Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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5-HYDROXY INDAZOLE DERIVATIVES FOR TREATING GLAUCOMA
The present invention is directed to substituted 3-(2-aminoethyl)-1H indazol
5-ols, some of which are novel, for lowering and controlling normal or
elevated
s intraocular pressure (IOP) and treating glaucoma.
Background of the Invention
The disease state referred to as glaucoma is characterized by a permanent loss
~o of visual function due to irreversible damage to the optic nerve. The
several
morphologically or functionally distinct types of glaucoma are typically
characterized
by elevated IOP, which is considered to be causally related to the
pathological course
of the disease. Ocular hypertension is a condition wherein intraocular
pressure is
elevated but no apparent loss of visual function has occurred; such patients
are
is considered to be a high risk for the eventual development of the visual
loss associated
with glaucoma. Some patients with glaucomatous field loss have relatively low
intraocular pressure. These so called normotension or low tension glaucoma
patients
can also benefit from agents that lower and control IOP. If glaucoma or ocular
hypertension is detected early and treated promptly with medications that
effectively
Zo reduce elevated intraocular pressure, loss of visual function or its
progressive
deterioration can generally be ameliorated. Drug therapies that have proven to
be
effective for the reduction of intraocular pressure include both agents that
decrease
aqueous humor production and agents that increase the outflow facility. Such
therapies are in general administered by one of two possible routes, topically
(direct
Zs application to the eye) or orally.
There are some individuals who do not respond well when treated with certain
existing glaucoma therapies. There is, therefore, a need for other topical
therapeutic
agents that control IOP.
It has been found that serotonergic compounds which possess agonist activity
at 5-HTz receptors effectively lower and control normal and elevated IOP and
are
useful for treating glaucoma, see commonly owned co-pending application,
PCT/US99/19888. Compounds that act as agonists at 5-HTZ receptors are well
known
3s and have shown a variety of utilities, primarily for disorders or
conditions associated
with the central nervous system (CNS). U.S. Patent 5,494,928 discloses certain
2
(indol-1-yl)-ethylamine derivatives that are 5-HT2~ agonists for the treatment
of
obsessive compulsive disorder and other CNS derived personality disorders.
U.S.
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Patent 5,571,833 discloses tryptamine derivatives that are 5-HTZ agonists for
the
treatment of portal hypertension and migraine. U.S. Patent 5,874,477 discloses
a
method for treating malaria using 5-HTZ,~ZC agonists. U.S. Patent 5,902,815
discloses
the use of 5-HTZA agonists to prevent adverse effects of NMDA receptor hypo-
s function. W098/31354A2 discloses 5-HT2B agonists for the treatment of
depression
and other CNS conditions. Agonist response at the 5-HTZA receptor is reported
to be
the primary activity responsible for hallucinogenic activity, with some lesser
involvement of the S-HTZC receptor possible [Psychopharmacology, Vol. 121:357,
1995].
~o
The present invention is directed to substituted 3-(2-aminoethyl)-1H indazol-
S-ols, some of which are novel. It is believed that these compounds will have
a high
affinity for and function as agonists at the serotonergic 5-HTZ receptor, and
will
thereby be useful for lowering and controlling normal or elevated intraocular
pressure
is (IOP) and treating glaucoma. When a phenolic moiety is included in this
substitution,
e.g. a hydroxyl group at indazole ring position five, such compounds can be
particularly sensitive to oxidation reactions well known to occur with
phenolic
compounds in general, including the related hydroxytryptamines [J. Phys. Chem.
103,
8606 (1999), Chem. Res. Toxicol. 11, 639 (1998), J. Org. Chem. 52, 2817
(1987), J.
2o Pharm. Sci. 77, 911 (1988)], which are of particular relevance to the
present
application. Protection of such hydroxy substituted phenols from oxidation can
be
accomplished by derivatization of the aryl hydroxy group to provide a suitable
ester,
carbamate, or carbonate. Though the ester, carbamate, or carbonate derivatives
do not
themselves possess a high affinity for the above mentioned receptors, they do
have
zs utility in the treatment of glaucoma since suitably protected phenols can
be cleaved in
vivo either by chemical hydrolysis or through the action of tissue esterases.
Such
cleavage would deliver the desired therapeutic agent, that is, the desired
novel 5
hydroxy-indazole compounds of the present invention. The concept of utilizing
such
derivatized phenolic compounds as chemical delivery agents is well known in
the art
30 [Drugs Pharm. Sci. 53, 221 (1992), Pharm. Res., 168 (1984)].
The chemical synthesis of 3-(2-dimethylamino-ethyl)-1H indazol-5-0l has
been reported with no comment on the utility of the compound [J. Amer. Chem.
Soc.
79, 5242 (1957); J. Amer. Chem. Soc. 80, 965 (1958)].
The chemical synthesis of 1-(2-aminopropyl)-1H-indazol-6-0l and other ring
substitution variants has been reported in published International Patent
Application
No. W098/30548 (1998). The utility cited for the compounds of this application
is
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for treating central nervous system diseases such as sexual disorders, genital
insufficiency, appetite regulation disorders, anxiety, depression, and sleep
disorders.
There is no contemplation of ophthalmic use noted in this application.
Published
International Patent Application No. W000/12482 (2000) discloses certain 1-
s (indazol-3-yl)-2-propylamine derivatives that are 5-HTZ agonists for the
treatment of
disorders of the central nervous system
Summary of the Invention
Io The present invention is directed to derivatives of 3-(2-aminoethyl)-1H-
indazol-5-0l, some of which are novel, which can be used to lower and control
IOP
is
associated with normal-tension glaucoma, ocular hypertension, and glaucoma in
warm
blooded animals, including man. The compounds are formulated in pharmaceutical
compositions suitable for topical delivery to the eye.
Description of the Preferred Embodiments
Compounds that are useful according to the present invention are represented
zo
by the following Formula I.
FORMULA I
,o
R
R_
I
wherein G is chosen from hydrogen, halogen, or Cl~alkyl;
R is hydrogen, C~.~alkyl, C(=O)W, or P(=O)(OX)(OY),
Zs R' and RZ are hydrogen;
R3 and R4 are independently chosen from hydrogen, Cl~alkyl or R3, R4 and the
carbon
atom to which they are attached can form a cyclopropyl ring, or furthermore,
RZ and
R3 together can be (CH2)m to form a saturated heterocycle;
when RZ and R3 are part of a heterocycle, R' can be hydrogen or C»alkyl;
so Rs can be hydrogen or C»alkyl;
when R, R', RZ, Rs, and G all are hydrogen R3 and R4 cannot both be hydrogen;
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W is C1_6alkyl, NR6R', N(R6)CH2(CH2)"C(=O) N(R')(R8), OC~_balkyl, Cl_6alkyl
(which can be substituted with halogen, hydroxyl, COZCI~alkyl, CON(Cl~alkyl)z,
C(=NH)NHZ, HC(=NH)NH2, NHZ), CZ.~alkenyl (substituted by phenyl, unsubstituted
or substituted with one or more of C»alkyl, Cl~alkoxy or halogen);
s R6, R', Rg are independently chosen from hydrogen or C»alkyl;
X and Y are independently chosen from hydrogen, C1_lo alkyl or X and Y can
together
form a lower alkyl chain of (CHZ)m;
mist-4;
n is 1 or 2;
~o and pharmaceutically acceptable salts and solvates of the compounds of
Formula I.
Compounds that are novel and which are useful according to the present
invention can be defined as follows:
~s G is chosen from hydrogen, halogen, or C1_4alkyl;
R is C(=O)W, or P(=O)(OX)(OY),
RI and RZ are hydrogen;
R3, R4 and the carbon atom to which they are attached can form a cyclopropyl
ring, or
furthermore, RZ and R3 together can be (CHZ)m to form a saturated heterocycle;
Zo when RZ and R3 are part of a heterocycle, R' can be hydrogen or C»alkyl;
Rs can be hydrogen or C1_4alkyl;
W is C1_6alkyl, NR6R', N(R6)CHZ(CHZ)"C(=O)N(R')(R8), OCI_balkyl, C,_balkyl
(which can be substituted with halogen, hydroxyl, COZCI_4alkyl,
CON(Cl~alkyl)Z,
C(=NH)NHZ, HC(=NH)NHZ, NHZ), CZ_4alkenyl (substituted by phenyl, unsubstituted
is or substituted with one or more of C»alkyl, Cl~alkoxy or halogen);
R6, R', R8 are independently chosen from hydrogen or C»alkyl;
X and Y are independently chosen from hydrogen, C1_~o alkyl or X and Y can
together
form a lower alkyl chain of (CHZ)m;
mist-4;
3o n is 1 or 2.
Preferred compounds are:
G is chosen from hydrogen, halogen, or C1_4alkyl;
R is hydrogen, C(=O)W, or P(=O)(OX)(OY),
3s R' and RZ are hydrogen;
R3 and R4 are independently chosen from hydrogen, C»alkyl or R3, R4 and the
carbon
atom to which they are attached can form a cyclopropyl ring;
Rs can be hydrogen or C~~ralkyl;
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W is C1_6alkyl, NR6R', N(R6)CH2(CH2)"C(=O)N(R')(R8), C~_6alkyl (which can be
substituted with halogen, hydroxyl, C02Cl.~alkyl, CON(C~_4alkyl)2, C(=NH)NH2,
HC(=NH)NH2, NH2), C2_4alkenyl (substituted by phenyl, unsubstituted or
substituted
with one or more of C~.~alkyl, Cl~alkoxy or halogen);
s R6, R', R8 are independently chosen from hydrogen or C»alkyl;
X and Y are independently chosen from hydrogen, C~_~o alkyl or X and Y can
together
form a lower alkyl chain of (CH2)m;
mis2or3;
nis 1 or2.
io
Most preferred compounds are:
G is chosen from hydrogen, halogen, or C»alkyl;
R is hydrogen or C(=O)W;
RI and RZ are hydrogen;
is R3 is hydrogen and R4 is methyl or R3, R4 and the carbon atom to which they
are
attached form a cyclopropyl ring;
RS is hydrogen;
W is C1_6alkyl, C~_6alkyl (which can be substituted with halogen, hydroxyl,
CON(C~.~alkyl)2, C(=NH)NH2).
Representative examples of preferred novel compounds of Formula I are:
3-(2-Aminopropyl)-1H indazol-5-0l;
3-(2-Aminopropyl)-1-methyl-1H indazol-5-0l;
2-(5-Methoxy-1 H-indazol-3-yl)-1-methyl-ethylamine;
2s 3-(2-Aminopropyl)-6-fluoro-1H indazol-5-0l;
3-(2-Aminopropyl)-7-methyl-1H indazol-5-0l;
3-(2-Aminopropyl)-6-fluoro-1-methyl-1H indazol-5-0l;
2-Methyl-propionic acid 3-(2-aminopropyl)-1H indazol-5-yl ester;
2,2-Dimethyl-propionic acid 3-(2-aminopropyl)-1H indazol-5-yl ester;
3o Cyclopropanecarboxylic acid 3-(2-aminopropyl)-1H indazol-5-yl ester;
N, N Diethyl-succinamic acid 3-(2-aminopropyl)-1H-indazol-5-yl ester.
It is recognized that compounds of Formula I can contain one or more chiral
centers. This invention contemplates all enantiomers, diastereomers and,
mixtures
ss thereof.
In the above definitions, the total number of carbon atoms in a substituent
group is indicated by the C; ~ prefix where the numbers i and j define the
number of
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carbon atoms; this definition includes straight chain, branched chain, and
cyclic alkyl
or (cyclic alkyl)alkyl groups.
It is important to recognize 'that a substituent may be present either singly
or
s multiply when incorporated into the indicated structural unit. For example,
the
substituent halogen, which means fluorine, chlorine, bromine, or iodine, would
indicate that the unit to which it is attached may be substituted with one or
more
halogen atoms, which may be the same or different.
to SYNTHESIS
The compounds of Formula I can be prepared by using one of several synthetic
procedures. For example, condensation of the suitably substituted indazol-3-
carboxaldehyde (1), which can be prepared from the corresponding indazole by
is known methods [J. Med. Chem. 38, 2331 (1995)], with the appropriate
nitroalkane
gives the corresponding nitroalkene (2), which can be reduced with, e.g.
LiAlH4, and
if desired dealkylated with, e.g. boron tribromide, to provide the desired
compounds 3
of Formula I.
2o SCHEME 1
CH3 CH,
CHO ~~ 'NH
NOi HO
/0 ~ \ /O
N ~ N ---~ ~N
G ~ H/ G ~ H/ ~ H
G
1 2 3
2s Another procedure for preparing compounds of Formula I is outlined in
Scheme 2. The appropriately substituted 2-fluoro-acetophenone (4), either
purchased
or prepared by known procedures, e.g. Tetrahedron 50, 1179 (1994), can be
reacted
with the desired aldehyde, such as acetaldehyde, in the presence of a strong
base, e.g.
lithium diisopropylamide, to give the aryl 13-hydroxyalkyl ketone (5) [Synth.
3o Commun. 10, 851 (1980)], which can be reacted with anhydrous hydrazine by
known
methods to provide the substituted indazole 6 [J. Med. Chem. 37, 2721 (1994)].
Conversion of the secondary alcohol moiety of 6 to the desired primary amine
can be
accomplished using the well known sequence involving initial activation by
formation
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of a sulfonate ester followed by displacement of this ester by reaction with
sodium
azide, and finally reduction of the azide with concomitant removal of any
phenol
protective groups, e.g. benzyl, to provide the desired amine (3). Alternately,
compound 4 can be reacted with an aldehyde, e.g. acetaldehyde, under acidic
s conditions to provide the chalcone intermediate, e.g. 7 [J. Chem. Soc., 2403
(1953)]
(Scheme 3). Addition of a suitably protected amine, e.g. benzylamine, to the
double
bond of 7 provides the desired amino ketone 8 [Chem. Pharm. Bull. 22, 1348
(1974)]
which when treated with hydrazine provides the substituted indazole 9 [J. Med.
Chem.
37, 2721 (1994)]; removal of the protective groups by hydrogenolysis provides
the
~o desired compounds 3.
SCHEME 2
CH3
CH3
RIO HOC-CHO RIO \ OH
\ O
-~ I O
/ F
G G
4 5
N=H,
CH3 CH3
~NH 1. Ms=O OH
HO z ,O
I \ \N E-- ~ R I \ \N
G / H 3.H= G / H
3 6
SCHEME 3
is
CH; CH3
~O N-P9
4 H3--~ RIO I \ O R-=~ R I \ H
O
H~ / / F
G F G
7 8
N=H4
CH3 CH3
.NHz .O H-P9
HO I \ \ H R \ \N
~N ~--- / ,
/ N
G ~ G H
3 9
_7_
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Yet another procedure for preparing compounds of Formula I, but beginning with
(5-
benzyloxy-1H indazol-3-yl)-acetic acid (10) [J. Amer. Chem. Soc. 79, 5246
(1959)]
as the starting material, is outlined in Scheme 4. Reaction of 10 with acetic
anhydride
in the presence of the appropriate base under Dakin-West conditions [Chem.
Soc.
s Rev, 17, 91 (1988)] provides intermediate 11 which upon reaction with O-
methyl-
hydroxylamine gives the oxime 12. Reduction of the 12, e.g. with borane, will
provide the desired compounds 13 of Formula I [Eur. J. Med. Chem. 27, 595
(1992),
Tetrahedron, 29, 223 (1988)].
1o SCHEME 4
0 0
O OH O CHI
Bzl~ ~ ~ \ N Ac.,O/base Bz~~ ~ ~ \ N
N ~ N
H
H
15 1~ 11
RONH,
CH3 N-OR
NH CH
2O HO ~ ~ \ N ~H,/Pd-C- BzI~O ~ ~ \ N
N ~ N
H
H
12
13
zs Compounds of Formula I, wherein R is C(=O)W, can be prepared by reacting
the appropriate indazole 3, or preferably a suitable amino-protected
intermediate, e.g.
14 (Scheme 5) with the desired activated acid derivative, such as an acid
halide or
active ester, or the like, to provide the esters 15. Removal of the N
protective group
from the intermediate 15 provides the desired compounds 16 of Formula I.
_g_
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SCHEME 5
CH3 CHI
HO 'NHz HO 'H Groupting
\ \N I \ \N
N ~ N
G H G H
3 14
CH3 CH3
NH ~ ~ Protecting
R O \ \ z R O \ H Group
I N E-- I \ N
N O ~ N
G 16 H G H
The compounds of this invention, Formula I, can be incorporated into various
s types of ophthalmic formulations for delivery to the eye (e.g., topically,
intracamerally, or via an implant). The compounds are preferably incorporated
into
topical ophthalmic formulations for delivery to the eye. The compounds may be
combined with ophthalmologically acceptable preservatives, surfactants,
viscosity
enhancers, penetration enhancers, buffers, sodium chloride, and water to form
an
to aqueous, sterile ophthalmic suspension or solution. Ophthalmic solution
formulations
may be prepared by dissolving a compound in a physiologically acceptable
isotonic
aqueous buffer. Further, the ophthalmic solution may include an
ophthalmologically
acceptable surfactant to assist in dissolving the compound. Furthermore, the
ophthalmic solution may contain an agent to increase viscosity, such as,
is hydroxymethylcellulose, hydroxyethylcellulose,
hydroxypropylmethylcellulose,
methylcellulose, polyvinylpyrrolidone, or the like, to improve the retention
of the
formulation in the conjunctival sac. Gelling agents can also be used,
including, but
not limited to, gellan and xanthan gum. In order to prepare sterile ophthalmic
ointment formulations, the active ingredient is combined with a preservative
in an
2o appropriate vehicle, such as, mineral oil, liquid lanolin, or white
petrolatum. Sterile
ophthalmic gel formulations may be prepared by suspending the active
ingredient in a
hydrophilic base prepared from the combination of, for example, carbopol-974,
or the
like, according to the published formulations for analogous ophthalmic
preparations;
preservatives and tonicity agents can be incorporated.
2s
The compounds are preferably formulated as topical ophthalmic suspensions
or solutions, with a pH of about S to 8. The compounds will normally be
contained in
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these formulations in an amount 0.01% to 5% by weight, but preferably in an
amount
of 0.25% to 2% by weight. Thus, for topical presentation 1 to 2 drops of these
formulations would be delivered to the surface of the eye 1 to 4 times per day
according to the discretion of a skilled clinician.
s
The compounds can also be used in combination with other agents for treating
glaucoma, such as, but not limited to, (3-blockers (e.g., timolol, betaxolol,
levobetaxolol, carteolol, levobunolol, propranolol), carbonic anhydrase
inhibitors
(e.g., brinzolamide and dorzolamide), al antagonists (e.g. nipradolol), a2
agonists
~o (e.g., iopidine and brimonidine), miotics (e.g., pilocarpine and
epinephrine),
prostaglandin analogs (e.g., latanoprost, travaprost, unoprostone, and
compounds set
forth in U.S. Patent Nos. 5,889,052; 5,296,504; 5,422,368; and 5,151,444,
"hypotensive lipids" (e.g., lumigan and compounds set forth in 5,352,708), and
neuroprotectants (e.g., compounds from U.S. Patent No. 4,690,931, particularly
~s eliprodil and R-eliprodil, as set forth in a pending application U.S.S.N.
06/203350,
and appropriate compounds from W094/13275, including memantine.
The preferred compounds of Formula I are described in Examples 1 and 2.
The most preferred compound is in Example 1. Examples of formulations
anticipated
zo to be suitable for delivery of this compound to the eye are provided.
EXAMPLE 1
3-(2-Aminopropyl)-1H indazol-5-0l hydrochloride
is Step A: 1-[5-Benzyloxy-3-(2-oxo-propyl)-indazol-1-yl]-ethanone
A mixture of (5-benzyloxy-1H indazol-3-yl)-acetic acid (2 g, 7.08 mmol) and
sodium acetate (0.99 g, 12 mmol) in acetic anhydride (6 ml) was stirred at 130
°C for
3 h. After cooling, water (15 ml) and ethyl acetate (15 ml) were added to the
reaction
mixture. The aqueous layer was separated and extracted with ethyl acetate (2 x
15
so ml). The combined extracts were washed with saturated aqueous NaHC03 (2 x
20
ml) and saturated aqueous NaCI (20 ml), dried (MgS04), and evaporated to a
residue
which was purified by chromatography (silica, 15% ethyl acetate in hexane) to
give a
yellow solid (0.48 g): 'H NMR (CD30D) 8 8.35-8.30 (m, 1H), 7.48-7.23 (m, 6H),
7.04-7.02 (m, 2H), 5.11 (s, 2H), 4.02 (s, 2H), 2.77 (s, 3H), 2.27 (s, 3H); ~3C
NMR
3s (CDC13 ) 8 203.64 (C), 170.47 (C), 156.20 (C), 145.04 (C), 136.52 (C),
128.64 (CH),
128.31 (CH), 127.60 (CH), 120.51 (CH), 116.74 (CH), 102.26 (CH), 70.68 (CH2),
42.76 (CH2), 29.59 (CH3), 22.77 (CH3), MS(APCI) m/z 323 ( M+H)+.
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Step B: 1-[5-Benzyloxy-3-(2-hydroxypropyl)-indazol-1-yl]-ethanone
To a solution of the product from Step A (0.13 g, 0.4 mmol) in methanol
(8 ml) was added NaBH4 (0.016 g, 0.4 mmol) and the mixture was stirred at room
temperature for 20 min. A saturated aqueous solution of NH4C1 (20 ml) and
ethyl
s acetate (20 ml) were added to the reaction mixture. The aqueous layer was
separated
and extracted with ethyl acetate (3 x 15 ml). The combined extracts were
washed
with a saturated aqueous solution of NaCI (2 x 15 ml), dried (MgS04), and
evaporated
to give an oil (0.12 g). MS(APC1) m/z 325 (M+H)+.
io Step C: 1-[3-(2-Azido-propyl)-5-benzyloxy-indazol-1-yl]-ethanone
A solution of the product from Step B (0.12 g, 0.37 mmol) and
methanesulfonyl chloride (0.04 ml, 0.48 mmol) in CH2C12 (5 ml) and under
nitrogen
was cooled to 0 °C and triethylamine (0.07 ml, 0.48 mmol) was added.
The resulting
mixture was stirred at 0 °C for 10 min followed by the addition of a
saturated aqueous
Is solution of NH4C1 (20 ml) and ethyl acetate (20 ml). The aqueous layer was
separated
and extracted with ethyl acetate (2 x 5 ml). The combined extracts were washed
with
a saturated aqueous solution of NaCI (2 x 15 ml), dried (MgSOa), and
evaporated to a
residue which was dissolved in DMF (3 ml). Sodium azide (0.08 g, 1.2 mmol) was
added and the mixture was stirred at 70 °C for 18 h. After cooling,
water (20 ml) and
2o ether (20 ml) were added and the aqueous layer was separated and further
extracted
with ether (3 x 20 ml). The combined extracts were washed with a saturated
aqueous
solution of NaCI (3 x 1 S ml), dried (MgS04), and evaporated to a residue that
was
purified by chromatography (silica, 10% ethyl acetate in hexane) to give a
yellow oil
(0.12 g). MS(ES) m/z 350 ( M+H)+.
2s
Step D: 3-(2-Aminopropyl)-1H indazol-5-0l hydrochloride
A solution of the product from Step C (0.12 g, 0.34 mmol) in methanol
(20 ml) was shaken under a hydrogen atmosphere (35 psi) in the presence of 10%
palladium-on-carbon (0.12 g) for 18h. The catalyst was removed by filtration
and the
3o filtrate was evaporated to a residue, which was purified by chromatography
to give an
oil. Treatment of the oil with a 1 N solution of HCl in ethanol gave the
hydrochloride
salt as a colorless semi-solid (0.013 g): ' H NMR (CD30D) 8 7.34-7.30 (m, 1
H), 7.05-
6.98 (m, 2H), 7.04-7.02 (m, 2H), 3.68-3.58 (m, 1H), 3.19-3.05 (m, 2H), 1.19-
1.16 (d,
J = 6 Hz, 3H), MS(ES) mlz 192 ( M+H)+. Treatment of the oil prepared by this
ss procedure with fumaric acid gave the fumarate salt as a gray solid; mp 227-
230°C.
Analysis: Calculated for C~oH13N30 . CaH4O4 . 0.3H20: C, 53.77; H, 5.63; N,
13.43.
Found: C, 53.83; H, 5.85; N, 13.34.
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EXAMPLE 2
3-(2-Aminopropyl)-1-methyl-1~1 indazol-5-0l fumarate
s
Step A. 1-(5-Benzyloxy-1H indazol-3-yl)-propan-2-one
A solution of the product from Step 1 of Example 1 (2.0 g, 6.2 mmol) and
sodium hydroxide (0.3 g, 7.5 mmol) in a mixture of methanol (15 ml) and water
(15
ml) was stirred for 18 h at room temperature. The reaction mixture was
extracted with
io ethyl acetate (4 x 30 ml) and the combined extracts were washed with brine,
dried
(MgS04), and evaporated to a residue which was purified by chromatography
(silica,
ethyl acetate/hexane, 1:1) to give a syrup (1.5 g): ESI/MS m/z 281 (M+H)+.
Step B. 1-(5-Benzyloxy-1-methyl-1H indazol-3-yl)-propan-2-one
~s To a solution of the product from Step A (1.2 g, 4.28 mmol) in DMF (10 ml)
was added iodomethane (0.53 ml, 8.6 mmol) and potassium carbonate (1.2 g, 8.6
mmol); this mixture was stirred for 16 h at 70°C. After adding water
(IS ml) and
ethyl acetate (15 ml) to the reaction mixture, the aqueous layer was separated
and
extracted with ethyl acetate (3 x 20 ml). The combined extracts were washed
with
zo brine, dried (MgS04), and evaporated to a residue which was puified by
chromatography (silica, ethyl acetate/hexane, 1:1) to give a viscous oil (0.6
g):
APCI/LCMS m/z 295 (M+H)+.
Step C. 3-(2-Aminopropyl)-1-methyl-1H indazol-S-of fumarate
zs The product of Step B (0.3 g, 0.1 mmol) was treated in the manner similar
to
that described in Example 1 Steps B through D to give an oil which was
converted to
the fumarate salt (0.071 g): mp 136-139°C; LCMS m/z 206 (M+H)+.
Analysis.
Calculated for CIIH~sN30 ' C4H4O4 ~ 0.1 H20: C, 55.76; H, 5.98; N, 13.00.
Found: C,
55.53; H, 6.11; N, 13.22.
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EXAMPLE 3
2-Methyl-propionic acid 3-(2-aminopropyl)-1H indazol-5-yl ester
Step A: 3-(2-(9-Fluorenylmethoxycarbonylamino)propyl)-1H indazol-5-0l
s To a mixture of dioxane and water (4:1, 10 mL) was added 3-(2-aminopropyl)
1H-indazol-5-0l (0.10 g, 0.36 mmol), 9-fluorenylmethoxycarbonyl chloride (0.13
g,
0.54 mmol) and sodium bicarbonate (0.9 g, 0.54 mmol). The reaction mixture
poured
into dilute sodium bicarbonate and the resulting mixture extracted with ether.
The
combined organic extracts were dried (MgS04) and concentrated to a residue
that was
Io purified by chromatography.
Step B: 2-Methyl-propionic acid 3-(2-(9
fluorenylmethoxycarbonylamino)propyl)-1H indazol-5-yl ester
To a solution of 3-(2-(9-fluorenylmethoxycarbonylamino)propyl)-1H-indazol-
is 5-0l (0.17 g, 0.41 mmol) and diisopropylehtylamine (0.06 g, 0.50 mmol), in
methylene chloride (10 mL) cooled at 0° C is added 2-methylpropionyl
chloride (0.05
g, 0.5 mmol) followed by 4-dimethylaminopyridine (0.05 g, 0.40 mmol). The
reaction
allowed to warm to room temperature and then is stirred at room temperature.
The
reaction mixture is added to dilute aqueous sodium bicarbonate and extracted
with
Zo ether. The combined ether extracts are washed, dried (MgS04) and
concentrated. The
residue is purified by chromatography.
Step C: 2-Methyl-propionic acid 3-(2-aminopropyl)-1H indazol-5-yl ester
A solution of 2-methyl-propionic acid 3-(2-(9-
is fluorenylmethoxycarbonylamino)propyl)-1H indazol-S-yl ester (0.16g, 0.33
mmol) in
a l: 4 mixture of piperidine and dimethylformamide (2.5 mL) is stirred at
ambient
temperature. The reaction mixture is poured into dilute aqueous sodium
bicarbonate
and the resulting mixture is extracted first with ethyl acetate and then with
methylene
chloride. The combined organic extracts are dried (MgS04) and concentrated.
The
3o residue is purified by chromatography.
Receptor and binding agonist activity according to this invention can be
determined using the following methods.
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METHOD 1
SHTZ Receptor Binding Assay
To determine the affinities of serotonergic compounds at the 5HT2 receptors,
s their ability to compete for the binding of the agonist radioligand
[1z'I]DOI to brain
SHTZ receptors is determined as described below with minor modification of the
literature procedure [Neuropharmacology, 26, 1803 (1987)]. Aliquots of post
mortem
rat or human cerebral cortex homogenates (400 ~1) dispersed in 50 mM TrisHCl
buffer (pH 7.4) are incubated with [l2sl]DOI (80 pM final) in the absence or
presence
io of methiothepin (10 ~M final) to define total and non-specific binding,
respectively, in
a total volume of 0.5 ml. The assay mixture is incubated for 1 hour at
23°C in
polypropylene tubes and the assays terminated by rapid vacuum filtration over
Whatman GFB glass fiber filters previously soaked in 0.3% polyethyleneimine
using
ice-cold buffer. Test compounds (at different concentrations) are substituted
for
is methiothepin. Filter-bound radioactivity is determined by scintillation
spectrometry on
a beta counter. The data are analyzed using a non-linear, iterative curve-
fitting
computer program [Trends Pharmacol. Sci., 16, 413 (1995)] to determine the
compound affinity parameter. The concentration of the compound needed to
inhibit
the [~ZSI]DOI binding by 50% of the maximum is termed the ICso or K; value.
2o
METHOD 2
SHTZ Functional Assay: Phosphoinositide (PI) turnover assay
The relative agonist activity of serotonergic compounds at the SHTZ receptor
Zs can be determined in vitro using the ability of the compounds to stimulate
the
production of [3H]inositol phosphates in [3H]myo-inositol-labeled A7r5 rat
vascular
smooth muscle cells by their ability to activate the enzyme phospholipase C.
These
cells are grown in culture plates, maintained in a humidified atmosphere of 5%
COZ
and 95% air and fed semi-weekly with Dulbecco's modified Eagle medium (DMEM)
3o containing 4.5 g/1 glucose and supplemented with 2mM glutamine, 10 ~g~'ml
gentamicin, and 10% fetal bovine serum. For the purpose of conducting the
phosphoinositide (PI) turnover experiments, the A7r5 cells are cultured in 24-
well
plates as previously [J. Pharmacol. Expt. Ther., 286, 411 (1998)]. Confluent
cells are
exposed for 24-30 hrs to 1.5 ~Ci [3H]-myo-inositol (18.3 Ci/mmol) in 0.5 ml of
3s serum-free medium. Cells are then rinsed once with DMEM/F-12 containing 10
mM
LiCI prior to incubation with the test agent (or solvent as the control) in
1.0 ml of the
same medium for 1 hr at 37°C, after which the medium is aspirated and 1
ml of cold
0.1 M formic acid added to stop the reaction. The chromatographic separation
of
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[3H]-inositol phosphates ([3H]-IPs) on an AG- 1-X8 column is performed as
previously described [J. Pharmacol. Expt. Ther. 286, 411 (1998)] with
sequential
washes with H20 and 50 mM ammonium formate, followed by elution of the total
[3H]-IPs fraction with 1.2 M ammonium formate containing 0.1 M formic acid.
The
s eluate (4 ml) is collected, mixed with 15 ml scintillation fluid, and the
total [3H]-IPs
determined by scintillation counting on a beta-counter. Concentration-response
data
are analyzed by the sigmoidal fit function of the Origin Scientific Graphics
software
(Microcal Software, Northampton, MA) to determine agonist potency (ECSO value)
and efficacy (Emax). Serotonin (SHT) is used as a positive control (standard)
agonist
io compound and the efficacy of test compounds is compared to that of SHT (set
at
100%). The concentration of the compound needed to stimulate the production of
[3H]-IPs by 50% of the maximum response is termed the ECSO value.
The above procedures were used to generate the data shown in Table 1.
~s
TABLE 1
SHTZ Receptor Binding and Functional Data
Compound ICso, ECso, Efficacy
nM nM
(Emax~
/~
3-(2-Aminopropyl)-1H indazol-5-0l2.5 1,210 97
hydrochloride
3-(2-Aminopropyl)-1-methyl-1H-- 778 98
indazol-5-0l fumarate
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EXAMPLE 4
Ingredients Amount (wt %)
3-(2-Aminopropyl)-1-methyl-1H 0.01-2~
indazol-
5-0l fumarate
Hydroxypropyl methylcellulose 0.5%
Dibasic sodium phosphate (anhydrous)0.2%
Sodium chloride 0.5%
Disodium EDTA (Edetate disodium)0.01%
Polysorbate 80 0.05%
Benzalkonium chloride 0.01
Sodium hydroxide / HydrochloricFor adjusting pH
acid
Purified water q.s. to 100%
EXAMPLE 5
Ingredients Amount (wt %)
3-(2-Aminopropyl)-1H indazol-5-0l0.01-2%
Methyl cellulose 4.0%
Dibasic sodium phosphate 0.2%
(anhydrous)
Sodium chloride 0.5%
Disodium EDTA (Edetate disodium)0.01
Polysorbate 80 0.05%
Benzalkonium chloride 0.01
Sodium hydroxide / HydrochloricFor adjusting pH
acid
Purified water q.s. to 100%
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EXAMPLE 6
Ingredients Amount (wt %)
3-(2-Aminopropyl)-1H indazol-5-0l0.01 - 2%
Guar gum 0.4- 6.0%
Dibasic sodium phosphate (anhydrous)0.2%-
Sodium chloride 0.5%
Disodium EDTA (Edetate disodium)0.01
Polysorbate 80 0.05%
Benzalkonium chloride 0.01
Sodium hydroxide / HydrochloricFor adjusting pH
acid
Purified water q.s. to 100%
EXAMPLE 7
Ingredients Amount (wt %)
2-Methyl-propionic acid 3-(2-aminopropyl)-0.01 - 2%
1H indazol-5-0l ester
White petrolatum and mineral oil Ointment consistency
and lanolin
Dibasic sodium phosphate (anhydrous)0.2%
Sodium chloride 0.5%
Disodium EDTA (Edetate disodium) 0.01
Polysorbate 80 0.05%
Benzalkonium chloride 0.01
Sodium hydroxide / Hydrochloric For adjusting pH
acid
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