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Cancer Patent Abstract
The invention is directed to compounds of formula (I) ##STR00001##
wherein X is O or NH; R' is a hydrocarbon chain; R.sup.3 and R.sup.4
are hydrogen, OH or a monosaccharide; R.sup.5 is hydrogen or a monosaccharide;
Q' is optionally present and may be a C.sub.1-10 hydrocarbon; X'
is optionally present and may be O, S or NR.sup.8; and Q.sup.3 may
be a hydrocarbon or hydrogen. The invention is also directed to
the use of the compounds for treating cancer, infectious diseases
and autoimmune diseases. The invention is also directed to syntheses
of the compounds of formula (I).
Cancer Patent Claims
What is claimed is:
1. A method of inducing the production of Th1 type cytokines in
a mammal suffering from a disease that is treatable by inducing
TH1 type responses, by administering to said mammal a therapeutically
effective amount of a compound of formula I ##STR00022## wherein
X is O or NH; R.sup.1 is selected from the group consisting of --(CH.sub.2).sub.11CH.sub.3,
--(CH.sub.2).sub.12CH.sub.3, --(CH.sub.2).sub.13CH.sub.3, --(CH.sub.2).sub.9CH(CH.sub.3).sub.2,
--(CH.sub.2).sub.10CH(CH.sub.3).sub.2, --(CH.sub.2).sub.11CH(CH.sub.3).sub.2
and (CH.sub.2).sub.11CH(CH.sub.3)--C.sub.2H.sub.5; R.sup.3 is OH
or a monosaccharide and R.sup.4 is hydrogen, or R.sup.3 is hydrogen
and R.sup.4 is OH or a monosaccharide; R.sup.5 is hydrogen or a
monosaccharide; Q.sup.1 is optionally present and is a C.sub.1-10
straight or branched chain alkylene, alkenylene, or alkynylene;
X' is optionally present and is O, S or NR.sup.8; Q.sup.2 is optionally
present and is a C.sub.1-10 straight or branched chain alkylene,
alkenylene or alkynylene; X'' is optionally present and is O, S
or NR.sup.8; Q.sup.3 is a straight or branched chain C.sub.1-10
alkyl, alkenyl or alkynyl, or is hydrogen, wherein each Q.sup.1,
Q.sup.2 or Q.sup.3 is optionally substituted with hydroxyl, halogen,
cyano, nitro, SO.sub.2, NHR.sup.8, or C(.dbd.O)--R.sup.9; and wherein
R.sup.8 is hydrogen, C.sub.1-5 alkyl, C.sub.1-5 alkoxy, halogen,
cyano, nitro, SO.sub.2 or C(.dbd.O)--R.sup.9; R.sup.9 is hydrogen,
C.sub.1-5 alkyl, C.sub.1-5 alkoxy or NHR.sup.10; R.sup.10 is hydrogen,
C.sub.1-5 alkyl or C.sub.1-5 alkoxy; and a pharmaceutically acceptable
salt or ester thereof, wherein the compound does not induce substantial
production of interleukin 4 (IL-4).
2. The method of claim 1, wherein said Th1 type cytokines comprise
interferon-.gamma. (IFN-.gamma.) and interleukin 12 (IL-12).
3. The method of claim 1, wherein the mammal is human.
4. The method of claim 1, wherein the disease is an infectious
disease selected from the group consisting of malarial infection,
HIV infection, hepatitis B virus infection, hepatitis C virus infection,
Mycobacterium infection, respiratory syncitial virus infection,
and Herpes virus infection.
5. The method of claim 1, wherein the disease is allergy.
6. The method of claim 1, wherein the disease is asthma.
7. The method of claim 1, wherein the disease is sarcoidosis.
8. The method of claim 1, wherein the disease is a cancer selected
from the group consisting of carcinoma of bladder, breast, colon,
esophagus, liver, lung, ovary, pancreas, prostate, kidney, renal,
stomach, testicles, cervix, thyroid, skin, squamous cell carcinoma,
small cell lung cancer, non-small cell lung cancer, leukemia, acute
lymphocytic leukemia, acute lymphoblastic leukemia, T cell lymphoma,
B cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy
cell lymphoma, Burkett's lymphoma, acute myelogenous leukemia, chronic
myelogenous leukemia, myelodysplastic syndrome, promyelocytic leukemia,
fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, glioma,
schwannoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma
pigmentosum, keratoctanthoma, thyroid follicular cancer, and Kaposi's
sarcoma.
9. The method of claim 1, wherein the disease is melanoma.
Cancer Patent Description
FIELD OF THE INVENTION
The invention is directed to novel synthetic C-glycolipids, which
are useful in treating cancer, infectious diseases and autoimmune
diseases. Specifically, the invention is directed to novel synthetic
analogs of .alpha.-C-galactosylceramides, which are potent mediators
of Natural killer T cells, and to methods of making the novel synthetic
analogs.
BACKGROUND OF THE INVENTION
Natural killer T (NKT) cells are lymphoid cells which are distinct
from mainstream T cells, B cells and NK cells (Arase et al., 1992,
Proc. Nat'l Acad. Sci. USA, 89:6506; Bendelac et al., 1997, Annu.
Rev. Immunol., 15:535). These cells are characterized by co-expression
of NK cell receptors and semi-invariant T cell receptors (TCR) encoded
by V.alpha.14 and J.alpha.281 gene segments in mice and V.alpha.24
and J.alpha.Q gene segments in humans. The activation of NKT cells
in vivo promptly induces a series of cellular activation events
leading to the activation of innate cells such as natural killer
(NK) cells and dendritic cells (DC), the activation of adaptive
cells such as B cells and T cells, the induction of co-stimulatory
molecules and the abrupt release of cytokines such as interleukin-4
(IL-4) and interferon-.gamma. (IFN-.gamma.) (Burdin et al., Eur.
J. Immunol. 29: 2014-2025, 1999; Carnaud et al., J. Immunol., 163:
4647-4650, 1999; Kitamura et al., J. Exp. Med., 189: 1121-1128,
1999; Kitamura et al., Cell Immunol., 199: 37-42, 2000; Aderem et
al., Nature, 406: 782-787, 2000). In addition, activated NKT cells
can themselves bring about killing mediated by Fas and perforin.
The full activation cascade can be recruited by the engagement of
NKT TCR. Alternatively, powerful T-helper-cell type 1 (Th1) functions
can be selectively triggered by cytokines such as interleukin-12
(IL-12) released by infected macrophages or DC. These functions
are believed likely to be correlated with the important role of
NKT cells in conditions such as autoimmune diabetes, rejection of
established tumours or the prevention of chemically induced tumours
(Yoshimoto et al., 1995, Science, 270: 1845; Hammond et al., J.
Exp. Med., 187: 1047-1056, 1998; Kawano et al., 1998, Proc. Natl.
Acad. Sci. USA, 95: 5690; Lehuen et al., J. Exp. Med., 188: 1831-1839,
1998; Wilson et al., Nature, 391: 177-181, 1998; Smyth et al., J.
Exp. Med., 191: 661-668, 2000). Finally, NKT cells are thought to
contribute to antimicrobial immunity through their capacity to influence
the Th1-Th2 polarization (Cui et al., J. Exp. Med., 190: 783-792,
1999; Singh et al., J. Immunol., 163: 2373-2377, 1999; Shinkai et
al., J. Exp. Med., 191: 907-914, 2000). These cells are therefore
implicated as key effector cells in innate immune responses. However,
the potential role of NKT cells in the development of adaptive immune
responses remains unclear.
Glycolipids are molecules typically found in plasma membranes of
animal and plant cells. Glycolipids contain an oligosaccharide which
is bonded to a lipid component. Sphingoglycolipids are complex glycolipids
which contain ceramide as the lipid component. One class of sphingoglycolipids
is alpha-galactosylceramides (.alpha.-GalCer), which contain D-galactose
as the saccharide moiety, and ceramide as the lipid moiety. .alpha.-GalCer
is a glycolipid originally extracted from Okinawan marine sponges
(Natori et al., Tetrahedron, 50: 2771-2784, 1994).
It has been demonstrated that .alpha.-GalCer can activate NTK cells
both in vitro and in vivo. .alpha.-GalCer has been shown to stimulate
NK activity and cytokine production by NKT cells and exhibit potent
antitumor activity in vivo (Kawano et al., 1997, Science 278: 1626-9;
Kawano et al. 1998, supra; Kitamura et al. 1999, supra). Kitamura
et al. (1999, supra) demonstrated that the immunostimulating effect
of .alpha.-GalCer was initiated by CD40-CD40L-mediated NKT-DC interactions.
As the immunoregulatory functions of .alpha.-GalCer were absent
in both CD1d-1- and NKT-deficient mice, this indicates that .alpha.-GalCer
has to be presented by the MHC class I-like molecule CD1d.
CD1 is a conserved family of non-polymorphic genes related to MHC
that seems to have evolved to present lipid and glycolipid antigens
to T cells and in this way participates in both an innate and an
adaptive pathway of antigen recognition (reviewed by Park et al.,
Nature, 406: 788-792, 2000; see also Calabi et al., Eur. J. Immunol.,
19: 285-292, 1989; Porcelli et al., Annu. Rev. Immunol., 17: 297-329,
1999). The CD1 family comprises up to five distinct genes (isotypes)
that can be separated into two groups on the basis of sequence homology.
Group 1, which comprises CD1a, CD1b, CD1c and CD1e, is present in
humans but absent from mouse and rat. Group 2, which includes CD1d,
is found in all species studied so far, including humans.
CD1 isotypes are expressed selectively by antigen-presenting cells
such as dendritic cells (DCs), macrophages and subsets of B cells,
but apart from CD1d expression in hepatocytes they are generally
not expressed in solid tissues (Porcelli et al., supra; Bendelac
et al., Annu. Rev. Immunol., 15: 535-562, 1997).
.alpha.-GalCer is recognized in picomolar concentrations by mouse
and human CD1d-restricted lymphocytes that express a semi-invariant
TCR and exert potent effector and regulatory functions (Kawano et
al., 1997, supra). CD1d/.alpha.-GalCer complex is, in turn, recognized
by the antigen receptors of mouse V.alpha.14 and human V.alpha.24
natural killer T (NKT) cells (Bendelac et al., Science, 268: 863-865,
1995; Bendelac et al., Annu. Rev. Immunol., 15: 535-562, 1997; Park
et al., Eur. J. Immunol., 30: 620-625, 2000).
.alpha.-GalCer has been demonstrated to activate murine NKT cells
both in vivo and in vitro, upon binding to CD1d (Kawano et al.,
1997, supra; Burdin et al., 1998, J. Immunol., 161:3271-3281), and
in human NKT cells in vitro (Spada et al., 1998, J. Exp. Med., 188:1529-1534;
Brossay et al., 1998, J. Exp. Med. 188:1521-1528). For example,
.alpha.-GalCer was shown to display NKT-mediated anti-tumor activity
in vitro by activating human NKT cells (Kawano et al., 1999, Cancer
Res., 59:5102-5105).
In addition to .alpha.-GalCer, other glycosylceramides having .alpha.-anomeric
conformation of sugar moiety and 3,4-hydroxyl groups of the phytosphingosine
(such as .alpha.-glucosylceramide [.alpha.-GlcCer], Gal.alpha.1-6Gal.alpha.1-1'Cer,
Gal.alpha.1-6Glc.alpha.1-1'Cer, Gal.alpha.1-2Gal.alpha.1-1'Cer,
and Gal.beta.1-3Gal.alpha.1-1'Cer) have been demonstrated to stimulate
proliferation of V.alpha.14 NKT cells in mice, although with lower
efficiency (Kawano et al., Science, 278: 1626-1629, 1997, supra).
By testing a panel of .alpha.-GalCer analogs for reactivity with
mouse V.alpha.14 NKT cell hybridomas, Brossay et al. (J. Immunol.,
161: 5124-5128, 1998) determined that nearly complete truncation
of the .alpha.-GalCer acyl chain from 24 to 2 carbons does not significantly
affect the mouse NKT cell response to glycolipid presented by either
mouse CD1 or its human homolog.
It has been also demonstrated that in vivo administration of .alpha.-GalCer
not only causes the activation of NKT cells to induce a strong NK
activity and cytokine production (e.g., IL-4, IL-12 and IFN-.gamma.)
by CD1d-restricted mechanisms, but also induces the activation of
immunoregulatory cells involved in acquired immunity (Nishimura
et al., 2000, Int. Immunol., 12: 987-994). Specifically, in addition
to the activation of macrophages and NKT cells, it was shown that
in vivo administration of .alpha.-GalCer resulted in the induction
of the early activation marker CD69 on CD4+ T cells, CD8+ T cells,
and B cells (Burdin et al., 1999, Eur. J. Immunol. 29: 2014; Singh
et al., 1999, J. Immunol. 163: 2373; Kitamura et al., 2000, Cell.
Immunol. 199:37; Schofield et al., 1999, Science 283: 225; Eberl
et al., 2000, J. Immunol., 165:4305-4311).
Various .alpha.-GalCer compounds have been shown in the prior art.
U.S. Pat. No. 5,780,441 describes mono- and di-glycosylated .alpha.-GalCer
compounds of the following structure:
##STR00002## wherein R.sup.1 is H or
##STR00003## R.sup.2 is H,
##STR00004## R.sup.3 and R.sup.6 are H or OH, respectively, R.sup.4
is H, OH or
##STR00005## R.sup.5 is H or
##STR00006## x is an integer from 19 to 23; and R.sup.7 is --(CH.sub.2).sub.11--CH.sub.3,
--(CH.sub.2).sub.12--CH.sub.3, --(CH.sub.2).sub.13--CH.sub.3, --(CH.sub.2).sub.9--CH(CH.sub.3).sub.2,
--(CH.sub.2).sub.10--CH(CH.sub.3).sub.2, --(CH.sub.2).sub.11--CH(CH.sub.3).sub.2,
--(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5, wherein at least
one of R.sup.1, R.sup.2, R.sup.4 and R.sup.5 is a glycosyl moiety.
The compounds are disclosed for use as antitumor agents, as bone
marrow cell-proliferation treating agents, and as immunostimulating
agents.
Recently, .alpha.-GalCer molecules have also been shown to have
activity against viral diseases. Kakimi, J. Exp. Med. 192: 921-930
(2000) discloses that natural killer (NKT) cells in the liver of
hepatitis B virus (HBV) transgenic mice were activated by a single
injection of .alpha.-GalCer, thereby inhibiting HBV replication.
.alpha.-GalCer has also been shown to be effective against microbial
infections. Gonzalez-Asequinaloza, Proc. Natl. Acad. Sci. USA 97:
8461-8466 (2000) discloses that the administration of .alpha.-GalCer
inhibits the development of malaria parasites, resulting in strong
antimalaria activity.
.alpha.-GalCer has also demonstrated inhibition of the onset and
recurrence of autoimmune type I diabetes. Sharif, Nature Medicine
7: 1057-1062 (2001) demonstrates that activation of NKT cells by
.alpha.-GalCer protects mice from type I diabetes and prolongs the
survival of pancreatic islets transplanted into newly diabetic mice.
See also Hong, Nature Medicine 9: 1052-1056 (2001). Sharif also
demonstrated that when administered after the onset of insulitis,
.alpha.-GalCer and IL-7 displayed a synergy, which is believed to
be due to the ability of IL-7 to render NKT cells fully responsive
to .alpha.-GalCer.
.alpha.-GalCer has also demonstrated antifungal activity. Kawakami,
Infection and Immunity 69: 213-220 (2001) demonstrates that upon
administration to mice, .alpha.-GalCer increased the serum level
of gamma interferon, resulting in inhibition of the fungal pathogen
Cryptococcus neoformans.
.alpha.-GalCer analogs have also demonstrated effectiveness against
autoimmune diseases. Miyamoto, Nature 413: 531-534 (2001) describes
use of .alpha.-GalCer analogs which induce TH2 bias of autoimmune
T cells by causing natural killer T (NKT) cells to produce IL-4,
leading to suppression of experimental autoimmune encephalomyelitis.
A synthetic analog of .alpha.-GalCer, KRN 7000 (2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-
-octadecanetriol, can be obtained from Pharmaceutical Research Laboratories,
Kirin Brewery (Gumna, Japan) or synthesized as described in Morita
et al., J. Med. Chem., 1995, 38: 2176-2187.
KRN 7000 has the structure:
##STR00007##
KRN 7000 has been shown to display activity against tumors in mice.
Kobayashi, et al., Oncol. Res. 7:529-534 (1995). In particular,
KRN 7000 has been shown to be effective in preventing cancer metastasis.
See, e.g., Nakagawa, Canc. Res. 58, 1202-1207 (1998) (KRN 7000 effective
in treating liver metastasis of adenocarcinoma colon 26 cells in
mice). KRN 7000 is also described in Kobayashi et al., 1995, Oncol.
Res., 7:529-534, Kawano et al., 1997, Science, 278:1626-9, Burdin
et al., 1998, J. Immunol., 161:3271, and Kitamura et al., J. Exp.
Med., 1999, 189: 1121, and U.S. Pat. No. 5,936,076.
Importantly, in addition to its ability to stimulate immune responses,
recent human trials have shown that .alpha.-GalCer is not cytotoxic
in humans. See Shimosaka et al. Cell Therapy: Filling the gap between
basic science and clinical trials, First Int'l Workshop 2001, abstract
pp. 21-22. Other studies have demonstrated that .alpha.-GalCer,
independently of its dosage, does not induce toxicity in rodents
and monkeys (e.g., Nakagawa et al., 1998, Cancer Res., 58: 1202-1207),
although a recent study showed the transient elevation of liver
enzyme activities immediately after .alpha.-GalCer treatment in
mice, suggesting a minor liver injury (Osman et al., 2000, Eur.
J. Immunol., 39: 1919-1928).
However, most mammals, including humans, have abundant amount of
.alpha.-galactosidase, an enzyme which digests .alpha.-GalCer by
catalyzing the degradation of .alpha.-D-galactoside bonds. As a
result, .alpha.-GalCer has a short half-life, and therefore its
in vivo therapeutic effect may be reduced.
Recently, it has been shown that the activity of .alpha.-GalCer
can be modified through formation of a truncated sphingosine chain.
The modified .alpha.-GalCer is effective in treating autoimmune
encephalomyelitis in mice. Miyamato et al., Nature 413:531-534 (2001).
Applicants have now discovered .alpha.-GalCer analogs which have
improved stability in vivo over .alpha.-GalCer.
Applicants have also discovered .alpha.-GalCer analogs which have
improved therapeutic efficacy over .alpha.-GalCer.
OBJECTS OF THE INVENTION
It is an object of the invention to form compounds having the pharmacological
activity of .alpha.-GalCer, and resistance to .alpha.-galactosidase,
resulting in improved stability in vivo.
It is also an object of the invention to form novel compounds for
treating cancers, infectious diseases and autoimmune diseases.
SUMMARY OF THE INVENTION
This invention is directed to novel C-glycolipid compounds of formula
(I)
##STR00008## wherein X is O or NH; R.sup.1 is selected from the
group consisting of --(CH.sub.2).sub.11CH.sub.3, --(CH.sub.2).sub.12CH.sub.3,
--(CH.sub.2).sub.13CH.sub.3, --(CH.sub.2).sub.9CH(CH.sub.3).sub.2,
--(CH.sub.2).sub.10CH(CH.sub.3).sub.2, --(CH.sub.2).sub.11CH(CH.sub.3).sub.2
and (CH.sub.2).sub.11CH(CH.sub.3)--C.sub.2H.sub.5; R.sup.3 is OH
or a monosaccharide and R.sup.4 is hydrogen, or R.sup.3 is hydrogen
and R.sup.4 is OH or a monosaccharide; R.sup.5 is hydrogen or a
monosaccharide; Q.sup.1 is optionally present and is a C.sub.1-10
straight or branched chain alkylene, alkenylene, or alkynylene;
X' is optionally present and is O, S or NR.sup.8; Q.sup.2 is optionally
present and is a C.sub.1-10 straight or branched chain alkylene,
alkenylene or alkynylene; X'' is optionally present and is O, S
or NR.sup.8; Q.sup.3 is a straight or branched chain C.sub.1-10
alkyl, alkenyl or alkynyl, or is hydrogen, wherein each Q.sup.1,
Q.sup.2 or Q.sup.3 is optionally substituted with hydroxyl, halogen,
cyano, nitro, SO.sub.2, NHR.sup.8, or C(.dbd.O)--R.sup.9; and wherein
R.sup.8 is hydrogen, C.sub.1-5 alkyl, C.sub.1-5 alkoxy, halogen,
cyano, nitro, SO.sub.2 or C(.dbd.O)--R.sup.9; R.sup.9 is hydrogen,
C.sub.1-5 alkyl, C.sub.1-5 alkoxy or NHR.sup.10; R.sup.10 is hydrogen,
C.sub.1-5 alkyl or C.sub.1-5 alkoxy; and pharmaceutically acceptable
salts or esters thereof.
A preferred compound of formula (I) is 3'S,4'S,5'R-3'-hexacosanoyl-4,5'-di-O-acetylnonadecyl-2,3,4,6-tetra-O-ace-
tyl-.alpha.-C-D-galactopyranoside (wherein X is O, R.sup.3 is OH,
R.sup.4 and R.sup.5 are hydrogen, R.sup.1 is --(CH.sub.2).sub.13CH.sub.3,
Q.sup.1-X'-Q.sup.2-X''-Q.sup.3 is --(CH.sub.2).sub.24--CH.sub.3,
which is also known as CRONY-101.
The invention is also directed to prodrugs and pharmaceutically
acceptable salts of the compounds described, and to pharmaceutical
compositions suitable for different routes of drug administration
comprising a therapeutically effective amount of a described compound
of the invention admixed with a pharmaceutically acceptable carrier.
The invention is also directed to methods of treating a disease
selected from the group consisting of cancers, autoimmune diseases
and infectious diseases (including HIV and Hepatitis C virus).
The invention is also directed to pharmaceutical compositions comprising
the compounds disclosed above, as well as methods of using these
compositions to treat cancer, infectious diseases and autoimmune
diseases.
The invention is also directed to methods of inducing the production
of Th1 type cytokines, such as IFN-.gamma. and IL-12, in a mammal
in need thereof, by administering to the mammal a therapeutically
effective amount of a compound of claim 1. In preferred embodiments,
the mammal is a human.
The invention is also directed to novel intermediate compounds
of formula (II)
##STR00009## wherein X is O or NH; R.sup.3 is OH or a monosaccharide
and R.sup.4 is hydrogen, or R.sup.3 is hydrogen and R.sup.4 is OH
or a monosaccharide; R.sup.5 is hydrogen or a monosaccharide; and
R.sup.1 is selected from the group consisting of --(CH.sub.2).sub.11CH.sub.3,
--(CH.sub.2).sub.12CH.sub.3, --(CH.sub.2).sub.13CH.sub.3, --(CH.sub.2).sub.9CH(CH.sub.3).sub.2,
--(CH.sub.2).sub.10CH(CH.sub.3).sub.2, --(CH.sub.2).sub.11CH(CH.sub.3).sub.2
and (CH.sub.2).sub.11CH(CH.sub.3)--C.sub.2H.sub.5; and salts or
esters thereof.
A preferred embodiment of formula (II) is the novel intermediate
compound 3'S,4'S,5'R,3'-amino-4,5'-di-O-acetylnonadecyl-2,3,4,6-tetra-O-acetyl-.al-
pha.-C-D-galactopyranoside, which is used as a scaffold for the
introduction of acyl chains C(.dbd.O)-Q'-X'-Q.sup.2-X''-Q.sup.3
in the synthesis of compounds of formula (I).
The invention is also directed to a method of synthesizing a C-glycolipid
compound of formula (I) by acylating a compound of formula (II)
at the amino nitrogen.
The invention is also directed to novel intermediate compounds
of formula
##STR00010## wherein R.sup.1 is selected from the group consisting
of --(CH.sub.2).sub.11CH.sub.3, --(CH.sub.2).sub.12CH.sub.3, --(CH.sub.2).sub.13CH.sub.3,
--(CH.sub.2).sub.9CH(CH.sub.3).sub.2, --(CH.sub.2).sub.10CH(CH.sub.3).sub.2,
--(CH.sub.2).sub.11CH(CH.sub.3).sub.2 and (CH.sub.2).sub.11CH(CH.sub.3)--C.sub.2H.sub.5;
R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are each independently selected
from the group consisting of alkyl, aryl, alkylaryl and trialkylsilyl;
R.sup.6 is selected from the group consisting of alkyl, aryl, alkylaryl,
trialkylsilyl, --(C.dbd.O)--O-alkyl, --(C.dbd.O)--O-aryl and --(C.dbd.O)--O-alkylaryl;
or a salt or ester thereof.
Preferred compounds are those wherein each of R.sup.2, R.sup.3,
R.sup.4 and R.sup.5 are alkylaryl, for example benzyl or 4,6-benzylidene.
Also preferred are compounds wherein R.sup.6 is benzyloxycarbonyl.
A preferred embodiment of formula (III) is the novel intermediate
compound 1-(2',3',4',6'-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-3-benzyloxycarb-
onylamino-1-nonadecene-4,5-diol.
The invention is also directed to a method of synthesizing a C-glycolipid
compound of formula (I) wherein X is O, R.sup.3 is OH and R.sup.4
is hydrogen, and R.sup.5 is hydrogen, from a compound of formula
(III), which is subjected to reduction of the carbon-carbon double
bond and deprotection of the amine and the sugar hydroxyl groups
to form a compound of formula (II), which may be used as a scaffold
for the introduction of acyl chains C(.dbd.O)-Q'-X'-Q.sup.2-X''-Q.sup.3
at the amino nitrogen.
The invention is also directed to a method of synthesizing a C-glycolipid
compound of formula (I) wherein X is O, R.sup.3 is OH, R.sup.4 is
hydrogen, and R.sup.5 is hydrogen, by reacting a compound of formula
(IV)
##STR00011## wherein each of R.sup.10, R.sup.11, R.sup.12 and R.sup.13
is independently selected from alkyl, aryl, alkylaryl or trialkylsilyl;
with a compound of formula V
##STR00012## wherein R.sup.14 is selected from alkyl, aryl, alkylaryl,
trialkylsilyl, --C(.dbd.O)--O-alkyl, --C(.dbd.O)--O-aryl or --C(.dbd.O)--O-alkylaryl;
and wherein R.sup.1 is selected from the group consisting of --(CH.sub.2).sub.11CH.sub.3,
--(CH.sub.2).sub.12CH.sub.3, --(CH.sub.2).sub.13CH.sub.3, --(CH.sub.2).sub.9CH(CH.sub.3).sub.2,
--(CH.sub.2).sub.10CH(CH.sub.3).sub.2, --(CH.sub.2).sub.11CH(CH.sub.3).sub.2
and (CH.sub.2).sub.11CH(CH.sub.3)--C.sub.2H.sub.5; to form a compound
of formula (VI)
##STR00013## and subjecting the compound of formula (VI) to
(a) deisopropylidenation to remove the ring structure;
(b) reduction of the C--C double bond; and
(c) deprotection of the sugar hydroxyl groups, thereby forming
the compound of formula (II), which may be used as a scaffold for
the introduction of acyl chains C(.dbd.O)-Q'-X'-Q.sup.2-X''-Q.sup.3
in the synthesis of compounds of formula (I).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1(A) is a bar graph showing malaria liver stage development
in wild type BALB/c mice treated intraperitoneally with 2 .mu.g
of .alpha.-C-GalCer, .alpha.-GalCer, or nothing two days before
challenge with live P. yoelii sporozoites;
FIG. 1(B) is a bar graph showing malaria liver stage development
in CD1d-deficient mice treated intraperitoneally with 2 .mu.g of
.alpha.-C-GalCer or nothing two days before challenge with sporozoites;
FIG. 1(C) is a bar graph showing malaria liver stage development
in J.alpha.18-deficient mice treated intraperitoneally with 2 .mu.g
of .alpha.-C-GalCer or nothing two days before challenge with sporozoites;
FIGS. 2(A) and (B) are bar graphs showing malaria liver stage development
in IFN-.gamma. (FIG. 2(A)) and IFN-.gamma. receptor (FIG. 2(B))
deficient mice treated intraperitoneally with 2 .mu.g of .alpha.-C-GalCer
or nothing two days before challenge with sporozoites;
FIG. 3(A) is a bar graph showing malaria liver stage development
in wild type BALBc mice treated intraperitoneally with different
doses of () .alpha.-C-GalCer or () .alpha.-GalCer three days before
challenge with live P. yoelii sporozoites;
FIG. 3(B) is a bar graph showing malaria liver stage development
in wild type BALBc mice treated intraperitoneally with 100 ng of
() .alpha.-C-GalCer or () .alpha.-GalCer at various times before
challenge with sporozoites;
FIG. 3(C) is a graph showing incidence of infection in wild type
BALBc mice treated intraperitoneally with 100 ng of (.diamond.)
.alpha.-C-GalCer, () .alpha.-GalCer or () nothing 3 days before
challenge with live P. yoelli sporozoites. Mice where then monitored
daily for the presence of blood stage parasites;
FIG. 4 depicts the lungs of C57BL/6 mouse treated intravenously
with different doses of .alpha.-C-GalCer or .alpha.-GalCer two days
before intravenous challenge with 5.times.10.sup.4 syngeneic B16
melanoma cells. The results in the study are expressed as the average
number of metastatic nodules thta had developed in the lungs +/-SD
of five mice;
FIGS. 5(A), (B) and (C) are graphs showing cytokine production
in wild type BALB/c mice treated intravenously with 1 .mu.g of .alpha.-C-GalCer
(.diamond.) or .alpha.-GalCer () or with nothing (). Serum samples
were obtained at the indicated time points (hr) after injection
for ELISA analyses of IL-4 (FIG. 5(A)), IFN-.gamma. (FIG. 5(B)),
and IL-12 (FIG. 5(C)) concentrations;
FIGS. 6(A) and (B) are bar graphs showing malaria liver stage development
in wild type (FIG. 6(A)) or IL-12-deficient (FIG. 6(B)) mice treated
intraperitoneally with 100 ng of .alpha.-C-GalCer or .alpha.-GalCer
or with nothing four days before challenge with live P. yoelii sporozoites.
DETAILED DESCRIPTION OF THE INVENTION
.alpha.-Galactosylceramide (.alpha.-GalCer) is a glycolipid ligand
for natural killer T (NKT) cells, which respond to the glycolipid
and produce both interferon (IFN)-.gamma. and interleukin (IL)-4.
The production of large amounts of both cytokines, which possess
opposite biological effects, i.e. Th1- and Th2-type response, hampers
.alpha.-GalCer from executing either desired effect. It has now
been discovered that synthetic C-glycoside analogs of .alpha.-GalCer
of general formula (I) act as an NKT cell ligand and display 100-1000
fold higher activity against tumor and malaria, by preferentially
inducing the production of Th1-type cytokines, IFN-.gamma. and IL-12,
in vivo. Administration of the .alpha.-C-GalCer to mice consistently
resulted in not only prolonged production of the Th1-type cytokines,
but also decreased population of the Th2 cytokine, IL-4, as compared
to .alpha.-GalCer. In two disease models requiring Th1-type responses
for control, namely malaria and melanoma metastases, .alpha.-C-GalCer
exhibited a 1000-fold and 100-fold more potent activity, respectively,
than .alpha.-GalCer.
Definitions
The term "monosaccharide" means a sugar molecule having
a chain of 3-10 carbon atoms in the form of an aldehyde (aldose)
or ketone (ketose). Suitable monosaccharides contemplated for use
in the invention include both naturally occurring and synthetic
monosaccharides. Sample monosaccharides include trioses, such as
glycerose and dihydroxyacetone; textroses such as erythrose and
erythrulose; pentoses such as xylose, arabinose, ribose, xylulose
ribulose; methyl pentoses (6-deoxyhexoses), such as rhamnose and
fucose; hexoses, such as glucose, mannose, galactose, fructose and
sorbose; and heptoses, such as glucoheptose, galamannoheptose, sedoheptulose
and mannoheptulose. Preferred monosaccharides are hexoses.
An "effective amount" of the compound for treating a
disease, e.g., a cancer, an infectious disease or an autoimmune
disease, is an amount that results in measurable amelioration of
at least one symptom or parameter of the disease in mammals, including
humans.
The term "prodrug" as used herein refers to any compound
that may have less intrinsic activity than the active compound or
"drug" but when administered to a biological system generates
the active compound or "drug" substance either as a result
of spontaneous chemical reaction or by enzyme catalyzed or metabolic
reaction.
As used herein, the term "pharmaceutically acceptable salts,
esters, amides, and prodrugs" refer to those salts (e.g., carboxylate
salts, amino acid addition salts), esters, amides, and prodrugs
of the compounds of the present invention which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of patients without undue toxicity, irritation, allergic
response, and the like, commensurate with a reasonable benefit/risk
ratio, and effective for their intended use, as well as the zwitterionic
forms, where possible, of the compounds of the invention.
The terms "treatment" or "treating" include
prophylactic or therapeutic administration of compounds of the invention,
for the cure or amelioration of disease or symptoms associated with
disease, and includes any benefits obtained or derived from the
administration of the described compounds.
The term "therapeutically effective" applied to dose
or amount refers to that quantity of a compound or pharmaceutical
composition that is sufficient to result in a desired activity upon
administration to a mammal in need thereof.
The terms "pharmaceutically acceptable" and "physiologically
acceptable" are used interchangeably, and as used in connection
with compositions of the invention refer to molecular entities and
other ingredients of such compositions that are physiologically
tolerable and do not typically produce untoward reactions when administered
to a human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal
or a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in mammals, and more particularly
in humans.
The term "carrier" applied to pharmaceutical compositions
of the invention refers to a diluent, excipient, or vehicle with
which a compound of the invention is administered. Such pharmaceutical
carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such
as peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water or aqueous solution, saline solutions, and aqueous dextrose
and glycerol solutions are preferably used as carriers, particularly
for injectable solutions. Suitable pharmaceutical carriers are described
in "Remington's Pharmaceutical Sciences" by E. W. Martin,
18th Edition.
Therapeutic Uses
In one embodiment, the compounds of the invention are useful for
the treatment of cancer, e.g. as anti-tumor agents for inhibiting
the growth of tumors, and for treatment of cell proliferative disorders.
The compounds of the invention may be used alone, or in combination
with chemotherapy or radiotherapy.
More specifically, the compounds of the invention are useful in
the treatment of a variety of cancers including, but not limited
to carcinoma such as bladder, breast, colon, kidney, liver, lung,
including small cell lung cancer, non-small cell lung cancer, esophagus,
gall bladder, ovary, pancreas, testicular, stomach, renal, liver,
cervix, thyroid, prostate, and skin, including squamous cell carcinoma;
hematopoietic tumors of lymphoid lineage, including leukemia, acute
lymphocitic leukemia, acute lymphoblastic leukemia, B cell lymphoma,
T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy
cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid
lineage, including acute and chronic myelogenous leukemias, myelodysplastic
syndrome and promyelocytic leukemia; tumors of mesenchymal origin,
including fibrosarcoma and rhabdomyosarcoma; tumors of the central
and peripheral nervous system, including astrocytoma, neuroblastoma,
glioma and schwannomas; other tumors, including melanoma, seminoma,
teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma,
thyroid follicular cancer and Kaposi's sarcoma.
Cell proliferative disorders for which the compounds are useful
include benign prostate hyperplasia, familial adenomatosis polyposis,
neuro fibromatosis, psoriasis, vascular smooth cell proliferation
associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis
and post-surgical stenosis and restenosis.
In another embodiment, the compounds of the invention are also
useful for treating infectious diseases, including parasitic, fungal,
yeast, bacterial, mycoplasmal and viral diseases (where a particular
class of cells can be identified as harboring the infective entity).
For example, the compounds may be useful in treating infections
from a human papilloma virus, a herpes virus such as herpes simplex
or herpes zoster, a retrovirus such as human immunodeficiency virus
1 or 2, a hepatitis virus (hepatitis A virus (HAV)), hepatitis B
virus (HBV) non-A, blood borne (hepatitis C) and other enterically
transmitted hepatitis (hepatitis E), and HBV associated delta agent
(hepatitis D)), influenza virus, rhinovirus, respiratory syncytial
virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, a bacterium
of the genus Salmonella, Staphylococcus, Streptococcus, Enterococcus,
Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba,
a malarial parasite, Trypanosoma cruzi, helminth infections, such
as nematodes (round worms) (Trichuriasis, Enterobiasis, Ascariasis,
Hookworm, Strongyloidiasis, Trichinosis filariasis); trematodes
(flukes) (Schistosomiasis, Clonorchiasis), cestodes (tape worms)
(Echinococcosis, Taeniasis saginata, Cysticercosis); visceral worms,
visceral larva migrans (e.g., Toxocara), eosinophilic gastroenteritis
(e.g., Anisaki spp., Phocanema ssp.), cutaneous larva migrans (Ancylostona
braziliense, Ancylostoma caninum).
In certain preferred embodiments, the compounds of the invention
are useful for treating infection with a hepatitis C virus.
In other preferred embodiments, the compounds of the invention
are useful for treating human immunodeficiency virus (HIV), and
in the prevention of infection by HIV, the treatment of infection
by HIV and the prevention and/or treatment of the resulting acquired
immune deficiency syndrome (AIDS).
In another preferred embodiment, the compounds of the invention
are useful for treating malaria in a mammal (e.g., human) by administration
of a compound of the invention.
In other embodiments, the compounds of the invention are useful
for treating autoimmune diseases, such as rheumatoid arthritis,
psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus,
myasthenia gravis, juvenile onset diabetes, glomerulonephritis,
autoimmune thyroiditis, Behcet's disease, and other disorders such
as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis,
psoriasis, ichthyosis, Graves ophthalmopathy and asthma.
The subjects to which the present invention is applicable may be
any mammalian or vertebrate species, which include, but are not
limited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats,
cats, dogs, hamsters, mice, rats, monkeys, rabbits, chimpanzees,
and humans. In a preferred embodiment, the subject is a human.
Modes of Administration
Modes of administration of compounds and compositions of the invention
include oral and enteral, intravenous, intramuscular, subcutaneous,
transdermal, transmucosal (including rectal and buccal), and by
inhalation routes. Preferably, an oral or transdermal route is used
(i.e., via solid or liquid oral formulations, or skin patches, respectively).
In some cases, the compounds can be pulsed with syngeneic dendritic
cells, followed by transferring intravenously into patients.
Pharmaceutical Compositions
Solid dosage forms for oral administration of compounds and compositions
of the invention include capsules, tablets, pills, powders, granules,
and suppositories. In such solid dosage forms, the active compound
of the invention can be admixed with at least one inert customary
excipient (or carrier) such as sodium citrate or dicalcium phosphate;
or (a) fillers or extenders, as for example, starches, lactose,
sucrose, glucose, mannitol, and silicic acid; (b) binders, as for
example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,
sucrose, and acacia; (c) humectants, as for example, glycerol; (d)
disintegrating agents, as for example, agar-agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain complex silicates,
and sodium carbonate; (e) solution retarders, as for example paraffin;
(f) absorption accelerators, as for example, quaternary ammonium
compounds; (g) wetting agents, as for example, cetyl alcohol, and
glycerol monostearate; (h) adsorbents, as for example, kaolin and
bentonite; and (i) lubricants, as for example, talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,
or mixtures thereof. In the case of capsules, tablets, and pills,
the dosage forms may also comprise buffering agents. Such solid
compositions or solid compositions that are similar to those described
can be employed as fillers in soft- and hard-filled gelatin capsules
using excipients such as lactose or milk, sugar as well as high
molecular weight polyethyleneglycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, pills, and
granules can be prepared with coatings and shells, such as enteric
coatings or other suitable coatings or shells. Several such coatings
and/or shells are well known in the art, and can contain opacifying
agents, and can also be of such composition that they release the
active compound or compounds in a certain part of the intestinal
tract in a delayed manner. Examples of embedding compositions which
can be used are polymeric substances and waxes. The active compounds
can also be used in microencapsulated form, if appropriate, with
one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically
acceptable emulsions, solutions, suspensions, syrups, and elixirs.
In addition to the active compounds, the liquid dosage forms can
contain inert diluents commonly used in the art, such as water or
other solvents, solubilizing agents and emulsifiers, as for example,
ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,
benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,
dimethylformamide, oils, in particular, cottonseed oil, groundnut
oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol,
tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters
of sorbitan or mixtures of these substances, and the like. If desired,
the composition can also include adjuvants, such as wetting agents,
emulsifying and suspending agents, sweetening, flavoring and/or
perfuming agents.
The composition may include a carrier, as defined herein. Suitable
carriers include macromolecules which are soluble in the circulatory
system and which are physiologically acceptable, as defined herein.
The carrier preferably is relatively stable in the circulatory system
with an acceptable plasma half life for clearance. Such macromolecules
include but are not limited to Soya lecithin, oleic acid and sorbitan
trioleate, with sorbitan trioleate preferred.
Suspensions, in addition to the active compounds, can contain suspending
agents, such as, ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol and sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar, tragacanth, and the like. Mixtures
of suspending agents can be used if desired.
Compositions for rectal administrations are preferably suppositories
which can be prepared by mixing the compounds of the present invention
with suitable nonirritating excipients or carriers such as cocoa
butter, polyethyleneglycol, or a suppository wax which are solid
at ordinary temperatures but liquid at body temperature and therefore,
melt in the rectum or vaginal cavity and release the active component.
Compositions suitable for parenteral injection can comprise physiologically
acceptable sterile aqueous or nonaqueous solutions, dispersions,
suspensions or emulsions, and sterile powders for reconstitution
into sterile injectable solutions or dispersions. Examples of suitable
aqueous and nonaqueous carriers, diluents, solvents or vehicles
include water, ethanol, polyols (propyleneglycol, polyethyleneglycol,
glycerol, and the like), suitable mixtures thereof, vegetable oils
(such as olive oil) and injectable organic esters such as ethyl
oleate. Proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersions and by the use of surfactants.
Dosage forms for topical administration of a compound of the invention
include ointments, powders, sprays and inhalants. The active component
can be admixed under suitable conditions (e.g., sterile conditions)
with a physiologically acceptable carrier and any preservatives,
buffers, or propellants as may be required. Ophthalmic formulations,
eye ointments, powders, and solutions are also contemplated as being
within the scope of this invention.
Effective Dosages
An effective amount for treating the diseases can easily be determined
by empirical methods known to those skilled in the art, such as
by establishing a matrix of dosages and frequencies of administration
and comparing a group of experimental units or subjects to each
point in the matrix. The exact amount to be administered to a patient
will vary depending on the particular disease, the state and severity
of the disease, and the physical condition of the patient. A measurable
amelioration of any symptom or parameter can be determined by a
physician skilled in the art or reported by the patient to the physician.
Clinically significant attenuation or amelioration means perceptible
to the patient and/or to the physician.
It will also be understood that the specific dosage form and dose
level for any particular patient will depend on a variety of factors
including the activity of the specific compound employed; the age,
body weight, general health, and sex of the individual being treated;
the time and route of administration; the rate of excretion; other
drugs which have previously been administered; and the severity
of the particular disease undergoing therapy.
The amount of the agent to be administered can range from between
about 0.01 to about 25 mg/kg/day, preferably from between about
0.1 to about 10 mg/kg/day and most preferably from between about
0.2 to about 5 mg/kg/day. It will be understood that the pharmaceutical
compositions of the present invention need not in themselves contain
the entire amount of the agent that is effective in treating the
disorder, as such effective amounts can be reached by administration
of a plurality of doses of such pharmaceutical compositions.
For example, the compounds of the invention can be formulated in
capsules or tablets, each preferably containing 50-200 mg of the
compounds of the invention, and are most preferably administered
to a patient at a total daily dose of 50-400 mg, preferably 150-250
mg, and most preferably about 200 mg.
Toxicity and therapeutic efficacy compositions containing compounds
of the invention can be determined by standard pharmaceutical procedures
in experimental animals, e.g., by determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic
and therapeutic effects is the therapeutic index and it can be expressed
as the ratio LD50/ED50. Compositions that exhibit large therapeutic
indices are preferred. While therapeutics that exhibit toxic side
effects can be used (e.g., when treating severe forms of cancer
or life-threatening infections), care should be taken to design
a delivery system that targets such immunogenic compositions to
the specific site (e.g., lymphoid tissue mediating an immune response,
tumor or an organ supporting replication of the infectious agent)
in order to minimize potential damage to other tissues and organs
and, thereby, reduce side effects.
As specified above, data obtained from the animal studies can be
used in formulating a range of dosage for use in humans. The therapeutically
effective dosage of compounds of the present invention in humans
lies preferably within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage can vary
within this range depending upon the dosage form employed and the
route of administration utilized. Ideally, a single dose should
be used.
Synthesis of Compounds of the Invention
In a first method of synthesizing the compounds of the invention,
Synthesis A, the compounds may be formed from commercially available
starting materials galactose penta acetate (1) and L-homoserine
(2), as shown below:
##STR00014##
As taught by Kolb et al, 1994, Chem. Rev. 94: 2483, hydroxy groups
are introduced into the homosphingosine moiety. As taught in Belica,
et al, 1998, Tetrahedron Lett. 39: 8225-8228, Yang et al., 1999,
Organic Letters 1: 2149-2151, and Yang, et al, 2001, Organic Letters
3: 197-200, the homosphingosine is linked to the galactose. The
alpha configuration is established using the method of Yang, et
al., 1999, Organic Letters 1: 2149-2151. The sphingosine is converted
to the ceramide using well-established methods.
The compounds of formula (I) may be formed from the compounds of
formula (II) by acylating a compound of formula (II) with a reactant
R.sup.x--C(.dbd.O)-Q'X'Q.sup.2X''Q.sup.3 to add the C(.dbd.O)-Q'X'Q.sup.2X''Q.sup.3
chain at the amino nitrogen position of (II). The acylation of an
amino group is well known to chemists skilled in the art of organic
synthesis. Suitable reactants include p-nitrophenyl carboxylates,
wherein R.sup.x is p-nitrophenyl as taught in Morita et al. J. Med.
Chem, 1995, 38: 2176-2187. Alternative R.sup.x groups include o-nitrophenyl,
o-N-succinimidyl, chloride, bromide, or mixed anhydrides.
The compounds of formula (II) can be formed from the compounds
of formula (III) by reducing the carbon-carbon double bond, and
then deprotecting the amine and hydroxyl groups of the sugar moiety
Compounds of formula (T) wherein X is NH may be formed according
to the methods taught by Savage et al., Org. Lett. 2002 Apr. 18
4(8): 1267-70.
In a second method of synthesizing the compounds of the invention,
Synthesis B, a sugar aldehyde, e.g. .alpha.-C-galactosyl aldehyde,
is coupled with the commercially available compound phytosphingosine.
.alpha.-C galactosyl aldehyde can be formed by the Bednarski procedure
from the starting material methyl galactoside.
The coupling reaction yields a compound of formula III, which is
then subjected to cleavage of an isopropylidene group, reduction
of the double bond, and deprotection, to yield the compound of formula
(II).
EXEMPLARY EMBODIMENTS OF THE INVENTION
The compounds of this invention and their preparation can be understood
further by the examples which illustrate some of the processes by
which these compounds are prepared or used. Theses examples do not
limit the invention. Variations of the invention, now known or further
developed, are considered to fall within the scope of the present
invention as hereinafter claimed.
1. Synthesis of CRONY-101 by Synthesis A Method
The .alpha.-GalCer derivative CRONY-111 may be synthesized according
to the following synthesis.
##STR00015##
The diastereoselective dihydroxylation of the optically active
olefin A, which is readily accessible from L-homoserine, would afford
the protected homophytosphingosine derivative B in a stereoselective
fashion. The synthetic route from commercially available L-homoserine
is shown in Scheme A2.
L-homoserine 1 was converted into methyl ester 2 via two steps
in 60% overall yield (Ozinskas, A. et al., J. Org. Chem. 1986, 51,
5047-5050; Shioiri, T. et al., Org. Synth., 1989, 68, 1). After
the primary alcohol was protected, the ester was reduced to an aldehyde
3 using diisobutylaluminum hydride (DIBAL) as the reducing reagent.
The aldehyde was then coupled to C.sub.15 long-chain Wittig phosphonium
salt using sodium hexamethyldisilazane (NaHMDS) in THF (-75.degree.
C.) to give Z-olefin 4 as the only product (Beaulieu, P. L. et al.,
Org. Chem. 1991, 56, 4196-4204; Imashiro, R. et al, Tetrahedron
1998, 54, 10657-10670). Sharpless dihydroxylation (Sharpless, K.
B. et al., J. Org. Chem. 1992, 57, 2768-2771), of the optically
active Z-olefin using AD-mix-.beta. gave ca. 7:3 mixture of 3S,4S,5R(5)
and 3S,4R,5S(6)dihydroxylated isomers, respectively. Their relative
and absolute configurations were confirmed by comparison of NMR
data of their cyclic carbamate derivatives.
##STR00016##
Acetonide formation was used to protect the 1,2-diols in compound
5 (Scheme 3), then the primary alcohol 8 was released by desilylation
of 7. Because the basic fluoride ion caused the cyclization, acetic
acid was added to Bu.sub.4NF solution as the buffer (Niu, C. et
al., J. Org. Chem. 1996, 61, 1014-1022) to afford 8 as the only
product, since there was no cyclic compound formed. The iodo compound
9 can be made by one skilled in the art using PPh.sub.3, iodine
and imidazole reflux in THF (Spak, S. J. et al., Tetrahedron 2000,
56, 217-224).
Based on the general idea of synthesis of the model .alpha.-C-galactoside
(Yang, G. et al., Org. Lett.; 1999, 1, 2149-2151), the synthesis
was continued by treatment of thioacetate 10 with hydrazinium acetate
in DMF under N.sub.2 to deprotect thioacetate (Park, W. K. C. et
al., Carbohydr. Lett. 1995, 1, 179-184). The freshly deprotected
thio derivative was subsequently treated with electrophile 9 to
provide thio-galactoside 11 in 95% overall yield (Scheme A4). Treatment
of .beta.-D-thio-galactoside 11 with NaOMe in MeOH followed by protection
using p-methoxybenzaldehyde.
##STR00017##
Dimethyl acetal (Johansson, R. et al., J. Chem. Soc. PerkinTrans.
1 1984, 2371-2374) and p-toluene sulfonic acid gave 4,6-O-(4-methoxybenzylidene)-.beta.-D-1-thio-galactoside
12 in 86% yield. Benzylation of 12 followed by oxidation of thiogalactoside
using MMPP gave sulfonyl galactoside 13 in good yield. N-benzlyation
could not be avoided in this step.
##STR00018##
The RB reaction using C.sub.2F.sub.4Br.sub.2/t-BuOH at reflux afforded
the product 14 (Scheme A5). The ratio of Z:E alkene isomers was
not determined because of peak broadening in the NMR. The intermediate
1-O-Methyl-2,3-dibenzyl .beta.-galactoside can be made in one step
by using chlorotrimethylsilane in methanol. Esterification of the
primary hydroxyl group at C6 afforded the benzoate 15 in 88% yield
(Scheme A5). Treatment of the acetonide 15 with 1N HCl/Et.sub.2O
in methanol generated the corresponding diol 16. Cyclic carbonation
of the diol using triphosgene (Burk, R. M. et al., Tetrahedron Lett.
1993, 34, 395-398) followed by silylation of the axial hydroxyl
group at C4 afforded the silyl ether 17. Pump addition (McCombie,
S. W. et al., Tetrahedron Lett. 1991, 32, 2083-2086) of 17 in CH.sub.2Cl.sub.2
(0.01M) to BF.sub.3. Et.sub.2O in CH.sub.2Cl.sub.2 solution (4:1,
0.05M) afford .alpha.-C-galactoside 18 and cyclized compound 19
(20%). Treatment of silyl ether 18 with 1N Bu.sub.4NF in THF afforded
product 20, which is identified by .sup.1H NMR (anomeric H: 3.95
ppm, J.sub.12=4.6 Hz) and TLC.
##STR00019##
The carbonyl groups were removed prior to debenzylation. Compound
20 was treated with NaOH and refluxed in 1:1 dioxane and H.sub.2O
to afford the oxazolidinone 21 (Scheme A6). Hydrolysis of 21 gave
the N-benzylamine 22, which was fully debenzylated by transfer hydrogenolysis
(10% Pd/C, cyclohexene) (Roush, W. R. et al., J. Org. Chem. 1985,
50, 3752-3757) to afford crude 23 in 80% overall yield. The fatty
##STR00020## amide chain was then introduced using p-nitrophenyl
hexadeconate as the acylating agent to afford the target 24 (Morita,
M. et al., J. Med. Chem. 1995, 38, 2176-2187). Final purification
was done by flash chromatography on silica gel eluting with CHCl.sub.3:MeOH
(4:1). The .sup.1H and .sup.13C NMR and optical rotation {[.alpha.].sup.25.sub.D
+40.8.degree. (c=1.3, pyridine)}, mp 175-178.degree. C., high resolution
FABMS m/z 856.7601 (C.sub.51H.sub.101O.sub.8N+H.sup.+ requires 856.7605)
obtained for a sample of 24. The mass spectrum and .sup.1H NMR of
fully acylated compound 25 further confirmed that 24 was the right
compound, namely, CRONY-101.
L-2-[(benzyloxycarbonyl)amino]-4-hydroxybutyric acid (1)
To a solution of L-homoserine 1 (4.0 g, 33.6 mmol) in 160 ml of
1N NaHCO.sub.3 was added 6.0 ml (37 mmol) of benzyl chloroformate.
The reaction mixture was stirred at 23.degree. C. for 24 h and then
extracted with ether (2.times.200 ml). The aqueous phase was ice
cooled, carefully acidified to pH 2-3 with 3N HCl, and extracted
with ethyl acetate (4.times.100 ml). The extract was dried over
Na.sub.2SO.sub.4, filtered, and evaporated to afford 6.52 g (77%)
product as a white solid. .sup.1H NMR (Me.sub.2CO-d.sub.6, 300 MHz)
.delta. 7.39-7.31(m, 1H, C.sub.6H.sub.5), 6.63 (d, J=7.7 Hz, 1H,
NH), 5.08(s, 2H, CH.sub.2Ph), 4.42(m, 1H, CH), 3.70(m, 2H, CH.sub.2O),
2.05(m, 1H), 1.91(m, 1H).
Methyl-L-2-[(benzyloxycarbonyl)amino]-4-hydroxybutyrate (2)
To a solution of above compound (5.7 g, 22.5 mmol) in 50 ml MeOH
was added dropwise 2M trimethylsilyldiazomethane in hexanes (22.5
ml, 25 mmol) at 0.degree. C. The reaction mixture was stirred at
rt overnight. Basic dowex resin was added, filtered and rinsed by
methanol. After evaporation of the methanol in room temperature,
the residue was purified by flash chromatography on florisil eluting
with 50% PE/EtOAc to afford 4.6 g (77%) 2 as a colorless oil. .sup.1H
NMR (CDCl.sub.3, 300 MHz): .delta. 7.35 (s, 5H, C.sub.6H.sub.5),
5.69(d, J=6.6 Hz, 1H, NH), 5.12(s, 2H, CH.sub.2Ph), 4.55(m, 1H),
3.76(s, 3H, OMe), 3.70(m, 2H), 2.81(br, 1H, OH), 2.15(m, 1H), 1.71(m,
1H). .sup.13C NMR(CDCl.sub.3, 75 MHz): .delta. 174.09, 153.72.136.21,
128.21, 128.61, 128.18, 67.42, 58.60, 52.77, 51.69, 35.33.
Methyl-L-2-[(benzyloxycarbonyl)amino]-4-O-(tert-butyldimethylsilyl)-butyra-
te
To a solution of 2 (4.19 g, 15.66 mmol) in 20 ml CH.sub.2Cl.sub.2
was added TBDMSCl (2.83 g, 18.8 mmol) followed by imidazole (2.55
g, 37.6 mmol). This reaction mixture was stirred at room temperature
for 2 h. The mixture was filtered, rinsed by CH.sub.2Cl.sub.2 and
washed with water. The solution was concentrated and purified by
column chromatography on silica gel eluting with EtOAc-PE (30%)
to afford 5.429 g (90%) product as a colorless oil. .sup.1H NMR
(CDCl.sub.3, 300 MHz): .delta. 7.34(s, 5H, C.sub.6H.sub.5), 5.93(d,
J=7.7 Hz, 1H, NH), 5.10(m, 2H, CH.sub.2Ph), 4.45(m, 1H), 3.73(s,
3H, OMe), 3.68(m, 2H), 2.00(m, 2H), 0.87(s, 9H), 0.04(s, 6H). .sup.13C
NMR(CDCl.sub.3, 75 MHz): .delta. 172.72, 156.06, 136.35, 128.56,
128.12, 128.02, 66.99, 60.16, 52.88, 52.47, 34.13, 26.12, 18.46,
-5.26.
L-2-[(benzyloxycarbonyl)amino]-4-O-(tert-butyldimethylsilyl)-butylaldehyde
(3)
To a solution of above compound (5.42 g, 15.66 mmol) in 20 ml THF
at -78.degree. C. was added 1M DIBAL in heptane (43 ml, 42 mmol).
The reaction mixture was stirred at -78.degree. C. for 3 h. The
resulting emulsion was slowly poured into 100 ml of ice-cold 1N
HCl with stirring over 10 min, and the aqueous mixture was extracted
with EtOAc (3.times.100 ml), dried over Na.sub.2SO.sub.4, filtered
and concentrated in vacuo. The residue was purified by column chromatography
on silica gel eluting with EtOAc-PE (20%) to afford 4.03 g (85%)
3 as a colorless oil. .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.
9.59 (s, 1H, CHO), 7.35(m, 5H, C.sub.6H.sub.5), 5.86(br, 1H, NH),
5.12(s, 2H, CH.sub.2Ph), 4.30(m, 1H), 3.69(t, 2H), 2.14(m, 2H),
0.86(s, 9H), 0.03(s, 3H), 0.02(s, 3H). .sup.13C NMR(CDCl.sub.3,
75 MHz), .delta. 199.01, 156.18, 136.41, 128.65, 128.29, 128.16,
67.20, 59.21, 59.13, 32.16, 26.09, 18.42, -5.21, -5.30.
Preparation of Z-olefin (4)
To a suspension of pentadecylphosphonium bromide (5.52 g, 9.8 mmol;
prepared from 1-bromopentadecane and triphenylphosphine, refluxed
in toluene for 5 days, 98%) in THF (20 ml) was added dropwise NaHMDS
(0.6M in toluene, 15 ml, 9.2 mmol) at -75.degree. C. under nitrogen
atmosphere. The solution was gradually warmed to 0.degree. C. and
stirred for 1 h. To this solution, which was cooled down to -75.degree.
C. again, aldehyde 3 (2.472 g, 7 mmol) in 8 ml THF was added dropwise
over 30 min. After the reaction mixture was stirred at rt for 2
h, the reaction was quenched by addition of saturated NH.sub.4Cl
(100 ml) and extracted with ether. The organic extract was washed
with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated
in vacuo. The residue was purified by column chromatography on silica
gel eluting with EtOAc-PE (10%) to afford 3.44 g (85%) Z-olefin
4 as a colorless oil. .sup.1H NMR (CDCl.sub.3, 500 MHz): .delta.
7.34-7.31(m, 5H, C.sub.6H.sub.5), 5.47(after decoupling, d, J=10
Hz, 1H, vinyl H next to CH.sub.2), 5.42(m, 1H, NH), 5.27(t, J=9.8
Hz, 1H, vinyl H next to CH), 5.09(m, 2H, CH.sub.2Ph), 4.58(m, 1H),
3.67(m, 2H), 2.11(m, 2H), 1.73(m, 2H), 1.25(s, 22H), 0.89(s, 12H),
0.05(s, 3H), 0.04(s, 3H). .sup.13C NMR(CDCl.sub.3, 75 MHz): .delta.
155.71, 136.96, 132.39, 129.96, 128.52, 128.16, 128.02, 66.59, 60.34,
60.31, 47.24, 38.06, 32.21, 29.99, 29.85, 29.69, 29.65, 29.55, 29.50,
27.96, 26.17, 22.99, 18.43, 14.42, -5.15.
Dihydroxylation of olefin (Z)-4 using AD-mix-.beta.
To a solution of AD-mix-.beta. (6.294 g) and methanesulfonamide
(0.427 g, 4.50 mmol) in t-BuOH/H.sub.2O (1:1, 10 ml) was added Z-4
(2.45 g, 4.49 mmol) at 0.degree. C. under nitrogen atmosphere. The
mixture was stirred at rt for 48 h, quenched with Na.sub.2S.sub.2O.sub.3
(6.7 g) and extracted with EtOAc. The organic extract was washed
with 1N KOH, H.sub.2O, brine and dried over Na.sub.2SO.sub.4. After
evaporation of the solvent under the reduced pressure, the diols
were purified by column chromatography (EtOAc/PE=30%) to give 6
(3,4 syn form, 0.5 g, 19% yield) and 5 (3,4-anti form, 1.7 g, 65%
yield) as a white solid.
(3S,4R,5S)-1-O-(tert-butyldimethylsilyl)-3-[(benzyloxycarbonyl)amino-4,5-n-
onadecanediol (6)
mp 39-40.degree. C. [.alpha.].sup.25 3.0.degree. (c 9, CHCl.sub.3).
.sup.1H NMR (CDCl.sub.3, 300 MHz): .delta. 6.36(s, 5H, C.sub.6H.sub.5),
5.29(d, J=8.8 Hz, 1H, NH), 5.01(s, 2H, CH.sub.2Ph), 4.16(m, 1H),
3.73(t, J=5.6 Hz, 2H), 3.59(br, 1H), 3.34(m, 2H), 3.04(d, J=4.0
Hz, 1H), 1.86(m, 2H), 1.73(m, 1H), 1.55(m, 1H), 1.26(s, 24H), 0.89(s,
12H), 0.06(s, 6H). .sup.13C NMR(CDCl.sub.3, 75 MHz): .delta. 157.64,
136.38, 128.68, 128.35, 128.22, 76.43, 71.53, 67.38, 60.08, 50.22,
49.86, 35.46, 33.52, 32.23, 30.10, 30.00, 29.96, 29.66, 26.11, 23.00,
18.43, 14.43, -5.20, -5.23.
(3S,4S,5R)-1-O-(tert-butyldimethylsilyl)-3-[(benzyloxycarbonyl)amino-4,5-n-
onadecanediol (5)
mp 40-43.degree. C. [.alpha.].sup.25 16.3.degree. (c 9, CHCl.sub.3).
.sup.1H NMR (CDCl.sub.3, 300 MHz): .delta. 7.38(s, 5H, C.sub.6H.sub.5),
5.61(d, J=8.0 Hz, 1H, NH), 5.08(s, 2H, CH.sub.2Ph), 4.09(m, 1H),
3.73(m, 3H), 3.57(m. 1H), 3.49(m, 1H), 2.11(br, 1H), 1.95-1.76(m,
12H), 1.26(s, 26H), 0.89(s, 12H), 0.07(s, 6H). .sup.13C NMR(CDCl.sub.3,
75 MHz): .delta. 156.41, 136.57, 128.58, 128.17, 128.12, 76.26,
73.22, 66.95, 59.98, 51.36, 33.77, 32.19, 32.05, 29.63, 26.12, 26.04,
22.96, 18.44, -5.20, -5.27.
(3S,4S,5R)-1-O-(tert-butyldimethylsilyl)-3-[(benzyloxycarbonyl)amino-4,5-O-
-isopropylidene-nonadecane (7)
To a solution of diol 5 (2.23 g, 3.85 mmol) in 30 ml CH.sub.2Cl.sub.2
was added 2,2-dimethoxy propane (2.37 ml, 19.3 mmol) followed by
PPTs (97 mg, 0.38 mmol). After the reaction mixture was stirred
at rt for 1.5 h, 50 ml saturated NaHCO.sub.3 was added and extracted
with CH.sub.2Cl.sub.2 (30 ml.times.2). The organic phase was dried
over Na.sub.2SO.sub.4. After evaporation of the solvent under the
reduced pressure, the residue was purified by column chromatography
(EtOAc/PE=10%) to give product 7 (2.287 g, 96% yield) as an oil.
.sup.1H NMR (CDCl.sub.3, 300 MHz): .delta. 7.34(s, 5H, C.sub.6H.sub.5),
5.17(d, J=8.8 Hz, 1H, NH), 5.07(s, 2H, CH.sub.2Ph), 4.13(m, 2H),
3.90(m, 1H), 3.78-3.70(m, 2H), 1.89(m, 2H), 1.56(m, 2H), 1.43(s,
3H), 1.33(s, 3H), 1.25(s, 24H), 0.88(s, 12H), 0.04(s, 3H), 0.03(s,
3H). .sup.13C NMR(CDCl.sub.3, 75 MHz): .delta. 155.86, 136.77, 128.47,
128.02, 107.78, 79.39, 77.96, 66.68, 60.25, 49.19, 34.40, 32.16,
29.92, 29.59, 29.12, 27.44, 27.01, 26.13, 25.61, 22.92, 18.39, 14.35,
-5.25, -5.28.
(3S,4S,5R)-3-[(benzyloxycarbonyl)amino-4,5-O-isopropylidene-nonadecanol
(8)
To a solution of above compound (3.31 g, 5.33 mmol) in 25 ml THF
was added 1M Bu.sub.4NF in THF (12 ml) followed by 0.5 ml acetic
acid. After the reaction mixture was stirred at rt overnight, 20
ml saturated NaHCO.sub.3 was added and extracted with CH.sub.2Cl.sub.2
(50 ml.times.2). The organic phase was dried over Na.sub.2SO.sub.4.
After evaporation of the solvent under the reduced pressure, the
residue was purified by column chromatography (EtOAc/PE=50%) to
give 8 (2.56 g, 90% yield) as a white solid. Mp 58-60.degree. C.
[.alpha.].sup.25 -3.67.degree. (c 3, CHCl.sub.3). .sup.1H NMR (CDCl.sub.3,
300 MHz): .delta. 7.36(s, 5H, C.sub.6H.sub.5), 5.11(s, 2H, CH.sub.2Ph),
4.86(br, 1H, NH), 4.12(m, 1H), 4.03-3.92(m, 2H), 3.72(m, 2H), 2.82(br,
1H, OH), 2.02(m, 2H), 1.52(m, 24H), 1.44(s, 3H), 1.33(s, 3H), 0.88(t,
J=6.6 Hz, 3H). .sup.13C NMR(CDCl.sub.3, 75 MHz): .delta. 156.91,
136.38, 128.72, 128.41, 128.22, 108.28, 79.78, 78.03, 77.44, 77.39,
77.02, 67.38, 59.06, 48.70, 35.36, 32.21, 30.16, 29.99, 29.90, 29.85,
29.65, 29.25, 27.04, 25.74, 22.98, 14.42.
(3S,4S,5R)-1-iodo-3-[(benzyloxycarbonyl)amino-4,5-O-isopropylidene-nonadec-
ane (9)
A mixture of 8 (2.5 g, 4.95 mmol), PPh.sub.3 (1.63 g, 6.1 mmol),
imidazole (0.87 g, 11.8 mmol) and iodine (2.03 g, 7.4 mmol) in THF
(50 ml) was stirred under reflux for 2.5 h. After evaporation of
the solvent, the crude product was dissolved in CH.sub.2Cl.sub.2
(100 ml) and solids were removed by filtration. An equal volume
of saturated aqueous NaHCO.sub.3 was added and the mixture was stirred
for 10 min. Iodine was added in portions and when the organic phase
remained iodine-colored, the mixture was stirred for an additional
10 min. Excess iodine was destroyed by the addition of saturated
aqueous Na.sub.2S.sub.2O.sub.3 solution. The organic layer was diluted
with CH.sub.2Cl.sub.2 (50 ml), separated, washed with water (50
ml), dried over Na.sub.2SO.sub.4. After evaporation of the solvent
under the reduced pressure, the residue was purified by column chromatography
(EtOAc/PE=20%) to give 9 (2.57 g, 87% yield) as a white solid. Mp
79-81.degree. C. .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta. 7.33(s,
5H, C.sub.6H.sub.5), 5.07(m, 34.05(m, 3H), 3.78(m, 1H), 3.23(m,
2H), 2.26(m, 2H), 1.89(m, 2H), 1.42(s, 3H), 1.30(s, 3H), 1.55-1.26(m,
24H), 0.88(t, J=6.6 Hz, 3H). .sup.13C NMR(CDCl.sub.3, 75 MHz): .delta.
155.88, 136.35, 128.59, 128.26, 128.21, 108.11, 79.57, 77.75, 67.11,
52.56, 36.72, 32.14, 30.35, 30.26, 29.91, 29.81, 29.76, 29.58, 29.15,
27.36, 26.99, 25.51, 22.91, 14.37.
(3'S,4'S,5'R)3'-[(benzyloxycarbonyl)amino-4',5'-O-isopropylidene-nonadecan-
ylthio]2,3,4,6-tetra-O-acetyl-.beta.-D-galactopyranose (11)
To a degassed solution of 2.02 g (4.98 mmol) .beta.-2,3,4,6-tetra-O-acetyl-galactosyl
thioacetate 10 in 15 ml DMF, NH.sub.2NH.sub.2.HOAc (0.47 g, 5.96
mmol) was added. This solution was degassed at room temperature
for 1 h. Iodide 9 (2.55 g, 4.14 mmol) was added, followed by triethyl
amine (0.64 ml, 6.58 mmol). After 2 h, 100 ml ethyl acetate and
50 ml water were added. The organic layer was washed with water
and brine, and dried over anhydrous sodium sulfate. After evaporation
of the organic solvent, the residue was purified by chromatography
on silica gel eluting with 50% EtOAc/PE to afford 3.2 g .beta.-thiogalactoside
11 (90% yield) as a sticky oil. .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 7.32 (s, 5H), 5.42 (d, J=3.0 Hz, 1H, H-4), 5.24(t, J=9.9
Hz, 1H, H-2), 5.10(m, 2H), 5.03(dd, J=3.3, 9.9 Hz, 1H, H-3), 4.83(d,
J=9.5 Hz, 1H, NH), 4.46(d, J=9.9 Hz, 1H, H-1), 4.13(m, 3H), 4.04(t,
J=5.8 Hz, 1H, H-5), 3.94(t, 1H), 3.79(m, 1H), 2.85-2.72(m, 2H, H--SCH.sub.2),
2.12(s, 3H, H--OAc), 2.05(s, 3H, H--OAc), 2.04(s, 3H, H--OAc), 1.98(s,
3H, H--OAc), 1.76(m, 1H), 1.54(m, 1H), 1.43(s, 3H), 1.32(s, 3H),
1.26(s, 24H), 0.88(t, J=6.6 Hz, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3):
.delta. 170.27, 170.21, 170.04 169.56, 155.77, 136.49, 128.60, 128.24,
128.12, 108.09, 84.30, 77.77, 77.45, 77.05, 76.96, 74.62, 72.05,
67.38, 67.32, 66.99, 61.43, 50.97, 32.44, 32.14, 30.26, 30.18, 29.92,
29.82, 29.28, 27.55, 26.95, 26.78, 26.75, 25.64, 22.91, 21.02, 20.87,
20.84, 14.35.
(3'S,4'S,5'R)3'-[(benzyloxycarbonyl)amino-4',5'-O-isopropylidene-nonadecan-
ylthio]4,6-O-benzylidene-.beta.-D-galactopyranose (12)
Into the solution of 2.31 g (2.71 mmol) of 2,3,4,6-tetra-O-acetyl-.beta.-thio-galactoside
11 and 50 ml methanol was added NaOMe (70 mg, 1.3 mmol). The mixture
was stirred at rt until a white precipate was formed. The precipate
was dissolved in EtOAc, then acidic resin was added until the pH
of the solution was neutral. The resin was filtered off and rinsed
by EtOAc. The solution was concentrated until completely dry to
afford 1.76 g of a white solid. To a mixture of above solid (1.75
g, 2.57 mmol), p-methoxybenzaldehyde dimethyl acetal (1.1 ml, 6.42
mmol), and 50 ml dry CH.sub.2Cl.sub.2 and 3 ml DMF was added p-toluene
sulfonic acid monohydrate (29 mg) at room temperature. After 2 h,
the mixture was neutralized with triethyl amine (1 ml) and concentrated.
The residue was chromatographed (SiO.sub.2, EtOAc/MeOH, 100% to
95%) to give 12 (1.72 g, 86% overall yield) as a white solid. .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta. 7.48(d, J=8.8 Hz, 2H), 7.29(m,
5H), 6.82(d, J=8.8 Hz, 2H), 5.43(s, 1H), 5.06(m, 3H), 4.17(d, 2H),
4.16(s, 1H), 4.05(m, 1H), 3.90(m, 3H), 3.75(s, 3H), 3.61(m, 2H),
3.39(s, 1H), 2.89(m, 1H), 2.68(m, 1H), 2.05(m, 2H), 1.80(m, 2H),
1.60-1.20(m, 30H), 0.88(t, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3):
.delta. 160.14, 156.19, 136.45, 130.37, 128.56, 128.16, 128.08,
127.75, 113.61, 108.03, 101.17, 85.69, 79.64, 77.76, 75.74, 73.88,
70.19, 69.33, 68.98, 67.00, 55.38, 50.59, 32.62, 32.09, 29.88, 29.79,
29.73, 29.53, 29.11, 27.50, 26.95, 25.63, 25.33, 22.87, 14.33.
(3'S,4'S,5'R)3'-[(benzyloxycarbonyl)benzylamino-4',5'-O-isopropylidene-non-
adecanylthio]4,6-O-benzylidene-2,3-di-O-benzyl-.beta.-D-galactopyranose
.beta.-S-galactoside 12 (1.49 g, 1.86 mmol) was dissolved in 20
ml THF and 5 ml DMF, NaH (0.6 g, 60% in mineral oil) was added,
the mixture was stirred at rt for 1/2 h, then 0.068 g (0.186 mmol)
tetra-butylammonium iodide was added followed by 0.89 ml benzyl
bromide (7.44 mmol). After the mixture was stirred at room temperature
overnight, the reaction was quenched with 10 ml of MeOH. The resulting
solution was added to 50 ml H.sub.2O and extracted by EtOAc (100
ml.times.3). The organic phase was washed by brine, and dried over
Na.sub.2SO.sub.4 and concentrated. The residue was chromatographed
on a column of silica gel (eluted with 30% EtOAc-petroleum ether)
to afford 1.62 g product (83%) as a colorless oil. MS: m/z 1094(M.sup.++Na.sup.+),
(calcd. C.sub.65H.sub.85O.sub.10SN, 1071). .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 7.48(d, J=8.8 Hz, 2H), 7.43-7.22(m, 20H), 6.88(d,
J=8.8 Hz, 2H), 5.46(s, 1H), 5.17(m, 2H), 4.78(m, 6H), 4.34(m, 4H),
4.16(d, J=3.3 Hz, 1H), 4.14(m, 1H), 3.95(m, 1H), 3.79(s, 3H), 3.59(dd,
J=3.3, 9.1 Hz, 1H), 3.50(m, 1H), 3.29(m, 1H), 2.70(m, 2H), 2.06(m,
2H), 1.47-1.13(m, 32H), 0.92(t, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3):
.delta. 160.06, 158.66, 139.19, 138.55, 138.41, 136.43, 130.64,
128.41, 128.33, 128.09, 127.84, 127.78, 127.67, 127.50, 127.34,
127.31, 127.21, 127.08, 113.57, 107.69, 101.33, 81.23, 79.64, 79.59,
79.42, 77.88, 75.79, 73.99, 71.77, 69.99, 69.40, 55.41, 32.14, 30.99,
30.08, 29.92, 29.85, 29.63, 29.57, 27.74, 25.54, 22.91, 14.35.
(3'S,4'S,5'R)3'-[(benzyloxycarbonyl)benzylamino-4',5'-O-isopropylidene-non-
adecanylsulfonyl]4,6-O-benzylidene-2,3-di-O-benzyl-.beta.-D-galactopyranos-
e (13)
A solution of MMPA (2.1 g, 4.26 mmol) in H.sub.2O (10 ml) was added
to a solution of thio-galactoside (1.52 g, 1.42 mmol) in EtOH (10
ml) and THF (10 ml), the mixture was kept at 60.degree. C. for 3
h. The mixture was concentrated in vacuo to dryness. The residue
was treated with 50 ml saturated NaHCO.sub.3 solution, and extracted
with EtOAc (50 ml.times.3), dried over Na.sub.2SO.sub.4 and evaporated
to dryness. The residue was purified by chromatography on silica
gel eluting with 40% EtOAc/PE to afford pure sulfone 13 (1.45 g,
93%) as a white solid. mp. 40-43.degree. C. MS: m/z 1121(M.sup.++NH.sub.4.sup.+),
(calcd. C.sub.65H.sub.85O.sub.12SN, 1103). .sup.1H NMR (CDCl.sub.3,
400 MHz heated at 55.degree. C.): .delta. 7.46-7.18(m, 22H), 6.88(d,
J=8.8 Hz, 2H), 5.39(s, 1H), 5.13(s, 2H), 4.95(d, 1H), 4.84(d, 1H),
4.73(s, 2H), 4.65(m, 1H), 4.42(t, J=9.6 Hz, 1H), 4.30(m, 2H), 4.24(s,
1H), 4.22(d, 2H), 4.11(d, 1H), 4.07(m, 1H), 3.91(dd, 1H), 3.79(s,
3H), 3.66(dd, 1H), 3.55(b, 1H), 3.32(s, 1H), 3.28(b, 1H), 3.00(b,
1H), 2.35(m, 1H), 2.20(b, 1H), 1.34(s, 3H), 1.25(s, 28H), 1.17(s,
3H), 0.89(t, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 160.39,
156.97, 138.64, 138.07, 136.21, 130.39, 128.81, 128.68, 128.60,
128.43, 128.06, 127.90, 127.72, 127.59, 113.86, 107.98, 101.59,
80.80, 78.93, 77.93, 77.42, 76.58, 75.78, 73.27, 73.11, 72.10, 70.71,
68.92, 55.56, 32.21, 30.00, 29.96, 29.92, 29.65, 28.03, 26.36, 25.67,
22.98, 14.41.
(3'S,4'S,5'R)3'-[(benzyloxycarbonyl)benzylamino-4',5'-O-isopropylidene]4,6-
-O-benzylidene-2,3-di-O-benzyl-.beta.-D-galactopyranosylidene Nonadecane
(14)
To a solution of 1.45 g 13 (1.32 mmol) in 10 ml t-BuOH and 10 ml
CF.sub.2BrCF.sub.2Br, 4 g 25% (by weight) KOH/Al.sub.2O.sub.3 (prepared
one day earlier) was added. This mixture was refluxed at 47.degree.
C. for 10 h. The solution was filtered through a pad of celite which
was washed by CH.sub.2Cl.sub.2. The residue was purified by column
chromatography on silica gel eluting with 25% EtOAc-PE to afford
0.6 g 14 (60% based on recovered starting material) as a colorless
oil. MS: m/z 1060(M.sup.++Na.sup.+), (calcd. C.sub.65H.sub.83O.sub.10N,
1037). .sup.1H NMR (300 MHz, CDCl.sub.3), .delta. 7.46 (d, J=8.8
Hz, 2H), 7.39-7.10(m, 20H), 6.87(d, J=8.8 Hz, 2H), 5.50(s, 1H),
5.40(t, 1H), 5.13(m, 2H), 4.97(d, 1H), 4.82-4.66(m, 5H), 4.52(m,
1H), 4.40-4.24(m, 3H), 4.09-3.99(m, 2H), 3.79(s, 3H), 3.72(m, 1H),
3.58(m, 1H), 3.48(m, 1H), 2.54(t, 2H), 1.41-1.12(m, 32H), 0.89(t,
3H).
Benzoate (15)
To a solution of 0.6 g 14 (Z+E, 0.578 mmol) in 10 ml MeOH, TMSCl
(73 .mu.l) was added at 0.degree. C. After the mixture was stirred
at 0.degree. C. for 30 min, 20 ml saturated NaHCO.sub.3 was added.
The mixture was extracted with CH.sub.2Cl.sub.2 (2.times.40 ml).
The organic phase was dried over Na.sub.2SO.sub.4, concentrated,
the residue was purified by column chromatography on silica gel
eluting with 35% EtOAc-PE to afford 0.36 g product (66%). .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta. 7.28(m, 20H), 5.08(m, 2H), 4.90(d,
1H), 4.68(s, 2H), 4.61(d, 1H), 4.56(d, 1H), 4.51(d, 1H), 4.35(d,
1H), 4.02-3.95(m, 4H), 3.84-3.81(m, 3H), 3.65(m, 1H), 3.58(m, 1H),
3.00(s, 3H), 2.59(br, 1H), 2.23(br, 1H), 1.51(m, 4H), 1.39(s, 3H),
1.33(s, 26H), 1.20(s, 3H), 0.89(t, 3H).
To a solution of above compound 0.36 g (0.378 mmol) in 10 ml CH.sub.2Cl.sub.2,
BzCl (66 .mu.l, 0.56 mmol) was added at 0.degree. C., followed by
Et.sub.3N (0.3 ml, 2.3 mmol). After the mixture was stirred at 0.degree.
C. for 2 h, 20 ml 10% ammonia solution was added. The mixture was
extracted with CH.sub.2Cl.sub.2 (2.times.40 ml). The organic phase
was dried over Na.sub.2SO.sub.4, concentrated, the residue was purified
by column chromatography on silica gel eluting with 25% EtOAc-PE
to afford 0.365 g product 15 (92%).
To a solution of 0.365 g 15 (0.347 mmol) in 10 ml MeOH, 1N HCl/Et.sub.2O
(1 ml) was added at 0.degree. C. After the mixture was stirred at
0.degree. C. for 2 h, 20 ml saturated NaHCO.sub.3 was added. The
mixture was extracted with CH.sub.2Cl.sub.2 (2.times.40 ml). The
organic phase was dried over Na.sub.2SO.sub.4, and concentrated,
the residue was purified by column chromatography on silica gel
eluting with 30% EtOAc-PE to afford 0.275 g product 16 (80%). .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta. 7.92(d, J=7.3 Hz, 2H), 7.53(t,
1H), 7.39-7.20(22H), 5.15(d, 1H), 4.94(m, 2H), 4.74-4.69(m, 3H),
4.63(br, 1H), 4.55(m, 2H), 4.43(br, 1H), 4.08-3.93(m 5H), 3.55(d,
1H), 3.42(m, 1H), 3.11(s, 3H), 2.17(br, 1H), 1.76(m, 2H), 1.47(m,
2H), 1.25(s, 26H), 0.89(t, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3):
.delta. 166.35, 157.80, 138.54, 136.34, 133.09, 130.21, 129.82,
129.71, 128.66, 128.60, 128.48, 128.43, 128.22, 128.04, 127.96,
127.79, 127.60, 103.18, 79.89, 79.05, 75.91, 75.58, 73.17, 72.60,
69.34, 68.17, 67.83, 64.57, 47.81, 33.80, 33.78, 32.18, 29.96, 29.60,
27.81, 25.89, 22.93, 14.30.
Cyclic Carbonate
To a solution of 0.27 g 16 (0.266 mmol) in 4 ml CH.sub.2Cl.sub.2
and pyridine 0.13 ml, 40 mg (0.133 mmol) triphosgene in 1 ml CH.sub.2Cl.sub.2
was dropwide added at -70.degree. C. After the addition was finished,
the reaction mixture was warmed up to room temperature. After 1.5
h, the mixture was diluted with CH.sub.2Cl.sub.2 (30 ml), quenched
with 20 ml saturated NH.sub.4Cl, then extracted with CH.sub.2Cl.sub.2
(20 ml.times.30). The organic phase was washed with 1N HCl, saturated
NaHCO.sub.3, and brine. The organic layer was dried over Na.sub.2SO.sub.4,
concentrated, the residue was purified by column chromatography
on silica gel eluting with 20% EtOAc-PE to afford 0.265 g product
(90%). .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.06(d, J=7.3
Hz, 2H), 7.58(t, 1H), 7.46(t, 2H), 7.36-7.24(m, 19H), 7.05(m, 1H),
5.16(m, 2H), 4.99(d, 1H), 4.71-4.49(m, 8H), 4.32(m, 1H), 4.09(m,
1H), 4.03(dd, 1H), 3.90(m, 1H), 3.82(m, 2H), 3.14-3.05(two singlets,
3H), 2.48(s, 1H), 1.85(m, 1H), 1.66(m, 3H), 1.46(m, 2H), 1.27(s,
24H), 0.89(t, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 166.41,
156.85, 153.62, 138.54, 138.47, 138.34, 138.32, 138.04, 136.19,
133.13, 129.82, 128.87, 128.72, 128.56, 128.35, 128.25, 128.13,
128.08, 127.97, 127.84, 127.75, 127.61, 127.57, 127.52, 101.95,
80.64, 79.96, 79.85, 77.48, 77.42, 77.11, 77.02, 76.90, 75.41, 72.53,
69.30, 68.24, 67.69, 64.35, 55.47, 48.21, 32.28, 32.20, 29.97, 29.79,
29.73, 29.61, 29.19, 28.92, 28.47, 25.66, 22.93, 14.30.
Silyl Ether (17)
To a solution of 260 mg above material (0.249 mmol) in 5 ml DMF,
i-Pr.sub.2SiHCl 0.13 ml (0.75 mmol) and 101 mg imidazole were added.
After the mixture was stirred at rt for 2 h, the solution was concentrated
and purified by column chromatography on silica gel eluting with
30% EtOAc-PE to afford 0.228 g 17 (87%) as a colorless oil. MS:
m/z 1173(M.sup.++NH.sub.4.sup.+), (calcd. C.sub.69H.sub.93O.sub.12SiN,
1155). .sup.1H NMR (300 MHz, CDCl.sub.3), .delta. 8.06(d, 7.3 Hz,
2H), 7.59(t, 1H), 7.47(t, 2H), 7.40-7.29(m, 19H), 7.04(m, 1H), 5.15(m,
2H), 5.01(d, 1H), 4.80(d, 1H), 4.65(m, 2H), 4.54-4.32(m, 7H), 3.99(m,
2H), 3.89(m, 1H), 3.80(m, 2H), 3.16-3.06(two singlets, 3H), 1.92(m,
1H), 1.69(m, 1H), 1.47(m, 2H), 1.27(s, 26H), 1.07(m, 14H), 0.89(t,
3H). .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 166.35, 156.88,
153.54, 138.68, 138.32, 133.15, 132.36, 130.36, 129.79, 128.91,
128.57, 127.47, 128.39, 128.22, 128.08, 127.90, 127.77, 127.70,
127.57, 127.51, 127.44, 127.40, 127.35, 101.95, 80.71, 80.29, 79.68,
77.43, 77.38, 77.11, 77.02, 76.98, 75.56, 73.16, 71.56, 70.55, 68.28,
64.55, 48.13, 32.21, 29.97, 29.93, 29.79, 29.71, 29.62, 29.26, 28.97,
25.57, 22.94, 17.95, 17.91, 17.84, 17.77, 14.31, 13.22, 13.16.
.alpha.-C-glycoside (20)
Syringe pump addition of a solution (92 mg, 0.079 mmol 17 in 6
ml CH.sub.2Cl.sub.2) to a solution of BF.sub.3.Et.sub.2O (50 .mu.l,
0.4 mmol) in 6 ml CH.sub.2Cl.sub.2 was carried out over a 5 h reaction
time. The mixture was then treated with 20 ml sat. NaHCO.sub.3,
and extracted with CH.sub.2Cl.sub.2 (20 ml.times.3). The organic
solvent was concentrated to afford a mixture of 18 and 19.
To the above crude products in 5 ml THF and 30 .mu.l acetic acid,
0.4 ml 1N Bu.sub.4NF was added. The reaction was stirred at rt for
1 h, the mixture was diluted with CH.sub.2Cl.sub.2, washed with
water. The organic was dried over Na.sub.2SO.sub.4, concentrated,
the residue was purified by column chromatography on silica gel
eluting with 20% EtOAc-PE to afford 61 mg product 20 (76%) and 18
mg side product 19 (20%). MS: m/z 1029(M.sup.++NH.sub.4.sup.+),
(calcd. C.sub.66H.sub.77O.sub.11N, 1011). .sup.13C NMR (75 MHz,
CDCl.sub.3): .delta. 166.54, 156.86, 153.59, 138.48, 138.30, 138.12,
136.17, 133.10, 130.36, 129.84, 128.89, 128.71, 128.61, 128.49,
128.41, 128.27, 128.23, 128.12, 127.95, 127.85, 127.68, 80.54, 79.76,
77.76, 77.50, 77.43, 76.22, 73.67, 73.03, 72.94, 70.30, 68.32, 67.53,
63.83, 55.20, 32.18, 29.96, 29.78, 29.66, 29.60, 29.23, 28.92, 25.52,
23.06, 22.93, 14.30.
Oxazolidinone (21)
Carbonate 20 (66 mg, 0.065 mmol) was dissolved in 5 ml dioxane:H.sub.2O
(1:1) and treated with NaOH 0.46 g and heated under reflux conditions
at 90.degree. C. overnight. The sample was concentrated in vacuo
and redissolved in CHCl.sub.3 and washed with saturated NH.sub.4Cl
solution. The aqueous layer was extracted with CHCl.sub.3 (20 ml.times.3).
The organic was dried over Na.sub.2SO.sub.4, concentrated, the residue
was dried in vacuo to afford 50 mg product 21 (96%). MS: m/z 774(M.sup.++H.sup.+),
(calcd. C.sub.47H.sub.67O.sub.8N, 773). .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta. 7.35-7.26(m, 15H), 4.84(d, J=15.0 Hz, 1H), 4.73(m, 2H),
4.67(d, J=10.0 Hz, 1H), 4.56(d, J=11.5 Hz, 1H), 4.21(t, J=8.5 Hz,
1H), 4.05(d, J=15.0 Hz, 1H), 3.96-3.87(m, 4H), 3.82(t, J=7.5 Hz,
1H), 3.66(d, J=10.0 Hz, 1H), 3.60(m, 1H), 3.54(dd, J=3.0, 8.5 Hz,
1H), 3.47(m, 1H), 2.53(br, 2H, OH), 2.36(br, 1H, OH), 1.98(m, 1H),
1.78(m, 1H), 1.69(m, 2H), 1.57(m, 2H), 1.42(m, 2H), 1.25(m, 22H),
0.88(t, J=6.5 Hz, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3): .delta.
157.97, 138.50, 138.06, 128.92, 128.67, 128.58, 128.16, 127.96,
127.85, 79.49, 78.14, 76.23, 74.51, 73.81, 72.76, 71.08, 68.91,
68.41, 63.28, 57.34, 46.80, 34.99, 32.17, 29.94, 29.59, 24.94, 24.45,
22.92, 22.13, 14.30.
Benzylamine (22)
The crude compound 21 (50 mg, 0.063 mmol) was dissolved in 5 ml
EtOH and 1 ml H.sub.2O and treated with KOH (0.5 g) at reflux overnight.
The cooled solution was diluted with saturated NH.sub.4Cl solution
and extracted with EtOAc (20 ml.times.3). The organic extracts were
dried over Na.sub.2SO.sub.4, filtered, and concentrated, the residue
was purified by column chromatography on silica gel eluting with
CHCl.sub.3:MeOH (4:1) to afford 39 mg product 22 (80%). MS: m/z
478(M.sup.++H.sup.+), (calcd. C.sub.46H.sub.69O.sub.7N, 477). .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta. 7.35-7.25(m, 15H), 4.76-4.70(m,
3H), 4.59(d, J=11.7 Hz, 1H), 3.97-3.85(m, 4H), 3.77(s, 2H), 3.69(dd,
J=3.6, 12.1 Hz, 1H), 3.60(m, 3H), 3.52(m, 1H), 3.30(t, J=6.6 Hz,
1H), 2.79(br, 5H), 1.88(m, 1H), 1.73(m, 2H), 1.57(m, 2H), 1.25(s,
25H), 0.89(t, J=6.9 Hz, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3):
.delta. 138.56, 138.19, 128.70, 128.58, 128.51, 128.17, 128.12,
127.95, 127.87, 127.43, 78.06, 76.48, 74.60, 74.42, 73.99, 73.84,
72.79, 71.34, 68.53, 68.50, 68.12, 67.70, 63.14, 60.97, 51.85, 34.66,
32.20, 30.18, 29.99, 29.63, 25.76, 25.69, 22.95, 21.91, 14.33.
3'S,4'S,5'R-3'-N-hexacosanoyl-4',5'-dihydroxynonadecyl-.alpha.-C-D-galacto-
pyranoside (23)
A solution of benzylamine 22 (39 mg, 0.052 mmol) in 1 ml MeOH was
treated with 10% Pd/C (40 mg), 1N HCl (52 .mu.l, 0.052 mmol), and
cylcohexene (0.2 ml). (Roush et al., J. Org. Chem. 1985 50, 3752-3757).
The resulting slurry was heated at reflux for 4 h, then cooled to
room temperature, filtered through a pad of celite and basic resin,
and concentrated to give 23 mg of crude 23. A solution of this material
in THF (1 ml) was treated with p-nitrophenyl hexacosanoate (75 mg,
0.144 mmol) (Morita et al., J. Med. Chem. 1995, 38 2176-2187) and
a crystal of DMAP. The resulting solution was stirred at rt for
48 h and concentrated. The residue was purified by column chromatography
on silica gel eluting with CHCl.sub.3:MeOH (4:1) to afford 23 mg
product 24 (60%) as a white solid. Mp: 175-178.degree. C. [.alpha.].sup.25
40.8.degree. (c 1.3, pyridine). FABMS (high-res.): m/z (calcd. C.sub.51H.sub.101O.sub.8N+H.sup.+,
856.7605. found 856.7601). .sup.1H NMR (500 MHz, C.sub.5D.sub.5N):
.delta. 8.47(d, J=8.8 Hz, 1H, NH), 6.78-6.00(br, 6H, OH), 5.14(m,
1H), 4.74(dd, J=5.5, 8.8 Hz, 1H), 4.52(m, 3H), 4.37(dd, J=4.3, 11.0
Hz, 1H), 4.25(m, 4H), 2.72(m, 1H), 2.59(m, 1H), 2.48(m, 3H), 2.33(m,
2H), 2.22(m, 1H), 1.94(m, 2H), 1.86(m, 3H), 1.71(m, 1H), 1.37(s,
64H), 0.88(t, J=6.4 Hz, 6H). .sup.13C NMR (100 MHz, C.sub.5D.sub.5N):
.delta. 173.36, 78.37, 76.90, 73.65, 72.53, 72.07, 70.46, 70.27,
62.61, 52.56, 36.86, 34.33, 32.00, 30.26, 30.07, 29.88, 29.70, 29.49,
26.42, 22.81, 14.15.
3'S,4'S,5'R-3'-N-hexacosanoyl-4',5'-di-O-acetylnonadecacyl-2,3,4,6-tetra-O-
-acetyl-.alpha.-C-D-galactopyranoside (25)
To a solution of 24 (6 mg, 5.86 .mu.mol) in 1 ml EtOAc, Ac.sub.2O
(15 .mu.l, 0.158 mmol) and DMAP (1 mg, 8.19 .mu.mol) were added.
The mixture was stirred at rt overnight. The residue was purified
by column chromatography on silica gel eluting with EtOAc:PE (40%)
to afford 5 mg product 25 (80%). MS: m/z (M.sup.++H.sup.+), 1108,
(M.sup.++Na.sup.+), 1130, (calcd. C.sub.63H.sub.113O.sub.14N, 1107).
.sup.1H NMR (500 MHz, C.sub.6D.sub.6): .delta. 5.56(m, 2H), 5.42(dd,
J=3.0, 9.0 Hz, 1H), 5.27(d, J=9.0 Hz, 2H), 5.16(d, J=10.0 Hz, 1H),
4.46(m, 2H), 4.33(m, 1H), 4.10(dd, J=5.0, 11.5 Hz, 1H), 3.74(m,
1H), 2.01(m, 3H), 1.83(s, 3H), 1.81(s, 3H), 1.78(s, 3H), 1.73(s,
3H), 1.70(s, 3H), 1.62(s, 3H), 1.45(m, 1H), 1.35-1.31(m, 74H), 0.90(m,
6H).
2. Synthesis of CRONY 101 by Method B
##STR00021##
The following convergent approach to the synthesis of the C-glycoside
analog of .alpha.-Galactosylceramide uses the one-pot Julia-Kocienski
olefination, to couple the very sensitive sugar aldehyde with a
similarly base-sensitive complete lipid sidechain. The sugar component
was the .alpha.-C-galactosyl aldehyde 38, which was prepared according
to the Bednarski procedure starting from methyl galactoside with
an overall isolated yield of 40% after five steps. The lipid side
chain was prepared from the commercially available phytosphingosine
31. The protection strategy was begun with benzyl carbamate formation,
followed by selective silylation of the primary alcohol. After routine
blocking the two secondary hydroxyl groups as an isopropylidene
ketal, the primary hydroxy was released for Mitsunobo transformation
to thioether. Oxidation of the sulfide 36 readily afforded sulfone
37 with an overall yield of 70% after six steps. The convergent
coupling of 37 and 38 was carried out by employing the Julia-Kocienski
olefination under an optimised condition to obtain 39 in 72% yield.
Finally, removal of protecting groups and simultaneous reduction
of the double bond according to standard procedures afford CRONY
101.
(2S,3R,4R)-2-amino-1,3,4-octadecanetriol (31)
(2S,3R,4R)-2-benzyloxycarbonylamino-1,3,4-octadecanetriol (32)
(Ozinskas et al., J. Org. Chem. 1986. 51: 4057-5050). To a suspension
of starting material 31 (Cosmoferm B.V., Delft, Netherlands) (6.35
g, 20 mmol) in aqueous NaHCO.sub.3 (1 N, 80 ml, 4 equiv.) and 1,4-dioxane
(30 ml) was added benzyl chloroformate (3.31 ml, 1.1 equiv.). The
reaction mixture was stirred at rt overnight, whereupon t.l.c (only
ethyl acetate or DCM-MeOH 5:1) indicated the reaction was finished.
The suspension was diluted with EtOAc and poured onto water. After
separation, the aqueous phase was extracted with EtOAc (3.times.).
The combined organic solutions were washed aqueous NH.sub.4Cl, brine,
dried over Na.sub.2SO.sub.4, and concentrated to afford a residue.
The residue was purified by flash column chromatography (Petroether-EtOAc,
2:1 to only EtOAc) to provide compound 32 (8.13 g, 90%) as a white
solid.
MS (ES, m/z): 452 (M+H).sup.+, 474 (M+Na).sup.+; .sup.1H NMR (500
MHz, CDCl.sub.3) 7.39-7.35 (m, 5 H), 5.48 (d, J=7.3 Hz, 1 H), 5.02
(s, 2 H), 3.85-3.83 (m, 2 H), 3.71-3.67 (m, 1 H), 3.60-3.56 (m,
2 H), 3.09 (d, J=5.5 Hz, 1 H), 2.91 (m, 1 H), 2.40 (d, J=5.5 Hz,
1 H), 1.62-1.18 (m, 26 H), 0.79 (t, J=6.8 Hz, 3 H); .sup.13C NMR
(75 MHz, CDCl.sub.3) 153.2, 136.3, 128.5, 128.2, 128.0, 76.4, 73.1,
67.2, 62.2, 53.6, 33.3, 32.0, 29.8, 29.5, 25.9, 22.8, 14.2.
(2S,3R,4R)-1-O-tert-butyldimethyl-2-benzyloxycarbonylamino-1,3,4-octadecan-
ctriol (33)
(Chaudhary et al., Tetrahedron Letters. 1979. 20: 99-102). To a
solution of triol 32 (1.6 g, 3.54 mmol) in anhydrous DCM (25 ml)
and DMF (5 ml) was added triethylamine (0.54 ml, 1.1 equiv.) at
0.degree. C., followed by t-BuMe.sub.2SiCl (587 mg, 1.1 equiv.)
and a catalytic amount of 4-DMAP (22 mg, 0.05 equiv.). After stirring
for 1 h at the same temperature, the mixture was diluted with DCM
and washed subsequently with water (2.times.), aqueous NH.sub.4Cl
and brine. The organic phase was dried (sodium sulfate), concentrated
and the residue purified by flash column chromatography (Petroether-EtOAc,
8:1 to 4:1) to provide diol 33 (1.92 g, 96%).
.sup.1H NMR (300 MHz, CDCl.sub.3) 7.39-7.34 (m, 5 H), 5.46 (d,
J=8.4 Hz, 1 H), 5.14 (s, 2 H), 3.99-3.91 (m, 2 H), 3.83-3.79 (m,
1 H), 3.67-3.61 (m, 2 H), 3.13 (d, J=7.7 Hz, 1 H), 2.64 (d, J=7.7
Hz, 1 H), 1.75-1.21 (m, 26 H), 0.93 (s, 9 H), 0.91 (t, J=9.9 Hz,
3 H), 0.13 (s, 3 H).
(2S,3R,4R)-1-O-tert-butyldimethyl-2-benzyloxycarbonylamino-3,4-Di-O-isopro-
pylidene-1,3,4-octadecanetriol (34)
(Kitamura et al., J. Am. Chem. Soc. 1984. 106: 3252-3257). A solution
of the diol 33 (3.33 g, 5.88 mmol) in dry DCM (60 ml) and 2,2-dimethoxypropane
(6.0 ml, 8.0 equiv.) containing a catalytic amount of PPTs (50 mg)
was stirred at room temperature for 3 h. The reaction mixture was
diluted with DCM, quenched with aqueous NaHCO.sub.3. After separation,
the aqueous phase was extracted with DCM (3.times.). The combined
organic solutions were washed with brine, dried over Na.sub.2SO.sub.4,
and concentrated to afford acetonide 34. The residue was employed
for the next step without further purification. MS (m/z): 606 (M+H).sup.+,
628 (M+Na).sup.+.
(2S,3R,4R)-2-benzyloxycarbonylamino-3,4-Di-O-isopropylidene-1,3,4-octadeca-
netriol (35)
(Corey et al., J. Am. Chem. Soc. 1984. 106: 3252-3257). To a solution
of the residue silylether 34 in dry THF (80 ml) was added tetrabutylammoniumfloride
(1.0 M in THF, 8 ml, 1.36 equiv.) under nitrogen at 0.degree. C.
After stirring for 1 h, saturated aqueous NH.sub.4Cl solution was
added whereby the reaction was quenched. After separation, the aqueous
phase was extracted with DCM (3.times.). The combined organic solutions
were washed with aqueous NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4,
concentrated, and the residue purified by flash column chromatography
(Petroether-EtOAc, 2:1) to provide alcohol 35 (2.68 g, 92% for two
steps) as a white solid.
MS (ES, m/z): 492 (M+H).sup.+, 514 (M+Na).sup.+; .sup.1H NMR (500
MHz, CDCl.sub.3) 7.36-7.31 (m, 5 H), 5.17 (d, J=8.5 Hz, 1 H), 5.12
(d, J=12.2 Hz, 1 H), 5.08 (d, J=12.2 Hz, 1 H), 4.16 (m, 1 H), 4.11
(t, J=7.0 Hz, 1 H), 3.91-3.86 (m, 1 H), 3.84 (m, 1 H), 3.71 (m,
1 H), 2.19 (t, J=5.1 Hz, 1 H), 1.60-1.21 (m, 26 H), 1.45 (s, 3 H),
1.33 (s, 3 H), 0.88 (t, J=6.9 Hz, 3 H); .sup.13C NMR (75 MHz, CDCl.sub.3)
155.8, 136.3, 128.4, 128.1, 127.9, 1, 78.0, 77.8, 67.0, 63.6, 51.9,
32.1, 29.9, 29.8, 29.7, 29.5, 29.5, 27.8, 26.8, 25.5, 22.8, 14.3.
2-[(3R,4R,5R)-3-benzyloxycarbonylamino-4,5-Di-O-isopropylidene-4,5-octadec-
anediolyl-thio]benzothiazole (36)
(Bellingham et al., Synthesis 1996, 285-296) A solution of DiPAD
(0.56 ml, 2.84 mmol, 1.1 equiv) in dry THF (1 mL) was added to a
solution of alcohol 35 (1.27 g, 2.58 mmol), Ph.sub.3P (747 mg, 2.2.84
mmol, 1.1 equiv.) and 2-mercaptanbenzothiazole (BTSH, 449 mg, 2.84
mmol, 1.1 equiv) in THF (80 ml) dropwise via syringe. After stirring
for 2 h at ambient temperature, the mixture was diluted with DCM
and poured onto sat. aq NaHCO.sub.3. The phases were separated and
the aqueous phase was extracted with DCM (3.times.). The combined
organic solutions were washed subsequently with aqueous NH.sub.4Cl,
brine, and dried over Na.sub.2SO.sub.4. The filtrate was concentrated
and the residue was purified by flash column chromatography (Petroether-EtOAc,
10:1 to 8:1) to provide thioether 36 (1.52 g, 92%) as a white solid.
MS (ES, m/z): 641 (M+H).sup.+, 663 (M+Na).sup.+; .sup.1H NMR (500
MHz, CDCl.sub.3) 7.70 (d, J=8.1 Hz, 1 H), 7.64 (d, J=7.7 Hz, 1 H),
7.29 (t, J=7.5 Hz, 1 H), 7.20 (t, J=8.1 Hz, 1 H), 7.17 (br s, 3
H), 7.04 (br s, 2 H), 5.54 (d, J=6.5 Hz, 1 H), 4.93 (d, J=12.5 Hz,
1 H), 4.87 (d, J=12.5 Hz, 1 H), 4.09 (br s, 3 H), 3.70 (d, J=13.2
Hz, 1 H), 3.50 (m, 1 H), 1.55-1.16 (m, 26 H), 1.41 (s, 3 H), 1.25
(s, 3 H), 0.79 (t, J=6.8 Hz, 3 H).
2-[(3R,4R,5R)-3-benzyloxycarbonylamino-4,5-Di-O-isopropylidene-4,5-octadec-
anediolyl-sulfonyl]benzothiazole (37)
(Bellingham et al., Synthesis 1996, 285-296). To a solution of
sulfide 36 (1.08 g, 1.69 mmol) in DCM (60 ml) was added NaHCO.sub.3
(709 mg, 5 equiv.) and MCPBA (948 mg, 2.5 equiv.). The mixture was
stired at ambient temperature overnight, whereupon t.l.c. indicated
that the oxidation was complete. The mixture was diluted with DCM
and quenched with aqueous sodium thiosulfate (20 ml, 10%). After
pouring onto sat. aq NaHCO.sub.3 and separation, the aqueous phase
was extracted with DCM (3.times.). The combined organic extracts
were dried over Na.sub.2SO.sub.4, concentrated, and the residue
purified by flash column chromatography (Petroether-EtOAc-CHCl.sub.3,
5:1:1) to provide sulfone 37 (1.08 g, 96%) as a white gel.
MS (ES, m/z): 673 (M+H).sup.+, 690 (M+NH.sub.4).sup.+; .sup.1H
NMR (500 MHz, CDCl.sub.3) 8.19 (d, J=7.9 Hz, 1 H), 7.97 (d, J=7.9
Hz, 1 H), 7.61 (t, J=7.6 Hz, 1 H), 7.57 (t, J=7.6 Hz, 1 H), 7.29
(br s, 3 H), 7.15 (br s, 2 H), 5.13 (d, J=8.8 Hz, 1 H), 4.86 (d,
J=11.8 Hz, 1 H), 4.77 (d, J=11.9 Hz, 1 H), 4.31 (m, 1 H), 4.26 (m,
1 H), 4.13 (m, 1 H), 4.04 (dd, J=15.1, 8.1 Hz, 1 H), 3.93 (d, J=13.4
Hz, 1 H), 1.55 (s, 3 H), 1.51-1.21 (m, 26 H), 1.38 (s, 3 H), 0.88
(t, J=7.0 Hz, 3 H); .sup.13C NMR (75 MHz, CDCl.sub.3) 166.3, 154.9,
152.5, 136.8, 135.8, 128.4, 128.2, 128.1, 128.0, 127.8, 127.6, 125.4,
122.3, 108.3, 77.9, 77.2, 66.9, 55.7, 47.9, 32.0, 29.8, 29.7, 29.6,
29.6, 29.5, 28.8, 27.4, 26.8, 25.2, 22.8, 14.2:
(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranoside)methanal (38)
(Kobertz et al., Tetrahedron Letters. 1992. 33: 737-740. The .alpha.-C-galactosyl
aldehyde 38 was prepared according to the Bednarski procedure starting
from methyl galactoside with an overall isolated yield of 40% after
five steps. .sup.1H NMR (500 MHz, CDCl.sub.3) 9.79 (s, 1 H), 7.38-7.17
(m, 20 H), 4.66-4.49 (m, 8 H), 4.33-4.30 (ddd, J=7.7, 4.4, 4.0 Hz,
1 H), 4.29 (d, J=4.4 Hz, 1 H), 4.12 (dd, J=6.2, 4.4 Hz, 1 H), 4.03
(dd, J=4.0, 2.6 Hz, 1 H), 3.87 (dd, J=10.6, 7.7 Hz, 1 H), 3.64 (dd,
J=10.6, 4.4 Hz, 1 H), 3.62 (dd, J=6.6, 2.6 Hz, 1 H).
Julia Coupling to give 39 1-(2',3',4',6'-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-3-benzyloxycarb-
onylamino-4,5-Di-O-isopropylidene-1-nonadecene-4,5-diol (39)
(Baudin et al., Tetrahedron Letters 1991 32: 1175-78; Blakemore
et al., Synlett 1998, 26-28). To a solution of sulfone 37 (185 mg,
0.28 mmol, 1.3 equiv.) in dry THF (5 mL) at -60 to -70.degree. C.
was added dropwise NaHMDS (1.0 M in THF, 0.56 mL, 0.56 mmol, 2 equiv.
based on sulfone) resulting in a bright yellow solution. After 45
min, the sugar aldehyde 38 (116 mg, 0.21 mmol) in THF (6 mL) was
added dropwise via syringe pump in a period of 1 h. The mixture
was stirred for 1 h at -60.degree. C., 1 h at -42.degree. C., then
gradually warmed to -10.degree. C. over 2 h. Stirring was continued
for 1 h at rt before the reaction was quenched with water (10 mL)
and diluted with Et.sub.2O (10 mL). The phases were separated and
the aqueous phase was extracted with Et.sub.2O (3.times.). The combined
organic phases were washed with water and brine, dried (Na.sub.2SO.sub.4)
and concentrated. The residue was purified by flash chromatography
(Petroether-EtOAc-CHCl.sub.3, 7:1:1) to provide olefin 39 (152 mg,
72%) as yellow thick oil.
MS (ES, m/z): 1010 (M+H).sup.+, 1027 (M+NH.sub.4).sup.+; E-9: .sup.1H
NMR (500 MHz, CDCl.sub.3) 7.24-7.17 (m, 25H), 5.87 (dm, J=16.4 Hz,
1 H), 5.83 (dm, J=16.1 Hz, 1 H), 5.12 (d, J=12.5 Hz, 1 H), 5.06
(d, J=11.7 Hz, 1 H), 4.90 (d, J=9.2 Hz, 1 H), 4.81 (d, J=11.4 Hz,
1 H), 4.68-4.60 (m, 4 H), 4.59 (br s, 1 H), 4.56 (d, J=11.4 Hz,
1 H), 4.50 (d, J=12.1 Hz, 1 H), 4.42 (d, J=12.1 Hz, 1 H), 4.38 (m,
1 H), 4.14 (m, 1 H), 4.05-4.00 (m, 3 H), 3.95 (br s, 1H), 3.64 (m,
1 H), 3.57 (m, 2 H), 1.58-1.22 (m, 26 H), 1.36 (s, 3 H), 1.29 (s,
3 H), 0.88 (t, J=6.8 Hz, 3 H); Z-9: .sup.1H NMR (500 MHz, CDCl.sub.3)
7.22-7.13 (m, 25H), 6.24 (m, 1 H), 6.09 (m, 1 H), 5.87 (dd, J=11.5,
4.6 Hz, 1 H), 5.07 (d, J=12.5 Hz, 1 H), 5.00 (d, J=12.8 Hz, 1 H),
4.90 (br s, 1 H), 4.84 (d, J=11.4 Hz, 1 H), 4.77 (d, J=12.2 Hz,
1 H), 4.70 (d, J=12.1 Hz, 1 H), 4.63 (d, J=11.7 Hz, 1 H), 4.58 (d,
J=11.7 Hz, 1 H), 4.52 (d, J=11.7 Hz, 1 H), 4.46 (br s, 2 H), 4.41
(d, J=12.1 Hz, 1 H), 4.31 (d, J=12.1 Hz, 1 H), 4.09 (m, 1 H), 3.94
(m, 1 H), 3.87 (m, 1 H), 3.80 (br s, 1H), 3.67-3.64 (m, 2 H), 3.29
(m, 1 H), 1.52-1.21 (m, 26 H), 1.40 (s, 3 H), 1.29 (s, 3 H), 0.88
(t, J=6.8 Hz, 3 H).
1-(2',3',4',6'-tetra-O-benzyl-.alpha.-D-galactopyranosyl)-3-benzyloxycarbo-
nylamino-1-nonadecene-4,5-diol (40).
DiPAD=diisopropyl azodicarboxylate MCPBA=meta chloroperbenzoic
acid PPTs=pyridinium p-toluenesulfonate DCM=dichloromethane THF=tetrahydrofuran
DMF=dimethylformamide
The following Examples illustrate the invention without limiting
its scope.
EXAMPLES
.alpha.-Galactosylceramide was synthesized by Kirin Brewery (Gumma,
Japan). The stock solution was dissolved in a 0.5% polysorbate-20
(Nikko Chemical, Tokyo), 0.9% NaCl solution at a concentration of
200 .mu.g/ml, and diluted in PBS just before injection into mice.
.alpha.-C-galactosylceramide was synthesized as described herein.
The stock solution was originally dissolved in 100% DMSO at a concentration
of 1 mg/ml. Before injection into mice, it was diluted to a concentration
of 200 .mu.g/ml in a 0.5% polysorbate-20 (Nikko Chemical, Tokyo),
0.9% NaCl solution, and diluted in PBS just before injection into
mice.
Six to eight-week-old female BALB/c mice were purchased from the
National Cancer Institute (Bethseda, Md.). Six to eight-week-old
female C57/BL6 mice were purchased from the Jackson Laboratory (Bar
Harbor, Me.). CD1d-deficient mice and J.alpha.18-deficient mice
were obtained as gifts. IFN-.gamma.-deficient mice of BALB/c background
were purchased from the Jackson laboratory (Bar Harbor, Me.). IFN-.gamma.-receptor-deficient
mice were bred and maintained in an animal facility. IL-12p40-deficient
mice of BALB/c and C57/BL6 background were purchased from the Jackson
Laboratory (Bar Harbor, Me.). All mice were maintained under pathogen-free
conditions.
P. yoelii (17NXL strain) was maintained by alternate cyclic passages
in Anopheles stephensi mosquitoes and Swiss Wesbter mice. Sporozoites
obtained from dissected salivary glands of infected mosquitoes 2
weeks after their infective blood were used for challenge of the
mice. Challenge of mice to determine the development of liver-stage
malaria infection was performed by an intravenous injection of 10,000
viable sporozoites into the tail vein. The outcome of the challenge
was determined 40-42 hours later by measuring the parasite burden
in the livers of the mice using a quantiative real-time RT-PCR method,
as taught in Bruna-Romero et al., Int. J. Parasitol. 31, 1449-1502,
2001. Challenge of mice toe determine the development of blood stage
malaria infection was performed by an intravenous injection of 75
viable sporozoites into the tail vein. Starting four days after
challenge, daily peripheral blood smears were obtained from each
mouse and examined miscroscopically for the presence of blood stage
parasites until day 17 post-challenge. Mice were considered positive
for parasitemia if at least one blood stage parasite was observed
during the time of examination.
The degree of liver stage develiopment in challenged mice was determined
by quantifying the amount of P. yoelii-specific 18S rRNA moelcules
in the livers of the mice by way of the real-time RT-PCR technique
of Bruna-Romero et al. A 2 .mu.g sample of total RNA prepared from
the livers of challenged mice was reverse-transcribed, and an aliquot
of the resulting cDNA (133 ng) was used for real-time PCR amplification
of P. yoelii 18S rRNA sequences. This amplification was performed
in a GeneAmp.RTM. 5700 Sequence Detection System (PE Applied Biosystems,
Foster City, Calif.). For this purpose, primers 5'-GGGGATTGGTTTTGACGTTTTTGCG-3'
(54 nM) and 5'-AAGCATTAAATAAAGCGAATACATCCTTAT-3' (60 nm) were used,
together with the dsDNA-specific dye SYBR Green I incorporated into
the PCR reaction buffer (PE Biosystems, Foster City, Calif.) in
order to detect the PCR product generated. The temperature profile
of the reaction was 95.degree. C. for 10 minutes followed by 35
cycles of denaturation of 95.degree. C. for 15 seconds and annealing/extension
at 60.degree. C. for 1 minute.
The development of melanoma lung metastases in C57/BL6 mice was
determined by first challenging mice intravenously with 5.times.10.sup.4
syngeneic B16 melanoma cells suspended in DMEM supplemented with
10% FCS. Two weeks after challenge the mice were sacrificed, the
lungs removed, and the number of metastatic nodules counted, as
described in Fujii et al., Natl. Immunol. 3, 867-874 (2002).
The serum concentrations of IFN-.gamma. and IL-4 were measured
2, 6, 12, 24, 48, and 72 hours after treatment with .alpha.-GalCer,
.alpha.-C-GalCer, or nothing by way of a sandwich ELISA (e-bioscience,
San Diego). The serum concentrations of IL-12p70 were also measured
at 2, 6, 12, 24, 48 and 72 hours after treatment by way of a sandwich
ELISA (Pharmingen, San Diego).
Biological Data
As reported in Gonzalez-Aseguinolaza, Proc. Nat'l Acad. Sci. USA
97, 8461-8466 (2000) .alpha.-GalCer, when administered to mice two
days before challenge with Plasmodium sporozoites, suppressed development
of malaria liver stages in a manner dependent on both CD1d-restricted
V.alpha.14+ NKT cells and IFN-.gamma./IFN-.gamma. receptor signaling.
To see if .alpha.-C-GalCer exhibited a similar behavior, wild type
mice were injected with either .alpha.-GalCer or .alpha.-C-GalCer
two days before challenge with live P. yoelii sporozoites, and the
degree of liver stage development was measured using a quantitative
real time RT-PCR assay. Mice treated with either .alpha.-GalCer
or .alpha.-C-GalCer showed virtually no liver stage development
as compared to untreated control mice, proving that .alpha.-C-GalCer
has in vivo anti-malaria activity similar to that of .alpha.-GalCer.
FIG. 1(A) demonstrates that .alpha.-C-GalCer displays anti-malaria
activity. Wild type BALB/c mice were treated intraperitoneally with
2 .mu.g of .alpha.-C-GalCer, .alpha.-GalCer or nothing two days
before challenge with live P. yoelii sporozoites, and then checked
for malaria liver stage development. The results are expressed as
the average +/-SD of 5 mice.
Thereafter, mice deficient in CD1d, J.alpha.18 (formerly know as
J.alpha.281), IFN-.gamma., or IFN-.gamma. receptor were injected
with .alpha.-C-GalCer, and liver stage development was measured.
As with .alpha.-GalCer, .alpha.-C-GalCer was unable to suppress
P. yoelii liver stages in the absence of these molecules (FIGS.
1B and C). FIGS. 1(B) and (C) demonstrate that .alpha.-C-GalCer's
anti-malaria activity requires CD1d molecules and V.alpha.14+ NKT
cells. CD1d- or J.alpha.18-deficient mice were tereated intraperitoneally
with 2 .mu.g of .alpha.-C-GalCer, .alpha.-GalCer or nothing two
days before challenge with live P. yoelii sporozoites, and then
checked for malaria liver stage development. The results are expressed
as the average +/-SD of 5 mice.
FIGS. 2(A) and (B) demonstrate that .alpha.-C-GalCer's anti-malaria
activity requires IFN-.gamma./IFN-.gamma. receptor. IFN-.gamma.-
or IFN-.gamma. rec |