Azetidine derivatives, compositions and methods of treating

BACKGROUND OF THE INVENTION
It is known that a selective subtype of excitatory amino acid receptors activated by N-methyl-D-aspartate (NMDA), Glutamate (GLU), and Aspartate (ASP) and competitively inhibited by 2-amino-5-phosphonovaleric acid (APV) are responsible for long term potentiation (LTP). Long term potentiation is defined as the increase in strength of synaptic transmission with repetitive use. LTP has been recognized as one type of synaptic plasticity and is believed to underlie behaviorally significant forms of memory storage and learning. The dysfunctions of excitatory amino acid receptors have been implicated in, e.g., the etiology of senile dementia of the Alzheimer type (SDAT), a disease characterized by severe memory loss, personality changes and symptoms of cortical disconnection; apraxias; asphasias; and agnosias. Glutamate receptors, particularly the NMDA subtype, have been shown to decrease in the neocortex of SDAT patients. The function of this receptor can also be allosterically inhibited by phencyclidine (PCP) and other dissociative anaesthetics, blocking LTP and resulting in learning and memory impairments.
The development of pharmacological agents with the ability to modulate the NMDA receptor in a positive manner has dual importance. In terms of basic neuroscience research it provides the necessary tools for the study of excitatory amino acid receptors, allowing discrimination of the actions of the NMDA receptor subtype from other glutamate receptors. In terms of clinical usefulness, such agents would be useful in treating memory and learning disorders, such as SDAT. The compounds are also effective as therapeutic agents in treating PCP toxicity and abuse.
SUMMARY OF THE INVENTION
This invention relates to novel azetidine dicarboxylic acids, carboxylic acid esters, mono- and diamides, acid alcohols, acid aldehydes, N-benzyl and N-acylated derivatives thereof, as well as to pharmaceutical compositions and methods of treating memory and learning disorders and dysfunctions of excitatory amino acid receptors in mammals, including humans, such as those discussed above. Another aspect of the invention relates to a method of utilizing the compounds and compositions as biological tools and materials for characterizing excitatory amino acid receptor systems. A further aspect of the invention relates to a method of utilizing the compounds and compositions in treating PCP toxicity.
More particularly, the present invention relates to a novel class of azetidine derivatives having the following formula: ##STR1## wherein A and A' are the same or different and selected from the group consisting of ##STR2## (CH.sub.2).sub.n OH, and C(OR).sub.2 H and wherein R and R' are the same or different and selected from the group consisting of hydrogen and lower alkyl, benzyl, and an amino acid; n=1-6; x is an hydrogen, group or a benzyl group; and pharmaceutically acceptable salts thereof.
In the above formula, R and R' can be lower alkyl. Lower alkyl means straight- or branched-chain saturated aliphatic radicals preferably having 1-6 carbon atoms. Exemplary of such lower alkyls are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-amyl, n-hexyl and the like. The preferred group of lower alkyl radicals are straight and branched chain C.sub.1-C.sub.4 alkyl radicals.
X in formula (I) is a benzyl group or hydrogen or acyl group, especially lower acyl or C.sub.1-6 acyl, and may particularly be formyl, acetyl, propionyl and butyal. Particularly preferred of the N-acyl derivatives is the N-acetyl derivative wherein X is acetyl.
The azetidine derivatives of the present invention can be 2,3 or 2,4-disubstituted azetidines, preferably 2,4-disubstituted azetidines.
The azetidine derivatives of the present invention exhibit stereoselectivity in their biological action. The cis-isomers have particularly been found to be the most biologically active.
The preferred compounds of the invention are C.sub.1-C.sub.4 straight or branched chain cis-2,4 disubstituted azetidine derivatives and their nontoxic, pharmaceutically acceptable salts.
Exemplary of such compounds are cis-azetidine-2,4-dicarboxylic acid dimethyl ester; cis-azetidine2,4-dicarboxylic acid t-butyl methyl ester; cis-azetidine-2,4-dicarboxylic acid mono t-butyl ester; cis-azetidine-2,4-dicarboxylic acid; cis-azetidine2,4-dicarboxylic acid di-t-butyl ester; cis-N-acetylazetidine-2,4-dicarboxylic acid; N-(cis-4-carboxyazetidine-2-carbonyl)glycine; cis-4-carbamoylazetidine-2-carboxylic acid; cis-azetidine-2,4-dicarboxamide; cis-azetidine-2,3-dicarboxylic acid; cis-azetidine-2,3-dicarboxamide; cis4-(hydroxymethyl)azetidine-2-carboxylic acid; cis-4-(dimethoxymethyl)azetidine-2-carboxylic acid.
The cis-azetidine derivatives of the present invention include pharmaceutically acceptable salts of the compounds of the present invention. Examplary of such salts are the oxalate, citrate, tartrate, hydrochloride salts, and the like.
The invention also relates to the process of preparing the aforementioned azetidine derivatives. In defining the processes, a lower alkyl group is defined as being C.sub.1 -C.sub.6, straight or branched chain radicals.
Stoichiometric amount is defined as the amount of reactant needed to react with an equivalent of another reactant. For example, when preparing a 2,4-azetidine dicarboxylic acid mono substituted ester, one would use 1/2 of a stoichiometric amount of the reactant lower alcohol.
Solvents for running the reaction are any organic, solvents. Particularly preferred solvents include carbon tetrachloride, benzene, acetone, toluene, methanol, dioxane, tetrahydrofuran, diethyl ether, hexane, and ethyl acetate. In terms of temperature ranges for running the reactions, -10.degree. C. to 100.degree. C. are preferred.
More particularly the invention is directed to a process for preparing azetidine-2,4-dicarboxylic acids and their (di)esters comprising
(a) reacting (I) ##STR3## with benzyl alcohol, a lower alcohol, or benzyl alcohol and a lower alcohol to produce a compound of the formula wherein R.sup.1 and R.sup.2 are the same or different and represent benzyl or a lower alkyl.
(b) reacting said compound of formula (III) with benzylamine to form ##STR4## wherein R.sup.1 and R.sup.2 are defined as above and ph is a phenyl group.
(c) separating cis- and trans-isomers of said compound of formula IV, and
(d) subjecting either of said isomers to N-debenzylation to form, respectively, ##STR5## wherein R.sup.3 and R.sup.4 are H and/or lower alkyl.
Moreover, a preferred embodiment of preparing azetidine-2,4-dicarboxylic acid (di)esters is wherein the lower alcohol is selected from the group consisting of C.sub.1 -C.sub.6, straight or branched chain alcohols.
This invention also relates to a process for preparing azetidine-2,3 dicarboxylic acids comprising
(a) irradiating a compound of the formula ##STR6## wherein Ph is a phenyl group, to produce a compound of the formula ##STR7## (b) reacting a compound of formula VIII with ozone to produce a compound of the formula ##STR8## (c) subjecting the compound- of formula IX to N-deacylation to form ##STR9##
A further aspect of the present invention is directed to a process for preparing azetidine-2,3-dicarboxylic acid diesters comprising
(a) irradiating a compound of formula ##STR10## wherein Ph is a phenyl group, to produce a compound of formula ##STR11## (b) reacting a compound of formula VIII with ozone to produce a compound of formula ##STR12## (c) esterifying the compound of formula VIII by reacting said compound with a lower alcohol, or benzyl alcohol and a lower alcohol, to produce a compound of formula ##STR13## wherein R.sup.5 and R.sup.6 are lower alkyl or benzyl. (d) subjecting said compound X to N-deacylation to form ##STR14## wherein R.sup.5 and R.sup.6 are H and/or lower alkyl.
Still a further aspect of the invention is directed to a process for preparing azetidine-2,3-dicarboxamides comprising:
(a) reacting a compound of formula IX ##STR15## wherein Ph is a phenyl group with an amine having the formula ##STR16## wherein R.sup.7 and R.sup.8 are each H, or lower alkyl, to form the compound ##STR17## (b) subjecting said compound XI to N-deacylation to form ##STR18##
This invention also relates to a process for preparing azetidine-2,4-(di)carboxamides comprising:
(a) reacting XII ##STR19## wherein Ph is a phenyl group, With a stoichiometric amount of MeAl(Cl)NH.sub.2 to form compounds of the formula ##STR20## (b) subjecting either of said XIII or XIV to N-debenzylation to form, respectively ##STR21##
This invention also relates to a process for preparing cis-4-(hydroxymethyl)azetidine-2-carboxylic acid which comprises
(a) reacting a compound ##STR22## wherein Ph is a phenyl group, with Me.sub.2 AlSeMe to form a compound of the formula ##STR23## (b) reacting a compound of formula XX with a reducing agent to form a compound ##STR24## (c) subjecting the compound of formula XXI to debenzylation to form the compound ##STR25##
A process for preparing cis-4-(hydroxy-methyl)azetidine-2,carboxylic acid ester which comprises
(a) reacting a compound of the formula ##STR26## wherein Ph is a phenyl group, sequentially with a reducing agent in a lower alcohol solvent, and then acid to form the compound ##STR27## wherein R is a lower alkyl group.
A process of preparing cis-4-(dialkoxymethyl)azetidine-2-carboxylic acid which comprises
(a) reacting a compound of formula ##STR28## wherein Ph is a phenyl group, first with (COCl).sub.2 and DMSO, and then triethylamine, to form a compound of the formula ##STR29## (b) reacting compound XXIV and a lower alcohol to form compound ##STR30## wherein R is a lower alkyl group
(c) subjecting the compound of formula XXV to N-debenzylation to form ##STR31## wherein R is a lower alkyl group.
The invention also relates to compositions of the compounds of formula I for carrying out the aforementioned methods of treatment in mammals, including humans, as well as for carrying out the biological tools and materials for characterizing excitatory amino acid receptor systems.
DETAILED DESCRIPTION OF THE INVENTION
The following examples further illustrate the present invention and will enable others skilled in the art to understand it more completely. It is to be understood, however, that the invention is not limited solely to the particular examples below. All percentages are by weight, unless otherwise specified.
MODE OF PREPARATION
The following examples explain in greater detail the preparation of the intermediates and the final products of the present invention.
General Procedures.
All reactions, if not described otherwise, are run under an ordinary atmosphere with exclusion of moisture. Column chromatography is performed on Merck No. 7734 Kieselgel 60 (0.063-0.200 mm) with ethyl acetate/hexanes as eluent if not stated otherwise. .sup.1 H NMR spectra are recorded on a Bruker AF 300 instrument at 300 MHz in CDCl.sub.3 or D.sub.2 O using TMS or sodium 3-trimethylsilyl-propanesulfonate as internal standards. Coupling constants are given in Hz; they are evaluated by first-order rules with an estimated accuracy of 0.5 Hz. IR spectra are measured on liquid film samples, KBr pellets, or nujol suspensions on an IBM IR/32 FTIR spectrometer. Mass spectra were recorded on Varian CH 5 and VG 70-G instruments. Elemental analyses were carried out by Galbraith Laboratories, Inc., Knoxville, Tenn. Melting points were determined on a Thomas Hoover capillary melting point apparatus and are uncorrected.
Cis- and trans-azetidine carboxylic derivatives are prepared by brominating glutaric anhydride in the presence of a catalyst to form 2,4-dibromoglutaric acid. Depending on the product desired, as will be explained in greater detail hereinbelow, the 2,4-dibromoglutaric acid can be further reacted with acetyl chloride to form 2,4-dibromoglutaric anhydride.

These two intermediates can then be reacted with a variety of alcohols to arrive at the compounds of the present invention.
EXAMPLE 1
Preparation of 2,4-Dibromoglutaric acid
The preparation is conducted in a well-ventilated hood because toxic fumes (Br2, HBr, CO) evolve. A 50 ml 3-necked flask equipped with thermometer, dropping funnel (its stem extending under the surface of the liquid by means of a piece of teflon tubing), reflux condenser and magnetic stirrer is charged with 10.1 g (88.5 mmol) of glutaric anhydride (Aldrich) and 136 mg (4.4 mmol) of red phosphorus (Aldrich), and heated in an oil bath to an inner temperature of approx. 100.degree. C. 9.9 ml (195 mmol) of bromine is added dropwise with stirring within 1 hour at a temperature of 105.degree.-110.degree. C., and the mixture is stirred for another 2 hours at 110.degree.-115.degree. C. (HBr evolution has become very slow at this point). After cooling to 50.degree. C., the viscous product is poured into 7.1 ml (180 mmol) of 95-97% formic acid, whereon vigorous gas (presumably CO) evolution takes place. After standing overnight, the solid is recrystallized from ether/chloroform (2 crops) and chloroform (another 2 crops), yielding a total of 11.3g (44%) of a tan product.
EXAMPLE 2
Preparation of 2,4-Dibromoglutaric anhydride
10.0g (34.5 mmol) of 2,4-dibromoglutaric acid and 4.9 ml (69 mmol) of acetyl chloride are refluxed for a period of 2 hours. Volatiles are removed at 10 torr up to 90.degree. C., and the residue bulb-to-bulb distilled (75.degree.-90.degree. C./0.25 torr), yielding 8.9g (95%) of a colourless, viscous oil. .sup.1 H NMR (CDCl.sub.3): dl isomer: .delta. 4.90 (t, 2H, J=6), 2.87 (t, 2H, J=6); meso isomer: 4.77 (dd, 2H, J=6, 10), 3.15 (dt, 1H, J=6 (t), 15 (d)), approx. 2.85 (1H, partially overlapping); dl/meso=5:1. IR (neat): 3007, 2942, 1817, 1775, 1439, 1242, 1109, 1038, 972, 932, 847, 691, 617 cm.sup.-1.
EXAMPLE 3
Preparation of cis-Azetidine-2,4-dicarboxylic acid dimethyl ester
1.14g (3.93 mmol) of 2,4-dibromoglutaric acid, 1 ml of methanol, 1.2 ml of CCl.sub.4, and 1 drop of conc. sulfuric acid are refluxed for 8 hours. Aqueous workup (CH.sub.2 Cl.sub.2 /sat. NaHCO.sub.3), filtration over silica gel, and evaporation yields 0.87g (70%) of dimethyl 2,4-dibromoglutarate. .sup.1 H NMR (CDCl.sub.3): .delta.3.82 (s, 6H); dl isomer: .delta.4.53 (dd, 2H, J=6.5, 8), 2.68 (dd, 2H, J=6.5, 8); meso isomer: .delta.4.41 (t, 2H, J=7.5), 2.89 (dt, 1H, J=7.5 (t), 14.5 (d)), 2.64 (dt, 1H, J=7.5 (t), 14.5 (d)); dl/meso=3:1.IR (neat): 3004, 2955, 1740, 1437, 1300, 1267, 1215, 1156, 851 cm.sup.-1.
Dimethyl 2,4-dibromoglutarate (1.98 mmol), 3 molar equivalents of benzylamine, and enough DMF to prepare an approx. 0.12 M solution of dimethyl-2,4-dibromoglutarate are stirred at 80.degree. C. for 2 hours. The solvent is distilled into a cold trap at 0.2-0.3 torr/40.degree. C. bath temperature, the residue taken up in CH.sub.2 Cl.sub.2, and the solution washed with saturated aq. NaHCO.sub.3. After drying over MgSO.sub.4 and evaporation, the residue is chromatographed using a silica gel column to yield, after a forerun, first the trans, then the cis-isomer of N-benzylazetidine-2,4-dicarboxylic acid dimethyl ester as amber, viscous oils.
trans isomer 36% yield. .sup.1 H NMR (CDCl.sub.3): .delta.7.3-7.2 (m, 5H), 4.22 (t, 2H, J=7), 3.87 (narrow AB system, 2H, J=13.5), 3.65 (s, 6H), 2.51 (t, 2H, J=7). IR (neat): 2953, 1736, 1437, 1354, 1200, 1030, 749, 698 cm.sup.-1.
cis isomer 32% yield. .sup.1 H NMR (CDCl.sub.3): .delta.7.37-7.22 (m, 5H), 3.88 (s, 2H), 3.64 (s, 6H), 3.63 (t, 2H, J=8.5), 2.50 (dt, 1H, J=8.5 (t), 10.5 (d)), 2.34 (dt, 1H, J=8 (t), 10.5 (d)). IR (neat): 2953, 1744, 1437, 1225, 1202, 1042, 700 cm.sup.-1.
79 mg (0.3 mmol) of cis-N-benzylazetidine-2,4-dicarboxylic acid dimethyl ester and 25 mg of Pd(OH).sub.2 (20% on C, 31% H.sub.2 O, Aldrich) in 10 ml of methanol are hydrogenated under 3 atm of hydrogen in a Parr shaker for 6 hours. The catalyst is filtered off and thoroughly washed, the solution evaporated, and the residue filtered over a short column to yield 23.4 mg (45%) of cis-azetidine-2,4-dicarboxylic acid dimethyl ester. .sup.1 H NMR (CDCl.sub.3): .delta.4.27 (dd, 2H, J=6.5, 8.5), 3.76 (s, 6H), 2.95 (dt, 1H, J=8.5 (t), 12 (d)), 2.60 (dt, 1H, J=6.5 (t), 12 (d)). IR (neat): 3345, 2955, 1740, 1437, 1231, 1042 cm.sup.-1. Mass spectrum: m/z 173 (7%), 114 (100), 82 (44), 59 (14), 55 (32), 54 (98). Calcd. for C.sub.7 H.sub.11 NO.sub.4 : 173.0688, found: 173.0688.
EXAMPLE 4
Preparation of trans- and cis-Azetidine-2.4-dicarboxylic acid t-butyl methyl ester
544 mg (2 mmol) of 2,4-dibromoglutaric anhydride, 0.5ml of CH.sub.2 Cl.sub.2, and 85 .mu.l (2.1 mmol) of methanol are stirred in a capped vial at room temperature for 5.5 hours, after which time the IR spectrum indicates the absence of starting materials. 5 ml of benzene and 10 .mu.l of DMF are added, the solution is then cooled in an ice bath, and 0.19 ml (2.2 mmol) of oxalyl chloride in 2 ml of benzene are added dropwise. Stirring is continued for 15 min. at 0.degree. C. and for 45 min. at room temperature, after which time gas evolution (HCl, CO, CO.sub.2) virtually ceases. Volatiles are evaporated in vacuo, and the residue is taken up in 2 ml of CH.sub.2 Cl.sub.2. This solution is added dropwise with ice cooling to a solution of 0.47 ml(5 mmol) of tert.-butanol, 0.35 ml (2.5 mmol) of triethylamine, and 12.2 mg (0.1 mmol) of 4-dimethylaminopyridine in 3 ml of CH.sub.2 Cl.sub.2. After stirring at 0.degree. C. and room temperature for 20 min. each, the mixture is washed with aqueous KHSO.sub.4 solution, dried over MgSO.sub.4, evaporated, and filtered over silica gel to afford 410 mg (57%) of t-butyl methyl 2,4-dibromoglutarate. .sup.1 H NMR (CDCl.sub.3): .delta.4.52, 4.41, 4.40, 4.27 (each t, 1H, J=7.5), 3.81 (s, 3H), 1.49 (s,9H); dl isomer: .delta.2.61 (t, 2H, J=7.5); meso isomer: .delta. 2.83 (dt, 1H, J=7.5 (t), I5 (d)), 2.58 (dt, 1H, J=7.5 (t), 15 (d)); dl/meso=2:1. IR (neat): 2980, 1742, 1437, 1370, 1277, 1150, 843 cm.sup.-1.
t-Butyl methyl 2,4-dibromoglutarate (1.08 mmol), 3 molar equivalents of benzylamine, and enough DMF to prepare an an approx. 0.12M solution of t-butyl methyl 2,4-dibromoglutarate are stirred at 80.degree. C. for 2 hours. The solvent is distilled into a cold trap at 0.2-0.3 torr/40.degree. C. bath temperature, the residue taken up in CH.sub.2 Cl.sub.2, and the solution washed with saturated aq. NaHCO.sub.3. After drying over MgSO.sub.4 and evaporation, the residue is chromatographed using silica gel to yield, after a forerun, first the trans isomer, then the cis-isomer of N-benzylazetidine-2,4-dicarboxylic acid t-butyl methyl ester as amber, viscous oils.
trans isomer 32% yield . .sup.1 H NMR (CDCl.sub.3): .delta. 7.35-7.2 (m, 5H), 4.20 (t, 1H, J=7), 4.09 (t, 1H, J=6.5), 3.89 (s, 2H), 3.64 (s, 3H), 2.46 (dd, 2H, J=6.5, 7), 1.42 (s, 9H). IR (neat): 2977, 1734, 1455, 1368, 1237, 1202, 1156, 1028, 745, 700 cm.sup.-1.
cis isomer: 30% yield. .sup.1 H NMR (CDCl.sub.3): .delta. 7.35-7.2 (m, 5H), 3.86 (narrow AB system, 2H, J=13.5), 3.63 (s, 3H), 3.57 (t, 1H, J=8.5), 3.49 (t, 1H, J=8.5), 2.44 (dt, 1H, J=8.5 (t), 10.5 (d)), 2.29 (dt, 1H, J=8 (t), 10.5 (d)), 1.37 (s, 9H). IR (neat): 2977, 1738, 1455, 1368, 1229, 1157, 1030, 847, 754, 708 cm.sup.-1.
The trans- and cis-azetidine-2,4-dicarboxylic acid t-butyl methyl esters are obtained in the same way as in example 3 from their respective stereo-chemically pure N-benzyl precursors.
cis-isomer in 48% yield. .sup.1 H NMR (CDCl.sub.3): .delta.4.21 (dd, 1H, J=6, 9), 4.10 (dd, 1H, J=6, 9), 3.76 (s, 3H), 3.12 (br. s., 1H), 2.96 (dt, 1H, J=9 (t), 12 (d)), 2.49 (dt, 1H, J=6 (t), 12 (d)), 1.48 (s, 9H). IR (neat): 3276, 2975, 1728, 1370, 1269, 1159, 1100, 1088, 1026, 833, 776, 764 cm.sup.-1.
trans-isomer in 59% yield. .sup.1 H NMR (CDCl.sub.3): .delta.4.35 (dd, 1H, J =6.5, 9), 4.13 (dd, 1H, J =5.5, 9), 3.77 (s, 3H), 2.81 (br. s., 1H), 2.72 (ddd, 1H, J =6.5, 9, 11.5), 2.60 (ddd, 1H, J =5.5, 9, 11.5), 1.49 (s, 9H). IR (neat): 3328, 2979, 1732, 1370, 1237, 1159, 1059, 845 cm.sup.-1.
EXAMPLE 5
Preparation of trans- and cis-Azetidine-2,4-dicarboxylic acid mono t-butyl ester
2.18g (8 mmol) of 2,4-dibromoglutaric anhydride, 0.83 ml (8 mmol) of benzyl alcohol, and 2 ml of chloroform are stirred for 1 hour at room temperature and for another hour under reflux. 20 ml of benzene and 0.1 ml of DMF are added, the solution is cooled in an ice bath, and 1.05 ml (12 mmol) of oxalyl chloride in 5 ml of benzene is added dropwise Stirring is continued for 40 min. at 0.degree. C. and 70 min. at room temperature, after which time gas evolution has virtually ceased. Volatiles are evaporated in vacuo, and the residue is taken up in 5 ml of CH.sub.2 Cl.sub.2. This solution is added dropwise with ice cooling to a solution of 1.4 ml (15 mmol) of tert.-butanol, 1.4 ml (10 mmol) of triethylamine, and 19.5 mg (0.16 mmol) of 4-dimethylaminopyridine in 15 ml of CH.sub.2 Cl.sub.2. After stirring at 0.degree. C. and room temperature for 20 min. each, the mixture is washed with aqueous KHSO.sub.4 solution, dried over MgSO.sub.4, evaporated, and filtered over silica gel to afford 2.65g (76%) of benzyl t-butyl 2,4-dibromoglutarate. .sup.1 H NMR (CDCl.sub.3): .delta.7.37 (br. s., 5H), 5.22 (2 narrow AB systems, 2H), 1.48 (s, 9H); dl isomer: .delta.4.54 (t, 1H, J=7.5), 4.39 (t, 1H, J=7.5), 2 63 (t, 2H, J=7.5); meso isomer: .delta.4.44 (t, 1H, J=7.5), 4.26 (t, 1H, J=7.5), 2.83 (dt, 1H, ,J=7.5 (t), 15 (d)), 2.59 (dt, 1H, J=7.5 (t), 15 (d)); dl/meso=1:2. IR (neat): 2980, 1736, 1456, 1370, 1266, 1148, 843, 752, 696 cm.sup.-1.
Benzyl t-butyl 2,4-dibromoglutarate (2.13 mmol), 3 molar equivalents of benzylamine, and enough DMF to prepare an approx. 0.12M solution of benzyl t-butyl 2,4-dibromoglutarate are stirred at 80.degree. C. for 2 hours. The solvent is distilled into a cold trap at 0.2-0.3 torr/40.degree. C. bath temperature, the residue taken up in CH.sub.2 Cl.sub.2, and the solution washed with saturated aq. NaHCO.sub.3. After drying over MgSO.sub.4 and evaporation, the residue is chromatographed using a silica gel column to yield-after a forerun-first the trans isomer, then the cis-isomer of N-benzylazetidine-2,4-dicarboxylic acid benzyl t-butyl ester as amber, viscous oils.
cis isomer: 33% yield. .sup.1 H NMR (CDCl.sub.3): 7.38-7.18 (m, lOH), 5.10, 5.03 (AB system, 2H, J=12.5), 3.88, 3.84 (AB system, 2H, J=13), 3.61 (t, 1H, J=8), 3.46 (t, 1H, J=8), 2.48 (dt, 1H, J=8 (t), 11.5 (d)), 2.28 (dt, 1H, J=8 (t), 11.5 (d)), 1.36 (s, 9H). IR (neat): 2979, 1738, 1455, 1368, 1229, 1159, 1028, 845, 745, 698 cm.sup.-1.
267 mg (0.7 mmol) of stereochemically pure trans- or cis-N-benzylazetidine-2,4-dicarboxylic acid benzyl t-butyl ester, 50mg of 10% Pd/C, and 20 ml of methanol are hydrogenated under 3 atm of hydrogen in a Parr shaker for 4 hours. The catalyst is filtered off and thoroughly washed, and the solution evaporated to dryness and dried in vacuo to afford as off-white solids the trans- or cis-azetidine-2,4-dicarboxylic acid mono-t-butyl ester.
cis isomer in 94% yield. .sup.1 H NMR (D.sub.2 O): .delta.4.98 (dd, 1H, J=8, 10), 4.75 (dd, 1H, J=8, 10), 3.15 (dt, 1H, J=10 (t), 12.5 (d)), 2.57 (dt, 1H, J=8 (t), 12.5 (d)), 1.51 (s, 9H). lR (KBr): 3434, 2998, 1738, 1617, 1563, 1385, 1368, 1248, 1161, 843 cm.sup.-1. Mass spectrum: m/z 100 (100%), 82 (33), 57 (81), 56 (20), 55 (17), 54 (37), 41 (39).
trans isomer in 92% yield. .sup.1 H NMR (D.sub.2 O): .delta.4.91 (dd, 1H, J =7, 10), 4.71 (dd, 1H, J =7.5, 10), 2.98-2.77 (m, 2H), 1.52 (s, 9H). IR (KBr): 3438, 2982, 1740, 1632, 1570, 1389, 1281, 1248, 1175, 1080, 779, 515 cm.sup.-1.
EXAMPLE 6
Preparation of trans- and cis-Azetidine-2,4-dicarboxylic acid
2.18g (8 mmol) of 2,4-dibromoglutaric anhydride, 2.1 ml (20 mmol) of benzyl alcohol, and 2 ml of benzene are stirred for 1 5 hours at room temperature 76 mg (0.4 mmol) of p-toluenesulfonic acid monohydrate and 8 ml of benzene are added, and the solution is refluxed under a Dean-Stark trap for 2 hours. Filtration over silica gel and evaporation yields 3.77g (100%) of dibenzyl 2,4-dibromoglutarate as a colourless oil. .sup.1 H NMR (CDCl.sub.3): .delta.7.37 (br. s., 10H), 5.21 (narrow AB system, 4H, J=12.5); dl isomer: .delta.4.55 (dd, 2H, J=6.5, 7.5), 2.69 (dd, 2H, J=6.5, 7.5); meso isomer: .delta. 4.42 (t, 2H, J=7.5), 2.90 (dt, 1H, J=7.5 (t), 14.5 (d)), 2.66 (dt, 1H, J=7.5 (t), 14.5 (d)); dl/meso=6:1. IR (neat): 3034, 1740, 1499, 1456, 1385, 1273, 1154, 750, 696 cm.sup.-1.
Dibenzyl-2,4-dibromoglutarate (7.93 mmol), 3 molar equivalents of benzylamine, and enough DMF to prepare an approx. 0.12M solution are stirred at 80.degree. C. for 2 hours. The solvent is distilled into a cold trap at 0.2-0.3 torr/40.degree. C. bath temperature, the residue taken up in CH.sub.2 Cl.sub.2, and the solution washed with saturated aq. NaHCO.sub.3. After drying over MgSO.sub.4 and evaporation, the residue is chromatographed using a silica gel column to yield - after a forerun - first the trans, then the cis-isomer of N-benzylazetidine-2,4-dicarboxylic acid dibenzyl ester as amber, viscous oils.
trans isomer 38% yield. .sup.1 H NMR (CDCl.sub.3): .delta.7.4-7.15 (m, 15H), 5.09 (narrow AB system, 4H), 4.25 (t, 2H, J=7), 3.89, 3.85 (AB system, 2H, J=13), 2.52 (t, 2H, J=7). IR (neat): 3033, 2953, 1732, 1497, 1455, 1345, 1171, 1028, 737, 698 cm.sup.-1.
cis isomer: 27% yield. .sup.1 H NMR (CDCl.sub.3): .delta.7.4-7.2 (m, 15H), 5.12, 5.04 (AB system, 4H,J=12.5), 3.91 (s, 2H), 3.68 (t, 2H, J=8), 2.58 (dt, 1H, J=8.5 (t), b 10.5 (d)), 2.38 (dt, 1H, J=8 (t), 10.5 (d)). IR (neat): 3033, 1744, 1497, 1455, 1173, 1028, 911, 735, 696 cm.sup.-1.
208 mg (0.5 mmol) of stereochemically pure transor cis-N-benzylazetidine-2,4-dicarboxylic acid dibenzyl ester and 20 mg of 10% Pd/C in 15 ml of methanol are hydrogenated as above. The reaction mixture is diluted with 50 ml of warm water, filtered, and evaporated to dryness to afford as off-white solids the trans- or cis azetidine-2,4-dicarboxylic acid in virtually quantitative yield.
cis isomer: .sup.1 H NMR (D.sub.2 O): .delta.4.87 (dd, 2H, J=8, 10), 3.19 (dt, 1H, J=10 (t), 12 (d)), 2.60 (dt, 1H, J=8 (t), 12 (d)). IR (KBr): 3443, 3158, 1727, 1630, 1563, 1404, 1246, 843, 779, 604, 542 cm.sup.-1. Elemental analysis: calcd. C 41.38, H 4.86, N 9.65, found: C 41.25, H 4.80, N 952. IR (nujol suspension): 3158, 2684, 2624, 2575, 2408, 1732, 1657, 1563, 1404, 1379, 1246, 1161, 1044, 1033, 978, 891, 868, 843, 779, 752, 727, 604 cm.sup.-1.
trans isomer .sup.1 H NMR (D.sub.2 O): .delta.2.88 (t, J =8.5); another signal is hidden under the HDO signal (approx. 4.77). In D.sub.2 O/DCl: 5.12 (t, 2H, J =8.5), 3.08 (t, 2H, J =8.5). IR (KBr): 3426, 3013, 1744, 1628, 1406, 1260, 997, 808, 776 cm.sup.-1.
EXAMPLE 7
Preparation of cis-Azetidine-2,4-dicarboxylic acid di-t-butylester
To 1.45 g (5mmol) of 2,4-dibromoglutaric acid in 1.5 ml of ether is added at -78.degree. C. 1.5 ml of liquefied isobutene, followed by 3 drops of conc. sulfuric acid. The solution is stirred at room temperature in a screw-capped vial (slight pressure formation; use adequate shielding) for 22 h, cooled, and poured into a mixture of saturated aq. NaHCO.sub.3 and ether. The organic phase is dried over MgSO.sub.4 and chromatographed on SiO.sub.2 (ethyl acetate (EtOAc)/hexanes 1:5). Following impure di-tert.butyl ester, two side products are eluted. The crude 2,4-dibromoglutaric acid di-tert.butyl ester is again chromatographed on SiO.sub.2 (EtOAc/hexanes 1:25) to remove slightly more polar minor impurities, resulting in 0.97 g (48%) of a colorless oil. .sup.1 H NMR (CDCl.sub.3): dl isomer: .delta.4.38 (dd, 2H, J=6.5, 8) 2.56 (dd, 2H, J=6.5, 8); meso isomer: .delta.4.28 (t, 2H, J =7.5) 2.77 (dt 1H, J=15 (d), 7 (t)), 2.55 (dt, 1H, J=14.5 (d), 7.5 (t)); both .delta.1.49 (s, 18H). dl/meso =1:2. IR (neat): 2980, 2934, 1733, 1478, 1458, 1424, 1395, 1370, 1302, 1260, 1223, 1146, 1036, 969, 843, 761 cm.sup.-1.
cis- N-Benzylazetidine-2,4-dicarboxylic acid di-tert.butyl ester is obtained by warming 958 mg (2.38 mmol) of 2,4-dibromoglutaric acid di-tert.butyl ester and 0.79 ml (7.2 mmol) of benzylamine in 18 ml of DMF to 80.degree. C. for 4 h.
The solvent is distilled into a cold trap at 0.2-0.3 torr/40.degree. C. bath temp; the residue taken up in CH.sub.2 Cl.sub.2, and the solution washed with saturated aq. NaHCO.sub.3. After drying over MgSO.sub.4 and evaporation, the residue is chromatographed using a silica gel column to yield, after a forerun, first the trans, and then the cis isomer of N-benzylazetidine-2,4-dicarboxylic acid di-tert-butyl ester.
trans isomer: 295 mg (36%). mp. 46.degree.-48.degree. C. .sup.1 H NMR (CDCl.sub.3): .delta.7.34-7.18 (m, 5H), 4.07 (t, 2H, J =7), 3.95, 3.88 (AB system, 2H, J =13), 2.41 (t, 2H, J =7), 1.41 (s, 18H). IR (neat): 3064, 3030, 2978, 2933, 983, 912, 840, 735, 700 cm.sup.-1.
cis isomer: 316 mg (38%). mp. 53.degree.-56.degree. C. .sup.1 H NMR (CDCl.sub.3): .delta.7.34-7.19 (m, 5H), 3.84 (s, 2H), 3.44 (t, 2H, J=8), 2.39 (dt, 1H, J =11 (d), 8.5 (t)), 2.23 (dt, 1H, J=10.5 (d), 8 (t)), 1.37 (s, 18H). IR (neat): 3063, 2978, 2932, 1735, 1495, 1479, 1455, 1393, 1368, 1233, 1156, 1061, 1019, 994, 915, 845, 737, 705 cm.sup.-1.
307 mg (884 .mu.mol) of cis-N-benzylazetidine-2,4-dicarboxylic acid di-tert-butyl ester in 25 ml of methanol is hydrogenated in a Parr shaker over 100 mg of 20% Pd(OH).sub.2 /C (containing 31% water) under 4 atm of hydrogen for 7.5 h. The catalyst is filtered off through a double paper filter and thoroughly washed with methanol. Evaporation and filtration over SiO.sub.2 (EtOAc/hexanes 1:1) affords 149 mg (66%) of cis-azetidine-2,4-dicarboxylic acid di-tert.butyl ester as a yellowish oil which solidifies spontaneously. mp. 47.degree.-48.5.degree. C. .sup.1 H NMR (CDCl.sub.3): .delta.4.04 (dd, 2H, J =5.5, 9), 3.2 (br., 1H), 2.97 (dt, 1H, J =12 (d), 9 (t)), 2.38 (dt, 1H, J =12 (d), 5.5 (t)), 1.48 (s, 18H). IR (neat): 3328, 2979, 2934, 1731, 1480, 1458, 1394, 1369, 1280, 1257, 1156, 1103, 1048, 1031, 892, 846, 734 cm.sup.-1.
EXAMPLE 8
Preparation of cis-N-Acetylazetidine-2.4-dicarboxylic acid
To 29.2 mg (0.2 mmol) of cis-azetidine-2,4 dicarboxylic acid and 65 .mu.l (0.8 mmol) of pyridine in 0.5 ml of water is added with stirring in several portions within 1.5h 150 .mu.l (1.6 mmol) of acetic anhydride. After 3.5 h at room temperature, the 50W-X8 resin (H.sup.+ form, washed with 10% HCl and distilled water before use) to remove pyridine (elution with water). The crude material obtained after evaporation is chromatographed on SiO.sub.2 (isopropanol/water/acetic acid 14:5:1) and evaporated. The product holds acetic acid tenaciously; after drying at 100.degree. C. (0.1 torr, overnight), 35.5 mg of an amber glass is obtained which still contains 4% of acetic acid (corrected yield: 91%). .sup.1 H NMR (D.sub.2 O):.delta.5.06 (dd, 2H, J =5.5, 10), 3.07 (dt, 1H, J=12(d), 10 (t)), 2.43 (dt, 1H, J= 12(d), 5.5(t)), 1.96 (s,3H).
EXAMPLE 9
Preparation of N-(cis-4-Carboxyazetidine-2-carbonyl)glycine
To 80.5 mg (0.2 mmol) of cis-N-benzylazetidine- 2,4-dicarboxylic acid benzyl methaneselenol ester, and 74.2 mg (0.22 mmol) of glycine benzyl ester tosylate in 1 ml of THF is added 84 .mu.l (0.6 mmol) of triethylamine, followed by 45.2 mg (0.22 mmol) of cuprous bromidedimethylsulfide complex. After stirring for 2 h at room temperature, the suspension is diluted with is evaporated and chromatographed on SiO.sub.2. EtOAc/hexanes (1:6) elutes 37.5 mg (46%) of the starting selenol ester cis-N-benzylazetidine-2,4-dicarboxylic acid benzyl methaneselenol ester, followed by 1.2 mg of cis-N-benzylazetidine-2,4-dicarboxylic acid dibenzyl ester. An unidentified impurity is eluted with EtOAc/hexanes (1:2), and finally the title compound with EtOAc/hexanes (2:1). Evaporation and drying in vacuo yields 25.0 mg (26%) of N-[cis-N-benzyl-4-(benzyloxycarbonyl)azetidine-2-carbon-yl]glycine benzyl ester as a colorless syrup. .sup.1 H NMR (CDCl.sub.3): .delta.7.72 (br. t, 1H, J =5.5), 7.4-7.2 (m, 15H), 5.17 5.04 (narrow AB system, 2H), 3.97, 3.84 (AB system, 2H, J=18.5, both parts split into doublets with J=5.5), 3.78, 3.73 (AB system, 2H, overlapping), 3.80 (t, 1H, overlapping), 3.67 (t, 1H, J=8.5), 2.67 (dt, 1H, J =11(d), 8.5 (t)), 2.23 (dt, 1H, J=11 (d), 8 (t)). IR (neat): 3371, 3064, 3032, 2923, 2851, 1746, 1679, 1520, 1498, 1456, 1390, 1357, 1262, 1188, 1063, 1029, 741, 698 cm.sup.-1.
18.5 mg (39 .mu.mol) of N-[cis-N-benzyl-4-(benzyloxycarbonyl)azetidine-2-carbonyl]glycine benzyl ester in 10 ml of methanol is hydrogenated in a Parr shaker over 34.5 mg of 20% Pd (OH).sub.2 /C (containing 31 % of water) under 4 atm of hydrogen for 4h, 5 ml of water is added, the catalyst is removed by filtration through a double paper filter and washed thoroughly with methanol/water (2:1), and the solution is evaporated. Filtration over SiO.sub.2 (isopropanol/water/conc ammonia 14:5:1), evaporation and drying in a vacuum desiccator over P.sub.2 O.sub.5 and KOH affords 6.7 mg (85%) of N-(cis-4-carboxyazetidine-2-carbonyl)glycine as a colorless glass. .sup.1 H NMR (D.sub.2 O):.delta.4.98 (t, 1H, J =8.5), 4.73 (t, 1H, overlapping), 3.86, 3.74 (AB system, 2H, J =17.5), 3.08 (dt, 1H, J =11.5 (d), 10 (t)), 2.55 (dt, 1H, J =I2 (d), 8.5 (t)).
Mode of Preparation of the mono- and diamide of cis-Azetidine-2,4-dicarboxylic acid
The following is a diagram for preparing monoand diamide of cis-azetidine-2,4-dicarboxylic acid: ##STR32##
EXAMPLE 10
Preparation of cis-1-Benzyl-4-carbamoylazetidine-2-carboxylic acid benzyl ester and cis-1-Benzylazetidine-2,4-dicarboxamide.
To 108 mg (2 mmol) of finely powdered ammonium chloride is added, under a dry argon atmosphere via syringe at 0.degree. C., 1 ml of trimethylaluminum (2 M in toluene). After stirring at 0.degree. C. for 20 minutes and at room temperature for 1 hour, a solution of 623 mg (1.5 mmol) of N-benzylazetidine-2,4-dicarboxylic acid dibenzyl ester in 1 ml of toluene is added via syringe, and the mixture stirred at 50.degree. C. for 2.5 hours. The mixture is then cooled in an ice bath, diluted with 10 ml of methylene chloride, and quenched by addition of 0.84 g (20 mmol) of NaF, followed by cautious dropwise addition of 0.36 ml (20 mmol) of water. After gas evolution has ceased, stirring is continued at room temperature for 20 minutes, and the fluoroaluminates are removed by suction filtration through a medium porosity sintered glass funnel and thoroughly washed with methylene chloride. Evaporation to a small volume and dilution with ethyl acetate/hexanes (1:5) causes preciptation of cis-N-benzylazetidine-2,4-dicarboxamide, which is collected on a sintered glass funnel and washed with the same solvent, to yield 80 mg (23%) of cis-N-benzylazetidine-2,4-dicarboxamide as a colorless solid. mp. 209.degree.-212.degree. C. (dec.) after filtration over SiO.sub.2 (ethyl acetate/methanol 4:1). .sup.1 NMR (CDCl.sub.3): .delta.7.4- 7.25 (m, 5H), 6.29 (br. s, 2H), 5.30 (br. s, 2H), 3.76 (s, 2H), 3.72 (t, 2H, J=8.5), 2.89 (dt, 1H, J=9 (t), 11.5 (d)), 2.17 (dt, 1H, J=8.5 (t), 11.5 (d)). IR (nujol): 3474, 3409, 3318, 3156, 1673, 1647, 1570, 1418, 1323, 1291, 1148, 1055, 762, 708 cm.sup.-1.
The mother liquid and washings are combined, evaporated, and chromatographed on silica gel to yield, in the sequence of elution, unreacted starting material (with ethyl acetate/hexanes 1:3; 22 mg, 43%), benzyl alcohol, and cis-1-benzyl-4-carbamoylazetidine-2-carboxylic acid benzyl ester (II) (with ethyl acetate), yielding 56 mg (11.5%) as a highly viscous film which partially solidifies on standing. .sup.1 H NMR (CDCl.sub.3): .delta. 7.4-7.2 (m, 10H), 6.95 (br. s, 1H), 5.58 (br. s, 1H), 5.07 (s, 2H), 3.82, 3.71 (AB system, 2H, J=13), 3.80 (t, 1H, J=8.5), 3.61 (t, 1H, J=8.5), 2.70 (dt, 1H, J=8.5 (t), 11 (d)), 2.25 (dt, 1H, J=8 (t), 11 (d)). IR (neat): 3438, 3063, 3031, 2961, 2925, 2892, 1742, 1686, 1580, 1497, 1455, 1175, 1061, 1022, 747, 698 cm.sup.- 1.
EXAMPLE 11
Preparation of cis-4-Carbamoylazetidine-2-carboxylic acid
53 mg (163 umol) of cis-1-benzyl-4-carbamoylazetidine-2-carboxylic acid benzyl ester (II) in 10 ml of methanol are hydrogenated in a Parr shaker over 18 mg of 10% Pd/C under a hydrogen pressure of 3.3 atm for 3 hours. 10 ml of water is added, the mixture filtered through a fluted filter, and the catalyst washed with several portions of methanol. Evaporation and drying in vacuo (0.1 torr, 4 hours) affords 22.6 mg (96%) of a colorless glass. .sup.1 H NMR (D.sub.2 O): .delta. 5.00 (t, 1H, J=9), 4.75 (t, 1H, J=9), 3.14 (dt, 1H, J=10 (t), 12 (d)), 2.52 (dt, 1H, J=8(t), 12 (d)). IR (nujol): 3399, 3141, 3141, 3019, 2537, 2409, 1688, 1615, 1563, 1391, 1294, 1217, 1165, 1115, 1048, 968, 887, 810, 762, 696, 637 cm.sup.-1. Mass Spectra: El: m/z 142(M-2H, 40%), 100 (100), 91 (14), 82 (27), 68 (28), 54 (50), 44 (95); CI (isobutane): m/z 145 (M+ H, 100%), 126 (7), 100 (24), 99 (15), 84 (15), 72 (22).
EXAMPLE 12
Preparation of cis-Azetidine-2,4-dicarboxamide
45.8 mg (196 .mu.mol) of cis-N-benzylazetidine-2,4-dicarboxamide in 10 ml of glacial acetic acid (no reaction occurs in methanol) are hydrogenated in a Parr shaker over 11 mg of 10% Pd/C under a hydrogen pressure of 4 atm for 7 hours. The mixture is filtered through a fluted filter and the catalyst washed with several portions of methanol. Evaporation and drying in vacuo yields a colorless film which crystallizes slowly on addition of a few drops of methanol. After standing in a freezer overnight, the crystals are isolated, washed with a small volume of ethyl acetate/methanol (5:1), and dried in vacuo; yield 16.7 mg (60%). Mp. 215.degree.-218.degree. C. (dec.) after strong shrinking and darkening from approx. 200.degree. C. on (measured with oil bath preheated to 190.degree. C.). .sup.1 H NMR (D.sub.2 O): .delta. 4.29 (t, 2H, J=8.5), 2.98 (dt, 1H, J=9 (t), 11 (d)), 2.23 (dt, 1H, J=8 (t), 11.5 (d)). IR (nujol): 3391, 3335, 3208, 3073, 1683, 1669, 1281, 1211, 1109, 1030, 843, 749, 722, 677 cm.sup.-1. Mass Spectrum: CI(isobutane): m/z 144 (M+H, 100%), 99 (56).
Mode of Preparation of cis-Azetidine-2.3-dicarboxylic acid and its Diamide
The following is a diagram for preparing cis-azetidine-2,3-dicarboxylic acid and its diamide. ##STR33##
EXAMPLE 13
Preparation of cis-N-(Benzyloxycarbonyl)azetidine-2.3-dicarboxylic acid;
A solution of pyridine (4.0 g., 0.050 mol) and NaBH.sub.4 (2.0 g, 0.026 mol) in 20 ml of absolute ethanol is treated with benzyl chloroformate (8.5 g, 0.05 mol) over about 1 hour followed by stirring for 1.5 hour at -78.degree. C. The mixture is added to ice-water and extracted with ether, and the extract is washed with 3% HCl solution and 3% NaOH solution. After the extract is dried and evaporated, 8.0 g of N-benzyloxycarbonyl1,2-dihydropyridine is obtained in 74% yield. A 5% solution of N-benzyloxycarbonyl-1,2-dihydropyridine is then irradiated using a Rayonet photochemical reactor (RP-300 lamps) until NMR spectrum shows consumption of the starting materials. The solvent is evaporated to give the crude product, N-(benzyloxycarbonyl)-2-azabicyclo[2.2.0]hex-5-ene (VIII). Pure (VIII) is obtained by passing the crude product through basic alumina with ether. (VIII) is then reacted with ozone in methanol at -78.degree. C. followed by reaction with peroxyacetic acid to form cis-N-(benzyloxycarbonyl)-azetidine-2,3- dicarboxylic acid.
EXAMPLE 14
Preparation of cis-Azetidine-2.3-dicarboxylic acid
cis-N-(Benzyloxycarbonyl) azetidine-2,3-dicarboxylic acid, 10% Pd/C and methanol are hydrogenated under 3 atm of hydrogen in a Parr shaker for 4 hours. The catalyst is filtered off and thoroughly washed. The solution is evaporated to dryness and dried in vacuo to arrive at cis-azetidine2,3-dicarboxylic acid.
EXAMPLE 15
Preparation of cis-azetidine-2,3-dicarboxamide
A solution of cis-N-(Benzyloxycarbonyl) azetidine 2,3-dicarboxylic acid and, e.g., ammonia are reacted with [(CH.sub.3).sub.2 N].sub.3 PCl.sub.2 to form the cis-N(benzyloxycarbonyl)azetidine-2,3-dicarboxamide (XI), as set forth by Appel et al., Chem. Ber. 116, 2037-2040 (1983). XI, 10% Pd/C and methanol are hydrogenated in a Parr shaker under 3 atm of hydrogen, for 4 hours. The catalyst is filtered off and thoroughly washed. The solution is evaporated to dryness and dried in vacuo to arrive at cis-azetidine-2,3-dicarboxamide.
Preparation of the various ester derivatives of the cis-azetidine-2,3-dicarboxylic acids and N-substituted cis-azetidines can be accomplished by conventional esterification reactions utilizing lower alcohols and as per se known in the art or described hereinabove.
Mode of Preoaration of cis-4-(Hydroxymethyl)-azetidine-2-carboxylic acid(ester)
The following is a diagram of the steps used in preparing cis-4-(hydroxymethyl)azetidine-2-carboxylic acid (ester) derivatives ##STR34##
EXAMPLE 16
Preparation of N-Benzylazetidine-2,4-dicarboxylic acid benzyl methaneselenol ester, and N-Benzylazetidine-2,4-dicarboxylic acid bis(methaneselenol) ester.
0.82 g (10.4 mmol) of selenium powder is dried at 140.degree. C./0.1 torr for 1.5 h. Under an argon atmosphere, 5 ml (10 mmol) of trimethylaluminum (2M in toluene) is added, and the mixture is refluxed for 3 h. After cooling and settling of the heterogeneous mixture, 3 ml of the supernatant solution is taken up in a syringe and added, with ice cooling and under argon to a solution of 1.66 g (4 mmol) of cis-N-benzylazetidine-2,4-dicarboxylic acid dibenzyl ester in 5 ml of dry (P.sub.2 O.sub.5) methylene chloride. After 1.5 h at 0.degree. C., TLC (SiO.sub.2, EtOAc/hexanes 1:5) indicates the presence, in order of increasing polarity, of cis-N-benzylazetidine-2,4-dicarboxylic acid bis (methaneselenol) ester, cis-N-benzylazetidine-2,4-dicarboxylic acid benzyl methaneselenol ester, cis-N-benzylazetidine-2,4dicarboxylic acid dibenzyl ester, and benzyl alcohol. The mixture is hydrolyzed cautiously at 0.degree. C. by slow and dropwise addition of 2.5 ml of saturated aqueous sodium sulfate solution (vigorous methane evolution). Stirring is continued for 5 min. at r.t., sufficient MgSO.sub.4 is added to bind the water and stirring continued for another 5 min. Solids are filtered off through a medium porosity sintered glass funnel and thoroughly washed with dry CH.sub.2 Cl.sub.2. After evaporation, the residue is chromatographed on silica gel (EtOAc/hexanes 1:5) with application of slight pressure to ensure reasonably rapid elution of the hydrolytically sensitive selenol ester. There is obtained, in order of elution: (a) 0.28 g of a mixture of cis-N-benzylazetidine-2,4-dicarboxylic acid benzyl methanselenol ester and cis-N-benzylazetidine-2,4-dicarboxylic acid bis (methaneselenol) ester, (b) 0.76 g (47%) of pure N-benzylazetidine-2,4-dicarboxylic acid benzyl methaneselenol ester; (c) 0.25 g (15%) of starting material cis-N-benzylazetidine-2,4-dicarboxylic acid dibenzyl ester, which can be recycled. cis-N-benzylazetidine2,4-dicarboxylic acid benzyl methanselenol ester: viscous oil which solidifies on standing; mp. 56.5.degree.-58.degree. C. .sup.1 H NMR (CDCl.sub.3): .delta. 7.37-7.20 (m, 10H), 4.97 (narrow AB system, 2H), 4.00, 3.70 (AB system, 2H, J=13), 3.80 (t, 1H, J=8), 3.73 (t, 1H, J=8.5), 2.53 (dt, 1H, J=11 (d), 8.5 (t)), 2.32 (dt, 1H, J=11 (d), 7.5 (t)), 2.09 (s, 3H). IR (neat): 3063, 3031, 2930, 2882, 1744, 1694, 1495, 1455, 1271, 1215, 1169, 1055, 747, 696 cm.sup.-1 cis-N-benzylazetidine-2,4dicarboxylic acid bis (methaneselenol) ester: viscous oil which solidifies on standing; mp. 68.5.degree.-71.5.degree. C. .sup.1 H NMR (CDCl.sub.3): .delta. 7.4-7.2 (m, 5H), 3.89 (s, 2H), 3.81 (t, 2H, J=8), 2.57 (dt, 1H, J=11 (d), 8.5 (t)), 2.11 (dt, 1H, overlapping), 2.10 (s, 6H). IR (neat): 3063, 3029, 2930, 2870, 1694, 1495, 1269, 1161, 1123, 1026, 866, 762, 710 cm.sup.1.
EXAMPLE 17
Preparation of N-Benzyl-4-(hydroxymethyl)-azetidine-2-carboxylic acid benzyl ester.
324 mg (0.8 mmol) of cis-N-benzylazetidine-2,4-dicarboxylic acid benzyl methaneselenol ester is dissolved in 2 ml of methylene chloride and diluted with 30 ml of absolute ethanol. 3 portions of 91 mg (2.4 mmol) each of NaBH.sub.4 are added at 0.degree. C. in intervals of 20 min., and stirring at 0.degree. C. is continued for another 40 min. The mixture is hydrolyzed by cautious addition of 1 ml of acetic acid (vigorous hydrogen evolution). 6 g of silica gel is added, the solvent evaporated, and the residue placed on top of a SiO.sub.2 column. A small amount of benzyl alcohol formed as a byproduct is first eluted with EtOAc/hexanes (1:2), and then N-benzyl-4-(hydroxymethyl)azetidine2-carboxylic acid benzyl ester is eluted with EtOAc/hexanes (1:1). 179 mg (72%) of a colorless oil is obtained. .sup.1 H NMR (CDCl.sub.3): .delta. 7.37-7.25 (m, 10H), 5.09 (s, 2H), 3.86, 3.65 (AB system, 2H, J=12.5), 3.73 (t, 1H, J=8.5) 3.39 (tt, 1H, J=8, 2.5) 3.24-3.13 (m, 2H), 2.58 (br. dd, J=11.5, 2), 2.35 (dt, 1H, J=11 (d), 8 (t)), 2.27 (dt, 1H, J=11 (d), 8 (t)). IR (neat): 3401, 3065, 3031, 2924, 2872, 1742, 1613, 1497, 1455, 1395, 1216, 1177, 1094, 1028, 749, 698 cm.sup.1. MS: m/z 311 (M.sup.+, 0.1%), 309 (0.2), 293 (0.3), 292 (0.2), 280 (33), 176 (26), 91 (100). C.sub.19 H.sub.20 NO.sub.2 (M.sup.+ -OH) calcd. 294.1494, found 294.1495.
EXAMPLE 18
Preparation of 4-(Hydroxymethyl)azetidine-2-carboxylic acid.
179 mg (0.575 mmol) of N-benzyl-4-(hydroxymethyl)azetidine-2-carboxylic acid benzyl ester and 60 mg of 20% Pd(OH).sub.2 /C (containing 31% of water) in 30 ml of methanol are hydrogenated in a Parr shaker under 4 atm of hydrogen for 3.5 h. The catalyst is filtered off through a double paper filter and washed with methanol, the solution evaporated, and the residue chromatographed on silica gel (isopropanol/water/conc. NH.sub.3 14:5:1) to yield, after evaporation and drying i.v., 53.6 mg (71%) of 4-(hydroxymethyl)azetidine-2-carboxylic acid as a colorless solid. .sup.1 H NMR (D.sub.2 O): .delta. 4.71 (t, 1H, J=9), 4.56 (m, 1H), 3.80 (m, 2H), 2.81 (dt, 1H, J=12 (d), 9.5 (t)), 2.43 (dt, 1H, J=12.5 (d), 8.5 (t)). IR (nujol): 3239, 3146, 2589, 2438, 1620, 1565, 1410, 1327, 1302, 1188, 1099, 1038, 999, 885, 828, 758, 725 cm.sup.-1.
EXAMPLE 19
Preparation of N-Benzyl-4-(hydroxymethyl)-azetidine-2-carboxylic acid ethyl ester
This compound is obtained, as the result of a concomitant transesterification, when NaBH.sub.4 reduction is applied directly to cis-N-benzylazetidine-2,4-dicarboxylic acid dibenzyl ester, which may serve as an internediate on the way to N-benzyl-4-(hydroxymethyl)azetidine-2-carboxylic acid ethyl ester. To 104 mg (0.25 mmol) of cis-N-benzylazetidine-2,4-dicarboxylic acid dibenzyl ester in 2.5 ml of absolute ethanol is added, at r.t. in small portions over a period of 1 h, 45 mg (1.25 mmol) of NaBH.sub.4. Quenching with 50 .mu.l of acetic acid is followed by filtration through a medium porosity sintered glass funnel, repeated washing of the solids with methylene chloride, and evaporation. The residue is chromatographed on SiO.sub.2 ; EtOAc/hexanes (1:3) elutes benzyl alcohol, EtOAc/hexanes (1:1) the product. The product is a colorless oil obtained in 39.8 mg (64%) yield. .sup.1 H NMR (CDCl.sub.3): .delta. 7.35-7.25 (m, 5H), 4.09 (q, 2H, J=7), 3.84, 3.67 (AB system, 2H, J=12.5), 3.66 (t, 1H, J=8.5), 3.38 (tt, 1H, J=8, 2.5), 3.25-3.15 (m, 2H), 2.81 (br., 1H), 2.37-2.21 (m, 2H), 1.20 (t, 3H, J=7). IR (neat): 3432, 3063, 2928, 2870, 1736, 1495, 1455, 1194, 1096, 1034, 961, 739, 704 cm.sup.-1.
Preparation of cis-4-(Dialhoxymethyl)azetidine-2-carboxylic acid
The following is a flow diagram of preparing the cis-4-(Dialhoxymethyl)azetidine-2-carboxylic acid derivatives. ##STR35##
EXAMPLE 20
Preparation of cis-4-(Dimethoxymethyl)azetidine-2-carboxylic acid
To 64 .mu.l (0.73 mmol) of oxalyl chloride in 1 ml of methylene chloride is added, dropwise at -60.degree. C., 106 .mu.l (1.5 mmol) of dimethyl sulfoxide in 0.5 ml of methylene chloride. After stirring at -60.degree. C. for 5 min., 45.7 mg (147 .mu.mol) of cis-N-benzyl-4-(hydroxymethyl)azetidine-2-carboxylic acid benzyl ester in 0.7 ml of methylene chloride is added dropwise, and the mixture is stirred at -60.degree. C. for 30 min. 0.63 ml (4.5 mmol) of triethylamine is added, and after another 10 min. at -60.degree. C. the mixture is quenched with water. Aqueous workup (sat. aq. NaHCO.sub.3 /methylene chloride), drying over MgSO.sub.4, evaporation, and filtration over SiO.sub.2 (EtOAc/hexanes 1:1) afford 41.6 mg of the very impure crude product cis-N-benzyl-4-formylazetidine2-carboxylic acid benzyl ester, which is difficult to purify in quantity, but can be used as such in the following step.
An analytical sample is obtained by HPLC (2 Waters 501 HPLC pumps with automated gradient controller and model 441 absorbance detector operating at 254 nm; Waters Millipore.RTM. 24.times.1 cm .mu.Porasil.TM. column; EtOAc/hexanes 35:65, 5 ml/min., retention time 4.8 min.) as a colorless oil. .sup.1 H NMR (CDCl.sub.3): .delta. 9.24(d,1H,J=3), 7.40-7.18 (m,10 H), 5.16 (s,2H), 4.07, 3.57 (AB system, 2H, J=12.5), 3.84 (t, 1H, J=8.5), 3.50 (dt, 1H, J=3(d), 8.5 (t)), 2.49-2.34 (m,2H). IR (neat): 3063, 3031, 2886, 1743, 1732, 1497, 1455, 1354, 1267, 1175, 1055, 750, 698 cm.sup.-1. MS (EI): m/z 280 (M--CHO, 31%), 91 (91%), 57 (100%); (CI, isobutane): m/z 310 (M+H, 100%), 280 (32%), 91 (35%).
28 .mu.l (0.36 mmol) of formamide and 34 .mu.l (0.36 mmol) of dimethyl sulfate are stirred for 2 h in a screw-capped vial at 80.degree.-85.degree. C. After cooling, 16.mu.l (0.4 mmol) of methanol is added, followed by 37.5 mg (121 .mu.mol) the above crude aldehyde, cis-N-benzyl-4-formylazetidine-2-carboxylic acid benzyl ester. This mixture is stirred at room temperature for 5 h and then chromatographed on SiO.sub.2 (previously treated with 10 weight % of conc. aq. ammonia; EtOAc/hexanes 1:6). After elution of a forerun and evaporation, 15.5 mg (30% from the alcohol) of cis-N-benzyl-4-(dimethoxymethyl)azetidine-2-carboxylic acid benzyl ester is obtained as a colorless oil. .sup.1 H NMR (CDCl.sub.3): .delta.7.4-7.2(m, 10 H), 5.04, 4.96 (AB system, 2H, J=12.5), 4.27 (d, 1H, J=6.5), 3.90, 3.70 (AB system, 2H, J= 13), 3.59 (t, 1H, J=8.5), 3.36 (s, 3H), 3.35 (s, 3H), 3.25 (dt, 1H, J =6.5 (d), 8 (t)), 2.19 (t, 2H, J=8).
12.5 mg (35 .mu.mol) of cis-N-benzYl-4-(dimethoxymethyl)azetidine-2-carboxylic acid benzyl ester in 10 ml of methanol is hydrogenated in a Parr shaker over 53 mg of 20% Pd(OH)2/C (containing 31% water) under 4 atm of hydrogen for 3 h. Filtration through a double paper filter, repeated washing with methanol and evaporation gives a crude product which is filtered over silica gel with isopropanol/water/conc. ammonia (17:2:1) to yield 3.0 mg (49%) of cis-4(dimethoxymethyl)azetidine-2-carboxylic acid as a colorless glass. .sup.1 H NMR (D.sub.2 O): .delta.4.73 (d, 1H, overlapping), 4.72 (t, 1H, overlapping), 3.53 (s, 3H), 3.50 (s, 3H), 2.80 (dt, 1H, J=12 (d), 9.5(t)), 2.50 (dt, 1H, J=12(d), 8(t)).
IN VITRO AND IN VIVO STUDIES
For ease of discussing and reporting the biological data, the following abbreviations have been devised for representative compounds of this invention.
Az1 cis-azetidine-2,4-dicarboxylic acid t-butyl methyl ester
Az2 trans-azetidine-2,4-dicarboxylic acid-t-butyl methyl ester
Az3 cis-azetidine-2,4-dicarboxylic acid mono-t-butyl ester
Az4 trans-azetidine-2,4-dicarboxylic acid mono-t-butyl ester
Az5 cis-azetidine-2,4-dicarboxylic acid
Az6 trans-azetidine-2,4-dicarboxylic acid
Az7 cis-azetidine-2,4-dicarboxylic acid dimethyl ester
Az8 cis-4-carbamoylazetidine-2-carboxylic acid
Az9 cis-azetidine-2,4-dicarboxamide
Az10 cis-N-benzylazetidine-2,4-dicarboxylic acid
Az11 cis-azetidine-2,4-dicarboxylic acid methyl ester
Az12 L-2-azetidinecarboxylic acid (known)
Az13 cis-4-(hydroxymethyl)azetidine-2-carboxylic acid
Characterization of the experimental model
Studies described hereinbelow on the usefulness of the compounds were performed on primary cultures of cerebellar granule cells prepared from 8-day old rats according to the procedures as set forth in Wroblewski et al., Neuropharmacology 24(9), 919-921 (1985). These cultures are characterized by the presence of four subtypes of excitatory amino acid receptors, classified on the basis of the coupling of their recognition sites to specific signal transduction systems, as well as according to the pharmacology of their recognition sites. The classification distinguishes between excitatory amino acid recognition sites coupled with PI hydrolysis (G.sub.p) and those coupled with the activation of Ca.sup.2+ (G.sub.C). This is disclosed in greater detail in Wroblewski et al. Proc. Natl. Acad. Sci. 184(14), 5068-5072 (1987) G.sub.P1 and G.sub.C1 receptors are activated by glutamate, aspartate, N-methyl-D-aspartate (NMDA), antagonized by 2-amino-5-phosphonovalerate (APV), and inhibited by Mg.sup.2+ . These two receptor subtypes correspond to the NMDA receptor, however, two distinct receptors must be postulated on the basis of their signal transducing mechanisms. The G.sub.P2 receptor is activated by glutamate and quisqualate, and is insensitive to Mg.sup.2 + and APV. The G.sub.C2 receptor is activated by kainate and preferentially antagonized by cis-piperidine-2,3-dicarboxylate. The G.sub.C2 receptor is also insensitive to Mg.sup.2+ inhibition. These two receptors correspond to the quisqualate and kainate receptors, respectively.
Cis-azetidine-2,4-dicarboxylic acid and derivatives selectively potentiate signal transduction at the G.sub.C1 and G.sub.P1 receptors. Referring to Table 1 the influx of calcium stimulated by NMDA, GLU, ASP and QUIS acting at the G.sub.C1 receptor is potentiated by cis-azetidine-2,4-dicarboxylic acid, while the action of KAIN at the G.sub.C2 receptor is not affected.
TABLE 1______________________________________Cis-azetidine-2,4-dicarboxylic acid potentiates calciuminflux stimulated by excitatory amino acid receptoragonistsAgonist 20 .mu.M Percent Activity______________________________________NMDA 188Glutamate (GLU) 169Aspartate (ASP) 144Quisqualate (QUIS) 124Kainate 104______________________________________
Stereoselectivity of the action of azetidines
TABLE 2______________________________________Action of cis and trans stereoisomers of azetidines oncalcium influx and PI hydrolysis stimulated byexcitatory amino acids Calcium Influx PI hydrolysis % of stimulation byAzetidine 10 .mu.M GLU 20 .mu.M GLU______________________________________Az1 (cis) 107 117Az2 (trans) 104 --Az3 (cis) 141 125Az4 (trans) 100 109Az5 (cis) 207 124Az6 (trans) 103 83*______________________________________ As disclosed above, the cis isomers are particularly more active than the trans. *This value shows antagonistic activity.
Structure-activity relationships
TABLE 3______________________________________Potentiation of calcium influx by cis-azetidinesAzeti-dine Group 1 Group 2 agonist % activity______________________________________Az1 .sub.- t-butyl ester methyl ester Glu 10 .mu.M 107Az3 .sub.- t-butyl ester acid Glu 10 .mu.M 141 Glu 20 .mu.M 136Az5 acid acid Asp 10 .mu.M 196 Asp 20 .mu.M 141 Glu 10 .mu.M 207 Glu 20 .mu.M 157Az7 methyl ester methyl ester Glu 50 .mu.M 68Az11 methyl ester acid Glu 10 .mu.M 122Az8 acid amide Asp 20 .mu.M 150Az9 amide amide Asp 20 .mu.M 106Az12 acid H Glu 20 .mu.M 110Az10 (N-benzyl) acid acid Asp 10 .mu.M 121Az13 alcohol acid Asp 10 .mu.M 159______________________________________
While not being bound to any theory, the cis-azetidines and derivatives thereof are believed to act by increasing the potency but not the efficacy of GLU action at the G.sub.C1 receptor.
Inhibition of Phencyclidine Action by cis-Azetidines
Phencyclidine (PCP), once used as an anaesthetic, produces strong psychotropic effects manifested in humans by a state of sensory isolation, hallucinations and confusional states. It is one of the most widely abused drugs in the United States. One of the possible mechanisms of action of PCP involves the noncompetitive inhibition of the signal transduction at excitatory amino acid receptors, through allosteric interaction at a site distinct from the primary transmitter site (see Proc. Natl. Acad. Sci. USA discussed above). However, cis-azetidine-2,4-dicarboxylic acid exhibits the ability to decrease the action of PCP on ASP-stimulated signal transduction as shown in Table 4. This action occurs at a site different than the binding site for PCP, since cis-azetidine-2,4-dicarboxylic acid failed to displace the specific binding of [.sup.3 H]PCP in radioligand binding studies.
TABLE 4______________________________________cis-azetidine-2,4-dicarboxylic acid prevents theinhibition of ASP-stimulated calcium influx byPhencyclidineConcentration nmol .sup.45 Ca.sup.2+ /mg protein ASP 50 .mu.M+ ASP 50 .mu.M Az5 50 .mu.M______________________________________No PCP 31.3 .+-. 3.4 64.5 .+-. 2.6PCP 20 nM 23.0 .+-. 1.7 57.2 .+-. 3.2PCP 50 nM 9.2 .+-. 0.5 32.2 .+-. 1.3PCP 100 nM 0.7 .+-. 0.1 13.3 .+-. 0.3______________________________________
Azxetidines increase the potency but not the efficacy of GLU action at the G.sub.C1 receptor. When calcium influx stimulated by GLU is measured in the absence and presence of Az5, the EC.sub.50 for GLU is decreased by 50 .mu.M Az5 from 51 .mu.M to 7 .mu.M. This indicates a 7-fold potentiation of the action of GLU.
These data suggest that cis-azetidines may interact with a specific recognition site different from the GLU recognition site but located within the same receptor complex. This interaction results in the potentiation of the action of the primary transmitter (GLU or ASP). Since cis-azetidines are inactive in the absence of the primary transmitter, they serve a modulatory function. It is possible that cis-azetidines interact with a positive allosteric modulatory site of the glutamatergic receptors.
IN VIVO STUDIES
Characterization of the in vivo model
The action of the NMDA receptor subtype can be estimated in vivo by measuring the changes in the content of cyclic GMP (cGMP) in the rat cerebellum after intracerebroventricular injections of NMDA. The increases in cGMP content induced by NMDA are antagonized by APV indicating receptor selectivity. In terms of its pharmacology and of its mechanism of signal transduction the active receptor in the above conditions can be compared to the C.sub.C1 reporter measured in cultures of cerebellar granule cells. See Novelli et al., J. Neuroscience 7(1), 40-47 (1987) for a more detailed explanation of the procedure.
Action of the cis-azetidine derivatives in vivo
In two series of experiments rats were injected icv with NMDA together with or without cis-azetidine-2,4-dicarboxylic acid. The results of the measurements of cGMP content in the cerebellum are shown in Table 5.
TABLE 5__________________________________________________________________________TIME 10 min TIME 5 minInjection cGMP % Control Injection cGMP % Control__________________________________________________________________________Saline 7.8 + 1.9 100 Saline 4.8 + 0.8 100NMDA 1 .mu.g 10.5 + 2.3 135 NMDA 2 .mu.g 5.6 + 0.8 136Az5 10 .mu.g 14.9 + 1.3 192 Az5 2 .mu.g 6.8 + 0.8 142NMDA + Az5 16.5 + 2.8 211 NMDA + Az5 16.1 + 2.9 334__________________________________________________________________________
In both series of experiments cis-azetidine-2,4-dicarboxylic acid potentiated the action of the endogenous receptor agonist as well as the action of injected NMDA.
The compounds of the present invention may be made into pharmaceutical compositions by combination with appropriate medical carriers or diluents, and may be formulated into preparations in solid, semisolid, liquid or gaseous form such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, aerosols in usual ways for oral or parenteral administration. The following methods and excipients are merely exemplary and are in no way limiting.
In pharmaceutical dosage forms, the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
In the case of oral preparations, the compounds may be used alone or combined with appropriate additives to make tablets, powders, granules or capsules, e.g. with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
Furthermore,they may be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
The compounds of the present invention may be formulated into preparations for injections by dissolving, suspending or emulsifying them in aqueous or non-aqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
In case of inhalations or aerosol preparations, the compounds of the invention in the form of a liquid or minute powder may be filled up in an aerosol container with gas or liquid spraying agents, and if desired, with conventional adjuvants such as humidifying agents added. They may also be applied as pharmaceuticals for non-pressurized preparation such as in a nebulizer or an atomizer.
The desirable dose of the azetidine derivatives of the present invention varies with the subject, drug form, method and period of administration. However, in order to obtain desirable effects, generally it is recommended to administer 0.1 mg to 100 mg/kg body weight of the compounds of the present invention. In terms of composition, compounds should be present between 0.1 to 100% by weight.
EXAMPLE 21
10 .mu.g of Az5 prepared according to Example 6 are dissolved in 5 .mu.l of 0.9% sterile saline. The composition is then administered to a mammal.

Number	Date	Country
29265	May 1981	EPX
169602	Jan 1986	EPX
221579	May 1987	EPX
318638	Nov 1974	DEX

Azetidine derivatives, compositions and methods of treating

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Wasserman et al., Stereochemical Effects in the Reactions of N-Alkyl-4-Substituted Azetidine 2 Carboxylic Acids with Oxalyl Chloride, Tetrahedron Letters, vol. 25, No. 29, pp. 3111-3114, 1984.
Baldwin et al., Gamma Lactam Analogues, J. Chem. Soc. Commun., 1985, pp. 194-196.
Baldwin et al., Gamma Lactam Analogues of Carbapenicillanic Acids, Tetrahedron vol. 42, No. 17, pp. 4879-4888, 1986.
Wroblewski et al., Phencyclidine is a Negative Allosteric Modulator, Proc. Nat. Acad. Sci. U.S.A. vol. 84, pp. 5068-5072, Jul., 1987.