U.S. patent application number 12/831111 was filed with the patent office on 2010-12-23 for novel synthetic ganglioside derivatives and compositions thereof.
This patent application is currently assigned to Seneb Biosciences, Inc.. Invention is credited to Shawn DeFrees, Zhi-Guang Wang.
Application Number | 20100324274 12/831111 |
Document ID | / |
Family ID | 23226244 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100324274 |
Kind Code |
A1 |
DeFrees; Shawn ; et
al. |
December 23, 2010 |
NOVEL SYNTHETIC GANGLIOSIDE DERIVATIVES AND COMPOSITIONS
THEREOF
Abstract
Novel synthetic gangliosides and pharmaceutical compositions
containing such synthetic gangliosides are described. Methods of
making the novel synthetic ganglioside compounds and compositions
as well as their use in the field of neuroprotection and cancer
treatment is also described.
Inventors: |
DeFrees; Shawn; (North
Wales, PA) ; Wang; Zhi-Guang; (Dresher, PA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP (SF)
One Market, Spear Street Tower, Suite 2800
San Francisco
CA
94105
US
|
Assignee: |
Seneb Biosciences, Inc.
|
Family ID: |
23226244 |
Appl. No.: |
12/831111 |
Filed: |
July 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10487841 |
Oct 1, 2004 |
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PCT/US2002/027935 |
Aug 29, 2002 |
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12831111 |
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60315831 |
Aug 29, 2001 |
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Current U.S.
Class: |
536/17.4 ;
536/17.9 |
Current CPC
Class: |
A61P 17/02 20180101;
C07H 3/06 20130101; A61P 25/16 20180101; A61P 25/22 20180101; A61P
25/28 20180101; A61P 25/00 20180101; A61P 9/10 20180101; C12P 19/28
20130101; C07H 17/02 20130101; A61P 25/24 20180101 |
Class at
Publication: |
536/17.4 ;
536/17.9 |
International
Class: |
C07H 15/26 20060101
C07H015/26; C07H 15/18 20060101 C07H015/18 |
Claims
1-22. (canceled)
23. A synthetic compound having a formula selected from the group
consisting of (Va), (Vb), (Vc) and (Vd): ##STR00062## wherein the
saccharide portion is selected from the group consisting of a
monosaccharide, a disaccharide, an oligosaccharide, a
polysaccharide, an N-acetylated derivative thereof and an
N-deacetylated derivative thereof; X is NR.sup.1R.sup.2; Y is --OH;
Z is O; each R.sup.1 and R.sup.2 are independently selected from
the group consisting of --C(=M)R.sup.3, and --C(=M)-p-R.sup.3;
wherein M and p are independently selected from O, NR.sup.4 and S;
R.sup.3 is H, alkyl, arylalkyl, haloalkyl, aryl, heteroaryl, or
heteroalkyl; R.sup.4 is H, alkyl, aryl, arylalkyl, heteroaryl,
heteroalkyl, or haloalkyl; R.sup.6 is H; R.sup.6' is
--NR.sup.9R.sup.10; R.sup.6'' is H; R.sup.7 and R.sup.8 are
independently H, and aryl, where, R.sup.7 and R.sup.8 are
optionally substituted with at least one group selected from the
group consisting of halo, haloalkyl, alkoxy, and thiohaloalkyl;
R.sup.7' and R.sup.8' are independently H, alkenyl,
--C(.dbd.O)NR.sup.9R.sup.10, aryl, heteroaryl, or a linked bicyclic
mixture of aryl and heteroaryl, where R.sup.7' and R.sup.8' are
optionally substituted with at least one group selected from the
group consisting of halo, haloalkyl, alkoxy, and thiohaloalkyl;
R.sup.9, R.sup.10, and R.sup.11 are independently alkyl,
cycloalkyl, aryl, arylalkyl, heteroaryl, or heteroalkyl, where
R.sup.9, R.sup.10, and R.sup.11 are optionally substituted,
preferably-substituted with at least one group selected from the
group consisting of halo, haloalkyl, alkoxy, and thiohaloalkyl; or
where R.sup.9 and R.sup.10 taken together with the nitrogen to
which they are attached form a heterocyclic ring comprising at
least one heteroatom selected from N, O and S; and all
pharmaceutically acceptable salts, hydrates, geometric isomers and
solvates thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/487,841, filed Feb. 17, 2004, which is a U.S. national phase
of PCT Application No. PCT/US2002/027935 filed Aug. 29, 2002, which
claims the benefit of priority under 35 U.S.C .sctn.119(e) to U.S.
Provisional Application No. 60/315,831, filed Aug. 29, 2001, which
applications are incorporated by reference, herein in their
entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] The term "carbohydrate" or "saccharide" embraces a wide
variety of chemical compounds having the general formula
(CH.sub.2O).sub.n, such as monosaccharides, disaccharides,
oligosaccharides and polysaccharides. Oligosaccharides and
polysaccharides are chains composed of monosaccharide units, which
are also generally referred to as sugars. The monosaccharide units
of an oligo- or polysaccharide can be arranged in various orders.
The linkage between any two saccharide units can occur in any of
approximately ten different ways. As a result, the number of
different possible stereoisomeric oligosaccharide or polysaccharide
chains is enormous. Saccharides are a key component of
glycosphingolipids found in cell membranes. Of interest with
respect to the present invention is a certain class of
glycoshingolipids known as gangliosides.
[0003] Gangliosides are glycosphingolipids, often found in cell
membranes, that consist of three elements. One or more sialic acid
residues are attached to an oligosaccharide or carbohydrate core
moiety, which in turn is attached to a hydrophobic lipid (ceramide)
structure which generally is embedded in the cell membrane. The
ceramide moiety includes a long chain base (LCB) portion and a
fatty acid (FA) portion. Gangliosides, as well as other glycolipids
and their structures in general, are discussed in, for example,
Lehninger, Biochemistry (Worth Publishers, 1981) pp. 287-295 and
Devlin, Textbook of Biochemistry (Wiley-Liss, 1992). Gangliosides
are classified according to the number of monosaccharides in the
carbohydrate moiety, as well as the number and location of sialic
acid groups present in the carbohydrate moiety. Mono
sialogangliosides are given the designation "GM",
disialogangliosides are designated "GD", trisialogangliosides "GT",
and tetrasialogangliosides are designated "GQ". Gangliosides can be
classified further depending on the position or positions of the
sialic acid residue or residues bound. Further classification is
based on the number of saccharides present in the oligosaccharide
core, with the subscript "1" designating a ganglioside that has
four saccharide residues (Gal-GalNAc-Gal-Glc-Ceramide), and the
subscripts "2", "3" and "4" representing trisaccharide
(GalNAc-Gal-Glc-Ceramide), disaccharide (Gal-Glc-Ceramide) and
monosaccharide (Gal-Ceramide) gangliosides, respectively.
[0004] Numerous types of gangliosides found in nature have been
isolated and identified and vary primarily in the basic saccharide
structure (e.g. G.sub.M3, G.sub.M2, G.sub.M1, G.sub.D1a, G.sub.D1b
and G.sub.T1). A variety of procedures are available for the
isolation and purification of such "natural" gangliosides from
organs and tissues, particularly from animal brain (Sonnino et al.,
1992, J. Lipid Res., 33:1221-1226; Sonnino et al., 1988, Ind. J.
Biochem. Biophys., 25:144-149; Svennerholm, 1980, Adv. Exp. Med.
Biol., 125:533-44) as well as bovine buttermilk (Ren et al., 1992,
J. Bio. Chem., 267:12632-12638; Takamizawa et al., 1986, J. Bio.
Chem., 261:5625-5630).
[0005] Gangliosides are normal components of plasma membranes and
are particularly abundant in the nervous system. In humans,
gangliosides are most abundant in the gray matter of the brain,
particularly in nerve endings. They are believed to be present at
receptor sites for neurotransmitters, including acetylcholine, and
can also act as specific receptors for other biological
macromolecules, including interferon, hormones, viruses, bacterial
toxins, and the like.
[0006] Certain gangliosides are found on the surface of human
hematopoietic cells (Hildebrand et al. (1972) Biochim. Biophys.
Acta 260: 272-278; Macher et al. (1981) J. Biol. Chem. 256:
1968-1974; Dacremont et al. Biochim. Biophys. Acta 424: 315-322;
Klock et al. (1981) Blood Cells 7:247) which may play a role in the
terminal granulocytic differentiation of these cells. Nojiri et al.
(1988) J. Biol. Chem. 263: 7443-7446. These gangliosides, referred
to as the "neolacto" series, have neutral core oligosaccharide
structures having the formula
[Gal.beta.-(1,4)GlcNAc.beta.(1,3)].sub.nGal.beta.(1,4)Glc, where
n=1-4. Included among these neolacto series gangliosides are
3'-nLM.sub.1
(NeuAc.alpha.(2,3)Gal.beta.(1,4)GlcNAc.beta.(1,3)Gal.beta.(1,4)-Glc.beta.-
(1,1)-Ceramide) and 6'-nLM.sub.1
(NeuAc.alpha.(2,6)Gal.beta.(1,4)GlcNAc.beta.(1,3)Gal.beta.(1,4)-Glc.beta.-
(1,1)-Ceramide).
[0007] It has been widely demonstrated that gangliosides are able
to enhance functional recovery both in the lesioned peripheral
nervous system (PNS) and the central nervous system (CNS), through
the involvement of specific membrane mechanisms and the interaction
with trophic factors, as pointed out from studies in vitro on
neuronal cultures (Ferrari, F. et al., Dev. Brain Res., 1983,
8:215-221; Doherty, P. et al., J. Neurochem., 1985, 44:1259-1265;
Skaper, S. D. et al., Mol. Neurobiol., 1989, 3:173-199).
Gangliosides have been used for treatment of nervous system
disorders, including cerebral ischemic strokes. See, e.g., Mahadnik
et al. (1988) Drug Development Res. 15: 337-360; U.S. Pat. Nos.
4,710,490 and 4,347,244; Horowitz (1988) Adv. Exp. Med. and Biol.
174: 593-600; Karpiatz et al. (1984) Adv. Exp. Med. and Biol. 174:
489-497.
[0008] As a result, attempts have been made to use gangliosides in
the treatment of disorders of the nervous system. This has led to
the development of synthetic gangliosides as well as natural
ganglioside containing compositions for use in the treatment of
disorders of the nervous system (U.S. Pat. Nos. 4,476,119,
4,593,091, 4,639,437, 4,707,469, 4,713,374, 4,716,223, 4,849,413,
4,940,694, 5,045,532, 5,135,921, 5,183,807, 5,190,925, 5,210,185,
5,218,094, 5,229,373, 5,260,464, 5,264,424, 5,350,841, 5,424,294,
5,484,775, 5,519,007, 5,521,164, 5,523,294, 5,677,285, 5,792,858,
5,795,869, and 5,849,717).
[0009] Gangliosides have also been implicated as playing a
significant role in certain types of cancer. Neuroblastoma is a
form of cancer that primarily afflicts children under the age of
five. Individuals suffering from neuroblastoma may have tumors
growing near the spinal cord, and very large tumors have been found
to cause paralysis in such patients. Gangliosides have been shown
to play a role in both the growth and the inhibition of the growth
of neuroblastoma-associated tumors (Basavarajappa et al., 1997,
Alcohol Clin. Exp. Res., 21(7):1199-203; Singleton et al., 2000,
Int. J. Dev. Neurosci., 2000, 18(8):797-80).
[0010] However, there still exists a need in the art for compounds
capable of acting as neuroprotective agents in a manner similar to
or better than the natural gangliosides for the prophylaxis,
treatment and cure of disorders of the nervous system. Further,
differences in the structure of ganglioside compounds can refine
the structure-function relationship of such compounds to provide
powerful tools for control of the growth of certain kinds of
tumors, including neuroblastoma tumors.
SUMMARY OF THE INVENTION
[0011] The present invention answers such a need by providing novel
synthetic gangliosides of formulae (I) and (Va)-(Vd):
##STR00001##
including pharmaceutically acceptable salts, isomers, hydrates,
solvates, and prodrugs thereof.
[0012] The invention further provides a novel synthetic ganglioside
of the formula
##STR00002##
in which Z can be O, S, C(R.sup.2).sub.2 and NR.sup.2, X can be H,
--OR.sup.3, --NR.sup.3R.sup.4, --SR.sup.3, and --CHR.sup.3R.sup.4,
and R.sup.1, R.sup.2 and R.sup.3 can be independently selected from
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl, --C(=M)R.sup.5, --C(=M)-Z--R.sup.5,
--SO.sub.2R.sup.5, and --SO.sub.3 functional moieties. Further, a
novel ganglioside of the present invention can have M and Z
independently selected from O, NR.sup.6 or S, and Y can be selected
from H, --OR.sup.7, --SR.sub.7, --NR.sup.7R.sup.8, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, and substituted or unsubstituted heterocycloalkyl
moieties. Further still, a novel ganglioside of the invention can
have R.sup.5, R.sup.6, R.sup.7 and R.sup.8 independently selected
from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycloalkyl moieties.
[0013] The present invention also provides a novel ganglioside
compound as described above, with the proviso that when X is
NHR.sup.4, in which R.sup.4 is selected from H and
--C(.dbd.O)R.sup.5, in which R.sup.5 is substituted or
unsubstituted alkyl, Y is OH; and Z is O, R.sup.5 is other than a
substituted or unsubstituted alkyl moiety.
[0014] The present invention also provides a novel ganglioside
compound in which the saccharide component can be
##STR00003##
and such saccharide moieties may or may not be deacetylated.
[0015] The invention further provides pharmaceutical compositions
including at least one compound of the invention and a
pharmaceutically acceptable carrier.
[0016] The invention still further provides a method for the
prevention and/or treatment and/or cure of a disorder of the
nervous system in an animal or human including the step of
administering to a patient in need thereof a therapeutically
effective amount of at least one compound or pharmaceutical
composition of the invention. Such patients in need of a compound
of the present invention may suffer from a disorder of the nervous
system, including Parkinson's disease, ischemia, stroke,
Alzheimer's disease, depression, anxiety, encephalitis, meningitis,
amyotrophic lateral sclerosis, trauma, spinal cord injury, nerve
injury, and nerve regeneration.
[0017] One embodiment of the invention provides a method for the
treatment of a glioma in a human and includes the step of
administering to the human in need thereof a therapeutically
effective amount of a compound of the present invention.
[0018] The present invention also provides a method of synthesizing
a synthetic ganglioside compound of the invention, wherein the
steps of synthesis of the saccharide moiety include contacting a
sphingoid acceptor molecule and a glucose molecule with a
galactosyltransferase enzyme and a galactose donor molecule to
form
##STR00004##
contacting the
##STR00005##
with a trans-sialidase enzyme and a sialic acid (NANA) donor
molecule to form
##STR00006##
contacting the
##STR00007##
with a N-acetyl galactose (GalNAc)-transferase enzyme and a GalNAc
donor molecule to form
##STR00008##
contacting the
##STR00009##
with a galactosyltransferase enzyme and a galactose (Gal) donor
molecule to form
##STR00010##
and contacting the
##STR00011##
with a fatty acid moiety under conditions sufficient to form a
ganglioside.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows a schematic diagram of two methods for
synthesis of the ganglioside GM2 by enzymatic synthesis using as
the starting material lactosylceramide obtained from bovine
buttermilk.
[0020] FIG. 2 shows a schematic diagram of two methods for
synthesizing the ganglioside GD.sub.2 from lactosylceramide
obtained from bovine buttermilk.
[0021] FIG. 3 shows three routes for synthesizing a GM2 ganglioside
using a plant glucosylceramide as the starting material.
[0022] FIG. 4 shows three routes for synthesizing GM2 and other
gangliosides starting from a glucosylceramide.
[0023] FIG. 5 shows a scheme used for synthesis of the ganglioside
GM2 from lactosylceramide via deacylation, two consecutive
enzymatic glycosylations, and final chemical acylation.
[0024] FIGS. 6-15 show attenuation of neuroblastoma cell growth
when the neuroblastoma cells are treated with various compounds of
the present invention.
[0025] FIG. 6 shows that compound 1003, at 50 .PHI.M, causes almost
100% growth inhibition in all cell lines (86-100%).
[0026] FIG. 7 shows that compound 1009 has a profile similar to
that for compound 1003 in four cell lines (77-89% growth inhibition
with 50 .PHI.M compound 1009) and in U-118 cells, the growth
inhibition with 50 .PHI.M compound 1009 is 21%.
[0027] FIG. 8 shows that compound 1011 has activity similar to
compound 1003, with the exception that the inhibition of 9 L cells
by 50 .PHI.M compound 1011 was 46%.
[0028] FIG. 9 shows that compound 1014, when used to treat Hs 683
and Sw1088 cells, inhibited proliferation 42% and 35%,
respectively, when used at a concentration of 50 .mu.M.
[0029] FIG. 10 shows that 50 .PHI.M compound 1081 inhibited
proliferation of 9 L 23%, U-118 cells 27%, Hs 683 cells 48%, and Sw
1088 cells 68%.
[0030] FIG. 11 shows that compound 1082 inhibited the growth of 9 L
cells 11-37%.
[0031] FIG. 12 shows that compound 1083, at 5 .PHI.M, inhibited
growth of 9 L and Hs 683 cells (27% and 32%, respectively). At 50
.PHI.M, compound 1083 inhibited growth of 9 L, Hs 683, U-118, and
Sw 1088 cells 26-54%.
[0032] FIG. 13 shows that compound 1084 strongly inhibited growth
in all cell lines at 50 .PHI.M compound (91-100%).
[0033] FIG. 14 shows that compound 1085 was very active in the cell
proliferation assay.
[0034] Compound 1085 demonstrated growth inhibition activity at 5
.PHI.M in all cell lines tested (15-88%), and strong growth
inhibition at 50 .PHI.M in all cell lines (95-100%).
[0035] FIG. 15 shows that compound 1086, at 50 .PHI.M, inhibits
growth of all cell lines 66-100%.
DEFINITIONS
[0036] In accordance with the invention and as used herein, the
following terms are defined with the following meanings, unless
explicitly stated otherwise.
[0037] The article "a" and "an" as used herein refers to one or to
more than one (i.e. at least one) of the grammatical object of the
article. By way of example, "an element" means one element or more
than one element.
[0038] The term "alkenyl" as used herein refers to a substituted or
unsubstituted trivalent straight chain or branched chain
unsaturated aliphatic radical that includes at least two carbons
joined by a double bond.
[0039] The term "alkynyl" as used herein refers to a straight or
branched chain aliphatic radical that includes at least two carbons
joined by a triple bond. If no number of carbons is specified,
"alkenyl" and "alkynyl" each refer to radicals having from 2-12
carbon atoms.
[0040] The term "cycloalkyl" as used herein refers to a substituted
or unsubstituted saturated aliphatic ring system, preferably a
mono-, bi-, or tricyclic saturated aliphatic ring system. Examples
include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, adamantyl, cyclooctyl,
[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane
(decalin), and [2.2.2]bicyclooctane
[0041] The term "aromatic" is intended to mean stable substituted
or unsubstituted mono-, bi-, tri-, polycyclic ring structures
having only carbon atoms as ring atoms including, but not limited
to, a stable monocyclic ring which is aromatic having six ring
atoms; a stable bicyclic ring structure having a total of from 7 to
12 carbon atoms in the two rings of which at least one of the rings
is aromatic; and a stable tricyclic ring structure having a total
of from 10 to 16 atoms in the three rings wherein the tricyclic
ring structure of which at least one of the ring is aromatic. Any
non-aromatic rings present in the monocyclic, bicyclic, tricyclic
or polycyclic ring structure may independently be saturated,
partially saturated or fully saturated. Examples of such "aromatic"
groups include, but are not limited to, phenyl and naphthyl.
[0042] The term "arylalkyl" as used herein refers to one, two, or
three substituted or unsubstituted aryl groups having the number of
carbon atoms designated appended to an alkyl group having the
number of carbon atoms designated. The direction of attachment of
an arylalkyl group to the remainder of the molecule may be through
either the aryl or alkyl portion of the group. Suitable arylalkyl
groups include, but are not limited to, benzyl, picolyl,
naphthylmethyl, phenethyl, benzylhydryl, trityl, and the like, all
of which may be optionally substituted.
[0043] As used herein the term "heteroaryl," "heteroaromatic" or
"aromatic heterocyclic ring system" refers to a monocyclic,
bicyclic or polycyclic, substituted or unsubstituted heterocyclic
ring system containing at least one aromatic ring.
[0044] The term "substituted" as used herein means that a hydrogen
atom has been replaced with another monovalent group (e.g. halo,
haloalkyl, hydroxy, thiol, alkoxy, thiohaloalkyl, amino, and the
like).
[0045] The terms "halo" or "halogen" as used herein refer to Cl,
Br, F or I. The term "haloalkyl" and the like, refer to an alkyl
group, as defined herein, wherein at least one hydrogen atom of the
alkyl group is replaced by a Cl, Br, F or I. A mixture of different
halo atoms may be used if more than one hydrogen atom is replaced.
For example, a haloalkyl includes chloromethyl (--CH.sub.2C1) and
trifluoromethyl (--CF) and the like.
[0046] The term "methylene" refers to --CH.sub.2--.
[0047] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents which would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is intended to also recite --OCH.sub.2--.
[0048] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include, but are not
limited to, groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below, such as
"heteroalkyl." Alkyl groups which are limited to hydrocarbon groups
are termed "homoalkyl".
[0049] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified, but not limited, by
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and further includes those
groups described below as "heteroalkylene." Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0050] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0051] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and at
least one heteroatom selected from the group consisting of O, N, Si
and S, and wherein the nitrogen and sulfur atoms may optionally be
oxidized and the nitrogen heteroatom may optionally be quaternized.
The heteroatom(s) O, N and S and Si may be placed at any interior
position of the heteroalkyl group or at the position at which the
alkyl group is attached to the remainder of the molecule. Examples
include, but are not limited to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0052] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like.
[0053] Examples of "heterocycles", "heterocyclic rings" or
"heterocyclic ring systems" include, but are not limited to,
acridinyl, azocinyl, benzimidazolyl, benzofuranyl,
benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl,
benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,
benzimidazalinyl, carbazolyl, 4aH-carbazolyl, carbolinyl,
chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl,
isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl,
isoindolyl, isoquinolinyl (benzimidazolyl), isothiazolyl,
isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl,
oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,
1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl,
oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,
phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl,
phthalazinyl, piperazinyl, piperidinyl, pteridinyl, purinyl,
pyranyl, pyrazinyl, pyroazolidinyl, pyrazolinyl, pyrazolyl,
pyridazinyl, pryidooxazole, pyridoimidazole, pyridothiazole,
pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl,
2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl,
quinoxalinyl, quinuclidinyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl,
6H-1,2,5-thiadazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,
1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,
thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl,
thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl. Also included are
fused ring and spiro compounds containing, for example, the above
heterocyclic ring structures.
[0054] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" is mean to
include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0055] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, hydrocarbon substituent which can be a
single ring or multiple rings (preferably from 1 to 3 rings) which
are fused together or linked covalently. The term "heteroaryl"
refers to aryl groups (or rings) that contain from one to four
heteroatoms selected from N, O, and S, wherein the nitrogen and
sulfur atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. A heteroaryl group can be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below.
[0056] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0057] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") are meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0058] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to:
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''')=NR'''',
--NR--C(NR'R'')=NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R', R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0059] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are varied and are
selected from, for example: halogen, --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R',
--NR--C(NR'R''R''')=NR'''', --NR--C(NR'R'').dbd.NR''', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and
--NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where R', R'', R''' and R'''' are
preferably independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-(C.sub.1-C.sub.4)alkyl, and
(unsubstituted aryl)oxy-(C.sub.1-C.sub.4)alkyl. When a compound of
the invention includes more than one R group, for example, each of
the R groups is independently selected as are each R', R'', R'''
and R'''' groups when more than one of these groups is present.
[0060] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q--U--, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2NR'-- or a
single bond, and r is an integer of from 1 to 4. One of the single
bonds of the new ring so formed may optionally be replaced with a
double bond. Alternatively, two of the substituents on adjacent
atoms of the aryl or heteroaryl ring may optionally be replaced
with a substituent of the formula
--(CRR').sub.s--X--(CR''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R''' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6)alkyl.
[0061] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
[0062] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When
compounds of the present invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amino, or magnesium salt, or
a similar salt. When compounds of the present invention contain
relatively basic functionalities, acid addition salts can be
obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of pharmaceutically acceptable acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, for
example, Berge et al., "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds
of the present invention contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0063] The neutral forms of the compounds are preferably
regenerated by contacting the salt with a base or acid and
isolating the parent compound in the conventional manner. The
parent form of the compound differs from the various salt forms in
certain physical properties, such as solubility in polar
solvents.
[0064] In addition to salt forms, the present invention provides
compounds, which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present invention. Additionally, prodrugs can be converted to
the compounds of the present invention by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present invention when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0065] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are encompassed within the scope of the present
invention. Certain compounds of the present invention may exist in
multiple crystalline or amorphous forms. In general, all physical
forms are equivalent for the uses contemplated by the present
invention and are intended to be within the scope of the present
invention.
[0066] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are encompassed within the scope of the present invention.
[0067] The compounds of the present invention may also contain
unnatural proportions of atomic isotopes at one or more of the
atoms that constitute such compounds. For example, the compounds
may be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C).
All isotopic variations of the compounds of the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention.
[0068] "Pharmaceutically acceptable acid addition salt" as used
herein refers to salts retaining the biological effectiveness and
properties of the free bases and which are not biologically or
otherwise undesirable, formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like, and organic acids such as acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicyclic acid and the like.
[0069] "Pharmaceutically acceptable base addition salts" as used
herein refers to those salts derived from inorganic bases such as
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Salts derived
from pharmaceutically acceptable organic nontoxic bases include
salts of primary, secondary, and tertiary amines, substituted
amines including naturally occurring substituted amines, cyclic
amines and basic ion exchange resins, such as isopropylamine,
trimethylamine, diethylamine, triethylamine, tripropylamine,
ethanolamine, 2-diethylaminoethanol, trimethamine,
dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,
hydrabamine, choline, betaine, ethylenediamine, glucosamine,
methylglucamine, theobromine, purines, piperizine, piperidine,
N-ethylpiperidine, polyamine resins and the like.
[0070] The term "biological property" as used herein means an in
vivo activity that is directly or indirectly performed by a
compound or pharmaceutical composition of the invention that is
often shown by in vitro assays. In the present invention, the
biological property is neuroprotection, including the prophylaxis,
treatment and/or cure of disorders of the nervous system.
[0071] The term "isomer" as used herein refers to a compound having
the same number and kind of atoms and hence the same molecular
weight as another compound, but differing in respect to the
arrangement or configuration of the atoms of the compound (e.g. cis
and trans isomers). The term "isomer" also includes stereoisomers,
diastereoisomers, enantiomers or mixtures thereof. The D-isomer is
preferred.
[0072] The term "substructure" as used herein refers to a portion
of a chemical compound. For example, a single aromatic ring of a
napthalene structure is herein referred to as a substructure of the
entire napthalene molecule.
[0073] The term "hydrate" as used herein refers to the product of
water with a compound of the invention such that the H--OH bond is
not split. A compound of the invention may form more than one
hydrate. However, the amount of water in a hydrate of the invention
is such that the compound remains stable. Preferably, a hydrate of
a compound of the invention contains about 0.1-10% water.
[0074] The term "prodrug" as used herein refers to a
pharmacologically inactive derivative or precursor of a compound of
the invention which upon biotransformation, either spontaneous or
enzymatic, within an organism releases a compound of the invention
as a pharmaceutically active drug. A prodrug derivative of a
compound of the invention contain groups cleavable under metabolic
conditions such as, for example, solvolysis under physiological
conditions or enzymatic degradation. According to the invention, a
compound of the invention resulting from the biotransformation of
its prodrug derivative are pharmaceutically active in vivo. Prodrug
derivatives of a compound of the invention may be designated as
single, double, triple, etc., corresponding to the number of
biotransformation steps required to release the pharmaceutically
active compound of the invention within the organism and/or
indicating the number of functionalities present in the prodrug
derivative. Prodrugs often offer advantages of solubility, tissue
compatibility, or delayed release in the mammalian organism (see,
Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam
1985 and Silverman, The Organic Chemistry of Drug Design and Drug
Action, pp. 352-401, Academic Press, San Diego, Calif., 1992).
[0075] As used herein, the term "saccharide" may be used
interchangeably with the term "carbohydrate" and refers to single
simple sugar moieties or monosaccharides as well as combinations of
two or more single sugar moieties or monosaccharides covalently
linked to form disaccharides, oligosaccharides, and
polysaccharides. The term "saccharide" also includes N-acetylated
and N-deacylated derivatives of such monosaccharides,
disaccharides, oligosaccharides, and polysaccharides. Saccharides
for use in the invention may be linear or branched. Examples of
suitable monosaccharides include, but are not limited to, known
aldoses and ketoses (i.e. aldehyde and ketone derivatives of
straight-chain polyhydroxy alcohols containing at least three
carbon atoms) including, for example, glyceraldehyde, erythrose,
threose, ribose (Rib), arabinose (Ara), xylose (Xyl), lyxose (Lyx),
allose, altrose, glucose (Glc), mannose (Man), gulose, idose,
galactose (Gal), talose, dihydroxyacetone, erythrulose, ribulose,
xylulose, psicose, fructose (Frc), sorbose, and tagatose. Other
examples of suitable monosaccharides include, but are not limited
to, fucose (Fuc), N-acetylneuraminic acid (also called sialic acid,
NANA, or NAN (Sia)), N-acetylglucosamine (GlcNAc), and
N-acetylgalactosamine (GalNAc). The cyclic hemiacetal and hemiketal
forms of the monosaccharides are contemplated within the defined
term. Other examples of suitable saccharides include, but are not
limited to, those illustrated in FIG. 1.
[0076] As used herein, the term "disaccharide" refers to a
saccharide composed of two monosaccharides linked together by a
glycosidic bond. Examples of disaccharides include, but are not
limited to, lactose (Lac) (glycosidic bond between Gal and Glc),
sucrose (Suc) (glycosidic bond between Frc and Glc), and maltose
(Mal), isomaltose and cellobiose (glycosidic bond between Glc and
Glc).
[0077] The term "oligosaccharide" includes an oligosaccharide that
has a reducing end and a non-reducing end, whether or not the
saccharide at the reducing end is in fact a reducing sugar. In
accordance with accepted nomenclature, an oligosaccharide is
depicted herein with the non-reducing end on the left and the
reducing end on the right. An oligosaccharide described herein may
be described with the name or abbreviation for the non-reducing
saccharide (e.g., Gal), followed by the configuration of the
glycosidic bond (a or (3), the ring bond, the ring position of the
reducing saccharide involved in the bond, and then the name or
abbreviation of the reducing saccharide (e.g., GlcNAc). The linkage
between two sugars may be expressed, for example, as 2,3,2-->3,
2-3, or (2,3).
[0078] The term "sphingoid," as used herein, includes sphingosines,
phytosphingosines, sphinganines, ceramides, and the like. Both
naturally occurring and synthetically produced compounds are
included.
[0079] The term "glycosphingolipid" is a carbohydrate-containing
derivative of a sphingoid or ceramide. The carbohydrate residue is
attached by a glycosidic linkage to 0-1 of the sphingoid.
[0080] The term "sialic acid" (abbreviated "Sia") refers to any
member of a family of nine-carbon carboxylated sugars. The most
common member of the sialic acid family is N-acetyl-neuraminic acid
(2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic
acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member
of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in
which the N-acetyl group of NeuAc is hydroxylated. A third sialic
acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano
et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al.
(1990) J. Biol. Chem. 265: 21811-21819. Also included are
9-substituted sialic acids such as a 9-O--C.sub.1-C.sub.6
acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetylNeu5Ac,
9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of
the sialic acid family, see, e.g., Varki (1992) Glycobiology
2:25-40; Sialic Acids: Chemistry, Metabolism and Function, R.
Schauer, Ed. (Springer-Verlag, New York (1992). The synthesis and
use of sialic acid compounds in a sialylation procedure is
described in, for example, international application WO 92/16640,
published Oct. 1, 1992.
[0081] As used herein, the term "linker" refers to any element,
atom, molecule, that serves to join one portion of a molecule to
another. Linkers are well known to those skilled in the art.
Linkers can be mono- or multifunctional.
[0082] As used herein, the term "donor" refers to any molecule that
serves to donate or provide a monosaccharide for addition to a
growing saccharide chain or acceptor molecule. Thus the sugar
moiety serves as one part of a donor molecule. Generally, the
monosaccharide moiety is transferred from the donor to an
"acceptor," as defined herein, by means of an enzymatic reaction.
Donor molecules include those known to those of skill in the art
and will vary depending upon the desired monosaccharide to be
transferred.
[0083] As used herein, the term "contacting" or "contact" in
relation to an enzyme and "donor" and an "acceptor" to form a
growing saccharide chain means bringing the enzyme and donor into
association with the "acceptor" or growing saccharide chain to
affect the addition of a new monosaccharide unit to the acceptor or
growing saccharide chain.
[0084] As used herein, the term "acceptor" refers to a molecule
capable of receiving a monosaccharide moiety from a donor, each as
defined herein. An "acceptor" may accept more than one
monosaccharide such that a linear or branched "saccharide," as
defined above, can be formed. Thus, the term "acceptor" includes a
molecule containing a growing saccharide chain.
[0085] As used herein, the term "non-immobilized" in reference to
an "acceptor", as defined herein, means that the acceptor is not
affixed or bound to a substrate. For example, an acceptor that is
in solution would be a "non-immobilized" acceptor.
[0086] The term "glycosyltransferase" as used herein refers to
enzymes that catalyze the transfer of sugar moieties from activated
donor molecules to specific acceptor molecules, each as defined
herein, forming glycosidic bonds. Examples of glycosyltransferases
include, but are not limited to, galactosyltransferase,
glucosyltransferase, fucosyltransferase, and GalNActransferase.
Further, glycosyltransferases may be classified according to the
stereochemistries of the reaction substrates and products as either
retaining, i.e., leading to retention of the anomeric configuration
(for instance UDP-glucose->.alpha.-glucoside), or inverting,
i.e., leading to inversion of the anomeric configuration (for
instance UDP-glucose->.beta.-glucoside) (Sinnott, M. L. (1990)
Chem. Rev. 90, 1171-1202). The classification groupings of families
of glycosyltransferases is explained by Coutinho, P. M. &
Henrissat, B. (1999) Carbohydrate-Active Enzymes server, which can
be found on the Internet at
<<afmb.cnrs-mrs.fr/.about.pedro/CAZY/db.html>>.
[0087] As used herein, the term "trans-sialidase" refers to an
enzyme that catalyzes the addition of a sialic acid to galactose by
means of an .alpha.-2,3 glycosidic linkage. Trans-sialidases may be
found in many Trypanosomy species and some other parasites.
Trans-sialidases of these parasite organisms retain the hydrolytic
activity of usual sialidase, but with much less efficiency, and
catalyze a reversible transfer of terminal sialic acids from host
sialoglycoconjugates to parasite surface glycoproteins in the
absence of CMP-sialic acid. Trypanosome cruzi, which causes Chagas
disease, has a surface trans-sialidase the catalyzes preferentially
the transference of .alpha.-2,3-linked sialic acid to acceptors
containing terminal .beta.-galactosyl residues, instead of the
typical hydrolysis reaction of most sialidases (Ribeirao et al.,
1997, Glycobiol., 7:1237-1246; Takahashi et al., 1995, Anal.
Biochem., 230:333-342; Scudder et al., 1993, J. Biol. Chem.,
268:9886-9891; Vandekerckhove et al., 1992, Glycobiol., 2:541-548).
T. cruzi trans-sialidase (TcTs) has activity towards a wide range
of saccharide, glycolipid, and glycoprotein acceptors which
terminate with .beta.-linked galactose residue, and synthesizes
exclusively an .alpha.2-3 sialosidic linkage (Scudder et al.,
supra). At a low rate, it also transfers sialic acid from synthetic
.alpha.-sialosides, such as
p-nitrophenyl-.alpha.-N-acetylneuraminic acid, but
NeuAc2-3Gal.beta.1-4(Fuc.alpha.1-3)Glc is not a donor-substrate.
Modified 2-[4-methylumbelliferone]-.alpha.-ketoside of
N-acetyl-D-neuraminic acid (4MU-NANA) and several derivatives
thereof can also serve as donors for TcTs (Lee & Lee, 1994,
Anal. Biochem, 216:358-364). Enzymatic synthesis of
3'-sialyl-lacto-N-biose I has been catalyzed by TcTs from
lacto-N-biose I as acceptor and
2'-(4-methylumbellyferyl)-.alpha.-D-N-acetyneuraminic as donor of
the N-acetylneuraminil moiety (Vetere et al., 2000, Eur. J.
Biochem., 267:942-949). Further information regarding the use of
trans-sialidase to synthesize .alpha.2,3-sialylated conjugates can
be found in European Patent Application No. 0 557 580 A2 and U.S.
Pat. No. 5,409,817. The intramolecular trans-sialidase from the
leech Macrobdella decora exhibits strict specificity toward the
cleavage of terminal Neu5Ac (N-acetylneuraminic acid)
.alpha.2.fwdarw.3Gal linkage in sialoglycoconjugates and catalyzes
an intramolecular trans-sialosyl reaction (Luo et al., 1999, J.
Mol. Biol., 285:323-332). Trans-sialidases primarily add sialic
acid onto galactose acceptors, but will transfer sialic acid onto
some other sugars. Transfer of sialic acid onto GalNAc, however,
requires a sialyltransferase. Further information on the use of
trans-sialidases can be found in PCT Application No. WO 93/18787;
Vetere et al., 1997, Eur. J. Biochem., 247:1083-1090.
[0088] As used herein, the term "sialyltransferase" refers to
enzymes that catalyze glycoside synthesis by inversion of the
configuration of the added sugar and which require sugar
nucleotides as the monosaccharide donor. An example of a
sialyltransferase is the enzyme from the trypanosome Trypanosoma
rangeli called TrSA (Buschiazzo et al., 2000, EMBO J.,
19:16-24).
DETAILED DESCRIPTION OF THE INVENTION
[0089] The invention provides a novel synthetic ganglioside of
formula (I):
##STR00012##
[0090] In formula (I):
[0091] the saccharide is as defined herein selected from the group
consisting of a monosaccharide, a disaccharide, an oligosaccharide,
a polysaccharide, an N-acetylated derivative thereof, and an
N-deacylated derivative thereof;
Z is O, S, or --NR.sub.1;
[0092] X is H, --OR.sub.1, --NR.sub.1R.sub.2, --SR.sub.1, or
--CHR.sub.1R.sub.2;
[0093] R.sub.1 and R.sub.2 are independently H, --CH.sub.2R.sub.3,
--C(=M)R.sub.3, --C(=M)-p-R.sub.3, --SO.sub.2R.sub.3, --SO.sub.3,
alkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, heteroalkyl, or
haloalkyl;
[0094] M is O, NR.sub.4 or S;
[0095] R.sub.4 is H, alkyl, cycloalkyl, aryl, arylalkyl,
heteroaryl, heteroalkyl, or haloalkyl;
[0096] p is O, --NR.sub.4, or S, where R.sub.4 is as set forth
above;
[0097] R.sub.3 is H, alkyl, cycloalkyl, arylalkyl, haloalkyl, aryl,
heteroaryl, or heteroalkyl;
[0098] Y is H, --OR.sub.1, --SR.sub.1, --NR.sub.1R.sub.2, branched
alkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, heteroalkyl, or
haloalkyl, where R.sub.1 and R.sub.2 is as set forth above; and
[0099] R.sub.5 is H, alkyl, cycloalkyl, alkenyl, aryl, arylalkyl,
heteroaryl, heteroalkyl, or haloalkyl;
[0100] and all pharmaceutically acceptable salts, isomers,
hydrates, prodrugs, and solvates thereof with the proviso that when
Z is O, Y is OH, and R.sub.5 is alkenyl, X is not any of
--NH.sub.2, --NH(alkyl), --NHC(.dbd.O)alkenyl,
--NHC(.dbd.O)fluoroalkyl, and --NHC(.dbd.O)alkyl.
[0101] The invention also provides a novel synthetic ganglioside of
formula (II):
##STR00013##
wherein: [0102] X is --OH, --OC(.dbd.O)--C.sub.1-C.sub.11alkyl, or
--OC(.dbd.O)-haloalkyl; and [0103] R.sub.5 is a C.sub.1-C.sub.18
alkyl; and and all pharmaceutically acceptable salts, isomers,
hydrates, prodrugs, and solvates thereof.
[0104] The invention further provides compounds of formula (II) as
set forth in Table 1 below and include their pharmaceutically
acceptable salts, isomers, hydrates, prodrugs, and solvates:
TABLE-US-00001 TABLE 1 1 ##STR00014## 2 ##STR00015## 6 ##STR00016##
7 ##STR00017## 8 ##STR00018## 9 ##STR00019## 10 ##STR00020## 11
##STR00021## 12 ##STR00022## where R is C18, C16, . . . C4
[0105] The invention also provides synthetic gangliosides of
formula (III):
##STR00023##
wherein: [0106] X is --OH, --OC(.dbd.O)--C.sub.1-C.sub.11alkyl, or
--OC(.dbd.O)-haloalkyl; and [0107] R.sub.5 is a C.sub.1-C.sub.18
alkyl; and and all pharmaceutically acceptable salts, isomers,
hydrates, prodrugs, and solvates thereof.
[0108] The invention further provides compounds of formula (III) as
set forth in Table 2 below and include their pharmaceutically
acceptable salts, isomers, hydrates, prodrugs, and solvates:
TABLE-US-00002 TABLE 2 14 ##STR00024## 15 ##STR00025## where R is
C18, C16, . . . C4
[0109] The invention also provides a synthetic ganglioside of
formula (IV):
##STR00026##
wherein: [0110] X is --OH, --OC(.dbd.O)--C.sub.1-C.sub.11alkyl, or
--OC(.dbd.O)-haloalkyl; and [0111] R.sub.5 is a C.sub.1-C.sub.18
alkyl; and and all pharmaceutically acceptable salts, isomers,
hydrates, prodrugs, and solvates thereof.
[0112] The invention further provides compounds of formula (IV) as
set forth in Table 3 below and include their pharmaceutically
acceptable salts, isomers, hydrates, prodrugs, and solvates:
TABLE-US-00003 TABLE 3 20 ##STR00027## 21 ##STR00028## where R is
C18, C16, . . . C4
[0113] The invention further provides a synthetic ganglioside of
formulae (Va), (Vb), (Vc) and (Vd):
##STR00029##
[0114] In formulae (Va), (Vb), (Vc), and (Vd), as set forth
above:
[0115] the saccharide is selected from the group consisting of a
monosaccharide, a disaccharide, an oligosaccharide, a
polysaccharide, an N-acetylated derivative thereof, and an
N-deacylated derivative thereof; preferably, the saccharide is
selected from the group consisting of:
##STR00030##
more preferably, the saccharide is:
##STR00031##
[0116] Z is O, S, or --NR.sub.1;
[0117] X is H, --OR.sub.1, --NR.sub.1R.sub.2, --SR.sub.1, or
--CHR.sub.1R.sub.2; preferably, --NR.sub.1R.sub.2;
[0118] R.sub.1 and R.sub.2 are independently H, --CH.sub.2R.sub.3,
--CH(halo).sub.2, --C(=M)R.sub.3, --C(=M)-p-R.sub.3,
--SO.sub.2R.sub.3, --SO.sub.3, alkyl, aryl, arylalkyl, heteroaryl,
heteroalkyl or haloalkyl; preferably, --CH(halo).sub.2 or
--C(=M)R.sub.3;
[0119] M is O, NR.sub.4 or S;
[0120] R.sub.4 is H, alkyl, aryl, arylalkyl, heteroaryl,
heteroalkyl, or haloalkyl;
[0121] p is O, --NR.sub.4, or S, where R.sub.4 is as set forth
above;
[0122] R.sub.3 is H, alkyl, arylalkyl, haloalkyl, aryl, heteroaryl,
or heteroalkyl;
[0123] Y is H, --OR.sub.1, --SR.sub.1, --NR.sub.1R.sub.2, alkyl,
aryl, arylalkyl, heteroaryl, heteroalkyl, or haloalkyl, where
R.sub.1 and R.sub.2 is as set forth above; preferably, --OR.sub.1,
where R.sub.1 and R.sub.2 is as set forth above; and
[0124] R.sub.6, R.sub.6N, R.sub.6O, R.sub.7 and R.sub.8 are
independently H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,
heterocycloalkyl, heteroaryl, thioalkyl, thioaryl, --CN,
--NR.sub.9R.sub.10,
[0125] --C(.dbd.O)R.sub.9, --C(.dbd.O)OR.sub.9, or
--C(.dbd.O)NR.sub.9R.sub.10, where R.sub.6, R.sub.6N, R.sub.6O,
R.sub.7 and R.sub.8 may be optionally substituted with at least one
group selected from the group consisting of halo, haloalkyl,
alkoxy, and thiohaloalkyl; and where R.sub.6N, R.sub.6O and
R.sub.7, or R.sub.6 and R.sub.7, or R.sub.7 and R.sub.8, or R.sub.6
and R.sub.8, or R.sub.6N, R.sub.6O, R.sub.7 and R.sub.8, or
R.sub.6, R.sub.7 and R.sub.8 may each independently be taken
together with the atoms to which they are attached to form a
substituted cycloalkyl, heterocycloalkyl, aryl or heteroaryl group;
preferably, where R.sub.6N, R.sub.6O and R.sub.7, or R.sub.6 and
R.sub.7, or R.sub.7 and R.sub.8, or R.sub.6 and R.sub.8, or
R.sub.6N, R.sub.6O, R.sub.7 and R.sub.8, or R.sub.6, R.sub.7 and
R.sub.8 may each independently be taken together with the atoms to
which they are attached forms an optionally substituted aryl or
heteroaryl; with the proviso that when R.sub.7 of formula (Vb) is
an alkyl group or a C.sub.11 alkenyl group, X is not a --NH-fatty
acid group and Y is not --OH; R.sub.9, R.sub.10, and R.sub.11 are
independently alkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, or
heteroalkyl, where R.sub.9, R.sub.10, and R.sub.11 may be
optionally substituted, preferably substituted with at least one
group selected from the group consisting of halo, haloalkyl,
alkoxy, and thiohaloalkyl; and where R.sub.9 and R.sub.10 taken
together with the nitrogen to which they are attached form a
heterocyclic ring at least one heteroatom selected from N, O and
S,
[0126] and all pharmaceutically acceptable salts, isomers,
hydrates, prodrugs, and solvates thereof.
[0127] In a preferred embodiment of the invention, the
##STR00032##
moiety of (Vb) is a conjugated hydrophobic moiety, including but
not limited to, mono-, bi- and polycyclic aromatic and
heteroaromatic rings systems, as defined herein. More preferably,
the invention provides a synthetic ganglioside of the following
formula:
##STR00033##
where at least one carbon of the
##STR00034##
moiety may be replaced with a heteroatom selected from the group
consisting of N, O and S and Q may be a substituent as defined
herein including, but not limited to, halo, hydroxy, alkoxy, thio,
thiol, hydrocarbon, and amino.
[0128] In another embodiment of the invention, the
##STR00035##
moiety of (Vb) is a cyclic moiety, including but not limited to,
mono-, bi- and polycyclic ring systems, as defined herein. Such
cyclic ring systems may be either homocyclic or heterocyclic, or a
mixture of both. Further, such cyclic ring systems may be
conjugated. Any such ring systems of the present invention may also
be unsubstituted, or mono-, bi- or polysubstituted. Examples of
synthetic gangliosides of the invention comprising such cyclic ring
systems include:
##STR00036##
[0129] In yet another embodiment of the invention, the
##STR00037##
moiety of (Vb) is an aromatic moiety, including but not limited to,
mono-, bi- and polycyclic aromatic ring systems, as defined herein.
Such aromatic ring systems may be either homocyclic or
heterocyclic, or a mixture of both. Such aromatic ring structures
may also be unsubstituted, or mono-, bi- or polysubstituted.
Examples of synthetic gangliosides of the invention comprising such
aromatic moieties include:
##STR00038##
[0130] In still another embodiment of the invention, the
##STR00039##
moiety of (Vb) is such that the double bond has been removed by
simultaneously substituting R.sub.9 for R.sub.6 and R.sub.10 for
R.sub.8, as defined herein. An example of such a substituted
embodiment of the present invention is illustrated by
##STR00040##
[0131] The invention further provides preferred compounds of
formula (Vb) where the
##STR00041##
moiety is selected from the group consisting of:
##STR00042##
[0132] Preferred compounds of formula (Vb) include, but are not
limited to:
##STR00043## ##STR00044## ##STR00045## ##STR00046##
[0133] The invention also encompasses all pharmaceutically
acceptable isomers, salts, hydrates, solvates, and prodrugs of each
of the compounds described above. In addition, such compounds can
exist in various isomeric and tautomeric forms, and all such forms
are meant to be included in the invention, along with
pharmaceutically acceptable salts, hydrates, and solvates of such
isomers and tautomers.
Methods of Preparation
[0134] According to the invention, synthetic ganglioside compounds
of formulae (I) and (Va)-(Vd) may be prepared using, unless
otherwise indicated, conventional methods and protocols in
chemistry and enzymology known in the art. For example, compounds
of the invention may be prepared by synthetic and enzymatic
processes as outlined in Schemes 1-6 set forth below.
A. Method of Preparing Saccharide
[0135] The saccharide portion of the compounds of the invention may
be prepared by any means known in the art including those methods
described in U.S. Pat. Nos. 5,922,577, 6,284,493 and 6,331,418,
each of which is incorporated herein in its entirety by reference.
Preferably, the saccharide portion of the compounds of the
invention is prepared enzymatically whereby a specific enzyme may
be used to affect transfer of a monosaccharide from a donor
molecule to an acceptor molecule, each as defined herein.
[0136] More specifically, disaccharides, oligosaccharides and
polysaccharides, as found in the synthetic ganglioside compounds of
the invention, may be prepared biosynthetically by use of
glycosyltransferases. Such glycosyltransferase reactions may be
carried out in the presence of an organic solvent, such as, for
example, methanol, ethanol, dimethylsulfoxide, isopropanol,
tetrahydrofuran, chloroform, and the like, either singly or in
combination. Alternatively, such glycosyltransferase reactions may
be conducted in a biological medium in vitro, such as a biological
buffer, a cell lysate, or on a chromatographic support, wherein the
glycosyltransferase is immobilized on the chromatographic support
and the other components of the reaction mixture are contacted with
the glycosyltransferase by contacting the components with the
choromatographic support in an aqueous medium.
[0137] Glycosyltransferase-mediated synthesis of saccharides found
in synthetic ganglioside compounds of the invention may also be
conducted in vivo. For example, whole-cell expression systems may
be used for glycosyltransferase-mediated synthesis. Cell types that
may be used for expression of glycosyltransferases and concomitant
production of saccharide structures include bacterial cells, yeast
cells, and insect cells, as would be understood by one of skill in
the art. The desired saccharide product can be isolated from the
cell in which it was synthesized by lysis of the cell, or by
isolation of cell culture medium when using a cell that secretes
the saccharide product into the culture medium. The saccharide
product may then be purified by means described elsewhere herein,
or it may be used without further purification in a lysate or cell
culture medium.
[0138] As would be understood by one of skill in the art, the
enzyme used may vary depending upon the monosaccharide to be
transferred. Examples of suitable enzymes include, but are not
limited to, glycosyltransferases, trans-sialidases, and
sialyltransferases. The choice of glycosyltransferase(s) used in a
given synthesis method of the invention will depend upon the
identity of the acceptor and donor molecules used as the starting
material and the nature of the desired end product. A method of the
invention can involve the use of more than one glycosyltransferase,
where more than one saccharide is to be added. Multiple
glycosyltransferase reactions can be carried out simultaneously,
i.e., in the same reaction mixture at the same time, or
sequentially.
[0139] To obtain sufficient amounts of glycosyltransferase for
large-scale in vitro reaction, a nucleic acid that encodes a
glycosyltransferase can be cloned and expressed as a recombinant
soluble enzyme by methods known to one of ordinary skill in the
art. The expressed enzyme may then be purified by means known to
one of ordinary skill in the art, or it may be used without further
purification in a lysate or cell culture medium.
[0140] By way of example, the saccharide moiety:
##STR00047##
may be prepared by contacting an acceptor molecule containing a
glucose (Glc) with a galactosyltransferase and a galactose (Gal)
donor molecule to form:
##STR00048##
which in turn can be contacted with a trans-sialidase and a NANA
donor molecule to form:
##STR00049##
which in turn can be contacted with a N-acetylated galactose
(GalNAc)-transferase and a GalNAc donor molecule to form:
##STR00050##
which in turn can be contacted with a galactosyltransferase and a
galactose (Gal) donor molecule to form the desired saccharide:
##STR00051##
[0141] If the acceptor is a ceramide, the enzymatic step is
typically preceded by hydrolysis of the fatty acid moiety from the
ceramide; a fatty acid moiety can be reattached after completion of
the glycosyltransferase reaction. The initial monosaccharide may be
added, depending on the desired end product, either a ceramide
glucosyltransferase (EC 2.4.1.80, for glucosylceramide) or a
ceramide galactosyltransferase (EC 2.4.1.45, for
galactosylceramide). For review of glycosphingolipid biosynthesis,
see, e.g., Ichikawa and Hirabayashi (1998) Trends Cell Biol.
8:198-202. Ceramide glucosyltransferases are available from various
sources. For example, the human nucleotide sequence is known
(GenBank Accession No. D50840; Ichikawa et al. (1996) Proc. Nat'l.
Acad. Sci. USA 93:4638-4643), so recombinant methods can be used to
obtain the enzyme. The nucleotide sequence of the human ceramide
galactosyltransferase also has been reported (GenBank Accession No.
U62899; Kapitonov and Yu (1997) Biochem. Biophys. Res. Commun. 232:
449-453), and thus the enzyme is easily obtainable. The acceptor
used in these reactions can be any of N-acylsphingosine,
sphingosine and dihydrosphingosine. Suitable donor nucleotide
sugars for the glycosyltransferase include UDP-Glc and CDP-Glc,
while the galactosyltransferase typically uses UDP-Gal as a
donor.
[0142] Methods of removing a fatty acid moiety from a
glycosphingolipid are known to those of skill in the art. Standard
carbohydrate and glycosphingolipid chemistry methodology can be
employed, such as that described in, for example, Paulson et al.
(1985) Carbohydrate Res. 137: 39-62; Beith-Halahmi et al. (1967)
Carbohydrate Res. 5: 25-30; Alais and Veyrieries (1990)
Carbohydrate Res. 207: 11-31; Grudler and Schmidt (1985)
Carbohydrate Res. 135: 203-218; Ponpipom et al. (1978) Tetrahedron
Lett. 1717-1720; Murase et al. (1989) Carbohydrate Res. 188: 71-80;
Kameyama et al. (1989) Carbohydrate Res. 193: c1-c5; Hasegawa et
al. (1991) J. Carbohydrate Chem. 10: 439-459; Schwarzmann and
Sandhoff (1987) Meth. Enzymol. 138: 319-341; Guadino and Paulson
(1994) J. Am. Chem. Soc. 116: 1149-1150 (including supplemental
material, which is also incorporated herein by reference). For
example, hydrolysis of the fatty acid moiety can be effected by
base hydrolysis. Once the glycosylation reactions are completed,
the same or a different fatty acid can be attached to the product
of the glycosylation reactions. Methods for coupling a fatty acid
include those known in the art.
[0143] Another possible biosynthetic method for the synthesis of
the saccharide portion of a compound of the invention is
exemplified in Scheme 1 below. In a preferred embodiment, the
acceptor molecule is non-immobilized. For example, the acceptor
molecule may be free in solution or otherwise not associated with
other acceptor molecules.
[0144] Additional saccharide residues may be added to a compound of
the invention without prior modification of the glycosylation
pattern of the glycosphingolipid starting material. Alternatively,
the invention provides methods of altering the glycosylation
pattern of a glycosphingolipid prior to adding the additional
saccharide residues. If the starting glycosphingolipid does not
provide a suitable acceptor for the glycosyltransferase which
catalyzes a desired saccharide addition, one can modify the
glycosphingolipid to include an acceptor by methods known to those
of skill in the art. For example, to provide a suitable acceptor
for a sialyltransferase, a suitable acceptor can be synthesized by
using a galactosyltransferase to attach a galactose residue to, for
example, a GalNAc or other appropriate saccharide moiety that is
linked to the glycosphingoid. In other embodiments,
glycosphingoid-linked oligosaccharides can be first "trimmed,"
either in whole or in part, to expose either an acceptor for the
sialyltransferase or a moiety to which one or more appropriate
residues can be added to obtain a suitable acceptor. Enzymes such
as glycosyltransferases and endoglycosidases are useful for the
attaching and trimming reactions.
[0145] Sialyltransferases and other glycosyltransferases can be
used either alone or in conjunction with additional enzymes. For
example, FIG. 2 shows a schematic diagram of two pathways for
synthesis of the ganglioside GD.sub.2 starting from
lactosylceramide. Each pathway involves the use of two different
sialyltransferases (an .alpha.2,3ST and an .alpha.2,8ST), as well
as a GalNAc transferase. In the preferred pathway, the fatty acid
is removed from the lactosylceramide by treatment with base (Step
1). Acetylation is then performed (Step 2), after which a sialic
acid is attached to the galactose residue in an .alpha.2,3 linkage
by an .alpha.2,3 sialyltransferase (Step 3). The sialylation steps
are performed, preferably in the presence of an organic solvent as
described herein, thereby driving the reaction nearly to
completion. A GalNAc residue is then added to the galactose in a
.beta.1,4 linkage using a GalNAc transferase (Step 5). Finally, a
fatty acid is added, e.g., by reaction with steroyl chloride, to
complete the ganglioside (Step 6).
##STR00052##
B. Method of Preparing Compounds of Formulae (I)-(Vd)
[0146] Compounds of the invention may be prepared by any means
known in the art. Preferred synthetic pathways are illustrated in
Schemes 2-5.
##STR00053## ##STR00054##
##STR00055##
[0147] Once synthesized, the compounds of the invention may be
isolated and purified by any means known in the art including, but
not limited to, chromatography (e.g., thin, ion-exchange, column),
filtration, membrane filtration (e.g., reverse osmotic membrane,
nanfiltration), recrystallization, distillation, and the like.
[0148] A compound of the invention is useful in the field of
neuroprotection. The term "neuroprotection" relates to any
prophylaxis (pre-onset), treatment (on-set) and/or cure
(post-onset) of indications resulting from the impairment or
destruction of neuronal cells. Such indications include Parkinson's
disease, ischemia, stroke, Alzheimer's, central nervous system
disorders (e.g., spinal cord injury), multiple sclerosis,
Huntington's disease, CABG, depression, anxiety, encephalitis,
meningitis, amyotrophic lateral sclerosis, trauma, spinal cord
injury, nerve injury, and nerve regeneration. A compound of the
invention is also useful in the treatment of cancers in general,
including liver, lung, colon, prostate, breast, pancreatic, and
cancers of the brain, such as glioma and neuroblastoma. Further, a
compound of the present invention is useful as an immunosuppressive
and immunostimulatory agent, and has applications in organ
transplantation, autoimmune disease, arthritis, Systemic Lupus
Erythematosus, irritable bowel disease, radiation toxicity and
inflammation, psoriasis, dermatitis, multiple sclerosis, trauma and
sepsis.
[0149] A compound of the invention can be used to stimulate or
suppress T-cells and B-cells, and can be used for antibody
suppression or stimulation. Methods of stimulating and suppressing
T-cells and B-cells is well-known in the art. Further, a compound
of the invention may be used in a method to inhibit or activate
membrane receptors, including G-protein coupled receptors, cell
surface membrane receptor systems, and nuclear membrane receptors.
A compound of the invention can further be used to treat type II
diabetes and as an ethryopoeitin replacement.
[0150] A compound of the present invention is also useful as an
inhibitor of platelet aggregation. Further, a compound of the
present invention is useful in AIDS treatment, by inhibiting viral
adhesion through G-protein coupled receptors, including CCRC5 and
CXC4. A compound of the invention is also useful in the treatment
of diseases such as Chagas disease, as well as diseases, disorders,
and conditions described in U.S. Pat. Nos. 4,476,119, 4,593,091,
4,639,437, 4,707,469, 4,713,374, 4,716,223, 4,849,413, 4,940,694,
5,045,532, 5,135,921, 5,183,807, 5,190,925, 5,210,185, 5,218,094,
5,229,373, 5,260,464, 5,264,424, 5,350,841, 5,424,294, 5,484,775,
5,519,007, 5,521,164, 5,523,294, 5,677,285, 5,792,858, 5,795,869,
and 5,849,717, each of which is incorporated by reference
herein.
[0151] One possible mechanism of action of a compound of the
invention is to stimulate nerve growth factors. Another possible
mechanism of action of a compound of the invention is to inhibit
growth of cancer cells, and in particular, neuroblastoma cells. For
example, it has been shown that administration of ganglioside GM3
to murine neuroblastoma cells can inhibit the growth of the
neuroblastoma cells (Zhang et al., 1995, Anticancer Res. 15:661-6).
Ganglioside and ganglioside-like compounds of the present invention
can be used in a similar inhibitory capacity.
[0152] According to the invention, isolated and purified compounds
of the invention for use in the field of neuroprotection or cancer
treatment are of an acceptable purity level. As would be understood
by one of skill in the art, acceptable purity levels would depend
upon the particular application. The compounds of the invention may
be purified to levels ranging from about 80-100% pure, preferably,
from about 90-100% pure, and more preferably about 95-100%
pure.
Pharmaceutical Compositions
[0153] The invention further provides a pharmaceutical composition
comprising at least one synthetic ganglioside compound of formulae
(I) and (Va)-(Vd), each as set forth above, and a pharmaceutically
acceptable carrier. Mixtures of synthetic gangliosides of the
invention are also contemplated for use in pharmaceutical
compositions.
[0154] Pharmaceutical compositions of the invention may be prepared
for storage or administration by any means known in the art. For
example, a pharmaceutical composition of the invention may be
prepared by mixing a compound of the invention, preferably having a
desired degree of purity, with a pharmaceutically or
physiologically acceptable carriers or agent. The amount of active
ingredient in these compositions is such that a suitable dosage in
the range indicated is obtained.
[0155] A pharmaceutically acceptable carrier or agent may be any
such carrier or agent known in the art. See, for example, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., (A. R.
Gennaro edit. 1985). The pharmaceutical composition of the
invention may further include a binder (e.g., acacia, corn starch
or gelatin), an excipient (e.g., microcrystalline cellulose), a
disintegrating agent (e.g., corn starch or alginic acid), a
lubricant (e.g., magnesium stearate), a sweetening agent (e.g.,
sucrose or lactose), a buffer (e.g., phosphate, citrate, acetate
and other organic acid salts), an antioxidant (e.g., ascorbic
acid), a low molecular weight (less than about ten residues)
peptide (e.g. polyarginine), a protein (e.g., serum albumin,
gelatin, or immunoglobulins), a hydrophilic polymer (e.g.,
polyvinylpyrrolidinone), an amino acid (e.g., glycine, glutamic
acid, aspartic acid, or arginine), a monosaccharide, a
disaccharide, and other carbohydrates (e.g. cellulose or its
derivatives, glucose, mannose or dextrins), a chelating agent
(e.g., EDTA), sugar alcohol (e.g., mannitol or sorbitol), a
counterion (e.g., sodium) and/or nonionic surfactants such as
TWEEN, Pluronics or polyethyleneglycol. Additional acceptable
adjuvants include those well known in the pharmaceutical field, and
as described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co., (A. R. Gennaro edit. 1985).
[0156] A compound or a pharmaceutical composition of the invention
may be administered in solid or liquid form depending upon the
desired application. Thus, a compound or pharmaceutical composition
of the invention may be administered in solid form such as, for
example, tablets, capsules, suppositories, in liquid form such as,
for example, elixirs for oral administration, sterile solutions,
sterile suspensions or injectable administration, and the like, or
incorporated into shaped articles. A compound or a pharmaceutical
composition of the invention may also be administered as sustained
release and timed release formulations. Other modes of
administration of a compound or composition of the invention
include, but not limited to, implantable medical devices (e.g.,
stents), inhalable formulations, sprays, transdermal, liposomes,
gels, intracraneal, and intrathecal.
[0157] A compound or pharmaceutical composition of the invention,
especially when administered in capsule form, may also contain a
liquid carrier such as, for example, water, saline, or a fatty oil.
Other materials of various types may be used as coatings or as
modifiers of the physical form of the compound or pharmaceutical
composition. For example, dissolution or suspension of the active
compound of the invention in a vehicle such as an oil or a
synthetic fatty vehicle like ethyl oleate, or into a liposome may
be desired.
[0158] According to the invention, such materials as well as
compounds of the invention are nontoxic to the recipients at the
dosages and concentrations employed, i.e. are pharmaceutically
acceptable.
[0159] In general, a compound of the invention, alone or as part of
a pharmaceutical composition as described herein, may be used as a
diagnostic or therapeutic agent for the prevention and/or treatment
of disorders of the nervous system including neurological diseases
such as, for example, Parkinson's disease, CABG, Alzheimer's
Disease, and stroke. Further, a compound of the invention, alone or
as part of a pharmaceutical composition as described herein, may be
used as a therapeutic agent for the treatment of certain types of
cancer, including neuroblastoma.
[0160] Compounds and pharmaceutical compositions of the invention
are suitable for use alone or as part of a multi-component
treatment regimen in combination with other therapeutic or
diagnostic agents such as, for example, other synthetic
gangliosides of the invention, natural gangliosides, other
synthetic gangliosides, anti-inflammatory compounds, analgesics,
other neurotrophic factors (e.g., growth factors). Coadministered
compounds and agents may act in a synergistic fashion to enhance
the neuroprotective activity of the compound of the invention.
[0161] The compounds and pharmaceutical compositions of the
invention may be utilized in vivo, ordinarily in mammals such as
primates, such as humans, sheep, horses, cattle, pigs, dogs, cats,
rats and mice, or in vitro. The biological properties, as described
above, of the compounds of the invention can be readily
characterized by methods that are well known in the art including,
for example, in vitro screening protocols and in vivo studies to
evaluate the neuroprotective activity of the tested compound or
pharmaceutical composition.
[0162] Subjects (animals or humans), preferably mammalian, in need
of treatment may be administered a therapeutically effective
amount, i.e., a dosage that will provide optimal efficacy, of a
compound of the invention, alone or as part of pharmaceutical
composition. As would be recognized by those of skill in the art, a
"therapeutically effective amount" and mode of administration will
vary from subject to subject and thus will be determined on a case
by case basis. Factors to be considered include, but are not
limited to, the subject (e.g. mammal) being treated, its sex,
weight, diet, concurrent medication, overall clinical condition,
the particular compounds employed, and the specific use for which
these compounds are employed. Therapeutically effective amounts or
dosages may be determined by either in vitro or in vivo methods. In
general, a "therapeutically effective amount" of a compound or
composition is an amount that will result in the prophylaxis,
treatment or cure of neuronal cell disorders. For example, a
therapeutically effective amount of a compound or composition of
the invention in the prophylaxis, treatment or cure of Parkinson's
disease will be that amount that results in slower progression of
the disease and/or development of motor skills. A therapeutically
effective amount of a compound or composition of the invention in
the prophylaxis, treatment or cure of Alzheimer's disease will be
that amount that results in, for example, improvement of the
subject's memory. A therapeutically effective amount of a compound
or composition of the invention in the prophylaxis, treatment or
cure of the lasting effects of eschemia/stroke will be that amount
that results in, for example, reduction of loss of neurological
function (e.g., speech, motor, etc.) and/or improvement of
sympathetic or parasympathetic pathways.
[0163] Modes of administration include those known in the art
including, but not limited to, oral, injection, intravenous (bolus
and/or infusion), subcutaneous, intramuscular, colonic, rectal,
nasal and intraperitoneal administration. Preferably, compounds of
the invention, alone or as part of a pharmaceutical composition are
taken orally.
[0164] For injection by hypodermic needle, it may be assumed the
dosage is delivered into the body's fluids. For other routes of
administration, the absorption efficiency may be individually
determined for each compound of the invention by methods well known
in pharmacology. Accordingly, as would be understood by one of
skill in the art, it may be necessary for the therapist to titer
the dosage and modify the route of administration as required to
obtain the optimal therapeutic effect. The determination of
effective dosage levels, that is, the dosage levels necessary to
achieve the desired result, will be within the ambit of one skilled
in the art. Typically, a compound of the invention is administered
at lower dosage levels, with dosage levels being increased until
the desired effect is achieved.
[0165] A typical dosage might range from about 0.1 mg/kg to about
1000 mg/kg, preferably from 0.1 mg/kg to about 100 mg/kg, more
preferably from about 0.1 mg/kg to about 30 mg/kg, more preferably
from about 0.1 mg/kg to about 10 mg/kg, and more preferably 0.1
mg/kg to about 3 mg/kg. Advantageously, the compounds of the
invention, alone or as part of a pharmaceutical composition, may be
administered several times daily, and other dosage regimens may
also be useful. A compound of the invention may be administered on
a regimen in a single or multidose (e.g. 2 to 4 divided daily
doses) and/or continuous infusion.
[0166] A compound of the invention, alone or as part of a
pharmaceutical composition, for administration may be sterilized
prior to administration. Sterility may be readily accomplished by
filtration through sterile membranes such as 0.2 micron membranes,
or by other conventional methods. A compound of the invention,
alone or as part of a pharmaceutical composition, typically may be
stored in lyophilized form or as an aqueous solution. pH may be a
factor for certain modes of administration. In such instances, the
pH typically will range between about 2-10, preferably, between
about 5-8, more preferably 6.5-7.5, i.e., physiological pH.
Screening Protocols
[0167] The compounds and pharmaceutical compositions of the
invention may be utilized in vivo, ordinarily in mammals such as
primates, such as humans, sheep, horses, cattle, pigs, dogs, cats,
rats and mice, or in vitro. The effectiveness of the compounds of
the invention as neuroprotective agents may be determined using
screening protocols known in the art. For example, the biological
properties, as described above, of the compounds of the invention
can be readily characterized by methods that are well known in the
art including, for example, in vitro screening protocols (e.g. cell
culture (MPTP (rat ventral mesophenthalic cells), NMDA (mouse
primary cortical neurons), ceramide (neuroblastoma-human)), CACO-2
(oral absorption), RBC lysis) and in vivo studies (e.g. mouse and
primate MPTP toxicity studies (IP, IV, and/or oral) for
effectiveness in the treatment of Parkinson's, rat Stoke studies
for effectiveness for treatment of neural damage due to stroke or
CABG, and dog studies for treatment of CABG) to evaluate
neuroprotective efficacy.
[0168] In the cell based assays, as described herein, the compounds
of the invention exhibited 50-100% greater neuroprotective activity
at lower concentrations ranging between about 0.1 to about 1
.mu.M.
[0169] The invention is now described with reference to the
following Example. This Example is provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to this Example, but rather should be construed to
encompass any and all variations which become evident as a result
of the teaching provided herein.
EXPERIMENTAL EXAMPLES
Example 1
General Procedure for Preparing the GM.sub.1 Aldehyde
[0170] GM.sub.1 (2.5 g, 1.62 mmol) was dissolved in 2500 mL of
methanol. This solution was cooled to -70.degree. C. and ozone
bubbled through the solution until the light blue color did not
disappear (about 30 mins). The ozone was removed by bubbling
nitrogen through the reaction mixture until the solution became
colorless. Then, 80 mL of dimethylsulfide was added and the
resulting mixture was stirred at room temperature for 2 h. The
solvent was evaporated with nitrogen to dryness. The residue was
co-evaporated with toluene (50 mL) and the residue dried on a high
vacuum pump for 1 h to yield a white solid containing the
aldehyde.
Example 2
Wittig Reaction Preparation of
##STR00056##
[0172] A suspension containing
3-chloro-2-fluoro-5-(trifluoromethyl)benzyl-triphenylphosphonium
bromide (2.58 g, 4.66 mmol), dimethylformamide (DMF) (50 mL) was
cooled to -40.degree. C. and 1M potassium tert-butyloxide in
tert-butylalcohol solution (4.49 mL) was then added. After 10
minutes, this reaction mixture was added slowly to a solution of
aldehyde dissolved in DMF (200 mL) and cooled to -40.degree. C.
After addition was complete, the reaction mixture was stirred at
room temperature for 1 h. The reaction mixture was then
concentrated on a rotovap and the residue chromatographed (silica,
CHCl.sub.3/MeOH 3:1 then, MeOH/H.sub.2O/NH.sub.4OH 60:40:7:1) to
afford 1.5 g (60% yield) of the desired product as a 70/30
cis/trans mixture. ESI-MS; calcd for
C.sub.67H.sub.106ClF.sub.4N.sub.3O.sub.31, 1559; found 1558
[M-1].sup.-. .sup.1H-NMR (500 MHz, 95% DMSO-d.sub.6+5% D.sub.2O)
.delta. 7.98 (d, J=6.0 Hz, 2H), 7.84 (d, J=6.0 Hz, 1H), 7.82 (d,
J=5.5 Hz, 2H), 7.60 (d, J=5.5 Hz, 1H), 7.34 (d, J=9.5 Hz, 2H), 6.64
(d, J=16 Hz, 1H), 6.48 (d, J=11.5 Hz, 2H), 5.93 (dd, J 11.5/11.5
Hz, 2H), 4.79 (d, J=8.5 Hz, 2H), 4.27 (d, J=8.0 Hz, 2H), 4.21 (d,
J=8.5 Hz, 2H), 3.00-4.00 (m), 1.98 (m, 2H), 1.86 (s, 3H,
COCH.sub.3), 1.78 (s, 3H, COCH.sub.3), 1.25 (m), 0.83 (t, 3H,
CH.sub.3).
Example 3
Wittig Reaction Preparation of
##STR00057##
[0174] The Wittig procedure of Example 2 was followed except that
the starting glide was changed. The desired product was obtained as
a white solid, (43% yield). ESI-MS; calcd for
C.sub.65H.sub.108N.sub.4O.sub.31, 1440; found 1439 [M-1].sup.-.
.sup.1H-NMR (500 MHz, 95% DMSO-d.sub.6+5% D.sub.2O) .delta. 8.46
(d, J=4 Hz, 1H), 7.70 (dd, J 6.5 and 9.6 Hz, 1H), 7.37 (d, J=8.0
Hz, 1H), 7.18 (dd, J 5.0 and 5.0 Hz, 1H), 6.64 (dd, J15.5 and 6.0
Hz, 1H), 6.57 (d, J=15.5 Hz, 1H), 4.82 (d, J=8.5 Hz, 1H), 4.27 (d,
J=8.0 Hz, 1H), 4.18-4.22 (2d, 2H), 3.10-3.93 (m), 2.02 (t, 2H),
1.86 (s, 3H, COCH.sub.3), 1.75 (s, 3H, COCH.sub.3), 1.36 (m, 2H),
1.22 (s), 1.06 (m, 2H, CH.sub.2), 0.83 (t, 3H, CH.sub.3).
Example 4
Wittig Reaction Preparation of
##STR00058##
[0176] The Wittig procedure of Example 2 was followed except that
the starting ylide was changed. The desired product was obtained as
a solid (21% yield), as a 50/50 cis/trans mixture. ESI-MS; calcd
for C.sub.64H.sub.111N.sub.3O.sub.31, 1417; found [M-1].sup.-.
Example 5
Wittig Reaction Preparation of
##STR00059##
[0178] The Wittig procedure of Example 2 was followed except that
the starting ylide was changed. The desired product was obtained as
a solid (45% yield). ESI-MS; calcd for
C.sub.68H.sub.109ClN.sub.6O.sub.31 1540; found 1539[M-1].sup.-.
.sup.1H-NMR (500 MHz, 95% DMSO-d.sub.6+5% D.sub.2O) .delta. 8.00
(d, J=9.0 Hz, 2H), 7.50 (d, J=9.0 Hz, 2H), 4.80 (d, J=8.5 Hz, 1H),
4.26 (d, J=8.0 Hz), 4.22 (d, J=7.5 Hz, 1H), 4.19 (d, J=8.0 Hz, 1H),
3.05-4.00 (m), 2.02 (m, 2H), 1.87 (s, 3H, COCH.sub.3), 1.75 (s, 3H,
COCH.sub.3), 1.21 (s), 0.83 (t, J=6.5 Hz, CH.sub.3).
Example 6
Wittig Reaction Preparation of
##STR00060##
[0180] The GM.sub.1 aldehyde (20 mg, 0.013 mmol) of Example 1 and
dioctylamine (6 mg, 0.024 mmol), was added with stirring to 2.5 mL
of dimethylformamide (DMF) at room temperature. Then
trans-2-phenylvinylboronic acid (9 mg, 0.045 mmol) in methanol (5
mL) was added. The resulting solution was stirred at room
temperature for three days. The reaction mixture was then
concentrated to dryness on a rotovap and the residue purified by
solid phase extraction using a 1 g HAX cartridge. The eluant was
then purified using HPLC to afford 9.5 mg (43% yield) of white
solid. ESI-MS; cacld for C.sub.83H.sub.144N.sub.4O.sub.31, 1693;
found 1692 [M-1].sup.-. .sup.1H-NMR (500 MHz, 95% DMSO-d.sub.6+5%
D.sub.2O) .delta. 8.05 (d, J=3.0 Hz, 1H), 7.70 (m 5H), 6.40 (m,
1H), 6.25 (dd, J 9.0 and 16 Hz, 1H), 4.80 (d, J=8.5 Hz, 1H), 4.28
(d, J=8.0 Hz, 1H), 4.22 (d, J=8.0 Hz, 1H), 4.16 (d, 4.2 Hz, 1H),
3.00-4.00 (m), 2.10 (m, 2H), 1.86 (s, 3H, COCH.sub.3), 1.60 (s, 3H,
COCH.sub.3), 1.19 (s), 0.83 (t, 3H, CH.sub.3).
Example 7
MPTP/VMC Assay (In Vitro) for Evaluation of Neuroprotective
Efficacy
##STR00061##
[0182] Ventral Mesophenthalic Cells (VMCs) are isolated from fetal
rat brain stems (15 days old). Cells are cultured for several days
(48 well plates) with controls on every plate. Cells are treated
with MPTP (10 .mu.M) for 24 hours which results in 30-50% cell
death. Toxin is then removed. Cells are then treated with a
compound of the invention in DMSO. After 24 hours, a tyrosine
hydroxylase immuno-stain and cell count is performed.
[0183] The controls are MPTP (10 .mu.M--30-50% cell kill) and
GM.sub.1 (30 .mu.M) or LIGA-20 (10 .mu.M)--30-50% protection.
Example 8
Sialylation of Lyso-Lactosyl Ceramide
[0184] This Example describes the reaction conditions for
sialylation of lyso-lactosyl ceramide. Lactosylceramide was
obtained from bovine buttermilk and the fatty acid moiety removed
by base hydrolysis to form lyso-lactosyl ceramide. A mixture of the
lyso-lactosyl ceramide (1.0 mg, 1.6 .mu.mol) and CMP-sialic acid
(2.46 mg, 65% purity, 2.40 .mu.mol in HEPES buffer (200 mM,
containing 8% MeOH, pH 7.5, 50 .mu.L) was sonicated for twenty
minutes. .alpha.2,3 sialyltransferase (10 .mu.L, 5 U/mL, 50 mU) was
then added followed by alkaline phosphatase (1 .mu.L,
1.0.times.10.sup.5 U/mL, 100 U). The reaction mixture was kept at
room temperature. After one day, a further portion of .alpha.2,3
sialyltransferase (10 .mu.L, 5 U/mL, 50 mU) was added. After four
more days, an additional portion of .alpha.2,3 sialyltransferase
(10 .mu.L, 5 U/mL, 50 mU) was added. After an additional one day at
room temperature, thin layer chromatography indicated that the
reaction was nearly complete.
Example 9
Synthesis of GM2 from Lactosylceramide Obtained From Bovine
Buttermilk
[0185] A schematic diagram of showing two pathways for synthesis of
the ganglioside GM.sub.2 from lactosylceramide obtained from bovine
buttermilk is shown in FIG. 1. In the pathway shown at left, the
fatty acid is not removed from the lactosylceramide prior to
sialylation, and the reaction is not carried out in the presence of
an organic solvent. The reaction at right, in contrast, is carried
out in the presence of an organic solvent, and with removal of the
fatty acid.
[0186] First, the fatty acid is hydrolyzed from the
lactosylceramide by treatment with a base and water (Step 1). A
sialic acid residue is then added by enzymatic transfer to the
galactose residue using an .alpha.2,3 sialyltransferase, preferably
an ST3GalIV (Step 2). This reaction can be carried out in the
presence of an organic solvent. A GalNAc residue is then attached
to the galactose in a .beta.1,4 linkage using a GalNAc transferase
(Step 3); this step may or may not be carried out in the presence
of an organic solvent. Finally, the fatty acid moiety is reattached
to the sphingosine to obtain the desired GM.sub.2 ganglioside. The
reaction typically proceeds nearly to completion due to the
presence of an organic solvent during the sialylation.
Example 10
Synthesis of Gangliosides from Plant Glucosyl Ceramid
[0187] This Example describes three alternative procedures for the
synthesis of the GM.sub.2 ganglioside using plant glucosylceramide
as the precursor (FIG. 3). In Route 1, .beta.1,4-galactosidase is
used to catalyze the transfer of a Gal residue to the
glycosylceramide. Simultaneously, an .alpha.2,3-sialyltransferase
is used in a sialyltransferase cycle to link a sialic acid residue
to the Gal. Next, a .beta.1,4-GalNAc transferase is added to the
reaction mixture, either with UDP-GalNAc or as part of a GalNAc
transferase cycle. In this step, the GalNAc residue is linked to
the Gal residue in an .alpha.2,3 linkage.
[0188] Route 2 differs from the synthesis shown in Route 1 in that
the addition of the Gal to the glycosylceramide is catalyzed by a
.beta.1,4-galactosyltransferase enzyme, using either a
galactosyltransferase cycle or UDP-Glc/Gal as the acceptor sugar.
Sialylation and addition of GalNAc are carried out as described
above to obtain GM.sub.2.
[0189] In Route 3, the fatty acid is first removed by treatment
with aqueous base prior to the glycosyltransferase steps. The
galactosylation, sialylation, and GalNAc transferase reactions are
carried out as in Route 2. Following the addition of the GalNAc
residue, a fatty acid is linked to the molecule. The fatty acid can
be the same as that originally found on the plant glucosylceramide,
or can be different. In the example shown in FIG. 4, an activated
C.sub.18 fatty acid is used, resulting in the synthesis of
GM.sub.2. Greater efficiency is generally observed when the fatty
acid is removed prior to the glycosylation reactions.
Example 11
Synthesis of Ganglioside GM.sub.2 from Glycosylceramide
[0190] This Example describes three alternative procedures for the
synthesis of the GM.sub.2 and other gangliosides using a
glucosylceramide as the precursor (FIG. 4). In Route 1, a
.beta.1,4-galactosidase is used to catalyze the transfer of a Gal
residue to the glycosylceramide. Simultaneously, an
.alpha.2,3-sialyltransferase is used in a sialyltransferase cycle
to link a sialic acid residue to the Gal. Next, a .beta.1,4-GalNAc
transferase is added to the reaction mixture, either with
UDP-GalNAc or as part of a GalNAc transferase cycle. In this step,
the GalNAc residue is linked to the Gal residue in an .alpha.2,3
linkage.
[0191] Route 2 differs from the synthesis shown in Route 1 in that
the addition of the Gal to the glycosylceramide is catalyzed by a
.beta.1,4-galactosyltransferase enzyme, using either a
galactosyltransferase cycle or UDP-Glc/Gal as the acceptor sugar.
Sialylation and addition of GalNAc are carried out as described
above to obtain GM.sub.2.
[0192] In Route 3, the fatty acid is first removed by treatment
with aqueous base prior to the glycosyltransferase steps. The
galactosylation, sialylation, and GalNAc transferase reactions are
carried out as in Route 2. Following the addition of the GalNAc
residue, a fatty acid is linked to the molecule. In the example
shown in FIG. 3, an activated C.sub.18 fatty acid is used,
resulting in the synthesis of GM.sub.2. Greater efficiency is
generally observed when the fatty acid is removed prior to the
glycosylation reactions.
[0193] After each synthetic route, additional glycosyltransferases
can be used to add additional saccharide residues in order to
obtain more complex gangliosides.
Example 12
Effect of Compounds of the Invention on Growth of Mammalian
Cells
Synthesis of Ganglioside Compounds of the Invention
[0194] Compounds 1003, 1009, 1011, 1014, 1081, 1082, 1083, 1084,
1085, and 1086 were made according to methods of the present
invention and stored in powder form until use.
Reagents
[0195] 9 L cells were obtained from Wake Forest University
(Winston-Salem, N.C.) and the other five cell lines from American
Type Culture Collection (ATCC, Manassas, Va.). Minimum essential
medium Eagles (MEM) and basal medium Eagles (BME) media, fetal
bovine serum (FBS), newborn bovine serum, and trypsin-EDTA solution
were obtained from Sigma Chemical Co., St. Louis, Mo. Dulbecco's
modified Eagle's medium (DMEM) and Liebovitz L-15 medium were
obtained from ATCC (Manassas, Va.). MTT dye reagents were obtained
from Promega Corporation, Madison, Wis.
Cell Culture
[0196] 9 L cells were grown in BME media with 10% newborn bovine
serum, 2 mM glutamine, and 1% penicillin/streptomycin at 37.degree.
C. in 5% CO.sub.2/95% air. The cell lines obtained from ATCC were
grown in the ATCC-recommended medium at 37.degree. C. in 5%
CO.sub.2/95% air. SK-N-MC (HTB-10) and U-87 (HTB-14) were grown in
MEM with Earles salts, 2 mM glutamine, 1 mM pyruvate, 0.1 M
non-essential amino acids (NEAA), and 10% FBS. U-118S (HTB-15) and
Hs 683 (HTB-138) cells were grown in DMEM, 4 mM glutamine, 4.5 g/L
glucose, 1.5 g/L sodium bicarbonate, and 10% FBS. SW 1088 (HTB-12)
cells were grown in Liebovitz L-15 medium with 10% FBS in a
humidified 37.degree. C. air environment (no added CO.sub.2).
Medium for each cell line was changed every third day, and cells
were passaged weekly using 0.25% trypsin-EDTA solution as the
dissociation agent.
Proliferation Assay
[0197] Cells at 80% confluence were harvested using 0.25%
trypsin-EDTA solution. The trypsinized cells were plated in 96-well
plates at 2000 cells per well (with the exception of 9 L cells,
which were plated at 1200 cells per well, as they grow very fast).
Working stocks of each of the ten compounds--1003, 1009, 1011,
1014, 1081, 1082, 1083, 1084, 1085, and 1086--were prepared in
dimethyl sulfoxide (DMSO). After the cells were allowed to attach
for 24 h, the cultures were fed and dosed with each of the ten
compounds--1003, 1009, 1011, 1014, 1081, 1082, 1083, 1084, 1085,
and 1086--at concentrations of 0.05, 0.5, 5, and 50 .mu.M. For each
concentration, replicates of six wells were used. Controls received
the same volume of DMSO diluted in medium that was added to the
test wells. The culture medium was renewed with fresh test compound
every three days. After seven days of culture, the viable cells
were measured using MTT reagent. The MTT assay was performed by
removing the medium from each well, adding 100 .PHI.L of fresh
medium and 15 .PHI.L of tetrazolium dye solution to each well and
incubating the cells at 37.degree. C. for 4 h. After 4 h, 100
.PHI.L of solubilization/stop solution was added to each well. The
plates were incubated at room temperature overnight, and the
intensity of the yellow color of each well was measured at 575 nm
on a Bio-Tek Instruments (Winooski, Vt.) microplate scanning
spectrophotometer.
[0198] It should be understood that the foregoing discussion and
examples merely present a detailed description of certain preferred
embodiments. It will be apparent to those of ordinary skill in the
art that various modifications and equivalents can be made without
departing from the spirit and scope of the invention. All the
patents, journal articles and other documents discussed or cited
above are herein incorporated in their entirety by reference.
* * * * *