U.S. patent application number 11/255975 was filed with the patent office on 2006-03-16 for delta2-1,2,3-triazoline anticonvulsants and their active metabolite analogues, the aminoalkylpyridines, are excitatory amino acid antagonists and antiischemic agents, useful in the treatment of cerebral ischemia resulting from stroke.
This patent application is currently assigned to K and K Bioscience, Inc.. Invention is credited to Pankaja K. Kadaba.
Application Number | 20060058357 11/255975 |
Document ID | / |
Family ID | 26936896 |
Filed Date | 2006-03-16 |
United States Patent
Application |
20060058357 |
Kind Code |
A1 |
Kadaba; Pankaja K. |
March 16, 2006 |
Delta2-1,2,3-triazoline anticonvulsants and their active metabolite
analogues, the aminoalkylpyridines, are excitatory amino acid
antagonists and antiischemic agents, useful in the treatment of
cerebral ischemia resulting from stroke
Abstract
Pharmaceutical compositions comprise as the active ingredient,
nonneurotixic antiischemic compounds that are highly effective by
the intraperitoneal route, and that are excitatory amino acid and
NMDA/sigma receptor antagonists and are selected from the group
consisting of those of the formulae, ##STR1## wherein R.sup.2 is
4-pyridyl, 3-pyridyl, or 2-oxo-1-pyrrolidino and R.sup.2 is 3,4- or
3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or
m-trifluoromethyl, p-methyl, p-methoxy, or hydrogen, and those of
the formulae, ##STR2## wherein R.sup.2 is 4-pyridyl or 3-pyridyl,
R.sup.3 is hydrogen, methyl or ethyl and R.sup.1 is 3,4- or
3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or
m-trifluoromethyl, p-methyl, p-methoxy or hydrogen.
Inventors: |
Kadaba; Pankaja K.; (Chadds
Ford, PA) |
Correspondence
Address: |
MCDERMOTT WILL & EMERY LLP
600 13TH STREET, N.W.
WASHINGTON
DC
20005-3096
US
|
Assignee: |
K and K Bioscience, Inc.
Chadds Ford
PA
|
Family ID: |
26936896 |
Appl. No.: |
11/255975 |
Filed: |
October 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10679420 |
Oct 7, 2003 |
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11255975 |
Oct 24, 2005 |
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09985318 |
Nov 2, 2001 |
6638954 |
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10679420 |
Oct 7, 2003 |
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60244930 |
Nov 2, 2000 |
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60307360 |
Jul 25, 2001 |
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Current U.S.
Class: |
514/340 |
Current CPC
Class: |
C07D 213/38 20130101;
C07D 403/04 20130101; A61K 31/44 20130101; A61K 31/4192 20130101;
C07D 401/04 20130101; A61K 31/4439 20130101 |
Class at
Publication: |
514/340 |
International
Class: |
A61K 31/4439 20060101
A61K031/4439 |
Claims
1-22. (canceled)
23. A 4-pyridyl methylamine compound of the following formulae,
##STR13## wherein R.sup.1 is p- or m-chloro, 3,4- or 3,5-dichloro,
p- or m-bromo, p- or m-fluoro, p-methyl or methoxyl.
24. A 1-(4-pyridyl)-1-ethylamine compound of the following
formulae, ##STR14## wherein R.sup.1 is 3,4- or 3,5-dichloro, 3,4-
or 3,5-difluoro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p-
or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or
hydrogen.
25. A 1-(4-pyridyl)-1-ethylamine compound according to claim 24
wherein R.sup.1 is 3,5-dichloro.
26. A 1-(4-pyridyl)-1-ethylamine compound according to claim 24
wherein R.sup.1 is m-bromo, m-fluoro, m-trifluoromethyl, or
3,4-difluoro.
27. A 1-(3-pyridyl)-1-ethylamine compound of the following
formulae, ##STR15## wherein R.sup.1 is 3,4- or 3,5-dichloro, p- or
m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl,
p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen.
28. A 1-(3-pyridyl)-1-ethylamine compound according to claim 27
wherein R.sup.1 is p-chloro, p-bromo or 3,4-dichloro.
29. A 1-(4-pyridyl)-1-propylamine compound of the following
formulae, ##STR16## wherein R.sup.1 is 3,4- or 3,5-dichloro, 3,4-
or 3,5-difluoro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p-
or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or
hydrogen.
30. A 1-(3-pyridyl)-1-propylamine compound of the following
formulae, ##STR17## wherein R.sup.1 is 3,4- or 3,5-dichloro, 3,4-
or 3,5-difluoro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p-
or m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or
hydrogen.
31. A .alpha.-phenyl-.alpha.-(4-pyridyl)methylamine compound of the
following formulae, ##STR18## wherein R.sup.1 is 3, 4 or
3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro,
m-trifluoromethyl, p-dimethylamino, p- or m-lower alkyl, p- or
m-lower alkoxy or hydrogen.
32. A .alpha.-phenyl-.alpha.-(3-pyridyl)methylamine compound of the
following formulae, ##STR19## wherein R.sup.1 is 3,4- or
3,5-dichloro, p- or m-chloro, p-bromo, m-trifluoromethyl, p-methyl,
p-methoxy or hydrogen.
33. A .alpha.,.alpha.-bis-(2-pyridyl)methylamine compound of the
following formula, ##STR20##
34. A non-neurotoxic antiischemic composition, effective by the
intraperitoneal route of administration in the treatment of global
ischemia and comprising as the active ingredient, an effective
amount of an antiischemic compound selected from the group
consisting of those of the formulae, ##STR21## wherein R.sup.2 is
4-pyridyl or 3-pyridyl, R.sup.3 is hydrogen, methyl or ethyl, and
R.sup.1 is 3,4- or 3,5-dichloro, 3,4- or 3,5-difluoro, p- or
m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl,
p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen, and a
pharmaceutical carrier.
35. A composition according to claim 34 wherein R.sup.2 is
4-pyridyl, R.sup.3 is hydrogen, methyl or ethyl, and R.sup.1 is
3,4- or 3,5-dichloro.
36. A composition according to claim 34 wherein R.sup.2 is
3-pyridyl, R.sup.3 is hydrogen, methyl or ethyl, and R.sup.1 is
p-chloro, p-bromo, or 3,4-dichloro.
37. A composition consisting of an effective amount of an
excitatory amino acid inhibitor (glutamate inhibitor) compound
according to claim 34 to yield and antiischemic composition
effective against global and focal ischemia.
38. (canceled)
39. A method for the treatment of cerebral ischemia resulting from
stroke in mammals, including man, which comprises administration
thereto of an effective dosage amount of a aminoalkylpyridine
antiischemic composition of claim 37.
40. A composition according to claim 34 wherein R.sup.2 is
3-pyridyl, R.sup.3 is hydrogen, methyl or ethyl, and R.sup.1 is
p-chloro, p-bromo, or 3,4-dichloro.
41. A composition consisting of an effective amount of an
excitatory amino acid inhibitor (glutamate inhibitor) compound
according to claim 34 to yield an antiischemic composition
effective against global and focal ischemia.
42. A composition according to claim 37, wherein a sufficient
amount of the aminoalkylpyridine in claim is contained in said
composition to provide a dosage amount ranging from about 25 mg/kg
to 200 mg/kg.
43. A method for the treatment of cerebral ischemia resulting from
stroke in mammals which comprises administration thereto of an
effective dosage amount of a triazoline or aminoalkylpyridine
antiischemic composition of claim 34.
44. A method for the treatment of cerebral ischemia resulting from
stroke in mammals, including man, which comprises administration
thereto of an effective dosage amount of a triazoline or
aminoalkylpyridine antiischemic composition of claim 34.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from Provisional
Application Ser. No. 60/244,930, filed Nov. 2, 2000, and
Provisional Application Ser. No. 60/307,360, filed Jul. 25, 2001,
the disclosures of these provisional applications being
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the drug potential of
anticonvulsants in the treatment of stroke, particularly, several
.DELTA..sup.2-1,2,3-triazoline and aminoalkylpyridine (AAP)
anticonvulsants that seem to work by impairing the excitatory amino
acid (EAA) L-glutamate (L-Glu) neurotransmission, as antiischemic
agents, useful in the treatment of stroke victims.
[0003] There is strong evidence that the "excitotoxic" action
resulting from the excessive accumulation of L-Glu plays a
prominent role in human epilepsy as well as brain ischemia/stroke,
leading to neuronal dysfunction and cell death. The
1,2,3-triazolines and the aminoalkylpyridine (AAP) metabolite
analogues are two groups of novel anticonvulsants discovered in the
Applicant's laboratories. These are very effective in the kindling
and in the maximal electroshock (MES) seizure models of epilepsy,
the best analogies to human partial seizures, where EAA
neurotransmission plays an important role. Thus it is logical to
expect that the anticonvulsant triazolines and AAP metabolite
analogues would evince beneficial therapeutic potential in cerebral
ischemia.
[0004] The ability of the triazolines and AAP compounds to afford
protection and reduce neuronal degeneration are assessed in animal
models of stroke, by utilizing the bilateral carotid occlusion
model in the gerbil and the middle cerebral artery occlusion (MCAO)
model in the rat. Post-ischemic gerbils undergo a predictable
pattern of behavioral changes and the effects of drugs in producing
alterations in this pattern are monitored by determining the post
ischemic changes in locomotor activity as well as by changes in
radial arm maze performance, and corroborated by post reperfusion
histopathological assessment. In the MCAO rat model, a focal stroke
model, drug effects are evaluated from their ability to reduce the
infarct volume following MCAO.
BACKGROUND ART
[0005] There is a desperate need for clinically effective
chemotherapeutic agents for intervention in and management of
cerebral ischemia resulting from stroke. In the U.S. alone, 1.1
million individuals suffer stroke annually; it is the most common,
and devastating neurological condition that kills more than a
quarter million Americans every year and the leading cause of
long-term intellectual and physical disability. In the past decade,
it has become increasingly evident from data from numerous
laboratories that EAA neurotransmission plays an important role in
ischemic brain injury occurring in stroke and other neurological
disorders (McCulloch, J., et al., Ed., "Frontiers in Pharmacology
and Therapeutics: Excitatory Amino Acid Antagonists.", Oxford, UK;
Blackwell Scientific Publishers, 287-326, 1991: Choi, D. W. &
Rothman, S. M., Annu. Revs., Neurosci., 13, 171-182, 1991; Takagi,
K., et al., J. Cereb. Blood Flow Metab., 13, 575-585, 1993; Graham,
S. H., et al., J. Cereb. Blood Flow Metab., 13, 88-97, 1993; Muir,
K. W., & Lees, K. R., Stroke, 26, 503-515, 1995). The excessive
accumulation of the excitatory neurotransmitter L-Glu, followed by
its excitotoxic action, has been strongly implicated in the cascade
of pathological mechanisms that cause neuronal dysfunction and cell
death in cerebral hypoxia-ischemia resulting from stroke, cardiac
arrest, or mechanical brain injury. Thus, the EAA neurotransmitter
systems may be considered potential therapeutic targets and
development of agents that are EAA antagonists may constitute novel
and effective therapies, as cytoprotective agents, in stroke.
SUMMARY OF THE INVENTION
[0006] It is accordingly one object of the present invention to
provide novel .DELTA..sup.2-1,2,3-triazolines and AAP compounds and
their method of preparation.
[0007] It is a further object of the present invention to provide
antiischemic/antistroke agents which comprise triazolines and AAP
compounds
[0008] A further object of the present invention is to provide a
method for the treatment of cerebral ischemia resulting from
stroke, by administration of an effective amount of the triazoline
and AAP compounds of this invention.
[0009] A further object of the present invention is to provide
triazolines and AAPs bearing three different pyridyl substituents
and a pyrrolidinone group, and methods for their use in the
treatment of neurological disorders such as cerebral ischemia
resulting from stroke and also in the treatment of epilepsy.
[0010] A still further object of the present invention is to
provide triazolines and AAP compounds, as inhibitors of the EAA
neurotransmitter L-glutamate. The triazolines and AAPs of this
invention afford pronounced protection in the maximal electroshock
seizure (MES) model in both mice and rats, by the intraperitoneal,
intravenous, and oral route, which is indicative of their action as
glutamate antagonists.
[0011] A still further object of the present invention is to
provide antiischemic compositions that contain as the essential
ingredient certain triazolines and AAPs and that are highly
effective by the intraperitoneal and intravenous routes, the
preferred routes of administration, in stroke victims, and use of
these triazolines and AAPs as effective antiischemic drugs in the
treatment of cerebral ischemia resulting from stroke.
[0012] Other objects and advantages of the present invention
include use of the triazolines and AAPs in the treatment of stroke
and epilepsy and also other neurological disorders such as
Parkinson's disease, by virtue of their action as EAA antagonists
and inhibitors of L-glutamate neurotransmission.
[0013] In satisfaction of the foregoing objects and advantages,
there are provided by this invention several triazolines and AAPs
which are useful as antiischemic/antistroke drugs. The various
groups of triazolines and AAPs substituted with the various pyridyl
groups and also the pyrrolidinyl group, may be characterized by the
following general formulae: ##STR3##
[1-(Phenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazolines]
[0014] ##STR4##
[1-(Phenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazolines]
[0015] ##STR5##
[1-(Phenyl)-5-(2-pyridyl)-.DELTA..sup.2-1,2,3-triazolines]
[0016] ##STR6##
[1-(Phenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-triazolines]
[0017] ##STR7##
[1-(N-Phenyl)-1-(4-pyridyl)-1-ethylamine](propyl)
[0018] ##STR8##
[1-(N-Phenyl)-1-(3-pyridyl)-1-ethylamine](propyl)
[0019] ##STR9##
[1-(N-Phenyl)-1-(2-pyridyl)-1-ethylamine](propyl)
[0020] wherein R.sup.1 is 3,4- or 3,5-dichloro, p- or m-chloro, p-
or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower
alkyl, p- or m-lower alkoxy or hydrogen.
[0021] Also provided by this invention are non-toxic antiischemic
compositions that are intraperitoneally and intravenously active
and comprise as the active ingredient, a compound selected from
those of the formulae (I-VII), wherein R.sup.1 is 3,4- or
3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or
m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or
hydrogen.
[0022] Also provided are methods for the administration of the
antiischemic compositions of this invention to mammals, including
animals and humans, in the treatment of cerebral ischemia resulting
from stroke, including both global ischemia and focal ischemia.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0023] As indicated above, this invention relates to several groups
of compounds belonging to the seven structures (I-VII) shown above,
which are useful as antiischemic drugs in the treatment of cerebral
ischemia resulting from stroke. In one group of triazolines (I) and
AAPs (V), a 4-pyridyl substituent is present, in a second group of
these compounds (II & VI), a 3-pyridyl substituent and in a
third group (III & VII), a 2-pyridyl substituent is present.
Also, in a fourth group of triazolines (IV), a 2-oxo-1-pyrrolidino
group is present. In all three groups of AAP compounds, the R.sup.2
group is methyl, ethyl or phenyl. The triazolines and AAPs of this
invention are further substituted on the phenyl rings by 3,4- or
3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or m-fluoro, p- or
m-trifluoromethyl, p- or m-lower alkyl, p- or m-lower alkoxy or
hydrogen. The triazolines and AAPs of this invention have potent
antiischemic activity and protect the brain from neuronal damage in
both global and focal ischemia, and are useful as
antiischemic/antistroke drugs in the treatment of cerebral ischemia
resulting from stroke in humans.
[0024] In one aspect of the present invention, three groups of
triazolines and two groups of AAPs, are provided which have potent
antiischemic activity and which have the general formulae
represented by structures I, II and IV, and V and VI, respectively.
In the above formulae, in structures I and II, the 5-substituent is
4-pyridyl or 3-pyridyl and R.sup.1 is 3,4- or 3,5-dichloro, p- or
m-chloro, p- or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl,
p- or m-lower alkyl, p- or m-lower alkoxy or hydrogen. In structure
IV, the 5-substituent is a 2-oxo-1-pyrrolidino group and R.sup.1 is
3,4- or 3,5-dichloro, p- or m-chloro, p- or m-bromo, p- or
m-fluoro, p- or m-trifluoromethyl, p- or m-lower alkyl, p- or
m-lower alkoxy or hydrogen. Structures V and VI, are 4-pyridyl and
3-pyridyl AAPs respectively, where R.sup.4 is methyl, ethyl or
phenyl and R.sup.1 is 3,4- or 3,5-dichloro, p- or m-chloro, p- or
m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower
alkyl, p- or m-lower alkoxy, or hydrogen. Several of these
triazolines and AAP compounds are potent anticonvulsants and are
already under U.S. patent protection (P. K. Kadaba, U.S. Pat. No.
4,511,572, 1985; U.S. Pat. No. 4,689,334, 1987; U.S. Pat. No.
4,820,721, 1989; U.S. Pat. No. 5,648,369, 1997; U.S. Pat. No.
6,083,964, 2000).
[0025] In a second aspect of this invention, there are provided
novel antiischemic compositions which are effective by the
intraperitoneal and intravenous routes and are non-toxic, and which
comprise as the active ingredient an effective amount of a compound
selected from those of the seven groups represented by structures
(I-VII), and having 4- or 3-pyridyl, or 2-oxo-1-pyrrolidinyl
substituent groups, and R.sup.4 and R.sup.1 are as described
above.
[0026] There are further provided by this invention, methods for
the administration of the antiischemic compositions to mammals
including animals and humans.
[0027] In a third aspect of this invention, there are provided
triazoline and AAP compounds of the formulae represented by the
structures (I-VII), and which exhibit pronounced and selective
activity in the MES test and the kindling model of epilepsy, and
are useful in the treatment of stroke.
Significance of Pronounced Selective Activity in the MES Test:
[0028] Both the triazolines and the AAP compounds of this invention
exhibit pronounced and selective anticonvulsant activity in the
maximal electroshock seizure (MES) test. While the triazolines show
activity in the subcutaneous Metrazole (scMet) test also, the AAPs
show hardly any activity in the scMet test. The activity of the
compounds of this invention in the MES test is of great
significance, because partial seizures in humans correlate
positively with experimental seizures elicited by the MES test
[Porter, R. J. and Pitlick, W. H., In "Basic and Clinical
Pharmacology", 4th Edn., B. G. Katzung Ed., Appleton & Lange,
C. A., 1989, pp 287-303]. Since antiepileptic drugs effective
against MES seizures alter ionic transport across excitable
membranes, the triazolines and the AAPs that evince significant
activity in the MES test, may be expected to attenuate EAA
neurotransmission. There is strong evidence that the excitatory
neurotransmitter glutamate plays a key role in EAA
neurotransmission along limbic circuits which are particularly
relevant to kindling epileptogenesis. Since the triazolines and the
AAPs are quite effective in the kindling model, both these classes
of compounds could be expected to be effective glutamate
antagonists.
1,2,3-Triazoline and AAP Anticonvulsants, and Their Mechanism of
Anticonvulsant Activity, as Inhibitors of Both Post-and Presynaptic
EAA Neurotransmission:
[0029] Previous studies in our laboratories had led to the
emergence of the 1,2,3-triazoline heterocycles represented by
structures I-IV, as a new class of anticonvulsant agents with a
unique mechanism of action quite different from the more
traditional anticonvulsants (Kadaba, P. K., J. Med. Chem., 31,
196-302, 1988; "Drugs of the Future", 15, 1013-1024, 1990; Kadaba,
P. K., and Slevin, J. T., Epilepsia, 29, 330, 1988; Kadaba, P. K.,
and Slevin, J. T., Pharmaceut. Res., 6, S-42, 1989; Kadaba, P. K.
and Slevin, J. T., 200th Nat. Meeting of the ACS, Washington, D.C.,
Abstracts, 56, MEDI 31, 1990; Kadaba, P. K., Stevenson, P. J.,
Nnane, I. P., and Damani, L. A., Bioorg. Med. Chem., 4, 165-178,
1996). The triazolines afford a high degree of protection in
seizure provocation by chemical (scMet) and electrical (MES)
stimuli and have good protective indices. They offer complete
protection against N-methyl D-aspartate (NMDA)-induced seizures in
the mouse at significantly low ED.sub.50 values: (Kadaba, P. K., et
al., Bioorg. Med. Chem., 4, 165-178, 1996). They show good response
on oral administration and a good margin of safety. They compare
very well with prototype antiepileptic drugs in both mice and rats.
Unlike the prototype drugs, one triazoline represented by structure
1, R.sup.1=p-Cl, offers complete protection against
stimulus-induced electrographic after-discharge seizures and
generalized convulsions, in both amygdala-kindled
(ED.sub.50=215.+-.61 mg/kg) and entorhinal-kindled rats
(ED.sub.50=423.+-.45 mg/kg), in non-sedative, non-neurotoxic doses
(Kadaba, P. K., "Drugs of the Future", 15, 1013-1024, 1990: Kadaba,
P. K. & Slevin, J. T., Pharmaceut. Res., 6, S-42, 1989).
[0030] Studies on the metabolism and in vivo and in vitro
pharmacology of triazolines represented by structure I and
potential metabolites, seem to indicate that the triazolines may be
functioning as prodrugs and act by a unique "dual-action"
mechanism; while the parent triazoline inhibits the presynaptic
release of glutamate (58% at 50 .mu.M and 83% at 100 .mu.M drug
concentration), the active .beta.-amino alcohol metabolite
displaces >90% of the binding of [.sup.3H]-Glu from glutamate
receptors, and 56% of the binding of [.sup.3H]MK-801, from the
MK-801 sites on the NMDA receptor ionophore complex (Kadaba, P. K.,
ACS Abstrs. MEDI 144, 1991; Kadaba, P. K. & Slevin, J. T.,
Epilepsia, 29, 330, 1988; Pharmaceut. Res., 6, S-42, 1989; ACS
Abstrs., 56, MEDI 31, 1990; Kadaba, P. K., et al., Bioorg. Med.
Chem. 4, 165-178, 1996). Furthermore, radioligand binding studies
at ion-channel binding sites using [.sup.3H]TBOB, indicates
significant activity at Cl.sup.- channels ranging from 50 to 63% at
10 .mu.M concentration, for triazolines belonging to structure I
(Kadaba, P. K., et al., Bioorg. Med. Chem. 4, 165-178, 1996).
Augmentation in Cl.sup.- influx is a useful membrane action that
reduces membrane excitability or alters circuit behavior to favor
inhibition, and thus might help suppress the firing of
glutamatergic neurons and hence glutamate release. Such drugs may
be most beneficial in the control of prolonged seizures such as in
status epilepticus where excessive neuronal firing occurs (Choi, D.
W., Cerebrovasc. Brain Metab. Revs., 2, 105-147, 1990). And indeed,
the complete protection afforded by triazoline I (R.sup.1=p-Cl)
against amygdala- and entorhinal-kindled seizures as well as
NMDA-induced convulsions is significant, in view of the current
concepts regarding the central role of EAA neurotransmission,
particularly L-Glu, in the kindling model of human partial
epilepsy.
[0031] Studies by the Applicant on the metabolism and pharmacology
of the triazoline anticonvulsants have led to the evolution and
discovery of the aminoalkylpyridines (AAPs) as a unique class of
orally active anticonvulsant agents, superior to the triazolines
themselves (Kadaba, P. K., et al., Bioorg. Med. Chem., 2, 165-178,
1996; Kadaba, P. K., U.S. Pat. No. 4,511,572, 1985; U.S. Pat. No.
4,618,681, 1986; U.S. Pat. No. 4,689,334, 1987; U.S. Pat. No.
4,820,721, 1981). Work on the aminoalkylpyridines indicate they are
non-toxic, and highly effective by the oral route, with protective
indices greater than 20. The AAPs also show high anticonvulsant
activity in the MES test and are practically inactive in the scMet
test (Deshmukh, T. R. & Kadaba, P. K., Med. Chem. Res., 3,
223-232, 1993; U.S. Pat. No. 5,648,369, 1997).
[0032] Radioligand binding and release studies indicate that the
ability of triazolines to impair presynaptic release of glutamate
is retained to the full extent or better in the corresponding AAP
compounds (V, R.sup.1=p-Cl) (74% at 50 .mu.M and 80% at 100 .mu.M
drug concentration as also the postsynaptic activity of the
.beta.-amino alcohol, albeit at a different site; the AAP compounds
weakly displace [.sup.3H]DTG, a o specific ligand, with Ki values
in the .mu.M range and show no affinity for the PCP sites (Kadaba,
P. K., ACS Abstrs. MEDI 073, 1992; Pharmaceut. Res., MNPC 5013, 11,
S-120, 1994a; Epilepsia, AES, Dec. 5, 1994b; Deshmukh, T. R., &
Kadaba, P. K., J. Pharm. Res. 9, S-109, 1992; Med. Chem. Res. 3,
323, 1993; Kadaba, P. K., & Deshmukh, T. R., ACS Abstrs., MEDI,
1069, 1993a; Amino Acios, June, 1993b; Kadaba, P. K., et al.,
Bioorg. Med. Chem., 2, 165-178, 1996). As o and PCP sites are two
distinct molecular entities (Kamenka, J. M. & Domino, E. F.,
(Eds), "Multiple Sigma and PCP Receptor Ligands: Mechanisms for
Neuromodulation and Neuroprotection?", NPP Books, P.O. Box 1491,
Ann Arbor, Mich., 48106, 1992) and the o receptor is not a
component of the NMDA receptor-ionophore complex, the potent
anticonvulsant activity of the AAPs seems to result from their
selective low-affinity interaction at o.sub.1 sites. The
selectivity of the AAPs for the o receptor sites with no activity
at the PCP sites, might also account for the absence of undesirable
toxic side effects in these compounds.
EAA Neurotransmitter Systems and the NMDA Receptor Complex in
Relation to Epilepsy and Cerebral Ischemia:
[0033] The role of EAAs and the NMDA receptor in health and disease
are extensively reviewed (Cavalheiro, E. A., Lehmann, J., and
Turski, L., Eds., "Frontiers in Excitatory Amino Acid Research", A.
R. Liss, New York, N.Y., 1988; Cotman, C. W., Bridges, R. J.,
Taube, J. S., Clark, A. S., Geddes, J. W., and Monaghan, D. T., J.
NIH Res., 1, 65, 1989; Dingledine, R., Boland, L. M., Chamberlin,
N. L., Kawasaki, K., Kleckner, N. W., Traynelis, S. F., and
Verdoom, T. A., CRC Crit. Rev. Neurobiol., 4, 1, 1988; Honore, T.,
Med Res. Rev. 9, 1, 1989; Johnson, G., Ann. Rep. Med. Chem., 24,
41, 1989). Overstimulation of the NMDA receptor by high levels of
glutamate has been implicated in both epilepsy (Cavalheiro, E. A.,
Lehmann, J., and Turski, L., Eds., "Frontiers in Excitatory Amino
Acid Research", A. R. Liss, New York, N.Y., 1988; Fisher, R. S. and
Coyle, J. T., Eds., "Neurotransmitters and Epilepsy", Wiley-Liss,
New York, N.Y., 1991) and stroke (Meldrum, B. S. and Garthwaite,
J., TIPS, 11, 379-385, 1990; Rothman, S. M. and Olney, J. W., Ann.
Neurol., 19, 105-111, 1986). Both diseases have been suggested to
have a common pathology, i.e., chronic or acute cell death
resulting from EAA-induced "excitotoxicity" (Greenamyre, J. T.,
Maragos, W. F., Albin, R. L., Pemmy, J. B., and Young, A. B., Prog.
Neuro Psychopharmacol & Biol. Psychiat., 12, 421, 1988; Mayer,
M. L., and Westbrook, G. L., Prog. Neurobiol., 28, 197, 1987; Choi,
D. S., Neuron, 1, 623, 1988; Simpson, M. D. C., Royston, M. C.,
Deakin, J. F. W., Cross, A. J., Mann, D. M. A., and Slater, P.,
Brain Res., 462, 76, 1988). Excessive accumulation of glutamate
leads to overactivation of the NMDA receptor resulting in excessive
intraneuronal Ca.sup.2+ which precipitates neurodegeneration and
neuronal death (Cotman, C. W., Bridges, R. J., Taube, J. S., Clark,
A. S., Geddes, J. W., and Monaghan, D. T., J. NIH Res., 1, 65,
1989). Evidence for the excitotoxic action of glutamate at the NMDA
receptor, derived from numerous studies of cultured cortical
neurons in vitro (Choi, D. S., Neuron, 1, 623, 1988), suggests an
influx of Ca.sup.2+ through the stimulated NMDA ionophore to be a
prerequisite for cell death to occur (Choi, D. S., Neuron, 1, 623,
1988; Hahn, J. S., Aizenman, E., and Lipton, S. A., Proc. Natl.
Acad. Sci., 85, 6556, 1988; Ogura, A., Miyamoto, M. and Kudo, Y.,
Exp. Brain Res. 73, 447, 1988). Agents that block the action of
glutamate and thus the overstimulation of the NMDA receptor thus
represent novel therapies, as neuroprotective agents, for both
epilepsy and cerebral ischemia resulting from stroke (Johnson, G.,
Ann. Rep. Med. Chem., 24, 41, 1989; Cotman, C. W., Bridges, R. J.,
Taube, J. S., Clark, A. S., Geddes, J. W., and Monaghan, D. T., J.
NIH Res., 1, 65, 1989). Thus, based on the ability of the
triazoline and the AAP anticonvulsants to effectively impair
glutamate neurotransmission, it appears logical to expect that
these compounds would provide beneficial drug candidates for
stroke-related ischemic brain damage.
EAAs and the Kindling Model of Epilepsy:
[0034] The kindling phenomenon mimics human epilepsy (Kalichman, M.
W., Neurosci. Biobehav. Rev., 6, 165, 1982) and there is increasing
evidence that EAAs may play an important role in kindling
mechanisms. EAAs may be critically involved in both epileptogenesis
and as a focus for the mechanism of action of anticonvulsants
(Meldrum, B. S., and Chapman, A. G., In "Glutamine, Glutamate, and
GABA in the Central Nervous System,", L. Hertz, et al., Ed., Alan
R. Liss, Inc., New York, 1983, pp 625-641; Cavalheiro, E. A.,
Lehmann, J., and Turski, L., Eds., "Frontiers in Excitatory Amino
Acid Research", A. R. Liss, New York, N.Y., 1988; Muir, K. W. and
Lees, K. R., Stroke, 26, 503-513, 1995). Enhanced activity at the
EAA synapse will lower the threshold and promote hyperactivity of
the postsynaptic neuron. Evidence for a causal connection between
EAA release and onset of hyperactivity has been provided by the use
of specific EAA receptor antagonists, APB, APV, and APH, in various
models of epilepsy (Cruczwar, S. J., and Meldrum, B. S., Eur. J.
Pharmacol., 83, 335, 1982).
EAAs and Cerebral Ischemia:
[0035] Brain regions such as the hippocampus and the dorsolateral
striatum that are enriched in EAA receptors are especially
vulnerable to ischemic lesions (Jorgensen, M. D. and Diemer, N. A.,
Acta Neurol. Scand., 66, 536-46, 1982) and selective brain
lesioning studies have supported a role for glutamate in ischemic
and hypoglycemic brain injury (Jorgensen, M. B., Johnson, F. F.,
and Diemer, N. H., Acta Neuropathol., 73, 189, 1987; Linden, T.,
Kalimo, H., and Weiloch, T., Acta Neuropathol., 74, 335, 1988).
Furthermore, ischemia-induced hippocampal damage is reduced by
prior local infusion of EAA receptor antagonists (Simon, R. P.,
Grffiths, T., Evans, M. C., Swan, J. H., and Meldrum, B. S., J.
Cereb. Blood Flow Metab., 4, 350-361, 1984; Simon, R. P., Swan, J.
H., Griffiths, T., and Meldrum, B. S., Science, 226, 850-852, 1984)
or by their systemic administration (Boast, C. A., Gerhardt, S. C.,
Pastor, G., Lehmann, J., Etienne, P. E., and Liebman, J. M., Brain
Res. 442, 345-348, 1988). Glutamate can trigger toxic neuronal
degeneration with considerable potency and speed; a 5-minute
exposure to 100 .mu.M Glu is sufficient to destroy large numbers of
cultured cortical neurons (Choi, D. W., Maulucci-Gedde, M. A.,
Kriegstein, A. R., J. Neurosci., 7, 357-368, 1987). Such brief
intense exposure likely accompanies several types of acute insults,
including hypoxia (Rothman, S. M., J. Neurosci., 4, 188-191, 1984),
ischemia (Simon, R. P., Griffiths, T., Evans, M. C., Swan, J. H.,
and Meldrum, B. S., J. Cereb. Blood Flow Metab., 4, 350-361, 1984;
Simon, R. P., Swan, J. H., Griffiths, T., and Meldrum, B. S.,
Science, 226, 850-852, 1984) and prolonged seizures (Ben-Ari, Y.,
Neuroscience, 14, 375-403, 1985).
[0036] In the hippocampus, the pattern of neuronal loss is similar
after an episode of ischemia or of status epilepticus or temporal
lobe epilepsy, the most common form of focal (partial) epilepsy.
Irreversible cell loss is common in the hilus of the hippocampal
area dentata and in the CA1 and CA3 pyramidal cell layers.
Prolonged (24 hours) electrical stimulation of the perforant path
fibers in vivo produces histopathological changes in the
hippocampal CA1 and CA3 pyramidal neurons similar to those elicited
by EAAs (Meldrum, B. S., and Corsellis, J. A. N., In "Greenfield's
Neuropathology", 4th Edn., J. H. Adams, et al., Ed., 1984, pp
921-950; Sloviter, R. S., Brain Res. Bull., 10, 675-697, 1983;
Sloviter, R. S., Science, 73, 1987). The increased activity in
excitatory hippocampal pathways is suggested as the cause for the
irreversible damages to cells, probably by the release of EAAs in
neurotoxic concentrations followed by Ca.sup.2+ influx through the
stimulated NMDA receptor-ion channel complex. The mitochondria in
selectively vulnerable hippocampal neurons show massive overloading
with Ca.sup.2+ during status epilepticus and after 2 hours of
reperfusion following cerebral ischemia (Griffiths. T.,
Neuroscience, 10, 385-395, 1983).
[0037] The compounds of the present invention are useful in
pharmaceutical compositions using conventional pharmaceutical
carriers or vehicles for administration to humans and animals in
unit dosage forms, such as tablets, capsules, pills, powders,
granules, suppositories, sterile parenteral solutions or
suspensions, sterile non-parenteral solutions or suspensions, oral
solutions or suspensions, oil in water or water in oil emulsions
and the like, containing suitable quantities of the active
ingredient.
[0038] Compositions for injection, may be prepared in unit dosage
form in ampules, or in multidose containers. The injectable
compositions may take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain various
formulating agents. Alternatively, the active ingredient may be in
powder form for reconstitution at the time of delivery with a
suitable vehicle, such water. In injectable compositions, the
carrier is typically comprised of sterile water, saline or another
injectable liquid. Also, various buffering agents, preservatives
and the like can be included.
[0039] Topical applications may be formulated in carriers such as
hydrophobic or hydrophilic bases to form ointments, creams,
lotions, in aqueous, oleaginous or alcoholic liquids to form paints
or in dry diluents to form powders.
[0040] Oral compositions may take such forms as tablets, capsules,
oral suspensions and oral solutions. The oral compositions may
utilize carriers such as conventional formulating agents, and may
include sustained release properties as well as rapid delivery
forms.
[0041] The dosage to be administered depends to a large extent upon
the condition and size of the subject being treated, the route and
frequency of administration, and the particular compound selected.
Such matters, however, are left to the routine discretion of the
physician according to principles of treatment well known in the
medical arts. The compositions of this invention for human delivery
per unit dosage, whether liquid or solid, comprise from about 0.01%
to as high as about 99% of the active compound, the preferred range
being from about 10-60%.
[0042] The invention described herein also includes a method of
treating a mammal in need of ischemia treatment comprising
administering to said mammal the claimed composition in an amount
effective to treat said condition. About 1 to 300 mg/kg of body
weight, preferably about 25 to 200 mg/kg, one to four times daily
is preferred.
[0043] The 5-pyridyl substituted triazoline compounds represented
by structures I, II and III of this invention may be prepared by
the reaction of diazomethane with Schiff bases as described in the
Applicant's previous patents on triazolines (P. K. Kadaba, U.S.
Pat. No. 4,511,572, 1985; U.S. Pat. No. 4,689,334, 1987, the
disclosures of which are hereby incorporated by reference), and
illustrated in Equation 1. ##STR10## where R.sup.2 is 4-, 3- or
2-pyridyl and R.sup.1 is 3,4- or 3,5-dichloro, p- or m-chloro, p-
or m-bromo, p- or m-fluoro, p- or m-trifluoromethyl, p- or m-lower
alkyl, p- or m-lower alkoxy or hydrogen.
[0044] In the method of preparation, the reaction between the
Schiff base and diazomethane is carried out by treating the
appropriate Schiff base with a dioxane solution of diazomethane at
room temperature, as described previously (P. K. Kadaba, U.S. Pat.
No. 4,511,572, 1985; U.S. Pat. No. 4,689,334, 1987).
[0045] The following examples are presented to illustrate the
invention, but it is not to be considered as limited thereto. In
the examples and throughout the specifications, parts are by weight
unless otherwise indicated.
EXAMPLE 1
Preparation of 1-Phenyl-5-(4-,3-, or
2-pyridyl)-1,2,3-triazolines
[0046] To a wet (undried) solution of diazomethane in p-dioxane
(0.06 mole), contained in an Earlenmeyer flask and kept cold in an
ice bath, is added the Schiff base (0.03 mole), and gently swirled
until complete solution resulted. The flask is then stoppered with
a clean cork, and allowed to stand at room temperature for 24-96
hours as necessary. At the end of the reaction, if crystals of the
triazoline have appeared, the reaction mixture is filtered, and the
filtrate cooled and diluted with water until a precipitate is
obtained. It is filtered, and crystallized from ethanol or acetone
or acetone-petroleum ether. The total yield of pure products ranges
from 60-80%.
[0047] The 1,2,3-triazolines that are prepared according to the
above described procedure are all patented (P. K. Kadaba, U.S. Pat.
No. 4,511,572, 1985; U.S. Pat. No. 4,689,334, 1987) and presented
in Table I along with their melting points and yields.
[0048] The 5-(2-oxo-1-pyrrolidino)-1,2,3-triazolines represented by
structure IV of this invention, may be prepared by reacting aryl
azides with N-vinylpyrrolidinone as described in the U.S. Patent
(P. K. Kadaba, U.S. Pat. No. 4,820,721, 1989, the disclosure of
which is hereby incorporated by reference), and shown in Equation
2. ##STR11##
[0049] where R.sup.1 is as defined above.
[0050] In the method of preparation, the reaction between the
N-vinylpyrrolidinone and the phenyl azide is carried out at room
temperature in ethanol as solvent, and allowed to stand in the dark
for several weeks, depending on the reactivity of the phenyl
azides. Refluxing the reaction mixture eliminates the
5-(2-oxo-1-pyrrolidino) group and yields the 1-phenyl triazole.
[0051] The following examples are presented to illustrate the
invention, but it is not to be considered as limited thereto. In
the examples and throughout the specifications, parts are by weight
unless otherwise indicated.
EXAMPLE 2
Preparation of
1-Phenyl-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-triazolines
[0052] To the N-vinylpyrrolidinone is added the appropriately
substituted phenyl azide, and the reaction mixture is allowed to
stand in the dark at room temperature, with periodic shaking, for
several weeks, depending on the reactivity of the azide. The
reaction is considered to be complete when the oily mixture has
almost solidified to a crystalline mass. The chunky mass of
crystals is triturated with small portions of ethanol, suction
filtered and washed several times with ether or an ether-petroleum
ether mixture, as the case may be, until all of the unreacted
N-vinylpyrrolidinone is removed. The triazolines are crystallized
from acetone or acetone-petroleum ether mixture. The yields of the
pure compounds range from 45% to 75%.
[0053] The 1-phenyl-5-(2-oxo-1-pyrrolidino)-1,2,3-triazolines
prepared according to the above procedure are patented (P. K.
Kadaba, U.S. Pat. No. 4,820,721, 1989) and are given in Table II
along with their melting points and yields.
[0054] The aminoalkylpyridines of this invention, represented by
structures V, VI and VII, may be prepared by the reaction of
pyridyl alkyl ketones with the appropriate anilines, followed by
sodium borohydride reduction of the resulting ketimines, according
to Equation 3, following the procedure described in an earlier
patent (P. K. Kadaba, U.S. Pat. No. 5,648,369, 1997, the disclosure
of which is hereby incorporated by reference). ##STR12##
[0055] In the above equation, R.sup.3 is 4-, 3- or 2-pyridyl group,
R.sup.4 is methyl or ethyl and R.sup.1 is as defined above
previously.
[0056] In the method of preparation, the pyridyl alkyl ketone is
condensed with the aniline in refluxing xylene, in the presence of
commercially available molecular sieves. The imine formed by this
reaction is then reduced by sodium borohydride in ethanol.
[0057] The following examples are presented to illustrate the
invention, but it is not to be considered as limited thereto. In
the examples and throughout the specifications, parts are by weight
unless otherwise indicated.
EXAMPLE 3
Preparation of 1-[N-(Phenyl)-1-(4-, 3-, or 2-pyridyl)]-1-ethyl (or
propyl) amine
[0058] A mixture of the appropriately substituted acetyl or
propionyl pyridine (0.06 mole) and the appropriately substituted
aniline (0.06 mole) in xylene (150 ml) is refluxed for 4 hours in
the presence of molecular sieves (75 g; Davison, grade 514,
effective pore size 4A.degree., 8-12 mesh beads). At the end of the
reaction, the molecular sieves are filtered, washed with benzene
and the combined filtrates rotary evaporated to remove xylene. The
syrupy residual material is crystallized from benzene or
benzene-petroleum ether mixture to yield the pure imines in yields
varying from 40% to 60%. The imines that are prepared according to
this procedure are given in Table III, along with their yields and
melting points.
Sodium Borohydride Reduction of Imines:
[0059] To a solution of the imine (0.03 mol) in ethanol (100 ml),
is added finely powdered sodium borohydride (0.15 mol) and the
reaction mixture refluxed with magnetic stirring for 2-4 hours.
[0060] The reaction mixture is then cooled in an ice bath and the
excess sodium borohydride is destroyed by slow addition of dilute
hydrochloric acid (1:1 mixture), until the reaction mixture is
acidic and no more hydrogen evolution is noticed. The white
inorganic solids that precipitate are dissolved by addition of
water and the solution then made basic with sodium hydroxide. It is
then cooled in the refrigerator for 1-2 days, when the
1-[N-(phenyl)]-1-(pyridyl)-1-alkylamines appear as white to beige
colored solids. They are filtered, washed well with water until the
filtrate is neutral, and recrystallized from a mixture of
acetone-petroleum ether or tertiary-butyl methyl ether and
petroleum ether. In Table IV, melting points and yields are given
for all the 1-[N-(phenyl)]-1-(pyridyl)-1-ethyl-(or propyl) amine
compounds that are prepared, including a number of new compounds,
not known before.
EXAMPLE 4
[0061] The 1,2,3-triazolines and the AAP compounds of this
invention are effective by the intraperitoneal, intravenous, and
oral routes of administration. These compounds are effective
antiischemic/antistroke agents that are useful in the treatment of
cerebral ischemia in humans, both focal and global, resulting from
stroke.
[0062] The potency of these compounds range from those which are
very highly potent to those of good medium potency, with no
accompanying toxicity, in both the gerbil model of global ischemia
and the rat model of focal ischemia.
[0063] There is a definite need for more effective, clinically
useful drugs in the management of stroke. Lipophilicity and
penetration of the blood brain barrier are important factors to be
taken into consideration when designing antistroke drugs, because
systemic drug administration as quickly as possible after an
episode of stroke is essential to prevent the onset and spread of
neuronal injury. The highly lipophilic triazolines and AAPs can
enter the brain in less than 15 minutes after intraperitoneal
administration, and reduce or prevent excitotoxicity as early as
possible in the ischemic brain. Furthermore, their oral activity,
especially that of the AAPs, makes them suitable candidates for
delayed post treatment of the stroke victims, without evincing
undue motor toxicity.
Evaluation of Antiischemic Activity:
[0064] A series of triazolines with different 5-substituents and
the corresponding metabolite analogue compounds, the AAPs, of this
invention, has been evaluated for antiischemic activity by the
intraperitoneal route using two reliable paradigms of brain injury
in stroke, the bilateral carotid occlusion model in the gerbil and
the middle cerebral artery occlusion (MCAO) model in the rat. These
two experimental procedures will indicate the potential of the
anticonvulsant triazolines and AAPs, for reducing or preventing
neuronal damage following cerebral ischemia.
[0065] The compounds are initially tested in the gerbil model of
global ischemia, to establish the presence or absence of
neuroprotective effect in the drug, when administered
intraperitoneally as a pretreatment compound. Compound's ability to
protect neurons from reperfusion injury in the gerbil is carried
out using histopathological and behavioral assessments. The more
active compounds are then advanced for further evaluation in the
rat MCAO model of reversible focal cerebral ischemia after ip
administration. This model is a clinically relevant model, as it
mimics stroke in humans. Compound evaluation in the rat model
consists of behavioral and histopathological studies and focused on
hippocampal damage in the CA1 and CA3 pyramidal neurons.
EXAMPLE 5
The Bilateral Carotid Occlusion Model of Global Ischemia in the
Gerbil to Study the Effects of Pretreatment Doses.
[0066] Male gerbils (50-60 gm, Tumblebrook Farm, West Brookfield,
Mass.) are housed in groups of three for at least one week prior to
instrumentation. Following surgery, the gerbils are singly housed
in order to avoid the possible accidental induction of ischemia by
cage mates. Food and water are available ad libitum in the home
cage. All gerbils are maintained under a 12 hr light/dark
cycle.
[0067] Transient ischemia is produced by occluding both common
carotids using surgically placed occluders (Chandler, M. J., et
al., J. Pharmacol. Meth 14, 137-146, 1985). A ventral midline
incision is made in the neck of gerbils anesthetized with
pentobarbital (40 mg/kg). Common carotids are exposed and separated
from the vagosympathetic nerve trunk. A loop of unwaxed dental
floss (Johnson and Johnson) is placed around each carotid. The ends
of the floss are each passed through one of the lumens of a double
lumen catheter (Dural Plastics and Engineering, Dural, NSW,
Australia). The catheter and dental floss are passed through the
dorsal musculature and exited at the dorsal surface of the neck.
The catheter is fixed in position, directly above the carotid
artery, using cyanoacrylate adhesive at the exit site. The dental
floss length is marked in order to assure that the animal does not
occlude the carotid during daily cleaning and exploratory activity.
The ventral incision is closed with 9 mm wound clips. After 48
hours following instrumentation, ischemia is produced by gently
pulling the looped dental floss until the artery is occluded.
Occlusion of the artery is associated with depression of
spontaneous motor activity, loss of consciousness, ptosis and a
change in breathing pattern. Complete interruption of blood flow
occurs under these conditions. The occlusion is maintained for 5
minutes, and then the dental floss is removed to allow complete
reperfusion, when these symptoms reverse. After reperfusion, the
catheter is trimmed flush with the surface of the neck.
[0068] During the 5 minutes of ischemia and for a minimum of 2
hours after reversal of carotid occlusion, rectal and cranial
temperature will be maintained at 36-37.degree. in all animals. The
rectal and cranial temperatures will be adjusted to the desired
levels by heating or cooling from a blanket-jacketed water bath, as
described previously (Busto, R., et al., Stroke, 20, 904-910, 1989;
Campos-Gonzales, R., and Kindy, M. S., J. Neurochem., 59,
1955-1958, 1992). All gerbils will be monitored for seizures that
may occur during postischemic reperfusion. Any gerbil demonstrating
motor activity that could be associated with seizures is
discarded.
[0069] Gerbils (n=6) are pretreated intraperitoneally, 30 minutes
prior to the initiation of carotid occlusion, with an appropriate
dose of the triazoline or AAP compound. If the highest dose of any
of the test compounds caused undue toxicity or mortality, then a
lower dose of drug is used (eg. 100, 150, and 200 mg/kg). All
animals are tested for postischemic locomotor activity using a
computer-controlled monitoring equipment. A brief description of
the equipment and the technique are as follows. The locomotor
activity arena consists of a walled, cylindrical drum of 2-foot
diameter that is equipped with two orthogonally placed photocell
detector systems. Interruption of each of the photocell detector
beams will define an activity count. All activity counts will be
recorded automatically by an IBM computer system. Each gerbil will
be tested 24 hours after reversal of carotid occlusion or
sham-ischemia.
[0070] After locomotor activity testing, gerbils are evaluated for
differences in patrolling behavior using a eight-arm radial maze.
In this procedure, animals are placed into the center start chamber
of the maze, the barrier removed and the amount of time and the
number of times, the animals make an error recorded, prior to
completion of exploration in all 8 arms of the maze. An error is
defined as the revisiting of an arm by entering to the extent of
the entire body without including tail by the animal. If the animal
persevers or fails to leave the arm for longer than fifteen
minutes, the session is terminated. In all the evaluations reported
here, animals never exceeded the fifteen minute cut-off point and
all eight-arms were successfully explored with differing degrees of
errors. In the control population of the animals, the number of
errors and exploration of the maze with no prior experience (naive)
was approximately 5 errors. Data are expressed as the mean (+/-S.E)
for groups of 6 gerbils. Following 5 minutes of bilateral carotid
occlusion and testing at 24 hours, gerbils make an average number
of errors of 28. When animals are pretreated with drug, there is a
dose-related decrease in the number of errors made (Table IV). The
threshold for protection in the maze test is lower than that seen
in the locomotor activity test. While not significantly different
from saline, a dose of 10 mg/kg slightly reduces the number of
errors in post ischemic gerbils.
[0071] All animals will then be subjected to seven day
post-reperfusion histopathological assessment after being
re-anesthetized with 60 mg/kg of pentobarbital. Histopathology is
determined using frozen sections fixed on treated slides and
stained with hematoxylin-eosin for cell body counting. Changes in
neuronal nuclei are determined for the dorsal hippocampus and for
the CA3 region as comparison. Histopathological evaluation of
gerbil brains seven days after 5 minutes of ischemia demonstrates,
the expected loss of CA1 hippocampus pyramidal cells (Kirino, T.,
Brain Res., 239, 57, 1982).
[0072] Table V presents the results of testing the compounds in the
gerbil model. This table identifies the specific compounds tested
by their chemical name, and provides the test model, the route of
administration, and the antiischemic activity as indicated by
neuronal density and radial maze errors and calculated as percent
protection afforded by the compound, when administered
intraperitoneally to the gerbils as a pretreatment dose.
EXAMPLE 6
MCAO Rat Model of Reversible Focal Cerebral Ischemia:
[0073] Males of a spontaneously hypertensive inbred strain of
Wistar rat (SHR) (250-350 gm., purchased from Harlan, Indianapolis,
Ind.) are used for the preparation of this model since they have
been found superior to others in producing consistent infarct
volumes and also because hypertension is a well-documented risk
factor in stroke (Brint, S., et al., J. Cereb. Blood Flow Metab.,
8, 474-485, 1988). The rats are maintained under conditions of
controlled lighting (12:12 light/dark cycle) and temperature
(22.degree. C.) and allowed free access to lab chow and tap water.
All experiments are performed during "lights on" hours. A method of
reversible focal ischemia in the rat is used, similar to the
original technique of Brint and colleagues (1988) as modified by
Aronowski and co-workers (Aronowski, J., et al., Stroke, 25,
2235-2240, (1994). It involves temporary occlusion of the MCA
(middle cerebral artery) and ipsilateral CCA (common carotid
artery) for two hours, to produce infarct volumes that are 50% of
those observed with permanent CCA-MCA occlusion (Pettigrew, L. C.,
et al., J. Cereb. Blood Flow Metab., 16, 1189-1202, 1996;
Smith-Swintosky, V. L., et al., J. Cereb. Blood Flow Metab., 16,
585-598, 1996).
[0074] Animals are fasted overnight prior to surgical preparation
for ischemia. Each rat is anesthetized with 500 mg/kg chloral
hydrate for isolation of MCA and CCA. A catheter is inserted into
the right femoral artery for sampling of blood and measurement of
mean arterial blood pressure (MABP) on a graphic recorder (Model
RS3400, Gould Electronics, Centerville, Ohio), to monitor
preischemic and 30 min postischemic blood glucose levels, blood
gases and hematocrits. A second catheter is inserted into the right
femoral vein for injection of drug or vehicle.
Electroencephalographic (EEG) and electrocardiographic (ECG)
activities are monitored through subdermal electrodes and displayed
on the graphic recorder. Thermistor probes are inserted into the
rectum and temporalis muscles to monitor body and brain
temperature, which is maintained at 36-37.degree. C. by external
warming. The left CCA is isolated through an anterior incision in
the neck. A second incision is made between the lateral canthus of
the left eye and the ipsilateral external auditory canal to expose
the underlying skull. Under direct visualization with a Zeiss
operating microscope, the left MCA is exposed through a 2-mm
burrhole drilled 2-3 mm rostral to the fusion of the zygomatic arch
and the squamosal bone. The dura is opened with a sharp needle and
an alloy wire (0.1 mm diameter) is inserted beneath the MCA just
superior to the inferior cortical vein. The MCA is elevated from
the cortical surface and reversibly occluded by compression against
the wire. Obstruction of blood flow in the MCA is confirmed by
direct microscopic observation. A bed of saline-soaked cotton is
fashioned to keep the MCA moist while it is being occluded. A
surgical clip is used to occlude the CCA for the two-hour period.
Ischemia is reversed by removing the clip from the CCA and
withdrawing the wire from beneath the MCA. The scalp and neck
incisions are sutured before the rat is returned to its cage and
given free access to water and chow.
[0075] Drug is administered intraperitoneally before ischemia or
during postischemic reperfusion. The doses selected are those shown
to be effective in preventing CA1 neuronal loss in pretreated
gerbils. Groups of sham-ischemic (n=10) and ischemic control (n=10)
animals are prepared for comparison to drug-treated SHRs. Other
groups of sham-ischemic animals are pretreated with the most
effective dose of each compound shown to prevent CA1 neuronal loss
in gerbils (n=6 in each group). Another group of six SHRs will be
pretreated with the same maximally effective dose of each
neuroprotective compound before the animals undergo two hours of
CCA-MCA occlusion. The post-treatment groups are given the same
dose immediately after reversal of ischemia, or following one,
three, or six hours of reperfusion (n=6 in each group). All animals
will undergo functional assessment of cognitive performance before
being euthanized for measurement of infarct volume 24 hours after
reversal of ischemia.
Measurement of Infarct Volume:
[0076] The size of the infarction resulting from two hours of MCAO,
is quantified using triphenyltetrazolium chloride (TTC) staining as
described by Bederson, J. B., and colleagues (Stroke, 17,
1304-1308, 1986). Twenty-four hours following MCAO, rats are
re-anesthetized with chloral hydrate (500 mg/kg body weight) and
perfused transcardially with heparinized saline. The brains are
removed and chilled at -20.degree. C. for 15 minutes before being
placed in a Rodent Brain Matrix (ASI Instruments). Seven serial
one-mm thick coronal sections through the rostral to caudal extent
of the infarction are obtained from each brain, beginning two mm
from the frontal pole (corresponding to approximately 10.2 mm from
the intra-aural line). This procedure reproducibly includes the
entire infarction observed in permanent focal ischemia
(Smith-Swintosky, V. L., et al., J. Cereb. Blood Flow Metab., 16,
585-598, 1996). The individual sections are immersed in 2% TTC and
incubated at 37.degree. C. for 10 minutes on each surface. The
TTC-stained sections are then placed in 10% formalin and kept in
darkness at 4.degree. C. for at least 24 hours. The infarct area in
each section is determined with a computer-assisted image analysis
system, consisting of a Power Macintosh computer (Apple Computer)
equipped with a Quick Capture frame grabber card (Data
Translations), Hitachi CCD camera mounted on an Olympus BX40
microscope, and NIH Image Analysis software, v. 1.55. The system is
calibrated against a Kodak Optical Density Standard. An optical
density threshold is taken from healthy gray matter in the
unaffected right cortex and used to create an artificial color
image to distinguish between infarcted and normal tissue. The
artificial color image and video photograph of each slice is used
to compute the area of the infarct (38.4 pixels/mm), which is
expressed as a fraction of the total area in the left hemisphere.
The total volume of the infarction is computed by multiplying the
infarct area in each coronal section by the number of slices (n=7)
and the thickness of each slice (one mm uniformly). All
measurements of infarct volume are performed by a single operator
blinded to treatment status. Statistical comparison of the ischemic
control animals to multiple groups of treated, ischemic rats will
be accomplished by ANOVA with Dunnett's post hoc test.
TABLE-US-00001 TABLE I
1-(Phenyl)-5-(pyridyl)-.DELTA..sup.2-1,2,3-triazolines Melting
Compound Point, .degree. C. Yield, % (1)
1-(Phenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline 160-161 68
(2) 1-(p-Chlorophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
151-152 82 (3)
1-(m-Chlorophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
109-111 80 (4)
1-(p-Fluorophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
139-140 91 (5)
1-(m-Fluorophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (6)
1-(p-Bromophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
158-160 51 (7)
1-(m-Bromophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (8)
1-(3,4-Dichlorophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
171-172 82 (9)
1-(3,5-Dichlorophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
(10) 1-(p-Trifluoromethylphenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-
149-150 58 triazoline (11)
1-(m-Trifluoromethylphenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-
68-71 42 triazoline (12)
1-(p-Methylphenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
157-158 97 (13)
1-(m-Methylphenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
(14)
1-(p-Methoxyphenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
148-148.5 45 (15)
1-(m-Methoxyphenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
(16)
1-(3,4-Difluorophenyl)-5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
(17) 1-(Phenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
113-114 27 (18)
1-(m-Chlorophenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
75-77 75 (19)
1-(3,5-Dichlorophenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
95-97 70 (20)
1-(p-Trifluoromethylphenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazol-
ine 138-140 50 (21)
1-(m-Trifluoromethylphenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazol-
ine 72-74 40 (22)
1-(p-Fluorophenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
104-106 60 (23)
1-(m-Fluorophenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
(24) 1-(m-Bromophenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
75-77 65 (25)
1-(m-Methylphenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
(26)
1-(m-Methoxyphenyl)-5-(3-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
(27)
1-(p-Chlorophenyl)-5-(2-pyridyl)-.DELTA..sup.2-1,2,3-triazoline 138
80 (28) 1-(Phenyl)-5-(2-pyridyl)-.DELTA..sup.2-1,2,3-triazoline
83-85 53 (29)
1-(p-Trifluoromethylphenyl)-5-(2-pyridyl)-.DELTA..sup.2-1,2,3-triazol-
ine
[0077] TABLE-US-00002 TABLE II
1-(Phenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-triazolines
Melting Point, .degree. C. Yield % (1)
1-(Phenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-triazoline
118.5-121 46 (2)
1-(p-Chlorophenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-triazol-
ine 126-128 62 (3)
1-(3,4-Dichlorophenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-
133-133.5 70 triazoline (4)
1-(p-Bromophenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-triazoli-
ne 129-131.5 60 (5)
1-(p-Fluorophenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-triazol-
ine 111-114 21 (6)
1-(p-Trifluoromethylphenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,-
3- 130-133 66 triazoline (7)
1-(m-Trifluoromethylphenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,-
3- 102-105 60 triazoline (8)
1-(p-Methylphenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-
110.5-113 55 triazoline (9)
1-(p-Methoxyphenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-
122-125 62 triazoline (10)
1-(3,5-Dichlorophenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-
triazoline (11)
1-(m-Chlorophenyl)-5-(2-oxo-1-pyrrolidino)-.DELTA..sup.2-1,2,3-triazo-
line
[0078] TABLE-US-00003 TABLE III Aminoalkylpyridine (AAP) Compounds
(Methyl, Ethyl or Propylamine Derivatives) Melting Compound Point,
.degree. C. Yield, % (1) N-(Phenyl)-4-pyridylmethylamine (2)
N-(p-Chlorophenyl)-4-pyridylmethylamine 91-94 26 (3)
N-(3,4-Dichlorophenyl)-4-pyridylmethylamine 99-101.5 31 (4)
N-(3,5-Dichlorophenyl)-4-pyridylmethylamine (5)
N-(m-Chlorophenyl)-4-pyridylmethylamine 82-84 18 (6)
N-(p-Bromophenyl)-4-pyridylmethylamine 92-94.5 32 (7)
N-(m-Bromophenyl)-4-pyridylmethylamine (8)
N-(p-Fluorophenyl)-4-pyridylmethylamine 67-70 33 (9)
N-(m-Fluorophenyl)-4-pyridylmethylamine (10)
N-(p-Methylphenyl)-4-pyridylmethylamine 71-73 38 (11)
N-(p-Methoxyphenyl)-4-pyridylmethylamine 74.5-76 33 (12)
1-[N-(p-Methylphenyl)]-1-(4-pyridyl)-1-ethylamine 93-95.5 72 (13)
1-[N-(p-Methoxyphenyl)]-1-(4-pyridyl)-1-ethylamine 74.5-76 70 (14)
1-[N-(Phenyl)]-1-(4-pyridyl)-1-ethylamine 126-128 60 (15)
1-[N-(m-Chlorophenyl)]-1-(4-pyridyl)-1-ethylamine 157-159.5 71 (16)
1-[N-(3,4-Dichlorophenyl)]-1-(4-pyridyl)-1-ethylamine 153.5-155
54.3 (17) 1-[N-(3,5-Dichlorophenyl)]-1-(4-pyridyl)-1-ethylamine
150-152.5 74 (18) 1-[N-(p-Bromophenyl)]-1-(4-pyridyl)-1-ethylamine
107-108.5 62 (19) 1-[N-(m-Bromophenyl)]-1-(4-pyridyl)-1-ethylamine
(20) 1-[N-(p-Fluorophenyl)]-1-(4-pyridyl)-1-ethylamine 85-87 60
(21) 1-[N-(m-Fluorophenyl)]-1-(4-pyridyl)-1-ethylamine (22)
1-[N-(3,4-Difluorophenyl)]-1-(4-pyridyl)-1-ethylamine 87-90.0 72
(23) 1-[N-(m-Trifluoromethylphenyl)]-1-(4-pyridyl)-1-ethylamine
149-151 72 (24) 1-[N-(Phenyl)]-1-(3-pyridyl)-1-ethylamine 132.5-134
77 (25) 1-[N-(m-Chlorophenyl)]-1-(3-pyridyl)-1-ethylamine 120-122
47 (26) 1-[N-(p-Fluorophenyl)-1-(3-pyridyl)-1-ethylamine (27)
1-[N-(m-Fluorophenyl)]-1-(3-pyridyl)-1-ethylamine (28)
1-[N-(p-Bromophenyl)]-1-(3-pyridyl)-1-ethylamine 128.5-130.5 47
(29) 1-[N-(m-Bromophenyl)]-1-(3-pyridyl)-1-ethylamine (30)
1-[N-(p-Methylphenyl)]-1-(3-pyridyl)-1-ethylamine 112-113.5 62 (31)
1-[N-(p-Methoxyphenyl)]-1-(3-pyridyl)-1-ethylamine 73-74.5 6.3 (32)
1-[N-(3,4-Dichlorophenyl)]-1-(3-pyridyl)-1-ethylamine 127-128.5 78
(33) 1-[N-(3,5-Dichlorophenyl)]-1-(3-pyridyl)-1-ethylamine (34)
1-[N-(Phenyl)]-1-(4-pyridyl)-1-propylamine 75-78 82 (35)
1-[N-(p-Chlorophenyl)]-1-(4-pyridyl)-1-propylamine 133-135 84 (36)
1-[N-(m-Chlorophenyl)]-1-(4-pyridyl)-1-propylamine 127.5-129.5 54
(37) 1-[N-(p-Bromophenyl)]-1-(4-pyridyl)-1-propylamine 128-130.5
74.5 (38) 1-[N-(m-Bromophenyl)]-1-(4-pyridyl)-1-propylamine (39)
1-[N-(p-Fluorophenyl)]-1-(4-pyridyl)-1-propylamine 94.5-97 45.6
(40) 1-[N-(m-Fluorophenyl)]-1-(4-pyridyl)-1-propylamine (41)
1-[N-(3,4-Difluorophenyl)]-1-(4-pyridyl)-1-propylamine 104-106.5 40
(42) 1-[N-(3,5-Difluorophenyl)]-1-(4-pyridyl)-1-propylamine (43)
1-[N-(p-Methoxyphenyl)]-1-(4-pyridyl)-1-propylamine 86-88 62 (44)
1-[N-(p-Methylphenyl)]-1-(4-pyridyl)-1-propylamine 117-119.5 74
(45) 1-[N-(m-Methoxyphenyl)]-1-(4-pyridyl)-1-propylamine (46)
1-[N-(m-Methylphenyl)]-1-(4-pyridyl)-1-propylamine (47)
1-[N-(3,4-Dichlorophenyl)]-1-(4-pyridyl)-1-propylamine 154-156 31
(48) 1-[N-(3,5-Dichlorophenyl)]-1-(4-pyridyl)-1-propylamine (49)
1-[N-(Phenyl)]-1-(3-pyridyl)-1-propylamine 90-92 39 (50)
1-[N-(p-Chlorophenyl)]-1-(3-pyridyl)-1-propylamine (51)
1-[N-(m-Chlorophenyl)]-1-(3-pyridyl)-1-propylamine (52)
1-[N-(p-Bromophenyl)]-1-(3-pyridyl)-1-propylamine (53)
1-[N-(m-Bromophenyl)]-1-(3-pyridyl)-1-propylamine (54)
1-[N-(p-Fluorophenyl)]-1-(3-pyridyl)-1-propylamine (55)
1-[N-(m-Fluorophenyl)]-1-(3-pyridyl)-1-propylamine (56)
1-[N-(3,4-Difluorophenyl)]-1-(3-pyridyl)-1-propylamine (57)
1-[N-(3,5-Difluorophenyl)]-1-(3-pyridyl)-1-propylamine (58)
1-[N-(p-Methylphenyl)]-1-(3-pyridyl)-1-propylamine 100-104 32 (59)
1-[N-(m-Methylphenyl)-1-(3-pyridyl)]-1-propylamine (60)
1-[N-(p-Methoxyphenyl)]-1-(3-pyridyl)-1-propylamine 94.5-96 74 (61)
1-[N-(m-Methoxyphenyl)]-1-(3-pyridyl)-1-propylamine (62)
1-[N-(3,4-Dichlorophenyl)]-1-(3-pyridyl)-1-propylamine (63)
1-[N-(3,5-Dichlorophenyl)]-1-(3-pyridyl)-1-propylamine (64)
1-[N-(Phenyl)]-1-(phenyl)-1-(4-pyridyl)methylamine 91.5-94 48 (65)
1-[N-(p-Methoxyphenyl)]-1-(phenyl)-1-(4-pyridyl)- 112-115 96.7
methylamine (66) 1-[N-(m-Methoxyphenyl)]-1-(phenyl)-1-(4-pyridyl)-
121.5-123 98 methylamine (67)
1-[N-(p-Chlorophenyl)]-1-(phenyl)-1-(4-pyridyl)- 135-137 71
methylamine (68) 1-[N-(m-Chlorophenyl)]-1-(phenyl)-1-(4-pyridyl)-
104-106 49 methylamine (69)
1-[N-(p-Methylphenyl)]-1-(phenyl)-1-(4-pyridyl)- 134-135.5 60
methylamine (70)
1-[N-3,5-Dichlorophenyl)]-1-(phenyl)-1-(4-pyridyl)- Sublimes 56
methylamine (71)
1-[N-(m-Trifluoromethylphenyl)]-1-(phenyl)-1-(4-pyridyl)- 123-125.5
70 methylamine (72) 1-[N-(p-Bromophenyl)]-1-(phenyl)-1-(4-pyridyl)-
methylamine (73)
1-[N-(p-Dimethylaminophenyl)]-1-(phenyl)-1-(4-pyridyl)- 149-151 63
methylamine (74)
1-[N-(3,4-Dichlorophenyl)]-1-(phenyl)-1-(4-pyridyl)- 130-132.5
methylamine (75) 1-[N-(p-Chlorophenyl)]-1-(phenyl)-1-(3-pyridyl)-
85-87 58 methylamine (76)
1-[N-(m-Chlorophenyl)]-1-(phenyl)-1-(3-pyridyl)- 77-80 methylamine
(77) 1-[N-(p-Methylphenyl)]-1-(phenyl)-1-(3-pyridyl)- 89.5-92.5 50
methylamine (78) 1-[N-(p-Methoxyphenyl)]-1-(phenyl)-1-(3-pyridyl)-
114-116 68.5 methylamine (79)
1-[N-(Phenyl)]-1-(phenyl)-1-(3-pyridyl)methylamine 113.5-115.5 66.8
(80) 1-[N-(3,4-Dichlorophenyl)]-1-(phenyl)-1-(3-pyridyl)- 108.5-111
48 methylamine (81) 1-[N-(p-Bromophenyl)]-1-(phenyl)-1-(3-pyridyl)-
114.5-17.5 22 methylamine (82)
1-[N-(m-Trifluoromethylphenyl)]-1-(phenyl)-1-(3-pyridyl)- 81-83.5
methylamine (83)
1-[N-(3,5-Dichlorophenyl)]-1-(phenyl)-1-(3-pyridyl)- 93-95 17
methylamine (84)
1-[N-(p-Chlorophenyl)]-1,1-(bis-2-pyridyl)methylamine 117-119.5
[0079] TABLE-US-00004 TABLE IV Results of Testing 1,2,3-Triazolines
and AAP Compounds in the Gerbil Model of Global Ischemia. Degree of
Protection in Terms of Neuronal Cell Count and Number of Radial Arm
Maze Errors at Three Different Drug Concentrations Degree of
Protection, % Neuronal Radial Cell Count, %.sup.a Maze Errors,
%.sup.b Test Concentrations, mg/kg Compound 100 150 200 100 150 200
(1) 1-(Phenyl)-5-(4-pyridyl)- 83.0 91.4 96.5 28.9 31.6 27.4
.DELTA..sup.2-1,2,3-triazoline (2) 1-(p-Chlorophenyl)-5-(4- 65.3
82.2 91.4 44.7 30.8 30.1 pyridyl)-.DELTA..sup.2-1,2,3-triazoline
(3) 1-(3,4-Dichlorophenyl)-5- 60.0 74.1 88.5 62.1 -- --
(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (4)
1-(p-Fluorophenyl)-5-(4- 54.9 67.8 77.3 72.2 58.3 45.9
pyridyl)-.DELTA..sup.2-1,2,3-triazoline (5)
1-(p-Trifluoromethylphenyl)-5- 47.0 46.5 62.5 75.9 -- --
(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (6)
1-(m-Chlorophenyl)-5-(4-pyridyl)- 60.1 71.2 87.4 44.7 25.7 21.3
.DELTA..sup.2-1,2,3-triazoline (7) 1-(p-Bromophenyl)-5-(4-pyridyl)-
25.6 39.5 47.9 73.5 61.7 54.2 .DELTA..sup.2-1,2,3-triazoline (8)
1-(3,5-Dichlorophenyl)-5- 30.6 41.3 49.8 72.2 56.6 48.1
(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (9)
1-(p-Chlorophenyl)-5-(3-pyridyl)- 47.9 53.7 59.7 58.7 52.3 45.3
.DELTA..sup.2-1,2,3-triazoline (10)
1-(m-Chlorophenyl)-5-(3-pyridyl)- 46.8 47.3 62.8 60.3 59.0 45.7
.DELTA..sup.2-1,2,3-triazoline (11) 1-(Phenyl)-5-(3-pyridyl)- 49.5
55.6 61.0 57.0 52.0 44.3 .DELTA..sup.2-1,2,3-triazoline (12)
1-(p-Chlorophenyl)-5- 45.6 36.9 63.5 60.7 58.3 42.7
(2-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (13)
1-(p-Bromophenyl)-5-(2-pyridyl)- 47.6 47.2 62.5 62.7 60.0 39.7
.DELTA..sup.2-1,2,3-triazoline (14)
1-(Phenyl)-5-(2-oxo-1-pyrrolidino)- 22.0 29.9 38.5 82.7 70.2 60.7
.DELTA..sup.2-1,2,3-triazoline (15) 1-(p-Chlorophenyl)-5-(2-oxo-1-
29.9 33.9 45.2 71.9 57.6 54.9
pyrrolidino)-.DELTA..sup.2-1,2,3-triazoline (16)
1-(p-Trifluoromethylphenyl)- 534.2 52.8 66.3 67.4 48.8 34.9
(2-oxo-1-pyrrolidino)- .DELTA..sup.2-1,2,3-triazoline (17)
1-(m-Trifluoromethylphenyl)- 25.8 38.2 45.8 76.3 63.4 52.2
5-(2-oxo-1-pyrrolidino)- .DELTA..sup.2-1,2,3-triazoline (18)
1-(p-Bromophenyl)-5- 6.4 17.2 21.4 82.2 76.2 74.1
(2-oxo-1-pyrrolidino)- .DELTA..sup.2-1,2,3-triazoline (19)
1-(p-Fluorophenyl)-5- 5.5 16.2 15.4 97.2 76.6 75.9
(2-oxo-1-pyrrolidino)- .DELTA..sup.2-1,2,3-triazoline (20)
1-(3,4-Dichlorophenyl)-5- 8.1 17.2 18.0 90.9 78.3 80.8
(2-oxo-1-pyrrolidino)- .DELTA..sup.2-1,2,3-triazoline (21)
1-(3,4-Dichlorophenyl)-5- 8.7 15.8 22.8 89.9 83.6 78.0
[N-methyl)-N-acetamide] (22) 1-[N-(p-Chlorophenyl)]-1- 0 6.6 14.5
100 88.5 82.4 (4-pyridyl)-1-ethylamine) (No effect) (No effect)
(23) 1-[N-(p-Bromophenyl)]-1- 0 15.6 28.1 100 81.2 71.3
(4-pyridyl)-1-ethylamine) (no effect) (no effect) (24)
1-[N-(3,4,-Dichlorophenyl)]-1- 26.4 32.9 45.2 74.7 66.3 65.1
(4-pyridyl)-1-ethylamine) (25) 1-[N-(3-Chlorophenyl)]-1- 29.6 39.6
38.9 82.6 77.6 82.2 (4-pyridyl)-1-ethylamine) (26)
1-[N-(3,5,-Dichlorophenyl)]-1- 29.7 32.7 44.1 87.1 84.5 72.7
(4-pyridyl)-1-ethylamine) (27) 1-[N-(3-Trifluoromethylphenyl)]-
15.1 14.6 11.7 103.8 109.1 111.0 1-(4-pyridyl)-1-ethylamine)
(compound has no protective effect) (28) 1-[N-(p-methylphenyl)]-1-
20.0 30.3 38.7 98.1 89.4 79.5 (4-pyridyl)-1-ethylamine) (29)
1-[N-(p-Chlorophenyl)]-1- 32.0 36.2 40.5 84.5 83.3 76.9
(3-pyridyl)-1-ethylamine) (30) 1-[N-(3,4-dichlorophenyl)]-1- 33.5
41.6 42.9 80.3 74.6 73.5 (3-pyridyl)-1-ethylamine) (31)
1-[N-(p-Bromophenyl)]-1- 35.7 19.7 6 81.8 101.1 114.0
(3-pyridyl)-1-ethylamine) (32) (+)1-[N-(p-Chlorophenyl)]-1- 10.7
14.8 19.1 98.6 92.6 91.2 (4-pyridyl)-1-1ethylamine) (33)
(-)1-[N-(p-Chlorophenyl)]-1- 7.6 15.0 16.5 97.5 81.1 79.0
(4-pyridyl)-1-ethylamine) .sup.aNeuronal cell count is directly
proportional to the surviving cells and is a measure of the degree
of protection afforded by the drug. .sup.bThe smaller the
percentage of errors, the better the protection afforded by drug.
The maze errors are indirectly proportional to the neuronal cell
count.
[0080] TABLE-US-00005 TABLE V Results of Testing
.DELTA..sup.2-1,2,3-Triazolines in the Rat Model of Focal Ischemia.
Degree of Protection in Terms of Reduction in Infarct Volume; Ip
Injection 30 min Prior to Ischemia, at 100, 150 and 200 mg/kg Dose
Reduction in Infarct Volume, % Test Concentrations of Drug,
Dissolved in 0.5% Carboxymethyl Cellulose, mg/kg Compound 100 150
200 (1) 1-(Phenyl)-5-(4-pyridyl)- 53.8 33.3 15.2
.DELTA..sup.2-1,2,3-triazoline (2) 1-(p-Chlorophenyl)-5- 16.9 -- --
(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (3)
1-(p-Fluorophenyl)-5- 0 -- --
(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (No reduction in infarct
volume)
[0081] The invention has been described herein with reference to
certain preferred embodiments. However, as obvious variations
thereon will become apparent to those of skill in the art, the
invention is not considered to be limited thereto. TABLE-US-00006
TABLE VI Results of Testing .DELTA..sup.2-1,2,3-Triazolines in the
Rat Model of Focal Ischemia. Degree of Protection in Terms of
Reduction in Infarct Volume; Post-Ischemic Ip Injection of Drug,
Three Times, at the Beginning, then 1 hr. and 2 hrs. of
Reperfusion. Ischemic Controls Treated 3 Times with Vehicle
Reduction in Infarct Volume When Drug is Administered Ip Compound
Three Times, Postischemic, % (1) 1-(p-Fluorophenyl)-5- 30
(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (25 mg/kg .times. 3) (2)
1-(3,4-Dichlorophenyl)- 34
5-(4-pyridyl)-.DELTA..sup.2-1,2,3-triazoline (50 mg/kg .times.
3)
* * * * *