Note: Descriptions are shown in the official language in which they were submitted.
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FUSED PYRROLO(2.3-C)CARBAZOLE-6-ONES WHdCH POTENTIATE AC3.'IVTrY OF GAMMA
TNTERFERON
FIELD OF INVENTION
This invention is related to pharmaceutical compounds which are referred to in
this
patent document as "fused pyrrolo(2,3-c]carbazole-6-ones." Also disclosed are
methods
for making these compounds. and methods for using the compounds.
BACKGROUND OF THE INVENTION
Human interferon-gamma ( IFN-y) is a natural human immunoregulatory protein.
It has been established as an agent effective in the treatment of tumors and
virus infections
in humans. The precise mechanisms by which IFN-y inhibits virus and tumor
growth in
vivo remain unknown. There is evidence that 1FN-y works by at least one of two
mechanisms: ( 1) by acting directly on the virus infected cell and the tumor
cell and/or (2)
by first activating cells of the immune system which then destroy the virus-
infected cell or
tumor cell [Interferons and other Regulatory Cytokines, E. De Maeyer and J. De
Maeyer-
Guignard, John Whey & Sons, New York ( 1988)].
One manifestation of a stimulated immune system is the enhanced expression on
the surface of immune cells of the proteins of the Major Histocompability
Complex
(MHC). The MHC is made of class I, II, and III genes that code for the
respective class I,
II, and III proteins. Class I and II proteins reside on the cell surface and
are involved in
controlling the immune response, whereas the class III proteins appear in the
serum and
are not involved in controlling the immune response. Class I and II proteins
on antigen
presenting cells e.g. monocytes, B Lymphocytes, dendritic cells, present
foreign antigens to
T Lymphocytes with subsequent destruction of the cell containing the foreign
antigen. The
enhanced expression of the class I and II proteins is essential for the immune
system to rid
an animal of virus-infected cells and enhance specific antibody production.
1FN-y is one
ofthe major regulators of the immune response due to its ability to enhance
the expression
of MHC class I and II proteins. An example of the benefit of MHC I enhancement
by
1FN-y is the enhancement of class I proteins on virus-infected cells. The
virus infected cell
presents synthesized viral antigens on its cell surface to the T cell receptor
on cytotoxic T
cells {CD4 cells) with the subsequent destruction of the virus infected cell
by the cytotoxic
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T cell. An example of the benefit of MHC II enhancement by IFN-y is the
enhancement of
class II proteins on monocytes. Monocytes can ingest invading microorganisms
and the
class II proteins on the monocyte surface present peptides derived from the
invading
microorganism. These peptides held by the class II proteins are presented to
the T cell
receptor on helper T cells (CD8) with subsequent secretion of Iymphokines by
the CD8
cell. The secreted lymphokines cause proliferation of the antibody
synthesizing B
lymphocytes which syntliesize large amounts of antibody against the invading
microorganism
It can be seem from the above examples that a compound that enhances the IF2T-
y
induction of MHC molecules would be useful in combination with IFN-y for the
treatment
of infections by microorgaiusms. Such a compound might permit a reduction in
the dose
of IFN-y, thereby advantageously giving the same therapeutic effect as with
IFN-y alone
but with fewer of the IF'N-y related side effects.
There are at Ieast three reports of compounds tliat potentiate the IFN-y-
induced
MHC expression [Coutinho. G.C., Dudrieu-Trautmann, O., Strosberg, A.D., and
Couraud, P.O., Catecholamines Stimulate the IFN-y-induced Class II MHC
Expression on
Bovine Brain Capillary Endothelial Cells, J. Immunol., I47, 2525-2529 (199/);
Zhu, J.,
Mix, E., Olsson, T., and Link, H.' "Influence of Ion Channel Modulation of in
Vitro
Interferon-y Induced MHC Class I and II Expression on Macrophages",
Immunopharmacology and Immunotoxicology, 17, 109-136 (1995); Mothes, T.,
Bendix,
U." Pfannschmidt, C." and Lehmann, L, ''Effect of Gliadin and Other Food
Peptides on
Expression ofMHC Class II Molecules by HT-29 Cells", Gut, 36, 548-552 (1995)].
SUMMARY OF THE INVENTION
Disclosed herein are a novel class of molecules represented by Formulae I and
II,
which we refer to as fused pynrolo[2,3-c]carbazole-6-ones.
FORMULA I
0
R5 N~R1
R3
O R4
N X
~2
R
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3
FORMULA II
0
Rj-N RS
R3
R4
N X
R2
Constituent numbers are defined, infra. Preferred methods for preparing these
compounds
are disclosed infra.
We have discovered that our fused py~olo[2,3-c]carbazole-6-one compounds
(numbering as designated for K-252a and K-252c set forth in Moody et. al, J.
Org.
Chem., 1992, 57, 2105) potentiate the activity ofhuman IFN-y in inducing the
expression
of MHC on the surface of receptive cells. The compounds of the invention show
ability
for enhancing the effectiveness of the immune system, and this in turn
provides,
beneficially, an enhancement in inhibiting virus andlor tumor growth. We have
also
discovered that the fused pyrrolo[2,3-c]carbazole-6-one compounds ofthe
invention are
useful for potentiating, preferably, neurotrophin-3 (NT-3) activity.
DETAIi,ED DESCRIPTION
We first describe the drawings.
i. Drawings
Figure 1 is a graph sliowing the enliancement of IFN-y-induced expression of
HLA-DR MHC II molecule by pyrrolo[2,3-c]-carbazole-6-ones of the invention.
Figure 2 is a schematic drawing outlining the chemical synthesis ofthe
pyrrolo[2,3-
- c]carbazole-6-ones of Formula I, Section V (A)-(D).
Figure 3 is a schematic drawing showing the synthesis of intermediates to
pyrrolo[2,3-c]carbazole-6-ones.
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y
Figure 4 is a scliematic drawing showing an alternate synthesis of
intermediates to
pytrolo[2,3-c]carbazole-6-ones.
Figure S is a schematic drawing showing the synthesis ofpyrrolo[2,3-
c]carbazole-
6-ones of Formula LI.
Figure 6 is a schematic drawing showing the synthesis ofpyrrolo[2,3-
c]carbazoie-
6-ones in which X is C=O from the corresponding compounds in which X is CHa.
Figure 7 is a schematic drawing showing the synthesis of intermediate I I to
pyrroio[2,3-c]carbazole-6-ones.
II. Fused pyrrolo[2,3-cJcarbazole-6-ones
The novel compounds of this invention, which are referred to as fused
pytroio[2,3-
c]carbazole-6-one derivatives are represented by the following Formulae:
O O
R6 N-R~ R'~_.N RS
3
R 4 R R4
N X )(
I and II
wherein:
a) R I is selected from the group consisting of H, alkyl of 1-4 carbons, aryl,
arylalkyI, heteroaryl, lieteroarylalkyl; C(=O)R9. where R9 is alkyl of I-4
carbons or
aryl; (CH2)"OR9, where n is an integer of 1-4; OR1°, where Rl°
is H or allcyl of I-4
carbons; (CH~)"OR''' _ where R 1'l is the residue of an amino acid after the
hydroxyl group of the carboxyl group is removed ; OR''', NR~RB; (CH2}aNR~Rg,
and O(CH2~NR7R8: and either
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" (I) R~ and R8 independently are H or alkyl of 1-4 carbons; or
(2) R~ and R8 are combined together to form a linking group of the
general formula -(CH2)2-X1-{CH2)2-, where X1 is O, S or CH2;
b) R2 is selected form tire group consisting ofH, S02R9, C02R9, C(=O)R9,
alkyl of I-8 carbons, alkenyl of 1-8 carbons, alkynyl of 1-8 carbons, and a
monosaccharide of ~-7 carbons. wherein each hydroxyl group of said
monosaccharide is independently selected from the group consisting of
unsubstituted liydroxyl and a replacement moiety replacing said hydroxyl group
selected from the group consisting of H, alkyl of 1-4 carbons,
alkylcarbonyloxy of
2-S carbons, and allcoxy of 1-4 carbons; wherein either
1) each alkyl of 1-8 carbons, allcenyl of 1-8 carbons, or alkynyl of 1-8
carbons is unsubstituted; or
2} each alkyl of 1-8 carbons, allcenyl of 1-8 carbons, or alkynyl of I-8
carbons independently is substituted with I-3 groups selected from the
group consisting of aryl of f>- I O carbons, heteroaryI, F, Cl, Br, I, CN,
N02, OH, OR9, O(CH2)aNR~RB, OCOR9, OCONHR9,
O-tetraliydropyranyl, NH2, NR~RB, NR10COR9~ NR10C02R9,
NR10CONR7R8, NHC(=NH)NH2, NR10S02R9; S(Oh,Rll, wherein
R11 is H, alkyl of 1-~ carbons, aryl of 6-10 carbons, or heteroaryI, and y is
I or 2; SR11, C02R~, CONR7R8, CHO, COR9, CH20R7, CHaOR9,
CH=NNR11R12, CH=NOR11, CH=NR9>
CH=NNHCH(N=NH)NH2. SO2NR12R13; wherein either
(la) R12 and R13, independently, are H, alkyl of 1-4 carbons,
aryl of 6-10 carbons,or heteroaryl; or
(2a) R 12 and R 13 are combined together to form a
.-(CH2)2-X 1-(CH2)2 ~g ~'ouP~
POOR 1 1 )2. NHR", NR'°R~'', OR 14, and a monosaccharide of
S-7 carbons wherein each hydroxyl group of said monosaccharide is
independently selected from the group consisting of unsubstituted
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~'
hydroxyl and a replacement moiety replacing said hydroxyl group selected
from the group consisting of H, alkyl of 1-4 carbons, alkyicarbonyloxy of
2-5 carbons, and alkoxy of 1-4 carbons;
c) each R3 and R4, independently, is selected from the group consisting of H,
aryl
of6-10 carbons, heteroaryl, F, CI, Br, I, CN, CFA, N02, OH, OR9,
O(CH2}nNR~RB. OCOR9, OCONHR9, NH2, (CH2~OR9, (CHa)~ORIO,
(CH2)nORI4, ORS''. NHR''', NR7R8, NR'(CH2~NR~RB,NRIOCOR9,
~lOC0~7R8, SRI I, S(O)yRI I, C02Rg, COR9, CONR~RB, CHO,
CH=NORl I, CH=NR9, CH=NNRI 1RI2, (CH2~SR9, (CH2~S(Oh,R9;
CH2SRI5, where RIS is alkyl of I-~I carbons; CH2S(Oh,RI4, (CH2~NR~RB,
(CH2~NHR14, all.-yi of I-8 carbons, alkenyl of I-8 carbons, and alhynyl of I-8
carbons; and either
I) each alkyl of I-8 carbons, allcenyl of 1-8 carbons or alkynyl of I-8
carbons is unsubstituted; or
2) each alkyl of I-8 carbons, alkenyI of I-8 carbons, or alkynyl of I-8
carbons is independently substituted as described in b)2) above;
d) RS is selected from the group consisting of alkyl of I-8 carbons, alkenyl
of I-8
carbons, and aikynyl of 1-8 carbons; and either
1) each alkyl, alkenyl, or allcenyl group is unsubstituted ; or
2) each alkyl, allcenyl, or alkynyl group is substituted with I-3 groups
selected from the group consisting of F, CI, Br, I, CN, CF3, N02, OIL
OR9, O(CH2~NR~RB, OCOR9, OCONHR9, NH2, (CH2~OR9,
(CH2)nORI4. NR7R8, NR'(CH2}nNR~RB.NRI0COR9,
NRIOCONR~RB, SRI I. S(O}yRI I, C02Rg, COR9, CONR~RB, CHO,
CH=NORI i, CH=NR9, CH=NNRI IR12, (CH2~SR9, (CH2~S(O~,R9,
CH2SR15, CH2S(O}yRl4 (CH2}nNR~RB, and (CH2}nNHR''':
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e) X is selected from the group consisting of -N-, -O-, -S-, -S(=O)-, -S(=O}Z-
,
alkylene of I-3 carbons, -C(=O)-, -C(R2)=C(RZ}-, -C(RZ}Z-, -CH=CH-, -CH(OH)-
CH(OH)-, -C(=NOR11)-, -C(OR11)(R11)_, _C(=O)CH(R15)-, -CH(R15)C(=O)-;
-CH2-Z-, -Z-CH2-, -CH2ZCHz- where Z is selected from the group consisting of
-C(OR11)(R")-, O, S, C(=O), and NR11; and alkylene of 1-3 carbons substituted
with a group selected from the group consisting of one RS substituent group,
SR'o,
OR'°, OR''', R''S, phenyl, naphthyl, and arylallcyl of 7-I4
carbons.
As used lierein, the term "aryl" means monocyclic and polycyclic aromatic
groups
including, for example, phenyl, naplitliyl, biphenyl, and xylyl groups. Aryl
groups may be
unsubstituted or substituted with, for example, alkyl and halogen groups.
Halogens
include fluorine, chlorine, bromine, and iodine. Preferred are aryl groups
which contain 6-
carbons. Phenyl and naphthyl groups are particularly preferred.
As used herein, the term '9ieteroaryl" means an aryl moiety which contains at
least
one basic nitrogen atom and 0-4 heteroatoms selected from O, S, and N.
Examples of
heteroaryl groups include pyrrolyl, pyranyl, thiopyranyl, furyl, imidazolyl,
pyridyi,
thiazolyl, triazinyl, phthalimido, indolyl. purinyl, and benzothiazolyl.
As used herein witli reference to the definition of R 14, the term "amino
acid"
means a molecule containing both an amino group and a carboxyl group. Tt
includes an
"a.-amino acid" which lias its usual meaning as a carboxylic acid which bears
an amino
functionality on the carbon adjacent to the carboxyl group. oc-Amino acids can
be
naturally occurring or non-naturally occurring. Amino acids also include
"dipeptides"
which are defined herein as two amino acids which are joined in a peptide
amide linkage.
Thus, constituents of dipeptides are not limited to a-amino acids, and can be
any molecule
containing both an amino group and a carboxyl group. Preferred are a.-amino
acids,
dipeptides such as Iysyl-~i-aianine, and aminoalkanoic acids of 2-8 carbons,
e.g., 3-
dimethylaminobutyric acid.
- Preferred "alkyl", "alkenyl", and "alkynyl" groups contain 1-4 carbon atoms.
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Preferred R' groups include H, alkyl of 1-4 carbons, substituted or
unsubstituted
phenyl, OR'°, and O(CH2)"NR'R°. Preferred phenyl substituents
include alkyl of 1-4 r
carbons, and halogen. Most preferred is H.
Preferred RZ groups include H, C(=O)R9, allcyl of 1-8 carbons, and alkyl of 1-
8
carbons substituted with one OR'', OH. OCOR9, NR'R8, NH2, NR'°COR9, or
NR'°R'''
group. Most preferred is H.
Preferred R; and R'' groups include H, lialogen, CN, OH, OR9, ORl'', NHa,
NR'R8,
(CHa)~OR'°, (CFiz)~OR'''. COR''. NR'°COR', NHR''', and
O(CHZ)~NR'R8. Most preferred
is H.
Preferred RS groups include H, and alkyl of 1-4 carbons. Most preferred is H.
Preferred X groups include -N-, -O-, -S-, allcylene of 1-3 carbons, -C=O-,
-CH2-Z- and -Z-CHZ-. Most preferred are -N-, -O-, -S-, and -CH2- groups.
IQ. Fused Pvrrolo[2,3-c] carbazole-6-one Utilities
Our fused pyrrolo[2,3-c]carbazole-6-ones have evidenced a panoply ofimportant
functional activities which find utility in a variety of settings, including
both research and
therapeutic arenas. For ease of presentation, and in order not to Limit the
range of utilities
for which these compounds can be characterized, the preferred activities of
the fused
pyrroio [2,3-c] carbazole-6-ones can be generally described as follows:
A. Enhancement of IFN-y induction of MHC molecules;
B. Potentiation of function and/or survival of trophic factor responsive
cells.
Enhancement of IFN-y induction of MHC molecules can preferably be established
using a
human monocyte cell line that responds to IFN-y; a particularly preferred cell
line is
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q
available from the American Type Culture Collection (ATCC), referred to as THP-
1,
under accession number ATCC TIB-202. 1FN-y is laiown to induce expression
ofthe
three MHC II heterodimers. HLA-DP, HLA-DQ and HLA-DR; in THP-!cells.
Potentiation of function and/or survival of trophic factor responsive cells,
e.g., cells of
neuronal lineage, can be preferably establislied using a cultured spinal cord
choIine
acetyltransferase ("ChAT") assay.
As used herein, the term 'poteutiation" when used to modify the terms
"function"
and "survival" means a positive alteration or change. Potentiation which is
positive can
also be referred to herein as an "enliancement" or "enhancing."
As used herein. the teens ''enhance" or "enhancing" or enhancement when used
to
modify the terms "fllnCt10I1" or wsmvival" or ''induction" means that the
presence of a
fused pyrrolo[2,3-c]carbazole-6-one has a comparatively greater effect on the
function
and/or survival of a tropliic factor responsive cell or, in the case of IFN-y,
induction of
MHC molecules, than a comparative cell not presented with the fused
pyrrolo[2,3-
c]carbazole-6-one.
As used herein the term 'neuron," "cell of neuronal lineage" and 'heuronal
cell"
includes, but is not limited to. a heterogeneous population of neuronal types
having
singular or multiple transmitters and/or singular or multiple functions;
preferably, these are
cholinergic neurons. As used herein, the phrase "choIinergic neuron" means
neurons of
the Central Nervous System (CNS) and Peripheral Nervous System (PNS) whose
neurotransmitter is acetylcholine; exemplary are basal forebrain and spinal
cord neurons.
As used herein a "trophic factor" is a molecule that directly or indirectly
affests the
survival or function of a trophic factor responsive cell. Exemplary trophic
factors include
CiIiary Neurotrophic Factor (CNTF), basic Fibroblast Growth Factor (bFGF),
insulin and
insulin-like growth factors (e.g., IGF-L, IGF-II, IGF-III), interferons,
interleukins,
cytokines, and the neurotrophiits. sncludtng Netve Growth Factor (NGF),
Neurotrophin-3
(NT-3), Neurotrophiii-=1/5 (NT--I/~) and Brain Derived Neurotrophic Factor
(BDNF).
A "trophic factor-responsive cell," as defined herein, is a cell which
includes a
receptor to which a trophic factor can specifically bind; examples include
neurons (e.g.,
cholinergic neurons) and non-neuronal cells (e.g., monocytes and neoplastic
cells).
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lo
As used herein, "tropliic factor- activity" and "trophic factor induced
activity" are
defined as any response which directly or indirectly results from the binding
of a trophic
factor (e:g., NT-3) to a cell comprising a trophic factor receptor.
As used in the pln~ases "trophic factor activity" and "trophic factor-induced
activity," the term "trophic factor" includes both endogenous and exogenous
trophic
factors, where "endogenous" refers to a tropluc factor already present and
"exogenous"
refers to a trophic factor added to a system. As defined, "trophic factor
induced activity"
includes activity induced by ( 1 ) endogenous trophic factors; (2) exogenous
trophic factors;
and (3) a combination of endogenous and exogenous trophic factors.
A. Enhancement of 1FN-y Induction of MHC Molecules
The compounds of the invention can be used to enhance IFN-y induction of
MHC molecules. IFN-y has shown e$'ectiveness in the treatment ofvirus
infections and
certain tumors. Due to its dose limiting side e$'ects, however, the utility of
IF'N-y has
been limited. In an immunocompromised situation, e.g., in a patient evidencing
viral
infection, enhancement of au 1FN-y mediated immune response would he
beneficial, given
that IFN-y induces expression of MHC molecules. Thus, the compounds ofthis
invention
which enhance the ability of endogenous IFN-y or exogenously administered IFN-
y to
induce MHC expression are of benefit.
As detailed in Section V below, the ability of a fused pyrrolo[2,3-c]carbazole-
6-
one to enhance 1FN-y induction of MHC molecules is preferably assessed using
the THP- I
cell line. This cell line evidences expression of the MHC II heterodimer HLA-
DR.
Computing the expression of HLA-DR in the presence of IFN-y and IFN-y plus one
or
more fused pyrrolo[2,3-c]carbazole-6-ones oftlie present invention provides a
rapid and
e$icient method for deterLniziation of enhancement of IFN-y induction of MHC
molecules
can be assessed.
The compounds of this invention can be used in the development of in vitro
models
of enhancement of expression of MHC molecules, function, identification, or
for the
screening of other synthetic compounds which have activities similar to that
of the fizsed
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pyrrolo[2,3-c]carbazole-6-ones. The compounds can be utilized in a research
environment
to investigate, refine and determine molecular targets associated with
fimctional responses.
For example, by radiolabelling a fused pyrolo[2,3-c]carbazole-6-one associated
with a
specific cellular fimction (e.g., HLA-DR induction), the target entity to
which the fused
pyrrolo[2,3-c]carbazole-6-one binds can be identified, isolated, and purified
for
characterization. In yet anotlier example, a fused pyrrolo[2,3-c]carbazole-6-
one can be
used as a screening tool to discover agents which have marginal activity, but
when
combined with at least one disclosed fused pyrrolo[2,3-c]carbazole-6-one are
capable of
enhancing IFN-y induction of MHC molecules. Because the disclosed fused
pyrrolo[2,3-
c]carbazole-6-ones are useful in enhancing 1FN-y induction of MHC molecules,
the
disclosed compounds beneficially lend tliemselves to utility as therapeutic
agents. Such
enhancement is ofvalue in an immunocompromised patient.
B. Potentiation of function and/or survival of trophic factor
responsive cells
The fused pyrrolo[2,3-c]carbazole-6-ones of this invention can be used to
enhance the fimction and/or survival of cells ofneuronal lineage. The fused
pyrrolo[2,3-
c]carbazole-6-ones can be utilized individually or with other fused
pyrrolo[2,3-
c]carbazole-6-ones, or in combination witli other beneficial molecules such as
indolocarbazoles whicli also liave the ability to potentiate the fimction
and/or survival of a
designated cell: In. situations wliere the fused pyrroio[2,3-c]carbazole-6-one
is intended to
enhance a biological activity, e.g., neurotrophin activity, exogenous
neurotrophins such as
NT-3 may be utilized in conjunction with the fused pyrrolo[2,3-c]carbazole-6-
one.
A variety of neurological disorders are characterized by neuronal cells which
are
dying, injured, fimctionally comprised, undergoing axonal degeneration, at
risk of dying,
etc. These disorders include, but are not limited to: Alzheimer's; motor
neuron disorders
(e.g., amyotrophic lateral sclerosis); Parkinson's; cerebrovascular disorders
{e.g., multiple
sclerosis; peripheral neuropathies (e.g., those affecting DRG neurons in
chemotherapy-
associated peripheral neuropathy); disorders induced by excitatory amino
acids; disorders
associated with concussive or penetrating injuries of the brain or spinal
cord.
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/~
As described in Section V below, the ability of a fused pyrrolo[2,3-
c]carbazole-6-
one to enhance the function and/or survival of cells of a neuronal lineage can
be
determined by employing a basal forebrain ChAT activity assay. ChAT catalyzes
the
synthesis of the neurotransmitter acetylcholine and is considered an enzymatic
marker for
a functional cholinergic neuron. A functional neuron is also capable of
survival.
Enhancement of a neurotrophiu such as NT-3 can be determined by comparing the
functional activity of the neurotropliin with or without the fused pyrolo[2,3-
c]carbazole-
6-one present.
Pharmaceutically acceptabie salts of the fused pyrrolo[2,3-c]carbazole-6-ones
also
fall within the scope of the present uivention. The term "pharmaceutically
acceptable
salts" as used herein means au inorganic acid addition salt such as
hydrochloride, sulfate,
and phosphate, or an organic acid addition salt such as acetate, maleate,
fizmarate, tartrate,
and citrate. Examples of pharmaceutically acceptable metal salts are alkali
metal salts such
as sodium salt and potassium salt, alkaline earth metal salts such as
magnesium salt and
calcium salt, aluminum salt, and zinc salt. Examples of pharmaceutically
acceptable
ammonium salts are ammonium salt and tetramethylammonium salt. Examples of
pharmaceutically acceptable organic amine addition salts are salts with
morpholine and
pipetidine. Examples of pharmaceutically acceptable amino acid addition salts
are salts
with lysine, glycine, and phenylalanine.
Compounds provided herein can be formulated into pharmaceutical compositions
by admixture with pharmaceutically acceptable nontoxic excipients and
carriers. Such
compositions can be prepared for use in parenteral administration,
particularly in the foam
ofliquid solutions or suspensions; or oral administration, particularly in the
form oftablets
or capsules; or iutranasally, particularly in the form ofpowders, nasal drops,
or aerosols;
or deimaIly, via, for example. trans-dermal patches.
The composition can be conveniently administered in unit dosage foam and may
be
prepared by any ofthe methods well known in the pliarmaceutical art, for
example, as
described in Renzirrgtorz's Plrarnrcrcezrtical Sciences (Mack Pub. Co.,
Easton, PA, 19$0).
Formulations for parenteral administration may contain as common excipients
sterile water
or saline, polyalkylene glycols such as polyethylene glycol, oils and
vegetable origin,
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I3
hydrogenated naphthaleues and the like. In particular, biocompatible,
biodegradable
lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-
polyoxypropylene
copolymers may be useful excipieuts to control the release of the active
compounds.
Other potentially useful pareuteral delivery systems for these active
compounds include
ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable
infusion systems,
and liposomes. Formulations for inhalation administration contain as
excipients, for
example, lactose, or may be aqueous solutions coutaiving, for example,
polyoxyethylene-
9-lauryl ether, glycocholate and deo~cycholate. or oily solutions for
administration in the
form ofnasal drops, or as a gel to be applied intranasally. Formulations for
parenteral
administration may also include glycocholate for buccai administration, a
salicylate for
rectal administration, or citric acid for vaginal administration. Formulations
for trans-
dermal patches are preferably lipophilic emulsions.
The compounds of this invention can be employed as the sole active agent in a
pharmaceutical composition. Alternatively, tliey can be used in combination
with other
active ingredients, e.g., synthetic iFN-y and/or other growth factors which
facilitate
potentiation ofNT-3 sucli as tliose disclosed in U.S. Patent No. 5,468,872 and
International Publication No. WO 95/0791 1 (publication date: March 23, 1995).
The concentrations of the compounds of this invention in a therapeutic
composition can vary. Tlie concentration will depend upon factors such as the
total
dosage of the drug to be administered, the chemical characteristics (e.g.,
hydrophobicity)
ofthe compounds employed. and the route ofadministration. The compounds ofthis
invention typically are provided in au aqueous pliysiological buffer solution
containing
about 0.1 to 10% w/v compound for parenteral administration. Typical dose
ranges are
from about 1 p.g/kg to about I g/kg ofbody weight per day; a preferred dose
range is
from about 0.01 mg/kg to I 00 mg/kg of body weight per day. A preferred dosage
of drug
to be administered is likely to depend on variables such as the type and
extent of
progression of the disease or disorder, the overall health status of the
particular patient,
the relative biological afficacv of the compound selected, and formulation of
the
compound excipient, and its route of adnmustration.
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ey
IV. General Description of the Synthetic Processes
Two synthetic routes were employed to prepare the fused pyrrolo[2,3-
c]carbazole-
6-one derivatives of the inventiou. In Metliod A (FIG 2) a 2-{aryl) or 2-
(heteroaryl)indole
derivative (1-4.) which is either uusubstituted or substituted at carbons 4-7
(inclusive) of
the indole ring (R') or substituted or unsubstituted in the (hetero)aryl
portion (R4) is
reacted with maleimide in the presence of a catalyst such as, trifluoroacetic
acid (TFA) to
give the fused pyrrolocarbazoie-6-one derivatives ofFormula I (Examples I-IV).
Additional Lewis acid cataIysts_ such as SnCl.~, A1C13, EtAICI2, or EtzAICI
may also be
used to effect the reaction. The reaction may also be run in a solvent such as
TFA,
toluene, CH2CIz or 1,2-dichloroethane.
The bi-aryl indole intennediates. 2,2'-biindoie 1 (X = N, R2, R3, R4 = H), 2-
{2-
furyl)indole 2 (X = O, R2, R3, R4 = H) and 2-(benzothienyl)indole 3 (X = S,
R2, R3, R4
= H) may be prepared using standard literature procedures (Hudkins, RL.; et.
al J.
Org.Chem., 1995, 60, 6218) {FIG 2). 2-(2-Tndenyl)indole 4 (FIG 3) (X = CH2,
R2, R-',
R4 = H), or 2-(2-indenyl)uidote derivatives substituted with an R' or R4
group, may be
prepared by reacting 1-carboxy-2-(tributylstannyl)indole 5, or 1-carboxy-2-
(tributylstannyl)indote substituted with au R3 group (Hudlcins, RL. et. a1 .I.
Org.Chem.,
1995, b0, 6218) with 2-bromoindeue 6 (J. Org. Chem., 1982, 47, 705) or a 2-
bromoindene substituted with an R'~ group. Alternatively, 2-(2-indenyl)indole
4 (X = CH2,
R2, R3, R4 = H), or 2-{2-iudeuyl)indole derivatives substituted with an R3 or
R4 group,
may be prepared by reaction of 1H-indole or a derivative thereof containing an
R3 group,
protected as a lzthium iudole-1-carbo~cylate intermediate (Tetrahedron Lett.
26:5935
(1985)), then treated with a strong base, such as t-Bui..i, then alkylated
with an
appropriate 2-indanone, or 2-indanoue derivative substituted with an R4 group
to give the
corresponding tertiary alcohol 7 (FIG 4). The resulting tertiary alcohol 7 is
treated with a
dilute acid {e.g., 2N HCl in acetone) to give the corresponding 2-(2-
indenyl)indole 4 (US
Patent 5,475,110). Using other Benzocycloallcan-2-ones such as 2-tetralone in
the
reaction sequence shown in Figure 4 will give 2-{2-(3,4-dihydronaphthyl)indole
8 (X =
CH2CH2, R2, R3, R4 = H) ( US Patent 5.475,110). Tlie palladium-catalyzed cross-
coupling methodology (Stille reaction) may be used to prepare other
dernratives, for
example, where X in Figure 3 has 1-3 carbons (inclusive), by coupling the
- 2-(trifluoromethanesuIfouate)- or the 2-iodo- or 2-bromovinyl derivative
ofthe
corresponding cyclic ketone with I-carboxy-2-tributylstannylindole to give 2-
(2-
benzocycloalkenyl)indoles.
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The starting 1 H-iudole derivative described previously is converted to a 1-
substituted indole derivative (R2 not H) by standard methodology, for example,
by
treatment of the 1H-indole with base and an alkylating agent to give a I-
substituted
indole. In these examples, the iudole derivative can be directly treated with
a strong base
(e.g., t-BuLi, sec-BuLi, n-BuLi, lithium diisopropylamide) followed by
alkylation with a 2-
indanone derivative to give the corresponding tertiary alcohol 7, which
includes R2
substituents in position one of the indole ring. The 2-{aryl)- or 2-
(heteroaryl)indole
derivative (1-3), 2-(2-indenyl)indole 4, or 2-(2-( 1,2-diliydronaphthyl)indole
8 may be
converted to intermediates which contain R' substituents in position one ofthe
indole ring
by the method described above for iudole derivatives.
Compounds of general formulae I and )Q which contain R' groups, not = H, may
be prepared by starting with the appropriate R' substituted maleimide (FIG 2,
Method A).
Compounds of general formulae I and LI. in which RI is hydrogen, can be
allcylated in the
presence ofbase (e.g., liydrides. alkoxides. hydroxides of alkali or alkaline
earth metals, or
oforgano-lithium compowids) by treatment with RIL in which L is a leaving
group such
as a halogen, mesylate or tosyiate to give a fused pyrrolocarbazole-6-one
derivative which
has an Rl group bound to the lactam nitrogen. Compounds of general formulae I
and II,
in which RS is hydrogen, may be converted to derivatives where one or two RS
groups
may be added by treatment of a fused pyrrolocarbazole-6-one derivative with
one
equivalent or an excess of a strong base ( e.g., liydrides. allcoxides,
hydroxides of alkali or
alkaline earth metals. or of oraano-lithium compounds) with R5L in which L is
a leaving
group such as a halogen. or by condensation with an RS containing ketone or
aldehyde
carbonyl derivative to give a fused pyrrolocarbazole-6-one derivative which
has one or
two an RS groups. The derivatives from the ketone or aldehyde condensation
reactions
would give vinyl derivatives at R5.
The indole derivatives are prepared using standard methodology (U.S. Patent
3,976,639; U.S. Patent 3,732.245; The Chemistry of Heterocyclic Compounds,
Indoles
Parts One and Two; HouIihan Ed.. Wiley-Interscience ( 1972)}. The 2-indanone
derivatives can be prepared usiua previously described procedures (see U. S.
Patent
4,192,888; U.S. Patent 4,128.666; J. ~Inz. Chenz. Soc. 89:4524 ( 1967);
Tetrahedron Lett.
43:3789 ( 1974); Chenz. Ber. 122: 1791 ( I 989); Can. J. Chem. 60:2678 (
1982); Helvetiea
Chimica Acta 70:179 I ( I 987); Chenz. Plzarnz. Bull. 33:3336 ( 1985 ); J.
Org. Chem.
55:4835 ( 1990}; Tetrahedron ~15: t 44 I ( 1989); Synthesis 818 ( 1981 )}.
CA 02241852 1998-06-29
WC 97131002 PCT/LTS97/02905
l~
In Method B (FiG 2), a 2-{heteroaryI)indoie derivative (1-3), or 2-
(aryl}indole
derivative such as 2-{2-indenyi)indole 4 is reacted with ethyl cis-(3-
cyanoacrylate in the
presence of a catalyst such as SnCL:;, AlCl3, EtAlCl2, Et2A1C1 or TFA in
CH2C12,
C2H4C12 or toluene as solvent to give the 6-oxo carbazole compounds offormula
I ofthe
invention.
Compounds of Foi~nula II are prepared as outlined in FIG 5. Addition of 2-
(aryl)-
or 2-(heteroaryl)indolyIzinc reagents (Tetrahedron Lett., 1994, 35, 793;
Tetrahedron
Lett., 1994. 35, 7123: Tetr-al~edroiz Lett.. 1993, 3=F, 5955: Tetrahedron
Lett., I993, 34,
6245) to succanimide. or an R' substituted succinimide derivative, via a
Reformatzsky type
reaction (Synthesis. 1975. 685) followed by dehydration would give compounds
ofthe
general structure 9. PalladiuFn catalyzed ring closure (US Patent 5,475,1 I0;
Tetrahedron
Lett., 1993, 34, 8361) would yield pyrrolo[2.3-c]carbazole compounds ofFormula
II.
Compounds in which X = (C=O) (general structure 10) are prepared by oxidation
of derivatives of general Formula I (or II) where X = CH2, using standard
oxidizing
reagents (e.g., Se02,. Cr03. Na2Cr07, or Mn02) (FIG 6). Alternatively, 2-(2-
indenyl}indole 4 may be oxidized to 2-(2-( t-oxoindenyl)indole and used to
prepare
compounds of Formula I or II where X = {C=O) by the methods shown in FIGS 2
and 5.
Alternatively, 2-(2-( 1-oxoindenyl))indole I 1 may prepared using the
palladium-catalyzed
cross-coupling methodology (F1G 3) by coupling I-carboxy-2-
tributylstannylindole 5 or
its derivatives with 2-(trifluorotnethanesulfonyl)oxyinden-I-one or 2-
bromoinden-I-one
12 (FIG 7) (J. Org. Chenz.. I994, 59, 3453) or one ofits derivatives.
Compounds ofthe
general structure 10 (X = (C=O)) may undergo a variety of olefination,
addition and
condensation reactions known to those skilled in the art of organic synthesis
to give
derivatives, for example, but not limited to, X = (C=C(R2)2), C{R2)2, C{ORI
I)(RI I}.
Fused pyrrolocarbazole-6-one derivatives where X is S(=O) or S(=O)2 may be
prepared
by oxidation of X = S derivatives in a mamier similar to X = (C=O).
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a ~7
EXAMPLES
V. Specific Description of the Synthetic Processes
The following examples are presented for purposes of illustration and are not
to be
construed as limiting the scope of the invention in any way.
A. Exanapte I: SH, 7H, 13H-Benzofurano[2,3-a]pyrrolo[2,3-
cJcarbazole-6(6H}one (X = O; R!, R2, R3, R4, RS = H)
A miocture of 2-(2-benzofuryl)indole 2 (250 mg; I.0 mmol) and maleimide {110
mg; I.2 mmol) in triffuoroacetic acid (2 mL) was heated in a sealed reaction
vial at 125 °C
for 18 h. The solid precipitated was collected. washed with TFA and dried to
give 150 mg
(48 %) of product. Recrystallization (THF} gave a tan solid; mp >300
°C., MS (ES+) 312
(M+}, 1H NMR {DMSO- d~) cS =1.00 (s, 2H). 4. I3 (s, 2H), 7.12 (t, 1H), 7.37
(t, 2H), 7.45
(t, 2H), 7.71 (d, 1H). 7.95 (d. 1H), 8.54 (d, 1H'), 10.12 (s, IH), 11.79 (s,
1H). Anal. calcd
for CzoFilzNzOz'0.4 HzO; C. 75. 18; H, 4.04; N, 8.77. Found: C, 75.29, H,
3.86, N, 8.60.
B. Example IZ: 5H, 7H, 13H-Benzothieno[2,3-a]pyrrolo[2,3-
c]carbazole-6(6H)one (X = S: R1, R2, R3, R4, RS = H)
A miacture of 2-(2-benzothienyl)indole 3 ( 100 mg, 0.4 mmol} and maleimide (40
mg; 0.4 mmol) in trifluoroacetic acid (2 mL) was heated in a sealed reaction
vial at 125 °C
for 16 h. The solid precipitated was collected, washed with cold methanol and
dried to
give 80 mg (58 %) of product. Recrystallization (THF) gave a tan solid; mp
>300 °C., MS
(ES+) 328 (M+), IH NlViR (DMSO- d~) 8 4.10 (s, 2H), 7.20 (t, 1H), 7.40-7.60
(m, 4H),
8.10 (d, 1H), 8.80 (m. 1H). l0.86 (s, 1H), 11.80 (s, 1H). Anal. calcd for
CzaHlzNzSO.
0.5 HzO; C, 71.20; H, 3.88: N, 8.30. Found: C, 70.86, H, 3.61, N, 8.39.
C. Example III: SH. 7H. 12H, 13H-lndolo[2,3-a]pyrrolo[2,3-
cJcarbazole-6(6H)one (X = N: R!. R2, R-'. R4, R~ = H)
(1} Method A
To a mi,~cture of 2-2'-biindole 1 (250 mg; 1.0 mmol) and
maleimide (110 mg; 1.2 mmol) suspended in toluene (50 mL) was added
trifluoroacetic
CA 02241852 1998-06-29
WO 97/31002 PCTlUS97/02905
1g
acid (0.5 mL). The solution was heated at refIux for 18 h., cooled to rt, and
concentrated
to approximately 20 mL. The solution was cooled in an ice bath, the solid
precipitate was
collected, washed with cold etlier and dried to give I50 mg (55 %} of product.
Purification by column chromatography (EtOAc: liexanes; 2:1} gave a brown-tan
solid,
mp >320 °C., MS (ES+) 3 I I (M+), ~H NMR (DMSO- ds) 8 4.00 (s, 2H),
7.17-7.22 (m,
2H), 7.37-7.42 (m, 2H), 7.67 (d. 2H}, 8.03 (d, IH), 8.58 {d, IH), 10.87 (s,
IH), 10.92
(s,lH), 11.18 (s, 1H). Anal. calcd for C~oH,~N~02' 0.5 HaO; C, 74.99; H, 4.41;
N, 13.12.
Found: C, 75.24, H,4.02, N. 13.05.
(2) Method I3
A mixture of 2-2'-biindole 1 ( I00 mg; 0.43 mmol) and ethyl
cis-[3-cyanoacrylate (50 mg; 0..4 imnol) ui metliyiene chloride ( 10 mL) was
added 25 mL
of SnCl.,. The mixture was stirred at rt for 30 min. The suspension was cooled
on an ice
bath, the solid collected, washed with cold ether and dried to give 36 mg (27
%) of
product. Purification by column chromatography (EtOAc: hexanes; 2:I) gave a
brown-tan
solid; mp >320 °C., MS (ES~} 3 I 1 (M+), ~H NMR (DMSO- d6) 8 4.00 (s,
2H), 7.17-7.22
(m, 2H), 7.37-7.42 (m, 2H), 7.67 (d, 2H), 8.03 {d, IH}, 8.58 (d, 1H}, 10.87
(s, 1H), 10.92
(s,lH}, 11.18 (s, 1H).
This compound showed identical spectral and analytical characteristics as that
prepared
by Method A.
I~. Example (V: 5H, 7H. 12H, I3H-Indeno[2,3-a]pyrrolo[2,3-
c)carbazole-6(6H)one (X = CHI; RI, R2, R3, R4, R5 = H)
(1) Method A
A mixture of 2-(2-indenyl)indole 4 (300 mg, I.3 mmol) and
maleimide ( 160 mg; 1.6 nunol} in trifluoroacetic acid {2 mL) was heated in a
sealed
reaction vial at 160 °C for 18 h. Tlie solution was evaporated and the
solid dissolved in
ethyl acetate, then waslied with water and dried {MgS04) to give brown solid
product.
The crude product was ctwomatographed (silica gel, EtOac: hexanes; 1:1) to
give a
product which was treated with THF and filtered. The concentrated residue was
trittutrated with MeOH to Give tl~e product: mp 275-280 °C., MS {ES+)
310 (M+), 'H '
- NMR (DMSO- d6) 8 4.00 (s, 2H), -I. 13 (s. 2H), 7. 17 (t, 1H), 7.25-7.42 (m,
3H), 7.50 (d,
1H), 7.7I (d, 1H), 8.00 (d, 1H), 8.37 (d, 1H), 10.75 (s, 1H), 11.33 (s, 1H).
IR (KBr)
1650-1700 cm l. Anal. calcd for C~,Hi.~N~O 0.7 H20; C, 78.1b; H, 4.81; N,
8.65. Found:
C,78.13,H,4.41,N,8.L0.
CA 02241852 1998-06-29
WO 97131002 PCT/US97/02905
(2) Method B
A mi,~-ture of 2-(2-indenyl)indole 4 {75 mg; 0.32 mmol) and ethyl
cis-(3-cyanoacrylate {81 mg: 0.64 mmol) in trifluoroacetic acid (1 mL) was
heated in a
sealed reaction vial at 120 "C for I li., followed by 4 h at 160 °C.
The mvmue was
evaporated at reduced pressure and the residue was triturated with ether. The
resulting
solid was chromatographed (silica gel; EtOAc: hexanes; 1:i) to give I2 mg
{12%) of tan
solid product; mp 275-280 "C._ MS (ES+) 310 (M+). This compound showed
identical
spectral data as tliat prepared in Method A.
E. Example V: Enhancement by Fused Pyrroio[2,3-c]carbazole-
6-ones of the Induction by LFN-y of the MHC II Antigen
EILA-DR
A human cell rule derived from liuman monocytes, THP- I {ATCC TIB 202) that
responds to IFN-y, was used to demonstrate enhancement of HLA-DR by the fused
pyrolo[2,3-c]carbazole-6-ones.
THP-1 cells were grown in RPMI 1640 medium containing 20 uM
mercaptoethanol and 10% fetal bovine serum at 37°C in an atmosphere
of5%C02:95%
air at 100% humidity. For determination of enhancement of HLA-DR by the
compounds
of the invention, cells were either left untreated as controls, treated with
IFN-y only at 100
units/ml, or treated with compounds of the uivention at 1 uM final
concentration for 30
min. prior to the addition of IFN-y at 100 units/mi. Duplicate cultures were
used in all
experiments. The treated THP-1 cells were incubated at 37°C for 48
hours and then
prepared for analysis of HLA-DR by Flow Cytometry. Induction of HL.A-DR was
performed by standard procedures as described in Lnterferons and Other Re
atory
Cytokines, Edward De Maeyer and Jacqueline De Maeyer Guignard, Chapter 9, John
Wiley & Sons, New York. 1988. Cells were prepared for flow cytometry and
analyzed for
HLA-DR by flow cytometry as instructed in Current Protocols in Immcmoloay.
Vol. I,
pages 5Ø1-5.8.8, Jolu~ Wiley & Sous. 1994. One million cells from each
treatment were
collected by centrifugation and washed twice with phosphate-buffered saline
(PBS). The
cells were resuspended in 100 u1 of PBS containing 10 ug ofpurified rabbit IgG
to block
non-specific sites on the cell surface. After 20 min. on ice, 20 u1 of anti-
HLA-DR
monoclonal antibody tagged with the fluorescent label FITC was added and the
cells left
on ice an additional 30 min to allow the antibody to bind to the HLA-DR The
cells were
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~D
then washed 2 times with PBS each wash and fixed in 0.5 mt of0.5%
paraformaldehyde.
The fixed cells were stored at .4°C until analyzed by flow
cytometry.
The enlxancement of E-1LA-DR by representative fused pyrrolo[2,3-c)carbazale-5-
ones is shown in FIG 1. Tlie enhancement of HLA-DR by IFN-y alone is
designated
100% on the Y axis. There is no sigiu$cant uiduction of HL.A-DR by the
representative
compounds alone at I uM (F1G t ). All of the representative compounds enhance
the
induction of HLA-DR by IFN-y above the induction by IFN-y alone, i.e. above
100%.
The percent enliancement above IFN-y alone by the four compounds is shown in
Table I.
For example. at: 2l.tM. the compound of Section V(D) (Example IV) enhanced IFN-
y
induction of HLA-DR by 60% over 1FN-y alone.
TAB LE 1
Compound of Conc. of Fused Conc. of Ig'N-y,Percent
Euample # Pyrrolo[2,3- Units/ml Enhancement
cjcarbazole-b-one, of
a VI HLA.-DR
1 - -
1 - 100* 100
1 100 1I5
1 ? I00 I08
U - _ 0
B - I00* I00
B t 100 133
B ? 100 153
ITI _ _ 0-
- 100* 100
100 135
100 130
- - 0
N - 100* 100
N 1 100 1S6
~ ? t00 160
arm-y alone is aennea as ~ uu ro.
CA 02241852 2005-05-06
F. Eiample vI. NT-3 Potentiadoa of ChAT Activity in Basal
Forebrain Cultures by Pyrrolo[2,3-c]carbazole-6-ones
The ability of pyrrolo[2,3-c]carbazole-6-ones to potentiate NT-3 activity in
basal
forebrain cultures was determined using chotine acetyltransferase (ChAT)
activity as a
measure of cholinergic neuron function or survival. The compounds alone had no
effect
on ChAT activity. However, in the presence ofNf-3, the representative
compounds gave
a dose dependent potentiation of ChAT activity to levels greater than those
elicited by
NT-3 alone. The results shown in Table 2 are the result of a single
application ofNT-3
and the compound to be tested on the day of culture initiation, indicating a
prolonged
effect on the stuvival or function of basal forebrain cholintrgic neurons. The
methods
employed are descn'bed in U.S. Patent 5,468,872, Columns 18 and 19.
Table 2
Compound of '/. Basui % NT 3
Eiample # (mean (mean
SD) SD)
laDnM 300nM 100nM
300aM
NT-3+I I84+3 221+2 113+2 136+1*
NT3 + II 2I2 + 4 228 + 13 0+ 140 +
13 2* 1
NT3+IZZ 180+3 203+2 114+2 129+1*
NT 3 (100 ng/ml) iaon~sed ChAT Activityr 163 ~ 5 % (rncaa ~ SD) over control
calt~s. Test c~ands ahe had basal (100°!0) level of aetMty.
*p < 0.05, statistically swim ooaaparod to NT-3 activhy alone by Duandt t
st~istics.
Those skdIed in the art will appreaate that numerous chaages and modifications
may be made to the prtfetred embodiments of the imrention and that such
changes and
modifications may be made without departing from the spirit of the invention.
It is
therefore intended that the appended claims coves all e~quNalent variations as
fall within
the true spirit and scope of the invention.
21
