Note: Descriptions are shown in the official language in which they were submitted.
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-1-
PHARMACEUTICALLY ACTIVE COMPOUND AND METHODS OF USE
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to N-(2-chloro-5-methylthiophenyl)-
N'-(3-methylsulfinylphenyl)-N'-methylguanidine, N-(2-chloro-5-
methylsulfinylphenyl)-1-(7-trifluoromethyl-1,2,3,4-
tetrahydroquinolinyl)carboximidamide, and N-(3-methylsulfinylphenyl)-N-
methyl-N'-(2-chloro-5-methoxyphenyl)guanidine and pharmaceutically
acceptable salts thereof, and pharmaceutical compositions and therapeutic
methods of treatment that comprise such compounds ("compounds of the
invention"). The compounds of the invention are particularly useful for the
treatment or prophylaxis of neurological injury and neurodegenerative
disorders.
2. Background
A number of substituted guanidines have been reported. See, e.g.,
U.S. Patent Nos. 1,411,731, 1,422,506, 1,597,233, 1,642,180, 1,672,431,
1,730,388, 1,756,315, 1,795,739, 1,850,682, 2,145,214, 2,254,009,
2,633,474, 3,117,994, 3,140,231, 3,159,676, 3,228,975, 3,248,426,
3,252,816, 3,283,003, 3,270,054, 3,301,755, 3,320,229, 3,301,775,
3,409,669, 3,479,437, 3,547951, 3,639,477, 3,681,457, 3,769,427,
3,784,643, 3,803,324, 3,908,013, 3,949,089, 3,975,533, 3,976,787,
4,060,640, 4,014,934, 4,161,541, 4,709,094, 4,906,779, 5,093,525, and
5,190,976; PCT applications WO 90/ 12575, WO 91 / 12797, WO 91 / 18868,
WO 92/ 14697, WO 94/ 14461, WO 94/27591, WO 95/ 14467, WO
95/20950, H.W. Geluk, et al., J. Med. Chem., 12:712 (1969), and N. L.
Reddy et al., J. Med. Chem., 37:260-267 (1994). WO 94/27591 discloses
inter alia N-(2-chloro-5-methylthiophenyl)-N'-(3-methylthiophenyl)-N'-
methylguanidine.
Nerve cell death (degeneration) can cause potentially devastating and
irreversible effects for an individual and may occur e.g. as a result of
stroke,
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-2-
heart attack or other brain or spinal chord ischemia or trauma.
Additionally, nerve cell death (degeneration) occurs with neurodegenerative
disorders such as Alzheimer's disease, Parkinson's disease, Huntington's
disease, Amyotrophic Lateral Sclerosis, Down's Syndrome and Korsakoff s
disease.
Therapies have been investigated to treat nerve cell degeneration and
related disorders, e.g., by limiting the extent of nerve cell death that may
otherwise occur to an individual.
The compound MK-801 has exhibited good results in a variety of in
10 vivo models of stroke. See B. Meldrum, Cerbrovascular Brain Metab. Rev.,
2:27-57 ( 1990); D. Choi, Cerbrovascular Brain Metab. Rev., 2:105-14?
(1990). See also Merck Index, monograph 3392, 11th ed., 1989. For
example, MK-801 exhibits good activity in mouse audiogenic tests, a
recognized model for evaluation of neuroprotective drugs. See, e.g., M.
Tricklebank et al., European Journal of Pharmacology, 167:127-135 ( 1989);
T. Seyfried, Federation Proceedings, 38(10):2399-2404 (1979).
However, MK-801 also has shown toxicity and further clinical
development of the compound is currently uncertain. See J. W. Olney et al.,
Science, 244:1360-1362 (1989); W. Koek et al., J. Pharmacol. Exp. Ther.,
252:349-357 ( 1990); F.R. Sharp et al., Society for Neuroscience Abstr.,
abstr.
no. 482.3 (1992).
It thus would be highly desirable to have new neuroprotection
therapies.
SUMMARY OF THE INVENTION
25 In a first preferred aspect, the present invention provides N-(2-chloro-
5-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine, and
pharmaceutically acceptable salts thereof, i.e. the compound of the following
structure (I):
Cl CH3
H
\ N II N /
NH
SCH3 S(O)CH3
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-3-
and pharmaceutically acceptable salts thereof. References herein to
"compound (I)" refer to the compound of the above structure as well as
pharmaceutically acceptable salts of N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine.
The invention also includes optically enriched mixtures of compound
(I). As can be seen from the above structure, the sulfur of the 3-
methylsulfinyl group is chiral. An optically enriched mixture of compound
(I) contains substantially more (e.g. about 60 mole%, 70 mole%, 80 mole%
or 90 mole% or more) of one enantiomer than the other enantiomer. For use
in the therapeutic methods of the invention, preferably a substantially pure
optically active mixture is employed, e.g. a mixture containing at least about
92 mole%, or 95 mole% or even 97 mole%, 98 mole% or 99 mole% or more
of one enantiomer of compound (I).
More specifically, the invention includes following optically active (-)-
N-(2-chloro-5-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-
methylguanidine (referred to herein, and shown below as compound (IA))
and (+)-N-(2-chloro-5-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-
methylguanidine (referred to herein, and shown below as compound (IB)).
CI
H
\ N~N \
(-)
~ /
$CH3 S(O)CH3
~3
H
\ N~ N \ (IB)
(+)
/ NH
SCH3 S(O)CH3
Compound (I) (which includes compounds I(A) and/or compound I(B))
has been found to exhibit potent neuroprotecdve activity. References herein
to "compound (I)" are intended to also refer to compound I(A) and/or
compound I(B) unless otherwise specified. Those optically active compounds
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/2i395
-4-
also can be identified as (R)-N-(2-chloro-S-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine (referred to herein, and shown
below as compound (IA)) and (S)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine, pursuant to the Cahn-Ingold-
Prelog convention.
Compound (I) also has been surprisingly found to exhibit significantly
decreased untoward behavioral effects in a number of in vivo assays,
including Irwin tests and rat rotarod motor coordination tests. See the
results of Examples 8 and 9 which follow.
In a further aspect, the invention provides the compound N-(2-chloro-
5-methylsulfmylphenyl)-1-(7-trifluoromethyl-1,2, 3,4-tetrahydroquinolinyl)
carboximidamide, and pharmaceutically acceptable salts thereof, i.e. the
compound of the following structure (II):
Ci
H
N~ N \ (II)
NH
CF3 S(O)CH3
and pharmaceutically acceptable salts thereof. References herein to
"compound (II)" refer to the compound of the above structure as well as
pharmaceutically acceptable salts of N-(2-chloro-5-methylsulfinylphenyl)-1-
(7-trifluoromethyl-1,2,3,4-tetrahydroquinolinyl)carboximidamide.
The invention includes both racemic mixtures and optically enriched
mixtures of compound (II). An optically enriched mixture contains
substantially more (e.g., about 60 mole%, 70 mole%, 80 mole%, 90 mole%
or 95 mole% or 98 mole% or more) of one enantiomer of compound (II) than
the other stereoisomer. For use in the therapeutic methods of the
invention, preferably a substantially pure optically active mixture is
employed, e.g. a mixture containing at least about 92 mole%, or 95 mole%
or even 97 mole%, 98 mole% or 99 mole% or more of one enantiomer of
compound (II).
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-5-
In a yet further aspect, the invention provides the compound N-(3-
methylsulfinylphenyl)-N-methyl-N'-(2-chloro-5-methoxyphenyl)guanidine,
and pharmaceutically acceptable salts thereof, i.e. the compound of the
following structure (III):
~3
/ ~ N
/ (III)
OCIi3 S(O)CH3
and pharmaceutically acceptable salts thereof. References herein to
"compound (III)" refer to the compound of the above structure as well as
pharmaceutically acceptable salts of N-(3-methylsulfinylphenyl)-N-methyl-
N'-(2-chloro-5-methoxyphenyl)guanidine.
The invention includes both racemic mixtures and optically enriched
mixtures of compound (III). An optically enriched mixture contains
substantially more (e.g., about 60 mole%, 70 mole%, 80 mole%, 90 mole%
or 95 mole% or 98 mole% or more) of one enantiomer of compound (III) than
the other stereoisomer. For use in the therapeutic methods of the
invention, preferably a substantially pure optically active mixture is
employed, e.g. a mixture containing at least about 92 mole%, or 95 mole%
or even 97 mole%, 98 mole% or 99 mole% or more of one enantiomer of
compound (III).
It has been found that compounds (I), (II) and (III) are each useful for
a number of therapeutic applications. In particular, the invention includes
methods for treatment and/or prophylaxis of neurological
conditions/injuries such as epilepsy, neurodegenerative conditions and/or
nerve cell death (degeneration) resulting from e.g. hypoxia, hypoglycemia,
brain or spinal chord ischemia, retinal ischemia, brain or spinal chord
trauma or post-surgical neurological deficits and the like as well as
neuropathic pain. The compounds of the invention are especially useful for
treatment of a person susceptible or suffering from stroke or heart attack or
neurological deficits relating to cardiac arrest, a person suffering or
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-6-
susceptible to brain or spinal cord injury, or a person suffering from the
effects of retinal ischemia or degeneration. Compounds (I), (II) and (III)
also
are each useful to treat and/or prevent various neurodegenerative diseases
such as Parkinson's disease, Huntington's disease, Amyotrophic Lateral
5 Sclerosis, Alzheimer's disease, Down's Syndrome, Korsakoff s disease,
cerebral palsy and/or age-dependent dementia. Compounds (I), (II) and (III)
will be further useful to treat and/or prevent migraines, shingles (herpes
zoster), epilepsy, emesis and/or narcotic withdrawal symptoms. Also, in
addition to treatment of retinal ischemia and related disorders, the
10 invention provides methods for treatment of optic nerve injury/damage. The
treatment methods of the invention in general comprise administration of a
therapeutically effective amount of compound (I), (II) and/or (III) to an
animal, including a mammal, particularly a human in need of treatment.
The invention also provides pharmaceutical compositions comprising
15 a therapeutically effective amount of compound (I), (II) and/or (III) and a
pharmaceutically acceptable carrier.
Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the time course effects of the hydrochloride salt of (~-
20 N-(2-chloro-5-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-
methylguanidine in the rotarod test of Example 8 which follows.
Figure 2 shows the time course effects of the acetate salt of (-)-N-(2-
chloro-5-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine
in the rotarod test of Example 8 which follows.
25 Figure 3 shows the time course effects of the acetate salt of (+)-N-(2-
chloro-5-methylthiophenyl)-N'-{3-methylsulfinylphenyl)-N'-methylguanidine
in the rotarod test of Example 8 which follows.
DETAILED DESCRIPTION OF THE INVENTION
Compound (I) may be suitably prepared as described in Example 1
30 which follows and which includes preparation of 3-methylsulfinyl-N-
methylaniline by oxidation of N-methyl-3-methylthioaniline. A variety of
oxidants may be employed, e.g. HaOa, sodium periodate and the like.
Hydrogen peroxide may be preferred because it can provide a final product
(compound (I)) that exhibits enhanced aqueous solution solubility relative to
CA 02306276 2000-04-07
WO 99/18962 PCTNS98/21395
_7_
the product of a periodate oxidation. An asymmetric oxidation also may be
conducted to provide the optically active sulfinyl (SO) moiety. See, for
example, the procedure disclosed in F. A. Davis et al., J. Org. Chem.,
57:7274-7285 ( 1992). See also the procedures set forth in Example 5 which
5 follows. Also, optically active compound (IA) or (IB), or an optically
precursor
compound such as 3-methylsulfinyl-N-methylaniline, where the sulfinyl
group is selectively either of the (R) or (S) configuration can be obtained by
column chromatography using an optically active binding material, or the
racemic compound (I) can be separated into optically active (+) and (-)
10 enantiomers by such column chromatography and optically active binding
material. See, for instance, the procedure disclosed in Example 2 which
follows.
After the oxidation step, the aniline salt then is suitably formed and
that salt reacted with 2-chloro-5-methylthiophenylcyanamide. The crude
15 compound (I) can be purified as desired, e.g. by column chromatography.
Compound (II) can be suitably prepared as generally described in
published PCT Application WO 97/30054 (PCT/US97/02678). In particular,
2-chloro-5-methylsulfinylphenylcyanamide can be reacted with 7-
trifluoromethyl-1,2,3,4-tetrahydroquinoline (the tetrahydroquinoline
20 typically in salt form such as a hydrochloride or mesylate salt) in a
suitable
solvent such as chlorobenzene or toluene under an inert atmosphere (e.g.
argon or nitrogen) with heating (e.g. reflux temperature) until reaction
completion, e.g. 2 or more hours. Additionally, 2-chloro-5-
methylthiophenylcyanamide can be reacted under similar conditions with 7-
25 trifluoromethyl-1,2,3,4-tetrahydroquinoline to form N-{2-chloro-5-
methylthiophenyl)-1-(7-trifluoromethyl-1,2,3,4-tetrahydroquinolinyl)
carboximidamide, which can be then oxidized to form the desired compound
{II). Suitable oxidants include e.g. HzOa and sodium periodate. See
Example 3 which follows for exemplary conditions. Optically active
30 stereoisomers of compound (II) can be produced as described above for
compound (I), i.e. an asymmetric oxidation may be conducted to provide the
optically active sulfinyl (SO) moiety (see F. A. Davis et al., supra), or an
optically precursor compound such as 2-chloro-5-methylsulfinylaniline,
where the sulfinyl group is selectively either of the (R) or (S) configuration
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/Z1395
_$_
can be obtained by column chromatography using an optically active
binding material. Racemic compound (II) also can be separated into
optically active (+) and (-) enantiomers by use of such column
chromatography and optically active binding material.
Compound (III) can be suitably prepared as generally described in
published PCT Application WO 94/27591 (PCT/US94/06008). In particular,
N-methyl-3-methylsulfinylaniline can be reacted with 2-chloro-5-
methoxyphenylcyanamide in a suitable solvent such as chlorobenzene or
toluene under an inert atmosphere (e.g. argon or nitrogen) with heating (e.g.
reflux temperature) until reaction completion, e.g. 2 or more hours.
Additionally, N-methyl-3-methylthioaniline can be reacted under similar
conditions with 2-chloro-5-methoxyphenylcyanamide to form N-(3-
methylthiophenyl)-N-methyl-N'-(2-chloro-5-methoxyphenyl)guanidine,
which can be then oxidized to form the desired compound (III). Suitable
oxidants include e.g. H2O2 and sodium periodate. See Example 4 which
follows for exemplary conditions. Optically active stereoisomers of
compound (III) can be produced as described above for compound (I), i.e. an
asymmetric oxidation may be conducted to provide the optically active
sulfinyl (SO) moiety (see F. A. Davis et al., supra), or an optically
precursor
compound such as N-methyl-3-methylsulfinylaniline, where the sulfinyl
group is selectively either of the (R) or (S) configuration can be obtained by
column chromatography using an optically active binding material. Racemic
compound (III) also can be separated into optically active (+) and (-)
enantiomers by use of such column chromatography and optically active
binding material.
As discussed above, the present invention includes methods for
treatment and/or prophylaxis of certain neurological disorders, including
the consequences of stroke, heart attack and traumatic head or brain
injury, epilepsy or neurodegenerative diseases comprising the
administration of an effective amount of compound (I), (II) and/or (III) of
the
invention to a subject including a mammal, particularly a human, in need of
such treatment. In particular, the invention provides methods for treatment
and/or prophylaxis of nerve cell death (degeneration) resulting e.g. from
hypoxia, hypoglycemia, brain or spinal cord ischemia, brain or spinal cord
CA 02306276 2000-04-07
WO 99118962 PCT/US98/21395
-9-
trauma, stroke, heart attack or drowning. Typical candidates for treatment
include e.g. heart attack, stroke and/or persons suffering from cardiac
arrest, neurological deficits, brain or spinal cord injury patients, patients
undergoing major surgery such as heart surgery where brain ischemia is a
potential complication and patients such as divers suffering from
decompression sickness due to gas emboli in the blood stream. Candidates
for treatment also will include those patients undergoing a surgical
procedure involving extra-corporal circulation such as e.g. a bypass
procedure. Neurodegenerative diseases that may be treated by a compound
of the invention include Parkinson's disease, Huntington's disease,
Amyotrophic Lateral Sclerosis, Alzheimer's disease, Down's Syndrome and
Korsakoff s disease.
The invention in particular provides therapeutic methods which
comprise administration of compound (I), (II) and/or (III) of the invention to
a patient that is undergoing surgery or other procedure where brain or
spinal cord ischemia is a potential risk. For example, carotid
endarterectomy is a surgical procedure employed to correct atherosclerosis
of the carotid arteries. Major risks associated with the procedure include
intraoperative embolization and the danger of hypertension in the brain
20 following increased cerebral blood flow, which may result in aneurism or
hemorrhage. Thus, an effective amount of compound (I), (II) and/or (III) of
the invention could be administered pre-operatively or peri-operatively to
reduce such risks associated with carotid endarterectomy, or other post-
surgical neurological deficits.
25 The invention further includes methods for prophylaxis against
neurological deficits resulting from e.g. coronary artery bypass graft surgery
and aortic valve replacement surgery, or other procedure involving extra-
corporal circulation. Those methods will comprise administering to a
patient undergoing such surgical procedures an effective amount of
30 compound (I), (II) and/or (III) of the invention, typically either pre-
operatively
or peri-operatively.
The invention also provides methods for prophylaxis and treatment
against neurological injury for patients undergoing myocardial infarction, a
procedure that can result in ischemic insult to the patient. Such methods
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-10-
will comprise administering to a patient undergoing such surgical procedure
an effective amount of compound (I), (II) and/or (III) of the invention,
typically either pre-operatively or peri-operatively.
Also provided are methods for treating or preventing neuropathic pain
such as may experienced by cancer patients, persons having diabetes,
amputees and other persons who may experience neuropathic pain. These
methods for treatment comprise administration of an effective amount of
compound (I), (II) and/or (III) of the invention to a patient in need of such
treatment.
The invention also provides methods for treatment and prophylaxis
against eye disorders and injury, including methods for treatment of
reduced flow of blood or other nutrients to retinal tissue or optic nerve,
methods for treatment of retinal ischemia and trauma and associated
disorders, and methods for treatment of optic nerve damage/injury.
Disorders associated with retinal or optic nerve injury or ischemia that may
be treated in accordance with the invention include e.g. diabetes,
significantly elevated intraocular pressures and glaucoma, diseases such as
retinal artery or vein occlusion, atherosclerosis, venous capillary
insufficiency, senile macular degeneration and cystoid macular edema. In
such methods, a compound of the invention can be administered
parenterally or by other procedure as described herein to a subject suffering
from or susceptible to ischemic insult or other injury or disorder that may
adversely affect visual function. Post-ischemic or post-injury administration
also may limit retinal damage. Intravitreal injection of a compound of the
invention also may be a preferred administration route to provide more
direct treatment to the injured retina or optic nerve.
The invention also provides methods for treatment of a subject
suffering from shingles as well as treatment of a person suffering from or
susceptible to migraines, particularly to alleviate the pain and discomfort
associated with those disorders. These methods comprise administration of
an effective amount of compound (I), (II) and/or (III) of the invention to a
patient in need of treatment.
The invention further provides a method of treating Korsako~'s
disease, a chronic alcoholism-induced condition, comprising administering
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-11-
to a subject including a mammal, particularly a human, compound (I), (II)
and/or (III) in an amount effective to treat the disease. Compounds of the
invention are each anticipated to have utility for the attenuation of cell
loss,
hemorrhages and/or amino acid changes associated with Korsakoffs
disease.
As discussed above, the invention also includes methods for treating
a person suffering from or susceptible to cerebral palsy, emesis, narcotic
withdrawal symptoms and age-dependent dementia, comprising
administering to a subject including a mammal, particularly a human,
compound (I), (II) and/or (III) in an amount effective to treat the condition.
For at least some therapeutic methods of the invention, it may be
preferred to use an optically enriched mixture of compound (I), (II) (III). In
particular, it may be preferred to use compound (IB), which appears to
exhibit enhanced therapeutic activity relative to the (-) enantiomer. See, for
instance, the results set forth in the examples which follow.
Compound (I), (II) or (III) of the invention may be used in therapy in
conjunction with other medicaments. For example, for treatment of a stroke
victim or a person susceptible to stroke, compound (I), (II) or (III) may be
suitably administered together with a pharmaceutical targeted for
20 interaction in the blood clotting mechanism such as streptokinase, tPA,
urokinase and other agents that lyse clots. Also, compound (I), (II) or (IiI)
may be administered together with agents such as heparin and related
heparin-based compounds, acenocoumarol or other known anticoagulants.
Compounds (I), (II) and (III) may be used singularly in a pharmaceutical
25 formulation, or two or all three of compounds (I), (II) and (III) may be
used in
combination, e.g. in the same or different pharmaceutical formulations, or
sequentially. However, preferably such a combination therapy will entail
substantially simultaneous administration of two or all three of compounds
(I), (II) and (III) to a patient, e.g. in a unitary pharmaceutical composition
30 containing the compounds.
The invention also includes methods for treatment of various pain,
e.g. migraines, chronic pain, and the like, comprising administering to a
subject, particularly a mammal such as a human, in need of such
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-12-
treatment, e.g. a subject that is suffering from migraines, chronic pain, or
other pain, an e~'ective amount of a compound of the invention.
The invention also includes methods for treatment of infections,
including Gram-negative and Gram-positive bacterial infections, comprising
administering a combination of 1) an aminoglycoside antibiotic, and 2) a
compound (I), (II) and/or (III) as defined herein. A wide variety of
aminoglycoside antibiotics are suitable for use in the formulations of the
invention. Typically, suitable aminoglycoside antibiotics contain two or
more amino sugars (aminoglycosides) connected to an amino-cyclitol
nucleus. Exemplary aminoglycoside antibiotics preferred for use in
formulations of the present invention include clinical agents such as
gentamycin, amikacin, kanamycin, streptomycin, parornoycin, neomycin,
netilmicin and tobramycin. Other suitable aminoglycosides include
seldomycins, sisomycins, aurimycin, lividomycins, streptothricins,
hybrimycins, coralinomycin, butirosin, strepomutins, nebramycins,
tenebrimycins, ribostamycins, destomycins, trehalosamines, myomycins,
fortimicins, mutamicins and kasugamycin. Suitable aminoglycoside
antibiotics are also disclosed in U.S. Patents Nos. 5,508,269; 4,645,760; and
4,380,625. It should be appreciated however that the present invention is
not limited by any particular aminoglycoside antibiotic, and the invention is
applicable to any aminoglycoside antibiotic now known or subsequently
discovered or developed. The aminoglycoside and compound (I), (II) and/or
(IiI} may be administered simultaneously, in the same or different
pharmaceutical formulations, or sequentially. Preferably, the components of
the combination are administered substantially simultaneously, e.g. in a
unitary pharmaceutical composition containing the two components.
Preferred methods and compositions that comprise an aminoglycoside in
combination with compound (I), (II) and/or (III) will be effective against
infections previously treated with aminoglycoside antibiotics, but with the
significant advantage of decreased occurrence of ototoxicity relative to use
of
an aminoglycoside antibiotic alone.
The invention also provides methods in vitro and in vivo binding
acfivity diagnostic methods using a radiolabelled form of compound (I), (II)
or (III), e.g., compound (I), (II) or (III) that is labeled with tritium, msI
or the
CA 02306276 2000-04-07
WO 99/18962 PGT/US98/21395
-13-
like. Such a labeled compound (I), (II) or (III) can be administered to a
mammal and the subject then scanned for binding of the compound.
Specifically, single photon emission computed tomography ("SPELT") can be
employed to detect such binding. Such an analysis of the mammal could
e.g. aid in the diagnosis and treatment of acute cerebral ischemia. That is,
labeled compound (I) will selectively bind to ischemic tissue of e.g. a
subject's brain to differentiate between ischemic and non-ischemic tissue
and thereby assess trauma or other injury to the brain. Accordingly, the
invention also includes compound (I), (II) or (III) that is in a radiolabelled
form such as tritium or 1251.
Compounds of the invention can be administered intranasally, orally
or by injection, e.g., intramuscular, intraperitoneal, subcutaneous or
intravenous injection, or by transdermal, intraocular or enteral means. The
optimal dose can be determined by conventional means. A compound of the
invention is suitably administered to a subject in the protonated and water-
soluble form, e.g., as a pharmaceutically acceptable salt of an organic or
inorganic acid, e.g., hydrochloride, sulfate, hemi-sulfate, phosphate,
nitrate,
acetate, oxalate, citrate, maleate, mesylate, etc.
A compound of the invention can be employed, either alone or in
combination with one or more other therapeutic agents as discussed above,
as a pharmaceutical composition in mixture with conventional excipient,
i.e., pharmaceutically acceptable organic or inorganic Garner substances
suitable for parenteral, enteral or intranasal application which do not
deleteriously react with the active compounds and are not deleterious to the
recipient thereof. Suitable pharmaceutically acceptable Garners include but
are not limited to water, salt solutions, alcohol, vegetable oils,
polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,
viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides,
petroethrai fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone,
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,
colorings, flavorings and/or aromatic substances and the like which do not
deleteriously react with the active compounds.
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
- 14-
For parenteral application, particularly suitable are solutions,
preferably oily or aqueous solutions as well as suspensions, emulsions, or
implants, including suppositories. Ampules are convenient unit dosages.
For enteral application, particularly suitable are tablets, dragees or
capsules having talc and/or carbohydrate Garner binder or the like, the
carrier preferably being lactose and/or corn starch and/or potato starch. A
syrup, elixir or the like can be used wherein a sweetened vehicle is
employed. Sustained release compositions can be formulated including
those wherein the active component is protected with differentially
degradable coatings, e.g., by microencapsulation, multiple coatings, etc.
Intravenous or parenteral administration, e.g., sub-cutaneous,
intraperitoneal or intramuscular administration are generally preferred.
It will be appreciated that the actual preferred amounts of a
compound of the invention used in a given therapy will vary according to the
particular compositions formulated, the mode of application, the particular
site of administration, etc. Optimal administration rates for a given protocol
of administration can be readily ascertained by those skilled in the art using
conventional dosage determination tests conducted with regard to the
foregoing guidelines. In general, a suitable effective dose of compound (I),
(II) or (III) will be in the range of from 0.01 to 100 milligrams per kilogram
of
bodyweight of recipient per day, preferably in the range of from 0.01 to 20
milligrams per kilogram bodyweight of recipient per day, more preferably in
the range of 0.05 to 4 milligrams per kilogram bodyweight of recipient per
day. The desired dose is suitably administered once daily, or several sub-
doses, e.g. 2 to 4 sub-doses, are administered at appropriate intervals
through the day, or other appropriate schedule. Such sub-doses may be
administered as unit dosage forms, e.g., containing from 0.05 to 10
milligrams of compounds) of the invention, per unit dosage, preferably from
0.2 to 2 milligrams per unit dosage.
As with prior guanidines such as those reported in U.S. Patent No.
1,411,713, compounds of the invention should have utility as rubber
accelerators.
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-IS_
The entire text of all documents cited herein are incorporated herein
by reference. The invention is further illustrated by the following non-
limiting examples.
Example 1 Preparation of (~)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine hydrochloride
Part 1. Preparation of 3-methylsulfinyl-N-methylaniline
To a well cooled (ice bath) solution of N-methyl-3-methylthioaniline
(30.4 g, 198.4 mmol) in acetone (250 ml) was added dropwise hydrogen
peroxide (25%, 92 ml) over a period of 15 minutes. The reaction mixture
was stirred for 2 hours and concentrated under reduced pressure to remove
acetone. The reaction mixture was diluted with water (200 ml) and
repeatedly extracted with ethyl acetate (3 x 100 ml). Combined organic
extracts were washed with sodium thiosulfate solution ( 10%, 50 ml), dried
and concentrated to afford the crude reaction mixture. The residue was
chromatographed on silica gel using gradually mixtures of hexanes:ethyl
acetate initially 4:1, and later 3:2. These elutions removed most of the
unreacted starting material and most of the sulfone formed. Finally elution
with ethyl acetate and later ethyl acetate containing 5% methanol eluted the
sulfoxide and the fractions were combined, concentrated under reduced
pressure to yield the sulfoxide as an oil (19 g, 5?%); TLC (CHCI3:MeOH;
19:1); Rf--0.48; 1H-NMR (CDCla) s 2.69 (s, 3H, S(O)Me), 2.86 (s, 3H, NMe),
6.67 (dd, 1H), 6.81 (dd, 1H), 6.92 (d, 1H), 7.27 (m, 1H) HPLC 98.6% @ 8.6
minutes.
Part 2. Preparation of 3-methylsulfmyl-N-methylaniline
hydrochloride
The free amine, ( 11.66 g, 68.90 mmol), obtained in Part 1 above, was
dissolved in dichloromethane ( 100 ml; cooled in ice cold water (5- lO~Cj) and
to that solution anhydrous hydrogen chloride in ether (1M, 80 ml) was
added slowly with vigorous stirring to enable thorough mixing. After the
addition was complete, the mixture was stirred for 30 minutes, and then
concentrated under reduced pressure and dried under high vacuum to
afford the hydrochloride salt as hygroscopic yellow fluffy solid in almost
quantitative yield. 1H-NMR (CD30D) b 2,87 (s, 3H, S(O)Me), 3.15 (s, 3H,
NMe) 7.65 (m, 1H), 7.77 (m, 1H), 7.83 (m, 1H); HPLC 98.6% @ 8.63 minutes.
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
- 16-
Part 3. Preparation of (J-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine hydrochloride
2-chloro-5-methylthiophenylcyanamide (9.28 g, 46.7 mmol) and
toluene (50 ml) was added to a solution of 3-methylsulfinyl-N-methylaniline
hydrochloride (7.17 g, 34.85 mmol) in dichloromethane (50m1). This
mixture was slowly heated in an oil bath to 135-140°C and the
dichloromethane distilled was collected. After the complete distillation of
dichloromethane the reaction mixture was maintained at 135-140°C for 1
hour, then cooled to room temperature and the solvent was removed under
reduced pressure. The residue was purified by silica gel column
chromatograph using initially chloroform and gradually to chloroform
containing 5% methanol as eluents. The title compound was obtained as a
light colored product upon concentration of fractions was dried under high
vacuum (5.45 g, 39%); mp: 148-154°C; HPLC 93.2 % @ 14.3 minutes; TLC
(CHCI3:MeOH; 4:1); Rf--0.33 1H-NMR (CDsOD) S 2.46 (s, 3H, SMe), 2.82 (s,
3H, S(O)Me), 3.53 (s, 3H, NMe), 7.19 (s, 1H), 7.21 (d, 1H), 7.40 (dd, 1H),
7.65
(m, 1H), 7.70 (m, 2H), 7.81 (d, 1H); Anal. Calcd. for C16H19C12N3OS2'1.5 HzO;
C:44.55, H: 5.14, N:9.27, S:14:37; Found: C: 44.23, H:4.75, N:9.35, S:14.09.
Example 2 Resolution of (~-N-(2-chloro-5-methylthiophenyl)-N'-{3-
methylsulfinylphenyl)-N'-methylguanidine (compound (I)) to provide(-)-N-(2-
chloro-5-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine
(compound (IA)) and (+)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine (compound (IB)).
2.25g of the free base of (~-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine was dissolved in ethanol and
chromatographed on a Chiralpak AD column (2 x 25 cm, Diacel Chemical
Ind.) at 40°C using 100% ethanol as eluent, and W detection at 300
nm.
The (-) enantiomer of compound (I), i.e. compound (IA), eluted first followed
by the (+) enantiomer of compound (I), i.e. compound (IB). The fractions
corresponding to pure enantiomers were pooled and evaporated to provide
1.07 g of compound (IA) and 1.01 g of compound (IB) (yields of 95% and
90%, respectively) as resins (free base) that crystallized at 4°C.
Analysis on
a Chiralpak AD analytical column (4.6 x 250 mm; 100% ethanol mobile
phase) showed that compound (IA) was obtained in 99.8% ee and compound
CA 02306276 2000-04-07
WO 99118962 PCT/US98/21395
- 17-
(IB) was obtained at 98.6% ee (retention times of 8.5 minutes and 16
minutes, respectively). Purifies were further evaluated by reversed phase
HPLC (Ultrasphere ODS column), 5 micron, 4.6 x 25 cm; 220 nm; 30 minute
linear gradient; 2 to 98% AcCN is water/0/ 1% TFA). Compound (IA) was
found to be 99.0% pure, and compound (IB) was found to be 98.7% pure.
Proton NMR showed trace of ethanol as only impurity.
Example 3 Preparation of N-(2-chloro-5-methysult3nylpheayl)-1-(1,2,3,
4-tetrahydroquiaoliayl)carboaumidamide
Part 1: Preparation of 7-trifluoromethyl-1,2,3,4-tetrahydroquinoline
hydrochloride
N \ HHh1 \
/ Methanol H~ ~ /
~3 ~3
A solution of 4-chloro-7-trifluoromethylquinoline (15 g, 64.8 mmole)
in methanol (75 ml) was added platinum (IV) oxide ( 1.5 g, 10% w/w) in a
250 ml Parr reaction vessel. The slurry was hydrogenated at 45-50 psi for
5-6 hours. The catalyst was filtered (Fire hazard!) and the clean filtrate was
concentrated in vacuo to afford the product as the hydrochloride salt, a
white solid, yield 15.3 g; mp: 164-168°C; HPLC 99% @ 20.6 minutes; 1H-
NMR (CDsOD) s 2.27 (m, 2H, CHa), 2.95 (t, 2H, CHa), 3.58 (t, 2H, CH2), 7.37
(d, 2H, ArH), 7.57 (d, 1H, ArH), 7.91 (s, 1H, ArH).
Part 2: Preparation of N-(2-chloro-5-methylthiophenyl)-1-(7-
trifluoromethyl-1,2,3,4-tetrahydroquinoline)carboxamidamide
/ ~ / N N \
\ ~~2
\ .HCI ~ Xylenes, heat \ NHHCI /
SMe SMe CF3
~3
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
- 18-
Condensation of 2-chloro-5-methylthiophenylcyanamide (2.4 g, 12
mmol) and 1,2,3,4-tetrahydroquinolinehydrochloride (2.0 g, 8.4 mmol) in
dichloromethane (5 ml) and xylenes (5 ml) was carned out as described in
Example 1, Part 3 above to provide N-(2-chloro-5-methylthiophenyl)-1-(7-
trifluoromethyl-1,2,3,4-tetrahydroquinoline)carboxamidamide: Yield 54%;
mp: 128-132°C; HPLC 96% @ 21.1 minutes; TLC (CHCIs:MeOH; 9:1);
Rf--0.57; 1H-NMR (CDsOD) s 2.07 (t, 2H, CH2), 2.47 (s, 3H, SMe), 2.89 (t, 3H,
CHa), 3.83 (t, 2H, CH2), 6.98 (m, 2H, ArH), 7.36 (m, 3H, ArH), 7.82 (s, 1H,
ArH) .
Part 3. Preparation of N-(2-chloro-5-methysulfinylphenyl)-1-
( 1,2,3,4-tetrahydroquinolinyl)carboxamidamide
To a solution of N-(2-chloro-5-methylthiophenyl)-1-(7-trifluoromethyl-
1,2,3,4-tetrahydroquinoline)carboxamidamide ( 1 g, 2.29 mmol) pepared in
Part 2 above of this Example in 10 ml of methanol and water (5 ml) sodium
I 5 periodate ( 1.3 g, 6.08 mmol) was added and the solution stirred
overnight.
Upon filitering the solid separated, the solid was washed with
dichloromethane ( 10 ml) and the filtrate was concentrated to remove
methanol and dichloromethane. The solution was made basic to pH ~ 12
with sodium hydroxide ( 1N, 25 ml) and extracted repeatedly with chloroform
(3 X 20 ml). Filtered to remove any solid separated, washed with organic
layer with water (20 ml), dried and concentrated the organic layer to give the
crude product. The free base (0.75 g), obtained was purified by column
chromatography eluting initially with methanol and gradually to chloroform
containing methanol (5%) to give the pure product. The pure product was
dissolved in methanol ( 10 ml), cooled in ice-water bath and was added 12 ml
of ethereal solution of hydrogen chloride (1M). After stirring for 30 minutes
the reaction mixture was concentrated, coevaporated twice with 10 ml of
dichloromethane to give the hydrochloride salt as a white solid and dried
under high vacuum. Yield 0.78 g (73%) mp: 144-148°C; HPLC 93% @ 16.7
minutes; TLC (CHCIs:MeOH; 9:1); Rf--0.63; 1H-NMR (CDaOD) s 2.18 (t, 2H,
CHa), 2.81 (s, 3H, SOMe), 2.89 (t, 3H, CHZ), 3.96 (t, 2H, CH2), 7.45 (s, 2H,
ArH), 7.75 (s, 4H, ArH).
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-19-
Example 4 Preparation of N-(2-chloro-5-methoxyphenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine hydrochloride
Part 1: N-Methyl-N-cyano-3-methylthioaniline
CH3 CH3
N
/ ~ CNBr / wCN
\ aq. NaHC03 \
SMe SMe
A suspension of N-methyl-3-methylthioaniline (1.53 g, 10 mmol) in
water (50 ml) containing sodium bicarbonate (0.9 g, 11 mmole) cyanogen
bromide ( 1.1 g, 11 mmol) was stirred overnight. The reaction mixture was
extracted repeatedly with ethyl acetate (3 X 15 ml) and the organic layer was
washed with water (2 X 10 ml), dried over magnesium sulfate and
concentrated to give the product, 1.5 g (84%); oil; TLC (Hexanes:EtOAc;
85:15); Rf--0.33; 1H-NMR (CDCIs) S 2.48 (s, 3H, SCHs), 3.32 (s, 3H, NMe),
6.83 (d, 1H, ArH), 6.93 (m, 2H, ArH), 7.26 (d, 1H, ArH).
Part 2: Preparation of N-(2-chloro-5-methoxyphenyl)-N'-(3-
methylthiophenyl)-N'-methylguanidine hydrochloride
~3
/ ~ \ N CI Cii3
.HCI I \~ xylenes / N N \
\ + / 115-12 °
O C, 3h \ NHHC1 /
OMe SMe
OMe SMe
A mixture of N-methyl-N-cyano-3-methylthioaniline, prepared in Part
1 of this Example above, ( 1.0 g, 5.61 mmol) and 2-chloro-5-methoxyaniline
hydrochloride ( 1 g, 5.15 mmol) in xylenes (5 ml) was heated to reflux for 3
hours. The cooled reaction mixture was concentrated and the residue was
treated with dichloromethane (5 ml) followed by ether (20 ml). The solid
separated was filtered, dried. Yield 542 mg (26%); mp: 194-196°C; HPLC
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-20-
98.7% @ 18.7 minutes; TLC (CHCIs:MeOH; 4:1) Rf--0.61; 1H-NMR (CDsOD) s
2.52 (s, 3H, SMe), 3.48 (s, 3H, NMe), 3.8 (s, 3H, OMe), 6.98 (m, 2H, ArH),
7.21 (d, 1H, ArH), 7.33 (m, 2H, ArH), 7.43 (m, 2H, ArH).
Part 3: Preparation of N-(3-chloro-5-methoxyphenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine hydrochloride
Cl CH3
/ N N \ N / N N \
M ato4
\ NI~HCI / e0I~H20 \ N1~HC1
OMe SMe OMe S(O)Me
To a solution of N-(2-chloro-5-methoxyphenyl)-N'-(3-
methylthiophenyl)-N'-methylguanidine hydrochloride, prepared in Part 2 of
this Example above, (0.4 g, 1.07 mmol) in methanol (5 ml) and water (3 ml)
was added sodium periodate (275 mg, 1.29 mmol) and the reaction mixture
was stirred at room temperature overnight. Upon filtering the solid
separated, the residue washed with dichloromethane ( 10 ml) and the filtrate
concentrated. The solution was made alkaline with sodium hydroxide ( 10
ml, 1N) and repeatedly extracted with dichloromethane (3 X 10 ml), washed
with organic layer with water (2 X S ml), dried and concentrated. The crude
product was purified by column using chloroform initially and gradually to
chloroform containing 12% methanol to isolate the title compound. Yield
300 mg (72%); mp: 72-74°; HPLC 99.3% @ 13.3 minutes; TLC (CHCIs:
MeOH; 4:1) Rf--0.38; 1H-NMR (CDsOD) 8 2.83 (s, 3H, SOMe), 3.48 (s, 3H,
NMe), 3.8 (s, 3H, OMe), 6.76 (m, 2H, ArH), 7.33 (m, 1H, ArH), 7.61 (m, 3H,
ArH), 7.78 (s, 1H, ArH).
Ermmple 5: Preparation of (R)-N-(2-chloro-3-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine acetate
To a solution of (R)-N-(2-chloro-3-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine ( 123 mg, 0.34 mmol) in
chloroform (5 ml) in ice bath (5-10°C) was added acetic acid in
chlorofom
(1M, 0.45 ml) was added slowly and stirred for 15 mins, concentrated under
reduced pressure followed by coevaporation with dichloromethane (3X5 ml)
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-21-
to obtain a white low melting solid of acetate salt (140 mg); 1H-NMR(CDsOD)
s 1.92 (s, 3H, MeCO), 2.47 (s, 3H, SMe), 2.82 (s, 3H, S(O)Me), 3.50 (s, 3H,
NMe), 7.11 {m, 2H, Ar), 7.37 (dd, 1H, Ar), 7.62-7.66 (m, 3H, Ar), 7.78 (m, 1H,
Ar); Anal. Calcd. For C18H22C1N3O3S2 0.5Ha0; C: 49.43, H: 5.26, N: 9.61;
Found: C: 49.63, H: 5.33, N: 9.49.
Example 6: Preparation of /8)-N-/2-chloro-3-methylthiophenyl)-N'-/3-
methylsulfinylphenyl)-N'-methylguanidine acetate
Following a similar procedure as disclosed in Example 5 above, (S)-N-
(2-chloro-3-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-
methylguanidine ( 125 mg, 0.34 mmol) was converted to the acetate salt ( 146
mg); 1H-NMR(CDsOD) s 1.94 (s, 3H, MeCO), 2.49 (s, 3H, SMe), 2.84 (s, 3H,
S(O)Me), 3.52 (s, 3H, NMe), 7.13 {m, 2H, Ar), 7.4 (dd, 1H, Ar), 7.64-7.68 {m,
3H, Ar), 7.80 {m, 1H, Ar); Anal. Calcd. For C18H22C1N3O3S2 0.5Ha0; C: 49.43,
H: 5.26, N: 9.61; Found: C: 49.15, H: 5.25, N: 9.41.
Example 7: Chiral Synthesis of (R)-N-{2-chloro-3-methylthiophenyl)-N'-(3-
methylsulf'mylphenyl)-N'-methylguanidine
A. Preparation of (R)-3-methylsulfinyl-N-methylaniline
hydrochloride
1. Oxidation Step: (R)-3-Methylsulfinyl-N-methylaniline
To a solution of (R)-(-)-(8,8-dichloro-10-camphorsulfonyl)oxaziridine
( 1.65 g, 5.53 mmol) in carbon tetrachloride (50 ml) under stirring was
added N-methyl-3-methylthioaniline ( 1.0 g, 7.17 mmol) in 5 ml of carbon
tetrachloride at room temperature. The reaction mixture was stirred for
overnight (-18 hours) and the reaction mixture was filtered to remove the
solid separated, washed with carbon tetrachloride ( 10 ml) and the combined
filtrate was concentrated . The residue was chromatographed on silica gel
using gradually a mixtures of hexanes:ethyl acetate initially 4:1, to remove
the unreacted starting material. Finally elution with chloroform and later
chloroform containing 2% methanol eluted the sulfoxide and the fractions
were combined, concentrated under reduced pressure to yield (R)-3-
Methylsulfinyl-N-methylaniline as an oil in quantitative yield. 0.9? gm; TLC
CHCI3:MeOH 19:1; Rf = 0.48; Purity 92% ee by 1H-NMR; 1H-NMR (CDC13)
s 2.71 (s, 3H, SOMe), 2.87 (s, 3H, NMe), 6.66 {dd, 1H), 6.81 (dd, 1H), 6.92
(s, 1H), 7.28 (m, 1H).
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-22-
2. Conversion to hydrochloride
The free amine of (R)-3-Methylsulfinyl-N-methylaniline (0.97g, 5.73
mmol), obtained as above, was dissolved in dichloromethane ( 15 ml), cooled
in an ice water bath and to this solution anhydrous hydrogen chloride in
methanol (1M, 6ml) was added slowly with vigorous stirring to enable
thorough mixing. Stirred for 30 mins as such, concentrated under reduced
pressure and dried under high vacuum to afford (R)-3-methylsulfinyl-N-
methylaniline hydrochloride as hygroscopic colorless fluffy solid ( 1.12 g) in
almost quantitative yield. 1H-NMR (CD30D} s 2.87 (s, 3H, S(O)Me), 3.15 (s,
3H, NMe) 7.65 (m, 1H, ), 7.77 (m, 1H), 7.83 (m, 1H).
B. Condensation Step: (R)-N-(2-chloro-3-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine hydrochloride
In a single necked round bottomed flask was placed a solution of (R)-3-
methylsulfmyl-N-methylaniline hydrochloride ( 1.12g, 5.64 mmol) in
dichloromethane (20 ml). With stirring 2-chloro-5-
methylthiophenylcyanamide ( 1.12g, 5.44 mmol) and toluene (20 ml) were
added. The flask was fitted with a Vigreaux column attached to a short
distillation setup for collecting the solvent distilled during the reaction.
This
mixture was slowly heated in an oil bath to 115-120oC and the
dichloromethane distilled was collected. After the complete distillation of
dichloromethane, the reaction mixture was maintained at 115-120oC for 1
hour, cooled to room temperature and the solvent was removed under
reduced pressure. The residue was purified by silica gel column
chromatography (charged with some charcoal for decolorization at the top of
the column) using initially chloroform and gradually changed to chloroform
containing 5% methanol as eluents. The almost colorless product, (R)-N-(2-
chloro-3-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine
hydrochloride, obtained upon concentration of fractions was dried under
high vacuum. 1.1 g (51%); TLC CHCI3:MeOH 4:1; Rf= 0.33; 1H-NMR
(CDCl3) s 2.40 (s. 3H, SMe), 2.72 (s, 3H, S(O)Me), 3.55 (s, 3H, NMe), 6.84
{2s,2H), (7.11 (d, 1H), ?.35 (m, 3H), 7.52 (s, 1H); Purity 72.7% ee by 1H-
NMR; Chiral HPLC 75.8% ee @ 32.6.
CA 02306276 2000-04-07
WO 99118962 PCT/US98/21395
-23-
C. Conversion of the hydrochloride salt to the free base
(R)-N-(2-chloro-3-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-
methylguanidine hydrochloride (0.56 g) was dissolved in chloroform (25 ml)
and methanol (3 ml) added to get a clear solution and this was treated with
sodium hydroxide (1N, 2X25 ml) in a separating funnel. The organic layer
was separated and the aqueous layer was extracted with chloroform (20 ml).
Combined organic layer was washed with water (25 ml) and passed through
a bed of sodium chloride to remove any solid suspensions and the filtrate
was concentrated to yield the free base of (R)-N-(2-chloro-3-
methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine (0.37 g.,
73% ); Chiral HPLC 76.5% ee @ 16.45; HPLC 97.7% @ 14.5 rains; 1H-NMR
(CD30D) b 2.46 (s. 3H, SMe), 2.77 (s, 3H, S(O)Me), 3.47 (s, 3H, NMe), 6.81
(dd, 1H), 6.87 (dd, 1H), 7.28 (d, 1H), 7.50 (m, 3H), 7.68 (s, 1H).
The free base obtained was crystallized from a mixture of hexanes
and dichloromethane to give white crystalline solid of (R)-N-(2-chloro-3-
methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine (0.15 g)
with increased enantiomeric purity; m. p. 118-120°C; Chiral HPLC
94.5%ee
@16.9 rains.
Example 8: Rat rotarod motor coordination test
A. Hydrochloride salt of (~-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine.
The effects of the hydrochloride salt of (~-N-(2-chloro-5-
methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine
(compound (I)) on motor coordination were assessed using the rotarod test.
Drug solutions were administered i.v. through the tail vein of test rats in a
volume of 5.0 ml/kg at a rate of 1 ml/min. The percentage of animals in
each treatment group that fell from the rotarod at each of the four time
points tested is summarized in the following Table I. Control was 20% C.D.
N in each of the Tables below specifies the number of animals in each test
group.
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-24-
Table I
Time Dose 15 min. 60 mina 120 min. 180 min. N
-
Control 29 14 0 14 7
8.0 mg/kg 38 50 25 25 8
12.0 mg/kg 100 100 75 50 4
The latency for falling from the rotarod was statistically analyzed
using analysis of variance (ANOVA), and a Neumann Keuls post-hoc was
applied when appropriate. Rats in the 12.0 mg/kg drug group had
significantly shorter latency for falling from the rotarod than the rats in
the
control group at the 15 minute, 60 minute and 120 minute time points. By
180 minutes, no statistically significant differences in falling times were
observed. The results (expressed as mean ~ s.e.m.) are shown in Figure 1 of
the drawings. In Figure 1, "**" designates P<0.01 and "***" designates
P<0.001.
The following behavioral effects also were observed with
administration of the hydrochloride salt of (+)-N-(2-chloro-5-
methylthiophenyl)-N'-(3-methylsulfmylphenyl)-N'-methylguanidine: animals
in both drug groups (i.e. 8.0 mg/kg and 12.0 kg/mg dosages) showed
increased locomotion at 120 minutes. Some animals displayed mild head
weaving. Two animals in the 12.0 mg/kg group died within 15 minutes
after injection, a third animal died 60 minutes after injection. Pre-testing
doses of 20.0 and 16.0 rng/kg were lethal.
B. Acetate salt of (-)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine (compound (IA)).
The acetate salt of (-)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine was also tested in the same
rotarod assay protocol as described in Part A above. Drug solutions were
administered i.v. through the tail vein in a volume of 5 ml/kg at a rate of 1
ml/min. The percentage of animals in each treatment group that fell from
the rotarod at each time point is summarized in the following Table II. In
Table II, Control ( 1 ) is 60 mM Acetate Buffer in 0.3M mannitol at 0.06 and
0.125 mg/kg doses and Control (2) is 60 mM Acetate Buffer in 0.3M
mannitol at 0.25 and 0.5 mg/kg doses.
SUBSTITUTE SHEET (RULE 26)
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-25-
Table II
Time Dose 15 min. 60 min. 120 min. 180 min. N
Control 0 13 13 0 8
( 1 )
Contol (2) 0 13 13 0 8
2.0 mg/kg 0 0 14 0 7
4.0 mg/kg 14 0 14 14 7
8.0 mg/kg 78 33 22 22 9
12.0 mg/kg 78 11 11 11 9
The latency for falling from the rotarod was statistically analyzed
using analysis of variance (ANOVA), and a Neuman Keuls post-hoc was
applied when appropriate. No statistical significance was found between
control animals and the animals in the 0.06 and 0.125 mg/kg groups at any
time point. Rats in the remaining groups at the 15 and 60 minute time
points had significantly shorter latencies for falling from the rotarod than
rats in the control groups. At 120 minutes, no statistically significant
differences in falling times were observed in the two low dose groups. Full
recovery was not achieved by the animals in the high dose groups even at
180 minutes. These results are summarized in Figure 2 of the drawings. In
Figure 2, Controls (1) and (2) are the same as specified above. The results
(expressed as mean ~ s.e.m.) are shown in Figure 2 of the drawings, where
"*"designates P<0.05 and "***" designates P<0.001 compared to group
controls.
C. Acetate salt of (+)-N-(2-chloro-5-methylthiophenyl)-N'-
(3-methylsulfinylphenyl)-N'-methylguanidine (compound (IB)).
The acetate salt of (+)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine was also tested in the same
rotarod assay protocol as described in Part A above. Drug solutions were
administered i.v. through the tail vein in a volume of 5 ml/kg at a rate of I
ml/min. The percentage of animals in each treatment group that fell from
the rotarod at each time point is summarized in the following Table III. In
Table III, Control (1) is 60 mM Acetate Buffer in 0.3M mannitol at 0.06 and
0.125 mg/kg doses and Control (2) is 60 mM Acetate Buffer in 0.3M
mannitol at 0.25 and 0.5 mg/kg doses.
SUBSTITUTE SHEET (RULE 26~
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-26-
Table Iii
Time Dose 15 min. 60 min. 120 min. 180 min. N
Control 0 13 13 0 8
( 1 )
Control 0 13 13 0 8
(2)
2.0 mg/kg 0 0 0 0 7
4.0 mg/kg 29 29 57 57 7
8.0 mg/kg 100 100 100 67 8
12.0 mg/kg 100 ~ 100 ~ 100 100 6
~
The latency for falling from the rotarod was statistically analyzed
using analysis of variance (ANOVA), and a Neuman Keuls post-hoc was
applied when appropriate. No statistical significance was found between
control animals and the animals in the 0.06 and 0.125 mg/kg groups at any
time point. Rats in the remaining groups at the 15 and 60 minute time
points had significantly shorter latencies for falling from the rotarod than
rats in the control groups. At 120 minutes, no statistically significant
differences in falling times were observed in the two low dose groups. Full
recovery was not achieved by the animals in the high dose groups even at
180 minutes. The results (expressed as mean + s.e.m.) are shown in Figure
3 of the drawings, where "***" designates P<0.001 compared to group
controls. Controls (1) and (2) are the same as specified above for Table 3.
The results for compounds of the invention can be compared to those
obtained for MK-801, i.e. (+)-5-methyl-10,11-dihydro-5H
dibenzo[a,d]cyclohepten-5,10-imine maleate. In the rotarod test for MK-
801, drug solutions were administered i.v. through the tail vein of each test
rat in a volume of 5 ml/kg at a rate of 1 ml/min. The control was 0.9%
saline solution. The percentage of animals in each treatment that fell from
the rotarod at each test point is summarized in the following Table IV:
T~~,~o n~
Time Dose 15 min. 60 min. 120~min. N
Control 13 13 13 8
0.01 mg/kg 38 13 25 8
0.05 mg/kg 86 57 29 7
SUBSTITUTE SHEET (RULE 26)
CA 02306276 2000-04-07
WO 99/18962 PCT/US98/21395
-27-
Example 9 Irwin general behavior screen.
The hydrochloride salt of (~-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine (compound (I)) was tested in the
Irwin general behavior screen with the lowest dose determined at which
definite behavioral effects were observed in the test rats, especially
excitation effects. See S. Irwin, Animal and Clinical Pharmacologic
Techniques in Drug Evaluation, pages 36-54 (Year Book Medical Publishers,
Chicago), which details the protocol of the Irwin behavior screen. The
following results were obtained: The following results were obtained: at dose
of 4 mg/kg i.v. administration, excitation ( 120-180') sterotypies (sniffing
60-
180~ fore-paw treading (120') loss of traction (2 of 3, 120; at dose of 16
mg/kg i.v. administration excitation (30-180 sterotypies (head-weaving to
180 fore-paw treading (to 180 loss of traction (to 180 hypothermia.
Example 10 Rat MCAO ischemia model.
A. The hydrochloride salt of (~-N-(2-chloro-5-methylthiophenyl)-
N'-(3-methylsulfinylphenyl)-N'-methylguanidine (compound (I)) was tested in
the rat MCAO ischemia model with % reduction in total lesion volume (mm3)
determined by the following protocol.
Rats were anaesthetized with isoflurane and focal ischemia was
induced by directly ligating the main trunk of MCA using a bipolar
electrocoagulor. Coronal sections (seven sections, 2.0 mm intervals) were
collected and incubated in 2,3,5-triphenyltetrazolium chloride (TTC) solution
for 15 minutes at 37°C, then fixed in 4% paraformaldehyde solution in
the
dark at 4~C for at least 48 hours. Seven sections of each brain were
photographed and analyzed. The areas that were not stained red with TTC
were measured as infarction.
The test animals were treated with the test compound and control
(mannitol) as follows. Ten minutes after the MCAO incident the test animals
were administered (i.v. infusion/ 10 minutes) with one third of the total dose
of the test compound (total doses specified below); 1 hour after that first
dosing the test animals were given another one third of the total dose of the
test compound; and then 2 hours after that first dosing the test animals
were given another one third of the total dose of the test compound.
Mannitol was administered to the control group at the first dosing only.
CA 02306276 2000-04-07
WO 99/18962 PCT/US98l2I395
-28-
The following results were obtained:
46% at dose of 9 mg/kg
32% at dose of 4.5 mg/kg
24% at dose of 2.25 mg/kg
B. The acetate salt of (-)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine (compound (IA)) was tested in the
rat MCAO ischemia model with % reduction in total lesion volume
determined by the same MCAO assay protocol specified in Part A above.
Sprague-Dawley rats with body weights of 270-320 grams were used in the
study.
The following results were obtained expressed as % reduction in total
average lesion volume (mm3) by the specified dosage (values below refer to
total dose administered; n designates number of test rats):
1% at 4.5 mg/kg (n=8)
22% at 9 mg/kg (n=8)
C. The acetate salt of (+)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfinylphenyl)-N'-methylguanidine (compound (IB)) was also tested
in the rat MCAO ischemia model with % reduction in total lesion volume
determined by the same protocol as specified in Part A above. Sprague-
Dawley rats with body weights of 270-320 grams were used in the study.
The following results were obtained expressed as % reduction in total
average lesion volume (mm3) by the specified dosage (values below refer to
total dose administered; n designates number of test rats):
23% at 2.25(mg/kg) (n=12)
32% at 4.5 mg/kg (n=8)
34% at 9.0 mg/kg (n=8)
E~mple 11 Rat pup hypoxia-ischemia model.
A. The hydrochloride salt of (~-N-(2-chloro-5-methylthiophenyl)-
N'-(3-methylsulfinylphenyl)-N'-methylguanidine was tested in the rat pup
hypoxia-ischemia model with % reduction in total lesion volume determined
at the speed test dose. A rat pup hypoxia-ischemia assay protocol of the
following protocol was employed.
8 day-old rat pups weighing 17-23 grams were anaesthetized with
isoffurane and the left common carotid artery ligated. Immediately prior to
CA 02306276 2000-04-07
WO 99/18962 PCTNS98/21395
-29-
the hypoxic-ischemia (H-I) insult a test group of animals was injected
intraperitoneally with 2 mg/kg, 5 mg/kg or 10 mg/kg of the test compound
and a control group was treated with a corresponding dose of mannitol.
Then, immediately after the H-I insult, the animals received a second
equivalent dose, i.e. the test group of animals was injected intraperitoneally
with 2 mg/kg (total 4 mg/kg administered in the two doses), 5 mg/kg (total
IO mg/kg administered in the two doses) or 10 mg/kg (total 20 mg/kg
administered in the two doses) of the test compound and a control group
was treated with a corresponding dose of mannitol. The animals were then
allowed to recover for 1-2 hours and then exposed to 7.8% Oa in nitrogen for
78 minutes in an incubator at 36.5~C. The rat pups were returned to
mothers immediately after the H-I insult. The animals were sacrificed two
days after H-I insult, their brain was removed and cut into four 2 mm thick
slices prior to staining with 2% 2,3,5-triphenyltetrazolium chloride (TTC)
solution. The sections were then fixed with 4% paraformaldehyde solution
in the dark at 4°C for at least 48 hours. Four sections from each brain
were
imaged and analyzed using the IPlrab system. The lesion area which was
not stained red with TTC was measured as infarction.
The following results were obtained:
100% at i.p. total dose of 20 mg/kg
60% at i.p. total dose of 10 mg/kg
B. The acetate salt of (+)-N-(2-chloro-5-methylthiophenyl)-N'-(3-
methylsulfmylphenyl)-N'-methylguanidine was also tested in the rat pup
hypoxia-ischemia model with % reduction in total lesion volume determined
by the same protocol as specified in Part A above, except the rat pups were
exposed to 7.8% 02 in nitrogen for 85 minutes. The following results were
obtained:
93% at i.p. total dose of 20 mg/kg
78% at i.p. total dose of 10 mg/kg
The foregoing description of the invention is merely illustrative
thereof, and it is understood that variations and modifications can be made
without departing from the spirit or scope of the invention as set forth in
the
following claims.
