), 1.89–1.96 (m, 2 H, H-5a, 6b), 2.24–2.30
(m, 1 H, H-5b), 2.51 (t, J = 7.4 Hz, 2 H, H-2¢), 4.00–4.10 (m, 2 H,
H-3, 4), 4.18 (m, 3 H, H-1a, 6¢¢), 4.26 (m, 2 H, H-4¢¢, 5¢¢), 4.56 (m,
1 H, H-3¢¢), 4.64–4.72 (m, 2 H, H-1b, 2¢¢), 5.27 (m, 1 H, H-2), 5.60
(d, J = 3.3 Hz, 1 H, H-1¢¢), 6.50 (br s, 6 H, OH), 8.65 (d, J = 8.5 Hz,
1 H, N-H).
13C NMR (100 MHz, pyridine-d5): d = 14.3 (C-18, C-26¢), 22.9,
25.7, 26.2, 29.5, 29.7, 29.9, 30.3, 32.1, 33.2, 33.5, 36.8, 52.2, 62.4,
66.6, 70.1, 70.7, 72.5, 72.6, 76.9, 101.2 (CH, H-1¢¢), 173.2 (NH-
CO).
(11) For selected examples, see: (a) Morita, M.; Natori, T.;
Akimoto, K.; Osawa, T.; Fukushima, H.; Koezuka, Y.
Bioorg. Med. Chem. Lett. 1995, 5, 699. (b) Takikawa, H.;
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(c) Figueroa-Pérez, S.; Schmidt, R. R. Carbohydr. Res.
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Wong, C. J. Org. Chem. 2002, 67, 4559. (e) Kim, S.; Song,
S.; Lee, T.; Jung, S.; Kim, D. Synthesis 2004, 847. (f) Xia,
C.; Yao, Q.; Schümann, J.; Rossy, E.; Chen, W.; Zhu, L.;
Zhang, W.; Libero, G. D.; Wang, P. G. Bioorg. Med. Chem.
Lett. 2006, 16, 2195. (g) Michieletti, M.; Bracci, A.;
Compostella, F.; De Libero, G.; Mori, L.; Fallarini, S.;
Lombardi, G.; Panza, L. J. Org. Chem. 2008, 73, 9192.
(h) Park, J.; Lee, J. H.; Ghosh, S. C.; Bricard, G.;
Venkataswamy, M.; Porcelli, S. A.; Chung, S. Bioorg. Med.
Chem. Lett. 2008, 18, 3906. (i) Boututeira, O.; Morales-
Serna, J. A.; Diaz, Y.; Matheu, M. I.; Castillón, S. Eur. J.
Org. Chem. 2008, 1851. (j) Morales-Serna, J. A.;
Boututeira, O.; Diaz, Y.; Matheu, M. I.; Castillón, S.
Carbohydr. Res. 2007, 342, 1595. (k) Veerapen, N.; Brigl,
M.; Garg, S.; Cerundolo, V.; Cox, L. R.; Brenner, M. B.;
Besra, G. S. Bioorg. Med. Chem. Lett. 2009, 19, 4288.
(12) For b-GalCer, see: Rai, A. N.; Basu, A. J. Org. Chem. 2005,
70, 8228.
(13) For a-C-glycoside analogues, see: (a) Yang, G.; Schmieg,
J.; Tsuji, M.; Franck, R. W. Angew. Chem. Int. Ed. 2004, 43,
3818; Angew. Chem. 2004, 116, 3906. (b) Wipf, P.; Pierce,
J. G. Org. Lett. 2006, 8, 3375. (c) Pu, J.; Franck, R. W.
Tetrahedron 2008, 64, 8618.
(14) For b-C-glycoside analogues, see: (a) Chaulagain, M. R.;
Postema, M. H. D.; Valeriote, F.; Pietraszkewicz, H.
Tetrahedron Lett. 2004, 45, 7791. (b) Postema, M. H. D.;
Piper, J. L.; Betts, R. L. Synlett 2005, 1345.
(15) For a-S-glycoside analogues, see: (a) Dere, R. T.; Zhu, X.
Org. Lett. 2008, 10, 4641. (b) Blauvelt, M. L.; Khalili, M.;
Jaung, W.; Paulsen, J.; Anderson, A. C.; Wilson, S. B.;
Howell, A. R. Bioorg. Med. Chem. Lett. 2008, 18, 6374.
(c) Rajan, R.; Mathew, T.; Buffa, R.; Bornancin, F.;
Cavallari, M.; Nussbaumer, P.; De Libero, G.; Vasella, A.
Helv. Chim. Acta 2009, 92, 918.
(16) Truncated analogues: (a) Fan, G. T.; Pan, Y.; Lu, K. C.;
Cheng, Y. P.; Lin, W. C.; Lin, S.; Lin, C. H.; Wong, C. H.;
Fang, J. M.; Lin, C. C. Tetrahedron 2005, 61, 1855.
(b) Tsujimoto, T.; Ito, Y. Tetrahedron Lett. 2007, 48, 5513.
Sphinganine analogues: (c) Ndonye, R. M.; Izmirian, D. P.;
Dunn, M. F.; Yu, K. O. A.; Porcelli, S. A.; Khurana, A.;
ESI-MS: m/z = 858 [M + H]+, 880 [M + Na]+, 896 [M + K]+.
HRMS (ESI): m/z [M + H]+ calcd for C50H100NO9: 858.7392; found:
858.7394.
Acknowledgment
This work was supported by the Fonds der Chemischen Industrie
and the Deutsche Forschungsgemeinschaft (Schwerpunktpro-
gramm 1179 Organokatalyse). We thank the companies Bayer AG,
BASF AG, Wacker Chemie and the former Degussa AG for the do-
nation of chemicals.
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Synthesis 2010, No. 17, 2979–2984 © Thieme Stuttgart · New York