U.S. patent application number 12/078908 was filed with the patent office on 2008-09-11 for method of inhibiting neurotrophin-receptor binding.
Invention is credited to Sandra Marone, Richard J. Riopelle, Gregory M. Ross, Igor L. Shamovsky, Donald F. Weaver.
Application Number | 20080221147 12/078908 |
Document ID | / |
Family ID | 26977631 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080221147 |
Kind Code |
A1 |
Ross; Gregory M. ; et
al. |
September 11, 2008 |
Method of inhibiting neurotrophin-receptor binding
Abstract
The present invention relates to compositions which inhibit the
binding of nerve growth factor to the p75.sup.NTR common
neurotrophin receptor and methods of use thereof. In one
embodiment, the compound which inhibits binding of nerve growth
factor to p75.sup.NTR comprises, particularly when bound to nerve
growth factor, at least two of the following: (1) a first
electronegative atom or functional group positioned to interact
with Lys.sup.34 of nerve growth factor; (2) a second
electronegative atom or functional group positioned to interact
with Lys.sup.95 of nerve growth factor; (3) a third electronegative
atom or functional group positioned to interact with Lys.sup.88 of
nerve growth factor; (4) a fourth electronegative atom or
functional group positioned to interact with Lys.sup.32 of nerve
growth factor; and (5) a hydrophobic moiety which interacts with
the hydrophobic region formed by Ile.sup.31, Phe.sup.101 and
Phe.sup.86 of nerve growth factor.
Inventors: |
Ross; Gregory M.; (Kingston,
CA) ; Shamovsky; Igor L.; (Kingston, CA) ;
Marone; Sandra; (Kingston, CA) ; Weaver; Donald
F.; (Kingston, CA) ; Riopelle; Richard J.;
(Kingston, CA) |
Correspondence
Address: |
GOWLING LAFLEUR HENDERSON LLP
SUITE 1600, 1 FIRST CANADIAN PLACE, 100 KING STREET WEST
TORONTO
ON
M5X 1G5
CA
|
Family ID: |
26977631 |
Appl. No.: |
12/078908 |
Filed: |
April 8, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11267129 |
Nov 7, 2005 |
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12078908 |
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10205601 |
Jul 26, 2002 |
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11267129 |
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09457606 |
Dec 8, 1999 |
6492380 |
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10205601 |
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09310883 |
May 17, 1999 |
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09457606 |
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Current U.S.
Class: |
514/296 ;
546/98 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 31/473 20130101; A61K 31/4745 20130101; A61P 25/28 20180101;
A61P 25/00 20180101; C07D 221/14 20130101 |
Class at
Publication: |
514/296 ;
546/98 |
International
Class: |
A61K 31/473 20060101
A61K031/473; C07D 221/06 20060101 C07D221/06; A61P 25/00 20060101
A61P025/00 |
Claims
1-42. (canceled)
43. A compound having Formula 4: ##STR00022## or a pharmaceutically
acceptable salt thereof; wherein X is NO.sub.2; and R.sub.1 is
CH.sub.2(CH.sub.2).sub.3COOH.
44. The compound of claim 43, wherein the compound having Formula 4
is N-(4-carboxybutyl)-3-nitro-1,8-naphthalimide or
N-(4-carboxybutyl)-4-nitro-1,8-naphthalimide.
45. The compound N-(2-carboxyethyl)-4-nitro-1,8-naphthalimide.
46. The compound N-(3-carboxypropyl)-3-nitro-1,8-naphthalimide.
47. A method of treating Alzheimer's disease, epilepsy, multiple
sclerosis, amyotrophic lateral sclerosis, stroke or pain in a
patient, the method comprising the step of administering to the
patient a therapeutically effective amount of a compound having
Formula 4: ##STR00023## or a pharmaceutically acceptable salt
thereof; wherein X is 3-NO.sub.2 or 4-NO.sub.2; and R.sub.1 is
CH.sub.2(CH.sub.2).sub.2COOH, or CH.sub.2(CH.sub.2).sub.3COOH.
48. The method of claim 47, wherein X is 3-NO.sub.2 or
4-NO.sub.2.
49. The method of claim 47, wherein the compound having Formula 4
is N-(3-carboxypropyl)-3-nitro-1,8-naphthalimide;
N-(4-carboxybutyl)-3-nitro-1,8-naphthalimide;
N-(3-carboxypropyl)-4-nitro-1,8-naphthalimide; or
N-(4-carboxybutyl)-4-nitro-1,8-naphthalimide.
50. The method of claim 47, wherein the compound having Formula 4
is N-(3-carboxypropyl)-3-nitro-1,8-naphthalimide.
51. A method of treating Alzheimer's disease, epilepsy, multiple
sclerosis, amyotrophic lateral sclerosis, stroke or pain in a
patient, the method comprising the step of administering to the
patient a therapeutically effective amount of
N-(2-carboxyethyl)-4-nitro-1,8-naphthalimide.
52. A method of inhibiting the binding of nerve growth factor to
the p75.sup.NTR receptor, comprising contacting cells expressing
the p75.sup.NTR receptor with an effective inhibiting amount of a
compound having Formula 4: ##STR00024## or a pharmaceutically
acceptable salt thereof; wherein X is NO.sub.2; and R.sub.1 is
CH.sub.2(CH.sub.2).sub.2COOH, or CH.sub.2(CH.sub.2).sub.3COOH.
53. The method of claim 52, wherein X is 3-NO.sub.2 or
4-NO.sub.2.
54. The method of claim 52, wherein the compound having Formula 4
is N-(3-carboxypropyl)-3-nitro-1,8-naphthalimide;
N-(4-carboxybutyl)-3-nitro-1,8-naphthalimide;
N-(3-carboxypropyl)-4-nitro-1,8-naphthalimide; or
N-(4-carboxybutyl)-4-nitro-1,8-naphthalimide.
55. The method of claim 52, wherein the compound having Formula 4
is N-(3-carboxypropyl)-3-nitro-1,8-naphthalimide.
56. A method of inhibiting the binding of nerve growth factor to
the p75.sup.NTR receptor, comprising contacting cells expressing
the p75.sup.NTR receptor with an effective inhibiting amount
N-(2-carboxyethyl)-4-nitro-1,8-naphthalimide. Y, Y.sub.1, Y.sub.2,
and Y.sub.3 are each, independently, N, O, S, C-L or N-L, where L
is H, alkyl or an electronegative atom or functional group; Z and
Z.sub.1 are each, independently, O, S, CH, C(O), N, NH, N-alkyl,
N-cycloalkyl or N--P, where P is a carbohydrate moiety; T.sub.1 and
T.sub.2 are each, independently, an sp.sup.2- or
sp.sup.3-hybridized carbon or nitrogen atom; d, h and c are each 0
or 1; and R.sub.1 is a monocyclic or polycyclic aryl or heteroaryl
group group which is substituted with at least one substituent
selected from the group consisting of hydroxyl and sulfonamide or
an alkyl or alkylamino group substituted with a carboxyl or
carbonate group; and at least one pharmaceutically acceptable
carrier or excipient.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
09/310,883, filed May 17, 1999, the entire teachings of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The neurotrophins are a family of structurally and
functionally related proteins, including Nerve Growth Factor (NGF),
Brain-Derived Neurotrophic Factor (BDNT), Neurotrophin-3 (NT-3),
Neurotrophin-4/5 (NT-4/5) and Neurotrophin-6 (NT-6). These proteins
promote the survival and differentiation of diverse neuronal
populations in both the peripheral and central nervous systems
(Hefti, 1986; Hefti and Weiner, 1986; Levi-Montalcini, 1987; Barde,
1989; Leibrock et al., 1989; Maisonpierre et al. 1990; Rosenthal et
al., 1990; Hohn et al., 1990; Gotz et al., 1994; Maness et al.,
1994) and are involved in the pathogenesis of diverse neurological
disorders. Neurotrophins exert many of their biological effects
through specific interactions with a class of transmembrane
receptor tyrosine kinases (trkA, trkB and trkC) (Kaplan et al.,
1991; Klein et al. 1991, 1992; Soppet et al., 1991; Squinto et al.,
1991; Berkemeier et al., 1991; Escandon et al., 1993; Lamballe et
al., 1991). Specificity of neurotrophin action results from their
selective interactions with the trk receptors. That is, trkA only
binds NGF (Kaplan et al., 1991; Klein et al. 1991); trkB binds BDNF
and NT-4/5 (Soppet et al., 1991; Squinto et al., 1991; Berkemeier
et al., 1991; Escandon et al., 1993; Lamballe et al., 1991; Klein
et al., 1992; Vale and Shooter, 1985; Barbacid, 1993); and trkC
exclusively binds NT-3 (Lamballe et al., 1991; Vale and Shooter,
1985). This is particularly evident when the trk receptors are
coexpressed with the common neurotrophin receptor p75.sup.NTR (For
review see Meakin and Shooter, 199; Barbacid, 1993; Chao, 1994;
Bradshaw et al., 1994; Ibanez, 1995).
[0003] The common neurotrophin receptor p75.sup.NTR is a
transmembrane glycoprotein structurally related to the tumor
necrosis factor and CD-40 receptors (Meakin and Shooter, 1992;
Ryden and Ibanez, 1996). As all neurotrophins bind to p75.sup.NTR
with similar affinity (Rodrizuez-Tebar et al. 1990; Hallbook et
al., 1991; Rodriguez-Tebar et al., 1992; Ibanez, 1995),
neurotrophin specificity is conventionally thought to be caused by
the binding selectivity for trk receptors which are differentially
expressed in different neuronal populations (Ibanez, 1995).
However, accumulated experimental data on neurotrophin activity
reveal important functional aspects of p75.sup.NTR (Heldin et al.,
1989; Jing et al. 1992; Herrmann, 1993; Barker and Shooter, 1994;
Dobrowsky et al. 1994, Matsumoto et al. 1995; Marchetti et al.,
1996; Washiyama et al., 1996). The common neurotrophin receptor
enhances functions and increases binding specificity of trk
receptors (Barker and Shooter, 1994; Mahadeo et al., 1994; Chao and
Hempstead, 1995; Ryden and Ibanez, 1996). In addition, p75.sup.NTR
possesses unique, trk-independent signaling properties which
involve ceramide production through activation of the sphingomyelin
cycle (Dobrowsky, et al. 1994), apoptosis (cell death) (Van der Zee
et al. 1996; Cassacia-Bonnefil et al., 1996; Frade et al. 1996),
and activation of the transcription factor NF.kappa.B (Carter et
al., 1996). Recently, p75.sup.NTR has been demonstrated to
participate in human melanoma progression (Herrmann et al. 1993;
Marchetti et al., 1996). Furthermore. NGF and NT-3 increase the
production of heparin by 70 W melanoma cells, which is associated
with their metastatic potential (Marchetti et al., 1996). Although
this effect has been shown to be mediated by the common
neurotrophin receptor, neither BDNF nor NT-4/5 appeared to be
active.
[0004] Due to the implication of NGF/p75.sup.NTR binding in various
disease states, a need exists for pharmaceutical agents and methods
of use thereof for interfering with the binding of NGF to the
p75.sup.NTR common neurotrophin receptor.
SUMMARY OF THE INVENTION
[0005] The present invention relates to the discovery of molecular
structural features which contribute to the ability of a compound
to inhibit the binding of NGF to the common neurotrophin receptor
p75.sup.NTR. Compounds which have these features are of use, for
example, for inhibiting binding of NGF to p75.sup.NTR. Such
compounds can also be used to treat a patient having a condition
which is mediated, at least in part, by the binding of NGF to
p75.sup.NTR.
[0006] In one embodiment, the present invention relates to
compositions which inhibit the binding of nerve growth factor to
the p75.sup.NTR common neurotrophin receptor and methods of use
thereof.
[0007] In one embodiment, the compound which inhibits binding of
nerve growth factor to p75.sup.NTR comprises at least two of the
following: (1) a first electronegative atom or functional group
positioned to interact with Lys.sup.34 of nerve or growth factor;
(2) a second electronegative atom or functional group positioned to
interact with Lys.sup.95 of nerve growth factor; (3) a third
electronegative atom or functional group positioned to interact
with Lys.sup.88 of nerve growth factor; (4) a fourth
electronegative atom or functional group positioned to interact
with Lys.sup.32 of nerve growth factor; and (5) a hydrophobic
moiety which interacts with the hydrophobic region formed by amino
acid residues of nerve growth factor, including Ile.sup.31,
Phe.sup.101 and Phe.sup.86. Such inhibitors, preferably, bind nerve
growth factor via at least two of the foregoing interactions.
[0008] In one embodiment, compounds which inhibit binding of nerve
growth factor to p75.sup.NTR have Formula 1,
##STR00001##
In Formula 1. D.sub.1, D.sub.2, E.sub.1, E.sub.2 and G are each,
independently, an sp.sup.2-hybridized carbon or nitrogen atom. One
of X.sub.1 and X.sub.2 is a hydrogen atom or absent, while the
other is an electronegative atom or an electronegative functional
group. R and R.sub.2 are each, independently, an electronegative
atom or an electronegative functional group, such as O, S,
CH.sub.2, or NR.sup.3, where R.sub.3 is H, alkyl, preferably
C.sub.1-C.sub.6-alkyl, or aryl, such as phenyl. R, R.sub.2 and one
of X.sub.1 and X.sub.2 can also each be, independently, an
electronegative atom or functional group, such as alkylcarbonyl;
alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; --OH; --CN;
--CO.sub.2H; --SO.sub.3H; --SO.sub.2H; --PO.sub.3H.sub.2;
--NO.sub.2; --ONO.sub.2, --CNO, --SH, --CNS, --OSO.sub.3H,
--OC(O)(OH); halomethyl, dihalomethyl or trihalomethyl group or a
fluorine, chlorine, bromine or iodine atom. Y is N, O, S, C-L or
N-L, where L is H, alkyl, preferably C.sub.1-C.sub.6-alkyl, or an
electronegative atom or functional group, such as, but not limited
to, alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl;
aminocarbonyl; --OH; --CN; --CO.sub.2H; --SO.sub.3H; --SO.sub.2H;
--PO.sub.3H; --NO.sub.2; --ONO.sub.2, --CNO, --SH, --CNS,
--OSO.sub.3H, --OC(O)(OH); halomethyl, dihalomethyl or
trihalomethyl groups or a halogen atom, such as a fluorine,
chlorine, bromine or iodine atom. Z and Z.sub.1 are each,
independently, O, S, CH, C(O), N, NH, N-alkyl, N-cycloalkyl and
N--P, where P is a carbohydrate moiety, such as a monosaccharide
group, for example, a fucosyl, glucosyl, galactosyl, mannosyl,
fructosyl, gulosyl, idosyl, talosyl, allosyl, altrosyl, ribosyl,
arabinosl, xylosyl or lyxosyl group. T.sub.1 and T.sub.2 are each,
independently, an sp.sup.2- or sp.sup.3-hybridized carbon or
nitrogen atom. a, b, and c are each 0 or 1, provided that at least
one of b and c is 1. R.sub.1 is a monocyclic or polycyclic aryl or
heteroaryl, monosaccharide or oligosaccharide, alkyl, cycloalkyl,
arylalkyl, alkylamino or alkoxy group which is substituted with at
least one substituent selected from the group consisting of
electronegative atoms and electronegative functional groups.
[0009] It will be appreciated that in this and the following
structures, the lines connecting the variables can be single or
double bonds. In addition, hydrogen atoms are added to the
variables as necessary to complete the valence of the atom.
[0010] In another embodiment, compounds have Formula 3,
In another embodiment, the NGF/p75.sup.NTR binding inhibitor has
Formula 3
##STR00002##
where D.sub.1, D.sub.2, X.sub.1, X.sub.2, Y, E.sub.1, E.sub.2,
T.sub.1, T.sub.2, R, G, R.sub.1, R.sub.2, and c have the meanings
given above for these variables in Formula 1. Y.sub.1, Y.sub.2, and
Y.sub.3 are independently selected from the identities given for Y
in Formula 1. E.sub.3 and E.sub.4 are each, independently, an
sp.sup.2-hybridized carbon or nitrogen atom, and d and h are,
independently, 0 or 1.
[0011] In another embodiment, compounds which inhibit the binding
of nerve growth factor to p75.sup.NTR have Formula 2,
##STR00003##
In Formula 2, D.sub.1, D.sub.2, E.sub.1, E.sub.2, E.sub.3, E.sub.4
and G are each, independently, an sp.sup.2-hybridized carbon or
nitrogen atom. One of X.sub.1 and X.sub.2 is a hydrogen atom or
absent, while the other is an electronegative atom or an
electronegative functional group. R, R.sub.2 and R.sub.4 are each,
independently, an electronegative atom or an electronegative
functional group, such as O, S, CH.sub.2, or NR.sub.3, where
R.sub.3 is H, OH, alkyl, preferably C.sub.1-C.sub.6-alkyl, or aryl,
such as phenyl. R, R.sub.2 and one of X.sub.1 and X.sub.2 can also
each be, independently, an electronegative atom or functional
group, such as alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl;
aminocarbonyl; --OH; --CN; --CO.sub.2H; --SO.sub.3H; --SO.sub.2H;
--PO.sub.3H.sub.2; --NO.sub.2; --ONO.sub.2, --CNO, --SH, --CNS,
--OSO.sub.3H, --OC(O)(OH); halomethyl, dihalomethyl or
trihalomethyl group or a fluorine, chlorine, bromine or iodine
atom. Y is N, O, S, C-L or N-L, where L is H, alkyl preferably
C.sub.1-C.sub.6-alkyl, or an electronegative atom or functional
group, such as, but not limited to, alkylcarbonyl;
alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; --OH; --CN;
--CO.sub.2H; --SO.sub.3H; --SO.sub.2H; --PO.sub.3H; --NO.sub.2;
--ONO.sub.2, --CNO, --SH, --CNS, --OSO.sub.3H, --OC(O)(OH);
halomethyl, dihalomethyl or trihalomethyl groups or a halogen atom,
such as a fluorine, chlorine, bromine or iodine atom. Z and Z.sub.1
are each, independently, O, S, CH, C(O), N, NH, N-alkyl,
N-cycloalkyl and N--P, where P is a carbohydrate moiety, such as a
monosaccharide group, for example, a fucosyl, glucosyl, galactosyl,
mannosyl, fructosyl, gulosyl, idosyl, talosyl, allosyl, altrosyl,
ribosyl, arabinosyl, xylosyl or lyxosyl group. T.sub.1, T.sub.2 and
T.sub.3 are each, independently, an sp.sup.2- or
sp.sup.3-hybridized carbon or nitrogen atom. When f is 0, T.sub.3
can further have the meanings given for Z and Z.sub.1, above a, b,
c, d, e, f, g, h and i are each 0 or 1, provided that at least one
of b and c is 1, at least one of d and e is 1 and at least one of f
and i is 1. R.sub.1 is a monocyclic or polycyclic aryl or
heteroaryl, monosaccharide or oligosaccharide, alkyl, cycloalkyl,
arylalkyl, alkylamine or alkoxy group which is substituted with at
least one substituent selected from the group consisting of
electronegative atoms and electronegative functional groups.
[0012] In another embodiment, a compound which inhibits the binding
of NGF to p75.sup.NTR has Formula 5,
##STR00004##
wherein D.sub.1, D.sub.2, X.sub.1, X.sub.2, E.sub.1, E.sub.2,
E.sub.3, T.sub.1, T.sub.2, T.sub.3, Z, G, R.sub.1, R.sub.2,
R.sub.4, b, e, f, i, and c have the meanings given for these
variables in Formula 2, Y.sub.1, Y.sub.2, and Y.sub.3 are
independently selected from the identities given for Y in Formula
2, and h is 0 or 1. E.sub.5 and E.sub.6 are each, independently, an
sp.sup.2-hybridized carbon or nitrogen atom, and g is 0 or 1. Ring
4 can be further unsubstituted or substituted with one or more
substituents, such as alkyl or aryl groups.
[0013] In another embodiment, the invention provides a
pharmaceutical composition comprising at least one compound of the
invention, or pharmaceutically acceptable salt thereof, in
combination with a pharmaceutically acceptable carrier or
excipient.
[0014] The invention also provides a method of inhibiting the
binding of nerve growth factor to the p75.sup.NTR receptor. The
method comprises contacting cells which express the p75.sup.NTR
receptor with a nerve growth factor/p75.sup.NTR binding inhibitor
of the invention in an amount which is sufficient to inhibit
binding of nerve growth factor to the p75.sup.NTR receptor. The
method can be practiced in vivo or in vitro.
[0015] In another embodiment, the invention relates to a method of
treating a condition in a patient which is mediated by the binding
of nerve growth factor to the p75.sup.NTR receptor. The method
comprises administering to the patient a therapeutically effective
amount of a nerve growth factor/p75.sup.NTR binding inhibitor of
the invention. Preferably, the compound to be administered
selectively inhibits the binding of nerve growth factor to
p75.sup.NTR in cells which do not express the NGF receptor
trkA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates examples of suitable configurations for
electronegative atoms in the NGF/p75.sup.NTR binding inhibitors of
the invention.
[0017] FIG. 2 illustrates examples of electronegative functional
groups.
[0018] FIG. 3 sets forth a synthetic pathway for certain compounds
of the invention: Pg=protecting group.
[0019] FIG. 4 sets forth a synthetic pathway for certain compounds
of the invention.
[0020] FIG. 5 sets forth a synthetic pathway for certain compounds
of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Nerve growth factor (also referred to hereinafter as "NGF")
is a neurotrophin implicated in the pathogenesis of Alzheimer's
disease, epilepsy and pain (Ben and Represa, 1990; McKee et al.
1991; Leven and Mendel, 1993; Woolf and Doubell, 1994; Rashid et
al. 1995; McMahon et al., 1995). The binding of NGF to its
receptors is determined by distinct sequences within its primary,
amino acid structure. While several regions of NGF participate in
the NGF/trkA interaction, mutation studies suggest that relatively
few key residues, namely those located in the NGF amino and
carboxyl termini, are required for high affinity binding.
[0022] NGF displays high and low affinity binding sites in sensor,
and sympathetic neurons and in pheochromocytoma PC12 cells (Sutter
et al., 1979; Landreth and Shooter, 1980; Schechter and Bothwell,
1981). The coexpression of the common neurotrophin p75.sup.NTR
receptor with trkA is required to form the high affinity binding
site (Hempstead et al., 1991; Barker and Shooter, 1994; Mahadeo et
al., 1994; Chao and Hempstead, 1995). Several models of the
trkA-p75.sup.NTR interaction have been proposed to explain high
affinity NGF binding (Bothwell, 1991; Chao, 1992b; Chao and
Hempstead, 1995; Wolf et al., 1995; Ross et al., 1996; Ross et al.,
1997). These models differ with respect to direct (conformational
model) or indirect (ligand-presentation model) interaction of
p75.sup.NTR with trkA. Direct trkA-p75.sup.NTR interaction is
consistent with much of the existing experimental data.
[0023] The hairpin loop at residues 29-35 of NGF is responsible for
recognition by p75.sup.NTR (Ibanez et al., 1992; Radziejewski et
al. 1992), while the amino and carboxyl termini are important
binding determinants for recognition by the trkA receptor (Shih et
al., 1994; Moore and Shooter, 1975: Suter et al., 1992; Burton et
al., 1992; Kahle et al., 1992; Luo and Neet, 1992; Drinkwater et
al., 1993; Treanor et al., 1995; Taylor et al. 1991; Shamovsky et
al., 1998; Shamovsky et al., 1999; WO 98/06048). Truncation of
either the amino or carboxyl terminus of NGF produces less active
NGF analogues; similarly most deletion or point mutations of the
amino terminus also lead to NGF analogues with diminished activity
(Shih et al., 1994; Burton et al., 1992, 1995; Kahle et al., 1992;
Drinkwater et al., 1993; Treanor et al., 1995; Taylor et al.,
1991). On the other hand, the NGF.DELTA.2-8 (NGF with residues 2-8
removed) and NGF.DELTA.3-9 deletion mutants are almost as active as
wild type NGF (Drinkwater et al., 1993). These NGF
structure-activity relationships in combination with the
considerable species variability (mouse, human, guinea pig and
snake) of the amino acid sequence of the NGF termini (McDonald et
al., 1991) are of potential value in understanding the NGF/trkA
interaction.
[0024] NGF exerts its biological activity as a non-covalent dimer
(Treanor et al., 1995; Burton et al., 1995; McDonald et al., 1991;
Ibanez et al., 1993; Bothwell and Shooter, 1977). Two 118 residue
NGF monomers are dimerized by hydrophobic and van der Waals
interactions between their three anti-parallel pairs of
.beta.-strands; consequently, the amino terminus of one NGF monomer
and the carboxyl terminus of the other are spatially juxtaposed
(McDonald et al., 1991). Furthermore, although a dimer has 2 pairs
of termini, only one pair of termini is required for trkA receptor
recognition (Treanor et al., 1995; Burton et al., 1995).
[0025] The X-ray crystallographic 3-dimensional structure of a
dimeric mouse NGF (mNGF) has been reported (McDonald et al., 1991).
However, within this structure, the amino terminus (residues 1-11)
and the carboxyl terminus (residues 112-118) remain unresolved for
both pairs of termini. High flexibility of the NGF termini makes it
difficult to experimentally determine their bioactive
conformations, particularly since transition metal ions commonly
used in X-ray crystallography (McDonald et al., 1991) have high
affinity for His residues (Gregory et al., 1993) which are present
in the NGF amino terminus (Bradshaw et al. 1994). Indeed,
conformational alterations in the receptor binding domains of NGF
caused by Zn.sup.2+ cations leading to its inactivation have been
described recently (Ross et al., 1997). Since the amino and
carboxyl termini are crucial for NGF bioactivity as mediated via
trkA and because of the significance of NGF in multiple neurologic
disease processes, the determination of the biologically active
conformation of these termini is an important and challenging
problem for computational chemistry.
[0026] The present invention relates to the discovery of molecular
structural features which contribute to the ability of a compound
to inhibit the binding of NGF to the common neurotrophin receptor
p75.sup.NTR. Compounds which have these features are of use, for
example, for inhibiting binding of NGF to p75.sup.NTR Such
compounds can also be used to treat a patient having a condition
which is mediated, at least in part, by the binding of NGF to
p75.sup.NTR.
[0027] Certain compounds which inhibit the binding of NGF to
p75.sup.NTR are disclosed in copending U.S. patent application Ser.
No. 09/292,450, incorporated herein by reference in its
entirety.
[0028] In one embodiment, the present invention provides compounds
which inhibit the binding of nerve growth factor (NGF) to the
p75.sup.NTR receptor. The compounds have at least two of the
following characteristics: (1) a first electronegative atom or
functional group positioned to interact with Lys.sup.34 of NGF; (2)
a second electronegative atom or functional group positioned to
interact with Lys.sup.95 of NGF; (3) a third electronegative atom
positioned to interact with Lys.sup.88 of NGF; (4) a fourth
electronegative atom or functional group positioned to interact
with Lys.sup.32 of NGF; and (5) a hydrophobic moiety which
interacts with the hydrophobic region formed by Ile.sup.31,
Phe.sup.101 and Phe.sup.86 of NGF. A compound having two or more of
these structural attributes is referred to herein as an
"NGF/p75.sup.NTR binding inhibitor". Preferably, the
NGF/p75.sup.NTR binding inhibitor has at least three of the
foregoing attributes when bound to NGF, more preferably at least
four such attributes. Most preferably, the NGF/p75.sup.NTR binding
inhibitor has each of the five foregoing attributes. Typically, an
NGF/p75.sup.NTR binding inhibitor of the invention interacts with
NGF via at least two of the foregoing interactions when bound to
NGF.
[0029] The term "electronegative atom", as used herein, refers to
an atom which carries a partial or full negative charge in a
particular compound under physiological conditions. The
electronegative atom can be, for example, an oxygen atom, a
nitrogen atom, a sulfur atom or a halogen atom, such as a fluorine,
chlorine, bromine or iodine atom. Preferably the electronegative
atom is an oxygen atom. The term "electronegative functional
group", as used herein, refers to a functional Croup which includes
at least one electronegative atom. Electronegative groups include
acid functional groups and other polar functional groups. For
example, suitable electronegative functional groups include, but
are not limited to, carbonyl, thiocarbonyl, ester, imino, amido
carboxylic acid, sulfonic acid, sulfinic acid, sulfamic acid,
phosphonic acid, boronic acid, sulfate ester, hydroxyl, mercapto,
cyano, cyanate, thiocyanate, isocyanate, isothiocyanate, carbonate,
nitrate and nitro groups. It is to be understood that, unless
otherwise indicated, reference herein to an acidic functional group
also encompasses salts of that functional group in combination with
a suitable cation.
[0030] An electronegative atom of the NGF/p75.sup.NTR binding
inhibitor bears a full or partial negative charge under
physiological conditions and can, therefore, interact
electrostatically with the positively charged side chain of an NGF
lysine residue. This will be an interaction, such as, for example,
a hydrogen bond, an ion/ion interaction, an ion/dipole interaction
or a dipole/dipole interaction. The hydrophobic region or moiety of
the NGF/p75.sup.NTR binding inhibitor can interact with a
hydrophobic region of NGF via a hydrophobic interaction. Without
being bound by theory, it is believed that compounds having the
disclosed structural features can interact with NGF in such a way
as to interfere with, and thereby inhibit, the binding of NGF to
p75.sup.NTR.
[0031] The ability of a compound to interact with the amino acid
residues of NGF specified above can be determined using a
structural model of NGF obtained using a energy-minimization
algorithm, as described in published PCT application WO 98/06048,
incorporated herein by reference in its entirety. For example, a
molecule will interact with the specified residues of NGF, as
discussed above, if it has at least 3 electronegative atoms (A, B
and C) such that at least one of the following two conditions is
satisfied: (i) atoms A and B are separated by 5-7 covalent bonds, B
and C are separated by 6-8 covalent bonds, and A and C are
separated by 10-14 covalent bonds and (ii) distance between A and B
is between 4.5 and 7.5 angstroms, and distance between B and C is
between 4.5 and 7.5 angstroms. The number of covalent bonds
separating atoms can be determined from the structural formula of a
molecule. Distance between atoms can be determined experimentally
(e.g., by X-ray crystallography, or NMR spectroscopy) or evaluated
theoretically using any molecular builder (e.g. SYBYL from Tripos
Inc. (St. Louis, Mo., USA) or QUANTA from Molecular Simulations
Inc. (San Diego, Calif., USA) as well as any molecular modeling
technique (e.g. AMBER from Oxford Molecular Group Inc./University
of California. San Francisco or CHARMm from Molecular Simulations
Inc.) or quantum chemical technique (e.g., MNDO from Oxford
Molecular Group Inc. (Campbell, Calif., USA) University of Zurich;
AMPAC from Semichem (Kansas City, Mo., USA); CADPAC from Oxford
Molecular Group Inc./Cambridge University; Gaussian-98 from
Gaussian Inc. (Carnegie, Pa., USA); or GAMESS from Iowa State
University). Examples of suitable configurations of groups A, B and
C are illustrated in FIG. 1, while a representative group of
electronegative functional groups is shown in FIG. 2.
[0032] Preferred NGF/p75.sup.NTR inhibitors of the invention
comprise a molecular scaffold or framework, to which the
electronegative atoms or functional groups are attached, either
directly or via an intervening moiety. The scaffold can be, for
example, a cyclic or polycyclic moiety, such as a monocyclic,
bicyclic or tricyclic moiety, and can include one or more
hydrocarbyl or heterocyclic rings. Preferably, the scaffold
includes two or more fused, planar, five- or six-membered rings.
The molecular scaffold presents the attached electronegative atoms,
electronegative functional groups or a combination thereof, in the
proper configuration or orientation for interaction with the
appropriate residues of NGF. In addition, the molecular scaffold,
such as polycyclic system, or a portion thereof, can serve as the
hydrophobic group which interacts with hydrophobic residues of NGF,
as described above.
[0033] In one embodiment, the NGF/p75.sup.NTR inhibitor is of
general Formula 1,
##STR00005##
In Formula 1, D.sub.1, D.sub.2, E.sub.1, E.sub.2 and G are each,
independently, an sp.sup.2-hybridized carbon or nitrogen atom. One
of X.sub.1 and X.sub.2 is a hydrogen atom or absent, while the
other is an electronegative atom or an electronegative functional
group. R and R.sub.2 are each, independently, an electronegative
atom or an electronegative functional group, such as O, S,
CH.sub.2, or NR.sub.3, where R.sub.3 is H, alkyl, preferably
C.sub.1-C.sub.6-alkyl, or aryl, such as phenyl. R, R.sub.2 and one
of X.sub.1 and X.sub.2 can also each be, independently, an
electronegative atom or functional group, such as alkylcarbonyl;
alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; --OH; --CN;
--CO.sub.2H; --SO.sub.3H; --SO.sub.2H; --PO.sub.3H.sub.2;
--NO.sub.2; --ONO.sub.2, --CNO, --SH, --CNS, --OSO.sub.3H,
--OC(O)(OH); halomethyl, dihalomethyl or trihalomethyl group or a
fluorine, chlorine, bromine or iodine atom. Y is N, O, S, C-L or
N-L, where L is H, alkyl, preferably C.sub.1-C.sub.6-alkyl, or an
electronegative atom or functional group, such as, but not limited
to, alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl;
aminocarbonyl; --OH; --CN; --CO.sub.2H; --SO.sub.3H; --SO.sub.3H;
--PO.sub.3H.sub.2; --NO.sub.2, --ONO.sub.2, --CNO, --SH, --CNS,
--OSO.sub.3H, --OC(O)(OH); halomethyl, dihalomethyl or
trihalomethyl groups or a halogen atom, such as a fluorine,
chlorine, bromine or iodine atom. Z and Z.sub.1 are each,
independently, O, S, CH, C(O), N, NH, N-alkyl, N-cycloalkyl and
N--P, where P is a carbohydrate moiety, such as a monosaccharide
group, for example, a fucosyl, glucosyl, galactosyl, mannosyl,
fructosyl, gulosyl, idosyl, talosyl, allosyl, altrosyl, ribosyl,
arabinosyl, xylosyl or lyxosyl group. T.sub.1 and T.sub.2 are each,
independently, an sp.sup.2- or sp.sup.3-hybridized carbon or
nitrogen atom. a, b and c are each 0 or 1, provided that at least
one of b and c is 1.
[0034] R.sub.1 is a monocyclic or polycyclic aryl or heteroaryl,
mono- or oligosaccharide, alkyl, cycloalkyl, arylalkyl, alkylamino
or alkoxy group which is substituted with at least one substituent
selected from the group consisting of electronegative atoms and
electronegative functional groups. Preferred monosaccharide groups
include fucosyl, glucosyl, galactosyl, mannosyl, fructosyl, gulosyl
idosyl, talosyl, allosyl, altrosyl, ribosyl, arabinosyl, xylosyl
and lyxosyl groups. The electronegative substituent can be bonded
to the aryl or heteroaryl ring system alkyl, cycloalkyl, or oligo-
or monosaccharide group either directly or indirectly via a
bridging group, for example, an alkylene group such as a
C.sub.1-C.sub.4-alkylene group or an oxyalkylene group. Suitable
directly bonded and alkylene bridged electronegative atoms and
functional groups include, but are not limited to, alkylcarbonyl;
alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; --OH; --CN;
--CO.sub.2H; --SO.sub.3H; --SO.sub.2H; --PO.sub.3H.sub.2;
--NO.sub.2; --ONO.sub.2, --CNO, --SH, --CNS, --OSO.sub.3H;
--OC(O)(OH): carboxyalkyl, nitroalkyl, --N,N-dialkylaminosulfonyl,
aminocarbonyl, alkoxycarbonyl, alkoxycarbonylalkyl,
cyanocarbonylalkyl, haloalkyl, such as fluoromethyl,
difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl or
trichloromethyl; alkylamido or a halogen atom, such as a fluorine,
chlorine, bromine or iodine atom. In one embodiment, R.sub.1 is
selected from the group consisting of groups including, but not
limited to, --(CH.sub.2).sub.aCOOH; --(CH.sub.2).sub.aNO.sub.2;
--(CH.sub.2).sub.aOH; --(CH.sub.2).sub.aPO.sub.3H.sub.2;
--(CH.sub.2).sub.aSO.sub.3H; --(CH.sub.2).sub.aSO.sub.2H;
--R.sub.4(CH.sub.2).sub.aCOOH; --R.sub.4(CH.sub.2).sub.aNO;
--R.sub.4(CH.sub.2).sub.aPO.sub.3H.sub.2;
--R.sub.4(CH.sub.2).sub.aSO.sub.2H;
--R.sub.4(CH.sub.2).sub.aSO.sub.3H; and
--R.sub.4(CH.sub.2).sub.aOH, where a is 1 to 12, preferably 1 to
about 4, and R.sub.4 is NH or O.
[0035] Rings 1 and 2 are each, independently, a five- or
six-membered ring and, preferably, are both planar.
[0036] It, is to be understood that compounds of Formula 1 and
Formulas 2, 3 and 5, below, will further include double bonds
between adjacent atoms as required to satisfy the valence of each
atom. That is, double bonds are added to provide the following
number of total bonds to each of the following types of atoms:
carbon: four bonds; nitrogen: 3 bonds; oxygen: two bonds; and
sulfur: two bonds.
[0037] The term "alkyl", as used herein, refers to a normal,
branched or cyclic aliphatic hydrocarbyl group, which can be
saturated or partially unsaturated. Preferred alkyl groups are
normal, branched and cyclic C.sub.1-C.sub.8-alkyl and -alkenyl
groups.
[0038] In another embodiment, the NGF/p75.sup.NTR binding inhibitor
of Formula 3
##STR00006##
where D.sub.1, D.sub.2, X.sub.1, X.sub.2, Y, E.sub.1, E.sub.2,
T.sub.1, T.sub.2, R, G, R.sub.1, R.sub.2, and c have the meanings
given above for these variables in Formula 1. Y.sub.1, Y.sub.2, and
Y.sub.3 are independently selected from the identities liven for Y
in Formula 1. E.sub.3 and E.sub.4 are each, independently, an
sp.sup.2-hybridized carbon or nitrogen atom, and d and h are each,
independently, 0 or 1.
[0039] In one embodiment of the compounds of Formula 3, R.sub.1 is
a mono- or polycyclic aryl or heteroaryl, oligo- or monosaccharide
group which is substituted with at least one electronegative atom
or electronegative group. The mono- or polycyclic aryl or
heteroaryl group is preferably substituted with an acid functional
group, such as --CO.sub.2H; --SO.sub.3H; --SO.sub.2H;
--PO.sub.3H.sub.2; --OSO.sub.3H; HOC(O)-alkyl; HOS(O).sub.2-alkyl;
HOS(O)-alkyl; (OH).sub.2P(O)-alkyl; and HOS(O).sub.2O-alkyl; where
the alkyl group is preferably a C.sub.1-C.sub.4-alkyl group. In
another embodiment, the electronegative atom or electronegative
functional group is selected from the group consisting of
alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl: --CN; --NO.sub.2;
--ONO.sub.2, --CNO, --SH, --CNS, nitroalkyl,
N,N-dialkylaminosulfonyl, aminocarbonyl, alkoxycarbonyl,
alkoxycarbonylalkyl, cyanocarbonylalkyl, fluoromethyl,
difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl,
trichloromethyl, acetamido and halogen atoms. R.sub.1 can also be
an alkylamino, alkyl or alkoxy group which is substituted with at
least one electronegative atom or functional group. For example, in
one embodiment, R.sub.1 is selected from the group consisting of
--(CH.sub.2).sub.aNO.sub.2; --(CH.sub.2).sub.aOH;
--(CH.sub.2).sub.aPO.sub.3H.sub.2; --(CH.sub.2).sub.aSO.sub.3H;
--(CH.sub.2).sub.aSO.sub.2H; --O(CH.sub.2).sub.aCOOH;
--O(CH.sub.2).sub.aNO.sub.2; --O(CH.sub.2).sub.aPO.sub.3H.sub.2;
--O(CH.sub.2).sub.aSO.sub.2H; --O(CH.sub.2).sub.aSO.sub.3H;
--O(CH.sub.2).sub.aOH; --NH(CH.sub.2).sub.aCOOH;
--NH(CH.sub.2).sub.aNO.sub.2; --NH(CH.sub.2).sub.aPO.sub.3H.sub.2;
--NH(CH.sub.2).sub.aSO.sub.2H; and NH(CH.sub.2).sub.aSO.sub.3H;
where a is 1 to 12, preferably 1 to about 4.
[0040] In another embodiment of the compounds of Formula 3, R.sub.1
is a phenyl group which is substituted by p-toluenesulfonamido or
hydroxyl; or R.sub.1 is a --NH(CH.sub.2).sub.aOH group, where a is
1 to about 4; a carboxyalkyl group, for example, a linear or
branched carboxy-C.sub.1-C.sub.8-alkyl group; an alkoxycarbonyl
group, such as a linear or branched C.sub.1-C.sub.8-alkoxycarbonyl
group or an alkylcarbonate group, such as a linear or branched
C.sub.1-C.sub.8-alkylcarbonate group. In this embodiment, ring atom
is an sp2-hybridized carbon atom, except for G, which is a nitrogen
atom; R and R.sub.2 are both O; and d, c and h are each 1.
[0041] Preferred compounds of Formula 3 are of the formula
##STR00007##
where X and R.sub.1 have the meanings given above for these
variables in Formula 1, R.sub.2 is O, CH, or NR.sub.3, where
R.sub.3 is H, alkyl, preferably C.sub.1-C.sub.6-alkyl or aryl and
ring 1 and 4 can, optionally independently be further substituted.
Suitable substituents include alkyl groups, preferably normal or
branched C.sub.1-C.sub.6-alkyl groups.
[0042] In another embodiment, the NGF/p75.sup.NTR binding inhibitor
is of Formula 2.
##STR00008##
In Formula 2, D.sub.1, D.sub.2, E.sub.1, E.sub.2, E.sub.3, E.sub.4
and G are each, independently, an sp.sup.2-hybridized carbon or
nitrogen atom. One of X.sub.1 and X.sub.2 is a hydrogen atom or
absent, while the other is an electronegative atom or an
electronegative functional group. R, R.sub.2 and R.sub.4 are each,
independently, an electronegative atom or an electronegative
functional group, such as O, S, CH.sub.2, or NR.sub.3, where
R.sub.3 is H, alkyl, preferably C.sub.1-C.sub.6-alkyl, or aryl,
such as phenyl. R, R.sub.2 and one of X.sub.1 and X.sub.2 can also
each be, independently, an electronegative atom or functional
group, such as alkylcarbonyl; alkylthiocarbonyl; alkoxycarbonyl;
aminocarbonyl; --OH; --CN; --CO.sub.2H; --SO.sub.3H; --SO.sub.2H;
--PO.sub.3H.sub.2; --NO.sub.2; --ONO.sub.2, --CNO, --SH, --CNS,
--OSO.sub.3H, --OC(O)(OH); halomethyl, dihalomethyl or
trihalomethyl group or a fluorine, chlorine, bromine or iodine
atom. Y is N, O, S, C-L or N-L, where L is H, alkyl, preferably
C.sub.1-C.sub.6-alkyl, or an electronegative atom or functional
group, such as, but not limited to, alkylcarbonyl;
alkylthiocarbonyl; alkoxycarbonyl; aminocarbonyl; --OH; --CN;
--CO.sub.2H; --SO.sub.2H; --SO.sub.2H; --PO.sub.3H.sub.2;
--NO.sub.2; --ONO.sub.2, --CNO, --SH, --CNS, --OSO.sub.3H,
--OC(O)(OH); halomethyl, dihalomethyl or trihalomethyl groups or a
halogen atom, such as a fluorine, chlorine, bromine or iodine atom.
Z and Z.sub.1 are each, independently, O, S, CH, C.dbd.O, N, NH,
N-alkyl, N-cycloalkyl and N--P, where P is a carbohydrate moiety,
such as a monosaccharide group, for example, a fucosyl, glucosyl,
galactosyl, mannosyl, fructosyl, gulosyl, idosyl, talosyl, allosyl,
altrosyl, ribosyl, arabinosyl, xylosyl or lyxosyl group. T.sub.1,
T.sub.2 and T.sub.3 are each, independently, an sp.sup.2- or
sp.sup.3-hybridized carbon or nitrogen atom. When f is 0, T.sub.3
can further have the meanings given for Z and Z.sub.1, above, a, b,
c, d, e, f and i are each 0 or 1, provided that at least one of b
and c is 1; at least one of d and e is 1 and at least one of f and
i is 1.
[0043] R.sub.1 is a monocyclic or polycyclic aryl or heteroaryl,
oligo- or monosaccharide, alkyl, cycloalkyl, arylalkyl alkylamino
or alkoxy group which is substituted with at least one substituent
selected from the group consisting of electronegative atoms and
electronegative functional groups. Preferred monosaccharide groups
include fucosyl, glucosyl, galactosyl, mannosyl, fructosyl,
gulosyl, idosyl, talosyl, allosyl, altrosyl, ribosyl, arabinosyl,
xylosyl and lyxosyl groups. The electronegative substituent can be
bonded to the aryl or heteroaryl ring system, or monosaccharide
group either directly or indirectly via a bridging group, for
example, an alkylene group such as a C.sub.1-C.sub.4-alkylene group
or an oxyalkylene group. Suitable directly bonded and alkylene
bridged electronegative atoms and functional groups include, but
are not limited to, alkylcarbonyl; alkylthiocarbonyl:
alkoxycarbonyl; aminocarbonyl; --OH; --CN; --CO.sub.2H;
--SO.sub.3H; --SO.sub.2H; --PO.sub.3H.sub.2; --NO.sub.2;
--ONO.sub.2, --CNO, --SH, --CNS, --OSO.sub.3H; --OC(O)(OH);
carboxyalkyl, nitroalkyl, N,N-dialkylaminosulfonyl, aminocarbonyl,
alkoxycarbonyl, alkoxycarbonylalkyl, cyanocarbonylalkyl, haloalkyl,
such as fluoromethyl, difluoromethyl, trifluoromethyl,
chloromethyl, dichloromethyl or trichloromethyl; alkyamido or a
halogen atom, such as a fluorine, chlorine, bromine or iodine atom.
In one embodiment, R.sub.1 is selected from the group consisting of
groups including, but not limited to, --(CH.sub.2).sub.2COOH;
--(CH.sub.2).sub.aNO.sub.2; --(CH.sub.2).sub.aOH;
--(CH.sub.1).sub.aPO.sub.3H.sub.2; --(CH.sub.2).sub.aSO.sub.3H;
--(CH.sub.2).sub.aSO.sub.3H; --R.sub.4(CH.sub.2).sub.aCOOH;
--R.sub.4(CH.sub.2).sub.aNO.sub.2;
--R.sub.4(CH.sub.2).sub.aPO.sub.3H.sub.2;
--R.sub.4(CH.sub.2).sub.aSO.sub.2H;
--R.sub.4(CH.sub.2).sub.aSO.sub.3H; and
--R.sub.4(CH.sub.2).sub.aOH, where a is 1 to 12, preferably 1 to
about 4, and R.sub.4 is NH or O.
[0044] Rings 1, 2 and 3 are each, independently, a five- or
six-membered ring and, preferably, are each planar.
[0045] In another embodiment, the compound is of Formula 5,
##STR00009##
wherein D.sub.1, D.sub.2, X.sub.1, X.sub.2, E.sub.1, E.sub.2,
E.sub.3, T.sub.1, T.sub.2, T.sub.3, Z, G, R, R.sub.1, R.sub.2,
R.sub.4, b, c, e, f and i have the meanings given for these
variables in Formula 2. Y.sub.1, Y.sub.2, and Y.sub.3 are
independently selected from the identities given for Y in Formula
2, and g and h are each, independently, 0 or 1. E.sub.5 and E.sub.6
are each, independently, an sp.sup.2-hybridized carbon or nitrogen
atom, and g is 0 or 1. Ring 4 can be further unsubstituted or
substituted with one or more substituents, such as alkyl or aryl
groups.
[0046] In one embodiment of the compounds of Formulas 1, 2, 3 and
5, R.sub.1 is selected from the group consisting of substituted
phenylene, naphthylene, quinolylene and other substituted aromatic
and heteroaromatic groups. R.sub.1, can also be a substituted
ethynyl or poly(ethynyl) group. Suitable identities for R.sub.1
include, but are not limited to, the groups shown below.
##STR00010## ##STR00011##
In each of these groups, J can be any of the electronegative atoms
or groups described in the definition of R.sub.1 in Formulas 1 and
2. Preferably, J is selected from the group consisting of --OH,
--CN--, --NO, --CO.sub.2H, --SO.sub.3H, --SO.sub.2H, --F, --Cl,
--B, --I, --PO.sub.3H.sub.2, --CF.sub.3,
--SO.sub.2N(CH.sub.3).sub.2, --C(O)NH.sub.2, --C(O)CH.sub.3,
--C(O)OCH.sub.3, --C(O)CN, --CH.sub.2F, --CH.sub.2Cl, --CF.sub.2H,
--CCl.sub.2H, --CCl.sub.3 and --NHC(O)CH.sub.3; R.sub.4 is NH or O,
and n is an integer from 0 to about 6.
[0047] Preferred compounds of Formula 1 are represented by Formulas
6-14, 16-18, 21-30 and 32-34, below. Preferred compounds of Formula
3 are represented by Formulas 15, 19, 20 and 31 below.
##STR00012## ##STR00013## ##STR00014##
In each of Formulas 6-34, R.sub.1, X and Y have the meanings given
above for these variables in Formula 1. In Formulas 6, and 9-15, Z
is selected from the group consisting of O, S, NH, N-alkyl,
N-cycloalkyl and N--P, wherein P is a carbohydrate moiety,
preferably a monosaccharide moiety, such as a fucosyl, glucosyl,
galactosyl, mannosyl, fructosyl, gulosyl, idosyl, talosyl, allosyl,
altrosyl, ribosyl, arabinosyl, xylosyl or lyxosyl group. In
Formulas 6, 7, 9, 10 and 12-17, R.sub.2 is selected from the group
consisting of O, S, CH, and NR.sub.3, wherein R.sub.3 is H, OH,
aryl or alkyl.
[0048] Preferred compounds of Formulas 2 and 5 are of Formulas
35-49 below.
##STR00015## ##STR00016##
In Formulas 32-46, the structural variables X, R.sub.1, R.sub.2, Z
and Y each have the identities given previously for Formula 2.
[0049] In another embodiment, the NGF/p75.sup.NTR binding inhibitor
is of general formula 50.
##STR00017##
In Formula 50, the structural variables D.sub.1, D.sub.2, X.sub.1,
X.sub.2, E.sub.1, E.sub.2, E.sub.3, T.sub.1, T.sub.2, T.sub.3, Z,
G, R, R.sub.1, R.sub.2, R.sub.4, b, and c have the meanings given
for these variables in Formula 2. T.sub.3 is an sp.sup.2- or
sp.sup.3-hybridized carbon or nitrogen atom, and is preferably an
sp.sup.2-hybridized carbon or nitrogen atom.
[0050] A preferred subset of compounds of Formula 3 is represented
by Formula 51,
##STR00018##
In Formula 51, X, Y and R.sub.1 each have the meanings given for
these variables in Formula 1. R.sub.2 is O, S, CH, or N--R.sub.3,
wherein R.sub.3 is H, OH, alkyl, preferably normal or branched
C.sub.1-C.sub.6-alkyl, or aryl, such as phenyl or substituted
phenyl.
[0051] In a preferred embodiment, the NGF/p75.sup.NTR inhibitor
exhibits greater NGF/p75.sup.NTR binding inhibition in cells which
express p75.sup.NTR but not trkA than in cells which express both
p75.sup.NTR and trkA. The binding of NGF to p75.sup.NTR in cells
which do not express trkA can, under certain conditions, mediate
apoptotic cell death. The p75.sup.NTR receptor has a greater
affinity for NGF in this proapoptotic state, that is, in cells
which do not express triad. Compounds which exhibit greater
NGF/p75.sup.NTR binding inhibitor in the absence of trkA
advantageously selectively inhibit or interfere with processes such
as apoptotic cell death, while having a smaller effect on other
p75.sup.NTR-mediated processes.
[0052] Preferred compounds which selectively inhibit the binding of
NGF to p75.sup.NTR in cells which do not express trkA include
compounds of Formulas 52 and 53, below.
##STR00019##
In Formulas 52 and 53, Q is selected from the group consisting of
C.sub.1-C.sub.5-alkylene; para- and meta-phenylene; cycloalkylene,
carbohydrate and para- and meta-CH.sub.2C.sub.6H.sub.2--. In
Formulas 51 and 52, R.sub.5, R.sub.6 and R.sub.7 are, preferably,
each, independently, H, --COOH or --NO.sub.2. More preferably, two
of R.sub.5, R.sub.6 and R.sub.7 are H and the other is --COOH or
--NO.sub.2.
[0053] The present invention also relates to a method of inhibiting
the binding of NGF to p75.sup.NTR. The method comprises contacting
NGF in the presence of p75.sup.NTR with an NGF/p75.sup.NTR binding
inhibitor, amount of a NGF/p75.sup.NTR inhibitor compound, thereby,
inhibiting binding of NGF to p75.sup.NTR. The method can be
practiced in vitro, for example, in a cell culture screening assay
to screen compounds which potentially bind, activate or inhibit
receptor function. In such a method, the inhibitor compound can
function by binding and eliminating any competing function of NGF
in the sample or culture. The inhibitor compounds can also be used
to control NGF activity in neuronal cell culture. The method can
also be practiced in vivo, for example, to inhibit one or more
processes mediated by binding of NGF to p75.sup.NTR.
[0054] In another embodiment, the invention provides a method of
treating a condition mediated by NGF/p75.sup.NTR binding in a
patient. The method comprises the step of administering to the
patient a therapeutically effective amount of a NGF/p75.sup.NTR
binding inhibitor, such as any of the inhibitors described above.
The condition to be treated can be any condition which is mediated,
at least in part, by binding of NGF to the p75.sup.NTR receptor.
Such conditions include, but are not limited to, Alzheimer's
disease, epilepsy, pain, multiple sclerosis, amyotrophic lateral
sclerosis, stroke and cerebral ischemia.
[0055] Preferably, the NGF/p75.sup.NTR binding inhibitor to be
administered selectively inhibits the binding of NGF to p75.sup.NTR
in cells which do not express trkA. In this embodiment, the
condition is mediated, at least in part, by the binding of NGF to
the p75.sup.NTR receptor in cells which do not express the trkA
receptor. Generally, such conditions are mediated by NGF-induced
apoptotic cell death.
[0056] The quantity of a given compound to be administered will be
determined on an individual basis and will be determined, at least
in part, by consideration of the individual's size, the severity of
symptoms to be treated and the result sought. The NGF/p75.sup.NTR
binding inhibitor can be administered alone or in a pharmaceutical
composition comprising the inhibitor, an acceptable carrier or
diluent and, optionally, one or more additional drugs.
[0057] The NGF/p75.sup.NTR binding inhibitor can be administered
subcutaneously, intravenously, parenterally, intraperitoneally,
intradermally, intramuscularly, topically, enteral (e.g., orally),
rectally, nasally, buccally, sublingually, vaginally, by inhalation
spray, by drug pump or via an implanted reservoir in dosage
formulations containing conventional non-toxic, physiologically
acceptable carriers or vehicles. The preferred method of
administration is by oral delivery. The form in which it is
administered (e.g., syrup, elixir, capsule, tablet, solution,
foams, emulsion, gel, sol) will depend in part on the route by
which it is administered. For example, for mucosal (e.g., oral
mucosa, rectal, intestinal mucosa, bronchial mucosa)
administration, nose drops, aerosols, inhalants, nebulizers, eye
drops or suppositories can be used. The compounds and agents of
this invention can be administered together with other biologically
active agents, such as analgesics, anti-inflammatory, agents,
anesthetics and other agents which can control one or more symptoms
or causes of a p75.sup.NTR-mediated condition.
[0058] In a specific embodiment, it may be desirable to administer
the agents of the invention locally to a localized area in need of
treatment; this may be achieved by, for example, and not by way of
limitation, local infusion during surgery, topical application,
transdermal patches, by injection, by means of a catheter, by means
of a suppository, or by means of an implant, said implant being of
a porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes or fibers. For example, the agent can
be injected into the joints.
[0059] The compound of the invention can, optionally, be
administered in combination with one or more additional drugs
which, for example, are known for treating and/or alleviating
symptoms of the condition mediated by p75.sup.NTR The additional
drug can be administered simultaneously with the compound of the
invention, or sequentially.
[0060] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically (or
prophylactically) effective amount of one or more NGF/p75.sup.NTR
binding inhibitors, preferably one or more compounds of Formulas 1,
2, 4 or 5, as described above, and a pharmaceutically acceptable
carrier or excipient. Suitable pharmaceutically acceptable carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The carrier and
composition can be sterile. The formulation should suit the mode of
administration.
[0061] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions (e.g., NaCl), alcohols,
gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols,
gelatin, carbohydrates such as lactose, amylose or starch,
cyclodextrin, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose,
polyvinyl pyrolidone, etc. The pharmaceutical preparations can be
sterilized and if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
coloring, flavoring and/or aromatic substances and the like which
do not deleteriously react with the active compounds.
[0062] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, polyvinyl
pyrollidone, sodium saccharine, cellulose, magnesium carbonate,
etc.
[0063] The composition can be formulated in accordance with the
routine procedures as a pharmaceutical composition adapted for
intravenous administration to human beings. Typically, compositions
for intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic to ease pain at the site
of the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampule or sachet indicating the
quantity of active agent. Where the composition is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water, saline or
dextrose/water. Where the composition is administered by injection,
an ampule of sterile water for injection or saline can be provided
so that the ingredients may be mixed prior to administration.
[0064] The pharmaceutical compositions of the invention can also
include an agent which controls release of the NGF/p75.sup.NTR
inhibitor compound, thereby providing a timed or sustained release
composition.
[0065] The present invention also relates to prodrugs of the
NGF/p75.sup.NTR binding inhibitors disclosed herein, as well as
pharmaceutical compositions comprising such prodrugs. For example,
compounds of the invention which include acid functional groups or
hydroxyl groups can also be prepared and administered as a
corresponding ester with a suitable alcohol or acid. The ester can
then be cleaved by endogenous enzymes within the patient to produce
the active agent.
[0066] In a further embodiment, the invention relates to the use of
an NGF/p75.sup.NTR binding inhibitor, such as any of the compounds
described above, for treating a condition mediated by binding of
NGF to p75.sup.NTR. The invention further relates to the use of
these compounds for the manufacture of a medicament for treating a
condition mediated by binding of NGF to p75.sup.NTR.
[0067] Representative syntheses of compounds of the invention are
set forth in the following examples. Other synthetic pathways that
can be used to prepare certain compounds of the invention are
illustrated in FIGS. 3 and 4.
EXAMPLES
Example 1 Synthesis of NGF/p75.sup.NTR Inhibitors
General Methods
[0068] Reagents and solvents were obtained from commercial sources
(Sigma, Aldrich, BDH). THF was dried by refluxing with benzophenone
and potassium and subsequently distilled. All other solvents were
utilized as they were received.
[0069] Thin layer chromatography (TLC) solvent systems used are
given in Table 1. These were developed by ascending TLC on
precoated aluminum backed sheets of silica gel 60 F254 (Merck). TLC
plates were developed using ultra-violet light, iodine crystal
and/or ninhydrin.
[0070] Melting points (mp) were determined on a Thomas Hoover
Unimelt melting point apparatus and are uncorrected.
[0071] NMR of final compounds were determined on an AVANCE 300 MHz
NUR spectrometer. All NMR samples were prepared in DMSO-d6 unless
otherwise indicated. Chemical shifts are reported as .delta. parts
per million using DMSO as an internal reference. Mass spectrometric
(MS) analyses were obtained on a Varian Instrument VG Quattro
multiple quadripole spectrometer using electrospray ionization
(ESI). The spectra were all obtained in the negative ion mode. IR
spectra were recorded on a Bomen MB-E120 FT-IR
spectrophotometer.
[0072] Abbreviations used herein are: HOAc, glacial acetic acid;
THF, tetrahydrofuran; DMSO-d.sub.6, deuterated dimethylsulfoxide;
CHCl.sub.3, chloroform: MeCN, acetonitrile; H.sub.2O, distilled
water; MeOH, methanol; EtOH, ethanol; TEA, triethylamine; EtOAc,
ethyl acetate.
TABLE-US-00001 TABLE 1 List of Solvent Systems. Solvent Code
Solvent System Solvent Ratio A MeOH:HOAc 5:1 B MeCN:H.sub.2O:MeOH
8:1:1 C MeCN:H.sub.2O:MeOH 4:1:1 D CHCl.sub.3:MeOH:HOAc 95:10:3 E
EtOH:HOAc 50:1
General Synthesis of Naphthalimide Derivatives
[0073] Method A. The naphthalimide series of compounds was prepared
through the condensation of stoichiometric amounts of
1,8-naphthalic anhydride or its derivative (I) with an appropriate
primary amine (II). The combined reagents were dissolved in glacial
acetic acid, dry THF, dry 1,4-dioxane or DMSO and placed under a
N.sub.2 atmosphere and refluxed. The progress of the reaction was
monitored by TLC. Final clear solutions were concentrated in vacuo
and the resulting crude material was either reprecipitated from
1,4-dioxane/1N HCl or HOAc/H.sub.2O and/or recrystallized from 95%
ethanol, THF or 1,4-dioxane. In the instances where the final
product precipitated out of the reaction solution, the completed
reaction mixture was cooled to room temperature, the solid
collected by filtration and washed with distilled water. This
precipitate was reprecipitated with 1,4-dioxane, 1N HCl or
HOAc/H.sub.2O and/or recrystallized from 95% ethanol, THF or
1,4-dioxane. Purification also included fractional
recrystallisation.
##STR00020##
where X and R.sub.1 are as previously defined.
[0074] Method B. Reaction conditions and purification procedures
were similar to those of method A. However, instead of
stoichiometric amounts of reagents, the anhydride (I) and the
primary amine(II) were combined in a 1:2 ratio with the optional
addition of 1 equivalent of anhydrous sodium acetate. During the
course of preparing the various naphthalimide derivatives, these
reaction conditions were found to lead to increased product yields.
Glacial acetic acid was the solvent of choice used under these
conditions.
TABLE-US-00002 TABLE 2 Synthesized Naphthalimide Derivatives
##STR00021## Compd. X R.sub.1 Name 200 3-NO.sub.2 CH.sub.2COOH
N-(1-carboxymethyl)-3-nitro-1,8- naphthalimide;
2-(1-carboxymethyl)- 5-nitrobenzo[d,e]isoquinoline-1,3- dione 201
3-NO.sub.2 CH.sub.2CH.sub.2COOH N-(2-carboxymethyl)-3-nitro-1,8-
naphthalimide; 2-(2-carboxymethyl)-5-
nitrobenzo[d,e]isoquinoline-1,3-dione 202 3-NO.sub.2
CH.sub.2(CH.sub.2).sub.2COOH N-(3-carboxypropyl)-3-nitro-1,8-
naphthalimide; 2-(3-carboxypropyl)-
5-nitrobenzo[d,e]isoquinoline-1,3- dione 203 3-NO.sub.2
CH.sub.2(CH.sub.2).sub.3COOH N-(4-carboxybutyl)-3-nitro-1,8-
naphthalimide; 2-(4-carboxybutyl)-5-
nitrobenzo[d,e]isoquinoline-1,3-dione 204 3-NO.sub.2
CH.sub.2(CH.sub.2).sub.4COOH N-(5-carboxypentyl)-3-nitro-1,8-
naphthalimide; 2-(5-carboxypentyl)-5-
nitrobenzo[d,e]isoquinoline-1,3-dione 220 4-NO.sub.2 CH.sub.2COOH
N-(1-carboxymethyl)-4-nitro-1,8- naphthalimide;
2-(1-carboxymethyl)- 6-nitrobenzo[d,e]isoquinoline-1,3- dione 221
4-NO.sub.2 CH.sub.2CH.sub.2COOH N-(2-carboxyethyl)-4-nitro-1,8-
naphthalimide; 2-(2-carboxyethyl)-6-
nitrobenzo[d,e]isoquinoline-1,3-dione 222 4-NO.sub.2
CH.sub.2(CH.sub.2).sub.2COOH N-(3-carboxypropyl)-4-nitro-1,8-
naphthalimide; 2-(3-carboxypropyl)-
6-nitrobenzo[d,e]isoquinoline-1,3- dione 223 4-NO.sub.2
CH.sub.2(CH.sub.2).sub.3COOH N-(4-carboxybutyl)-4-nitro-1,8-
naphthalimide; 2-(4-carboxybutyl)-6-
nitrobenzo[d,e]isoquinoline-1,3-dione 224 4-NO.sub.2
CH.sub.2(CH.sub.2).sub.4COOH N-(5-carboxypentyl)-4-nitro-1,8-
naphthalimide; 2-(5-carboxypentyl)-6-
nitrobenzo[d,e]isoquinoline-1,3-dione
Naphthalimide Derivatives:
Method A:
N-(1-carboxymethyl)-3-nitro-1,8-naphthalimide (200)
[0075] 3-nitro-1,8-naphthalic anhydride (1.0 g, 0.0041 mol) and
glycine (0.31 g, 0.0041 mol) and 50-60 mls of glacial acetic acid
were added to a 100 ml round-bottom flask equipped with a reflux
condenser, heating mantle and stir plate. The system was placed
under a N.sub.2 atmosphere and heated to a gentle reflux. The
progress of the reaction was monitored by TLC. After four days, the
clear dark amber solution was concentrated under vacuum and the
crude brown material reprecipitated with 1,4-dioxane/1N HCl. The
precipitate was filtered through a Buchner funnel and washed with
water. Successive fractional recrystallizations in ethanol afforded
0.35 g (28%) of 200 as a beige powder: M. Pt. 260-262.degree. C.;
TLC R.sub.f 0.83 (A): R.sub.f 0.82 (B): R.sub.f 0.23 (D): .sup.1H
NMR (DMSO-d.sub.6); IR (cm.sup.-1): 2750-3100 (OH), 3077
(C.dbd.CH), 2665 (C--H), 1732 (C.dbd.O), 1711 (bs, C.dbd.O), 1670
(C.dbd.O), 1597 (C.dbd.C), 1538 (N.dbd.O), 1509 (C.dbd.C), 1430
(C.dbd.C), 1372 (C--O), 1341 (N--O), 1244 (C--O), 787 (C.dbd.CH).
MS m/z (rel intensity) 299 (97) 255 (100), 281 (93), 172 (26).
N-(2-carboxyethyl)-3-nitro-1,8-naphthalimide (201)
[0076] 3-nitro-1,8-naphthalic anhydride (1.0 g, 0.0041 mol) and
.beta.-alanine (0.37, 0.0041 mol) were refluxed as per 200 for 48
hours. Precipitate that formed during the course on the reaction
was filtered and washed with water. The filtrate was concentrated
under vacuum and the crude brown material reprecipitated with
1,4-dioxane/1N HCl. The precipitate was filtered through a Buchner
funnel and washed with water. Both solids were combined and
triturated with hot ethanol and 1,4-dioxane. Any undissolved
material was removed by filtration. The combined ethanol and
1,4-dioxane fractions were concentrated in vacuo. Crystallization
from ethanol afforded 0.38 g (30%) of 201 as a beige powder: M. Pt.
246-248.degree. C.; TLC R.sub.f 0.74 (A): R.sub.f 0.84 (B): R.sub.f
0.67 (D): .sup.1H NMR (DMSO-d.sub.6); IR (cm.sup.-1): 2800-3130
(OH), 3071 (C.dbd.CH), 2646 (C--H), 1707 (bs, C.dbd.O), 1663
(C.dbd.O), 1625 (C.dbd.C), 1597 (C.dbd.C), 1537 (NMO), 1439
(C.dbd.C), 1420 (C.dbd.C). 1369 (C--O), 1347 (N--O), 1243 (C--O),
789 (C.dbd.CH). MS m/z (rel intensity) 313 (100) 241 (42).
N-(3-carboxypropyl)-3-nitro-1,8-naphthalimide (202)
[0077] 3-nitro-1,8-naphthalic anhydride (1.0 g, 0.0041 mol) and
4-aminobutyric acid (0.42 g. 0.0041 mol) were refluxed as per 200
for 48 hours. Resultant solution was purified as per 201 to afford
0.55 g (42%) of 202 as a fluffy beige solid: M. Pt. 200-202.degree.
C.; TLC R.sub.f 0.88 (A): R.sub.f 0.85 (B): R.sub.f 0.75 (D):
.sup.1H NMR (DMSO-d.sub.6); IR (cm.sup.-1): 2875-3100 (OH), 3062
(C.dbd.CH), 2560 (C--H), 1785 (C.dbd.O), 1702 (C.dbd.O), 1658
(C.dbd.O), 1625 (C.dbd.C), 1595 (C.dbd.C), 1538 (N.dbd.O), 1438
(C.dbd.C), 1418 (C.dbd.C), 1372 (C--O), 1334 (N--O), 1244 (C--O),
789 (C.dbd.CH). MS m/z (rel intensity) 327 (100) 241 (40).
N-(4-carboxybutyl)-3-nitro-1,8-naphthalimide (203)
[0078] 3-nitro-1,8-naphthalic anhydride (0.5 g, 0.0020 mol) and
5-aminopentanoic acid (0.24 g, 0.0020 mol) were refluxed in DMSO as
per 200 for 24 hours. The dark orange brown solution was diluted
with water until a beige precipitate formed. The precipitate was
filtered through a Buchner funnel and washed copiously with water.
The process was repeated on the concentrated filtrate. Combined
solids were recrystallized in 1,4-dioxane/1N HCl. The filtered
product was dried in air for a short time and then in vacuo for
48-72 hours to afford 0.43 g (63%) 203 as a beige feathery solid:
M. Pt. 2033-205.degree. C.: TLC R.sub.f 0.92 (A): R.sub.f 0.89 (C);
R.sub.f 0.26 (D): .sup.1H NMR (DMSO-d.sub.6); IR (cm.sup.-1):
2700-3130 (OH), 3066 (C.dbd.CH), 1784 (C.dbd.O), 1706 (bs,
C.dbd.O), 1659 (C.dbd.C), 1625 (C.dbd.C), 1596 (C.dbd.C), 1541
(N.dbd.C), 1458 (C.dbd.C), 1438 (C.dbd.C), 1351 (N--O), 1245
(C--O), 789 (C.dbd.CH). MS m/z (rel intensity) 341 (100), 241 (16),
216 (24).
N-(5-carboxypentyl)-3-nitro-1,8-naphthalimide (204)
[0079] 3-nitro-1,8-naphthalic anhydride (1.0 g, 0.0041 mol) and
6-aminohexanoic acid (0.54 g, 0.0041 mol) were refluxed in 100 ml
THF and 10 ml DMSO as per 200 for 24 hours. The dark-brown solution
was concentrated and manipulated as per 203 to yield 1.0 g (67%)
204 as beige powder: M. Pt. 192-194.degree. C.; TLC R.sub.f 0.70
(A): R.sub.f 0.91 (B): R.sub.f 0.63 (D): .sup.1H NMR (DMSO-d.sub.6)
.delta. 1.36 (m, 2H), 1.54 (m, 2H), 1.64 (m. 2H), 2.22 (t. J=7.3
Hz. 2H), 4.02 (t. J=7.3 Hz, 2H), 8.02 (dd, J=7.1, 8.3 Hz, 1H), 8.63
(d, J=7.1 Hz. 1H), 8.73 (d, J=8.3 Hz, 1 Hz), 8.89 (d, J=2.3 Hz,
1H), 9.43 (d, J=2.3 Hz, 1H); IR (cm.sup.-1): 2700-3175 (OH), 3075
(C.dbd.CH), 2688 (C--H), 1706 (bs, C.dbd.O), 1663 (C.dbd.O), 1625
(C.dbd.C), 1598 (C.dbd.C), 1533 (N.dbd.O), 1437 (C.dbd.C), 1419
(C.dbd.C), 1348 (N--O), 1244 (C--O), 791 (C.dbd.CH). MS m/z (rel
intensity) 355 (100).
N-(2-carboxyethyl)-4-nitro-1,8-naphthalimide (221)
[0080] 4-nitro-1,8-naphthalic anhydride (0.5 g, 0.0020 mol) and
.beta.-alanine (0.18 g, 0.0020 mol) were refluxed for 4 days and
the final mixture purified as per 200. Successive fractional
recrystallisations in CHCl.sub.3 afforded 0.24 g (38%) 221 as an
beige solid: M. Pt. 224-225.degree. C.; TLC R.sub.f 0.77 (A):
R.sub.f 0.80 (C): R.sub.f 0.46 (D): .sup.1NMR (DMSO-d.sub.6); IR
(cm.sup.-1): 2800-3175 (OH), 3077 (C.dbd.CH), 2637 (C--H), 1788
(C.dbd.O), 1705 (bs, C.dbd.O), 1657 (C.dbd.C), 1624 (C.dbd.C), 1594
(C.dbd.C), 1534 (N.dbd.O), 1440 (C.dbd.C), 1408 (C.dbd.C), 1346
(N--O), 1229 (C--C), 789 (C.dbd.CH). MS m/z (rel intensity) 314
(16), 313 (100). 274 (50).
N-(3-carboxypropyl)-4-nitro-1,8-naphthalimide (222)
[0081] 4-nitro-1,8-naphthalic anhydride (0.5 g, 0.0020 mol) and
4-aminobutyric acid (0.21 g. 0.0020 mol) were refluxed for 5 days
in dry THF as per 200. The dark orange amber solution was
concentrated to 20 ml in vacuo and diluted with 1N HCl until a
beige precipitate formed. The precipitate was filtered through a
Buchner funnel and washed with water. The crude material was
reprecipitated from 1,4-dioxane, 1N HCl and filtered. Successive
fractional recrystallisations in CHCl.sub.3 afforded 0.22 g (34%)
of 222 as a beige solid: M. Pt. 179-180.degree. C.; R.sub.f 0.85
(A): TLC R.sub.f 0.81 (B): R.sub.f 0.48 (D): .sup.1H NMR
(DMSO-d.sub.6); IR (cm.sup.-1): 2780-3200 (OH). 3075 (C.dbd.CH),
2688 (C--H). 1778 (C.dbd.O), 1705 (bs, C.dbd.O), 1655 (C.dbd.O),
1624 (C.dbd.C), 1584 (C.dbd.C), 1534 (N.dbd.O). 1439 (C.dbd.C),
1409 (C.dbd.C), 1344 (N--O), 1231 (C--O), 789 (C.dbd.CH). MS m/z
(rel intensity) 328 (20), 327 (100), 274 (27), 241 (36).
N-(4-carboxybutyl)-4-nitro-1,8-naphthalimide (223)
[0082] 4-nitro-1,8-naphthalic anhydride (0.5 g, 0.0020 mol) and
5-aminopentanoic acid (0.24 g, 0.0020 mol) were refluxed for 3 days
in dry THF as per 200. The dark orange amber solution was purified
as per 222 to afford 0.27 g (40%) of 223 as a orange powder: M. Pt.
198-200.degree. C.; TLC R.sub.f 0.84 (A): R.sub.f 0.88 (C): R.sub.f
0.46 (D): .sup.1H NMR (DMSO-d.sub.6). IR (cm.sup.-1): 2825-3130
(OH), 3077 (C.dbd.CH), 2670 (C--H). 1704 (bs, C.dbd.O). 1660
(C.dbd.O), 1624 (C.dbd.C), 1583 (C.dbd.C), 1529 (N.dbd.O), 1435
(C.dbd.C), 1411 (C.dbd.C). 1345 (N--C), 1233 (C--C), 787
(C.dbd.CH). MS m/z (rel intensity) 342 (19), 341 (100), 274 (20).
241 (40).
N-(5-carboxypentyl)-4-nitro-1,8-naphthalimide (224)
[0083] 4-nitro-1,8-naphthalic anhydride (0.5 g, 0.0020 mol) and
6-aminohexanoic acid (0.27 g, 0.0020 mol) were refluxed for 3 days
in dry THF and the final mixture purified as per 223.
Crystallization from THF/1N HCl afforded 0.65 g (95%) of 224 as a
beige solid: M. Pt. 168-169.degree. C.; TLC R.sub.f 0.85 (A):
R.sub.f 0.81 (B): R.sub.f 0.50 (D): .sup.1H NMR (DMSO-d.sub.6); IR
(cm.sup.-1): 2775-3175 (OH), 3078 (C.dbd.CH), 2670 (C--H), 1716
(bs, C.dbd.O), 1661 (C.dbd.O), 1624 (C.dbd.C). 1594 (C.dbd.C).
11-21 (N.dbd.O), 1136 (C.dbd.C), 1409 (C.dbd.C), 339 (N--O), 1261
(C--O). 785 (C.dbd.CH). MS m/z (rel intensity), 365 (21), 355
(100).
Method B:
N-(1-carboxymethyl)-4-nitro-1,8-naphthalimide (220)
[0084] 4-nitro-1,8-naphthalic anhydride (1.0 g, 0.0041 mol),
glycine (0.38 g, 0.0082 mol), anhydrous sodium acetate (0.51 g,
0.61 mol) and 70-80 mls of glacial acetic acid were added to a 100
ml round-bottom flask equipped with a reflux condenser, heating
mantle and stir plate. The system was placed under a N.sub.2
atmosphere and heated to a gentle reflux. After 3 days, the dark
amber solution was concentrated to 15-20 ml under vacuum with a
rotary evaporator. Ensuing precipitate was filtered and washed
twice with 5 ml 1N HCl and twice with 5 ml water. Crystallization
from 1,4-dioxane/H.sub.20 afforded 0.6 g (50%) 220 as a beige
solid: M. Pt. 263-264.degree. C.; TLC R.sub.f 0.81 (A): R.sub.f
0.84 (C): R.sub.f 0.35 (D): .sup.1H NMR (DMSO-d.sub.6) .delta. 4.74
(s, 2H), 8.10 (dd, J=8.4, 8.0 Hz, 1H), 8.55 (d, J=7.6 Hz, 1H), 8.63
(d, J=8.0 Hz, 1H), 8.65 (d, J=8.0, 1H), 8.73 (d, J=8.4 Hz. 1H); IR
(cm.sup.-1): 2850-3150 (OH), 3077 (C.dbd.CH), 2671 (C--H), 1711
(C.dbd.O), 1674 (bs, C.dbd.O), 1625 (C.dbd.C), 1583 (C.dbd.C), 1531
(N.dbd.O), 1429 (C.dbd.C), 1334 (N--O), 1232 (C--O), 785
(C.dbd.CH). MS m/z (rel intensity) 299 (100), 255 (280).
Example 2 Assessment of NGF/p75.sup.NTR Binding Inhibition
[0085] The radio-iodination and receptor binding of NGF (Sutter et
al., 1979) was performed with modifications (Ross et al., 1997) as
follows: Evaluation of the ability of NCP compounds to inhibit TrkA
and p75.sup.NTR binding was determined by the binding of
.sup.125I-NGF to PC12 cells (rat pheochromocytoma cells expressing
TrkA and p75.sup.NTR; obtained from ATCC) and PC12.sup.nnr5 (rat
pheochromocytoma cells expressing p75.sup.NTR only; obtained from
Dr. L. Greene, Columbia University, NY). The p75.sup.NTR is in a
low affinity state and a high affinity state, respectively, in
these cell types (Ross et al., 1998). PC12 and PC12.sup.nnr5 cells
were grown in RPMI (Sigma) with 10% heat inactivated donor horse
serum and 5% fetal calf serum. Cells were harvested by replacing
the medium with calcium, magnesium-free balanced salt solution
(Gey's solution) and incubating at 37.degree. C. for 15 minutes.
Cells were pelleted b centrifugation and suspended in HKR buffer
(10 mM Hepes [pH 7.35] containing 125 mM NaCl, 4.8 mM KCl, 1:3 mM
CaCl.sub.2, 1.2 mM MgSO.sub.4, 1.2 mM KH.sub.2PO.sub.4, 1 g/L
glucose and 1 g/L BSA) at a cell concentration of
2.times.10.sup.6/mL and kept at 4.degree. C. Triplicate tubes were
set up for total binding, non-specific binding and binding in the
presence of candidate competitor molecule (i.e., a tube for each
data point). Each tube contained .sup.125I-NGF (at 1 nM), 400,000
cells (for a final cell concentration of 10.sup.6/mL) and NGF (50
mM, to define non-specific binding), as required. The tubes were
incubated for 2 h at 4.degree. C. and specific binding evaluated by
measuring specifically bound DPM (Ross et al., 1997). Data were
analysed and the results expressed as receptor binding observed in
the presence of competitor as a percentage of receptor binding in
the absence of a competitor.
TABLE-US-00003 TABLE 3 Results of in vitro binding inhibition
assays PC12 nnr5 Compound (50 .mu.M) % of Max % of Max 200 92, 87,
74 = 84 58, 63, 64 = 62 201 27, 32, 41 = 33 30, 4, 34 = 23 202 21,
22, 30 = 24 16, 0, 0 = 5 203 36, 41, 35 = 37 33, 0, 18 = 17 204 41,
41, 36 = 39 12, 0, 47 = 20 220 NS 37, 43, 49 = 43 21, 17, 26 = 21
221 29, 32, 31 = 31 41, 37, 42 = 40 222 NS NT NT 223 27, 33, 33 =
31 40, 41, 47 = 43 224 31, 34, 22 = 29 36, 35, 39 = 36 NS: Not
Soluble @ 100 .mu.M DMSO NT: Not Tested
[0086] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various chances in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
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