WO1996040155A1 - Use of specific acpa derivative (npc-17742) for the treatment of ischemia - Google Patents

Abstract

A method of treating a mammal for a disease selected from the group consisting of focal ischemia, global ischemia and neuronal cell ischemia associated with spinal injuries and head trauma; said method comprising administering to the mammal a therapeutically effective amount of the 2R, 4R, 5S isomer of the compound 2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid or any pharmaceutically acceptable salt or hydrate of the isomer wherein the isomer or salt or hydrate of the isomer is substantially free from other stereoisomers of the compound.

Description

SE OF SPECIFIC ACPA DERIVATIVE (NPC- 17742) FOR THE TREATMENT OF ISCHEMIA

1. Field of the invention The present invention relates to a new use of one of the eight isomers of 2-amino-4,5-(l,2-cyclohexyl)-7- phosphonoheptanoic acid. One of these isomers is a compound which is a known excitatory amino acid (EAA) neurotransmitter receptor antagonist has been found to be useful as a means for treating global ischemia. The inventive isomer is unexpectedly more potent in in vitro assays and in in vivo efficacy studies.

2. Description of Related information The compound 2-amino-4,5-(l,2-cyclohexyl)-7- phosphonoheptanoic acid (ACPA) was described as an excitatory amino acid receptor antagonist in United States Patent No. 4 761,405, which issued on August 2, 1988. ACPA is a compound that has three asymmetric carbon atoms and thus eight steroisomers. In U.S. Patent No. 4,761,405 ACPA is reported as a racemic mixture of the eight isomers, and the in vitro and in vivo data presented to show efficacy as an excitatory amino acid receptor antagonist were generated by testing the racemic mixture.

The United States Patent No. 5,256,814 to Gregory S. Hamilton discloses one particular isomer of ACPA, namely the 2R,4R, 5S isomer that is substantially free from the other ACPA isomers. This compound was disclosed as being an effective and potent excitatory amino acid antagonists that possessed superior properties over the other seven isomers.

Ische ic damage to the central nervous system (CNS) may result from either global or focal ischemic conditions. Global ischemia occurs under conditions in which blood flow to the entire brain ceases for a period of time, such as may result from cardiac arrest. Focal ischemia occurs under conditions in which a portion of the brain is deprived of its normal blood supply, such as may result from thromboembolytic occlusion of a cerebral vessel, traumatic head injury, edema, and brain tumors.

Both global and focal ischemic conditions have the potential for producing widespread neuronal damage, even if the ischemic condition is transient. Although some permanent neuronal injury may occur in the initial minutes following cessation of blood flow to the brain, most of the damage in global and focal ischemia occurs over hours or even days following the ischemic onset. Much of this neuronal damage is attributed to secondary consequences of reperfusion of the tissue, such as the release of vasoactive products by damaged endothelium, and the release of cytotoxic products (free radicals, leukotrienes, etc.) by damaged tissues.

Several drug strategies have been proposed for treatment of stroke and other neuronal conditions related to ischemia. Anti-coagulants, such as heparin, have been examined, but with mixed results. Similarly, antivasoconstriction agents, such as flunarazine, excitatory neurotransmitter antagonists, such as MK-801 and AP7, and anti-edemic compounds have shown mixed results, with no clear benefits to outweigh a variety of side effects, including neurotoxicity or increased susceptibility to infection.

Unfortunately, drugs which have been proposed to date for the treatment of stroke and other ischemic- related conditions of the brain are either (i) relatively ineffective, (ii) effective only at dosage levels where undesired side effects are observed, and/or (iii) produce systemic effects, such as hypotension, which compromise the potential effectiveness of the drug. SUMMARY OF THE INVENTION

The present invention relates to a new use of the 2R, 4R, 5S isomer of ACPA that is substantially free from the other ACPA isomers. Substantially free is defined to mean at least about 95% pure.

More particularly, the present invention relates to a method of treating a mammal for a disease treated by direct neuronal protection selected from the group consisting of focal ischemia, global ischemia and neuronal cell ischemia associated with spinal injuries, stroke, cardiac arrest, head trauma, or drowning; said method comprising administering to a mammal a therapeutically effective amount of the 2R,4R, 5S isomer of the compound 2-amino-4,5-(1,2-cyclohexyl)-7- phosphonoheptanoic acid or any pharmaceutically acceptable salt or hydrate of the isomer wherein the isomer or salt or hydrate of the isomer is substantially free from other stereoisomers of the compound. Another embodiment relates to a method of treating a mammal to decrease brain injury caused by global ischemia, which comprises: administering to a subject an effective amount of the 2R,4R, 5S isomer of the compound 2-amino-4,5-(1,2-cyclohexy1)-7-phosphonoheptanoic acid or any pharmaceutically acceptable salt or hydrate of the isomer wherein the isomer or salt or hydrate of the isomer is substantially free from other stereoisomers of the compound.

DETAILED DESCRIPTION OF THE INVENTION

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system (CNS) . Once released into the synapse, L-glutamate can stimulate the

N-methyl-D-aspartate (NMDA) receptor, a subtype of an excitatory amino acid receptor. It has been discovered that excessive activation of the NMDA receptor has been implicated in a variety of acute as well as chronic neuropathological processes such as ischemia, epilepsy and Huntington's disease. Thus, considerable effort has focused on finding novel therapeutic agents to antagonize the postsynaptic effects of L-glutamate medicated through the NMDA receptor.

As described in U.S. Patent 5,256,814, the 2R, 4R, 5S isomer of ACPA, namely 2-amino-4,5-(l,2-cyclohexyl)-7- phosphonoheptanoic acid) is a potent, and selective, competitive NMDA receptor antagonist. This compound inhibits ³H-CGS 19755 receptor binding to the NMDA receptor in rat brain membranes (IC₅₀ = 182 4+39 nM) with a similar potency to the competitive NMDA receptor antagonists CGS 19755 and D(-) CPP (also disclosed in the ^,814 patent). It has been found that the 2R, 4R, 5S compound lacks significant affinity for over 200 other neurotransmitter receptor binding and uptake sites, including other excitatory amino acid receptors such as the AMPA and Kainate receptors. In functional testing, this compound is a competitive NMDA receptor antagonist. In the presence of 10 μM glycine, this compound produced a rightward shift in the potency of NMDA to enhance ³H- TCP binding, without a corresponding decrease in the maximal response to NMDA. In Xenopus oocytes injected with poly(A⁺)RNA from rat brain, the 2R, 4R, 5S isomer reduced the NMDA mediated inward cation current in a concentration dependent manner, with a Schild analysis consistent with competitive interaction. These in-vitro data demonstrate that this compound is a potent, and selective, competitive NMDA receptor antagonist.

It has been unexpectedly discovered that in mammals, the 2R, 4R, 5S isomer potently antagonizes NMDA and maximal electroshock (MES) induced seizures and death, with a similar potency to CGS 19755. In contract, 2R, 4R, 5S isomer was 6.4 fold more potent than CGS 19755 in inhibiting pentylenetetrazol (PTZ)-induced clonic and tonic seizures. The 2R,4R, 5S isomer also potently prevents cocaine induced seizures, and controls spontaneous convulsions in epileptic primates.

The anticonvulsant effects of the 2R, 4R, 5S isomer occur at doses substantially below those which produce sedation and ataxia, or impairment of performance in a schedule-controlled operant paradigm. In drug discrimination studies, rats trained to discriminate 2R, 4R, 5S isomer from saline, the competitive NMDA receptor antagonist CGS 19755, but not the competitive NMDA receptor antagonist LY274614 substituted for 2R, 4R, 5S isomer. The non-competitive NMDA receptor antagonists MK-801 and PCP did not substitute for 2R, 4R, 5S isomer.

These data suggest that NMDA antagonists do not represent a homogenous group of compounds with regard to their anticonvulsant actions, discriminative stimulus effects and side effect liability. Furthermore, these data show that the pharmacological profile of the 2R, 4R, 5S isomer is distinct from other competitive and non- competitive NMDA receptor antagonists.

The starting material for preparing the compound of the present invention is 1R, 2S- Methyl (hydrogen)-1,2- cis-cyclohex-4-ene diacetate 2. This compound is prepared from the known meso diester via enantioselective enzymatic hydrolysis utilizing the enzyme porcine pancreas lipase. Y. Nago, et al, J. Orσ. Chem. (1985), 50, 4072. The use of this enzyme to obtain the chiral product 2. is known in the literature. Y. Nago, et al., Chem. Lett. (1989) 239.

-OOOMc L bfMM

COO c

^-^\_r_«— COOH \^^ »— COO t The preparation of the compound of the invention starting from the known compound 2. is summarized in

Scheme 1 of U.S. Patent 5,256,814, the contents of which are incorporated herein by reference in their entirety.

After reduction of the double bond in 2. with hydrogen in the presence of a palladium catalyst, diborane is used to selectively reduce the carboxylic acid group to the corresponding hydroxyethyl side chain. The alcohol is converted to its tert-butyldimethylsilyl ether, and the ester moiety is reduced to the corresponding aldehyde using diisobutylaluminum hydride. The aldehyde was then converted to the hydantoin by reaction with sodium cyanide and ammonium carbonate; the silyl group is removed from the oxygen atom during this process. The alcohol is converted first to methanesulfonyl derivative by treatment with methanesulfonyl chloride and pyridine; which is then reacted with lithium bromide in N,N-dimethylformamide to obtain the bromide. The Bromide is then reacted with the sodium salt of diethylphosphite in tetrahydrofuran to obtain the phosphonate, which by enzymatic hydrolysis with the enzyme D-hydantoinase obtained from cells of Agrobacterium radiobacter results in conversion to the carbamoyl acid. R. Oliver, et al., Biotechnol. Bioen in (1981) 23, 2173; J. Takahashi, et al., J. Ferment Techno1. (1979) 57, 328. The free amino acid obtained from chemical hydrolysis of the carbomoyl acid is then deprotected with iodotri ethylsilane to obtain the compound useful in this invention, 2R, 4R, 5S ACPA.

An alternate method for synthesizing the current isomer also can be used and is summarized in Scheme II, of U.S. Patent 5,256,814, also incorporated by reference herein. Pharmaceutically acceptable acid and base addition salts of the inventive isomer are formed with moderately strong organic or inorganic acids or bases by known methods. Exemplary of the salts included in this invention are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, and the tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate, quinate, and nitrate salts thereof. Also included in the invention are pharmaceutical compositions comprising the particular isomer of ACPA and suitable carriers in pharmaceutical dosage forms such as capsules, tablets, injectable preparations, ointments, creams, topical reservoirs such as transdermal patches, and suppositories. Solid or liquid carriers can be used. Solid carriers include starch, lactose, calcium, sulfate, dehydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline and water. Suppositories are prepared from standard bases such as polyethylene glycol and cocoa butter.

The method of this invention for treating global ischemia comprises administering internally to a subject expected to be benefitted thereby with an effective amount of the 2R, 4R, 5S ACPA isomer. Doses of this isomer included in the present methods and pharmaceutical compositions are an efficacious, nontoxic quantity selected from the range of 0.01 to 100 mg/kg of active compound, preferably 0.1 to 50 mg/kg. Persons skilled in the art using routine clinical testing are able to determine optimum doses. The desired dose is administered to a subject from 1 to 6 or more times daily, orally, rectally, parenterally, or topically and may follow a higher initial amount administered as a bolus dose.

In methods of treating stroke, particularly acute ischemic stroke, and global ischemia caused by drowning, head trauma and so forth, an active compound of the present isomer can be co-administered with one or more agents active in reducing the risk of stroke, such as aspirin or ticlopidine (preferably ticlopidine, which has been demonstrated to reduce the risk of a second ischemic event) . Co-administration can be in the form of a single formulation (combining, for example, a compound of the present isomer and ticlopidine with pharmaceutically acceptable excipients, optionally segregating the two active ingredients in different excipient mixtures designed to independently control their respective release rates and durations) or by independent administration of separate formulations containing the active agents.

If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.

The compounds of this invention are generally administered as a pharmaceutical composition which comprises a pharmaceutical excipient in combination with a compound of the present isomer. The level of the drug in a formulation can vary within the full range employed by those skilled in the art, namely, from about 0.01 percent weight (% w) to about 99.99% w of the drug based on the total formulation and about 0.01% w to 99.99% w excipient. Preferably, the formulation will be about 3.5 to 60% by weight of the pharmaceutically active compound, with the rest being suitable pharmaceutically excipients.

The unexpected greater potency of the present isomer is shown by the data in Tables 1 and 2, below. The data reported were obtained using mixtures which were enriched in the stated isomer but were less than about 95% pure.

Also, it is believed that cis isomer A is the 2R, 4R, 5S isomer, if subsequent analysis shows it to be other than the 2R, 4R, 5S isomer. In these tables CPP is 4-(3- phosphonopropyl)-2-piperazine carboxylic acid and CGS-

19755 is cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid.

TABLE 1

POTENCY TO INHIBIT NMDA-INDUCED SEIZURES OR IMPAIR ROTOROD PERFORMANCE

FOLLOWING ICV ADMINISTRATION

Compound (mg/kg)

CGS-19755 2.64 17 6.4

(±) CPP 2.20 10 4.5 cis isomer A 1.05 32 30.5 cis isomer B 25-50 >150 cis isomer C 32.5 >150 >4.5 cis isomer D 39.5 >250 >3.8 trans isomers E + F >100 >100 — trans isomers G + H >100 >100 —

The procedure for determining the ED_J0 for inhibiting NMDA-induced seizures and impairing rotorod performance is described in Example 3. The Table 1 data show that the cis isomer A namely (2R, 4R, 5S isomer) is at last 25-fold more potent than the next most potent isomer in producing these in vivo effects.

TABLE 2

POTENCY TO INHIBIT SPECIFIC BINDING OF [3H]CGS-19755 TO RAT FOREBRAIN MEMBRANES

Compound IC10 (uM)

(+) CPP 0.19 ± 0.015 cis isomer A 0.27 ± 0.012 cis isomer B 5.55 + 1.25 cis isomer C 2.39 + 0.85 cis isomer D 2.64 + 0.85 The procedure for determining the binding potencies reported in Table 2 is described in Example 4. The Table

2 data show that the cis isomer A is approximately 8-fold more potent than cis isomer C, the next most potent isomer.

The following examples are illustrative of preferred embodiments of the invention and are not to be construed as limiting the invention thereto. All percentages are based on the percent by weight of the final system or formulation prepared unless otherwise indicated and all totals equal 100% by weight.

Examples

The following examples are illustrative of preparation and testing of the 2R, 4R, 5S isomer of ACPA.

In these Examples, the compounds designated by roman and arabic numerals were discussed in Schemes I and II, above, respectively.

Example l

Compound I: 1R, 2D-Methyl (hydrogen)-cis-1,2- cyclohexane diacetate.

A solution of 1R, 2S-Methyl (hydrogen)-1,2-cis- cyclohex-4-ene diacetate (41g; 0.19 mol) in ethanol (210 ml) was treated with 6g of 5% palladium on carbon and hydrogenated in a Paar hydrogenator for 5 hr at 55 psi pressure of hydrogen. The solution was filtered through Celite to remove the catalyst and concentrated to deliver 39.4g (95)) of the cyclohexane half-ester as a colorless oil.

^!H NMR (CDCl_j) : d 1.25-1.53 (m,8H); 2.21-2.28 (m,6H); 3.67 (s, 3H) . IR (neat): 2923, 2862, 2666, 1736, 1704, 1440, 1293, 1242, 1165, 1113, 1016, 946 cm¹.

Compound II:

Diborane (187 ml of a 1.0M solution in THF; 0.187 mol) was added in a dropwise manner to a cooled (0° C) solution of the half ester (36.83 g; 0.17 mol) in THF (200 ml) . After the addition was complete the reaction mixture was allowed to slowly come to room temperature overnight. The reaction was quenched by the addition of 500 ml of IN HCI and stirred for an additional 45 minutes. The product was extracted into 3 x 300 ml of ethyl acetate and the organic layers were washed with brine, dried (MgS0₄) and freed of solvent. The hydroxy ester was obtained as a colorless oil (32.8g; 96%).

*H NMR (CDC1₃) : d 1.23-1.76 (m, 11H) ; 2.12-2.27 (m, 3H) ; 3.70 (m, 5H) .

IR (neat): 3428, 2926, 2862, 1736, 1437, 1291, 1167, 1054, 1013 cm"¹.

Compound III (Silyl ether III) :

A mixture of hydroxy ester II (8.4g; 42 mmol) , tert- butyldimethylsilyl chloride (7.83 g; 50.4 mmol); dimethylaminopyridine (518 mg; 4.2 mmol) and triethylamine (4.67 g; 46.2 mmol) in dimethylformamide

(30 ml) was stirred overnight at room temperature. Water (30 ml) was added and the product was extracted into 3 x 50 ml of ethyl acetate. The combined organic layers were washed with brine, dried (MgS0₄) and concentrated. The crude product was purified by column chromatography, eluting with 3-5% ethyl acetate in hexane, to obtain 11.2 g (80%) of the product as a clear liquid.

*H NMR (CDC1₃) : d 0.01 (s, 6H) ; 0.84 (s, 9H) ; 1.25- 1.57 (m, 11H) ; 2.20 (m, IH) ; 2.23 (d, IH) ; 3.54-3.61 (m plus s, 5H) .

IR (neat): 2926, 2857, 1740, 1463, 1434, 1388, 1360, 1255, 1105, 1100, 1007, 938, 825 cm¹.

Compound IV (Aldehyde IV) :

A solution of ester III (2.50 g; 8.0 mmol) in 40 ml of toluene was cooled to -78° C and then poured into 30 ml of an ice-cold saturated aqueous solution of sodium- potassium tartrate. The layers were separated and the aqueous layer was washed with 2 x 20 ml of ethyl acetate. The combined organic layers were washed with brine, dried (MgS0₄) and freed of solvent. The residue was purified on a silica gel column eluting with 1-5% ethyl acetate in hexane to obtain 1.63 g (72%) of the product as a light oil.

^lH NMD (CDC1₃) : d 0.05 (s, 6H) ; 0.89 (s, 9H) ; 1.25- 1.74 (m, 11H) ; 2.23-2.38 (m, 3H) ; 3.65 (m, 2H) ; 9.76, (d, IH) .

IR (neat): 2931, 2707, 1730, 1468, 1388, 1255, 1100, 836, 776 cm¹.

Compound V (Hydroxy hydantoin V) :

Aldehyde IV (8.0g; 28.1 mmol) was dissolved in 65 ml of ethanol, and a solution of sodium cyanide (3.09 g;

61.8 mmol) and ammonium carbonate (13.5 g; 140 mmol) in water (65 ml) was added. This mixture was sealed in a glass tube and heated in an oil bath at 90° C for 18 hours. After cooling the reaction mixture was poured into a 500 ml beaker and acidified to pH 1 with HCI. After stirring at room temperature for one hour the mixture was cooled in an ice-bath and the precipitate was collected by vacuum filtration. The collected solid was washed with ice-cold water until the washings were pH 5- 6, then washed with cold ether and ethyl acetate. The solid product was dried under vacuum to obtain 3.71 g (68%) of the hydroxy hydantoin as a white solid, mp 209.

^lH NMR (DMS0-d6) : d 1.04 1.69 (m, 14H) ; 3.30-3.42 (m, 2H) ; 3.96 (d, IH) ; 4.32 (t, IH) ; 8.02 (d, IH) ; 10.57 (br d, IH) .

IR (KBr) : 3309, 2928, 2757, 1730, 1715, 1421, 1314,

1203, 1059 cm"¹.

Compound VI (Mesylate VI) .

Hydroxy hydantoin V (4.8 g; 20 mmol) was dissolved in pyridine (30 ml) . After cooling this solution to 0° C, methanesulfonyl chloride (2.75 g; 22 mmol) was added neat in a slow, dropwise fashion. After the addition was complete the reaction mixture was stirred for 15 minutes at 0° C and 2 hours at room temperature. The pyridine was removed in vacuo and the residue was dissolved in 80 ml of chloroform. The chloroform phase was washed with 30 ml of IN HCI, dried (MgS0₄) and freed of solvent to obtain 6.0 g (94%) of the product as a white solid, mp 150° C.

^lH NMR (DMS0-d6) : d 1.28-1.78 (m, 14H) ; 3.14 (s, 3H) ; 3.96 (t, IH) ; 4.18 (m, 2H) ; 8.03 (d, IH) ; 10.58 (d, IH) .

IR (KBr); 3286, 2928, 1769, 1416, 1357, 1177 cm¹. Compound VII (Bromide VII) :

Mesylate VI (6.0 g; 18.87 mmol) was dissolved in 50 ml of N,N-dimethylformamide containing 5.21 g (60 mmol) of lithium bromide. This mixture was stirred overnight at 45° C under an argon atmosphere. After cooling to 0° C, cold water (80 ml) was added to the reaction mixture and the resulting precipitate was collected by filtration and washed with 2 x 30 ml of ice-cold water and 2 x 30 ml of ether. The product was dried under vacuum to obtain 4.55 g (80%) of bromide VII as a white solid.

^lH NMR (DMS0-d6); d 1.22-1.71 (m, 14H) ; 3.61 (m, 2H) ; 3.99 (d, IH) ; 8.04 (d, IH) ; 10.58 (d, lh) .

IR (KBr): 3541, 3304, 3219, 2926, 2363, 1772, 1728, 1427, 774, 648 cm"¹.

Compound VIII (Phosphonoethyl hydantoin VIII) :

Diethyl phosphite (2.12g; 15.34 mmol) in tetrahydrofuran (10 ml) was added dropwise to a stirred slurry of sodium hydride (506 mg of an 80% oil dispersion; 16.87 mmol) in tetrahydrofuran (20 ml), at room temperature and under an argon atmosphere. After the addition was complete and gas evolution had ceased the mixture was stirred for 1.5 hours at room temperature. Bromide VII (1.5 g; 4.95 mmol) was added via syringe as a dimethylformamide solution (20 ml) . The mixture was stirred for one hour at room temperature and then heated to 80° C for 24 hours. After cooling, the reaction was quenched by the addition of saturated aqueous ammonium chloride (20 ml) and extracted into 100 ml of ethyl acetate. The organic phase was washed with 3 x 50 ml of brine, dried (MgS0₄) and freed of solvent. The residue was purified on a silica gel column, using a gradient elution from 1% methanol in methylene chloride to 10% methanol in methylene chloride. The product (1.50 g; 84%) was obtained as a white foamy glass.

^!H NMR (CDC1₃) : d 1.10-2.01 (m, 22H) ; 4.02-4.16 (m, 5H) ; 7.15-7.26 (d, IH) ; 9.64 (b, IH) .

IR (CDC₃) : 3440, 3155, 2934, 2263, 1772, 1725, 1468, 1383, 1211, 1095 cm¹.

Compound IX (Carbamoyl acid IX) .

Hydantoin VIII was converted to the carbamoyl derivative by reaction with the enzyme D-hydantoinase. Carbamoyl IX was obtained in yields of ca. 60% as a white crystalline solid after recrystallization from water. X- -ray diffraction analysis of this compound verified that it was of the 2R, 4R, 5S configuration.

•H NMR (CDC1₃) : d 1.27-2.01 (m, 22H) ; 3.49 (s, 2H) ;

4.17-4.22 (m, 4H) ; 4.48 (d, IH) ; 6.58 (br, IH) .

Electron impact mass spectrum: m/e 379 (MH⁺) , 364, 336, 335, 291, 363.

Compounds X and XI (Amino acid X and final product XI) .

Carbamoyl acid IX is chemically cleaved to the free amino acid X using nitrous acid as described in the literature cited above. Cleavage of the phosphonate esters of X to give XI is achieved using either bro o- or iodotrimethylsilane. Compound X (1 mmol) in methylene chloride/acetonitrile (10 ml) is treated with the halotrimethylsilane (10 mmol) . This mixture is stirred for 16 hours at room temperature and then evaporated to dryness. The residue is partitioned between water and chloroform. The aqueous layer is made neutral (pH 7) and concentrated in vacuo; the product can be purified by ion exchange on a Doxex 40%-Xb column. Compound XI is obtained as the hydrate.

Example 2

Compound 5_ (dehydro half-ester 5_) :

1-Methyl hydrogen (1S,2R)-1, 2-cyclohex-4- enedicarboxylate (10 g; 55.34 mmol) was dissolved in ethanol (150 ml) and hydrogenated in the presence of 5% palladium on carbon for four hours at 50 psi pressure of hydrogen. The reaction mixture was filtered through Celite to remove the catalyst and concentrated in vacuo to obtain the product as a white solid, 9.48 g (95%), mp 60-62° C.

'HNMR (CDC1₃) : d 1.37-2.15 (m, 8H) ; 2.85 (br t, 2H) ; 3.70 (S, 3H) ; 10.15 (br, IH) .

IR (neat): 1743, 1697 cm'¹.

Compound .6 ( (-)-lR, 2S-lactone 6_:

Half ester 5. (1.04 g; 5.59 mmol) was dissolved in 20 ml of dry tetrahydrofuran in a 100 ml flask. After cooling this solution to -78° C, with stirring and under an argon atmosphere, lithium triethylborohydride (18 ml of a 1.0M solution in tetrahydrofuran, 18 mmol, 3.2 eq) was added dropwise via syringe. The reaction mixture was allowed to slowly warm to room temperature over the course of four hours and was quenched by the addition of 30 ml of IN HCI. After stirring the mixture for an additional 1 hour the product was extracted into 3 x 50 ml of ether. The ether portions were washed with brine. dried (MgS0₄) and freed of solvent. The product was purified on a silica gel column eluting with 5% ethyl acetate in hexane to obtain the product as a clear oil, 550 mg (70%) .

^lH NMR (CDC1₃) : d 1.18-1.31 (m, 3H) ; 1.55-1.70 (m, 3H) ; 1.78-1.83 (m, IH) ; 2.15 (d, IH) ; 2.40-2.50 (m, IH) ; 2.60-2.68 (m, IH) ; 3.95-4.00 (d, IH) ; 4.20 (d of d, 2H) .

IR (neat): 1770 cm'¹.

Compound 1_ (Hydroxy vinylphosphonate 2) '

Tetraethyl methylenebisphosphonate (4.32 g; 14.99 mmol) was added dropwise as a tetrahydrofuran solution (10 ml) to a stirred suspension of sodium hydride (660 mg of a 60% oil dispersion; 16.5 mmol) in THF (15 ml). After gas evolution had ceased the mixture was stirred at room temperature for 2 hours and then cooled to -78° C. In a separate flask, a solution of lactone 6 (1.0 g; 7.14 mmol) in toluene (15 ml) , cooled to -78° C, was treated with diisobutylaluminum hydride in toluene via syringe (5.7 ml of a 1.5M solution; 8.55 mmol). After stirring this mixture for 45 minutes, it was quenched by the addition of 50 ml of methanol, and this mixture was added via canula to the THF solution of the anion. The resulting mixture was stirred for 4 hours at 78° C; tert- butyldimethylsilyl chloride (1.18 g; 7.5 mmoll) in 15 ml of THF was then added and the mixture was stirred overnight while slowly warming to room temperature. It was quenched by the successive addition of saturated aqueous ammonium chloride (20 ml) and IN HCI (50 ml) . The product was extracted into ethyl acetate and the organic phase was washed with brine, dried (mgS0₄) and freed of solvent. The product was purified on a silica gel column utilizing a gradient elution (40% hexane in ethyl acetate to pure ethyl acetate) . The product was obtained as a clear viscous oil, 1.40 g (71%).

^lH NMF (CDC1₃) : d 1.25-2.05 (m + t, 15H) ; 2.65-2.75 (m, IH) ; 3.48 (d, 2H0; 3.98-4.15 (m, 4H) ; 5.65-5.81 (m, IH) ; 6.83-7.03 (m, IH) .

IR (neat): 3400, 2982, 2926, 2861, 1627, 1447, 1391, 1229, 1041, 956 cm¹.

Compound 8 (Hydroxy phosphonoethyl compound 8 ) :

A mixture of vinylphosphonate 1_ (2.70 g) and 5% palladium on carbon (600 mg) in ethanol (30 ml) was hydrogenated at 50 psi pressure of hydrogen for 4 hours.

The reaction mixture was filtered through Celite and concentrated to obtain 8 as a clear oil, 2.70 g (99%).

^lH NMR (CDC1) : d 1.21-1.84 (m, 20H) ; 2.60 (br, IH) ; 3.47-3.60 (m, 2H) ; 4.05-4.14 (m, 4H) .

IR (neat): 3400, 2980, 2924, 2859, 1448, 1391, 1230, 1054, 959, 828, 784 cm"¹.

Compound (Phosphonoethyl aldehyde 9_) :

A solution of oxalyl chloride (1.34 g; 10.55 mmol) in methylene chloride (25 ml) was cooled to -60° C and treated with 2 ml of DMSO (28.18 mmol). After stirring for 10 minutes, alcohol 8. (2.63 g; 9.46 mmol) was added as a methylene chloride solution (5 ml) . After stirring this mixture for an additional 15 minutes triethylamine (6.6 ml; 47.4 mmol) was added and the mixture was stirred for 5 minutes at 60° C and 30 minutes at room temperature. Water (50 ml) was added, the layers were separated, and the organic phase was washed with brine, dried (MgS0₄) and the solvent removed in vacuo. The crude product was purified on a silica gel column eluting with ethyl acetate to obtain 2.20 g (84%) of the aldehyde as a clear thick oil.

^JH NMR (CDC1₃) : d 1.29-1.77 (m, 13HO; 2.43-255 (m, IH) ; 4.06-4.12 (m, 4H) ; 9.79 (s, IH) .

IR (Neat): 3456, 2980, 2931, 3859, 2715, 1720, 1450, 1391, 1242, 1036, 959, 831, 789 cm¹.

Compounds 11a and b (N-acetyl amino esters 11a and b) :

A solution of methyl 2-acetlamino-2- (dimethoxyphosphinyl) acetate (1.82 g; 7.61 mmol) in THF (50 ml) was added to a stirred slurry of sodium hydride

(304 mg of a 60% oil dispersion; 7.60 mmol) in 20 ml of THF at room temperature. After stirring this mixture for 1 hour, the aldehyde was added as a THF solution (20 ml) . The mixture was stirred at room temperature for 24 hours, quenched by the addition of 20 ml of saturated aqueous ammonium chloride, and acidified with IN HCI until the pH was 2. It was extracted into ethyl acetate and the ethyl acetate portions were washed with brine, dried and freed of solvent. The residue was purified on a silica gel column eluting with ethyl acetate. Dehydro N-acetyl amino ester .10. was obtained contaminated with unreacted phosphonate reagent; 640 mg of this impure product was obtained.

This impure material was hydrogenated in the presence of 5% palladium on carbon (200 mg) in 30 ml of ethanol under 50 psi pressure of hydrogen for 4 hours. The mixture was filtered through Celite and freed of solvent. Purification of the crude residue on a silica gel-60 column, eluting with 5% methanol in ethyl acetate, delivered a total of 260 mg (8.7% overall) of a pure mixture of 11a and lib. ^!H NMR (CDC1₃) ( lla + lib) : d 1. 15-1. 88 (m, 20H) ; 2 . 02 (s , 3H) ; 3 .73 (s , 3H0 ; 4 . 00-4 . 19 (m, 4H) ; 4 . 55-4 . 68 (m, IH) ; 5. 98 and 6. 38 (br d' s , IH total) .

Enzymatic resolution of lla and lib compounds lla and 12.

The mixture of esters lla and lib (260 mg total) was dissolved in pH 7.4 phosphate buffer and stirred at room temperature. Subtilisin A (Novo Biolabs, 30 mg) was added and the mixture was stirred overnight. The pH was brought back to 7.4 by the dropwise addition of 0.2M NaOH. The mixture was diluted with water, transferred to a separatory funnel, and extracted with 3 x 75 ml of ethyl acetate. The ethyl acetate portions were dried (MgS0₄) and freed of solvent to deliver 100 mg of lla.

The aqueous phase was made acidic (pH 1) with IN HCI and extracted with 3 x 75 ml of ethyl acetate. After drying and removal of the solvent 50 mg of acid .12. was obtained.

lla: ^!H NMR (CDC1³) : d 1.24-1.88 (m, 20H) ; 2.04 (s, 3H) ; 3.75 (s, 3H) ; 4.03-4.15 (m, 4H) ; 4.57 (t of d, IH) ; 6.34 (br d. IH) .

8.: ^!H NMR (CDC1₃) : d 1.24-1.84 (m, 20H) ; 2.07 (s,

3H) ; 4.05-4.10 (m, 4H) ; 4.66-4.68 (m, IH) ; 6.46 (br d, IH) .

Hydrolysis of lla and 12. to 2R, 4R, 5S and 2S, 4R, 5S isomers, respectively.

Compounds lla and ,12. were each refluxed vigorously in 6N HCI overnight. The solutions were concentrated in vacuo and in each case the crude material was dissolved in ethanol and treated with propylene oxide to obtain the free bases. Small amounts of each of the two final products were obtained. HPLC analysis of the products. using racemic NPC 12626 as a reference, confirmed their identities as two diastereomeric cis isomers of NPC ACPA.

Example 3

Anticonvulsant studies: The potency of the isomers of APCA to inhibit NMDA-induced seizures were evaluated in U.S. Patent 5, 256, 814 using male CF-1 mice. All drugs were dissolved in isotonic saline and the pH adjusted to neutrality using NaOH. The test compounds were injected intracerebroventricularly (i.e.v.) in a final volume of 5 ml. N-Methyl-D-aspartate (250 mg/kg of body weight) was dissolved in saline (1% v/w of body weight) and injected intraperitoneally (i.p.) 15 minutes following the administration of the test agent. Mice were observed for 30 minutes following the administration of the chemical convulsant and scored as present or absent. NMDA-induced seizures were defined as presence of tonic/clonic activity accompanied by hindlimb extension and death.

Rotorod: Animals used for seizure studies were evaluated for motor impairment using the rotorod test. Before use all animals were tested for their ability to maintain equilibrium for 60 seconds on a 1-inch knurled bar rotating at a speed of 6 to 7 rpm. Each animal was given three separate trials. Animals performing the task in any of the three trials were used for further study.

Animals were tested for impairment 10 minutes after i.c.v. administration of the test agent and 5 minutes before administration of the seizure-inducing agent. Each animal was tested on a maximum of three trials; completion of any one of the trials was screened as passing.

Example 4 Binding studies: The potency of compounds to inhibit the specific binding of [³H]CGS-19755 (specific activity 50.5 Ci/mmol, New England Nuclear, Boston, MA) to NMDA receptors was performed as described by Ferkany et al (J. Pharmacology and Experimental Therapeutics. 1989, 250:100-109). Briefly, animals were sacrificed by decapitation, the forebrain removed immediately and the "buffy coat" prepared as described by Enna and Snyder (Enna, S. and Snyder, S.H., Molecular Pharmacology. 1977, 13:422-433) . Crude synaptic membranes were stored frozen (-20° C) until used. On the date of the assay, membranes were thawed to room temperature and homogenized in 20 vol (w/v/) of assay buffer. The homogenate was centrifuged (48,000 x g; 20 min; 4° C) , the supernatant decanted and the pellet resuspended as before. The procedure was repeated until the tissue had been washed 4 times to remove endogenous inhibitors.

For assay, the final pellet was resuspended in sufficient buffer (Tris HCI, 0.05M, pH 7.1, 23° C) to yield a tissue concentration of 0.2 mg of protein per ml. Two-milliliter portions of the suspension were added in duplicate to tubes containing ligand (final concentration = 4.62 x 10"' M) and the compound of interest (40 ml). After an incubation of 20 min (23° C) , the reaction was terminated by centrifugation, the supernatant was decanted and the pellets washed rapidly and superficially with 2 x 2.5 ml of ice-cold buffer. Tissue was solubilized in Protosol (0.5 ml, New England Nuclear, Boston, MA) and after the addition of scintillant, radioactivity was determined using standard procedures.

Non specific binding was determined using L- glutamate at a final concentration of 10"³ M. Routinely, IC_jnS were determined using 8-12 concentrations of inhibitor. Example 5

The neuroprotectant effects of the 2R, 4R, 5S isomer were examined in several animal models of ischemia including the bilateral occlusion of the carotid artery in the Mongolian gerbil, transient focal ischemia in the cat and a canine global ischemia model. Following bilateral occlusion of the carotid artery of the Mongolian gerbil, the 2R, 4R, 5S isomer afforded complete protection of the CA1 region of the hippocampus following a five minute period of global ischemia.

The effects of the 2R, 4R, 5S isomer and CGS 19755 were directly compared in a model of transient focal ischemia in the cat. The 2R, 4R, 5S isomer (5 mg/kg bolus and 2.5 mg/kg per hour throughout reperfusion) , or CGS 19755 (40 mg/kg intravenous bolus) , in a randomized fashion, decreased the injury volume of the ipsilateral caudate nucleus and was less in cats treated with NMDA receptor antagonists compared to cats treated with saline. Recovery of somatosensory evoked potential amplitude was incomplete and similar in all groups. Thus, the 2R, 4R, 5S isomer and CGS 19755 produced similarly efficacy in decreasing early post-ischemic brain injury in a model of transient focal ischemia in the cat.

The 2R, 4R, 5S isomer was also tested in a canine global ischemia model. Brain injury shows injury in this model is thought to correlate with cardiac arrest induced brain- injury. This model shows reproducible results in reperfusion injury, as observed by the presence of a dense and persistent tissue acidosis, incomplete recovery of high energy phosphates, progressive decay of cerebral blood flow and poor restoration of integrated neuronal activity. The 2R, 4R, 5S isomer administered at a bolus dose of 5mg/kg and 2.5 mg/kg/hr during ischemia throughout reperfusion, significantly decreased brain injury following global ischemia in the dog. There was a 35 and 50% recovery in sensory evoked potentials (as compared to saline treated dogs) 60 to 180 minutes following global ischemia. In addition, there was a significant recovery in cerebral blood flow and ATP in dogs administered the 2R, 4R, 5S isomer, as compared to corresponding saline treated dogs. The efficacy observed within the 2R, 4R, 5S isomer, is of particular importance as other NMDA receptor antagonists (i.e. CGS 19755) tested in this model are not efficacious. These data indicate that the 2R, 4R, 5S isomer should have clinical utility in the treatment of cardiac arrest, stroke, head trauma, drowning and so forth.

The 2R, 4R, 5S isomer is a potent, and selective, competitive NMDA receptor antagonist. The overall pharmacological profile of the 2R, 4R, 5S isomer is distinct from other competitive and non-competitive NMDA receptor antagonists, suggesting that NMDA receptor antagonists may not represent a homogenous group of compounds. The potent neuroprotectant activity of the 2R, 4R, 5S isomer in decreasing brain injury following dog global ischemia model indicates that the 2R, 4R, 5S isomer has utility in the treatment of clinical disorders associated with the global ischemia. The results are set forth in Table I.

Table !

_ISC_HEMI_A " DURATION OF REPERFUSION (min) Materials and Methods Male dogs (7 to 10 kg) were anesthetized with intravenous fentanyl (50 μg/kg) and pentobarbital (6 mg/kg, then 3 mg/kg per hour) and mechanically ventilated with supplemental oxygen. Muscle paralysis was achieved with pancuronium bromide (0.1 mg/kg). Systematic arterial and venous catheters were placed for pressure and microsphere measurements and for infusion of fluids and drugs. Temporalis muscles were fully retracted from the skull, and a superior sagittal sinus catheter was placed through a midline skull burr hole near the injection of the coronal sutures. The left lateral ventricle was cannulated through a second burr hole with a Silastic ventricular drain catheter (Cordis) for infusion of mock cerebrospinal fluid and measurement of intracranial pressure (ICP) . An epidural thermistor was inserted through an additional burr hole for continuous monitoring of brain temperature. An electrode for measuring the somatosensory evoked potential (SEP) was secured with dental acrylic into a burr hole contralateral to the ventricular catheter. Primary cortical wave amplitude in response to forelimb stimulation was measured. Animals were placed in a cradle equipped with a warm water blanket and fiberglass insulation to maintain normothermic epidural temperature.

Arterial and sagittal sinus blood samples were analyzed for P0₂, PC0₂, and pH levels with a Radiometer ABL electrode system. Oxygen content was measured with a CO-Oximeter (No. 282, Instrumentation Laboratories). Arterial blood pressure and ICP were measured with

Statham transducers. Blood flow was measured by the radiolabeled-microsphere technique with microspheres 16±0.5 μm in diameter (Dupont-New England Nuclear Products) . Cerebral 0₂ uptake (CMR0₂) was calculated by multiplying the arteriosagittal sinus 0₂ content difference by blood flow to the cerebral hemispheres. ²¹P magnetic resonance spectra were obtained using a Vivospec spectrometer (Otsuka Electronics) with a 1.89-T horizonal superconducting magnet (25-cm bore; Oxford Instruments) and a 5-cm diameter surface coil. Spectral areas for β-ATP, phosphocreatine, and inorganic phosphate were analyzed by planimetry and expressed as a percentage of the respective area in the control spectra for each animal. Intracellular pH was determined by known methods using constants derived from our titration data: pH=6.73+log[ (α-3.07)/(5.68-α) ] where α is the chemical shift of inorganic phosphate relative to phosphocreatine. An external standard (dimethyl[2- oxopropyl]-phosphonate) placed over the coil served as a marker for the spectral position when the phosphocreatine peak disappeared. Intracellular [HC0₂-] was calculated from the Henderson-Hasselbalch equation using a pK, of 6.12, pH; as measured by magnetic resonance spectroscopy (MRS) , and sagittal sinus PC0₂ with a solubility coefficient of 0.0314 mmol/L per millimeter of mercury. Changes in sagittal sinus PC0₂ were assumed to approximate changes in tissue PC0₂.

After baseline measurements were made, drug or vehicle was administered intravenously over 15 minutes. An intravenous solution of 50% dextrose was then infused to maintain plasma glucose at approximately 400 to 500 mg/dL. Global incomplete ischemia was produced by infusion of warmed cerebrospinal fluid from a heated reservoir system into the lateral ventricular catheter. ICP was maintained at 10 to 15 mm Hg below mean arterial pressure, while arterial pressure spontaneously changed. This procedure produces a relatively constant CBF over the 30-minute ischemic period. At the end of ischemia, the glucose infusion was discontinued. The fluid reservoir was disconnected and ICP rapidly decreased toward baseline values. After 3 hours of reperfusion, the anesthetized dogs were killed with intraventricular potassium chloride injection. MRS spectra were analyzed in 15-minute epochs in duplicate before ischemia, in three 5-minute epochs during treatment, in one 6-minute and three 8-minute epochs during ischemia, in four 5- inute epochs during the first 20 minutes of reperfusion, and in 15-minute epochs for the remainder of the reperfusion. Sagittal sinus PC0₂and SEP were measured at the midpoint of each MRS spectrum. CBF and CMR0₂were measured at baseline at 18 minutes of ischemia, and at 8, 30, 90, and 180 minutes of reperfusion.

For further details, see Patricia D. Hum et al. "Deferoxamine Reduces Early Metabolic Failure Associated with Severe Cerebral Ischemic Acidosis In Dogs", (1955) American Heart Association, pages 688-694. The results are set forth in Table II.

Table II

Dog model of global ischemia

A canine global ischemia model which reproducibly results in reperfusion injury, as observed by the presence of a dense and persistent tissue acidosis, incomplete recovery of high energy phosphates, progressive decay of cerebral blood flow and poor restoration of integrated neuronal activity was utilized and measurements of the high energy phosphates, ATP and phosphocreatine, in a large area of the cerebral hemispheres, as well as intracellular pH and tissue bicarbonate during ischemia and reperfusion were made. Regional cerebral blood flow was also measured by the radiolabelled microsphere technique during ischemia and at 4 reperfusion time points.

The 2R, 4R, 5S isomer administered at a bolos dose of 5mg/kg and 2.5 mg/kg/hr during ischemia and throughout reperfusion significantly decreased brain injury following global ischemia in the dog. As discussed above, there was 35 and 50% recovery in sensory evoked potentials (as compared to saline treated dogs) 60 and 180 minutes following global ischemia. In addition, there was a significant recovery in cerebral blood flow and ATP in dogs administered the 2R, 4R, 5S isomer, as compared to corresponding saline treated dogs. Importantly, in this model, CGS 19755 is not effective.

The results are set forth in Table III.

TABLE III

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a mammal for a disease selected from the group consisting of focal ischemia, global ischemia and neuronal cell ischemia associated with spinal injuries, stroke, cardiac arrest, head trauma or drowning; said method comprising administering to the mammal a therapeutically effective amount of the 2R, 4R, 5S isomer of the compound 2-amino-4,5-(l,2-cyclohexyl)-7- phosphonoheptanoic acid or any pharmaceutically acceptable salt or hydrate of the isomer wherein the isomer or salt or hydrate of the isomer is substantially free from other stereoisomers of the compound.

2. A method of treating a mammal to decrease brain injury caused by global ischemia, which comprises: administering to a subject an effective amount of the 2R, 4R, 5S isomer of the compound 2-amino-4,5-(l,2- cyclohexyl)-7-phosphonoheptanoic acid or any pharmaceutically acceptable salt or hydrate of the isomer wherein the isomer or salt or hydrate of the isomer is substantially free from other stereoisomers of the compound.

3. The method of claim 2 wherein the global ischemia was caused by stroke, cardiac arrest, head trauma or drowning.

4. The method of claim 2 wherein the effective amount is administered intravenously either alone or in combination with other drugs.

5. A method for treating or preventing nerve cell death comprising: administering to a mammal exhibiting symptoms of nerve cell death or susceptible to nerve cell death, an effective amount of a compound comprising 2R, 4R, 5S isomer of the compound 2-amino-4,5- (1,2- cyclohexyl)-7-phosphonoheptanoic acid or any pharmaceutically acceptable salt or hydrate of the isomer wherein the isomer or salt or hydrate of the isomer is substantially free from other stereoisomers of the compound.

6. The use of the compound 2R, 4R, 5S isomer of 2- amino-4,5- (1,2-cyclohexyl)-7-phosphonokeptanoic acid or any pharmaceutically acceptable salt or hydrate of the isomer wherein the isomer or salt or hydrate of the isomer is substantially free from other stereoisomers of the compound in the preparation of a composition for treating a disease in a mammal selected from the group consisting of focal ischemia, global ischemia and neuronal cell ischemia.

7. The use of the compound of claim 6 for treating global ischemia to decrease brain injury.

8. The use of the compound of claim 6 for treating global ischemia caused by stroke, cardiac arrest, head trauma or drowning.

9. The use of the compound of claim 6 for treating a mammal exhibiting symptoms of nerve cell death or susceptible to nerve cell death.