U.S. patent application number 10/046373 was filed with the patent office on 2002-07-25 for novel 3, 5, and/or 6 substituted analogues of swainsonine, processes for their preparation and their use as therapeutic agents.
This patent application is currently assigned to GlycoDesign Inc.. Invention is credited to Carver, Jeremy, Dennis, James, Marino-Albernas, Jose, Shah, Rajan, Tropper, Francois Daniel, Tvaroska, Igor.
Application Number | 20020099065 10/046373 |
Document ID | / |
Family ID | 26702665 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020099065 |
Kind Code |
A1 |
Shah, Rajan ; et
al. |
July 25, 2002 |
Novel 3, 5, and/or 6 substituted analogues of swainsonine,
processes for their preparation and their use as therapeutic
agents
Abstract
The invention related to novel 3, 5, and/or 6 swainsonine
analogues, processes for their preparation and their use as
therapeutic agents. The invention also relates to pharmaceutical
compositions containing the compounds and their use as
therapeutics.
Inventors: |
Shah, Rajan; (Toronto,
CA) ; Carver, Jeremy; (Toronto, CA) ;
Marino-Albernas, Jose; (Vancouver, CA) ; Tvaroska,
Igor; (Toronto, CA) ; Tropper, Francois Daniel;
(Toronto, CA) ; Dennis, James; (Toronto,
CA) |
Correspondence
Address: |
Atten: Douglas P. Mueller
MERCHANT & GOULD P.C.
P.O. Box 2903
Minneapolis
MN
55402-0903
US
|
Assignee: |
GlycoDesign Inc.
Toronto
CA
|
Family ID: |
26702665 |
Appl. No.: |
10/046373 |
Filed: |
October 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10046373 |
Oct 29, 2001 |
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09506209 |
Feb 17, 2000 |
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09506209 |
Feb 17, 2000 |
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08941689 |
Oct 1, 1997 |
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60027791 |
Oct 1, 1996 |
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60027585 |
Oct 3, 1996 |
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Current U.S.
Class: |
514/290 ;
514/278; 514/299; 514/79; 546/112; 546/18; 546/23; 546/94 |
Current CPC
Class: |
A61P 37/04 20180101;
C07D 471/04 20130101; A61P 35/00 20180101; A61P 31/00 20180101 |
Class at
Publication: |
514/290 ;
514/299; 514/79; 514/278; 546/18; 546/23; 546/94; 546/112 |
International
Class: |
A61K 031/675; A61K
031/4747; A61K 031/4745; C07D 471/14; C07D 471/02 |
Claims
We claim:
1. A a compound of the formula I 46wherein (1)R.sup.1 R.sup.2 and
R.sup.3 are the same or different and represent hydrogen, alkyl,
cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic hydrocarbons,
aryl, or R.sup.1 and R.sup.2 together or R.sup.2 and R.sup.3
together form a carbocyclic or heterocyclic ring; (2) W, W' and W"
are the same or different and represent hydroxyl, alkoxy, thiol,
thioalkyl, thioaryl, halo or amino, or one or more of W and W" and
W' and W" together form a carbocyclic or heterocyclic ring; or one
or more of R.sup.1 and W, R.sup.2, and W' R.sup.3 and W" form a
spiro ring system; (3) X, X', Y, Y', Z, and Z' are the same or
different and represent hydrogen, alkyl, cycloalkyl, alkenyl,
alkynyl, unsaturated monocyclic hydrocarbons, aryl, alkosyl,
aryloxy, hydroxy, thiol, thioaryl, amino, ammonium, halogen,
carboxylic acid or esters or thioesters thereof, ketone, aldehyde,
carbonate, carbamate, amide, azide, imide, imine, imidazole,
acetal, ketal, nitrile, diaro, nitro, hydrazino, hydrazide,
hydrazone, hydroxarric acid, hydroxylamine, epoxide, alkoxy or
arylosxy amines, sulfate, sulfonic or sulfinic acid or esters
thereof, sulfonamide, phosphate or phosphonate acids or esters
thereof, silyl, sulfoxide, sufone, oxime, guanidino, phosphonate,
thiomide, thiocarbamate, thiocyanate, thioketone, thiourea,
thioethers, triazole, urea, xanthate, cyano, nitrile, --SR.sup.9
wherein R.sup.9 is alkyl, cycloalkyl, alkonyl, alkynyl, or
unsaturated monocyclic hydrocarbons, and --OR where R is alkyl is
alkyl, cycloalkyl, alknyl, alkynyl, or alkynyl, or unsaturated
monocyclic hydrocarbons; or (i) X and Y, X' and Y,X' and Y, or X'
and Y' may together form a carbocyclic, or heterocyclic ring, or Y
and Z, Y and Z', or Y' and Z' may together form a carocyclic, or
heterocyclic ring; (ii) one or more of X and X' together, Y and Y'
together, and Z and Z' together may form a spiro ring; or (iii) one
or more of X and X' together, Y and Y' together, and Z and Z'
together represent .dbd.O, .dbd.S, or .dbd.NR.sup.4 is hydrogen,
alkyl, cycloalkyl, alkonyl, alkynyl, unsaturated monocyclic
hydrocarbons, aryl, alkosy, hydrosyl, or .dbd.CR.sup.5R.sup.6 are
the same or different and represent hydrogen, alkyl, cycloalkyl,
alkenyl, alkynyl, unsaturated monocyclic hydrocarbons, or aryl;
with the proviso that X, X', Y, Y', and Z, Z' cannot all be
hydrogen, and salts and optically active and racemic forms of a
compound of the formula 1.
2. A compound of the formula I wherein when R.sup.1, R.sup.2,
R.sup.3, X, X', Y, Y' are hydrogen, W, W', and W" are not
hydroxyl.
3. A compound of the formula I wherein when R.sup.1, R.sup.2,
R.sup.3, X, X', Z, Z' are hydrogen, and W, W', and W" are hydroxyl,
Y and Y' together cannot be .dbd.O or one of Y and Y' cannot be
alkoxy.
4. A compound of the formula I as claimed in claim 1 wherein W, W',
and W" are the same and represent hydroxyl.
5. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 are the same and represent
hydrogen.
6. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 are the same and represent hydrogen,
and W, W', and W" are the same and represent hydroxyl.
7. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W', and W"
represent hydroxyl, and Z and Z' represent hydrogen.
8. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W', and W"
represent hydroxyl, and X and X' represent hydrogen.
9. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W', and W"
represent hydroxyl, and X, X', and Z and Z' represent hydrogen.
10. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W', and W"
represent hydroxyl, and Y, and Y', represent hydrogen.
11. A compound of the formula I as claimed in claim 1 wherein one
of Y and Y' represents methyl, ethyl, phenyl, benzyl,
trifluoromethyl, hydroxymethyl, or benzyloxymethyl.
12. A compound of the formula I as claimed in claim 1 wherein one
of X and X' represents thiomethyl, fluoromethyl, or methoxy.
13. A compound of the formula I as claimed in claim 1 wherein W"
and R.sup.3 are the same and represent halogen.
14. A compound of the formula I as claimed in claim 1 wherein Y and
Y' are the same and represent halogen.
15. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W' and W"
represent hydroxyl, Z and Z' represent hydrogen, one of X and X',
which may be substituted, is alkyl, aryl, alkoxy, hydroxyl, thiol,
thioalkyl, thioaryl, amino, halogen, carboxylic acid esters, thiol
esters, benzyl, pyridinyl, --CH.sub.2OR.sup.52 where R.sup.52
represents alkyl or aryl, and the other of X and X' is hydrogen, or
X and X' together represent .dbd.O, and, one of Y and Y', which may
be substituted, is alkyl, aryl, alkoxy, hydroxyl, thiol, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
CH.sub.2OR.sup.52 where R.sup.52 represents alkyl or aryl, benzyl,
or and the other of Y and Y' is hydrogen.
16. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W' and W"
represent hydroxyl, Y and Y' represent hydrogen, X and X', which
may be substituted, are the same or different and represent
hydrogen, alkyl, aryl, alkoxy, hydroxyl, thiol, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
benzyl, or pyridinyl, or --CH.sub.2OR.sup.52 where R.sup.52
represents alkyl or aryl, and Z and Z' are the same or different
and represent alkyl, aryl, alkoxy, hydroxyl, thiol, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
benzyl, or pyridinyl, or CH.sub.2OR.sup.52 where R.sup.52 presents
alkyl or aryl, with the proviso that at least one of X and X' and
at least one of Z and Z' cannot be hydrogen.
17. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W, W' and W"
represent hydroxyl, and X and X' represents hydrogen, Y, Y', Z, and
Z' are the same or different and represent hydrogen, alkyl, aryl,
alkoxy, hydroxyl, thiol, thioalkyl, thioaryl, amino, halogen,
carboxylic acid esters, thiol esters, benzyl, pyridinyl, or
--CH.sub.2OR.sup.50 where R.sup.52 represents alkyl or aryl, with
the proviso that at least one of Y and Y' and one of Z and Z'
cannot be hydrogen.
18. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W, W' and W"
represent hydroxyl, and X, X', Z and Z represent hydrogen, and one
of Y and Y' represent hydrogen, alkyl, aryl, hydroxyl, thiol,
thioalkyl, thioaryl, amino, halogen, carboxylic acid esters, thiol
esters, benzyl, pyridinyl, or --CH2OR.sup.50 where R.sup.50
represents alkyl or aryl, and the other of Y and Y' represents
hydrogen, alkyl, aryl, hydroxyl, thiol, thioalkyl, thioaryl, amino,
halogen, carboxylic acid esters, thiol esters, benzyl, pyridinyl,
or --CH2OR.sup.50 where R.sup.50 represents alkyl or aryl.
19. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W, W' and W"
represent hydroxyl, and Y, Y', Z and Z' represent hydrogen, and one
of X and X' represents hydrogen, alkyl, aryl, alkoxy, hydroxyl,
thio, thioalkyl, thioaryl, amino, halogen, carboxylic acid esters,
thiol esters, benzyl, pyridinyl, or --CH.sub.2OR.sup.50 where
R.sup.50 represents alkyl or aryl, and the other of X and X'
represent hydrogen, alkyl, aryl, alkoxy, hydroxyl, thiol,
thioalkyl, thioaryl, amino, halogen, carboxylic acid esters, thiol
esters, benzyl, pyridinyl, or --CH2OR.sup.52 where R.sup.52
represents alkyl or aryl, or X and X' together represent
.dbd.O.
20. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W, W' and W"
represent hydroxyl, and Z and Z' represent hydrogen, and X and Y,
X' and Y', X' and Y, or X and Y' together form a 6 member
heterocyclic ring containing one or two of O, S, or N.
21. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W' and W"
represent hydroxyl, X, X', Z and Z' represent hydrogen, and one of
Y and Y' represents methyl, ethyl, phenyl, or benzyl which may be
substituted, preferably trifluoromethyl, hydroxymethyl,
benzyloxymethyl, and the other of Y and Y' represent hydrogen.
22. A compound of the formula I as claimed in claim 1 wherein
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W represents
hydroxyl, and W' and W" represent halogen, preferably fluoro; X,
X', Z and Z' represent hydrogen, and one of Y and Y' represents
methyl, ethyl, phenyl, benzyl, trifluoromethyl, hydroxymethyl, or
benzyloxymethyl, and the other of Y and Y' represents hydrogen.
23. A compound of the formula I as claimed in claim 1 wherein one
of Y and Y' is hydrogen and the other of Y and Y' is methyl, and
one of Z and Z' is hydroxymethyl, --COCH.sub.2CH.sub.3, --CN,
--CH.sub.2NH.sub.2, --CH.sub.2NHAc, or --CH.sub.2NHCR.sup.60.dbd.NH
where R.sup.60 is alkyl or aryl.
24. A compound of the formula I as claimed in claim 1 wherein one
of Y and Y' and one of Z and Z' represents alkyl, aryl, alkoxy,
hydroxyl, thiol, thioalkyl, benzyl, pyridinyl, or --CH2OR.sup.52
where R.sup.52 represents alkyl or aryl, which may be substituted,
and the other of Y and Y' and Z and Z' represents hydrogen.
25. A compound of the formula I as claimed in claim 1 wherein one
of Z and Z' is --CONR.sup.70R.sup.71 where R.sup.70 and R.sup.71
are the same or different and represent hydrogen, alkyl, or aryl,
--COOH, --COOC.sub.2H.sub.5, methyl, or CH.sub.2OH, or Z and Z'
together form a spiro ring.
26. A compound of the formula I as claimed in claim 1 wherein X and
Y form a carbocyclic or heterocyclic ring of the formula
R.sup.75--R.sup.76--R.s- up.77--R.sup.78--R.sup.79 where R.sup.75
and R.sup.79 are part of the swainsonine skeleton and one or more
of R.sup.76, R.sup.77, and R.sup.78 represeent CH, CH.sub.2, O, S,
or N.
27. A compound of the formula I as claimed in claim 1 which is
(5R)-5-methylswainsonine, (5R)-5-methylswainsonine formate salt,
(5S)-5-methylswainsonine (5R)-8-Epi-5-methylswainsonine,
(5S)-5-ethylswainsonine, (5S,6S)-6
hydroxymethyl-5-methylswainsonine;
(5R)-5-benzyloxymethylswainsonine,
(5R-6R)-6-hydroxymethyl-5-methylswains- onine,
(5R)-5-hydroxymethylswainsonine, (5S)-5-hydroxymethylswainsonine,
(5R,(6R)-6-hydroxymethyl-5-methyl swainsonine,
(5S)-5-benzyloxymethylswai- nsonine, or
(5S)-5-benzyloxymethylswainsonine.
28. A pharmaceutical formulation comprising a compound of the
formula I as claimed in any one of claims 1 to 27 as an active
agent, and a pharmaceutically acceptable carrier, excipient or
diluent.
29. A method for stimulating the immune system, treating
proliferative disorders, or microbial infections in a patient
comprising administering an effective amount of a compound of the
formula I as claimed in any one of claims 1 to 27.
30. Use of a compound of the formula I as claimed in any one of
claims 1 to 27 in the preparation of a medicament of for
stimulating the immune system, and/or for treating proliferative
disorders, and microbial infections.
31. A method for stimulating the immune system, treating
proliferative disorders, or microbial infections in a patient
comprising administering an effective amount of a compound of the
formula I as claimed in claim 1 wherein R.sup.1, R.sup.2, and
R.sup.3 represent hydrogen, W, W' and W" represent hydroxyl, Y, Y',
Z and Z' represent hydrogen, and one of X and X' represents methyl,
phenyl, benzyl, hydroxymethyl, and the other of X and X' represent
hydrogen.
32. Use of a compound of the formula I as claimed in claim 1
wherein R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W' and
W" represent hydroxyl, Y, Y', Z and Z' represent hydrogen, and one
of X and X' represents methyl, phenyl, or benzyl which may be
substituted, and the other of X and X' represent hydrogen; R.sup.1,
R.sup.2, and R.sup.3 present hydrogen, W, W' and W" represent
hydroxyl, X, X', X and X' represent hydrogen, and one of Y and Y'
represents methyl, ethyl, phenyl, or benzyl which may be
substituted, and the other of Y and Y' represents hydrogen;
R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W, W' and W"
represent hydroxyl, and Y and Y' represent hydrogen, X and X' are
the same or different and represent hydrogen, alkyl, aryl, alkoxy,
hydroxyl, thiol, thioalkyl, thioaryl, amino, halogen, carboxylic
acid esters, thiol esters, benzyl, or pyridinyl, or
--CH.sub.2OR.sup.52 where R.sup.52 represents alkyl or aryl, which
may be substituted, and one of Z and Z' are the same or different
and represent alkyl, aryl, alkoxy, hydroxyl, thio, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
benzyl, or pyridinyl, or CH.sub.2OR.sup.52 where R.sup.52
represents alkyl or aryl, with the proviso that at least one of X
and X' and at least one of Z and Z' cannot be hydrogen, in the
preparation of a medicament for stimulating the immune system,
and/or for treating proliferative disorders, and microbial
infections.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel analogues of
swainsonine, processes for their preparation and their use as
therapeutic agents.
BACKGROUND OF THE INVENTION
[0002] Carbohydrate structures present on human tumor cells have
been associated with cancer invasion and metastasis. (Dennis et.
al., Science 236:582, 1987; Demetriou et. al., J. Cell Biol.
130:383, 1995). these structures include the GlcNAc .beta.(T1.6)
branched N- and O-linked carbohydrate side chains of cell-surface
glycoproteins. The Golgi enzymes required for their synthesis are
.beta.(T1.6) N-acetylglucosaminyltransfe- rase V (i.e. GlcNAc-TV)
and core 2 .beta.(T1-6) N-acetylglucosaminyltransf- erase (i.e.
core 2 GlcNAc-T), respectively. These enzymes are up-regulated in
human carcinomas (Fernandes et al., Cancer Res. 51:718-723, 1991),
a phenomenon that has been associated with the activation of the
ras signaling pathway (Dennis et al., Science 236:582-585, 1987;
Dennis et al Oncogene 4:853-860, 1989) Furthermore, overexpression
of GlcNAc-TV in epithelial cells results in morphological
transformation and tumor formation in mice (Demetriou et al, J.
Cell Biol. 130:383-392, 1995). Therefore, GlcNAc-TV as well as
enzymes supplying acceptor substrates to GlcNAc-TV (i.e. GlcNAc-TI,
.alpha. mannosidase II and core 2 GlcNac-T of the O-linked pathway)
are targets for anti-cancer pharmaceuticals.
[0003] Although there are no inhibitors of GlcNAc-TV presently
available, a lead .alpha.-mannosidase II inhibitor, swainsonine has
been tested in preclinical and human trials. Swainsonine is an
indolizidine alkaloid found in Australian Swainsona canescens
(Colegate et al., Aust J Chem 32:2257-2264, 1979), North American
plants of the genera Astragalus and Oxytropis (Molyneux R J and
James L F., Science 215:190-191, 1981), and also the fungus
Rhizoctonia leguminicola (Schneider et al., Tetradredron 39;29-31,
1983). Swainsonine's ability to inhibit .alpha.-mannosidase II
activity appears to be responsible for its interesting
immunomodulating and cancer suppression activity. Swainsonine is
believed to function as an enzyme inhibitor because it can mimic
the glycosylium cation intermediate generated during the hydrolytic
cleavage of mannopyranosides. (Goss, P. E. et al., Clin. Cancer
Res. 1:935-944, 1995).
[0004] The swainsonine blockage of .alpha.-mannosidase II is prior
to ClcNAc-TV and prevents expression of GlcNAc .beta.(T1-6)
branched N-linked carbohydrates. Swainsonine-treated murine tumor
cells have been found to be less metastatic in both
organ-colonization and spontaneous metastasis assays in mice
(Dennis J. W., Cancer Res. 46:5131-5136, 1986 and Humphries et al.,
Proc. Natl. Acad. Sci. USA 83:1752-1756, 1986). Swainsonine has
also been shown to block tumor cell invasion through extracellular
matrix in vitro (Yegel et al., Int. J. Cancer 44:685-690, 1989 and
Seftor et al., Melanoma Res. 1:53-54, 1991). Swainsonine
administered either orally or by mini-osmotic pumps to athymic nude
mice inhibited the growth rate of human MeWo melanoma and HT29m
colon carcinoma tumor xenografts in the mice (Dennis et al., J.
Natl. Cancer Inst. 81:1028-1033, 1989 and Dennis et al., Cancer
Res., 50:1867-1872, 1990).
[0005] Phase 1 clinical trials of swainsonine have been done which
indicate that it has efficacy in the treatment of human tumors.
(Goss et. al, Cancer Res., 54:1450, 1995). Although side-effects in
humans are mild, some of these may be associated with swainsonine's
inhibition of lysosomal storage of carbohydrates.
[0006] Swainsonine has positive effects on cellular immunity in
mice (reviewed in Humphries M. J. and Olden K., Pharmacol Ther.
44:85-105, 1989, and Olden et al., Pharmacol Ther 50:285-290,
1991)). In particular, swainsonine has been shown to alleviate both
chemically-induced and tumor-associated immune suppression (Hino et
al., J. Antibiot. (Tokyo) 38:926-935, 1985), increase NK cell
(Humphries et al., Cancer Res. 48:1410-1415, 1988), and LAK cell
activities (Yagita M and Saksela E. Scand. J. Immunol. 31:275-282,
1990), and increase splenic and bone marrow (BM) cell proliferation
(White et al., Biochem. Biophys. Res. Commun. 150;615.625, 1988;
Bowlin et al. Cancer Res 49, 4109-4113, 1989, and White et al.,
Cancer Commun. 3:83-91, 1991). SW has also been shown to be
hemorestorative in mice following treatment with both
cycle-specific and nonspecific chemotherapeutic agents (Oredipe et
al., J. Natl. Cancer Inst. 83:1149-1156, 1991).
[0007] Japanese Patent Application No. J61277685 describes
indolizidine derivatives which are reported to be useful as immune
regulators, which can be administered orally or parenterally at a
dose of about 0.1-100 ml/kg a day. It is also reported that the
indolizidine derivatives may be used in combination with antitumour
agents, antimicrobial agents or antiinflammatories.
[0008] Carbonoyloxy substitutions at the 2 and 8 carbons of
swainsonine have been reported to reduce inhibitor activity by 2-3
orders of magnitude for Jack Bean and MDAY-D2 tumor cell lysosomal
mannosidases in vitro. However, 2-p-nitrobenzoyloxy, 2-octanoyloxy-
and 2-butanoyloxy-derivatives of swainsonine retained full activity
as inhibitors of Golgi oligosaccharide processing inviable MDAY-D2
tumor cells. Inhibition of oligosaccharide processing was reduced
by the esterase inhibitor diethyl p-nitrophenyl phosphate,
suggesting that while the compounds are relatively poor inhibitors
of mannosidase in vitro, the compounds enter cells at a rate
comparable to that of swainsonine and are converted to swainsonine
by cellular esterases. The more lipophilic esters,
2-benzoyloxy-swainsonine, 2-tobuoyloxy-swainsonine,
8-palmitoyloxy-swainsonine and 8-myristinoyloxy-swainsonine, showed
IC.sub.50 values at least 10 times higher for inhibition of Golgi
oligosaccharide processing, probably due to less efficient entry of
the compounds into tumor cells. The anti-metastatic activities of
swainsonine and two analogs were tested and shown to correlate with
the IC.sub.50 values for inhibition of Golgi oligosaccharide
processing in cultured tumor cells. In vivo, SW and the analogues
were administered intraperitoneally to mice and found to have
comparable activities as stimulators of bone marrow cell
proliferation. (Dennis, J. W. et al. Biochemical Pharmacology
46:1459-1466, 1993).
[0009] Selected swainsonine analogues, in particular 2-substituted
analogues, and methods for preparing the analogues are described in
U.S. Pat. No. 5,466,809.
SUMMARY OF THE INVENTION
[0010] The present inventors have studied the profiles of reactions
catalyzed by mannosidase inhibitors, atomic charge distributions in
the mannopyranosyl cation (an intermediate in the reaction
catalyzed by mannosidases) and swainsonine derivatives, and the
chemical topography of the mannosidase II binding pocket. They
found that analogues of swainsonine which more closely mimick the
true transition state specifics (i.e. mannopyranosyl cation) rather
than the mannosylium cation intermediate, provided improved
inhibitory potency. In particular, selective derivatization of
swainsonine at one or both of positions 3 and 5 with electron
withdrawing groups provided analogues of swainsonine which are
ideally suited for use as drugs and prodrugs having improved
pharmacological properties. The present inventors have also found
that selective derivatization at the 6 position also provides
analogues which having improved pharmacological properties.
[0011] The present invention therefore relates to a compound of the
formula I 1
[0012] wherein
[0013] (1) R.sup.1, R.sup.2 and R.sup.3 are the same or different
and represent hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl,
unsaturated monocyclic hydrocarbons, aryl, or R.sup.1 and R.sup.2
together or R.sup.2 and R.sup.3 together form a carbocyclic or
heterocylic ring;
[0014] (2) W, W' and W" are the same or different and represent
hydroxyl, alkoxy, thiol, thioalkyl, thioaryl, halo or amino, or one
or more of W and W" and W' and W" together form a carbocyclic or
heterocyclic ring;
[0015] or one or more of R.sup.1 and W, R.sup.2 and W', and R.sup.3
and W" form a spiro ring system;
[0016] (3) X, X', Y, Y', Z, and Z' are the same or different and
represent hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl,
unsaturated monocyclic hydrocarbons, aryl, alkoxy, aryloxy,
hydroxyl, thiol, thioaryl, amino, ammonium, halogen, carboxylic
acid or esters or thioesters thereof, ketone, aldehyde, carbonate,
carbamate, amide, azide, imide, imine, imidazole, acetal, ketal,
nitrite, diazo, nitro, hydrazine, hydrazide, hydrazone, hydroxamic
acid, hydroxylamine, epooxide, alkoxy or aryloxy amines, sulfate,
sulfonic or sulfinic acid or esters thereof, sulfonamide, phosphate
or phosphonate acids or esters thereof, silyl, sulfoxide, sulfone,
oxime, guanidino, phosphonate, thioamide, thiocarbamate,
thiocyanate, thioketone, thiourea, thioethers, triazole, urea,
xanthate, cyano, nitrile, --SR.sup.9 wherein R.sup.9 is alkyl,
cycloalkyl, alkenyl, alkynyl, or unsaturated monocyclic
hydrocarbons, and --OR where R is alkyl is alkyl, cycloalkyl,
alkenyl, alkynyl, or unsaturated monocyclic hydrocarbons; or
[0017] (i) X and Y, X' and Y, X' and Y, or X' and Y' may together
form a carbocyclic, or heterocyclic ring, or Y and Z, Y and Z', or
Y' and Z' may together form a carbocyclic, or heterocyclic
ring;
[0018] (ii) one or more of X and X' together, Y and Y' together,
and Z and Z' together may form a spiro ring; or
[0019] (iii) one or more of X and X' together, Y and Y' together,
and Z and Z' together represent .dbd.O, .dbd.S, or .dbd.NR.sup.4
wherein R.sup.4 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl,
unsaturated monocyclic hydrocarbons, aryl, alkoxy, hydroxyl, or
.dbd.CR.sup.5R.sup.6 wherein R.sup.5 and R.sup.6 are the same or
different and represent hydrogen, alkyl, cycloalkyl, alkenyl,
alkynyl, cycloalkenyl, cycloalkynyl unsaturated monocyclic
hydrocarbons, or aryl;
[0020] with the proviso that X, X', Y, Y', and Z, Z' cannot all be
hydrogen,
[0021] and salts and optically active and racemic forms of a
compound of the formula I.
[0022] In an embodiment of the invention, compounds of the formula
I are provided wherein when R.sup.1, R.sup.2, R.sup.3, X, X', Y, Y'
are hydrogen, W, W', and W" are not hydroxyl. In another embodiment
of the invention, compounds of the formula I are provided wherein
when R.sup.1, R.sup.2, R.sup.3, X, X', Z and Z' are hydrogen, and
W, W', and W" are hydroxyl, Y and Y' together cannot be .dbd.O or
one of Y and Y' cannot be alkoxy.
[0023] The present invention also provides a process for the
preparation of a compound of the formula I as defined herein, and a
pharmaceutical formulation comprising a compound of the formula I
as an active agent.
[0024] The invention further relates to a method for stimulating
the immune system, treating proliferative disorders, or microbial
infections in a patient comprising administering an effective
amount of a compound of the formula I of the invention. The
invention also relates to the use of a compound of the formula I in
the preparation of a medicament for stimulating the immune system,
and/or for treating proliferative disorders, and microbial
infections.
[0025] The present invention also relates to the use of a compound
of the formula I which is esterified at free hydroxyls as a
prodrug.
[0026] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings. In addition, reference is made herein to various
publications, which are hereby incorporated by reference in their
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The invention will be better understood with reference to
the drawings in which:
[0028] FIG. 1 is a schematic diagram of a reaction for preparing a
novel analogue of the invention;
[0029] FIG. 2 is a schematic diagram of a reaction for preparing a
novel analogue of the invention;
[0030] FIG. 3A is a schematic diagram of a reaction for preparing a
novel analogue of the invention;
[0031] FIG. 3B is a schematic diagram of a reaction for preparing a
novel analogue of the invention;
[0032] FIG. 4 is a schematic diagram of a reaction for preparing a
novel analogue of the invention;
[0033] FIG. 5 is a schematic diagram of a reaction for preparing a
novel analogue of the invention;
[0034] FIG. 6 is a schematic diagram of a reaction for preparing a
novel analogue of the invention; and
[0035] FIG. 7 is a schematic diagram of a reaction for preparing a
novel analogue of the invention.
DETAILED DESCRIPTION OF THE INVENTION
I. Compounds of the Invention
[0036] Hereinabove and in the following the term "alkyl", alone or
in combination, refers to a branched or linear hydrocarbon radical,
typically containing from 1 through 20 carbon atoms, preferably 1
through 15. Typical alkyl groups include but are not limited to
methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, tert-butyl,
pentyl, hexyl, and the like.
[0037] The term "alkenyl", alone or in combination, refers to an
unsaturated branched or linear group typically having from 2 to 20
carbon atoms and at least one double bond. Examples of such groups
include but are not limited to ethenyl, 1-propenyl, 2-propenyl,
1-butenyl, 1,3-butadienyl, hexenyl, pentenyl, and the like.
[0038] The term "alkynyl", alone or in combination, refers to an
unsaturated branched or linear group having 2 to 20 carbon atoms
and at least one triple bond. Examples of such groups include but
are not limited to ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,
2-butynyl, 1-pentynyl, and the like.
[0039] The term "cycloalkyl" refers to cyclic hydrocarbon groups
and includes but is not limited to cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
[0040] The terms "cycloalkenyl" and "cycloalkynyl" refer to
unsaturated monocyclic hydrocarbons having one endocyclic double or
triple bond. Compound of the formula I having more than one such
multiple bond are cycloalkadienyl, cycloalkatrienyl, etc. The
inclusive term for any cyclic hydrocarbons having any number of
such multiple bonds is unsaturated monocyclic hydrocarbons.
Examples of unsaturated monocyclic hydrocarbons are cyclohexene,
cyclopentadiene, and cyclooctadiene.
[0041] The term "aryl", alone or in combination, refers to a
monocyclic or polycyclic group, preferably a monocyclic or bicyclic
group. An aryl group may optionally be substituted as described
herein. Examples of aryl groups and substituted aryl groups are
phenyl, benzyl, p-nitrobenzyl, p-methoxybenzyl, biphenyl, and
naphthyl.
[0042] The term "alkoxy" alone or in combination, refers to an
alkyl or cycloalkyl linked to the parent molecular moiety through
an oxygen atom. the term "aryloxy" refers to an aryl linked to the
parent molecular moiety through an oxygen atom. Examples of alkoxy
groups are methoxy, ethoxy, propoxy, vinyloxy, allyloxy, butoxy,
pentoxy, hexoxy, cyclopentoxy, and cyclohexoxy. Examples of aryloxy
groups are phenyloxy, O-benzyl, i.e. benzyloxy, O-nitrobenzyl and
O-p-methyl-benzyl, 4-nitrophenyloxy, 4-chlorophenyloxy, and the
like.
[0043] The term "halo" or "halogen", alone or in combination,
refers to a member of the family fluorine, chlorine, bromine, or
iodine.
[0044] The term "amino", alone or i combination, refers to a
chemical functional group where a nitrogen atom (N) is bonded to
three substituents being any combination of hydrogen, alkyl,
cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic hydrocarbons,
or aryl with the general chemical formula --NR.sup.7R.sup.8 where
R.sup.7 and R.sup.8 can be any combination of hydrogen, alkyl,
cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic hydrocarbons,
or aryl. Optionally one substituent on the nitrogen atom can be a
hydroxyl group (--OH) to give an amine known as a hydroxylamine.
Examples of amino groups are amino (--NH.sub.2), methylamine,
ethylamine, dimethylamine, cyclopropylamine, benzylamine,
allylamine, and hydroxylamine, cyclohexylamino
(--NHCH(CH.sub.2).sub.5), piperidine (--N(CH.sub.2).sub.5) and
benzylamino (--NHCH.sub.2C.sub.6H.sub.5). Some amines may contain
the basic skeletal structure of swainsonine to give analogues such
as: 2
[0045] The term "thioalkyl", "thiocycloalkyl", "thioalkynyl",
"thiocycloalkenyl", "thiocycloalkynyl", "thiocycloacetylenyl" alone
or in combination, refers to a chemical functional group where a
sulfur atom (S) is bonded to an alkyl, cycloalkyl, alkenyl,
alkynyl, or unsaturated monocyclic hydrocarbon group. the compounds
have the general chemical formula --SR.sup.9 where R.sup.9 is an
alkyl, cycloalkyl, alkenyl, alkynyl, or unsaturated monocyclic
hydrocarbon group. Examples of thioalkyl groups are thiomethyl,
thioethyl, thiopropyl, thiopropenyl, thiobutyl, thiohexyl,
thiocyclopentyl, thiomethoxymethyl, thiocyclohexyl, thioallyl,and
thiochloromethyl.
[0046] The term "thioaryl", alone or in combination, refers to a
chemical functional group where a sulfur atom (S) is bonded to an
aryl group with the general chemical formula --SR.sup.10 where
R.sup.10 is an aryl group which may be substituted. Example of
thioaryl groups are thiophenyl, para-chlorothiophenyl, thiobenzyl,
4-methoxy-thiophenyl, 4-nitro-thiophenyl, and
para-nitrothiobenzyl.
[0047] A "carboxylic acid" chemical functional group, alone or in
combination, has the formula --COOH and examples of compounds of
the formula I containing one carboxyl group are the following:
3
[0048] Esters of carboxylic acids have the chemical functional
group R.sup.11COOR.sup.12 where R.sup.11 represents the primary
skeleton structure of a compound of the formula I and R.sup.12 is
alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic
hydrocarbons, or aryl. A lactone is a cyclic ester where R.sup.11
and R.sup.12 represent the same tether. Preferred carboxylic acid
esters (--CO.sub.2R.sup.12) include methyl esters
(--CO.sub.2CH.sub.3), ethyl esters (--CO.sub.2CH.sub.2CH.sub.3),
propyl esters (--CO.sub.2CH.sub.2CH.sub.2CH- .sub.3), allyl esters
(--CO.sub.2CH.sub.2CH=50CH.sub.2), butyl esters
(--CO.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3) and benzyl esters
(--CO.sub.2CH.sub.2C.sub.6H.sub.5). Examples of compounds of the
invention bearing only one ester group are the following: 4
[0049] Where, for example R' is CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.3CH.sub.3, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3,
CH.sub.2C.sub.6H.sub.5.
[0050] Other examples of esters of compounds of the formula I of
the invention include the following: 5
[0051] Where, for example, R is methyl, ethyl, propyl, propenyl,
butyl, pentyl, hexyl, phenyl or benzyl.
[0052] Thioesters have the general formula R.sup.13COSR.sup.14
where R.sup.13 represents the primary skeleton of a compound of the
formula I, and R.sup.14 is an alkyl, cycloalkyl, alkenyl, alkynyl,
unsaturated monocyclic hydrocarbons, or aryl. Examples of
thioesters are analogous to those provided for the carboxylic acid
esters presented above.
[0053] The term "amides", alone or in combination, refers to a
chemical functional group of the formula
R.sup.15CONR.sup.16R.sup.17 where R.sup.15 represents the primary
skeleton of compounds of the formula I, and R.sup.16, and R.sup.17
are any combination of hydrogen, alkyl, cycloalkyl, alkenyl,
alkynyl, unsaturated monocyclic hydrocarbons, or aryl. Examples of
amide substituents for the compounds of the formula I include:
--CONH.sub.2, --CONHCH.sub.3, --CON(CH.sub.3).sub.2,
--CONHCH.sub.2CH.sub.3, --CON(CH.sub.2CH.sub.3).sub.2,
--CONHCH.sub.2CH.sub.2CH.sub.3, --CONHCH.sub.2CH.dbd.CH.sub.2,
--CONHC.sub.6H.sub.5, --CONHCH.sub.2C.sub.6H.sub.5,
--CONHCH.sub.2CH.sub.2OH, --CON(CH.sub.2CH.sub.2OCH.sub.3).sub.2,
and 6
[0054] Examples of compounds of the formula I containing an amide
substituent include: 7
[0055] where R and R" are permutations of hydrogen, methyl, ethyl,
hydroxyethyl, propyl, hydroxypropyl, butyl, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, phenyl and benzyl for example,
or, where R and R" of the basic amide formula may form a lactam
ring such as: 8
[0056] Other lactam rings of interest include structures where both
R and R' of the basic amide formula form parts of the basic
swainsonine skeleton of a compound of the formula I such as: 9
[0057] where R" for example may be hydrogen, methyl, ethyl,
hydroxylethyl, propyl, butyl, hexyl or benzyl.
[0058] Thioamides have the general formula 10
[0059] represents the primary skeleton of a compound of the formula
I, and R.sup.19 and R.sup.20 may be any combination of hydrogen,
alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic
hydrocarbons, or aryl. A thiolactam is a cyclic thioamide where
R.sup.18 and R.sup.19 represent the same tether. Examples of
thioamides are analogous to those described for amides above.
[0060] Sulfonamides have the general formula 11
[0061] represents the general structure of the compounds of the
formula I, R.sup.22 and R.sup.23 may be any combination of
hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated
monocyclic hydrocarbons, or aryl. Examples of thioamides are
analogous to those described for amides above.
[0062] Hydrazides have the general formula
R.sup.24C(O)NR.sup.25NR.sup.26R- .sup.27 where R.sup.25 represents
alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic
hydrocarbons, or aryl, one of R.sup.24, R.sup.26 or R.sup.27
represents the primary skeleton of a compound of the formula I, and
the other of R.sup.24, R.sup.26 and R.sup.27, may be any
combination of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl,
unsaturated monocyclic hydrocarbons, or aryl. Examples of hydrazide
substituent groups where R.sup.24 represents the primary skeleton
of a compound of the formula I include hydrazide
(--C(O)NHNH.sub.2), dimethyl hydrazide (--C(O)NHN(CH.sub.3).sub.2)
or benzyl hydrazide (--C(O)NHNHCH.sub.2C.sub.- 6H.sub.5). Examples
of hydrazide substituent groups where R.sup.26 R.sup.27 represent
the primary skeleton of a compound of the formula I include
CH.sub.3C(O)NHNH--, CH.sub.3CH.sub.2C(O)NHNH--,
CH.sub.3CH.sub.2CH.sub.2CH.sub.2C(O)NHNH-- or
C.sub.6H.sub.5C(O)NHNH--.
[0063] Hydrazines have the general formula 12
[0064] where R, R', R", and R'" can be any combination of hydrogen,
alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic
hydrocarbons, or aryl. Examples of hydrazine substituents include:
--NHNH.sub.2, --NHNHCH.sub.2C.sub.6H.sub.5 and
--NHN(CH.sub.3).sub.2.
[0065] Hydrazones have the general formula 13
[0066] where one of R, R', R" and R'" represents the primary
skeleton of a compound of the formula I, and the other of R, R', R"
and R'" can be any combination of hydrogen, alkyl, cycloalkyl,
alkenyl, alkynyl, unsaturated monocyclic hydrocarbons, or aryl.
Examples of compounds of the formula I with hydrazone substituents,
which may or may not be cyclic and form part of the swainsonine
skeleton of a compound of the formula I include the following:
14
[0067] Ureas have the general formula: 15
[0068] where one of R, R', R" and R'" represents the primary
skeleton of a compound of the formula I, and the other of R, R', R"
and R'" can be any combination of hydrogen, alkyl, cycloalkyl,
alkenyl, alkynyl, unsaturated monocyclic hydrocarbons, or aryl.
Examples of urea substituents and urea containing compounds of the
formula I include: 16
[0069] Thioureas have the general formula: 17
[0070] where one of R, R', R" and R'" represents the primary
skeleton of a compound of the formula I, and the other of R, R', R"
and R'" can be any combination of hydrogen, alkyl, cycloalkyl,
alkenyl, alkynyl, unsaturated monocyclic hydrocarbons, or aryl.
Examples of thiourea substituents and thiourea containing compounds
of the formula I are analogous to those provided above for
ureas.
[0071] The term "ketones" refers to a chemical functional group of
the formula R.sup.28COCR.sup.29, where R.sup.28 represents the
primary skeleton of a compound of the formula I and R.sup.29 is
alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic
hydrocarbons, or aryl. Examples of ketones which may be used in the
compounds of the invention include methyl ketones (--COCH.sub.3),
methylene methyl ketones (--CH.sub.2COCH.sub.3), ethylene methyl
ketones (--CH.sub.2CH.sub.2COCH.s- ub.3), ethyl ketones
(--COCH.sub.2CH.sub.3), propylketones, vinylketones, butylketones
(--COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), hexylketones,
cyclohexyketones, cyclopentylketones, phenylketones and
benzylketones. Compounds of the formula I containing cyclic ketone
groups include the following: 18
[0072] Thioketones have the general formula: 19
[0073] where R represents the primary skeleton of a compound of the
formula I and R' is alkyl, cycloalkyl, alkenyl, alkynyl,
unsaturated monocyclic hydrocarbons, or aryl. Examples for
thioketones are analogous to those given above for ketones.
[0074] Carbamates have the general formula: 20
[0075] where one of R and R" represents the primary skeleton of a
compound of the formula I, and the other of R and R' are hydrogen,
alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic
hydrocarbons, or aryl, and R' is hydrogen alkyl, cycloalkyl,
alkenyl, alkynyl, unsaturated monocyclic hydrocarbons, or aryl, or
R and R' represent the same compound of the formula I. Examples of
carbamate substituents (--NR'CO.sub.2") for compounds of the
formula I include O-allyl carbamates
(--NHCO.sub.2CH.sub.2CH.dbd.CH.sub.2), O-ethyl carbamates
(--NHCO.sub.2CH.sub.2CH.sub.3), O-tert-butyl carbamates
(--NHCO.sub.2C(CH.sub.3).sub.3) and O-benzyl carbamates
(--NHCO.sub.2CH.sub.2C.sub.6H.sub.5). Examples where both R and R"
form part of the primary skeleton of a compound of the formula I
(i.e. swainsonine) include: 21
[0076] Thiocarbamates have the general formula: 22
[0077] where one of R and R" represents the primary skeleton of a
compound of the formula I, and the other of R and R" represents
hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated
monocyclic hydrocarbons, or aryl and R' is hydrogen, alkyl,
cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic hydrocarbons,
or aryl. Examples for thiocarbamates are analogous to those given
above for carbamates.
[0078] Xanthates have the general formula: 23
[0079] where R and/or R' are the primary structure of a compound of
the formula I. Examples of xanthates (--SC(S)OR') where R contains
the primary skeleton of a compound of the formula I (i.e.
swainsonine) include O-methyl xanthates (--SC(S)OCH.sub.3), O-ethyl
xanthates (--SC(S)OCH.sub.2CH.sub.3) or O-benzyl xanthate
(--SC(S)OCH.sub.2CH.sub.3- ). Examples of xanthates (--OCS.sub.2R)
where R' contains the primary skeleton of a compound of the formula
I (i.e. swainsonine include S-methyl xanthates
(--OCS.sub.2CH.sub.3), S-ethyl xanthates
(--OCS.sub.2CH.sub.2CH.sub.3) or S-benzyl xanthate
(--OCS.sub.2CH.sub.2CH.sub.3).
[0080] Sulfoxides have the general formula R.sup.30SOR.sup.31 where
R.sup.30 and/or R.sup.31 represent the primary skeleton of a
compound of the formula I. Examples of sulfoxides of interest
include those where R.sup.30 represents the primary skeleton of a
compound of the formula I (i.e. swainsonine), and R.sup.31 includes
for example, methyl sulfoxides (--SOCH.sub.3), methylene methyl
sulfoxides (--CH.sub.2SOCH.sub.3), ethylene methyl sulfoxides,
(--CH.sub.2CH.sub.2SOCH.sub.3), ethyl sulfoxides
(--SOCH.sub.2CH.sub.3), butyl sulfoxides
(--SOCH.sub.2CH.sub.2CH.sub.2CH.sub.3), hexylsulfoxides,
cyclohexylsulfoxides, cyclopentylsulfoxides, allyl sulfoxides,
phenylsulfoxides and benzylsulfoxides. Other sulfoxides of interest
include those where both R.sup.30 and R.sup.31 form part of the
primary skeleton of a compound of the formula I (i.e. swainsonine)
to give cyclic sulfoxides such as: 24
[0081] Sulfones have the general formula: 25
[0082] where R and/or R' represent the primary skeleton of a
compound of the formula I. Examples of sulfones are analogous to
the sulfoxides described above.
[0083] Epoxides (or oxiranes are 3-membered cyclic ethers having
the general formula: 26
[0084] where one of R, R', R" and R'" represents the primary
skeleton of a compound of the formula I, and the other of R, R', R"
and R'" can be any combination of hydrogen, alkyl, cycloalkyl,
alkenyl, alkynyl, unsaturated monocyclic hydrocarbons, or aryl.
Some examples of epoxides include: 27
[0085] Similarly, 4-, 5- and 6- membered saturated cyclic ethers
which may be used in the compounds of the formula I include
trimethylene oxide ((CH.sub.2).sub.3O), tetrahydrofuran
((CH.sub.2).sub.4O), and tetrahydropyran ((CH.sub.2).sub.5O)
rings.
[0086] Ammonium salts have the general formula: 28
[0087] where one of R, R', R" and R'" is a primary skeleton of a
compound of the formula I and the other of R, R', R", R'" is
hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, unsaturated
monocyclic hydrocarbons, or aryl, and X.sup.- is a suitable
counterion such as chloride (Cl.sup.-), bromide (Br.sup.-) or
acetate (CH.sub.3CO.sub.2.sup.-). Examples of ammonium salts
include trimethylammonium chloride (--N(CH.sub.3).sub.3Cl)- ,
methylpiperidylammonium bromide (--N(CH.sub.3)(CH.sub.2).sub.5Br)
or benzyldiethyl ammonium chloride
(--N(CH.sub.2C.sub.6H.sub.5)(CH.sub.2CH.s- ub.3).sub.2Cl).
[0088] Thiols (also known as mercaptans) have the general formula
R.sup.37--SH where R.sup.37 is a primary skeleton of a compound of
the formula I. Nitro compounds have the general formula
R.sup.38NO.sub.2 where R.sup.38 is a primary skeleton of a compound
of the formula I. Organic azides have the general formula
R.sup.39--N.sub.3 where R.sup.39 is a primary skeleton of a
compound of the formula I.
[0089] Hydroxylamines have the general formula
R.sup.40--NR.sup.41(OH), where R.sup.40 is the primary skeleton of
a compound of the formula I and R.sup.41 is hydrogen, alkyl,
cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic hydrocarbons,
or aryl. Examples of hydroxylamine substituents (--NR.sup.75(OH))
include hydroxylamino (--NH(OH)), N-methylhydroxylamine
(--N(OH)CH.sub.3)), N-ethylhydroxylamine (--N(OH)CH.sub.2CH.sub.3))
or N-benzylhydroxylamine (--N(OH)(CH.sub.2C.sub.6H.sub.5)).
[0090] Alkoxy or aryloxy amines have the general formula
R.sup.42--NR.sup.43OR.sup.42 is a primary skeleton of a compound of
the formula I, R.sup.43 is hydrogen, alkyl, cycloalkyl, alkenyl,
alkynyl, unsaturated monocyclic hydrocarbons, or aryl, and R.sup.44
is alkyl or aryl. Examples of alkoxy or aryloxy amines substituents
(--NR(Or.sup.44)) include methoxylamine (--NH(OCH.sub.3)), N-ethyl
methoxylamine (--N(OCH.sub.3)CH.sub.2CH.sub.3)) or N-benzyl
ethoxylamine (--N(CH.sub.2CH.sub.3)(CH.sub.2C.sub.6H.sub.5)).
[0091] Nitriles have the general formula R.sup.45 --C N where
R.sup.45 is the primary skeleton of a compound of the formula I.
Thiocyanates have the general formula R.sup.46 --SCN where R.sup.46
is the primary skeleton of a compound of the formula I.
[0092] Imines (also known as Schiff bases) have the general formula
29
[0093] where R is a primary skeleton of a compound of the formula I
and R' and R" can be any combination of hydrogen, alkyl,
cycloalkyl, alkenyl, alkynyl, unsaturated monocyclic hydrocarbons,
or aryl.
[0094] Sulfinic acids have the general formula R.sup.47--SO.sub.2H
where R.sup.47 is a primary skeleton of a compound of the formula
I. Sulfonic acids have the general formula R.sup.48--SO.sub.3H
where R.sup.48 is a primary skeleton of a compound of the formula
I.
[0095] Sulfonic acid esters have the general formula: 30
[0096] Examples of sulfonic acid esters where R' contains the basic
skeleton of a compound of the formula I i.e. the swainsonine
skeleton, include tosylates (p--CH.sub.3C.sub.6H.sub.4SO.sub.3--)
and mesylates (CH.sub.3SO.sub.3--). Other examples were R contains
the basic skeleton of a compound of the formula I i.e. the
swainsonine skeleton, include methyl esters (--SO.sub.3CH.sub.3),
ethyl esters (--SO.sub.3CH.sub.2CH.su- b.3) or benzyl esters
(--SO.sub.3CH.sub.2C.sub.6H.sub.5).
[0097] Triazoles have the general formula: 31 32
[0098] The term "carbocyclic" or "carbocyclic ring system" refers
to molecular rings where the framework is constructed by joining
carbon atoms solely and includes but is not limited to any stable
3- to 7- membered monocyclic or bicyclic or 7- to 14-membered
bicyclic or tricyclic or up to 26-membered polycyclic carbon ring,
any of which may be saturated, partially unsaturated, or aromatic.
Examples of carbocyclic rings include substituted or unsubstituted
cycloalkyl, monocyclic unsaturated hydrocarbons, and aryl as
described herein, including but not limited to benzene and
napthalene.
[0099] Heterocyclic rings are molecular rings where one or more
carbon atoms have been replaced by hetero atoms (atoms not being
carbon) such as for example, oxygen (O), nitrogen (N) or sulfur
(S), or combinations thereof. Examples of heterocyclic rings
include ethylene oxide, tetrahydrofuran, thiophene, piperidine
(piperidinyl group), pyridine (pyridinly group), and caprolactam. A
carbocyclic or heterocyclic group may be optionally substituted at
carbon or nitrogen atoms with for example, alkyl, phenyl, benzyl or
thienyl, or a carbon atom in the heterocyclic group together with
an oxygen atom may form a carbonyl group, or a heterocyclic group
may be fused with a phenyl group.
[0100] A spiro ring is defined as two rings originating from the
same atom (the spiro center). a spiro ring is schematically
represented below: 33
[0101] Some examples of swainsonine analogues of the invention
containing spiro rings include: 34
[0102] One or more of R.sup.1, R.sup.2, R.sup.3, W, W', W", X, X',
Y, Y', Z, and/or Z', alone or together, which contain available
functional groups as described herein may be substituted with one
or more of the following: alkoxy, hydroxy, thiol, thiol, --SR.sup.9
wherein R9 is alkyl, cycloalkyl, alkenyl, alkynyl, or unsaturated
monocyclic hydrocarbons, thioaryl, amino, ammonium halogen,
carboxylic acid or ester, ketone, aldehyde, carbonate, carbamate,
amide, azide, imide, imine, imidazole, acetal, ketal, nitrile,
diazo, nitro, hydrazine, hydrazide, hydrazone, hydroxamic acid,
oxime, hydroxylamine, sulfate, sulfonic or sulfinic acid or ester,
sulfonamide, phosphate or phosphate acids or ester,s silyl,
sulfoxide, sulfone, oxime, guanidino, phosphonate, thioamide,
thiocarbamate, thiocyanate, thioketone, thiourea, triazole, cyano,
nitrile, urea or xanthate. The term "one or more" used herein
preferably refers to from 1 to 3 substituents, most preferably 1 to
2 substituents. Examples of substituted radicals are described
herein, and include but are not limited to --CH.sub.2R.sup.50 where
R.sup.50 is alkyl, aryl, amino, --CR.sup.51 where R.sup.51 is
halogen, in particular trifluoromethyl, and --CH.sub.2OR.sup.52
where R.sup.52 is alkyl or aryl.
[0103] In one embodiment of the invention, compounds of the formula
I are provided where W, W' and W" are the same and represent
hydroxyl. In another embodiment of the invention, a compound of the
formula I is provided wherein R.sup.1, R.sup.2, and R.sup.3 are the
same and represent hydrogen. In a still further embodiment of the
invention, compounds of the formula I are provided where R.sup.1,
R.sup.2, and R.sup.3 are the same and represent hydrogen, and W, W'
and W" are the same and represent hydroxyl.
[0104] In particular the invention contemplates compounds of the
formula I where:
[0105] (a) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W,
W' and W" represent hydroxyl, and Z and Z' represent hydrogen;
[0106] (b) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W,
W' and W" represent hydroxyl, and X and X' represent hydrogen;
[0107] (c) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W,
W', and W" represent hydroxyl, and X, X', Z and Z' represent
hydrogen;
[0108] (d) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W,
W', and W" represent hydroxyl, Y and Y' represent hydrogen;
[0109] (e) one of Y and Y' represents methyl, hydroxymethyl, ethyl,
phenyl, benzyl, benzyloxymethyl, or fluoromethyl;
[0110] (f) one of X and X' represents methyl which may be
substituted such as thiomethyl, fluoromethyl, or methoxy;
[0111] (g) W" and R.sup.3 are the same and represent halogen,
preferably fluoro; and
[0112] (h) Y and Y' are the same and represent halogen, preferably
fluoro.
[0113] Preferably the compounds of the formula I of the invention
are those where:
[0114] (a) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W'
and W" represent hydroxyl, Z and Z' represent hydrogen, one of X
and X', which may be substituted, is alkyl, aryl, alkoxy, hydroxyl,
thiol, thioalkyl, thioaryl, amino, halogen, carboxylic acid esters,
thiol esters, benzyl, pyridinyl, --CH.sub.2OR.sup.52 where R.sup.52
represents alkyl or aryl, and the other of X and X' is hydrogen, or
X and X' together represent .dbd.O, and one of Y and Y', which may
be substituted, is alkyl, aryl, alkoxy, hydroxyl, thiol, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
--CH.sub.2OR.sup.52 where R.sup.52 represents alkyl or aryl,
benzyl, or and the other of Y and Y' is hydrogen;
[0115] (b) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W'
and W" represent hydroxyl, Y and Y' represent hydrogen, X and X',
which may be substituted, are the same or different and represent
hydrogen, alkyl, aryl, alkoxy, hydroxyl, thiol, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
benzyl, or pyridinyl, or --CH.sub.2OR.sup.52 where R.sup.52
represents alkyl or aryl, and Z and Z' are the same or different
and represent alkyl, aryl, alkoxy, hydroxyl, thiol, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
benzyl, or pyridinyl, or CH.sub.2OR.sup.52 where R.sup.52 represent
alkyl or aryl, with the proviso that at least one of X and X' and
at least one of Z and Z' cannot be hydrogen;
[0116] (c) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W,
W.sup.40 and W.sup.41 represent hydroxyl, X and X' represents
hydrogen, Y, Y', Z, and Z' are the same or different and represent
hydrogen, alkyl, aryl, alkoxy, hydroxyl, thiol, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
benzyl, or pyridinyl, or --CH.sub.2OR.sup.52 where R.sup.52
represents alkyl or aryl, which may be substituted, with the
proviso that at least one of Y and Y' and at least one of Z and Z'
cannot be hydrogen: most preferably one of Y and Y' and one of Z
and Z' represents alkyl, aryl, hydroxyl, thiol, thioalkyl, benzyl,
pyridinyl, or --CH.sub.2OR.sup.50 where R.sup.50 represents alkyl
or aryl, which may be substituted, and the other of Y and Y' and Z
and Z' represents hydrogen;
[0117] (d) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W'
and W" represent hydroxyl, X, X', Z and Z' represent hydrogen, and
one of Y and Y', which may be substituted, represent alkyl, aryl,
hydroxyl, thiol, thioalkyl, thioaryl, amino, halogen, carboxylic
acid esters, thiol esters, benzyl, or pyridinyl, or
--CH.sub.2OR.sup.52 where R.sup.52 represents alkyl or aryl, and
the other of Y and Y', which may be substituted, represents
hydrogen, alkyl, aryl, hydroxyl, thiol, thioalkyl, thioaryl, amino,
halogen, carboxylic acid esters, thiol esters, benzyl, or
pyridinyl, or --CH.sub.2OR.sup.52 where R.sup.52 represents alkyl
or aryl;
[0118] (e) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W'
and W" represent hydroxyl, Y, Y', Z and Z', represent hydrogen, one
of X and X' represents alkyl, aryl, alkoxy, hydroxyl, thiol,
thioalkyl, thioaryl, amino, halogen, carboxylic acid esters, thiol
esters, benzyl, or pyridinyl, or --CH.sub.2OR.sup.52 where R.sup.52
represents alkyl or aryl, which may be substituted, and the other
of X and X', which may be substituted, represent hydrogen, alkyl,
aryl, alkoxy, hydroxyl, thiol, thioalkyl, thioaryl, amino, halogen,
carboxylic acid esters, thiol esters, benzyl, or pyridinyl, or
--CH.sub.2OR.sup.52 where R.sup.52 represents alkyl or aryl, or X
and X' together represent .dbd.O;
[0119] (f) R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W'
and W" represent hydroxyl, Z and Z' represent hydrogen, and X and
Y, X', and Y', X' and Y, or X and Y' together form a 6 member
heterocyclic ring containing one or two of O, S, or N.
[0120] Particularly preferred compounds of the invention are
compounds of the formula I where:
[0121] 1. One of Y and Y' and one of Z and Z' represents alkyl,
aryl, alkoxy, hydroxyl, thiol, thioalkyl, benzyl, pyridinyl, or
--CH2OR.sup.52 where R.sup.52 represents alkyl or aryl, which may
be substituted, and the other of Y and Y' and Z and Z' represents
hydrogen.
[0122] 2. R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W'
and W" represent hydroxyl, X, X', and Z' represent hydrogen, and
one of Y and Y' represents methyl, ethyl, phenyl, or benzyl which
may be substituted, preferably trifluoromethyl, hydroxymethyl, and
benzyloxymethyl, and the other of Y and Y' represents hydrogen;
[0123] 3. R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, and W
represents hydroxyl, and W' and W' represent halogen, preferably
fluoro; X, X', Z and Z' represent hydrogen, and one of Y and Y'
represents methyl, ethyl, phenyl, or benzyl, which may be
substituted, preferably trifluoromethyl, and hydroxymethyl, and
benzyloxymethyl and the other Y and Y' represents hydrogen;
[0124] 4. one of Y and Y' is hydrogen and the other of Y and Y' is
methyl, and one of Z and Z' is hydroxymethy, --COCH.sub.2CH.sub.3,
--CN, --CH.sub.2NH.sub.2, --CH.sub.2NHAc, or
--CH.sub.2NHCR.sup.60.dbd.NH where R.sup.60 is alkyl or aryl;
[0125] 5. one of Z and Z' is --CONR.sup.70R.sup.71 are the same or
different and represent hydrogen, alkyl, or aryl, --COOH,
--COOC.sub.2H.sub.5, methyl, or CH.sub.2OH, or Z and Z' together
form a spiro ring; or
[0126] 6. X and Y form a carbocyclic or heterocyclic ring of the
formula R.sup.75 R.sup.76 R.sup.77 R.sup.78 R.sup.79 where R.sup.75
and R.sup.76 are part of the swainsonine skeleton and one or more
of R.sup.76, R.sup.77, and R.sup.78 represent CH, CH.sub.2, O, S,
or N.
[0127] Selected compounds of the formula I are the following:
[0128] (5R)-5-methylswainsonine, (5R)-5-methylswainsonine formate
salt, (5S)-5-methylswainsonine, (5R)-8-Epi-5-methylswainsonine,
(5S)-5-ethylswainsonine,
(5S,6S)-6-hydroxymethyl-5-methylswainsonine;
(5R)-5-benzyloxymethylswainsonine,
(5R,6R)-6-hydroxymethyl-5-methylswains- onine, (5R) 5
hydroxymethylswainsonine, (5S)-5-hydroxymethylswainsonine,
(5R,6R)-6-hydroxymethyl-5-methyl swainsonine,
(5S)-5-benzyloxymethylswain- sonine, ethyl
(1R,2R,5S,6S,8S,8aR)-1,2,8-trihydroxy-5-methyloctahydro-6-in-
dolizinecarboxylate, 35
[0129] It will be appreciated that, owing to the asymmetrically
substituted carbon atoms in formula I, a compound of formula I may
exist in, and be isolated in, optically active and racemic forms.
It is to be understood that the present invention encompasses a
compound of formula I as a mixture of diastereomers, as well as in
the form of an individual diastereomer, and that the present
invention encompasses a compound of formula I as a mixture of
enantiomers, as well as in the form of an individual enantiomer. It
will be appreciated that the (S)-isomer and the (R)-isomer are
convertible by facile epimerization of the chiral centers, and that
a preparation containing compound of formula I as a mixture of the
(S)- and (R)-isomers of the formula I is within the scope of the
invention.
[0130] Therefore, the present invention contemplates all optical
isomers and racemic forms thereof of the compounds of the invention
and the formulas of the compounds shown herein are intended to
encompass all possible optical isomers of the compounds so
depicted.
[0131] The present invention also contemplates salts and esters of
the compounds of the formula I of the invention. In particular, the
present invention includes pharmaceutically acceptable salts. By
pharmaceutically acceptable salts is meant those salts which are
suitable for use in contact with the tissues of human and lower
animals without undue toxicity, irritation, allergic response and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art
and are described for example, in S. M. Berge, et. al., J.
Pharmaceutical Sciences, 1977, 66:1-19. Representative acid
addition salts include acetate, adipate, alginate, ascorbate,
aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphersulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, fumarate,
glucoheptonate, glycerophosphate, hemisulfate, heptonate,
hexanoate, hydrobromide, hydrochloride, hydroiodide,
2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl
sulfate, malate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate,
palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, stearate, succinate,
sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate,
valerate salts, and the like. Representative alkali or alkaline
earth metal salts include sodium, lithium, potassium, calcium,
magnesium, and the like, as well as nontoxic ammonium, quarternary
ammonium, and amine cations, including, but not limited to
ammonium, tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, triethylamine, ethylamine, and the
like.
II. Processes for Preparing Compounds
[0132] The compounds of the formula I of the present invention can
be prepared by utilizing procedures and techniques well known and
appreciated by one of ordinary skill in the art. By way of
illustration, descriptions of some methods that may be used to
prepare compounds of the formula I of the invention are set forth
herein.
[0133] Compounds of the Formula I wherein R.sup.1, R.sup.2, and
R.sup.3 represent hydrogen, W, W' and W" represent hydroxyl, and X,
X', Z and Z' represent hydrogen may be synthesized in a variety of
ways by adapting common synthetic organic chemistry practices to
known synthetic intermediates. For example, as shown in schematic
form in FIG. 1, the known (B. P. Bashyal, Tetrahedron,
43(13):3083-3093 (1987) azido alcohol 2 can be oxidized to provide
the corresponding aldehyde which is then reacted (Wittig reaction)
with a variety of commercially available or custom synthesized
phosphoranes or phosphonate derivatives (3 and 4 respectively) to
give .alpha.,.beta.-unsaturated ketone of the type 5 where the
group X can be any chemical group such as methyl, chloromethyl,
trifluoromethyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl,
sec-butyl, tert-butyl, cyclobutyl, pentyl, hexyl, phenyl, benzyl,
etc. Catalytic hydrogenation of 5 followed by removal of the
isopropylidene protecting group under standard conditions then give
the desire 5-substituted swainsonine derivatives. Further
functional group manipulations such as oxidation, reduction,
nucleophilic additions or substitutions etc. of the new
substituents can further increase the number of derivatives from
the limited number of Wittig reagents originally used to construct
the core molecule. Alternatively, reduction of the ketone 5 to the
corresponding amino alcohols 6 can give mixtures of 5- and 5'
substituted swainsonine derivatives after a Mitsunobu style
cyclization reaction to close the 6-membered ring, followed by the
usual catalytic hydrogenation to cyclize the 5-membered ring, and
final isopropylidene removal FIG. 2). The concept of displacing a
leaving group, generated from an alcohol function, with an amine as
in 6 in order to cyclize the 6-membered ring of swainsonine can be
performed by a variety of strategies that include, for example,
protection of the amino group with a benzyloxycarbonyl (CBZ) group
followed by mesylation of the free hydroxyl and nucleophilic
displacement of the mesyl group by the amine generated during a
catalytic hydrogenation step that simultaneously cyclizes both
rings into the required compounds of the formula I, and which is
apparent to anyone skilled in the art of synthetic organic
chemistry.
[0134] There are many possible synthetic routes that can be used to
prepare 5, 5'-disubstituted swainsonine analogues using some of the
concepts described above. For example, reduction of the azido group
in 2 followed by protection of the resulting amine with a suitable
protecting group such as fluorenylmethoxycarbonyl (Fmoc) gives the
alcohol 7. Oxidation followed by a Wittig reaction using
phosphoranes or phosphonates as described above gives the ketone 8.
Selective reduction of the alkene with palladium oxide and
subsequent removal of the amine protecting group using piperidine
for example, leads to the formation of a cyclic imine which can be
reacted with any nucleophile to provide the 5,5'-disubstituted
swainsonine analogues after final catalytic hydrogenation and
isopropylidene removal (FIG. 3B).
[0135] Alternatively, substituents at the 5 position of swainsonine
can be introduced by reacting the known amide 9 with suitable
Wittig reagents. Further modifications such as oxidative
hydroboration or simple reduction of the new alkene 10 can also
give a number of new 5-substituted derivatives (FIG. 4).
[0136] 3,5-disubstituted swainsonine analogues i.e. compounds of
the Formula I wherein R.sup.1, R.sup.2, and R.sup.3 represent
hydrogen, W, W' and W" represent hydroxyl, and Z and Z' represent
hydrogen may also be prepared by a variety of methods. For example,
temporary protection of the amine 11 with an Fmoc group followed by
mild reduction of the anomeric benzyl group under neutral
conditions gives the corresponding hemiacetal 12 which then
spontaneously temporarily generates a cyclic iminium complex 13
upon deprotection of the amine using a mild base such as piperidine
which then quickly reacts with any nucleophile present to give the
protected 3,5-disubstituted swainsonine analogue 14. Mild acid
treatment then removes the isopropylidene protecting group to give
the desired 3,5-disubstituted swainsonine analogues (FIG. 3A).
[0137] Synthetic routes for preparing compounds of the formula I
wherein R.sup.1, R.sup.2, and R.sup.3 represent hydrogen, W, W' W"
represent hydroxyl, and Z and Z' represent hydrogen; R.sup.1,
R.sup.2, and R.sup.3 represent hydrogen, W, W' W" represent
hydroxyl, and X and X' represent hydrogen; and R.sup.1, R.sup.2,
and R.sup.3 represent hydrogen, and W, W' and W" represent
hydroxyl, and Z and Z' represent hydrogen; R.sup.1, R.sup.2, and
R.sup.3 represent hydrogen and W, W' W" represent hydroxyl, are
shown in FIG. 3A; FIGS. 4, 5 and 6; and FIG. 7, respectively.
[0138] Reactive groups used for preparing the compounds of the
invention, may be blocked using appropriate protective groups.
Appropriate blocking and deblocking schemes are known to the
skilled artisan (See T. W. Greene, Protective Groups in Organic
Synthesis, John Wiley & Sons, N.Y., 1981). In general,
particular protective groups are selected which adequately protect
the reactive groups in question during subsequent synthetic steps
and which are readily removable under conditions which will not
cause degradation of the desired product. By way of example,
ethers, acetals, ketals, and esters can be used to protect isolated
hydroxyl groups. In particular, suitable protective groups which
may be used in the process of the invention include O-benzyl,
O-p-methoxybenzyl, O-acetoxy, O-haloacetoxy, O-benzoyloxy, and
O-allyl. Removal of the protective groups may be carried out using
procedures known in the art. For example, a p-methoxybenzyl group
may be removed using ceric ammonium nitrate in acetronitrile and
water.
[0139] Appropriate methods for replacing a free hydroxyl group by
alkoxy, halo, or amino in blocked/deblocked compounds to produce
compounds where for example W, W', and/or W' are alkoxy, aryloxy,
halo or amino, are well known to the skilled artisan. In
particular, a free hydroxyl group may be converted to an alkoxy or
aryloxy group by reacting with alkyl or aryl halide in the presence
of a base. To replace the free hydroxyl by a halo group, the
compound with the free hydroxyl is first reacted with triflic
anhydride, mesyl chloride or tosyl chloride, in the presence of a
base like pyridine, to block the hydroxyl with a leaving group such
as triflate, mesyl, or tosyl, respectively. the blocked hydroxyl is
then replace by O-benzoate, with inversion, by treatment with
sodium benzoate in dimethyl formamide (DMF). The O-benzoate is then
de-esterified, blocked again by a suitable leaving group like
triflate which, on treatment with tetrabutylammonium halide, is
replaced, with inversion, by the respective halide, or on treatment
with sodium azide is replaced, with inversion, by azido, and
subsequently by amino on reduction.
[0140] An alkoxy group may be added by dissolving a compound with a
free hydroxyl in DMF and adding it to a flask under an inert
atmosphere containing a base (e.g. sodium hydride) at low
temperature (0.degree. C. to 10.degree. C.) After stirring a few
minutes benzyl bromide in DMF is added dropwise at low temperature,
for example 0.degree. C. to 10.degree. C. The reaction mixture is
further stirred at room temperature for 2 24 hours. Conventional
work-up of the reaction mixture yields the alkoxy (benzyl)
compound.
[0141] A halo group, for example, fluoro, may be added by
dissolving a compound with a free hydroxyl in dichloromethane (DCM)
together with a base like pyridine. After cooling at low
temperature (-10.degree. C. to -60.degree. C.), an appropriate
amount of triflic anhydride, or mesyl chloride, or tosyl chloride
is added dropwise. The reaction is allowed to stir at a temperature
between 0.degree. C. to 25.degree. C. Conventional work-up of the
reaction mixture yields the esterified compound. Treatment of this
derivative with sodium benzoate in DMF is carried out immediately,
which replaces the leaving group with O-benzoate with inversion.
The free hydroxyl is generated by treatment with a base (e.g.
sodium methoxide) and then reblocked by a suitable leaving group
such as triflate (repeating the above described method). To obtain
a fluoro derivative with inversion, the triflate is treated with
anhydrous tetraalkylammonium fluorides (preferably tetra n-butyl)
or potassium fluoride in a suitable solvent (e.g. diethyl ether,
tetrahydrofuran or crown ether).
[0142] For the introduction of an amino group, the triflate may be
treated either with sodium azide or benzyl amine in DMF. The
product may be obtained with an azido or benzyl amine group, with
inversion, which on reduction with palladium on carbon in a
hydrogen atmosphere gives the free amino group.
[0143] Appropriate methods for introducing a thiol group in
compounds of the formula I (e.g. where W, W', W", X, X', X', Y, Y',
Z, and/or Z' are thiol) are well known to the skilled artisan. For
example, a thiol group may be added by nucleophilic substitution of
an alkyl halide or sulfonyl ester for example using sodium
sulfhydridio (NaSH) or, by nucleophilic substitution of a halide or
sulfonate ester using thioacetic acid to give a thioacetate group
which can then be deblocked to a free thiol upon treatment with
sodium methoxide in methanol by converting the same to a Bunte salt
using thiosulfate (S.sub.2O.sub.3.sup.2-) and later hydrolyzing the
Bunte salt with an acid or, by treating the hydroxyl group with a
fluoropyridinium salt and N,N-dimethyl thiocarbamate (Hojo:
Yoshino: Mukaiyama Chem. Lett. (1977) 133:437) or, by oxidizing a
hydroxyl to a ketone then converting it to a thioketone with
Lawson's reagent and reducing to a thiol with sodium borohydride.
For a review, see (Wardell, in Patai "The Chemistry of the thiol
Group, pt 1: Wiley: New York, 1974, pp. 179-211).
[0144] Methods for introducing a thioalkyl or a thioaryl group in
compounds of the formula I (e.g. where W, W', W", X, X', X', Y, Y',
Z, and/or Z' are thioalkyl or thioaryl) are well known to the
skilled artisan. For example, by nucleophilic substitution of an
alkyl halide or sulfonyl ester for example with alkyl or aryl
thiolate salts or with alkyl or aryl thiols in the presence of a
base such as 1,8-diazabicyclo[5.4.0] undecene (DBU), by alkylating
thiols with alkyl or aryl halides or sulfonate esters or, by
treating a hydroxyl group with an alkyl or aryl halide in the
presence of tetramethyl thiourea followed by sodium hydride
(Fujisaka; Fujiwara; Norisue; Kajigaeshi Bull. Chem. Soc. Jpn.
1985, 58:2529) or, by treating an alcohol with tributyl phosphine
and N (thioaryl)succinimide in benzene (Waters Tetrahedron Lett.
1977, p. 4475 and references cited within). For a review, see
Peach, in Patai "The Chemistry of the Thiol Group, pt 1: Wiley:
N.Y. 1974, pp 721-735.
[0145] In addition, appropriate methods for replacing a blocked or
deblocked hydroxyl group with a hydrogen in compounds of the
formula I are well known to the skilled artisan. For example, alkyl
halides or sulfonyl esters such as tosylates can be selectively
reduced with lithium aluminum hydride or a variety of other metal
hydride reducing agents in different solvents such as
III. Utility of Compounds of the Invention
[0146] the compounds of the formula I are inhibitors of
oligosaccharide processing and in particular are inhibitors of
mannosidase. General mannosidase inhibition may be tested by
measuring the inhibition of Jack Bean, .alpha.mannosidase, and
lysosomal .alpha.-mannosidase. Mannosidase inhibition may also be
tested using an L-PHA toxicity assay. The assay is based on the
finding that the specific binding of the toxic plant lectin L-PHA
to transformed cell lines such as MDAY-D2 tumor cells is a specific
measure of inhibition of oligosaccharide processing. The
measurement of IC.sub.50 in the L-PHA toxicity assay reflects the
ability of the compound to enter into cells and to effect
inhibition of oligosaccharide processing. It is a general screen
for activity in cells which measures cell entry, inhibition of the
target enzyme, .alpha.-mannosidase II in the Golgi, and the
resulting cellular phenotype.
[0147] Therefore, a compound of the invention may be tested for its
ability to inhibit N-linked oligosaccharide processing by growing
transformed cells in the presence of L-PHA and the compound;
measuring the amount of proliferation of the cells; and determining
the ability of the compound to inhibit N-linked oligosaccharide
processing by comparing the amount of proliferation of the cells
with the amount of proliferation observed for the cells grown in
the presence of L-PHA alone.
[0148] Transformed cells which may be used in this assay include
MDAY-D2, L1210, CHO, B16, melanoma tumor cells, and human tumor
cells such as SW 480, LS174T, HT-29, WiDr, T2, MDA-231, MCF7,
BT-20, Hs578T, K562, Hs578T, SK-BR-3, CY 6T, MDA-468, 1123, 11157,
11358, 111334, 111155, 1128, H460, Hmosol, H187, H510A, N417, H146,
H1092, H82 (Restifo, N. P. et al, J. Exper. Med. 177:265 272,
1993).
[0149] The amount of proliferation of the cells may be measured
using conventional techniques. For example, cell proliferation may
be measured by measuring incorporation of labeled thymidine. More
particularly, radioactively labeled thymidine may be added for
about 2-5 hours, preferably 3-4 hours and the cells can be
harvested and radioactivity counted using a scintillation
counter.
[0150] the conditions for carrying out the above assay will be
selected having regard to the nature of the compound and the cells
employed. For example, if the transformed cells are MDAY-D2 tumor
cells a concentration of about 1-4.times.10.sup.4 cells, preferably
2.times.10.sup.4 may be used. The MDAY-D2 cells are generally
cultured for about 10 to 30 hours, preferably 18 to 20 hours,
followed by addition of L-PHA at a concentration of about 10-50
.mu.g/ml, preferably 20-30 .mu./ml, most preferably 25
.mu.g/ml.
[0151] The following L-PHA assay may be used to assay for
inhibition of oligosaccharide processing(i.e. Golgi
.alpha.-mannosidase II) in viable cells. MDAY-D2 tumor cells are
inoculated into 96 well micro-test plates at 2.times.10.sup.4
cells/well, containing serial dilutions of the compound to be
tested in MEM plus 10% FCS. The cells are cultured for 18-20 hours,
followed by the addition of L-PHA at 25 .mu.g/ml for an additional
24 hours. Cell proliferation is measured by adding 0.5 .mu.Ci/well
of .sup.3II-thymidine for 3-4 hours, harvesting onto glass fibre
disks using a Titertek harvester, and counting the disks in a
liquid scintillation counter. The apparent IC.sub.50 values for the
test compounds are the drug concentrations showing 50% protection
from L-PHA toxicity; that is 50% .sup.3H-thymidine incorporated
compared with cells grown in the absence of L-PHA.
[0152] The ability of the compounds of the formulae I in which the
free hydroxyls have been esterified, to be converted into more
active compounds in cells can be measured by performing the L-PHA
toxicity assay in the presence of an esterase inhibitor such as
diethyl p-nitrophenyl phosphate. For example, the esterase
inhibitor diethyl p-nitrophenyl phosphate can be added to MDAY-D2
cells in the above described assay method about 4 hours prior to
the .alpha.-mannosidase inhibitors. An increase in IC.sub.50 in the
L-PHA toxicity assay in the presence of diethyl p-nitrophenyl
phosphate indicates that the compound requires activation by
esterases and would accordingly be useful as a prodrug. This method
may be used to screen for prodrugs and can be used to identify
substances which inhibit all steps in the N-linked oligosaccharide
pathway prior to .beta.-4 Gal-transferase.
[0153] The compounds of formula I have valuable pharmacological
properties and they provide immunostimulatory, antimicrobial and
cancer suppressing effects. In particular, the compounds are useful
in the prevention, treatment and prophylaxis of tumor growth and
metastasis of tumors. The anti-metastatic effects of the compounds
of the invention may be demonstrated using a lung colonization
assay. for example, melanoma cells treated with a compound may be
injected into mice and the ability of the melanoma cells to
colonize the lungs of the mice may be examined by counting tumor
modules on the lung after death. Suppression of tumor growth in
mice by the compound administered orally or intravenously may be
examined by measuring tumor volume.
[0154] The compounds of the formula I have particular application
in the prevention of tumor recurrence after surgery i.e. as an
adjuvant therapy.
[0155] The compounds of the invention are especially useful in the
treatment of various forms of neoplasia such as leukemias,
lymphomas, melanomas, adenomas, sarcomas, and carcinomas of solid
tissues in patients. In particular the composition may be useful
for treating malignant melanoma, pancreatic cancer, cervico-uterine
cancer, cancer of the kidney, stomach, lung, rectum, breast, bowel,
gastric, liver, thyroid, neck, cervix, salivary gland, leg, tongue,
lip, bile duct, pelvis, modiastinum, urethra, bronchogenic,
bladder, esophagus and colon, and Kaposi's Sarcoma which is a form
of cancer associated with HIV-infected patients with Acquired
Immune Deficiency
[0156] The compounds of the formula I may be used to stimulated
bone marrow cell proliferation, and they may be used as
hemorestorative agents, in particular following chemotherapy or
radiotherapy. The myeloproliferative activity of a compound of the
formula I may be determined by injecting the compound into mice,
sacrificing the mice, removing bone marrow cells and measuring the
ability of the compound to stimulate bone marrow proliferation by
directly counting bone marrow cells and by measuring clonogenic
progenitor cells in methylcellulose assays.
[0157] The compounds of the invention are immune modulators and in
particular they have immunostimulatory properties. Therefore, the
compounds of the formula I may be used in cases where a patient has
been immunocompromised such as patients infected with HIV, or other
viruses or infectious agents including bacteria and fungi, in
patients undergoing bone marrow transplants, and in patients with
chemical or tumor-induced immune suppression.
[0158] The compounds also have an antiviral effect in particular on
membrane enveloped viruses such as retroviruses, influenza viruses,
cytomegaloviruses and herpes viruses. The compounds of the
invention may also be used in the treatment of inflammation.
[0159] The following compounds of the invention show significant
inhibition of Golgi .alpha.-mannosidase II activity over lysosomal
mannosidase activity:
[0160] (a) Compounds of the formula I wherein R.sup.1, R.sup.2, and
R.sup.3 represent hydrogen, W, W' and W" represent hydroxyl, Y, Y',
Z and Z' represent hydrogen , and one of X and X' represents methyl
, phenyl, or benzyl which may be substituted, preferably
fluoromethyl or hydroxymethyl, and the other of X and X' represent
hydrogen;
[0161] (b) Compounds of the formula I where R.sup.1, R.sup.2, and
R.sup.3 represent hydrogen, W, W' and W" represent hydroxyl, X, X',
Z and Z' represent hydrogen, and one of Y and Y' represents methyl,
ethyl, phenyl, or benzyl which may be substituted, preferably
trifluoromethyl, hydroxymethyl, and benzyloxymethyl, and the other
of Y and Y' represents hydrogen; nd
[0162] (c) Compounds of the formula I wherein R.sup.1,R.sup.2, and
R.sup.3 represent hydrogen, W, W' and W" represent hydroxyl, and Y
and Y' represent hydrogen, X and X' are the same or different and
represent hydrogen, alkyl, aryl, alkoxy, hydroxyl, thiol,thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
benzyl, or pyridinyl, or --CH.sub.2OR.sup.52 where R.sup.52
represents alkyl, or aryl, alkoxy, hydroxyl, thiol, thioalkyl,
thioaryl, amino, halogen, carboxylic acid esters, thiol esters,
benzyl, or pyridinyl, or CH.sub.2OR.sup.52 where R.sup.52
represents alkyl or aryl, with the proviso that at least one of X
and X' and at least on of Z an Z' cannot be hydrogen.
[0163] Particularly preferred compounds are
(5S)-5-(hydroxymethyl)swainson- ine, (5S)-5-methylswainsonine,
(5S)-5-ethylswainsonine, (5R)-5-methylswainsonine,
(5S)-5-benzyloxymethylswainsonine,
(5R)-5-benzyloxymethylswainsonine, or
(5)-5-hydroxymethyl)swainsonine. These compounds are particularly
well suited for incorporation into pharmaceutical compositions for
use in treating the conditions mentioned herein since they have
improved pharmacological properties and selectivity avoid the
clinical side which can be exhibited by swainsonine.
[0164] The term "patient" refers to a warm-blooded animal such as a
mammal which is afflicted with a particular disease state or
condition as described herein. Examples of the animals within the
scope of the meaning of the term are dogs, cats, rats, mice,
horses, bovine cattle, sheep, and humans.
[0165] The compounds of the formula I may be converted using
customary methods into pharmaceutical compositions. The
pharmaceutical compositions contain the compounds either alone or
together with other active substances. Such pharmaceutical
compositions can be for oral, topical, rectal, parental, local,
inhalant, or intracerebral use. They are therefore in solid or
semisolid form, for example pills, tablets, creams, gelatin
capsules, capsules, suppositories, soft gelatin capsules, liposomes
(see for example U.S. pat. Ser. No. 5,376,452), gels, membranes,
and tubelets. For parenteral nd intracerebral uses, those forms for
intramuscular or subcutaneous administration can be used, or forms
for infusion or intravenous or intracerebral injection can be used,
and can therefore be prepared as solutions of the compounds or as
powders of the active compounds to be mixed one or more
pharmaceutical acceptable excipients or diluents, suitable for the
aforesaid uses and with an osmolarity which is compatible with the
physiological fluid. For local use, those preparations in the form
of creams or ointments for topical use or in the form of sprays
should be considered;for inhalant uses, preparation in the form of
sprays, for example nose sprays, should be considered.
[0166] The pharmaceutical compositions can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to patients, and such that
an effective quantity of the active substance is combined in a
mixture with a pharmaceutically acceptable vehicle. Suitable
vehicles are described, for example, in Remington's Pharmaceutical
Sciences (Remington's Pharmaceutical Science, Mack Publishing
Company, Easton, Pa., USA 1985). On this basis, the pharmaceutical
compositions include, albeit not exclusively, the compounds in
association with one or more pharmaceutically acceptable vehicles
or diluents, and contained in buffered solutions with a suitable pH
and iso osmotic with the physiological fluids.
[0167] The compounds are indicated as therapeutic agents either
alone or in conjunction with other therapeutic agents or other
forms of treatment (e.g. chemotherapy or radiotherapy). For
example, the compounds may be used in combination with
anti-proliferative agents, antimicrobial agents, immunostimulatory
agents, or anti-inflammatories. In particular the compounds may be
used in combination with anti-viral and/or anti-proliferative
agents such as interferons. The compounds of the invention may be
administered concurrently, separately, or sequentially with other
therapeutic gents or therapies.
[0168] In general, a dosage range of the compounds in the
composition is envisaged for administration in human medicine of
form about 0.001 to 50 mg/kg of body weight daily. In the case of
intravenous compositions, the dosage is for example about .1 to 0.6
mg/kg/day, and for oral composition the dosage is about 0.5 to 6
mg/kg/day.
[0169] Amounts of drug administered to produce serum levels
10-1000.times. the IC.sub.50 for inhibition of oligosaccharide
processing in the L-PHA assay are preferably employed.
[0170] It will also be appreciated that it may be necessary to
deviated from the amounts mentioned and in particular to do so as a
function of the body weight of the animal to be treated, the
particular disease to be treated, the nature of the administration
route and the therapy desired. In addition , the type of animal and
its individual behavior towards the medicine or the nature of its
formulation and the time or interval at which it is administered
may also indicate use of amounts different form those mentioned.
Thus it may in suffice, in some cases, to manage with less than the
above-mentioned minimum amounts whilst in other cases the upper
limit mentioned must be exceeded. Where major amounts are
administered, it may be advisable to divide these into several
administrations over course of the day.
[0171] The following non-limiting examples are illustrative of the
present invention:
EXAMPLES
Example 1
Synthesis of 5-Substituted Swainsonine Analogues
[0172] A. Synthesis of (5S)-5-Methylswainsonine(GD28) and
5(R)-5-Methylswainsonine(GD20) 36
[0173] Benzyl
4-azido-4,6,7,9-tetradeoxy-2,3-O-isopropylidene-.alpha.
-D-manno-nona-6-enopyranoside-8-ulose (GDLZ1).
[0174] Pyrdinium chlorochromate (776 mg. 3.6 mmol) was added to a
vigorously stirred suspension of flame-dried 3.lambda. molecule
sieves (3 g) and benzyl
4-azido-2,3-O-isopropylidene-.alpha.-D-mannopyranoside (500 mg.
1.49 mmol) in anhydrous methylene chloride (50 mL). After 30 min
the oxidation was complete, the slurry was loaded on top of a
silica gel column (50 g) and the product eluted with 1:1 ethyl
acetate:hexanes. Aldehyde-containing eluent was concentrated, the
residue dissolved in anhydrous benzene (30 mL), the solution cooled
to 0.degree. c., and triphenylphosphoranylidene-2-propanone (1.2 g
3.77 mmol) in anhydrous tetrahydrofuran (30 mL) added. The solution
was stirred at room temperature overnight. The reaction mixture was
concentrated to dryness, the residue chromatographed (16 g silica
gel, 1:3 ethyl acetate:hexanes), and gave GDLZ1 (340 mg, 61%) as a
spontaneously crystallizing syrup.
[0175] Benzyl
4-azido-4,6,7,9-tetradeoxy-2,3-O-isopropylidene-D/L-glycero--
D-manno-.alpha.-nona-6-enopyranoside (GDLZ18).
[0176] Sodium borohydride (150 mg) was added to a stirred solution
of crude GDLZ17 (1.46 g, .about.3.91 mmol) in 1.1 methylene
chloride:methanol. After 30 min the reaction was complete, the
solution was concentrated to dryness, the residue chromatographed
(34 g silica gel, 1:2 ethyl acetate:hexanes), and gave GDLZ18 (1.38
g, 94%).
[0177] Benzyl
4-(N-benzyloxycarbonyl)amino-4,6,7,9-tetradeoxy-2,3-O-isopro-
pylidene-D/L-glycero-D-manno-.alpha.-nonapyranoside (GDLZ21).
[0178] A suspension of GDLZ21 (550 mg, 1.47 mmol) and 10% palladium
on charcoal (50 mg) in 95% ethanol was stirred under hydrogen of
atmospheric pressure overnight. The catalyst was filtered off,
sodium hydrogencarbonate (0.5 g) was added, and to the stirred
suspension benzyl chloroformate (0.5 mL) was added in portions over
a period of 2 h. The mixture was filtered, the filtrate
concentrated, and chromatographed (2:3 ethyl acetate:hexanes). The
product (649 mg, 90%) was obtained as a slowly crystallizing
syrup.
[0179] Benzyl
4-(N-benzyloxycarbonyl)amino-4,6,7,9-tetradeoxy-2,3-O-isopro-
pylidene-8-O-tosyl-D/L-glycero-D-manno-.alpha.-nonapyranoside
(GDLZ22).
[0180] 4-Toluenesulfonyl chloride (140 mg, 0.73 mmol) was added to
a solution of GDLZ21 (43 mg, 88 .mu.mol) in anhydrous pyridine and
left stirring overnight. The solution was cooled to 0.degree. C.
and excess reagent hydrolyzed with water (1 mL). The solution was
transferred into water (50 mL) and the product extracted with ethyl
acetate (4.times.10 mL). The combined extracts were neutralized
with NaHCO.sub.3 solution, washed with water, dried, and
concentrated. The residues was purified by column chromatography
(10 g silica gel, 2:3 ethyl acetate:hexanes) and gave GDLZ22 (49
mg, 87%) as a colourless syrup.
[0181] Benzyl
4,6,7,8,9-pentadeoxy-4,8-(N-benzyloxycarbonyl)amino-2,3-O-is-
opropylidene-L-and -D-glycero-D-manno-.alpha.-nonapyranoside
(GDLZ29A and GDLZ29B).
[0182] Solid potassium tert-butoxide (100 mg) was added to a
stirred solution of GDLZ22 (380 mg, 594 .mu.mol) in anhydrous
tetrahydrofuran (20 mL). Overnight the reaction was complete. The
suspension was concentrated, the residue transferred into water (50
mL) and the products extracted with ethyl acetate (4.times.20 mL).
The combined extracts were washed with water, dried, and
concentrated. The isomeric mixture was resolved and the isomers
purified by column chromatography (18 g silica gel, 1:4 ethyl
acetate:hexanes). The faster migrating isomer (GDLZ29 A, 78.5 mg,
28%), the slower migrating isomer (GDLZ29B, 87.2 mg, 31%), and
unresolved mixture (47.3 mg, 17%) were obtained as colourless
syrups.
[0183] (5S)-1,2-O-Isopropylidene-5-methylswainsonine (GDLZ27).
[0184] Palladium on charcoal (10%, 80 mg) was added to a solution
of compound GDLZ29A (84 mg, 180 mmol) in 95% ethanol (6 mL), and
the suspension stirred under hydrogen of atmosphere pressure. After
1.5 h the starting material was consumed, indicating that the first
step of reduction (removal of the benzylcarbamate) was complete.
The suspension was then acidified by addition of 1M hydrochloric
acid (180 .mu.l) and further hydrogenolized overnight. Ion-exchange
resin (Dowex 1.times.8 100, OH.sup.--form, 1 g) was added, stirred
for 5 min, and filtered off together with the catalyst. The
filtrate was concentrated and gave the crude acetonide (38 mg,
.about.92%).
[0185] (5S)-5-Methylswainsonine (GD 28).
[0186] Compound GDLZ27 (25 mg, 110 mmol) was dissolved in aqueous
70% trifluoroacetic acid (3 mL) and stirred at room temperature
overnight. The solution was concentrated to dryness, the residue
dissolved in water (0.5 mL), and the free base generated by
addition of a strongly basic ion-exchange resin (BioRad, AG 1-X8
20-50 mesh, OH.sup.--form). The solution was removed from the
resin, and the resin extracted with water (5.times.0.5 mL). The
extracts were pooled, filtered and freeze-dried. The product was
obtained as a white solid (13.0 mg, 63%).
[0187] (5R)-1,2-O-Isopropylidene-5-methylswainsonine (GDLZ34).
[0188] Palladium on charcoal (10%, 37 mg) was added to a solution
of compound GDLZ29B (210 mg, 0.45 mmol) in 95% ethanol (14 mL), and
the suspension stirred under hydrogen of atmosphere pressure. After
3 h the starting material was consumed. The suspension was
acidified by addition of 1M hydrochloric acid (450 ml) and further
hydrogenolized for 3 days. Ion-exchange resin (Dowex 1.times.8 100,
OH.sup.--form, 3 g) was added and stirred for 5 min. The suspension
was filtered and the filtrate concentrated to give the crude
acetonide (136 mg, quantitative).
[0189] (5R)-5-Methylswainsonine Formate Salt (GD 19). 37
[0190] (5R)-1,2-O-Isopropylidene-5-methylswainsonine (63 mg, 0.28
mmol) was dissolved in 50% aqueous trifluoroacetic acid and the
solution stirred at room temperature for three days. The solution
was concentrated, and the residue purified by reversed phase HPLC
using a 20 mM ammonium formate buffer (pH 3.5). Product-containing
fractions were freeze-dried several times to remove all traces of
buffer and yielded (5R)-5-methylswainsonine as the formate salt
(GD19).
[0191] (5R)-5-Methylswainsonine (GD 20).
[0192] An aqueous solution of GD19 was eluted through a column of
freshly regenerated basic ion-exchange resin (DOWEX 1.times.8-100
mesh, hydroxyl form). Eluent containing the free base (GD20) was
combined and concentrated.
[0193] B. Synthesis of (5S)-5-Ethylswainsonine 38
[0194] 1-Bromo-2-butanol (GDLZ103)
[0195] Carbontetrabromide (26.6 g, 80 mmol) and triphenylphosphine
(21 g, 80 mmol) were added successively to 1,2-butanediol (7.2 g,
80 mmol) in anhydrous pyridine at 0.degree. C., and the solution
stirred at room temperature overnight. The mixture was concentrated
and the residual oil dropwise added to vigorously stirred 1:5 ethyl
acetate:hexanes (220 mL). The solution was decanted from the
precipitate, concentrated, chromatographed (40 g silica gel, 1:3
ethyl acetate:hexanes), and gave GDLZ103 (11.5 g, 90%).
[0196] 2-Butanonyltriphenylphosphonium Bromide (GDLZ105)
[0197] Pyridinium chlorochromatic (20 g, 93 mmol) was added to a
suspension of flame-dried 3 .ANG. molecular sieves (15 g) and
GDLZ103 (11.5 g, 75 mmol) in anhydrous methylene chloride, and the
mixture stirred for 1 h., the brown slurry was loaded on top of a
silica gel column (40 g), and the product eluted using 1:5 ethyl
acetate:hexanes. The ketone-containing eluates were combined,
concentrated, and the residue reacted with triphenylphosphine (12
g, 46 mmol) in chloroform (20 mL). The solution formed overnight
was added slowly into stirred diethyl ether (300 mL) and the
precipitate filtered off. The residue was recrystallized from
methylene chloride (50 mL) by addition of hexanes. GDLZ105 (3.539
g, 11.4%) was obtained as colourless crystals.
[0198] Triphenylphosphoranylidene-2-butanone (GDLZ113)
[0199] Finely powdered GDLZ105 (2.5 g, 6.05 mmol) was added to
aqueous 10% sodium carbonate solution (150 mL), and stirred
vigously overnight. The solid formed was filtered off, thoroughly
washed with water, and dried in vacuo in a desiccator overnight.
GDLZ113 (1.9 g, 94%) was obtained as an electrostatic, off-white
powder.
[0200] Benzyl
4-Azido-4,6,7,9,10-pentadeoxy-2,3-O-isopropylidene-.alpha.-D-
-manno-dec-6-enopyranoside-8-ulose (GDLZ102)
[0201] A slurry of flame-dried 3.ANG. molecule sleves (1 g). benzyl
4-azido-2,3-O-isopropylidene-.alpha.-.GAMMA.)-mannopyranoside (133
mg, 0.04 mmol), and pyridinium chlorochromatc (0.8 g, 3.71 mmol) in
anhydrous methylene chloride (40 ml.) was vigorously stirred for 30
min. The mixture was loaded on top of a silica gel column (16g.)
and the product eluted using 1:1 anhydrous benzene-tetrahydrofuran
(10 mL), and GDLZ113 (180 mg, 0.541 mmol) added. After stirring
overnight the mixture was concentrated, the residue chromatographed
(12 g silica gel, 1:5 ethyl acetate;hexanes), and gave GDLZ102 (95
mg, 61%) as a slightly yellow syrup.
[0202] Benzyl-4-
azido-4,6,7,9,10-pentadeoxy-2,3-O-isopropylidene-D/L-glyc-
ero-D-manno-.alpha.-dec-6-enopyranoside (GDLZ115).
[0203] Sodium borohydride (55 mg. 1.45 mmol) was added to a
solution of GDLZ102 (800 mg. 2,06 mmol) in methanol (25 mL), and
the mixture stirred for 30 min. The solution was concentrated to
dryness, the residue chromatographed (1:5.fwdarw.1:2 ethyl
acetate:hexanes), and gave GDLZ115 (560 mg, 70%) as a colourless
syrup.
[0204]
Benzyl-4-(N-benzyloxycarbonyl)imino-4,6,7,9,10-pentadeoxy-2,3-O-iso-
propylidene-D/L-glycero-D-manno-.alpha.-decopyranoside
(GDLZ116).
[0205] 10% Palladium on charcoal (-100mg) was added to a solution
of GDLZ115 (560 mg, 1.44 mmol) in methanol (25 mL), in ethanol (25
mL), and the mixture stirred under hydrogen of atmospheric pressure
overnight. The catalyst was filtered off, sodium hydrogencarbonate
(0.5 g) was added, and the mixture stirred while benzyl
chloroformate (500 .mu.L) was added in portions via a syringe over
a period of 1 h. The suspension was then filtered, the filtrate
concentrated, and the residue chromatographed (1:2 ethyl
acetate:hexanes). GDLZ116 (500 mg, 69.5%) was obtained as a
colourless foam.
[0206]
Benzyl-4-(N-benzyloxycarbonyl)amino-4,6,7,9,10-pentadeoxy-2,3-O-iso-
propylidene-8-O-tosyl-D/L-glycero-D-manno-.mu.-decopyranoside
(GDLZ117).
[0207] A solution of GDLZ116 (500 mg, 1.02 mmol) and
4-toluenesulfonyl chioride (430 mg, 2.3 mmol) in anhydrous pyridine
(25 mL) was stirred at room temperature overnight. Excess reagent
was hydrolysed by addition of water (1 mL). The solution was
transferred into water (300 mL), and the product extracted using
ethyl acetate (4.times.50 mL). The combined extracts were washed
with 1M hydrochloric acid, neutralised with aqueous sodium
bicarbonate solution, washed with water, dried and concentrated.
The residue was chromatographed (1:2 ethyl acetate:hexanes) and
gave GDLZ117 (586 mg, 89%) as a colourless syrup.
[0208]
Benzyl-4,6,7,8,9,10-hexadeoxy-4,8-(N-benzyloxycarbonyl)imino-2,3-O--
isopropylidene-L- and -D-glycero-D-manno-a-D-decopyranoside
(GDLZ118A and GDLZ118B). was concentrated and the residue
chromatographed (1:4.fwdarw.1:2 ethyl acetate:hexanos). The
products were rechromatographed, and the fast migrating isomer
(GDLZ118A, 130.2 mg, 30%), as well as the slow migrating isomer
(GDLZ118B, 139 mg, 32%) were obtained as colourless syrups.
(5S)-5-Ethyl-1,2-O-isopropylideneswainsoni- ne (GDLZ118A (139 mg,
288 .mu.mol) in 95% ethanol and the mixture stirred under hydrogen
of atmospheric pressure overnight. Tlc showed complete turnover of
the carbamate . The mixture was acidified by addition of 1 M
hydrochloric acid (250 .mu.L) and further hydrogenolysed. Overnight
a new product was former (tlc). The catalyst was filtered off and
the filtrate eluted through a column of basic ion-exchange resin (8
mL, AG 1X8 20-50 mesh, OH.sup.--form). The eluatc containing
GDLZ119 was concentrated and the residue purified by HPLC (50%
MeOH-50% aqueous 20 mM ammonium formate p119.0,7 mL/min, Waters ODS
25.times.100 mm, uv detection 206 nm). The product was obtained as
a formate salt (7 mg, 8.5%).
[0209] (5S)-5-Ethylswainsonine (GD 38).
[0210] A solution of GDLZ119 (7 mg, 29 .mu.mol) in 1:1
tetrahydrofuren:6 M hydrochloric acid (1 mL) was stirred overnight.
The solution was concentrated, the residue taken up in water (1 mL)
and eluted through a column of basic ion-exchange resin (10 mL, OH
.sup.--form). The eluate containing the free base was freeze-dried
and gave GDLZ125 (2,8 mg, 48%) as a colourless form.
C. Synthesis of (5R)- and (5S)-5-(Hydroxymethyl)swainsonine
[0211] 39
[0212] 3-Benzyloxy-1- bromo-2-propanol (GDLZ132)
[0213] Triphenylphosphine (6.82 g, 26 mmol) and carbontetrabromide
(8.63 g, 26 mmol) were added to a solution of
3-benzyloxy-1,2-propanediol (4.72 g, 25.9 mmol) in anhydrous
pyridine at 30.degree. C., and stirred overnight at room
temperature. The solution was concentrated and the residue dropwise
added to vigorously stirred 1:5 ethyl acetate:hexanes (200 mL) to
precipitate triphenylphosphineoxide. The supernatant was decanted,
concentrated, and the residue dropwise added to vigorously stirred
1:5 ethyl acetate:hexanes (200 mL) ti precipitate
triphenylphosphineoxide. The supernatant was decanted,
concentrated, and the residue chromatographed (60 g silica gel, 1:3
ethyl acetate:hexanes). The product was obtained as a colourless
oil (5.1 g, 80%).
[0214] 3- Benzyloxyacetonyl triphenylphosphoniumbromide
(GDLZ135)
[0215] Pyridunium chlorochromate (8.5 g, 39.4 mmol) was added to a
slurry of compound GDLZ132 (5.1 g. 20.8 mmol) and freshly
flame-dried 3 .ANG.molecule sleves (9 g) in anhydrous methylene
chloride, and the suspension stirred under argon at room
temperature. The reaction was complete after 1 h. The brown slurry
was directly loaded on top of a silica gel column (50 g) and the
product eluted using 1:2 ethyl acctare:hexanes. Product-containing
eluent was concentrated. The residue was diluted with chloroform
(10 mL) and reacted with triphenylphosphine (5.5 g) overnight at
room temperature. The thick solution obtained was dropwise diluted
into vigorously stirred diethyl ether (200 mL), and compound
GDLZ135 precipitated as an off-white solid. The product was
filtered by suction and dried in vacuo overnight. The product (7.68
g, 73%) was further reacted without purification.
[0216] 3-Benzyloxy-1-triphenylphosphoranylidene-2-propanone
(GDLZ138)
[0217] Finely powdered compound GDLZ135 (1.0 g, 1.98 mmol) was
added to an aqueous 10% solution of sodium carbonate (30 mL) and
the mixture vigorously stirred at room temperature. Overnight, the
starting material dissolved and the precipitated. The suspension
was filtered by suction and the off-white residue thoroughly washed
with water. The residue was then dried in vacuo over Dricrite. The
product (817 mg, 97%) was used for the next reaction without any
further purification.
[0218] Benzyl (b
4-azido-9-O-benzyl-4,6,7-trideoxy-2,3-O-isopropylidene-.m-
u.-D-manno-nona-6-enopyranoside-8-ulose (GDLZ150)
[0219] A suspension of flame-dried 3 .ANG. molecular sieves (2 g),
pyridinium chiorochromatc (1.5 g, 6.96 mmol), and benzyl
4-azido-2,3-O-isopropylidene-.mu.-D-mannopyranoside (466 mg, 1.39
mmol) was stirred at room temperature for 30 min. The brown mixture
was then loaded on top of a silican gel column (18 g, 1:2 ethyl
acetate;hexanes) and the aldehyde eluted. Product-containing
fractions were combined and concentrated. The residue was dissolved
in anhydrous benzene (20 mL), elide GDLZ138 (1.0 g, 2.3 mmol) was
added, and the mixture stirred at room temperature overnight. The
suspension formed was concentrated, the residue chromatographed (35
g silica gel, 1:3 ethyl acetate:hexanes), and gave GDLZ150 (500 mg,
75%) as a colcurless syrup.
[0220] Benzyl
4-N-(benzyloxycarbonyl)amino-9-O-benzyl-4,6,7-trideoxy-2,3-O-
-isopropylidene-D/L-glycero-D-glycero-D-manno-.mu.-nonapyranoside
IGDLZ166 )
[0221] A solution of GDLZ150 (834 mg, 1.74 mmol) and sodium
borohydride (66 mg, 1.74 mmol) in methanol (20 mL) was stirred at
room temperature for 2h. The solvent was evaporated and the residue
chromatographed (33 g silica gel, 1:3 ethyl acetate hexanes). The
reduced glycoside (554 mg, 66%) was then dissolved in 95% ethanol
(20 mL), 10% palladium on charcoal (.about.50 mg) was added, and
the solution basicified with two drops of trierhylamine. The
mixture was then stirred under hydrogen of atmospheric pressure for
3 h. The catalyst was filtered off, sodium hydrogencarbonate (400
mg) was added to the filtrate and stirred while benzyl
chloroformate (250 .mu.L, 1.75 mmol) was added in portions over a
period of 2 h. Solids were filtered off, the filtrate concentrated
and the residue chromatographed (1:1 EtOAc-hexanes), GDLZ166 (557
mg, 54.8% from GDLZ150) was obtained as a colourless syrup.
[0222] Benzyl
4-N-(benzyloxycarbonyl)amino-9-O-benzyl-4,6,7-trideoxy-2,3-O-
-isopropylidene-8-O-tosyl-D/L-glycero-D-manno-.alpha.-nonapyranoside
(GDLZ166
[0223] 4-Toluenesulfonyl chloride (500 mg, 2.6 mmol) was added to a
solution of GDLZ166 (557 mg, 953 .mu.mol) in anhydrous pyridine (15
mL) and the mixture stirred at room temperature overnight. Excess
reagent was hydrolysed by addition of water (1 mL), and the
solution was transferred into water (200 mL). The product was
extracted using ethyl acetate (3.times.50 mL), the combined
extracts were washed with 1 M hydrochloric acid, neutralised with
sat. aqueous NaHCO.sub.3, washed with water, dried, and
concentrated. The crude product was chromatographed (30 g silica
gel, 1:3.fwdarw.1:2 ethyl acetate:hexanes) and gave pure GDLZ169
(574 mg, 80.8%).
[0224] Benzyl
9-O-benzyl-4,6,7,8-tetradeoxy-4,8-(N-benzyloxycarbonyl)imino-
-2,3-O-isopropylidene-L- and
-D-glycero-D-manno-.alpha.-nonspyranoside (GDLZ170A and
GDLZ170B)
[0225] Potassium tert-butoxide (90 mg, 802 .mu.mol) was added to a
solution of GDLZ169 (574 mg, 770 .mu.mol) in anhydrous
tetrahydrofuran (25 mL), and the mixture stirred at room
temperature under argon overnight. The suspension was transferred
into ethyl acetate (100 mL), washed with water and brine, dried,
and concentrated. The isomeric mixture was purified by column
chromatography (33 g silica gel, 1:3.fwdarw.1:2 ethyl
acetate:hexanes), and the diastereomers resolved using preparative
HPLC (22:78 ethyl acetate:hexanes, 8 mL/min, 25.times.100 mm Waters
Silica). The faster migrating isomer GDLZ170A (141.5 mg, 32%) and
the slower migrating isomer GDLZ170B (157.6 mg, 35.7%) were
isolated as colourless syrups.
[0226] (5R)-5-Hydroxymethyl-1,2-O-isopropylideneswainsonine
(GDLZ162)
[0227] Compound GDLZ170A (38 mg, 66 .mu.mol) was hydrogenated in
95% ethanol using 10% palladium on charcoal (.about.30 mg) and
hydrogen of atmospheric pressure under neutral conditions for the
removal of the carboxybenzyl group. Overnight the reaction was
complete, the mixture was acidified with 1 M hydrochloric acid (80
.mu.L) and further stirred under hydrogen for 3 days. The
suspension was filtered, the filtrate concentrated, and the product
isolated and purified by HPLC (30:70 0.1% NH.sub.3 in MeOH-0.1%
NH.sub.3 in water, 2 mL/min, 10.times.150 mm Beckman Ultrapsphere
ODS). GDLZ162 (7.5 mg, 46.7%) was obtained as a colourless
syrup.
[0228] (5S)-5-Hydroxymethyl-1,2-O-isopropylideneswainsonine
(GDLZ167)
[0229] Compound GDLZ170b (57.6 mg, 100 .mu.mol) was hydrogenated in
95% ethanol using 10% palladium on charcoal (.about.100 mg) and
hydrogen of atmospheric pressure. The carboxybenzyl group was
removed overnight. the mixture was acidified with 1 M hydrochloric
acid (150 .mu.L) and further stirred under hydrogen for 2 days. The
suspension was filtered, the filtrate concentrated, and the product
isolated and purified by HPLC (30:70 0.1% NH.sub.3 in MeOH-0.1%
NH.sub.3 in water, 2 mL/min, 10.times.150 mm Beckman Ultrapsphere
ODS). GDLZ167 (9.0 mg, 37%) was obtained as a colourless syrup.
[0230] (5R)-5-(Hydroxymethyl)swainsonine (GDLZ168/GD45)
[0231] A solution of GDLZ162 (7.1 mg, 29.2 .mu.mol) in 2:1
tetrahydrofuran:6 M hydrochloric acid (1.5 mL) was stirred at room
temperature overnight. The solution was concentrated, the residue
dissolved in water (1 mL), and passed through a column of basic
ion-exchange resin to generate the free base. The crude product was
purified by HPLC (3:97 0.1% NH.sub.3 in MeOH-0.1% NH.sub.3 in
water, 2 mL/min, 10.times.150 mm Beckman Ultrapsphere ODS) and gave
pure GDLZ168 (3.7 mg, 62.3%).
[0232] (5S)-5-(Hydroxymethyl)swainsonine (GD 46)
[0233] A solution of GDLZ167 (9.0 mg, 37 .mu.mol) in 1:1
tetrahydrofuran:6 M hydrochloric acid (2 mL) was stirred at room
temperature overnight. The solution was concentrated, the residue
dissolved in water 1:1 MeOH:water (2 mL), basified using basic
ion-exchange resin, and passed through a C-18 Sep-Pak-column
(Waters). The crude product was purified by HPLC (7:93 0.1%
NH.sub.3 in MeOH-0.1% NH.sub.3 in water, 2 mL/min, 10.times.150 mm
Beckman Ultrapsphere ODS) and gave GDLZ172 (5.7 mg, 75.8%).
Example 2
Synthesis of 5,6-Disubstituted Swainsonine Analogues
[0234]
Benzyl-4-amino-4-deoxy-2,3-O-isopropylidene-.alpha.-D-mannopyranosi-
de (1):
[0235] To a stirred, cooled (ice bath at 0.degree. C.) solution of
benzyl-4-azido-4-deoxy-2,3-O-isopropylidene-.alpha.-D-mannopyranoside
(1.0 g, 3.0 mmol) in dry THF (10 mL) was added LiAlH.sub.4 (140 mg,
3.8 mmol) in small portions. The reaction was then allowed to warm
to room temperature slowly. After four hours, TLC indicated
complete consumption of the starting material and formation of a
single new product. The reaction was quenched with 5% NH.sub.4Cl
(10 mL) and then worked-up by liquid-liquid extraction
(H.sub.2O/CH.sub.2Cl.sub.2). Upon drying (MgSO.sub.4) and
concentrating the combined organic extracts, the desired amine 1
was obtained as a white crystalline solid (0.90 g, 2.9 mmol) in 97%
yield.
[0236]
Benzyl-4-benzyloxycarbonylamino-4-deoxy-2,3-O-isopropylidene-.alpha-
.-D-mannopyranoside (2)
[0237] To a stirred, cooled (ice bath at 0.degree. C.) solution of
1 (2.15 mg, 0.70 mmol) in (1:1) THF10% NaHCO.sub.3 (15 mL) was
added benzychloroformate (0.11 mL, 0.75 mmol) dropwise. The
reaction was then allowed to warm to room temperature slowly. After
stirring for three added and the mixture stirred under H.sub.2
(balloon) for a further four days. This reaction gives a mixture of
components which include the desired product (70:30 mixture of (5R,
6R) and (5S, 6S) isomers) as the major components. The structure
and ratio of these isomers was inferred by .sup.1H-NMR.
[0238] Ethyl
(1R,2R,5S,6S,8S,8aR)(5S,6S)-1,2,8-trihydroxy-5-methyloctahydr-
o-6-indolizine carboxylate [6-carboethoxy-5-methylswainsonine
(GD0036)] 40
[0239] Isolation of (GD0036) was achieved by de-acetonation of the
isomeric mixture 5, derivatization of the resulting triols (6) as
the TMS ethers, purification of 7 by preparatory GC, followed by
acidic hydrolysis of the TMS groups. This isomer (GD0036), which
have an .sup.1H-NMR consistent with the structure, was submitted
for enzyme assay.
[0240] (5R,6R)-6-hydroxymethyl)-5-methylswainsonine acetonide
(8):
[0241] To a solution of isomers 5 (64 mg, 021 mmol) in dry THF was
added 20 mg of LiAlH.sub.4 in small portions. This suspension was
stirred overnight. At that time the starting material had been
completely consumed as inferred by TLC, Methanol (1 mL) was added
to this mixture and the methanol was removed by rotary evaporation.
The dry Celite/product was then charged onto a SiO.sub.2 gel
column. Elution with 27:2:1 EtOAc:MeOH:10%, NH.sub.4OH gave the
purified isomers along with mixed fractions which were
re-chromatographed. The total recovery of (5R,6) 8 from all
fractions was 12 mg. The total recovery of (5S,6S) 8 from all
fractions was 15 mg. The structure of these products was confirmed
by .sup.1H-NMR.
[0242] (5S,6S), and (5R,6R)-6-hydroxymethyl-5-methylswainsonine
41
[0243] A solution of the acetonide 8 (approx. 5 mg) in the THF-6M
(2 mL) was stirred at room temperature for two days. The solutions
were then concentrated and passed through a hydroxide ion exchange
resin column using methanol as an eluent. The free base was then
purified using HPLC. Recovery of each isomer was approximately 2-3
mg. These samples, (5S,6S) 6 hydroxymethyl-5-methylswainsonine
(GD0040), and (5R,6R)-6-hydroxymethyl- -5-methylswainsonine
(GD0044) were submitted for enzyme assay.
Example 3
(5R)-5-Benzyloxymethylswainsonine (GD42)
[0244] 42
[0245] (5R)-5-Benzyloxymethyl-1,2-O-isopropylideneswainsonine
(GDLZ177).
[0246] Sodium hydride (10 mg, 60% in mineral oil) was added in
portions to a stirred solution of crude
(5R)-5-hydroxymethyl-1,2-O-isopropylideneswai- nsonine (30.7 mg,
<0.126 mmol) in anhydrous DMF containing benzyl chloride (200
ml). After stirring for two days methanol (1 ml) was added, and the
solution concentrated. The residue was purified by reversed phase
HPLC and gave GDLZ177 (7.0 mg, .about.17%).
[0247] (5R)-5-Benzyloxymethylswainsonine (GD42)
[0248] Compound GDLZ177 (7.0 mg, 21 mmol) was dissolved in
tetrahydrofuran (1 ml), 6 M hydrochloric acid (1 ml) was added, and
the solution stirred at room temperature for two days. The solution
was concentrated to dryness, the residue purified by reversed phase
HPLC, and gave GD42 (2.3 mg, 38%) as a colourless residue.
[0249] (5S)-5-Benzyloxymethyl-1,2-O-isopropylideneswainsonine
(GDLZ252).
[0250] Sodium hydride (7 mg, 60% in mineral oil) was added in
portions to a stirred solution of
(5S)-5-hydroxymethyl-1,2-O-isopropylidene swainsonine (20.5 mg, 85
mmol) in anhydrous DMF containing benzyl chloride (100 ml). After
stirring for two days methanol (1 ml) was added, and the solution
concentrated. The residue was purified by reversed phase HPLC and
gave GDLZ252 (10.4 mg, 36%).
(5S)-5-Benzyloxymethylswainsonine (GD91)
[0251] 43
[0252] Compound GDLZ252 (6.0 mg, 18 mmol) was dissolved in
tetrahydrofuran (1 ml), 6 M hydrochloric acid (1 ml) was added, and
the solution stirred at room temperature for two days. The solution
was concentrated to dryness, the residue purified by reversed phase
HPLC, and gave GD91 (4.9 mg, 93%) amorphous material.
Example 4
[0253] (1S, 2S, 3R/S, 8R,
8aS)-3-cyano-1,2,8-Trihydroxyoctahydroindolizine (GD 92) 44
[0254] A process for preparing 3-cyano swainsonine is outlined and
shown schematically below.
[0255] (1R, 3S, 4S, 5S,
6R)-7-Aza-3-benzyloxy-4,5-O-isopropylidenedioxy-2-- oxa-biscyclo
[4,4,0]-decane (GDSC1009)
[0256] To a solution of benzyl
E-4-azido-4,6,7-trideoxy-6-ene-2,3-O-isopro-
pylidene-.alpha.-D-manno-octadialdopycanoside (GDSC1005) (1.6 g,
4.46 mmol) in ethanol (25 mL) was added 10% palladium on carbon
(800 mg, 50% wet). The reaction mixture was stirred under a
hydrogen atmosphere overnight. The catalyst was removed by
filtration and the solvent was removed under a reduced pressure.
The residue was purified by silica gel chromatography (1 to 10%
methanol in dichloromethane gradient) to give GDSC1009 1.24 g (3.97
mmol) in 87.6% yield.
[0257] (1R, 3S, 4S, 5S,
6R)-7-[N-(tert-Butyloxycarbonyl)amino]-3-benzyloxy-
-4,5-O-isopropylidenedioxy-2-oxa-biscyclo [4,4,0]-decane
(GDSC1117)
[0258] To a solution of (1R, 3S, 4S, 5S,
6R)-7-Aza-3-benzyloxy-4,5-O-isopr- opylidenedioxy-2-oxa-biscyclo
[4,4,0]-decane (GDSC1009) (1.40 g, 4.41 mmol) in dioxane/water(1:1,
20 mL) was added 1 N NaOH (10 mL). Di-tert-butyl dicarbonate (2.89
g, 13.23 mmol) was added to this solution and the mixture was
stirred at room temperature for 4 h. The reaction mixture was
partitioned between dichloromethane (50 mL) and water (60 mL). The
organic payer was washed with saturated NaCl (50 ml), dried over
MgSO.sub.4 and filtered. The filtrate was evaporated to dryness
under reduced pressure and the residue was purified by column
chromatography using 0-2% methanol in dichloromethane gradient as
eluent to give GDSC1117 (1.38 g, 74.9%).
[0259] (1R, 3S, 4S, 5S,
6R)-7-[N-(tert-Butyloxycarbonyl)amino]-3-hydroxy-4-
,5-O-isopropylidenedioxy-2-oxa-biscyclo [4,4,0]-decane
(GDSC1127)
[0260] To a solution of (1R, 3S, 4S, 5S,
6R)-7-[N-(tert-Butyloxycarbonyl)a-
mino]-3-hydroxy-4,5-O-isopropylidenedioxy-2-oxa-biscyclo
[4,4,0]-decane (GDSC1127)
[0261] To a solution of (1R, 3S, 4S, 5S,
6R)-7-[N-(tert-Butyloxycarbonyl)a-
mino]-3-benzyloxy-4,5-O-isopropylidenedioxy-2-oxa-biscyclo
[4,4,0]-decane (GDSC1117) (1.2 g, 2.86 mmol) in ethanol (50 mL) was
added 10% palladium on carbon (1.2 g, 50% wet) and a catalytic
amount of acetic acid. The flask was evacuated by aspiration and
purged with hydrogen three times. The resulting heterogeneous
mixture was stirred under a balloon of hydrogen for 8 h. The
catalyst was removed by filtration and the solvent was removed
under reduced pressure. The residue was then subjected to column
chromatography on silica gel, eluting with a gradient of 1-5%
methanol in chloromethane to afford GDSC1127 0.810 g (2.46 mmol) in
86% yield as an .alpha.,.beta. anomeric mixture. A minor product of
this reaction was purified and identified as the deacetonated
product of GDSC1127 (ca. 6%).
[0262] Synthesis of GDSC1147
[0263] A solution GDSC1127 (0.5 g, 1.52 mmol) in DMSO (3 mL) and
acetic anhydride (3 mL) was stirred at room temperature under argon
for 14 h., the solution was diluted with dichloromethane (25 mL),
then poured into a saturated NaHCO.sub.3 solution (25 mL). The
organic layer was dried (Na.sub.2SO.sub.4), filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography using 1% methanol in dichloromethane as
eluent to give GDSC1147 (0.457 g) in 92% yield.
[0264] Synthesis of GDSC1149
[0265] GDSC1147 (215 mg, 0.657 mmol) was dissolved in 0.5 ml of
dichloromethane. To this solution was added 4 mL of 80% TFA in
water. The reaction mixture was stirred at room temperature for 12
h. The reaction mixture was concentrated under reduced pressure and
coevaporated twice with water (10 mL). After being evaporated under
high vacuum overnight, the residue was dissolved in 5 mL of 0.2 N
sodium methoxide and stirred at room temperature under argon for 20
h. The reaction mixture was concentrated on a rotary evaporator.
The orange residue was dissolved in a minimum amount of water and
subjected to sequential purifications using Dowex 50WX.times.2-200
(H') and an AG 1-X8 (OH') ion exchange resins followed by a C-18
column purification to give GDSC1149 (96 mg, 78%) as a crystalline
compound.
[0266] Synthesis of 3-cyano Swainsonine (GDSC3027)
[0267] GDSC1147 (110 mg, 0.334 mmol) was dissolved in a mixture of
0.3 mL of dichloromethane, 3.2 mL of TFA and 0.8 mL of water. The
reaction mixture was stirred at room temperature for 12 h. The
reaction mixture was concentrated on a rotary evaporator. The
residue was dissolved in water (4 mL) and stirred with AG 1-X8(OH)
ion exchange resin (0.5 g dry resin). After 30 min, the mixture was
filtered and the filtrate was concentrated. The residue was
dissolved in methanol (4 ml.) and stirred with potassium cyanide
(217 mg, 3.34 mmol) for 12 h. The mixture was concentrated. The
residue was dissolved in 1 mL of water and filtered through an AG
1-X8OH ion exchange resin column (1.0 g), rinsing with water (12
mL). The filtrate was concentrated and residue was purified with a
C-18 column (1 g) using 10-50% methanol in water as eluents to give
GDSC3027 (56 mg) as 1:1 .alpha., .beta.mixture of 3 -cyano
swainsonine. 45
EXAMPLE 5
[0268] Inhibition of Golgi .alpha.-mannosidase II and Lysosomal
.alpha.-mannosidase by Compounds of the Invention
[0269] The compounds of the invention and swainsonine were added
(10.mu.l) into 96 well Elisa plates followed by the addition of 200
mM sodium acetate 5.6 and 25 .mu.l of 10 mM p-nitrophenyl
.alpha.-D-mannospyranosid- e. 15 .mu.l of .alpha.-mannosidase
(Sigma 38 U/ml) was added to each well and the plates were
incubated for 60 min at 37.degree.C. The reaction was stopped by
the addition of 50 .mu.l of 0.5M of sodium carbonate and formation
of p-nitrophenol was measured with a plate set at 405 nM. The
effects of the compounds and swainsonine on lysosomal mannosidase
were measured by adding (10 .mu.l) of the compounds into 96 well
Elisa plates followed by the addition of 200 mM sodium acetate pH
5.0 and 25 .mu.l of 10 mM p-nitrophenyl .alpha.-D-mannospyranoside.
15 .mu.l of lysosomal mannosidase (about 8 mM/mL) was added to each
well and the plates were incubated for 60 min at 37.degree. C. The
reaction was stopped by the addition of 50 .mu.l of 0.5M sodium
carbonate and formation of p-nitrophenol was measured with a plate
set at 405 . The mannosidase 11and lysosomal mannosidase activity
of compounds of the formula 1 of the invention are shown in Table
1.
[0270] While the present invention has been described with
reference to what are presently considered to be the preferred
examples, it is to be understood that the invention is not limited
to the disclosed examples. To the contrary, the invention is
intended to cover various modifications and equivalent arrangements
included within the spirit and scope of the appended claims.
[0271] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
1TABLE 1 Lysosomal MannosidaseII Mannosidase Compound Activity
(.mu.M) Activity (.mu.M) Swainsonine 0.080 .+-. 0.033 0.080 .+-.
0.079 (5S)-5-Hydroxymethyl- 0.638 .+-. 0.22 1.485 .+-. 0.455
swainsonine (GD 46) (5S)-5- 0.738 .+-. 0.32 0.316 .+-. 0.0759
Methylswainsonine (GD 28) (5S)-5-Ethylswainsonine 0.768 .+-. 0.12
0.360 .+-. 0.3132 (GD 38) (5R)-5- 0.847 .+-. 0.33 2.296 .+-. 1.128
Methylswainsonine (GD 20) (5S)-5-benzyloxymethyl 0.989 .+-. 0.44
3.23 .+-. 0.268 swainsonine (GD 91) (5R)-5-benzyloxymethyl 1.140
.+-. 0.33 0.164 .+-. 0.0491 swainsonine (GD42) 3-hydroxymethyl-
1.28 .+-. 0.8 (3) 1.70 .+-. 0.5 (9) swainsonine (EJH-4-264)
3-hydroxymethyl- 1.83 .+-. 1.3 (3) 39.13 .+-. 16.3 (10) swainsonine
(EJH-4-263) (5R)-5-Hydroxymethyl 2.051 .+-. 0.65 0.705 .+-. 0.309
swainsonine GD45 (5R)-5- 2.455 .+-. 1.53 1.45 .+-. 0.071
Methylswainsonine formate salt (GD19) (5R)-8-cpl-5- 17.223 .+-.
3.87 28.157 .+-. 7.847 methylswainsonine GD37
Ethyl(1R,2R,5S,6S,8S,8aR)- 18.700 .+-. 4.95 120.275 .+-. 32.173
1,2,8-trihydroxy-5- methyloctahydro-6- indolizinecarboxylate (GD
36) (5S,6S)-6- 19.88 .+-. 7.29 62.378 .+-. 17.892 hydroxymethyl-5-
methylswainsonine (GD40) (5R,6R)-6- 25.167 .+-. 10.41 24.482 .+-.
7.618 hydroxymethyl-5- methylswainsonine (GD44) (1S,2S,3R/S,8R,
30.850 .+-. 12.23 30.150 .+-. 1.202 8aS)-3-cyano-1,2,8-
Trihydroxyoctahydro-5- indolizine (GD 92) ethyl(5R,6R)-5- 1000 1500
.+-. 707 methylswainsonine-6- carboxylate (GD84)
(1S,2R,8R,8aS)-1,2,8- 1030 1087 Trihydroxyhexahydo-
3(2II)-indolizinone (GD 35) (1S,8R,8aS)- No detectable No
detectable 1,5,6,7,8,8a-Hexahydro- inhibition inhibition
1,2,3,8-indolizinetetraol
* * * * *