U.S. patent number 4,657,899 [Application Number 06/849,696] was granted by the patent office on 1987-04-14 for antagonists of specific excitatory amino acid neurotransmitter receptors.
This patent grant is currently assigned to Nova Pharmaceutical Corporation. Invention is credited to Maria E. Guzewska, Robert L. Hudkins, Waclaw J. Rzeszotarski.
United States Patent |
4,657,899 |
Rzeszotarski , et
al. |
April 14, 1987 |
Antagonists of specific excitatory amino acid neurotransmitter
receptors
Abstract
The invention pertains to novel, potent anticonvulsants,
analgesics and cognition enhancers achieving their action through
the antagonism of specific excitatory amino acid neurotransmitter
receptors. In particular, the invention is directed to
.omega.-[2-phosphonoalkyleneyl)phenyl]-2-aminoalkanoic acids having
general formula: ##STR1## Wherein R.sub.1 and R.sub.2 are the same
or different and are selected from the group consisting of
hydrogen, lower alkyl, halogen, --CH.dbd.CH--CH.dbd.CH.dbd., amino,
nitro, trifluoromethyl or cyano; n and m=0, 1, 2, or 3; and the
pharmaceutically acceptable salts and derivatives thereof. Examples
of specific preferred compounds of general formula are selected
from the group consisting of:
4-[2-phosphonomethylphenyl]-2-aminobutanoic acid, ethyl
3-[2-(2-diethylphosphonoethyl)phenyl]-2-acetamido-2-carboethoxypropanoate,
3-[2-(2-phosphonomethyl)phenyl]-2-aminopropanoic acid, ethyl
3-[2-(3-bromopropyl)phenyl]-2-acetamido-3-carboethoxypropanoate,
ethyl
3-[2-(3-diethylphosphonopropyl)phenyl]-2-acetamido-2-carboethoxypropanoate
, ethyl 3-[2-(3-phosphonopropyl)-phenyl]-2-aminopropanoic acid,
ethyl
5-[2-(diethylphosphonomethyl)-phenyl]-2-acetamido-2-carboethoxypentanoate,
and 5-[2-phosphonomethylphenyl]-2-aminopentanoic acid.
Inventors: |
Rzeszotarski; Waclaw J.
(Millersville, MD), Hudkins; Robert L. (Baltimore, MD),
Guzewska; Maria E. (Baltimore, MD) |
Assignee: |
Nova Pharmaceutical Corporation
(Baltimore, MD)
|
Family
ID: |
25306304 |
Appl.
No.: |
06/849,696 |
Filed: |
April 9, 1986 |
Current U.S.
Class: |
514/120; 514/114;
558/192; 562/11; 987/162; 987/165; 514/113; 558/190; 558/193 |
Current CPC
Class: |
A61P
25/04 (20180101); C07F 9/3882 (20130101); C07F
9/3834 (20130101); A61P 25/08 (20180101) |
Current International
Class: |
C07F
9/00 (20060101); C07F 9/38 (20060101); C07F
009/38 (); A61K 031/04 (); A61K 031/135 (); A61K
031/195 () |
Field of
Search: |
;260/52.5D
;514/120,113,114 ;558/198,190,192,193 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Obrycki et al, J. Org. Chem, vol. 33, No. 2, Feb. 1968, pp.
632-636. .
Doak et al, Antibiotics and Chemotherapy, vol. 8 (1958) pp.
342-348..
|
Primary Examiner: Evans; J. E.
Attorney, Agent or Firm: Breneman, Georges, Hellwege &
Yee
Claims
What is claimed is:
1. A potent selective excitatory amino acid neutrotransmitter
receptor antagonist having the general formula: ##STR6## wherein
R.sub.1 and R.sub.2 are the same or different and are selected from
the group consisting of hydrogen, lower alkyl, halogen, amino,
nitro, triflouromethyl or cyano, or taken together are
--CH.dbd.CH--CH.dbd.3 CH--; n an m=0, 1, 2, or 3; and the
pharmaceutically acceptable salts and the 2-acetamido-2-carboethoxy
esters thereof.
2. The potent selective excitatory amino acid neurotransmitter
receptor antagonist of claim 1 wherein R.sub.1 R.sub.2
=--CH.dbd.CH--CH.dbd.CH-- and n=1 and m=2 and the
2-acetamido-2-carboethoxy esters thereof.
3. The compound according to claim 1 that is
4-[2-phosphonomethylphenyl]-2-amino-butanoic acid.
4. The compound according to claim 1 that is ethyl
3-[2-(2-diethylphosphonoethyl)-phenyl]-2-acetamido-2-carbethoxypropanoate.
5. The compound according to claim 1 that is
3-[2-(2-phosphonoethyl)-phenyl]-2-aminopropanoic acid.
6. The compound according to claim 1 that is
3-[2-phosphonomethylphenyl]-2-amino-propanoic acid.
7. The compound according to claim 1 that is
3-[2-(3-phosphonopropyl)-phenyl]-2-aminopropanoic acid.
8. The compound according to claim 1 that is
5-[2-phosphonomethylphenyl]-2-amino-pentanoic acid.
9. A pharmaceutical composition for relieving pain which comprises
a pain relieving effective amount of one or more compounds of claim
1 with a pharmaceutically acceptable carrier and/or diluent.
10. A process of relieving pain in an animal in need thereof which
comprises administering said compound of claim 1 parenterally,
nasally, orally, rectally or a combination thereof to said animal
in need thereof.
11. A pharmaceutical composition for treatment of convulsions or
epilepsy which comprises an effective amount of one or more
compounds of claim 1 with a pharmaceutically acceptable carrier
and/or diluent.
12. A process for treating convulsions or epilepsy which comprises
administering said compound of claims 1 parenterally, orally,
nasally, rectally or a combination thereof to said animal in need
thereof.
13. A pharmaceutical composition of enhancing cognition which
comprises a cognition enhancing amount of one or more compounds of
claim 1 with a pharmaceutically acceptable carrier and/or
diluent.
14. A process for enhancing cognition which comprises administering
said compound of claim 1 parenterally, orally, nasally, rectally or
a combination thereof to said animal in need thereof.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention pertains to novel, potent anticonvulsants,
antiepileptics, analgesics and cognition enhancers achieving their
action through the antagonism of specific excitatory amino acid
(EAA) neurotransmitter receptors. In particular, the invention is
directed to .omega.-[2-(phosphonoalkylenyl)phenyl]-2-aminoalkanoic
acids, their pharmaceutically acceptable salts and derivatives, and
to the methods of synthesizing the same.
2. Description of the Prior Art
While L-glutamate and L-aspartate were initially thought merely to
participate in brain metabolism, sufficient molecular
pharmacological, biochemical and electrophysiological evidence now
exists to suggest that these amino acids are neuroexcitatory
transmitters [D. R. Curtis, A. W. Duggar, D. Felix, G. A. R.
Johnston, A. K. Tebecis and J. C. Watkins. Brain Res. 41:283-301
(1972)].
For many years following the initial characterization of the
neuro-excitotoxic actions of amino acis, it was tacitly assumed
that all compounds of this type (agonists and antagonists) acted
upon the same receptor. The discovery of relatively selective
antagonists of different actions of EAA or of actions of different
EAA compounds, has changed this perception, and it is now accepted
that multiple recognition sites for EAA are present in the
vertebrate central nervous system [J. C. Watkins and R. H. Evans.
Ann. Rev. Pharmacol. Toxicol. 21:165-204 (1981)]. Defined by
prototypical agonists or antagonists, these include:
1. receptors activated by L-Glutamate (Glu) and the
conformationally restricted Glu analog, quisqualic acid (Quis), and
antagonized selectively by glutamic acid diethylester,
2. receptors responsive to the synthetic analogue of L-aspartate
(Asp), N-methyl-D-aspartate (NMDA), the isoxazole neurotoxin,
ibotenic acid (Ibo), the pyridinedicarboxylic acid neurotoxin,
quinolinic acid (Quin) and, probably, to Asp itself. These
receptors are antagonized by D(-)-2-amino-5-phosphonopentanoic acid
(AP5), D(-)-2-amino-7-phosphonoheptanoic acid (AP7), and the
divalent cation, Mg++,
3. receptors activated by the pyrrolidine neuroexcitotoxin, kainic
acid (KA), for which no specific antagonists have yet been
identified and,
4. receptors antagonized by L(+)-2-amino-4-phosphonobutyric acid
(LAP4). Originally identified as an EAA antagonist by
electrophysiological means, LAP4 inhibits the response at the
lateral perforant pathway synapses of the hippocampus to an
unidentified endogenous excitatory substance. The possibility that
Glu is this neurotransmitter is minimal and recent evidence
suggests that the N-blocked dipeptide, N-acetylaspartyl-L-glutamate
may function in this capacity [J. M. H. ff-French-mullen, K. J.
Koller, R. Zaczek, Li Hori, J. T. Coyle and D. O. Carpenter. Proc.
Nat. Acad. Sci. USA 82, 3897-4001 (1985)].
EAA's, possibly acting through one or more of these receptors, have
been implicated in the etiology of various pathological conditions
affecting the CNS. Thus, KA [K. Biziere, J. T. Slevin, R. Zaczek,
J. C. Collins and J. T. Coyle. In: Advances in Pharmacology and
Therapeutics (H. Yoshida, Y. Hagihara and S. Ebashi, eds) Pergamon,
New York. pp. 271-276 (1982)], NMDA [R. Zaczek, J. Collins and J.
T. Coyle. Neurosci. Letts 24:181-186 (1981)] and the endogenous
excitatory amino acid Quin [R. Schwarcz, W. O. Whetsell and R. M.
Mango. Science 219:316-318 (1983)] have been used to produce in
animal models a syndrome analogous to human epilepsy and other
convulsive disorders, and the anatomical and neurochemical lesions
and deficiencies produced by such chemicals in animals with these
compounds are similar to the characteristics seen postmortem in the
brains of patient's dying of Huntington's disease [J. Coyle, and R.
Schwarcz. Nature 263:244-246 (1976)] and epilepsy. Kainate
administration can produce a limbic structure lesion that mimicks
Ammon's Horn Sclerosis, an abnormality frequently found in temporal
lobe epilepsy. Research on this model of temporal lobe epilepsy has
suggested that endogenous EAA's may play a role in this disorder,
that is particularly resistant to existing antiepileptics [J. V.
Nadler, B. W. Perry, C. W. Cotman. Nature 271:676-677 (1978)]. In
addition to Huntington's disease and epilepsy, it has been
suggested that EAA's may contribute to Alzheimer's disease [A. C.
Foster, J. F. Collins and R. Schwarcz. Neuropharmac. 22:1331-1341
(1983)], E. Roberts. In: Strategies for the development of an
Effective Treatment for Senile Dementia (E. Crook and L. Gershon,
eds.) Mark Power Assoc., New Camarin, Conn. pp. 247-230 (1981)],
the neuronal death following stroke and other factors leading to
cerebral ischemia, [R. P Simon, J. H. Swan, T. Griffiths and B. S.
Meldrum, Science, 226, 850-852, (1984); S. Rothman. J. Neuroscience
4:1884-1891 (1984)] and hereditary olivopontocerebellar atrophy [J.
T. Coyle, TINS 5:287-288 (1982)].
Because of the conceptual link between EAA activity at specific
brain receptors in vitro and in vivo, excitotoxic lesions caused by
EAA in animals, and the pathogeneis of the above neurodegenerative
diseases, it is logical to explore pharmacologic means to
antagonize endogenous excitatory and excitotoxic neurotransmitters.
The development of antagonists of exogenous excitotoxins such as KA
is also logical, since there is presumably and yet undiscovered
specific endogenous substance that acts at brain KA receptors. The
advent of potent and selective antagonists of EAA's exemplified by
.alpha.-amino-.omega.-phosphonoalkylenylcarboxylic acids (the most
potent and selective being D(-)-2-amino-7-phosphonoheptanoic acid,
D(-)AP7 has provided a point of departure for the pharmacologic
intervention of EAA action at their receptors.
Besides interfering with the neurotoxic and convulsive actions of
NMDA, the exogenous excitotoxin, IBO, and the endogenous
excitotoxin Quin (but not KA) [A. C. Foster and G. E. Fagg. Brain
Res. Rev. 7:103-184 (1984); A. C. Foster, J. F. Collins and R.
Schwarcz. Neuropharmac. 22:1331-1341 (1983); R. Schwarcz, J. F.
Collins and D. A. Parks, Neurosci. Letts 33:85-90 (1982)], AP7
(i.c.v. and i.v.) protects against audiogenically-induced seizures
in genetically susceptible mice [M. J. Croucher, J. F. Collins and
B. S. Meldrum. Science 216:899-901 (1982)]. I.v. AP7 suppresses
photically-induced myoclonus in the baboon [B. S. Meldrum. M. J.
Croucher, G. Badman and J. F. Collins, Neurosi. Letts 39:101-104
(1983)], increases threshold current for electroshock induced
seizures of mice and prevents chemically induced seizures in
rodents [S. J. Czuczwar and G. Meldrum. Eur. J. Pharmac. 83:335-338
(1982)]. Very recently, AP7 (intrahippocampally) has been reported
to markedly reduce or eliminate ischemic brain damage in the rodent
carotid artery occlusion model of stroke [R. P. Simon, J. H. Swan,
T. Griffiths and B. S. Meldrum. Science 226:850-852 (1984)], and
another, less potent, EAA antagonist .delta.-D-glutamyl glycine,
has been shown to protect cultured at hippocampal neurones from
degeneration under conditions of oxygen depletion while blocking
the toxicity of exogenously applied Glu and Asp [S. Rothman. J.
Neuroscience 4:1884-1891 (1984)]. Recently, kainate and quisqualate
receptor antagonists have also been shown to posses anticonvulsant
activity [M. J. Croucher, B. S. Meldrum, A. W. Jones and J. C.
Watkins. Brain Res. 377:111-114 (1984)]. Finally, and
significantly, several lines of circumstantial evidence link
excitatory amino acids, especially glutamate, with the onset of
age-associated neurodegenerative diseases, including Alzheimer's
disease [J. T. Greenamyre, J. B. Penney, A. B. Young, C. D'Amato,
S. P. Hicks, I. Schoulson, Science 227:1496-1498 (1985)], and with
tardive dyskinesia [J. W. Olney. In: Excitotoxins (K. Fuxe, R.
Roberts, and R. Schwarcz, eds)].
SUMMARY OF THE INVENTION
The present invention provides a potent, selective excitatory amino
acid neutrotransmitter receptor antagonist having the general
formula: ##STR2## wherein R.sub.1 and R.sub.2 are the same or
different and are selected from the group consisting of hydrogen,
lower alkyl, halogen, --CH.dbd.CH--CH.dbd.CH--, amino, nitro,
trifluoromethyl or cyano; n and m=0, 1, 2 or 3; and the
pharmaceutically acceptable salts and derivatives thereof.
DETAILED DESCRIPTION OF THE INVENTION
The structure and formulation of the novel compounds of the
invention was the result of the extensive research investigation
into the antagonism of heterogenic excitatory amino acid
neurotransmitter receptors.
Defined by prototypical agonists or antagonists, these include:
1. receptors activated by L-Glutamate (Glu) and the
conformationally restricted Glu analog, quisqualic acid (Quis), and
antagonized selectively by glutamic acid diethylester,
2. receptors responsive to the synthetic analogues of L-aspartate
(Asp), N-methyl-D-aspartate (NMDA), the isoxazole neurotoxin,
ibotenic acid (Ibo), the pyridinedicarboxylic acid neurotoxin,
quinolinic acid (Quin) and, probably, to Asp itself. These
receptors are antagonized by D(-)-2-amino-5-phosphonopentanoic acid
(AP5), D(-)-2-amino-7-phosphonoheptanoic acid (AP7), and the
divalent cation, Mg++,
3. receptors activated by the pyrrolidine neuroexcitotoxin, kainic
acid (KA), for which no specific antagonists have yet been
identified and,
4. receptors antagonized by L(+)-2-amino-4-phosphonobutyric acid
(LAP4). Originally identified as an EAA antagonist by
electrophysiological means, LAP4 inhibits the response at the
lateral perforant pathway synapses of the hippocampus to an
unidentified endogenous excitatory substance. The possibility that
Glu is this neurotransmitter is minimal and recent evidence
suggests that the N-blocked dipeptide, N-acetylaspartyl-1-glutamate
may function in this capacity [J. M. H. ff-French-mullen, K. J.
Koller, R. Zaczek, Li Hori, J. T. Coyle and D. O. Carpenter, Proc.
Nat. Acad. Sci. (USA) 82, 3897-4001 (1985)].
The structure of novel compounds provides potent antagonists having
greater affinity toward one of the receptors or no affinity to some
of them rendering the compound selective. This would therefore
permit one to selectively antagonize one EAA receptor in the tissue
also containing other EAA receptors. As a result of the greater
affinity and selectivity of the present invention fewer side
effects are exhibited by the novel compounds.
The high affinity and selectivity of such compounds e.g.:
3-[2-(2-phosphonoethyl)phenyl]-2-aminopropanoic acid or
3-[2-(2-phosphonomethyl)phenyl]-2-aminopropanoic acid, has been
demonstrated in receptor binding studies and in mice by their
ability to provide protection in pentylenetetrazol (PTZ) induced
seizures.
The novel compounds of the invention can be readily prepared by the
following synthetics routes: ##STR3##
In route 1, leading to compounds of examples I and IV, the reaction
of isochroman with a solution of hydrobromic and acetic acids in a
sealed tube gives the required intermediate o-(2-bromoethyl)benzyl
bromide in high yield (Anderson, E. L.; Holliman, F. G. J. Chem.
Soc., 1950, 1037). The reaction of this compound with
triethylphosphite gives the compound of example I in 70% yield. The
compound of example II, ethyl
4-[2-(diethylphosphono-methyl)-phenyl]-2-acetamido-2-carboethoxy-butanoate
was prepared by reacting the bromophosphonate described in example
I with the sodium salt of diethylacetamidomalonate. Hydrolysis in
6N HCl gives the compound of example III. Alternatively, reacting
the intermediate o-(2-bromoethyl)benzyl bromide with the sodium
salt of diethyl acetamidomalonate gives the compound of example IV.
Reaction of this compound with triethylphosphite gives the compound
of example V in 75% yield. Hydrolysis in 6N HCl gives the compound
of example VI. ##STR4## In route 2, the commercially available
.alpha.,.alpha.'-dibromo-o-xylene reacts with triethylphosphite
giving the compound of example VII. Reaction of this intermediate
with the sodium salt of diethyl acetamidomalonate yields the
compound of example VIII. Hydrolysis in 6N HCl gives the compound
of example IX. ##STR5## In route 3, it is necessary to synthesize
the required intermediate o-(3-bromopropyl)benzyl bromide [Rieche,
A.; Gross, H. Chem. Ber., 1962, 91]. Chloromethylation of
3-phenylpropanol gives chloromethyl 3-phenylpropyl ether in high
yield. Friedel-Crafts cyclization with AlCl.sub.3 in CS.sub.2 gives
2-benzoxepine. Reaction of this compound with a solution of
hydrobromic acid-acetic acid in a sealed tube yields the required
common intermediate o-(3-bromopropyl)benzyl bromide. Reaction of
this compound with the sodium salt of diethyl acetamidomalonate
yields the compound of example X. Reaction of this compound with
triethylphosphite gives the phosphonomalonate compound of example
XI. Hydrolysis in 6N HCl gives the compound of example XII.
Alternatively, reacting the intermediate o-(3-bromopropyl)benzyl
bromide with triethylphosphite gives the compound of example XIII.
Reacting this compound with the sodium salt of diethyl
acetamidomalonate gives the compound of example XIV. Hydrolysis in
6N HCl gives the compound of example XV.
The preparation of compounds for administration in pharmaceutical
preparations may be in a variety of well known methods known to
those skilled in the art of pharmacy. More specifically the novel
compounds may be formulated as an acid salt, i.e., HCl salt,
sulfate, phosphate, nitrate, methanesulfonate, tartrate or a base
salt and other pharmaceutically acceptable salts and
compositions.
In parenteral administration of the novel compounds and
compositions of the invention the compounds may be presented in
aqueous injection solutions which may contain antioxidants,
buffers, bacteriostats, etc. Extemporaneous injection solutions may
be prepared from sterile pills, granules or tablets which may
contain diluents, dispersing and surface active agents, binders and
lubricants.
In the case of oral administration, fine powders or granules of the
compound may be formulated with diluents and dispersing and surface
active agents, and may be prepared in a draft in water or in a
syrup, in capsules or cachets in the dry state or in a non-aqueous
suspension, when a suspending agent may be included. The compounds
may also be administered in tablet form along with optional binders
and lubricants, or in a suspension in water or a syrup or an oil or
in a water/oil emulsion and may include flavoring, preserving,
suspending, thickening and emulsifying agents. The granules or
tablets for oral administration may be coated and other
pharmaceutically acceptable agents and formulations may be utilized
as known to those skilled in the art.
The following examples are illustrative of compounds of the
invention but are not to be construed as limiting the invention
thereto.
EXAMPLES
PREPARATION EXAMPLES
EXAMPLE I
Diethyl 2-(2-Bromoethyl)benzylphosphonate
In a round bottom flask equipped for distillation, 15.0 g (54 mmol)
of 2-(2-bromoethyl)benzyl bromide and 9.0 g (54 mmol) of
triethylphosphite were heated on an oil bath with stirring at
90.degree.-100.degree. C. When ethyl bromide ceased distilling off
(1 h) the remaining volatile by-products and triethylphosphite were
removed from the mixture by distillation under vacuum. The viscous
oil which remained was chromatographed on a column of silica gel
with hexane-ethyl acetate (1:1) as eluant. The combined fractions
were concentrated under reduced pressure to yield 12.5 g (70%) of
the product as a yellow oil. IR(neat): 2987, 1249, 1170, 1064, 964,
802 cm.sup.-1. .sup.1 H NMR(CDCl.sub.3) .delta. 1.2 (t, 6H);
3.0-4.35 (m, 10H); 7.2 (s, 4H).
EXAMPLE II
Ethyl
4-[2-(diethylphosphonomethyl)phenyl]-2-acetamido-2-carboethoxybutanoate
To 1.03 g (44.8 mmol) of sodium in 50 mL of dry ethanol was added
9.72 g (44.8 mmol) of solid diethyl acetamidomalonate portionwise.
This solution was stirred at reflux under nitrogen for 2 h. After
cooling to room temperature the solvent was removed under reduced
pressure yielding a tan solid. This solid was dried under vacuum
about 2 h. The sodium salt of diethyl acetamidomalonate was then
suspended in 50 mL of dry toluene and 15.0 g (44.8 mmol) of diethyl
2-(2-bromoethyl)benzylphosphonate in 25 mL of toluene was added
dropwise. This solution was stirred at reflux under nitrogen for 36
h. After cooling the solution to room temperature the solid
precipitate was removed by filtration and washed with 20 mL of
toluene. The combined toluene solutions were concentrated under
reduced pressure to yield a viscous oil. This oil was
chromatographed on a column of silica gel with ethyl acetate as
eluant. The combined fractions were concentrated under pressure to
give 7.2 g (34%) of the product as a clear viscous oil. IR(neat):
1745, 1676 (C.dbd.O) cm.sup.-1. .sup.1 H NMR(CDCl.sub.3) .delta.
1.0-6 (m, 12H); 1.8-3.2 (complex m, 9H); 3.7-4.6 (m, 8H); 6.8-7.4
(m, 5). Anal. Calcd. for C.sub.22 H.sub.34 NO.sub.8 P0.5H.sub.2 O:
C, 54.99; H, 7.34; N, 2.92. Found: C, 54.75; H, 7.37; N, 2.97.
EXAMPLE III
4-[2-Phosphonomethylphenyl]-2-aminobutanoic acid
A solution of 2.5 g (5.3 mmol) of ethyl
4-[2-(diethylphosphonomethyl)-phenyl]-2-acetamido-2-carboethoxybutanoate
in 50 mL of 6N HCl was stirred at vigorous reflux for 12 h. After
cooling to room temperature the reaction mixture was concentrated
at reduced pressure yielding an oil. This oil was washed with three
50 mL portions of water then dissolved in 95% ethanol and a slight
excess of propylene oxide added. The precipitated acid was
collected by filtration and recrystallized from dilute ethanol
yielding 1.26 g (77%) of the product as a white solid: mp
247.degree.-249.degree. C. IR(KBr): 1725, 1620 cm.sup.-1 ; .sup.1 H
NMR (D.sub.2 O) .delta. 2.0-3.4 (6H unresolved), 3.9-4.3 (m, 1H),
7.4 (s, 4H); Anal. Calcd. for C.sub.11 H.sub.16 NO.sub.5
P0.5H.sub.2 O: Calcd: C, 46.62; H, 6.05; N, 4.94. Found: C, 46.73;
H, 6.04; N, 4.79.
EXAMPLE IV
Ethyl
3-[2-(2-bromoethyl)phenyl]-2-acetamido-2-carboethoxypropanoate
To a solution of 0.41 g (18 mmol) Na in 100 mL of dry ethanol was
added portionwise 3.9 g (18 mmol) of solid
diethylacetamidomalonate. This mixture was stirred at reflux under
nitrogen for 2 h then cooled to 0.degree.-10.degree. C. Then, 5.0 g
(18 mmol) of 2-(2-bromoethyl)benzyl bromide was rapidly added in
one portion. The reaction was stirred for 2 h at
0.degree.-10.degree. C. then 24 h at room temperature. The
precipitated inorganic salt was removed by filtration and
discarded. The solvent was removed under reduced pressure yielding
a golden oil. This oil was chromatographed on a reverse phase
column (C-18) with methanol-water (1:1) as eluant. The combined
fractions were concentrated under reduced pressure to yield 5.6 g
(75%) of the product as a white solid, mp 86.0.degree.-86.5.degree.
C. IR(nujol): 1785, 1637 cm.sup.-1 (C.dbd.O). .sup.1 H
NMR(CDCl.sub.3) .delta. 1.2 (t, 6H); 1.9 (s, 3H); 2.8-3.5 (m, 4H),
3.6 (5, 2H); 4.2 (q, 4H); 6.8 (s. 1H); 7.2 (m, 4H). Anal. Calcd.
for C.sub.18 H.sub.24 NO.sub.5 Br: C, 52.18; H, 5.84; N, 3.38.
Found: C, 52.26; H, 5.86; N, 3.34.
EXAMPLE V
Ethyl
3-[2-(2-diethylphosphonoethyl)phenyl]-2-acetamido-2-carboethoxy-propanoate
A solution of 5 g (1.2 mmol) of ethyl
3-[2-(2-bromoethyl)phenyl]-2-acetamido-2-carboethoxypropanoate in
10 mL of P(OEt).sub.3 was stirred at reflux for 4 h. The excess
P(OEt).sub.3 and the volatile by-products were removed from the
mixture by distillation under vacuum. The remaining viscous oil was
initially purified by column chromatography (C-18, MeOH:H.sub.2 O;
4:1), then by preparative HPLC (C-18, MeOH:H.sub.2 O, 7:3) giving
0.48 g (86%) of the product as a clear viscous oil. IR(Nujol)
1745.9, 1676.5 cm.sup.-1 (C.dbd.O); .sup.1 H NMR(CDCl.sub.3)
.delta. 1.1-1.5 (m, 12H); 1.8-3.1 (complex, m, 7H); 3.7 (s, 2H);
3.8-4.4 (m, 8H); 6.6 (s, 1H), 7.0-7.3 (m, 4H) Anal. Calcd. for
C.sub.22 H.sub.34 NO.sub.8 P: C, 56.04; H, 7.27; N, 2.97. Found: C,
55.91; H, 7.31, N, 2.84.
EXAMPLE VI
3-[2-(2-Phosphonoethyl)phenyl]-2-aminopropanoic acid
A solution of 7.9 g (16.8 mmol) of ethyl
3-[2-(2-diethylphosponoethyl)-phenyl]-2-acetamido-2-carboethoxypropanoate
in 40 mL of 6N HCl was stirred at vigorous reflux for 14 h. After
cooling to room temperature the reaction mixture was concentrated
at reduced pressure yielding an oil. This oil was washed with four
25 mL portions of water then dissolved in 20 mL 95% ethanol and
propylene oxide added dropwise. The precipitated crude acid was
collected by filtration. Recrystallization from dilute ethanol
yielded 4.1 g (90%) as a white solid, mp 241.degree.-243.degree. C.
IR(Nujol): 1712.5 cm.sup.-1 (C.dbd.O). .sup.1 H NMR(D.sub.2 O)
.delta. 1.5-2.2 (m, 2H); 2.6-3.3 (m, 4H); 3.9-4.2 (t, 1H); 7.2 (m,
4H). Anal. Calcd. for C.sub.11 H.sub.16 NO.sub.5 P: C, 48.35; H,
5.90; N, 5.13. Found: C, 48.69; H, 6.16; N, 4.95.
EXAMPLE VII
Diethyl 2-(bromomethyl)benzylphosphonate
In a round bottom flask equipped for distillation, 20.0 g (75.8
mmol) of .alpha.,.alpha..sub.1 -dibromo-o-xylene and 12.6 g (75.8
mmol) of triethylphosphite were heated with stirring at
70.degree.-75.degree. C. When ethyl bromide ceased distilling off
(about 2-3 h), the remaining volatile by-product and
triethylphosphite were removed from the mixture by distillation
under vacuum. The remaining oil was chromatographed on a column of
silica gel with ethyl acetate as eluant. The fractions were
combined and concentrated under reduced pressure to yield a yellow
oil. IR(neat): 1249; 1164.8; 1038.8; 966.8; 794.5 cm.sup.-1. .sup.1
H NMR (CDCl.sub.3) .delta. 1.0-1.4 (m, 6H); 3.1 (s, 1H); 3.5 (s,
1H); 3.6-4.2 (m, 4H); 4.6 (s, 1H); 7.2 (s, 4H).
Anal. Calculated for C.sub.12 H.sub.18 PO.sub.3 Br: C.
EXAMPLE VIII
Ethyl
3-[2-(diethylphosphonomethyl)phenyl]-2-acetamido-2-carboethoxy-propanoate
To a solution of 0.43 g (18.8 mmol) Na in 50 mL of dry ethanol was
added 4.09 g (18.8 mmol) of solid diethyl acetamidomalonate
portionwise. This mixture was stirred at reflux under nitrogen for
2 h, then cooled to room temperature. Then, 6.05 g (18.8 mmol) of
2-(diethylphosphonomethyl)benzyl bromide in 40 mL of ethanol was
added dropwise and the mixture stirred for 24 h. The salt which
precipitated was removed by filtration and the solvent concentrated
at reduced pressure to yield a viscous oil. This oil was
chromatographed on a column of silica gel with ethyl acetate as
eluant. The combined fractions were concentrated under reduced
pressure to yield 6.67 g (79%) of the product as a clear oil.
IR(neat): 1745.9, 1676.5, 1501.6, 1375.6, 1247.1, 1649.1, 969.4
cm.sup.-1. .sup.1 H NMR (CDCl.sub.3) .delta. 1.0-1.4 (m, 12H); 2.0
(s, 3H); 2.9 (s, 1H); 3.3 (s, 1H); 3.8-4.4 (m, 10H); 5.8-7.4 (m,
5H). Anal. Calcd. for C.sub.21 H.sub.32 NPO.sub.8 0.5H.sub.2 O: C,
54.06; H, 6.92; N, 3.00. Found: C, 53.73; H, 7.11; N, 3.27.
EXAMPLE IX
3-[2-Phosphonomethylphenyl]-2-aminopropanoic acid 532
A solution of 4.5 g (9.8 mmol) of ethyl
3-[2-(diethylphosphonomethyl-phenyl]-2-acetamido-2-carboethoxypropanoate
in 50 mL of 6N HCl was stirred at vigorous reflux for 12 hours.
After cooling to room temperature the reaction mixture was
concentrated at reduced pressure yielding an oil. This oil was
washed with three 50 mL portions of water then dissolved in 95%
ethanol and an excess of propylene oxide added. The precipitated
acid was collected by filtration and recrystallized from dilute
ethanol yielding 1.6 g (63%) of the product as a white solid: mp
259.degree.-261.degree. C.; IR(nujol): 1722; 1625; 1128; 1049
cm.sup.-1. .sup.1 H NMR(D.sub.2 O) .delta. 2.8-3.7 (unresolved,
4H), 4.2 (m, 1H), 77.3 (s, 4H); Anal. Calcd. for C.sub.10 H.sub.14
NO.sub.5 P, 0.25H.sub.2 O: C, 45.55; H, 5.54; N, 5.32. Found: C,
45.57, H, 5.55; N, 5.38.
EXAMPLE X
Ethyl
3-[2-(3-bromopropyl)phenyl]-2-acetamido-2-carboethoxypropanoate
To a stirred solution of 0.61 g (27 mmol) Na in 40 mL of dry
ethanol was added portionwise 5.86 g (27 mmol) of solid diethyl
acetamidomalonate. This mixture was stirred at reflux under
nitrogen for 2 h, then cooled to 0.degree.-10.degree. C. on an ice
bath. Then, 8.0 g (27 mmol) of 2-(3-bromopropyl)benzyl bromide in
40 mL of dry ethanol was rapidly added. The reaction mixture was
stirred for 2 h at 0.degree.-10.degree. C., then 24 h at room
temperature. The precipitated inorganic salt was removed by
filtration, and was washed with 20 mL of ethanol and discarded. The
combined solvents were removed under reduced pressure yielding an
orange colored oil. This oil was chromatographed on a column of
silica gel with hexane-ethyl acetate (3:1) as eluant. The combined
fractions were concentrated under reduced pressure to yield an oil
which solidified upon standing, yield 9.5 g (82%), mp
73.degree.-74.5.degree. C. IR(nujol): 1745, 1648 cm.sup.-1
(C.dbd.O). .sup.1 H NMR(D.sub.2 O) .delta. 1.3 (t, 6H); 1.9-2.4 (m,
5H); 2.7 (t, 2H); 3.4 (t, 2H); 3.75 (5, 2); 4.3 (q, 4); 6.6 (s,
1H); 7.0-7.3 (m, 4H). Anal Calcd. for C.sub.19 H.sub.26 NO.sub.3
Br: C, 53.28; H, 6.12; N, 3.27; Br, 18.66. Found: C, 53.33; H,
6.13; N, 3.23; Br, 18.67.
EXAMPLE XI
Ethyl
3-[2-(3-diethylphosphonopropyl)phenyl]-2-acetamido-2-carboethoxy-propanoat
e
A solution of 7.5 g (17.5 mmol) of ethyl
3-[2-(3-bromopropyl)phenyl]-2-acetamido-2-carboethoxypropanoate in
20 mL of freshly distilled triethylphosphite was stirred at reflux
for 6 h. The excess P(OEt).sub.3 and the volatile by-products were
removed from the mixture by distillation under vacuum. The
remaining viscous oil was chromatographed on a column of silica gel
with ethyl acetate as eluant. The combined fractions were
concentrated under reduced pressure to yield 4.2 g (49%) of the
product as a viscous yellow oil. IR(neat): 1746, 1680 cm.sup.-1
(C.dbd.O). .sup.1 H NMR(CDCl.sub.3) .delta. 1.1-2.1 (complex m,
19H); 2.4-2.7 (t, 2H); 3.6 (5, 2H); 3.8-4.3 (m, 8H); 6.55 (s, 1H);
6.9-7.2 (m, 4H).
EXAMPLE XII
3-[2-(3-Phosphonopropyl)phenyl]-2-aminopropanoic acid
A solution of 3.0 g (6.9 mmol) of ethyl
3-[2-(diethylphosphonopropyl)phenyl]-2-acetamido-2-carboethoxypropanoate
in 25 mL of 6N HCl was stirred at vigorous reflux 12 h. After
cooling to room temperature the reaction mixture was concentrated
at reduced pressure yielding an oil. The oil was washed with three
25 mL portions of water then dissolved in 25 mL 95% ethanol and
propylene oxide added dropwise. The precipitated acid was collected
by filtration. Recrystallization from dilute ethanol yield 0.76 g
(38%) as a white solid mp>95.degree. C. (dec.). IR(nujol):
1717.6 cm.sup.-1 (C.dbd.O). .sup.1 H NMR(D.sub.2 O) .delta. 1.1-2.0
(complex m, 4H); 2.6-3.1 (m, 4H); 3.35-3.65 (m, 1H); 7.3 (m, 4H).
Anal. Calcd. for: C.sub.12 H.sub.18 NO.sub.5 P.H.sub.2 O: C, 47.21;
H, 6.60; N, 4.58. Found: C, 47.47; H, 6.72; N, 4.53.
EXAMPLE XIII
Diethyl 2-(3-bromopropyl)benzylphosphonate
In a round bottom flask equipped for distillation, 11.36 g (38.9
mmol) of 2-(3-bromopropyl)benzyl bromide and 6.46 (38.9 mmol) of
freshly distilled triethylphosphite were heated with stirring at
100.degree.-110.degree. C. on an oil bath. When ethyl bromide
ceased distilling off (about 2 h) the remaining volatile
by-products and the triethylphosphite were removed from the mixture
by distillation under vacuum. The remaining oil was chromatographed
on a column of silica gel with hexame-ethyl acetate (1:1) as
eluant. The combined fractions were concentrated under reduced
pressure to yield 11.2 g (83%) of the product as a clear oil.
IR(neat): 2985, 1496, 1450, 1391, 1252, 1162, 104, 967, 843, 802,
758 cm.sup.-1. .sup.1 H NMR(CDCl.sub.3) .delta. 1.2 (t, 6); 1.8-2.3
(m, 2H); 2.7-3.55 (m, 4H); 3.8-4.2 (m, 2H); 7.1-7.4 (m, 4H).
EXAMPLE XIV
Ethyl
5-[2-(diethylphosphonomethyl)phenyl]-2-acetamido-2-carboethoxypentanoate
To 0.48 g (21 mmol) of sodium in 50 mL of dry ethanol was added
4.56 g (21 mmol) of solid diethyl acetamidomalonate portionwise.
This solution was stirred at reflux under nitrogen for 2 h. After
cooling to room temperature the solvent was removed under reduced
pressure yielding a tan solid. This solid was dried under vacuum
about 2 h. The sodium salt of diethyl acetamidomalonate was then
suspended in 50 mL of dry toluene and 9.0 g (21 mmol) of diethyl
2-(3-bromopropyl)benzylphosphonate in 25 mL of dry toluene was
added dropwise. This solution was stirred at reflux under nitrogen
for 20 h. After cooling to room temperature the solid which
precipitated was removed by filtration and washed with toluene. The
combined toluene solutions were concentrated under reduced pressure
to yield a dark oil. This oil was chromatographed on a column of
silica gel with ethyl acetate as eluant. The combined fractions
were concentrated under reduced pressure yielding 4.2 g (42%) of
the product as a yellow viscous oil which solidified upon standing,
mp 76.degree.-79.degree. C. IR(neat) 1745.9, 1680 cm.sup.-1
(C.dbd.O). .sup.1 H NMR(CDCl.sub.3) .delta. 1.0-1.4 (m, 12H); 2.0
(s, 3H); 2.2-3.3 (m, 6H); 3.7-4.4 (m, 8H); 6.8 (5, 1H); 7.0-7.3 (m,
4H). Anal. Calcd. for C.sub.23 H.sub.36 NO.sub.8 P: C, 56.90; H,
7.48; N, 2.89. Found: C, 56.27; H, 7.51; N, 2.86.
EXAMPLE XV
5-[2-Phosphonomethylphenyl]-2-aminopentanoic acid
A solution of 3.8 g (7.8 mmol) of Ethyl
5-[2-diethylphosphonomethyl)phenyl]-2-acetamido-2-carboethoxypentanoate
in 25 mL of 6N HCl was stirred at vigorous reflux for 12 h. After
cooling to room temperature the reaction mixture was concentrated
at reduced pressure yielding an oil. This oil was washed with three
25 mL portions of water then dissolved in 25 mL of 95% ethanol and
propylene oxide was added dropwise. The precipitated crude acid was
collected by filtration. Recrystallization from dilute ethanol
yielded 1.7 g (76%) of the product as a white solid,
mp>152.degree. C. (dec.). IR(nujol): 1717.6 cm.sup.-1 (C.dbd.O).
.sup.1 H NMR(D.sub.2 O) .delta. 1.6-1.9 (broad, 4H); 2.8-3.3 (m,
4H); 3.45 (m, 1H); 7.3.7.7 (m, 4H). Anal. Calcd. for C.sub.12
H.sub.18 NO.sub.5 P. 0.5H.sub.2 O: C, 48.65; H, 6.46; N, 4.73.
Found: C, 48.56, H, 6.46; N, 4.72.
EXAMPLE XVI
In vitro Receptor Binding Assays
The potency of the compounds described in examples III, V, VI, IX,
XII and IV to inhibit the specific binding of various excitatory
amino acid ligands to rat brain membranes was examined using
standard in vitro ligand binding techniques. Specifically,
compounds were evaluated for potency to inhibit the specific
binding of [.sup.3 H]kainic acid, [.sup.3 H]KA,
RS-.alpha.-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
[.sup.3 H]AMPA, [.sup.3 H]DL(.+-.)2-amino-7 phosphono heptanoic
acid [.sup.3 H]AP7.
The methods were as follows: rat forebrain membranes were prepared
as described by Enna and Synder (Mol. Pharmacol, 13, 422-453, 1977)
and the final pellet was washed three additional times by
centrifugation (45,000 g; 10 min; 4.degree. C.) with intermittent
resuspensions (20 vol; w/v) in fresh buffer appropriate to the
assay. For the [.sup.3 H]AP4 assay, tissue was used immediately.
For all other procedures, tissue was stored frozen (-40.degree. C.)
until use. All assays were performed using triplicate incubations.
Radioactivity was determined using conventional liquid
scintillation counting after solubilizing the pellet in 1 mL
Protosol (New England Nuclear, Boston, MA) and following the
addition of 6 mL of Enconofluor (New England Nuclear, Boston,
MA).
Specific [.sup.3 H]AP4 (specific activity (S.A.)=26.1 Ci/mmol, New
England Nuclear, Boston, MA) binding was studied according to the
method of Butcher et al. (Brit. J. Pharmacol., 80, 355-364, 1983)
using HEPES KOH buffer (0.05 M; pH 7.1). Incubations (2mL) were
conducted for 45 min at 37.degree. C. and the reaction was
terminated by centrifugation (45,000 g; 10 min; 4.degree. C.). The
supernatant was decanted and the pellet washed rapidly and
superfically with 2.times.3.5 ml of ice cold buffer. Final ligand
concentration in the assay was 50 nM and L-glutamate (10-.sup.3 M)
was used to define non specific binding.
Specific [.sup.3 H]AMPA (S.A.=25.6 Ci/mmol, New England Nuclear)
binding was examined according to the method of Murphy et al.,
(Soc. Neurosci. Abs., 11, 109, 1985) using Tris HCL buffer (0.05M,
pH 6.9; 23.degree. C.) containing 100 mM KSC. Following
pretreatment of tissue with Triton-X-100 (0.05%; v/v) for 30 min
(37.degree. C.) incubations (2 mL) were conducted for 60 min at
4.degree. C.). The supernatant was decanted and the pellet washed
rapidly and superficially with 2.times.3.5 mL of ice cold buffer.
Final ligand concentration in the assay was 16 nM and L-glutamate
(10-.sup.3 M) was used to define nonspecific binding.
Specific [.sup.3 H]AP7 (S.A.=58.4 Ci/mmol, New England Nuclear)
binding was examined as described by Ferkany and Coyle (Life Sci.,
33, 1295-1305, 1983) using Tris citrate buffer (0.05M; pH 7.5;
23.degree. C.). Following preincubation of the tissue (30 min;
37.degree. C.), incubation (2 ml) were conducted for 90 min at
37.degree. C. and the reaction was terminated by centrifugation
(45,000 g; 10 min; 4.degree. C.). The supernatant was decanted and
the pellet washed rapidly and superficially wth 2.times.3.5 mL of
ice cold buffer. Final ligand concentration in the assay was 500 nM
and L-glutamate (10-.sup.3 M) was used to define nonspecific
binding.
Specific [.sup.3 H]KA (S.A.=60 Ci/mmol, New England Nuclear)
binding was examined according to the methods of London and Coyle
(Mol. Pharmacol., 15, 492-505, 1979) using Tris HC; buffer (0.05M;
pH 7.4; 23.degree. C.). Incubations 2 mL) were performed for 90 min
at 4.degree. C.) and the reaction terminated by centrifugation
45,000 g; 10 min; 4.degree. C.). The supernatant was decanted and
the pellet washed rapidly and superfically with 2.times.3.5 ml of
ice cold buffer. Final ligand concentration in the assay was 5 nM
and L-glutamate (10-M) was used to define nonspecific binding.
Results are reported in Table 1. When tested at final concentration
of 100 uM compounds III, V, VI, IX, XII and IV inhibited less than
20 percent of specifically bound [.sup.3 H]KA or [.sup.3 H]AMPA.
Similarly, compound IX failed to inhibit the specific binding of
[.sup.3 H]AP4 and [.sup.3 H]AP7 when tested at 100 uM
concentration. Compounds III, VI, XII and XV inhibited the specific
binding of [.sup.3 H]AP4 and [.sup.3 H]AP7 in a concentration
dependent manner with the order of potency in each assay being
XV>III>XII.gtoreq.VI. Whereas compounds III, XII and XV were
equipotent to the .alpha.-amino-.omega.-phosphono acid, DL(.+-.)AP7
to inhibit both specific [.sup.3 H]AP4 and [.sup.3 H]AP7 binding,
compound VI was 3-10 fold less potent in this regard. Further,
compound VI effectively discriminated between the two assays and
was more potent to inhibit the specific binding of [.sup.3 H]AP7
than the binding of [.sup.3 H]AP4.
TABLE I ______________________________________ Potency of Example
Compounds to Inhibit Specific [.sup.3 H] Excitatory Amino Acid
Binding to Rat Brain Membranes IC.sub.50 (uM) Example [.sup.3 H]AP4
[.sup.3 H]AP7 [.sup.3 H]Kainate [.sup.3 H]AMPA
______________________________________ XV 1.03 2.29 >>100
>>100 III 6.8 6.1 >>100 >>100 XII 9.5 N.T.
>>100 >>100 VI 16.7 52.5 >>100 >>100 IX
>>100 >>100 >>100 >>100 V >>100
>>100 >>100 N.T. AP7 5.1 6.8 >>100 >>100
______________________________________ Methods have been described
in the text. Values shown are the means of at least three separate
determinations performed in triplicate and using eight
concentrations of drug. Where values are >>100 uM, this
indicates the highest concentration of drug tested and, that less
than 20 percent o the specifically bound ligand was displaced.
EXAMPLE XVII
Protection Against Maximal Electroshock Seizures (MES)
The anticonvulsant properties of compounds III, V, VI, IX, XII and
XV and, of the reference compound DL(.+-.)P7 against seizures
induced by maximal electroshock were evaluated.
For testing, electrodes were clipped to the ears of male CF-1 mice
(20-25 g; Charles Rivers), and a current of 0.5 mA was delivered
for 0.2 seconds to produce seizures. Anticonvulsant activity was
indicated by abolition of the extensor component of the seizure and
was defined as hindlimb extension that did not exceed the 90 degree
angle with the plane of the body. Data was calculated as the
percent of mice not displaying hindlimb extension as described.
Drugs were disolved in a solution of propylene glycol and distilled
water (5:95; v/v). For i.c.v. administration, drugs were
administered in a final volume of 5 uL, fifteen minutes prior to
testing. For i.p. administration, drugs were delivered in a volume
of 12.5 ml/kg, thirty minutes prior to testing.
Results are reported in Table 2. As expected, the reference
compound AP7 afforded dose-dependent protection against MES-induced
seizures with calculated ED.sub.50 's of 8.4 ug (n=8) and 127 mg/kg
(n=16) following i.c.v. and i.p. injection, respectively. When
tested at a mole dose equivalent to 1 times or twice the ED.sub.50
of the reference compound, examples III, V, VI, XII and XV were
without effect on MES-induced convulsions by either route of
administration. Example IX afforded limited protection against
MES-induced seizures with an estimated ED.sub.50 of 15 ug (i.c.v.)
and ED.sub.25 of 500 mg/kg (i.p). Higher doses of the example IX
could not be tested due to the appearance of marked ataxia in some
animals.
TABLE II ______________________________________ Potency of Example
Compounds to Antagonize Maximal Electroshock of
Pentylenetetrazol-Induced Seizures in Male CF-1 Mice ED.sub.50 MES
PTZ i.c.v. i.p. i.c.v. i.p. Example (ug) (mg/kg) (ug) (mg/kg)
______________________________________ AP7 8.3 127 1.8 199 VI
>100 N.T. 3.2 >>350 IX 16 500* 24 >>250 V
>>100 N.T. >>28 350** III N.T. >>250 >>6
>>500 XII >>23 >>155 >>5 >>300 XV
>>22 >>150 >>5 >>300
______________________________________ Methods have been described
in the text. Where ED.sub.50 values are shown dose response curves
were generated using at least 5 concentrations of th indicated
agent with 6-8 animals at each drug concentration. Where ED.sub.50
is shown as (>>) this indicates the maximum drug dose tested
an the fewer than 20 percent of the tested animals were protected.
*highest drug dose tested; ED.sub.25 **highest drug dose tested; 50
percent of animals protected from seizures 3 of 7 animals dead
prior to end of observation period.
EXAMPLE XVIII
Protection Against Pentylenetetrazol-induced Seizures (PTZ)
The anticonvulsant properties of compounds III, V, VI, IX, XII and
XV and, of the reference compound, DL(.+-.)AP7 against seizures
induced by pentylenetetrazol (PTZ) were examined.
For testing, PTZ was dissolved in saline (0.9%; w/v) and
administered to male CF-1 mice (Charles Rivers; 20-25 g) at a dose
of 85 mg/kg fifteen minutes (i.c.v.) or thirty minutes (i.p.) after
the administration of the test compound. Mice were observed for ten
minutes following the administration of PTZ and seizures were
scored as present or absent. Data were expressed as the percent of
animals showing seizures activity.
For testing, examples V, VI, XLL, XV and the reference compound
were dissolved in propylenegylcol and water (95:5, v/v) whereas
examples III and IX were dissolved in 0.2M bicarbonate. Drugs were
administered in a volume of 5 uL or 12.5 mL/kg for i.c.v. and i.p.
administration, respectively.
Results of testing are shown in Table 2. When administered at drug
amounts equal to 1 times or 2 times the ED.sub.50 of the reference
compound to attenuate PTZ-induced seizures, examples III, XII and
XV were devoid of activity followed i.c.v. or i.p. administration.
Example V, intermediary compound to the synthesis of example VI,
provided limited seizure protection (4 of 7 animals) following i.p.
injection of 350 mg/kg. Administration of higher doses (500 mg/kg)
of example V resulted in mortality in 40 percent of the tested
animals and seizure protection was not scored.
Example VI was equipotent to the reference compound to attentuate
PTZ-induced convulsions following i.c.v. administration (Table 2).
However, following i.p. administration at doses up to 350 mg/kg.
example VI failed to significantly protect animals in this seizure
model.
Example IX was similarly potent to protect mice from PTZ-induced
seizure activity when administered intraventricularly having an
ED.sub.50 10-fold greater than the reference compound and 6-fold
greater than example VI. As was the case for example VI, example IX
was essentially devoid of anticonvulsant activity when administered
via intraperitoneal injection.
Compounds VI and IX are potent anticonvulsants in the PTZ-induced
seizure model following i.c.v. administration and are distinguished
from the reference compound by their selectivity to confer
protection in this model vis-a-vis MES-induced seizure
activity.
* * * * *