Note: Descriptions are shown in the official language in which they were submitted.
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Title: NOVEL DERIVATIVES OF SWAINSONINE, PROCESSES FOR THEIR
PREPARATION, AND THEIR USE AS THERAPEUTIC AGENTS
FIELD OF THE INVENTION
5 The present invention relates to novel 8a substituted derivatives of
swainsonine, processes
for their preparation, and their use as therapeutic agents.
BACKGROUND OF THE INVENTION
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.
10 130:383, 1995). These structures include the GIcNAc (3(T1-6) branched N and
O-linked
carbohydrate side chains of cell-surface glycoproteins. The Golgi enzymes
required for their
synthesis are (3(Tl-6) N acetylglucosaminyltransferase V (i.e. GIcNAc-TV) and
core 2 (3(T1-6)
N acetylglucosaminyltransferase (i.e. core 2 GIcNAc-T), respectively. These
enzymes are
up-regulated in human carcinomas (Fernandes et al., Cancer Res. 51:718-723,
1991), a
15 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 GIcNAc-TV in epithelial cells results in morphological
transformation and
tumor formation in mice (Demetriou et al, J. Cell Biol. 130:383-392, 1995).
Therefore,
GIcNAc-TV as well as enzymes supplying acceptor substrates to GIcNAc-TV (i.e.
GIcNAc-TI, oc
2 0 -mannosidase II and core 2 GIcNAc-T of the O-linked pathway) are targets
for anti-cancer
pharmaceuticals.
A lead a-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
25 Oxytropis (Molyneux RJ and James LF., Science 215:190-191, 1981), and also
the fungus
Rhizoctonia leguminicola (Schneider et al., Tetrahedron 39;29-31, 1983).
Swainsonine's ability to
inhibit a-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 giycosylium cation intermediate generated during the
hydrolytic cleavage
3 0 of mannopyranosides. (Goes, P.E. et al., Clin. Cancer Res. l: 935-944,
1995).
The swainsonine blockage of a-mannosidase II is prior to GIcNAc-TV and
prevents
expression of GIcNAc (3(Tl-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.,
3 5 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,
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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 xenograRs in the mice (Dennis et al.,
J. Natl.
Cancer Inst. 81:1028-1033, 1989 and Dennis et al., Cancer Res., 50:1867-
1872,1990). 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).
Swainsonine has positive effects on cellular immunity in mice (reviewed in
Humphries
M.J. and Olden K., Phanmacol 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 lnst.
83:1149-1156, 1991).
Selected indolizidine derivatives and swainsonine analogues have been reported
in the
literature (Dennis, J. W. et al. Biochemical Pharmacology 46:1459-1466,
Japanese Patent
Application No. J61277685, U.S Patent No. 5,466,809, U.S Patent No 5,650,4I3,
W096/40683,
W098/14446, and W098/14445).
SUMMARY OF THE INVENT10N
The present invention relates to a compound of the formula I
_
I
wherein
(a) R,, RZ and R3 are each independently hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, or R, and Rz together form a carbocyclic or heterocylic
ring;
(b) W and W" and W' are each independently hydrogen, hydroxyl, alkoxy, thiol,
3 5 thioalkyl, thioaryl, halo or amino, or W and W' together form a
carbocyclic or
heterocyclic ring; and
(c) X represents alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, or aryl,
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and salts and optically active and racemic forms of a compound of the formula
I.
The present invention also provides a process for the preparation of a
compound of the
formula I as defined herein.
The compounds of the invention have valuable pharmacological properties and
they
provide antimicrobial, cancer suppressing effects, hemorestorative,
chemoprotective,
radioprotective, and immunomodulatory properties. Therefore, the invention
contemplates a
pharmaceutical composition comprising a compound of the fonmula I as an active
agent.
The invention further relates to a method for stimulating the immune system,
stimulating
hematopoietic progenitor cell growth, treating proliferative disorders or
microbial or parasitic
infections, or conferring protection against chemotherapy and radiation
therapy 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 fonmula I in the
preparation of a
medicament for stimulating the immune system, stimulating hematopoietic
progenitor cell growth,
or conferring protection against chemotherapy and radiation therapy, and/or
for treating
proliferative disorders, and microbial or parasitic infections.
The present invention also relates to the use of a compound of the formula 1
which is
esterified at free hydroxyls as a prodrug.
These and other aspects of the present invention will become evident upon
reference to
the following detailed description and attached drawing.
2 0 BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
Figure 1 is a schematic diagram of a reaction for preparing a compound of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
I. COMPOUNDS OF THE INVENTION
2 5 Hereinabove and in the following the term "alkyl", alone or in
combination, refers to a
branched or linear hydrocarbon radical, typically containing from I through 20
carbon atoms,
preferably I through 10 carbon atoms, more preferably 1 to 6 carbon atoms.
Typical alkyl
groups include but are not limited to methyl, ethyl, 1-propyl, 2-propyl, 1-
butyl, 2-butyl, tert-
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.
3 0 The teen "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, 1-
hexenyl, 2-hexenyl, I-pentenyl, 2-pentenyl, and the like.
The term "alkynyl", alone or in combination, refers to an unsaturated branched
or linear
3 5 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,
I-pentynyl, and the
like.
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The term "cycloalkyl" refers to cyclic hydrocarbon groups and includes but is
not limited
to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and
cyclooctyl.
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
5 described herein. Examples of aryl groups and substituted aryl groups are
phenyl, benzyl, p
nitrobenzyl, p-methoxybenzyl, biphenyl, and naphthyl.
The term "alkoxy" alone or in combination, refers to an alkyl or cycloalkyl
linked to the
parent molecular moiety through an oxygen atom. Examples of alkoxy groups are
methoxy,
ethoxy, propoxy, vinyloxy, allyloxy, butoxy, pentoxy, hexoxy, cyclopentoxy,
cyclohexoxy, and the
10 Iike.
The teen "halo" or "halogen", alone or in combination, means fluoro, chloro,
bromo, or
iodo.
The tenor "amino", alone or in combination, refers to a chemical functional
group where a
nitrogen atom (N) is bonded to three substituents being any combination of
hydrogen, alkyl,
15 cycloalkyl, alkenyl, alkynyl, or aryl with the general chemical formula
NR,,Rs where R, and Rs
can be any combination of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, 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 (-NH2), methylamine,
ethylamine,
dimethylamine, 2-propylamine, butylamine, isobutylamine, cyclopropylamine,
benzylamine,
2 0 allylamine, hydroxylamine, cyclohexylamino (-NHCH(CH2)5), piperidine (-
N(CH2)g) and
benzylamino (-NHCH2C6H5).
The team "thioalkyl", alone or in combination, refers to a chemical functional
group
where a sulfur atom (S) is bonded to an alkyl. Examples of thioalkyl groups
are thiomethyl,
thioethyl, and thiopropyl.
2 5 The tenor "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
-SR6 where R6 is
an aryl group which may be substituted. Examples of thioaryl groups and
substituted thioaryl
groups are thiophenyl, pare-chlorothiophenyl, thiobenzyl, 4-methoxy-
thiophenyl, 4-nitro-
thiophenyl, and pare-nitrothiobenzyl.
3 0 The term "carbocyclic" 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
3 5 unsaturated hydrocarbons, and aryl as described herein, including but not
limited to benzene and
napthalene.
Heterocyclic rings are molecular rings where one or more carbon atoms have
been
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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
(pyridinyl 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.
One or more of R,, R2, R3, W, W', W', and/or X, alone or together, which
contain
available functional groups as described herein, may be substituted with for
example one or more
of the following: alkyl, alkoxy, hydroxyl, aryl, cycloalkyl, alkenyl, alkynyl,
thiol, thioalkyl,
thioaryl, amino, or halo. The term "one or more" used herein preferably refers
to from 1 to 3
substituents, most preferably I to 2 substituents.
In an embodiment of the invention, compounds of the formula I are provided
where R,,
Rz, and R3, are the same and represent hydrogen; W, W', and W' are the same
and represent
hydroxyl; R,, RZ, and R3 are the same or different and represent alkyl,
alkenyl, alkynyl, or aryl,
preferably alkyl or aryl, most preferably alkyl; R, and RZ together form a
cat~bocyclic or
heterocyclic ring; W, W', and W' represent hydroxyl, alkoxy, thiol, thioalkyl,
thioaryl, halo, or
amino; W and W' together form a carbocyclic or heterocyclic ring; or X
represents alkyl, alkenyl,
alkynyl, aryl, cycloalkyl or alkoxy, preferably alkyl or alkoxy, more
preferably alkyl.
2 0 Preferred compounds of the formula I of the invention are those where R,,
R2, and R3
represent hydrogen, and W, W', and W ' represent hydroxyl, and X represents
methyl, ethyl,
phenyl, benzyl, or methoxy.
Particularly preferred compounds of the invention are (IS, 2R, 8R, 8aR~8a-
methyl-1,2,8
trihydroxyindolizidine, (IS, 2R,8R, 8aR)-8a-ethyl-1,2,8-trihydroxy-
indolizidine, (IS, 2R, 8R, 8aR)
8a-propyl-I, 2, 8-trihydroxyindolizidine, and (IS, 2R, 8R, 8aR)-8a-butyl-1, 2,
8
trihydroxyindolizidine.
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
3 0 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 enantiomers
and diastereomers
are convertible by facile epimerization of the chiral centers, and that a
preparation containing a
compound of formula I as a mixture of isomers of the formula I is within the
scope of the
3 5 invention.
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
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intended to encompass all possible optical isomers of the compounds so
depicted.
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 humans 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. Illustrative of such salts are the
salts with inorganic acids
such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and
like acids; with organic
carboxylic acids, such as for example acetic, propionic, glycolic, lactic,
pyruvic, malonic, succinic,
fumaric, malic, tartaric, citric, ascorbic, malefic, hydroxymaleic, and
dihydroxymaleic, benzoic,
phenylacetic, 4-aminobenzoic, 4-hydroxybenzoic, anthranilic, cinnamic,
salicylic, 4-
aminosalicylic, 2-phenoxybenzoic, 2-acetoxybenzoic, mandelic, and like acids;
and with organic
sulfonic acids such as methanesulfonic acid, and p-toluenesulfonic acid. Such
salts can be obtained
by standard procedures from an amine of this invention and the appropriate
acid.
Crystalline forms of the compounds of the formula I of the invention are also
contemplated.
II. PROCESSES FOR PREPARING COMPOUNDS
The compounds of the formula I of the present invention can be prepared by
utilizing
2 0 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.
Compounds of the Formula I may be synthesized in a variety of ways by adapting
common synthetic organic chemistry practices to known synthetic intermediates.
For example,
compounds of the formula I where X is alkyl may be synthesized by protecting
swainsonine
acetonide (I) at the 8 position with a protecting group; converting the
protected swainsonine
acetonide to an N-oxide; reacting the N-oxide with trifluoroacetic anhydride;
reacting the acylated
N-oxide with a nucleophile under basic conditions for example, an alkyl
magnesium bromide; and
removing the protecting groups.
In an embodiment of the invention, {IS, 2R, 8R, 8aR~8a-methyl-1, 2, 8-
trihydroxyindolizidine is prepared by converting swainsonne acetonide to (1S,
2R, 8R, 8aR)-8-
methoxymethoyl-1,2-(isopropylidenedioxy)indolizidine by adding tetrabutyl-
ammonium iodide,
methoxy methyl chloride, and sodium hydride in THF; reacting (1S, 2R, 8R,
8aRr8-
methoxymethoxy-I, 2-(isopropylidenedioxy)indolizidine with hydrogen peroxide
in
dichloromethane-ethanol to yield (1S, 2R, 8R, 8aR~8-methoxymethoxy-1,2-
(isopropylidenedioxy)indolizidine N oxide (3); reacting the N-oxide with
trifluoroacetic anhydride
followed by an alkylmagnesium bromide to yield (1S, 2R, 8R, 8aR)-8-
methoxymethoxy-8a-alkyl-
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1, 2-(isopropylidenedioxy~indolizidine (4); and removing the protecting groups
to yield (IS, 2R,
8R, 8aR)-8a-methyl-1, 2, 8-trihydroxyindolizidine. This reaction is
illustrated in the scheme shown
in Figure 1.
Reactive groups used in processes 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, 2"° Ed,
T.W. Greene, and P.G.M. Wats, John Wiley & Sons, New York, 1991). 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, silyl ethers,
orthoesters, 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-
para-
methoxybenzyl, O-acetoxy, O-benzoyloxy, O-pivaloyl, O-allyl, methoxymethyl,
isopropylidene,
benzylidene, methylidene, acetylidene, I-methoxy ethylidene, 1,3-(1,1,3,3
tetraisopropyldisiloxanylidene, O-trimethy) silyl, O-t-butyl dimethylsilyl.
Removal of the
protective groups may be carried out using procedures known in the art.
Methods or references to methods for transformation of groups such as hydroxyl
to
groups such as halo, amino, or alkoxy, with or without inversion of
configuration are known to
those skilled in the art and can be found in for example "Organic Functional
Group Preparations"
2 0 2"d Ed., S.R. Sandier and W. Kare, Academic Press; "Comprehensive Organic
Transformations",
R.C. Larock, VCH Publishers, 1989, "Advanced Organic Chemistry" 4'" Ed. By J.
March, Wiley
lnterscience, 1992; and "Compendium of Organic Synthetic Methods" John Wiley &
Sons.
Representative methods are described herein.
A free hydroxyl group may be converted to an alkoxy group in blocked/deblocked
2 5 compounds to produce compounds where for example one or more of W, W', and
W ' are alkoxy,
by reacting with an alkyl halide in the presence of a base.
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°C to 10°C). After stirring for a few minutes,
benzyl bromide in DMF is added
3 0 dropwise at low temperature, for example 0°C to 10°C. The
reaction mixture is further stirred at
room temperature for 2 to 24 hours.
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°C to -60°C), an appropriate amount of triflic
anhydride, or mesyl chloride, or
3 5 tosyl chloride is added dropwise. The reaction is allowed to stir at a
temperature between 0°C to
25°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
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_g_
leaving group with O-benzoate with inversion of configuration at the carbon
center. 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. To obtain a fluoro derivative with
inversion, the triflate is
treated with anhydrous tetra alkyl ammonium fluorides (preferably tetra n-
butyl) or potassium
fluoride in a suitable solvent (e.g diethyl ether, tetrahydrofuran or crown
ether). Alternatively, the
hydroxyl group is simply treated with (diethyl amino) sulfur trifluoride
(DAST) in a one-step
fluorination reaction.
For the introduction of an amino group, the triflate may be treated either
with sodium
azide, phthalimide, 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 (azide and benzylamine), or treatment with hydrazine or methyl
amine (phthalimido)
gives the free amino group.
Appropriate methods for introducing a thiol group in compounds of the formula
I 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 sulfhydride
(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 (S203z ) 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
ofthe Thiol Group, pt 1: Wiley: New York, 1974, pp. 179-211).
Methods for introducing a thioalkyl or a thioaryl group in a compound of the
formula I
are also 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;
3 0 Kajigaeshi Bull. Chem. Soc. Jpn. 1985, 58:2529) or, by treating an alcohol
with tributyl
phosphine and an 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
l: Wiley: New York, 1974, pp 721-735.
In addition, appropriate methods for replacing a blocked or deblocked hydroxyl
group
3 5 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 ether. A
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large list of methods able to achieve this transformation is provided in J.
March "Advanced
Organic Chemistry. Reactions, Mechanisms and Structure" 4th Edition, 1992, pp
438- 446 and
references cited therein.
Some alkyl or aryl groups, particularly those which may contain unsaturations
or other
5 chemical functional groups such as hydroxyl, or alkoxy for example, can be
further derivatized by
chemical processes such as oxidation, hydroxylation, hydrolysis, alkylation,
reduction, carbon-
carbon chain elongation by Grignard or Wittig reactions for example to
introduce new or
additional functional groups in any final compound. Such transformations can
be achieved by
anyone skilled in the art of synthetic organic chemistry.
If necessary, the products of the processes described above may be purified by
conventional methods such as column chromatography.
Compounds of the formula I with available hydroxyl groups can be convened to
epi-
isomers by SN2 inversion. For example, a free hydroxyl may be reacted with
mesyl chloride and
pyridine to give O-mesyl (methyl sulphonyl), which on treatment with sodium
benzoate in DMF
15 (dimethyl formamide) produces a compound where the free hydroxyl group is
replaced by epi-O-
benzoate. Alternatively, a Mitsunobu reaction can be used to provide the
epibenzoate (O.
Mitsunobu, Synthesis, January 1981, p. I-28). Deesterification using NaOMe in
methanol results
in a compound of the formula I where the free hydroxyl is replaced by
epihydroxyl.
The compounds of the formula I described above may be converted into salts
using
2 0 conventional procedures.
Compounds of the formula I with free hydroxyl groups may also be converted
into esters
using conventional procedures. For example, a compound of the formula I may be
dissolved in
DCM and pyridine. After cooling (0°C to 5°C) benzoic anhydride
or benzoyl chloride in DCM
and pyridine is added dropwise. The reaction is allowed to stir at room
temperature for 2 to 24
2 5 hours. Conventional work-up yields the esterified derivatives.
Optical antipodes of the compounds of the formula I may be prepared from the
corresponding racemic forms by standard resolution techniques, involving, for
example, the
separation of diastereomeric salts of those compounds of the formula 1
characterized by the
presence of a basic amino group, and an optically active acid, or by synthesis
from optically active
3 0 precursors.
lll. UTILITY OF COMPOUNDS OF THE INVENTION
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, a-mannosidase, or lysosomal oc-
mannosidase.
3 5 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
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measurement of ICS 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, cx-
mannosidase II in the Golgi,
and the resulting cellular phenotype.
5 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.
Transformed cells which may be used in this assay include MDAY-D2, L1210, CHO,
B 16, melanoma tumor cells, and human tumor cells such as SW 480, LS 174T, HT-
29, WiDr, T2,
MDA-231, MCF7, BT-20, Hs578T, K562, Hs578T, SK-BR-3, CY 6T, MDA-468, H23,
H157,
H358, H 1334, H i 155, H28, H460, Hmesol, H 187, HS 10A, N417, H 146, H 1092,
H82 (Restifo, N.
P. et al, J. Exper. Med. 177:265-272, 1993).
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.
2 0 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 x 10° cells, preferably 2 x
10' 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 pg/ml,
preferably 20-30 p/ml,
2 5 more preferably 25 pg/ml.
The following L-PHA assay may be used to assay for inhibition of
oligosaccharide
processing (i.e. Golgi a-mannosidase II) in viable cells. MDAY-D2 tumor cells
are inoculated
into 96 well micro-test plates at 2 x 10° 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
3 0 of L-PHA at 25 ug/ml for an additional 24 hours. Cell proliferation is
measured by adding 0.5
pCi/well of 'H-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 ICs° values for the
test compounds are the drug concentrations showing 50% protection from L-PHA
toxicity; that is
50%'H-thymidine incorporated compared with cells grown in the absence of L-
PHA.
3 5 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
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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
oc-mannosidase
inhibitors. An increase in ICS in the L-PHA toxicity assay in the presence of
diethyl p-nitrophenyl
phosphate indicates that the compound requires activation by esterases and
would accordingiy be
useful as a prodrug.
The compounds of the fonmula 1 have valuable pharmacological properties and
they
provide immunostimulatory, antimicrobial and anti-cancer 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
10 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 nodules 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.
Examples of protocols for confirming the activities of the compounds of the
invention are included
15 in the Example section.
The compounds of the invention may be 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, ovarian cancer, cervico-uterine cancer,
cancer of the
2 0 kidney, stomach, lung, rectum, breast, bowel, gastric, liver, thyroid,
head and neck such as
unresectable head and neck cancers, lymphangitis carcinamatosis, cervix,
salivary gland, leg,
tongue, lip, bile duct, pelvis, mediastinum, urethra, bronchogenic, bladder,
esophagus and colon,
non-small cell lung cancer, and Kaposi's Sarcoma which is a form of cancer
associated with HIV-
infected patients with Acquired Immune Deficiency Syndrome (AIDS). The
compounds may also
25 be used for other anti-proliferative conditions such as arthrosclerosis and
viral infections, in
particular AIDS or hepatitis C.
The compounds of the formula I may be used to stimulate bone marrow cell
proliferation
(hemorestoration), 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,
3 0 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.
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
35 where a patient has been immunocompromised such as patients infected with
HIV or hepatitis C,
or other viruses or infectious agents including bacteria, fungi, and
parasites, in patients undergoing
bone man: ow transplants, and in patients with chemical or tumor-induced
immune suppression.
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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. In
particular, they may be useful
in the treatment of arthritis and asthma. The compounds may render
carbohydrate structures on
neutrophils unable to bind selectins. Selectins present at the site of damage
interact with the
carbohydrate structures on neutrophils in such a way that the neutrophils roll
along the epithelial
wall, stick, infiltrate, and cause tissue damage.
The compounds of the invention may be used to protect against the lethality of
various
chemotherapeutic agents as well as against lethal doses of irradiation. The
compounds may also be
used in the prevention of tumor recurrence after surgery i.e. adjuvant
therapy.
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
animals within the
scope of the meaning of the term are dogs, cats, rats, mice, horses, bovine
cattle, sheep, and
humans.
The compounds 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,
parenteral, 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,
2 0 liposomes (see for example, U.S. Patent Seria) No. 5,376,452), gels,
membranes, and tubelets. For
parenteral and 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 with one or more pharmaceutically acceptable excipients or diluents,
suitable for the
2 5 aforesaid uses and with an osmolarity which is compatible with the
physiological fluids. 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, preparations in the form of sprays,
for example nose
sprays, should be considered.
The pharmaceutical compositions can be prepared by en r se known methods for
the
3 0 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 Sciences, Mack Publishing
Company,
Easton, Pa., USA 1985). On this basis, the pharmaceutical compositions
include, albeit not
3 5 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.
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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). The
compounds of the invention may be administered concurrently, separately, or
sequentially with
other therapeutic agents or therapies.
5 In particular, the compounds of the invention may be used in combination
with anti-
proliferative agents, antimicrobial agents, immunostimulatory agents, or anti-
inflammatories. For
example, the compounds may be used in combination with anti-viral and/or anti-
proliferative
agents such as a Thl cytokine. Thl cytokines include interleukins-2 and 12 (IL-
2, IL-12), and the
interferons-a, (3, y (IFN-a, IFN-[3, IFN-y), and inducers thereof. The
compounds of the invention
10 can be used with poly (LC.), poly (LC.~LC, tumor necrosis factor (TNF), or
transforming growth
factor (TGF). The compounds may be administered to a patient being treated
with a
myelosuppressive agent, or a bone marrow transplant recipient. The compounds
can be used in
combination with chemotherapeutic agents including doxorubicin, 5-
fluorouracil,
cyclophosphamide, and methotrexate, with isoniazid for the prevention and
treatment of peripheral
15 neuropathy, and with NSAID for the prevention and treatment of
gastroduodenal ulcers.
The percentage of active ingredient in each pharmaceutical composition and the
effective
amount of the active ingredient used to practice the present invention for
treatment of the disclosed
conditions will be decided by the attending physician or veterinarian. Such
amount of the
compound as determined by the attending physician or veterinarian is referred
to herein as the
2 0 "effective amount". In general, a dosage range of the compounds in the
composition is envisaged
for administration in human medicine of from 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 compositions the dosage is about 0.5 to 10 mg/kg/day, more preferably 1.5
to 9 mg/kg/day.
Amounts of drug administered to produce serum levels 10-1000x the ICso for
inhibition of
25 oligosaccharide processing in the L-PHA assay are preferably employed. It
will also be
appreciated that it may be necessary to deviate 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 behaviour towards the medicine or the nature of its formulation
and the time or
3 0 interval at which it is administered may also indicate use of amounts
different from those
mentioned. Thus it may 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 the course of the day.
3 5 A compound of the invention may be used as a vaccine adjuvant to induce a
potent
immune response to itself and/or induce immunity to antigens, particularly
antigens that are
normally poor immunogens. A compound of the invention may augment vaccine
immunogenicity
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through activation of antigen presenting cells, such as monocytes or
macrophages, to release
cytokines that can promote T-cell help for B cell and CTL response..As a
result, the compound
may induce a more favorable antibody response with high titers, which appear
earlier in the course
of immunization and persist over time, as well as increase memory responses
and CD8+ MHC
5 Class I-restricted CTL. A compound of the invention may be contained in a
vaccine or it may be
administered separately. A compound of the invention may be used to enhance
immunogenicity of
antigens that induce T cell responses (e.g. T cell antigens), and in
particular they may be used to
enhance the immunogenicity of carbohydrate antigens associated with cancers or
infectious
diseases. Examples of vaccines which may employ a compound of the invention to
augment
immunogenicity include cancer vaccines (e.g. breast cancer vaccines), and
vaccines for chronic
infectious diseases.
The following non-limiting examples are illustrative of the present invention:
Example 1
Synthesis of (1S, ZR, 8R, 8aR~8-Methoxymethoxy-1, 2-
(isopropylidenedioxy)indolizidine (2).
To a stirred solution of swainsonine acetonide 1 (107 mg, 0.50 mmol) in dry
THF (2.5 mL) was
added sodium hydride (60% in mineral oil; 40 mg, 1.0 mmol) and the mixture was
stirred for 20
min. Tetrabutylammonium iodide (37 mg, 0.10 mmol) and chloromethyl methyl
ether (76 L, 1 . 0
mmol) were added. The reaction mixture was heated at reflux for 16 h. TLC
showed the reaction
was complete. The reaction mixture was concentrated and the residue was
purified by column
20 chromatography (hexane/EtOAc = I:1), giving 109 mg (84%) of the title
compound as a pale
yellow oil. ~H NMR (CDCI3, ref TMS, 500 MHz) 8: 4.84 (d, J= 6.5 Hz, 1 H), 4.69
(d, J= 6.5 Hz,
1 H), 4.63 (dd, J = 6.2, 4.7 Hz, I H), 4.58 (dd, J = 6.2, 4.0 Hz, I H), 3.76
(ddd, J = 10.9, 9.4, 4.7
Hz, 1 H), 3.40 (s, 3 H), 3.13 (d, J = I 0.7 Hz, 1 H), 2.97 (br d, J = 10.5 Hz,
1 H), 2. I 6 (m, I H),
2.10 (dd, J= 10.7, 4.3 Hz, 1 H), 1.83 (td, J= 11.0, 3.4 Hz, 1 H), 1.59-1.70
(m, 3 H), 1.47 (s, 3 H),
2 S 1.31 (s, 3 H), 1.18-1.26 (m, 1 H); MS (CI, CH,) m/z: 258 (M+H), 242, 228,
196.
Synthesis of (IS, 2R, 8R, 8aRr8-Methoxymethoxy-1, 2-
(isopropylidenedioxy)indolizidine N
oxide (3). To a stirred solution of 2 (240 mg, 0.93 mmol) in dichloromethane-
ethanol (1:1, 1 mL)
was added hydrogen peroxide (30%, 0.4 mL). The mixture was heated at
65°C (bath temperature)
for 14.5 h. TLC showed the reaction was complete. 10% Palladium on carbon (16
mg) was added ,
3 0 and the mixture was stirred at room temperature for 2.5 h. The mixture was
filtered and the filtrate
was dried twice over anhydrous sodium sulfate, filtered, and concentrated. The
residue was dried
in vacuo overnight, giving 167 mg of the title compound as a pale yellow
syrup, which was used in
the next step without further purification.
Synthesis of (1S, 2R, 8R, 8aR~8-Methoxymethoxy-8a-methyl-1, 2-
(isopropylidenedioxy~
35 indolizidine (4). To a stirred solution of the crude N-oxide 3 (167 mg,
0.61 mmol) in dry
dichloromethane (1.5 mL) at -10°C was added trifluoroacetic anhydride
(0.17 ml, 1.22 mmol) over
20 min. The mixture was stirred at 0°C for 2.5 h and then at room
temperature for 0.5 h. The
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reaction mixture was concentrated. The residue was dried in vacuo for 0.5 h
and dissolved in dry
THF (4 mL). To the resulting solution was added methylmagnesium bromide (3.0 M
solution in
diethyl ether; 0.82 mL, 2.4 mmol) at -78°C over 10 min. Stirring was
continued at -78°C for 2.5 h.
Water (10 mL) was added and the mixture was warmed to room temperature. The
aqueous layer
was extracted with ethyl acetate (4x10 mL). The combined organic layers were
dried over
anhydrous sodium sulfate and concentrated. The residue was purified by column
chromatography
(CHZCIZ:MeOH = 20:1), giving 104 mg (41%, three steps) of the title compound
as a yellow oil.
'H NMR (CDCl3, ref TMS, 500 MHz) b: 4.84 (d,J=6.4 Hz, I H), 4.65 (d, J = 6.6
Hz, 1 H), 4.61
(dd, J= 5.6, 5.1 Hz, 1 H), 4.27 (d, J= 6.0 Hz, 1 H), 3.83 (dd, J= 11.5, 4.7
Hz, I H), 3.39 (s, 3 H),
2.90 (d, J= 10.4 Hz, 1 H), 2.60-2.67 (m, 2 H), 2.33 (m, 1 H), 1.89 (m, I H),
1.60 (m, 2 H), 1.50 (s,
3 H), 1.40-1.47 (m, 1 H), 1.30 (s, 3 H), 0.77 (s, 3 H); MS (ES) m/z: 272(M+H),
210.
Synthesis of (1S, 2R, 8R, 8aRr8a-Methyl-1, 2, 8-trihydroxyindolizidine (5). To
a stirred
solution of 4 (97 mg, 0.36 mmol) in THF (4 mL) was added 6 N HCl (4 mL).
Stirring was
continued for 63.5 h. The reaction mixture was concentrated under reduced
pressure. The residue
was dried in vacuo, diluted with methanol (2 mL), neutralized with
concentrated ammonium
hydroxide and filtered. The crude product was purified by preparative HPLC,
giving 35.4 mg
(54%) of the title compound as a white crystalline solid. 'H NMR (DSO, ref
HDO, 500 MHz) S:
4.29 (ddd, J 2,, = 6.4 Hz, J 2,3 = 3.5 Hz, J Z,s = 8.0 Hz, H-2), 3.91 (dd, J
g,~~q = 4.8 Hz, J 8,~,~ _
11.8 Hz, H-8), 3.71 (d, J ,,2 = 6.4 Hz, H-1 ), 2.89 (dd, J 3 , 2 = 8.0 Hz, J 3
, 3 = I 1.2 Hz, H-3 ) ,
2 0 2.67 (dd, J 3 ,2 = 3.5 Hz, J 3 , 3 = 1 I .2 Hz, H-3 ), 2.43 (m,J ~,s., =
12.5 Hz, J s~q,b"~ = 5.5 Hz, J
su,,sn, = 2.0 Hz, H-5eq), 2.39 (m, J s"~,s~q = 12.5 Hz, J s,~,b", = 12.2 Hz, J
5"~,~ = 3.6 Hz, H-5ax),
1.63 (m, J ~~q,~ = 4.7 Hz, J ~~q,~ = 2.5 Hz, J ~~~,~,~ =12.6 Hz, H-7eq), 1.44-
1.57 (m, H-6ax & H-
6eq), I .39 (m, J ~"~,6"~ = 12.6 Hz, J ~"~,~q = 4.7 Hz, J ~"~,~~ = 12.6 Hz, H-
7ax), 0.83 (s, CH3); '3C
NMR (DZO, ref external 1, 4-dioxane, 125 MHz) 8 76.4 (C-I), 68.1 (C-2), 67.9
(C-8), 65.3 (C-8a),
2 5 55.8 (C-3), 42.6 (C-5), 26.8 (C-7), 21.1 (C-6), 9.3 (CH3); MS (ES) m/z:
210 (M+Na), 188 (M+H),
170, 152.
Example 2
Inhibition of Golgi a-mannosidase II and Lysosomal a-mannosidase
The test compound, (1S,2R,8R,8aR)-8a-methyl-1,2,8-trihydroxyindolizidine, is
3 0 prepared by 0.4 serial dilution of a 40 pM stock. Present in each
determination is 10 pl diluted test
compound, 25 pl of 10 mM paranitrophenyl mannopyranoside, 200 mM sodium
acetate, pH 5.6
and 15 pl of purified rat liver Golgi mannosidase II. After incubating the
reaction for 60 minutes at
37°C, the reaction is quenched with 50 pl of 0.5M sodium carbonate.
Absorption is read at 405
nm. After subtracting the blank from positive controls and samples, the
samples are normalized
3 5 against the positive control mean using a variable slope, sigmoidal curve
fit, with bottom = 0, top =
100. The signal is proportional to the amount of products from the uninhibited
reaction. The
calculated ICs° for inhibition of purified Golgi mannosidase II by the
test compound is 18.214 t
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4.437 pM.
The effects of the test compound on lysosomal mannosidase were measured by
adding
(lOpl) of the compound into 96 well Elisa plates followed by the addition of
200 mM sodium
acetate pH 5.0 and 25 ul of 10 mM p-nitrophenyl a-D-mannospyranoside. IS pl of
lysosomal
mannosidase ( about SmM/mL) was added to each well and the plates were
incubated for 60 min at
37°C. The reaction was stopped by the addition of 50 pl of O.SM sodium
carbonate and formation
of p-nitrophenol was measured with a plate set at 405. The calculated ICso for
inhibition of
lysosomal mannosidase by swainsonine hydrochloride is 9.388 t 3.021 pM.
Example 3
Reuresentative In V~vo and In Vitro Protocols for Testing Compounds of the
Invention
A. Administration otTest Compound for the Inhibition of Lung Metastasis
B16F10 melanoma tumor cells are cultured for 48 hours in the presence or
absence of the
test compound (0.36 pg/ml) before injection of lOs cells into the lateral tail
veins of C57BL mice.
Lung nodules are counted on day 24 after injection of tumor cells as described
in Dennis, JW,
Cancer Res. 46:5131-5136, 1986.
B. Inhibition ottumor cell colonization otthe lung
Mice are given drinking water with or without 5.0 pg/ml of the test compound 2
days
before tumor cells are injected into the lateral tail vein and maintained on
the test compound for
periods of I-17 days. Lung nodules are counted on day 24 after injection
oftumor cells.
2'0 C. Inhibition of human tumor growth in mice
Athymic nude mice injected subcutaneously with MeWo, a human melanoma tumor
cell
line, are treated with once daily ip injections of sterile saline or 20
pg/mouse of test compound in
sterile saline. Tumor size is measured twice weekly with calipers and tumor
weights are measured
4 weeks after tumor cell injection as per the method of Dennis, JW (Cancer
Res. 50:1867-1872,
2 5 1990).
D. Determining Synergy of a Test Compound with the interferon-inducing agent
Poly (LC.)
for inhibition of solid tumor growth
Mice are provided with drinking water either with or without test compound
(3.0 pg/ml) 2
days before 105 MDAY-D2 tumor cells are injected. Tumor diameters are measured
with calipers
3 0 twice weekly, then on day 15 after tumor cell injection, tumors are
excised and weighed. The
tumor growth rate and tumor weight on day 15 in mice given test compound
supplemented
drinking water and/or two i.p. injections of poly (LC.) are compared as
described in Dennis JW
Cancer Res. 46:5131-5136, 1986.
E. Enhancement of the Anti-proliferative effect of Interferon in vitro
3 5 HT29m, SN 12C 11 human carcinoma cells or MeWo melanoma cells are seeded
into 5%
FBS in MEM tissue culture medium at 103/ml in the presence and absence of a
test compound
(approximately l.2ug/ml) either with or without 1000 IU/ml of human interferon
alpha-2 (intronA,
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Schering-Plough). The cells are cultured at 37°C in a 5% COZ atmosphere
and on day 5 the cell
number is determined. The method is as described by Dennis, J.W. JNCI 81:1028-
1033, 1989.
F. In Vitro Progenitor Cell Assay
At specified times after treatment with between 0.7 and 5.0 pg/ml of test
compound,
control, and treated mice are killed by cervical dislocation. Bone marrow (BM)
and spleen cells
from each are processed according to the procedures of the GIBCO-BRL Mouse
Bone Marrow
Stem Cell Proliferation Kit (Cat. # 3827SA, Grand Island, NY). The potential
colonies that form in
the semi-solid medium are the CFU-GEMM; the CFU-GM, and the BFUs. The plates
are
incubated for 10-14 days at 37°C in a humidified atmosphere of 5% COZ
and 95% air, and colonies
consisting of at least 40 cells are enumerated using an inverted microscope
(20X magnification) to
demonstrate stimulation of hematopoietic progenitor cell growth.
G. Bone Marrow Proliferation Assay
Mice are treated with either 3 pg/ml of test compound in their drinking water
or injected
with 20 pg/mouse of test compound daily for 2-6 days. Proliferation is
assessed by the
incorporation of [3H]-thymidine (5 pCi/ml) for 18 hours at 37°C into
cultures containing equal
numbers of freshly isolated BM cells in complete medium. The radiolabeled
cells are collected
with the aid of a cell harvester onto glass filters, and radioactivity is
determined using a liquid
scintillation counter. Cellularity of the bone marrow is also determined by
using the Coulter
counter to directly count BM cells after they are flushed from the tibias and
femurs.
2 0 H. In vivo progenitor assay: Spleen Colony Formation Assay
Mice (10-14 weeks old) are x-irrradiated for a total whole body exposure of
700cGY. The
irradiated mice are maintained on sterile drinking water {approximately 3
pg/ml) and are given
antibiotics to minimize mortality from infection. The number of BM stem cells
is estimated by the
method of Till and McCulloch (Biochim Biophys Acta 1980 Nov 26;605(4):431-59),
which is
2 5 based on the ability of intravenously injected progenitor stem cells to
form colonies in the spleens
of recipient mice previously exposed to a lethal dose of whole-body
irradiation. The number of
CFUs is proportionai to the number of pluripotent hematopoietic stem cells
present in the
hematopoietic graft. Ten days after transplantation, recipient mice are
sacrificed, their spleens are
removed and fixed in Bouin's solution, and grossly visible colonies are
counted.
3 0 I. Bone marrow transplant and repopulation
Prior to transplantation with bone marrow cells, mice are pre-treated with
either a lethal
dose of a chemotherapeutic agent or a lethal dose of x-irradiation, as
described in White et al
(Cancer Communications 3:83, 1991) and Oredipe et al. (JNCI 83:1149, 1991).
Mice aged 10-14
weeks, are irradiated using Phillips RT 250 x-ray machines (two opposing
therapeutic 250 Kvp x-
3 5 ray machines, 235 KV, 15 mA, filtration 0.25 copper and 0.55 aluminum,
with a half layer of 0.99
mm copper). Irradiation occurs with a dose rate of 126 cGy/min (63 cGy/min X
2) for 5 minutes
and 33 seconds, for a total whole body exposure of 700 cGy. This level of
irradiation exposure is
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within the range described as being lethal for mice. After x-irradiation,
animals are infused with
lOs bone marrow cells freshly prepared from either contml or test compound-
treated donor mice.
The test compound-treated donor mice receive approximately 20 pg/ml of test
compound for 6
days. Recipient mice are monitored for survival over a period of 30 to 50
days.
J. Thl immune response: Natural Killer (NK) and lymphokine-activated killer
(LAK) cell
assays
Human peripheral blood mononuclear cells (PBMCs) are isolated from whole blood
using
standard methods (Rees et al; J. Immunol Meths., 62:79-85, 1983; or Sedman et
al, Br. J. Surg. 75:
976-981, 1988). The PBMCs are seeded into six-well plates in 5 ml cultures at
a concentration of
1.5 million cells per ml either alone (control) or with varying concentrations
of test compound,
together with 1000 International Units (IU)/ml of IL-2 for three days for the
LAK assay or 1000
1U/ml interferon-alpha overnight for the NK assay. The NK cell activity of the
cultured PBMCs is
measured in a Crs~ release assay using the K562 cell line (NK cell-sensitive)
as target cells. LAK
cell activity is measured using Crs~-labeled Daudi cell line (NK cell-
resistant) as targets.
K. Activity in mouse models of hepatitis
Drug activity against viral hepatitis may be determined by infecting mouse
strains with
mouse hepatitis virus-3 (MHV-3). Previous studies with MHV-3 have focused on
mouse strains
which develop fulminant hepatitis (Balb\cJ) or display resistance (A/J) to MHV-
3 {Yuwaraj et al.,
1996).
2 0 The CH3/HeJ strain, which develops chronic hepatitis in response to MHV-3
infection is
treated with either saline or test compound {20 pg/mouse/day) alone or in
combination with IFN.
Before and during treatment, the levels and activation status of STATs is
measured as well as
serum cytokine levels, viral load and survival.
L. Activity in patients with chronic hepatitis C
2 5 The response to treatment with a test compound or test compound plus
interferon-alpha in
patients with chronic hepatitis C can be monitored by a decrease in viral load
and serum liver
alanine aminotransferase (ALT) measured during treatment, for example at 3, 6,
and 12 months.
Improvement in liver histology can also be assessed by performing biopsies
before and after
treatment.
3 0 The test compound is administered orally, twice daily, at doses between 50
and 200 pg/kg
either alone, or in combination with alpha-interferon, which is administered
at doses of 1 to 3 MU
three times weekly. During this time, the test compound may be administered
continuously or
intermittently (e.g. 2 weeks on, one week off). The response in patients
receiving test compound is
compared to the response in patients receiving placebo or alpha-interferon.
3 5 Detection of hepatitis C viral RNA in serum, liver, and peripheral blood
mononuclear
cells is performed by the reverse transcriptase-polymerise chain reaction
method (RT-PCR), using
primer specific for the highly conserved, 5'-untranslated region (UTR) for
qualitative or, with
CA 02352858 2001-05-30
WO 00/37465 PCT/CA99/01210
-19-
appropriate internal control RNA, quantitative detection. The second method is
a signal
amplification or branched chain DNA (bDNA) assay. Viral nucleic acids are
hybridized to
microtiter plates and reacted with virus-specific extender probes followed by
bDNA polymers.
For improvement in liver histology, the Histologic Activity Index based on a
scoring
5 system developed by ICnodell et al (Hepatology 1981, 1:431-435), assigns
grades in four
categories: periportal necrosis, interlobular necrosis, portal inflammation
and fibrosis.
Alternatively, a system based on grading hepatic inflammation (0-4) and
staging fibrosis (0-4) can
be used (Scheuer PJ, J. Hepatol 1991; 13:372-374).
M. Hemorestoration/Chemoprotection
10 Cellular and animal models of hemorestoration/chemoprotection are described
in Oredipe
et al, 1991, supra, and White et al, 1991, supra.
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
15 and equivalent arrangements included within the spirit and scope of the
appended claims.
All publications, patents, aid 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.
