HK1024241A - 3-alkoxyisoxazol-4-yl-substituted 2-amino carboxylic acid compounds - Google Patents

Description

3-alkoxyisoxazol-4-yl-substituted-2-aminocarboxylic acid compounds

Technical Field

The present invention relates to a novel class of (3-alkoxyisoxazol-4-yl) -substituted-2-aminocarboxylic acid derivatives and their sulfur-containing analogs. The compounds are ligands for Excitatory Amino Acid (EAA) receptors, in particular AMPA and/or NMDA (N-methyl-D-aspartate) receptors, useful for the treatment of cerebral ischaemia, Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, schizophrenia, pain, depression and anxiety.

Background

According to the results of extensive studies on the excitatory mechanisms of the Central Nervous System (CNS) in the last 30 years, it is currently agreed that (S) -glutamic acid (Glu) is the major EAA neurotransmitter in the CNS (Lodge, D. excitatory amino acids are in health and disease. J.Wiley & Sons: Chichester, 1988; Wheal, H.; Thomson, A. excitatory amino acid and synaptic transmission. Academic Press: London, 1991; Meldrum, B.S. excitatory amino acid antagonist. Black well Sci.Publ.: Oxford, 1991; Kroggaard-Larsen, P.; Hansen, J.J. excitatory amino acid receptor: design of agonists and antagonists. E.Horwood: Chichester, 1992). There are at least four heterogeneous receptor families classified by the large number of receptor-mediated glutamate-operated neurotransmission (Monaghan, d.t. et al, ann.rev. pharmacol. toxicol.1989, 29,365- & 402; Watkins, j.c.; Krogsgaard-Larsen, p.; honoree, t.trends pharmacol. sci.1990, 11, 25-33; Simon, r.p. excitatory amino acids. thiemedd. pub.: New York, 1992) which regulate glutamate-operated neurotransmission.

There is very strong evidence supporting the notion that extreme excitation through modulation of the EAA receptor ("excitotoxicity") is a major important factor in cerebral ischemia secondary to stroke, head injury, asphyxia, subarachnoid hemorrhage, cardiac arrest and other conditions (Lodge, d.1988 supra; Meldrum, b.s.,1991 supra). It has been shown in animal models that the damage caused by various ischemic diseases can be suppressed by administering the glutamate-antagonist. Therefore, while the relative importance of the different classes of EAA receptors in the phenomena underlying ischemic insult is unclear, it is generally believed that antagonists of EAA receptors are potential therapeutic agents for such diseases.

Cumulative evidence from different routes of neurochemical and pharmacological studies suggests that disorders of the EAA receptor mechanism, which may include "excitotoxicity", play a role in Huntington's disease (Young, A.B.; et al, Science 1988, 241, 981-flaked 983), epilepsy (Krogsgaard-Larsen, P.; Hansen, J.J.,1992 supra), Parkinson's disease (Klockgether, T.; Turski, L.trends. Neurosci.1989, 12,285-286), and Alzheimer's disease (Greenamyre, J.T.; Maragos, W.F.Cerebrasovc.Brain. Metab.Rev.1993, 5, 61-94; Francis, P.T., et al, J.Neurochem.1993, 60, 1589).

In addition, central EAA receptors may be involved in synaptic mechanisms underlying schizophrenia (Reynolds, G.P.trends. Pharmacol. Sci.1992, 13, 116-), pain and anxiety (Drejer, T.In: design of excitatory amino acid receptors: agonists and antagonists (Krogsgaard-Larsen, P.; Hansen, J.J. eds.) E.Horwood: Chichester,1992, pp.352- & 375) and depression (Trullas, R., Skolnick, P., Eur.J.Pharmacol.1990, 185,1-10 and Trullas et al, Eur.J.Pharmacol.1991, 203,379- & 385). Thus, it appears that a reduction in the function of the EAA receptor (EAA hypoactivity) may play a role in, for example, schizophrenia (Deutsch, S.I. et al, Clin. neuropharmacol.1989, 12,1-13) and in a part of the clinical symptoms seen in Alzheimer's disease (Greenamyre, J.T. et al; prog. neuro-Psychopharmacol & biol. Psychiaat.1988, 12, 421-430). "excitotoxicity" is likely to be involved in the complex mechanisms associated with Alzheimer's disease as well as low activity of EAA (Greenamyre, J.T.; 1988, supra; Greenamyre, J.T.; Maragos, W.F.,1993, supra).

In addition, ligands for the EAA receptor are believed to be useful in the treatment of cerebral ischemia, huntington's disease, epilepsy, parkinson's disease, alzheimer's disease, anxiety, schizophrenia, depression, and pain.

To date, most EAA agonists tested in model systems have shown more or less pronounced neurotoxicity, and subsequent clinical use of this class of compounds may be limited (Carlsson, m.; Carlsson, a. trends.neurosci.1990, 13, 272-.

On the other hand, partial EAA agonists that show an appropriate balance between agonist and antagonist may have a comparable therapeutic effect in the above indications (Greenamyre, J.T.; supra 1988; Christensen, I.T. et al, drug. Des. Del.1989, 5, 57-71; Francis, P.T. et al, J.Neurochem.1993, 60, 1589-. Based on their EAA antagonist profile, partial agonists may exhibit therapeutically useful neuroprotective effects while being sufficiently antagonistic to prevent total blockade of neurotransmission modulated by specific EAA receptors.

ATPA, the 5-tert-butyl analogue of AMPA ((RS) -2-amino-3- (3-hydroxy-5-methylisoxazol-4-yl) propionic acid) has been disclosed to have systemic activity, but, however, it has not been reported to show neurotoxic effects in animals (Ornstein, P.L. et al, J.Med.Chem.1993, 36, 2046. sup. 2048; Lauridsen, J.; Honor. T.; Kroggaard-Larsen, P.J.Med.Chem.1985, 28, 668. sup. 672).

Like AMPA itself, some mono-and bicyclic AMPA analogs have been found to exhibit selective agonism at the AMPA receptor (Hansen, J.J.; Krogsgaard-Larsen, P.Med.Res.Rev.1990, 10, 55-94; Krogsgaard-Larsen, P.Hansen, J.J.,1992, supra). One of these analogues, (RS) -2-amino-3- (3-hydroxy-5-phenylisoxazol-4-yl) propionic acid (APPA), in which the methyl group of AMPA is substituted by a phenyl group, shows a weak but unique partial agonist profile (Christensen, i.t. et al, 1989, supra).

ACPA ((RS) -2-amino-3 (3-carboxy-5-methylisoxazol-4-yl) propanoic acid) is described as a potent AMPA receptor agonist (Madsen, U. and Wong, E.J.mad.chem.1992, 35, 107-111).

Furthermore, WO-a 195012587 discloses a class of (5-arylisoxazol-4-yl) -or (5-arylisothiazol-4-yl) -substituted 2-aminocarboxylic acid compounds as EAA-receptor ligands.

As seen from the above non-neurotoxicity, ligands of CNS-active EAA receptors with good penetration into the CNS are highly desirable in the treatment of the various diseases already mentioned, and therefore, it is an object of the present invention to provide such new drugs.

Summary of The Invention

It has now been found that a novel class of (3-alkoxyisoxazol-4-yl) -substituted-2-aminocarboxylic acid derivatives and their sulphur-containing analogues are ligands for EAA receptors, in particular AMPA and/or NMDA receptors.

Furthermore, the invention relates to a composition of the general formula IOr II and pharmaceutically acceptable salts thereof:orWherein R is₁Is hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_2-6Cycloalkyl (en) yl, cycloalkyl (en) yl-C_1-6Alk (en/yn) yl or phenyl-C_1-6Alk (en/yn) yl, said phenyl being CF₃Halogen, C_1-6Alkyl or alkoxy optionally substituted; a is a bond or selected from C_1-6Alkylene radical, C_2-6Alkenylene or C_2-6Alkynylene and cycloalkylene spacers. B is-CR_a(NR_bR_c)-COOR₅Group, wherein R_a-R_cAre each hydrogen or C_1-6Alkyl, and R₅Is defined as R₁Or pivaloyloxymethyl, or B is a group of formula III:wherein R is₂、R₃And R₄Are respectively selected from:

a) hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, cycloalk (en) yl-C_1-6Alk (en/yn) yl, phenyl-C_1-6Alkyl, thienyl-C_1-6Alkyl, and

b) c in which one or more carbon atoms are replaced by N, O and/or S_1-6Alkyl radical, C_2-6Alkenyl and C_2-6An alkynyl group; or R₃And R₄Are connected to form C₂-C₆Alkylene radical, C₂-C₆Alkenylene or C₂-C₆Alkynylene; or R₄And R₂Linked to form C optionally mono-or di-substituted by hydroxy or methyl₁-C₃Alkylene radical, C₂-C₃Alkenylene or C₂-C₃Alkynylene, or form CH₂-O-CH₂(ii) a E is COOR₆Wherein R is₆Is defined as R₅Or E is tetrazol-5-yl, 1,2, 4-triazol-3-yl or 1,2, 3-triazol-4-yl; x is O or S; y is O or S.

In another aspect, the invention relates to a process for the preparation of the novel compounds of formula I or II.

In a further aspect, the invention relates to pharmaceutical compositions comprising the novel compounds of formula I or II together with a suitable pharmaceutically acceptable carrier or diluent.

In a further aspect, the invention relates to the use of a compound of formula I or II for the preparation of a pharmaceutical composition for the treatment of cerebral ischaemia, Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, schizophrenia, pain, depression or anxiety.

Some of the compounds of the invention were found to be ligands of AMPA receptors with affinity at micromolar concentrations, and some were found to bind to NMDA receptors. In addition, some of the compounds of the invention were found to be agonists, while another part were found to be antagonists. Thus, the compounds of the present invention are useful for the treatment of cerebral ischemia, huntington's disease, epilepsy, parkinson's disease, alzheimer's disease, schizophrenia, pain, depression and anxiety. Wherein R is₅And/or R₆Said compound other than hydrogen being R₅And R₆A prodrug of said corresponding compound being hydrogen.

Detailed Description

Some of the compounds of formula I or II may exist in the form of their optical isomers, and such optical isomers are also encompassed by the present invention.

In the formulae I and II, the term C_1-6Alkyl means a straight or branched chain alkyl group having from 1 to 6C atoms, including groups such as methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-2-propyl, and the like. Likewise, C_2-6Alkenyl and C_2-6Alkynyl denotes such straight-chain or branched radicals having 2 to 6C atomsA cluster of and C_1-6Alkylene radical, C_2-6Alkenylene and C_2-6Alkynylene denotes such a branched or straight chain divalent group. Cycloalkyl denotes such a group having 3 to 7 carbon atoms, and the term C_1-6-the alkoxy marker has C as defined above_1-6Alkyl radical, C_2-6Alkenyl or C_2-6Such groups of alkynyl moieties.

The term "alk (en/yn) yl" means that the group may be alkyl, alkenyl or alkynyl.

The term bond (defined for a) means that B can be attached directly to the 4-position of the isoxazole ring.

Halogen means fluorine, chlorine, bromine or iodine.

Some of the compounds of formula I or II may exist as pharmaceutically acceptable salts thereof, which are also encompassed by the present invention.

The salts of the compounds of the general formula I or II are salts with non-toxic organic acids, such as maleic acid, fumaric acid, benzoic acid, ascorbic acid, oxalic acid, tartaric acid, lactic acid and malic acid, or inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid, or they may be salts of inorganic bases, such as alkali metal salts, such as sodium, potassium or lithium salts, alkaline earth metal salts, such as calcium or magnesium salts, or ammonium or organic bases.

In the formulae I and II, A is preferably a bond or C₁-C₃Alkylene, most preferably methylene.

Preferably B is-CR_a(NR_bR_c)-COOR₅Wherein R is_b-R_cIs hydrogen, and R_aIs hydrogen or C_1-6Alkyl, suitably methyl, or a group of formula III, wherein R is₂、R₃And R₄Is hydrogen or C_1-6Alkyl, or R₄And R₂Are connected to form C₁-C₃An alkylene group. Most preferably, B is-CH (NH)₂) -COOH or wherein R₂、R₃And R₄Are each a hydrogen group of formula III.

Preferably, E is COOH, triazolyl or tetrazolyl, preferably COOH. Another subclass contains where E is COOR₆Wherein R is₆Is not H. According to a preferred subclass of said compounds of the invention, X and Y are O. Other subclasses are, wherein X is O and Y is S; y is O and X is S; and compounds in which X and Y are both S.

R₁Preferably C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6Alkynyl group. Particularly suitable R₁The radicals are methyl, ethyl, propyl, butyl and propargyl.

In a preferred embodiment of the invention, the compound is a compound of formula I, wherein A is a bond or C₁-C₃Alkylene, B being-CH (NH)₂) -COOH or a group of formula III, wherein R₃、R₄And R₂Are each hydrogen, X and Y are both oxygen, and R is₁Is C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6Alkynyl group. Particularly suitable R₁The radicals are methyl, ethyl, propyl, butyl and propargyl.

According to the invention, the compounds of formula I or II are prepared by the following process. For simplicity, the reactions a) to e) and g) to h) are shown only in formula I, and the same procedure can be used for formula II.

a) To obtain a compound in which B is-CR_a(NR_bR_c)-COOR₅A compound of formula (I) wherein R_a-R_cAnd R₅As previously defined, and at least R_b、R₅And R₆One is hydrogen, deprotecting the compound of formula iv:wherein R is₁A, X and Y are as previously defined, R_a’-R_c', E' and R₅Are respectively defined as R_a-R_cAnd E and R₅Or they are protecting groups, with the proviso that at least E', R₅' and R_cOne of' is a protecting group;

b) to obtain a compound in which B is-CR_a(NR_bR_c)-COOR₅A compound of formula (I) wherein R_b、R_cAnd R₅Are both hydrogen, deprotecting the compound of formula v:wherein R is₁A, Y, X and E' are as previously defined;

c) to obtain compounds of formula I in which B is a group of formula III, addition-elimination of compounds of formula VI with compounds of formula VII is carried out:in these formulae, R₁-R₄A, X, Y and E' are as previously defined;

d) to obtain compounds of formula I in which B is a group of formula III, in which R₄And R₂Is linked to form C optionally mono-or di-substituted by hydroxy or methyl_1-3Alkylene radical, C₂-C₃Alkenylene or C₂-C₃Alkynylene, a compound of formula VIII:wherein R is₁、R₃A, X, Y and E' are as previously defined; r₄And R₂Linked to form a group as defined above and BOS is t-butoxycarbonyl, reaction with 3, 4-diethoxy-3-cyclobutene-1, 2-dione and subsequent ring closure and deprotection;

e) to obtain a radical in which B is of the formula III and R₂-R₄Of formula i, with one or more compounds of formula ix other than hydrogen, alkylating a compound of formula ix:wherein R is₁、R₂、R₃、R₄A, X, Y and E' are as previously defined, but at least R₂-R₄One of which is hydrogen;

f) to obtain compounds of formula I or II, compounds having the general formula X are used other than R₁' canR being other than hydrogen₁' is R₁Of an alkylating agent R₁' Z alkylation:wherein A, X, Y and E' are as previously defined, and except that in R_b、R_cAnd R₅In which B' is B, except that hydrogen is replaced by a protecting group, to obtain a mixture of formulae xi and xii:orWherein A, X, Y, E 'and B' are as defined above, and then isolating and deprotecting said compound;

g) to obtain wherein R₅And/or R₆Esterifying a compound of formula XIII or XIV, other than hydrogen, with:wherein R is₁、R₂、R₃、R₄A, X, Y and R_a-R_cAs previously defined;

h) to obtain a compound in which B is-CR_a(NR_bR_c)-COOR₅A compound of formula I of the group, wherein R_a、R_b、R_cAnd R₅All hydrogen, E is COOH, especially enantiomerically pure compounds:subjecting a compound of formula XV to Sch ǒ llkopf bis-lactim amino acid synthesis and subsequent deprotection of the resulting bislactim ether of formula XVI:formula (III) X, Y, R_aAnd R₁As previously defined, and a 'is defined as a, except that a' may not be a bond.

In the process of the invention, preferred protecting groups are as follows:

for E = COOH: 4, 5-dihydro-4, 4-dimethyloxazol-2-yl, C_1-6An alkyl or benzyl group; for R₅= hydrogen: C_1-6Alkyl and for R_b= hydrogen: C_1-6An alkylcarbonyl group.

One step of deprotection according to process a) is carried out by treating the compound of formula IV with a suitable aqueous acid, suitably 0.5-12N aqueous hydrochloric acid, 48% aqueous HBr or saturated aqueous HBr in acetic acid. The deprotection process can also be carried out in a continuous step by using an aqueous acid and an aqueous base, suitably successively in an aqueous acid, such as 0.5-12N hydrochloric acid, an aqueous base, such as 1-8N sodium hydroxide, and an aqueous acid, such as 0.5-12N hydrochloric acid, or in an aqueous base, such as 1-8N sodium hydroxide, and an aqueous acid, such as 0.5-12N hydrochloric acid.

The starting material of formula iv is suitably prepared from 3-alkoxy-4-methylisoxazole-5-carboxylic acid (WO95/12587, a1) by complete deprotection in aqueous acid, optionally esterification of the 3-hydroxy-4-methylisoxazole-5-carboxylic acid and subsequent alkylation with the appropriate halide or directly by alkylation, according to the deprotection conditions described above. Subsequently, by bromination of the 4-methylisoxazole group and subsequent alkylation with an amino acid precursor such as diethyl acetamidomalonate. Other 4-alkylisoxazoles can be prepared by chain-extension reactions such as alkylation with cyanide or diethyl malonate and subsequent conversion to primary alkyl halides or aldehydes. The halide may be treated as described above. The aldehydes can be used as starting materials in the preparation of the compounds of the general formula V.

In b), one step of the deprotection is carried out by treating the compound of formula V with a suitable aqueous acid or aqueous base, suitably in aqueous 0.5-8N hydrochloric acid. As mentioned above for process a), the deprotection can also be accomplished in a continuous step by using an aqueous acid and an aqueous base. By using Ba (OH)₂Hydantoin rings may be cleaved in aqueous solution, 10-70% aqueous sulfuric acid, or by using enzymes such as hydantoinases. The hydantoin ring cleavage can be performed either before or after the deprotection of the E-group. After complete deprotection of the hydantoin intermediate, the R₁The group may have toReintroduced by alkylation.

The hydantoin rings in the compounds of formula V are conveniently formed according to the method described in Ware, E., chem.Rev.1950, 46, 403-470. Cleavage of hydantoin rings is conveniently accomplished in a manner similar to that described in Curry, K.et al J.Med.chem.1988, 31,864-867, G.K.et al J.Med.chem.1987, 30,2062-2067, Gruneward, G.L.et al J.Med.chem.1980, 23,754-758, Hiroi, K.et al chem.pharm.Bull.1968, 16,444-447 or Stark, G.R.et al J.biol.chem.1963, 238, 214-226. The starting materials for the preparation of the compounds of the formula V are obtained in the same way as the starting materials obtained in process a) outlined above. If A is a bond, the aldehyde can be prepared from the bromomethyl compound by bromination and subsequent conversion to the aldehyde.

The addition-elimination reaction according to process c) is conveniently carried out in an organic protic solvent, such as an alcohol, preferably at room temperature in the presence of a suitable inorganic base, such as aqueous NaOH. Intermediates of formula VII can be prepared by the methods described in Cohen, S.et al J.Amer.chem.Soc.1966, 88,1533-1536, EP-A2-0496561 or Kinney, W.A.et al J.Med.chem.1992, 35, 4720-4726.

Intermediates of formula VI are readily obtained by the Gabriel synthesis of primary amines as described in Sheehan, J.C. et al J.Am.chem.Soc.,1950, 72,2786-88. The alkyl halide starting material used in this synthesis is conveniently obtained as described in connection with the use of the starting material in process a) above.

The deprotection is conveniently accomplished by the use of aqueous acids or aqueous bases, preferably 0.5-8N HCl or aqueous 0.5-8N NaOH, both at room temperature and at elevated temperature.

In process d), the reaction and subsequent ring closure and deprotection are carried out as described by Kinney et al, EP-A2-0496561.

The starting material of formula VIII is obtained by reacting 4-bromomethylisoxazole, obtained as described in connection with the starting material in process a), with the mono-BOC-protected alkylenediamine obtained with reference to EP-A2-0496561.

The alkylation of the compound of the formula IX according to process e) is conveniently carried out in an inert organic solvent such as a suitable alcohol, ketone or dimethylformamide, preferably in the presence of a suitable base such as sodium hydride, potassium carbonate or triethylamine, as described by Kinney, W.A. EP-A2-0496561. The starting material of the formula IX is obtainable by process c).

In process f), the deprotection of the compounds of formulae XI and XII is carried out as described in process a) or by using hydrochloric acid solution in diethyl ether or other non-aqueous deprotection methods. The starting material X is obtained as described above in connection with the starting material in process a).

In process g), the esterification can be accomplished by methods well known in the art, such as treatment with an acidic solution of an alcohol. The starting materials can be prepared as described under process a) to e) or h).

The resolution of the compounds of formula I is conveniently accomplished by the diastereomeric salt formation of an optically active acid or base such as 1-phenylethylamine. In some cases, the resolution is conveniently accomplished by the formation of diastereomeric compounds and subsequent separation of the diastereomers by flash chromatography or crystallization. Certain diastereomers can be conveniently prepared by asymmetric synthesis using the Sch ǒ llkopf's bis-lactim amino acid synthesis in method h). In this synthesis, the starting material is the alkyl halide obtained as described above for the starting material in process a). The protecting group of the 5-carboxyisoxazole group is preferably a 2-oxazoline group, which is prepared from the corresponding 5-cyanoisoxazole by condensation with an amino alcohol (WO95/12587, A1).

Salts of the compounds of the present invention can be readily prepared by methods well known in the art by reacting the compound with an equivalent amount of an acid or base in a water-miscible solvent such as acetone or ethanol, isolating the salt by concentration and cooling, or directly isolating the desired salt after reaction with an excess of the acid or base in a water-immiscible solvent such as diethyl ether or chloroform. These salts may also be prepared by metathesis of the appropriate salts in conventional manner.

The compounds of formula i and the pharmaceutically acceptable acid addition salts thereof may be administered by any suitable route, for example, orally or parenterally, and the compounds may be presented for such administration in any suitable form, for example, as tablets, capsules, powders, syrups, or solutions or dispersions for injection.

The effective daily dose of the compound of formula I or a pharmaceutically acceptable salt thereof is from 10. mu.g/kg body weight to 50mg/kg body weight.

Examples

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention in any way.

All melting points were determined on a B ü chi SMP-20 instrument, uncorrected. Recording on a Brucker250MHz spectrometer¹H NMR and¹³c NMR spectrum (C: (C NMR)¹H NMR 250.13MHz and¹³c NMR 62.90MHz), TMS was used as an internal standard unless otherwise indicated.

Mass spectra were obtained from a Quattro MS-MS system on a VG Biotech, Fisons instrument (connected to an HP1050 mode HPLC system). 20-50 μ l of a sample (10 μ g/ml) dissolved in a mixture of acetonitrile/water = 1: 1 acetic acid or in a mixture of acetonitrile/water/ammonia (25%) = 25: 1 (zwitterion) was injected via an autosampler into the electron jet source at a flow rate of 30 μ l/min. Spectra were recorded under standard conditions to obtain molecular weight information ((M + H)⁺) Or ((M-H)). The background is subtracted.

Analytical HPLC was carried out on a 150X 4.6mm Lichrocart 250-4(Merck) column which was eluted at 35 ℃ with 1 ml/min methanol/0.01M ammonium acetate (pH8) = 3: 2. The apparatus used comprised a L6200 HPLC pump, a L5025 column incubator and a L4000A UV-VIS detector (set at 230 nm). The purity of the diastereomer, expressed as diastereomer excess (de), was calculated from the peak area.

Sumicrohiral OA-Chiral HPLC analysis was performed on a 5000 column using 1 ml/min 5mM CuSO at ambient temperature₄(aq) elution. The equipment used included an AS2000 autosampler, an L6200 HPLC pump, a T6300 column incubator, an L4250 UV-VIS detector (set at 240nm), and a D6000 computer interface, all from Merck-Hitachi. The enantiomeric purity, expressed as enantiomeric excess (ee), was calculated from the peak area.

Example 1

(RS) -2-amino-3- (5-carboxy-3-methoxyisoxazol-4-yl) propionic acid hydrate (Compound 1)

1) 3-hydroxy-4-methylisoxazole-5-carboxylic acid

3-ethoxy-4-methylisoxazole-5-carboxylic acid (15g,88mmol) and 47% HBr (aq) (150mL) were boiled for 6 hours at reflux. The solution was cooled and the crystallized target compound (8.7g, 69%) was collected by filtration: melting point 257 ℃ and 259 ℃. The acidic filtrate was added to water (100mL) and extracted with diethyl ether (6X 400 mL). The organic extract was washed with brine (100mL) and dried (MgSO)₄) And concentrated in vacuo to give the crude target compound (3.0g, 24%) in 93% total yield. The mixture of the two products will be used in the next step.

2) 3-hydroxy-4-methylisoxazole-5-carboxylic acid ethyl ester

3-hydroxy-4-methylisoxazole-5-carboxylic acid (6.0g,42mmol) and a saturated HCl solution in ethanol (110mL) were boiled under reflux for 4 hours. The solution was concentrated in vacuo and the residue was redissolved in EtOAc and dried (MgSO)₄) And evaporated in vacuo to give the crude title compound (7.2g, 100%). A small sample was recrystallized (EtOAc/heptane) to give colorless crystals: melting point 133-. The crude product was used in the next step without further purification.

3) 3-methoxy-4-methylisoxazole-5-carboxylic acid ethyl ester

Ethyl 3-hydroxy-4-methylisoxazole-5-carboxylate (1.0g,5.8mmol), methyl iodide (0.4mL,5.8mmol) and K at 40 ℃₂CO₃(1.6g,11.7mmol) in DMF (40mL) was heated for 1 hour. The mixture was poured onto an ice/water mixture (100mL) and extracted with ether (3X 100 mL). The organic extracts were washed with water (2X 50mL), brine (50mL), dried (MgSO)₄) And evaporated in vacuo (0.8g, 74%). To obtain the crude product equivalent to 17.5mmol of starting material, the procedure was repeated, which was subjected to flash chromatography (silica gel, eluent: dichloromethane/ether = 9: 1) to afford the crude title compound as a yellow oil (1.4g, 43%), which was used in the next step without further purification.

4)4- (bromomethyl) -3-methoxyisoxazole-5-carboxylic acid ethyl ester

Ethyl 3-methoxy-4-methylisoxazole-5-carboxylate (1.3g,7.0mmol), NBS (1.4g,7.9mmol), dibenzoyl peroxide (catalytic amount) and tetrachloromethane (40mL) were boiled under reflux for 10 hours. The mixture was cooled, filtered and concentrated in vacuo to afford the crude title compound as a yellow oil (1.8g, 97%). The crude product was used in the next step without further purification.

5) 2-acetylamino-2 (ethoxycarbonyl) -3- [5- (ethoxycarbonyl) -3-methoxyisoxazol-4-yl ] propionic acid ethyl ester

A mixture of diethyl acetamidomalonate (1.6g,7.4mmol) and potassium tert-butoxide (0.9g,8.0mmol) in N-methylpyrrolidone (30mL) was stirred at room temperature for 30 min. Ethyl 4- (bromomethyl) -3-methoxyisoxazole-5-carboxylate (1.8g,6.8mmol) in N-methylpyrrolidone (10mL) was added (temperature 22-28 ℃ C.), and the resulting mixture was stirred at room temperature for 1.5 hours. The reaction mixture was poured onto an ice/water mixture (100mL) and the aqueous phase was extracted with EtOAc (3X 150 mL). The organic extract was washed with aqueous potassium tert-butoxide, water (100mL) and brine (100mL), dried (MgSO)₄) And vacuum concentration. Flash chromatography (silica gel, eluent: EtOAc/heptane = 1: 1) afforded the crude target compound (1.8g, 66%). A small sample was recrystallized (EtOAc/heptane) to give colorless crystals: melting point 78-80 ℃. The crude product was used in the next step without further purification.

6) (RS) -2-amino-3- (5-carboxy-3-methoxyisoxazol-4-yl) propionic acid hydrate (Compound 1)

2-acetylamino-2 (ethoxycarbonyl) -3- [5- (ethoxycarbonyl) -3-methoxyisoxazol-4-yl in 0.5M HCl (100mL) at reflux]A suspension of ethyl propionate (1.2g,3.0mmol) was boiled for 48 hours. The mixture was cooled, washed with dichloromethane (100mL) and diethyl ether (2 × 100mL), filtered and concentrated in vacuo. Water (5mL) was added and the pH adjusted to about 3 by the addition of NaOH (0.1M and 1M). The aqueous phase was reduced in vacuo (2mL) and the precipitate was collected by filtration. The precipitate was stirred in water (2mL) at room temperature for 24 hours to afford compound 1(70mg, 10%) after filtration: melting point 222-]；¹H NMR(DMSO-D₆)δ2.88(dd,1H),3.01(dd,1H),3.85-3.96(m,1H),3.90(s,3H)；¹³C NMR(DMSO-d₆)d22.70,52.38,57.32,103.25,159.43,165.95,170.66(2C)；MS((M+H)⁺) m/z 231. Analysis (C)₈H₁₀N₂O₆·0.25H₂O) calcd for C40.94, H4.51, N11.94; found C41.01, H4.37, N11.91.

The following compounds were prepared in the same manner:

(RS) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (Compound 2)

Melting point 238-;¹H NMR(DMSO-D₆)d 1.34(t,3H),2.90(dd,1H),3.03(dd,1H),3.96(dd,1H),4.23(q,2H)；¹³CNMR(DMSO-d₆)δ14.46,22.41,51.89,65.63,103.34,159.22,164.97,169.75,170.40；MS((M+H)⁺) m/z 245. Analysis (C)₉H₁₂N₂O₆) Calcd for C44.27, H4.95, N11.47; found C44.10, H4.92, N11.34.

(RS) -2-amino-3- (5-carboxy-3-isopropoxyisoxazol-4-yl) propionic acid (Compound 3)

Melting point 242 and 243 ℃ (decomposition);¹H NMR(DMSO-D₆)d 1.32(dd,6H),2.88(dd,1H),3.01(dd,1H),3.96(dd,1H),4.79(h,1H)；¹³CNMR(DMSO-d₆)δ21.57,21.77,22.35,51.82,73.13,103.56,159.22,164.91,169.08,170.36；MS((M+H)⁺) m/z 259. Analysis (C)₁₀H₁₄N₂O₆) Calcd for C46.51, H5.46, N10.85; found C46.37, H5.46, N10.83.

(RS) -2-amino-3- (5-carboxy-3-hydroxyisoxazol-4-yl) propionic acid hydrate (Compound 4)

Melting point 175-177 ℃;¹H NMR(DMSO-D₆)δ3.00(d,2H),3.88(t,1H)；¹³C NMR(DMSO-d₆)δ23.07,52.07,105.84,159.41,162.11,169.89,170.78；MS((M+H)⁺) m/z 217. Analysis (C)₇H₈N₂O₆·0.25H₂O) calcd for C38.10, H3.88, N12.70; found C37.72, H3.98, N12.52.

Example 2

(RS) -2-amino-3- (5-carboxy-2, 3-dihydro-2-methyl-3-oxoisoxazol-4-yl) propionic acid hydrate (Compound 5)

2, 3-dihydro-2, 4-dimethyl-3-oxoisoxazole-5-carboxylic acid ethyl ester

Ethyl 3-hydroxy-4-methylisoxazole-5-carboxylate (2.0g,11.7mmol) and K in ethanol (50mL) were combined at 40 deg.C₂CO₃The mixture (4.0g,29mmol) was heated for a total of 26 hours, after 1 hour methyl iodide (0.8mL,13mmol) was added and 3 more times over the remaining 25 hours. The solution was filtered and evaporated in vacuo (according to¹H NMR to obtain a 1: 1 mixture of the target compound and ethyl 3-methoxy-4-methylisoxazole-5-carboxylate). Flash chromatography (silica gel, eluent: dichloromethane/diethyl ether = 9: 1, then 1: 1) afforded ethyl 3-methoxy-4-methylisoxazole-5-carboxylate (0.40, 18%) and the title compound (0.45g, 21%) as a yellow oil. The latter small sample was recrystallized (EtOAc/heptane) to give colorless crystals: melting point 64-65 ℃. The crude target compound was used in the next step without further purification.

(RS) -2-amino-3- (5-carboxy-2, 3-dihydro-2-methyl-3-oxoisoxazol-4-yl) propionic acid hydrate (Compound 5)

The title compound (70mg, colorless crystals, 72%) was obtained by a method similar to that of example 1, steps 2) -6) using the product of 1) above. Melting point 211 and 212 deg.C (decomposition);¹H NMR(DMSO-D₆)δ2.87(dd,1H),2.97(dd,1H),3.43(s,3H),3.92(dd,1H)；¹³C NMR(DMSO-d₆)δ23.10,32.32,51.79,106.51,158.59,162.37,166.64,170.35；MS((M+H)⁺) m/z 231. Analysis (C)₈H₁₀N₂O₆·0.25H₂O) calcd for C40.94, H4.51, N11.94; found C40.93, H4.55, N11.71.

The following compounds were prepared in the same manner:

(RS) -2-amino-3- (5-carboxy-2-ethyl-2, 3-dihydro-3-oxo-isoxazol-4-yl) propionic acid monohydrate (Compound 6)

¹H NMR(D₂O,1, 4-dioxane d 3.70) δ 1.28(t,3H),3.19(d,2H),4.01(q,2H),4.18(t, 1H);¹³C NMR(D₂o,1, 4-dioxane δ 67.40) δ 12.87,23.85,42.31,53.27,110.57,159.88,162.65,166.67,172.55; MS ((M + H)⁺) m/z 245. Analysis (C)₉H₁₂N₂O₆·H₂O) calcd for C41.22, H5.38, N10.68; found C41.28, H4.74, N10.27.

Example 3

(S) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (Compound (S) -2)

(R) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (Compound (R) -2)

1)5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxy-4-methylisoxazole

3-ethoxy-4-methylisoxazole-5-carbonitrile (2.6g,17.1mmol), 5.4M NaOMe in MeOH (0.6mL,3.4mmol), and EtOH (80mL) were stirred at room temperature for 30 min. Acetic acid (2.2mL,39.3mmol) and2-amino-2-methylpropan-1-ol (1.8mL,18.8mmol), and the resulting mixture was boiled under reflux for 20 h. The reaction mixture was cooled, water (100mL) was added and extracted with EtOAc (3X 100 mL). The organic extracts were washed with 1M NaOH (50mL), brine, and dried (MgSO)₄) And vacuum evaporation. The residue was dissolved in EtOH (60mL), a solution of KOH (1.8g,32mmol) in water (12mL) was added, and the mixture was stirred at room temperature for 20 hours. EtOH was removed in vacuo, water (80mL) was added and the aqueous phase was extracted with EtOAc (3X 100 mL). The organic extracts were washed with brine and dried (MgSO)₄) And vacuum evaporation. Flash chromatography (silica gel, eluent: EtOAc/heptane/triethylamine = 75: 25: 1) afforded the crude title compound as a yellow oil (2.0g, 52%).

2)4- (bromomethyl) -5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxyisoxazole

5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxy-4-methylisoxazole (2.0g,8.9mmol), NBS (1.75g,9.8mmol) and tetrachloromethane (150mL) were boiled under reflux for 5 hours. The mixture was cooled, filtered and concentrated in vacuo. Flash chromatography (silica gel, eluent: toluene/EtOAc/triethylamine = 100: 10: 1) afforded the title compound as a yellow oil (2.0g, 74%).

3) (2S,5R) -2, 5-dihydro-2- { [5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxyisoxazol-4-yl ] methyl } -5-isopropyl-3, 6-dimethoxypyrazine and (2R,2R) -2, 5-dihydro-2- { [5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxyisoxazol-4-yl ] methyl } -5-isopropyl-3, 6-dimethoxypyrazine

A1.6M solution of butyllithium in hexane (1.9mL,3.0mmol) was added to a pre-cooled (-78 deg.C) solution of (2R) - (-) -2, 5-dihydro-2-isopropyl-3, 6-dimethoxypyrazine (0.5mL,2.8mmol) in anhydrous tetrahydrofuran (8 mL). Stirring was continued for 10 min at-78 ℃ and 4- (bromomethyl) -5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxyisoxazole (0.85g,2.8mmol) dissolved in tetrahydrofuran (5mL) was added. The resulting mixture was stirred at-78 ℃ for 4.5 hours and the reaction was allowed to proceedThe mixture was warmed to room temperature and concentrated in vacuo. The residue was dissolved in diethyl ether (40mL) and poured onto an ice/water mixture (40 mL). The layers were separated and the aqueous phase was extracted with ether (2X 40 mL). The organic extracts were washed with brine and dried (MgSO)₄) And vacuum evaporation. Flash chromatography (silica gel, eluent: heptane/EtOAc = 3: 1) afforded (2S,5R) -target compound as a yellow oil (0.65g, 57%) de = 99.2% (retention time about 38 min). Further elution provided (2R,5R) -target compound as a yellow oil (38mg, 3%).

4) (2R,5S) -2, 5-dihydro-2- { [5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxyisoxazol-4-yl ] methyl } -5-isopropyl-3, 6-dimethoxypyrazine and (2S,5S) -2, 5-dihydro-2- { [5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxyisoxazol-4-yl ] methyl } -5-isopropyl-3, 6-dimethoxypyrazine

The target compound was obtained by the method of the above-described step 3) using (2S) - (+) -2, 5-dihydro-2-isopropyl-3, 6-dimethoxypyrazine as a starting material. Flash chromatography (silica gel, eluent: heptane/EtOAc = 3: 1) afforded (2R,5S) -target compound as a yellow oil (0.8g, 54%) de > 99.2% (retention time about 38 min). Further elution provided (2S,5S) -target compound as a yellow oil (60mg, 4%).

5) (S) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (Compound (S) -2)

Mixing (2S,5R) -2, 5-dihydro-2- { [5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxyisoxazol-4-yl]A suspension of methyl } -5-isopropyl-3, 6-dimethoxypyrazine (0.6g,1.5mmol) in 1M trifluoroacetic acid (200mL) was boiled under reflux for 5h, the reaction mixture (2mL) was concentrated in vacuo, and the residue was dissolved in water (50mL) and washed with EtOAc (3X 50 mL). The aqueous phase was filtered and evaporated to dryness in vacuo. The residue was treated with water (10mL) and the precipitate formed was stirred at room temperature for 24 hours, collected by filtration and recrystallized (water) to afford compound (S) -2(0.12g, 33%) as colorless crystals: melting point 259 and 261 deg.C (decomposition); ee > 99% (retention time about 30 minutes);¹H NMR(DMSO-D₆)δ1.34(t,3H),2.90(dd,1H),3.03(dd,1H),3.96(dd,1H),4.23(q,2H)；MS((M+H)⁺) m/z 245. Analysis (C)₉H₁₂N₂O₆) Calcd for C44.27, H4.95, N11.47; found C44.45, H4.96, N11.46.

6) (R) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (Compound (R) -2)

0.25M HCl (74mL,7.4mmol) was added to (2R,5S) -2, 5-dihydro-2- { [5- (4, 5-dihydro-4, 4-dimethyl-1, 3-oxazol-2-yl) -3-ethoxyisoxazol-4-yl]Methyl } -5-isopropyl-3, 6-dimethoxypyrazine (0.6g,1.5mmol) and MeOH (7mL) in a stirred solution and the resulting mixture stirred at room temperature for 2 h. The pH was adjusted to about 7 by adding aqueous ammonia (0.5M) and the methanol was removed in vacuo. The pH was adjusted to 8-9 by adding aqueous ammonia (0.5M) and the aqueous phase was extracted with EtOAc (4X 50 mL). The organic extracts were washed with brine and dried (MgSO)₄) And concentrated in vacuo. The residue was suspended in 1M HCl and the mixture was boiled under reflux for 4.5 h. The reaction mixture was concentrated in vacuo (2mL), the residue was dissolved in water (50mL) and washed with EtOAc (3X 50 mL). The aqueous phase was filtered, evaporated to dryness in vacuo and the residue treated with water (10 mL). The precipitate formed was stirred at room temperature for 2 hours, collected by filtration and recrystallized (water) to afford compound (R) -2(0.13g, 36%) as colorless crystals: melting point 258-; ee > 99% (retention time about 50 minutes);¹H NMR(DMSO-D₆)δ1.34(t,3H),2.90(dd,1H),3.03(dd,1H),3.96(dd,1H),4.23(q,2H)；MS((M+H)⁺) m/z 245. Analysis (C)₉H₁₂N₂O₆) Calcd for C44.27, H4.95, N11.47; found C44.56, H4.95, N11.53.

Example 4

(RS) -2-amino-3- [ 3-ethoxy-5- (1H-1,2, 4-triazol-3-yl) isoxazol-4-yl ] propionic acid hydrate (Compound 7)

N- [ (dimethylamino) methylene ] -3-ethoxy-4-methylisoxazole-5-carboxamide

A solution of 3-ethoxy-4-methylisoxazole-5-carboxamide (3.5g,21mmol) in N, N-dimethyl-formamide dimethyl acetal (15mL) was stirred at 120 ℃ for 15 minutes. After cooling, the title compound (4.2g, 91%) was collected as colorless crystals.

3- (3-ethoxy-4-methylisoxazol-5-yl) -1H-1,2, 4-triazole

N- (dimethylamino) methylene 3-ethoxy-4-methylisoxazole-5-carboxamide (1.8g,8.0mmol) was added to a solution of hydrazine hydrate (0.6mL,12.4mmol) in acetic acid (15 mL). The reaction mixture was stirred at 90 ℃ for 15 minutes and then left to crystallize at room temperature to afford the pure target compound (1.2g, 77%): melting point 194 ℃ and 196 ℃. Water (40mL) was added and the aqueous phase was extracted with EtOAc (3X 30 mL). The organic extracts were washed with brine and dried (MgSO)₄) And concentrated in vacuo to afford the crude target compound (0.3g, 20%). The two products were combined.

3- (3-ethoxy-4-methylisoxazol-5-yl) -1-trityl-1H-1, 2, 4-triazole

To 3- (3-ethoxy-4-methylisoxazol-5-yl) -1H-1,2, 4-triazole (1.1g,5.7mmol), triethylamine (2.5mL,18mmol) and DMF (20mL) was added trityl chloride (1.6g,5.7mmol) in DMF (5 mL). The mixture was stirred at room temperature for 5 hours and poured into an ice/water mixture (200 mL). The aqueous phase was extracted with diethyl ether (3X 200mL), and the organic extracts were extracted with Na₂CO₃Aqueous (10%) (200mL) and brine (200mL) washes. Drying (Na)₂SO₄) The solution was concentrated in vacuo to give the crude title compound (2.5 g). A small sample was recrystallized (EtOAc) to give a colorless crystal of the single isomer: melting point 181-. The crude product was used in the next step without further purification.

3- [4- (bromomethyl) -3-ethoxyisoxazol-5-yl ] -1-trityl-1H-1, 2, 4-triazole

A mixture of 3- (3-ethoxy-4-methylisoxazol-5-yl) -1-trityl-1H-1, 2, 4-triazole (2.4g,5.5mmol) and NBS (1.1g,6.2mmol) was boiled in tetrachloromethane (150mL) at reflux for 3 hours. The reaction mixture was cooled, filtered and concentrated in vacuo to afford crude target compound (2.8 g). The crude product was used in the next step without further purification.

2-acetylamino-3- [ 3-ethoxy-5- (1-trityl-1H-1, 2, 4-triazol-3-yl) isoxazol-4-yl ]2- (ethoxycarbonyl) propionic acid ethyl ester

A mixture of diethyl acetylaminomalonate (1.3g,6.0mmol) and potassium tert-butoxide (0.73g,6.5mmol) in N-methylpyrrolidone (30mL) was stirred at room temperature for 30 minutes. 3- [4- (bromomethyl) -3-ethoxyisoxazol-5-yl in N-methylpyrrolidone (20mL) was added]-1-trityl-1H-1, 2, 4-triazole (2.8g,5.4mmol) (temperature 22-28 ℃) and the resulting mixture stirred at room temperature for 2 hours. The reaction mixture was poured onto an ice/water mixture (250mL) and the aqueous phase was extracted with EtOAc (3X 250 mL). The organic extracts were washed with aqueous potassium tert-butoxide and brine, dried (Na)₂SO₄) And concentrated in vacuo. Flash chromatography (silica gel, eluent: EtOAc/heptane/triethylamine = 50: 2) afforded the title compound (2.2g, 62%): melting point 145-149 ℃.

(RS) -2-amino-3- [ 3-ethoxy-5- (1H-1,2, 4-triazol-3-yl) isoxazol-4-yl ] propionic acid hydrate (Compound 7)

2-acetylamino-3- [ 3-ethoxy-5- (1-trityl-1H-1, 2, 4-triazol-3-yl) isoxazol-4-yl in 1M HCl (150mL)]A suspension of ethyl-2- (ethoxycarbonyl) propionate (1.5g,2.3mmol) in 1M HCl (150ml) was boiled under reflux for 24 h. The solution was cooled, washed with diethyl ether (2X 150mL) and dichloromethane (150mL), filtered and concentrated in vacuo. Water (5mL) was added, the pH was adjusted to about 3.5 by addition of NaOH (0.1M and 1M), and filtration afforded compound 7(0.35g, 56%): melting point 225-;¹H NMR(DMSO-D₆)δ1.38(t,3H),2.94(dd,1H),3.18(dd,1H),3.58(dd,1H),4.30(q,2H),8.64(s,1H)；¹³C NMR(DMSO-d₆)δ14.52,23.60,53.30,65.93,104.25,146.18,150.87,158.44,169.47,170.51；MS((M+H)⁺) m/z 268. Analysis (C)₁₀H₁₃N₅O₄·0.25H₂O) calcd for C44.20, H5.01, N25.77; found C44.42, H5.29, N25.52.

Example 5

(RS) -2-amino-3- [ 3-ethoxy-5- (5-tetrazolyl) isoxazol-4-yl ] propanoic acid (Compound 8)

Prepared by a method analogous to that in example 4 from ethyl 2-acetamido-3- [ 3-ethoxy-5- (tetrazol-5-yl) isoxazol-4-yl ] -2- (ethoxycarbonyl) propionate.

Example 6

(RS) -2-amino-3- (3-benzyloxy-5-carboxyisoxazol-4-yl) propionic acid (Compound 9)

A solution of (RS) -2-amino-3- (5-carboxy-3-hydroxyisoxazol-4-yl) propionic acid (3.5g,11.8mmol) and HCl in ethanol (50ml) was boiled at reflux for 2.5h and evaporated to dryness in vacuo to give ethyl (RS) -2-amino-3- (5-ethoxycarbonyl-3-hydroxyisoxazol-4-yl) propionate (4.15g, 100%).

A mixture of di-tert-butyl dicarbonate (3.1g,14mmol), triethylamine (3.8g,37mmol) and 1, 4-dioxane (15ml) was added to a solution of (RS) -ethyl 2-amino-3- (5-ethoxycarbonyl-3-hydroxyisoxazol-4-yl) propionate (4.15g,11.7mmol) in water/1, 4-dioxane (1: 1) (50ml) and the resulting mixture was stirred at room temperature for 16 h. The 1, 4-dioxane was evaporated in vacuo and the aqueous phase was acidified with dilute aqueous HCl. The aqueous phase was extracted with ethyl acetate and the organic extract was washed with water, brine and dried (MgSO)₄) And vacuum concentration. Flash chromatography (SiO)₂Eluent: heptane/ethyl acetate/acetic acid (1: 1, 4%)) ethyl (RS) -2-tert-butoxycarbonylamino-3- (5-ethoxycarbonyl-3-hydroxyisoxazol-4-yl) propionate was obtained as an oil (4.1g, 92%).

Ethyl (RS) -2-tert-butoxycarbonylamino-3- (5-ethoxycarbonyl-3-hydroxyisoxazol-4-yl) propionate (3.2g,8.6mmol), K₂CO₃A mixture of (2.4g,17.2mmol) in acetone (40ml) was heatedTo reflux temperature. Benzyl bromide (2.2g,12.9mmol) was added and the mixture was boiled under reflux for 1.5 h. Vacuum concentrating, and subjecting to flash chromatography (SiO)₂Eluent: heptane/ethyl acetate (2: 1)) to give (RS) -2- [ (tert-butoxycarbonyl) amino group]-3- [ 3-benzyloxy-5- (ethoxycarbonyl) isoxazol-4-yl]Ethyl propionate (1.64g, 41%) and (RS) -2- [ (tert-butoxycarbonyl) amino]-ethyl 3- (2-benzyl-5-ethoxycarbonyl-2, 3-dihydro-3-oxoisoxazol-4-yl) propionate (0.7g, 18%).

Reacting (RS) -2- [ (tert-butoxycarbonyl) amino group]-3- [ 3-benzyloxy-5- (ethoxycarbonyl) isoxazol-4-yl]A mixture of ethyl propionate (0.65g,1.4mmol) and 1M NaOH (50ml) was boiled under reflux for 16 h. The mixture was cooled (5 ℃), acidified with dilute HCl solution and concentrated in vacuo. The residue was recrystallized from water to give (RS) -2-amino-3- (3-benzyloxy-5-carboxyisoxazol-4-yl) propionic acid (0.1g, 23%): melting point 209-;¹H NMR(DMSO-D₆)δ2.95(dd,1H),3.05(dd,1H),3.99(t,1H),5.26(s,2H),7.31-7.52(m,5H)；MS((M+H)⁺) m/z 307. Analytically calcd C54.89, H4.62, N9.15; found C54.31, H4.56, N8.97.

The following compounds were prepared in the same manner:

(RS) -2-amino-3- (3-propoxy-5-carboxyisoxazol-4-yl) propionic acid (Compound 10)

Melting point 250 ℃ and 251 ℃ (decomposition);¹H NMR(D₂o, dioxane, 1M NaOD) d 0.95(t,3H),1.76(se,2H),2.78(dd,1H),2.90(dd,1H),3.42(dd,1H),4.17(t, 2H);¹³C NMR d12.3,24.4,29.8,58.3,74.9,111.5,164.3,166.6,173.9,184.8；MS((M+H)⁺) m/z 259. Analytically calculated values C46.51, H5.46, N10.85; found C46.43, H5.41, N10.54.

(RS) -2-amino-3- (3-butoxy-5-carboxyisoxazol-4-yl) propionic acid (Compound 11)

Melting point 238-;¹H NMR(D₂o, dioxane, 1M NaOD) d 0.95(t,3H),1.43(se,2H),1.76(qui,2H),2.8(dd,1H),2.91(dd, 1H)),3.44(dd,1H),4.25(t,2H)；¹³C NMR (D2O, dioxane, 1M NaOD) D13.79, 19.30,27.90,31.03,56.39,71.27,109.65,162.39,164.72,172.02,182.92; MS ((M + H)⁺) m/z 273. Analytically calculated C48.53, H5.92, N10.29; found C48.80, H5.99, N10.34.

(RS) -2-amino-3- (3-allyloxy-5-carboxyisoxazol-4-yl) propionic acid (Compound 12)

Melting point 239-;¹H NMR(DMSO-D₆)d 2.93(dd,1H),3.06(dd,1H),3.99(dd,1H),4.73(d,2H),5.29(dd,1H),5.44(dd,1H),6.05(dq,1H)；

furthermore, the following compounds were prepared analogously:

(RS) -2-amino-3- [3- (trans-2-but-en-oxy) -5-carboxyisoxazol-4-yl) ] propionic acid

(RS) -2-amino-3- [3- (3-methyl-2-but-en-oxy) -5-carboxyisoxazol-4-yl) ] propionic acid

Example 7

(RS) -2-amino-3- (2-benzyl-5-carboxy-2, 3-dihydro-3-oxoisoxazol-4-yl) propionic acid, hydrochloride monohydrate (Compound 13)

Reacting (RS) -2- [ (tert-butoxycarbonyl) amino group]A mixture of ethyl-3- (2-benzyl-5-ethoxycarbonyl-2, 3-dihydro-3-oxoisoxazol-4-yl) propionate (0.9g,1.9mmol) and 1M HCl was boiled under reflux for 5 h. The mixture was evaporated to dryness in vacuo (0.56g, 80%): melting point 146-;¹H NMR(DMSO-D₆)δ3.08(dd,1H),3.19(dd,1H),4.17(brs,1H),5.16(s,2H),7.24-7.45(m,5H)；MS((M+H)⁺) m/z 307. Analytically calculated values C46.60, H4.76, N7.77; found C46.88, H4.81, N7.96.

Example 8

(RS) -2-amino-3- (5-benzyloxycarbonyl-3-ethoxyisoxazol-4-yl) propionic acid benzyl ester hydrochloride (Compound 14)

Di-tert-butyl dicarbonate (1.1g,4.9mmol), NaHCO₃A mixture of (1.1g,13mmol) and 1, 4-dioxane (3ml) was added to a solution of (RS) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (1.0g,4.1mmol) in water/1, 4-dioxane (1: 1) (10ml) and the resulting mixture was stirred at room temperature for 16 h. The 1, 4-dioxane was evaporated in vacuo and the aqueous phase was acidified with dilute aqueous HCl. The aqueous phase was extracted with ethyl acetate and the organic extracts were washed with water and brine. Drying (MgSO)₄) Vacuum concentrating and subjecting to flash chromatography (SiO)₂Eluent: ethyl acetate/ethanol/acetic acid (3: 1, 4%)) to obtain (RS) -2- [ (tert-butoxycarbonyl) amino group]-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (1.4g, 100%).

To (RS) -2- [ (tert-butoxycarbonyl) amino group]To a mixture of (1.4g,4.1mmol) of (E) -3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (1.4g,4.1mmol), benzyl bromide (1.4g,8.2mmol) in benzene/tetrahydrofuran (4: 1) was added 1, 8-diazabicyclo [ 5.4.0%]Undec-7-ene (1.3g,8.6mmol) and the resulting mixture was boiled under reflux for 3 h. The mixture was filtered and evaporated in vacuo. Flash chromatography (SiO)₂Eluent: ethyl acetate in heptane (1: 3)) gave (RS) -2- [ (tert-butoxycarbonyl) amino group as an oil]-benzyl 3- (5-benzyloxycarbonyl-3-ethoxyisoxazol-4-yl) propionate (1.9g, 86%).

Reacting (RS) -2- [ (tert-butoxycarbonyl) amino group]A mixture of benzyl-3- (5-benzyloxycarbonyl-3-ethoxyisoxazol-4-yl) propionate (1.9g,3.6mmol) and a saturated solution of HCl in ether (40ml) was boiled under reflux for 2 h. The crystals formed were collected by filtration, stirred with ethyl acetate and collected by filtration (0.53g, 32%): melting point 142-144 ℃;¹HNMR(DMSO-D₆)δ1.32(t,3H),3.17(dd,1H),3.25(dd,1H),4.17-4.32(m,3H),5.09(dd,2H),5.39(s,2H),7.24-7.53(m,10H)；MS((M+H)⁺) m/z 425. Analytically calculated values of C59.93, H5.48, N6.08; found C59.70, H5.49, N6.26.

Example 9

(RS) -2-amino-3- (3-ethoxy-5-ethoxycarbonylisoxazol-4-yl) propionic acid ethyl ester, oxalate salt (Compound 15)

A mixture of (RS) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionic acid (2.0g,8.2mmol) and HCl in solution in ethanol (35ml) was boiled under reflux for 3h to obtain ethyl (RS) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionate. To ethyl (RS) -2-amino-3- (5-carboxy-3-ethoxyisoxazol-4-yl) propionate (0.6g) was added a dilute NaOH solution and the aqueous phase was extracted with ethyl acetate. The organic extracts were washed with brine and dried (MgSO)₄) Filtered and evaporated to dryness in vacuo. The residue was dissolved in acetone (6ml) and a solution of oxalic acid (0.14g,1.6mmol) in acetone (6ml) was added. The precipitate formed was collected by filtration (110mg, 10%): melting point 159-161 deg.C;¹H NMR(DMSO-D₆)δ1.11(t,3H),1.32(t,3H),1.36(t,3H),3.02(dd,1H),3.11(dd,1H),3.98-4.16(m,3H),4.32(q,2H),4.37(q,2H)；MS((M+H)⁺) m/z 301. Analytically calculated C46.15, H5.69, N7.18; found C46.38, H5.69, N7.36.

Example 10

(RS) -2-amino-3- (5-butoxycarbonyl-3-ethoxyisoxazol-4-yl) propionic acid butyl ester, oxalate salt (Compound 16)

The compound was obtained in a similar manner to that described in example 9, using a solution of HCl in butanol. Melting point 120-;¹H NMR(DMSO-D₆)δ0.84(t,3H),0.92(t,3H),1.14-1.31(m,2H),1.31-1.51(m,4H),1.37(t,3H),1.62-1.75(m,2H),3.01(dd,1H),3.13(dd,1H),3.98-4.09(m,3H),4.16-4.36(m,4H)；MS((M+H)^-) m/z 357. Analytically calculated values C51.11, H6.79, N6.28; found C51.06, H6.82, N6.35.

Example 11

[4- (2-amino-3, 4-dioxocyclobut-1-en-1-yl) aminomethyl ] -3-ethoxyisoxazole-5-carboxylic acid (Compound 17)

3-ethoxy-4-methylisoxazole-5-carboxylic acid ethyl ester

Acetyl chloride (25ml,0.35mol) was added to EtOH (250ml) at 0 ℃ and the solution was stirred for 20 minutes at 0 ℃.A solution of 3-ethoxy-4-methylisoxazole-5-carboxylic acid (WO95/12587, A1) (18g,0.10mol) in EtOH (20ml) was added and the resulting mixture was boiled under reflux for 4 h. The mixture was cooled and NaHCO was added₃(200ml) and extracted with diethyl ether (3X 300 ml). The organic extract was dried (MgSO)₄) And concentrated in vacuo to afford the crude title compound (18g, 86%).

4-Bromomethyl-3-ethoxyisoxazole-5-carboxylic acid ethyl ester

Ethyl 3-ethoxy-4-methylisoxazole-5-carboxylate (18g,91mmol), NBS (17.5g,100mmol), dibenzoyl peroxide (1g,4.1mmol) in tetrachloromethane (500ml) was boiled under reflux for 16 h. The mixture was cooled, filtered and concentrated in vacuo to afford the crude title compound (24.5g, 97%).

3-ethoxy-4-phthalimidomethylisoxazole-5-carboxylic acid ethyl ester

A solution of 4-bromomethyl-3-ethoxyisoxazole-5-carboxylic acid ethyl ester (5g,17.9mmol) in DMF (85ml) was added to a suspension of potassium phthalimide (3.6g,19.7mmol) in DMF (125ml) at 90 ℃. The resulting mixture was stirred at 90 ℃ for 40 minutes, then cooled and concentrated in vacuo. Water (250ml) was added and the aqueous phase was extracted with diethyl ether (2X 200 ml). The organic extract was dried over (MgSO)₄) And concentrated in vacuo to give the crude product which was recrystallized (EtOH) to give the title compound (3.70g, 60%): the melting point is 93-94 ℃.

4-aminomethyl-3-ethoxyisoxazole-5-carboxylic acid hydrochloride

A solution of 3-ethoxy-4-phthalimidomethylisoxazole-5-carboxylic acid ethyl ester in 1M NaOH was boiled under reflux for 45 minutes. The mixture was cooled, concentrated HCl added and extracted with ether (3X 400 ml). The organic extracts were concentrated in vacuo, 1M HCl (600ml) was added and boiled under reflux for 1 h. After cooling, the mixture was washed with diethyl ether (3 × 600ml) and concentrated in vacuo to give the crude product which was recrystallised (acetic acid) to give the title compound (1.5g, 82%): melting point 215-.

[4- (2-amino-3, 4-dioxocyclobut-1-en-1-yl) aminomethyl ] -3-ethoxyisoxazole-5-carboxylic acid

To a solution of 4-aminomethyl-3-ethoxyisoxazole-5-carboxylic acid hydrochloride (1.2g,5.4mmol) and 3-amino-4-ethoxy-cyclobut-3-ene-1, 2-dione (0.60g,5.9mmol) in EtOH (300ml) was added 1M NaOH (12 ml). The resulting suspension was stirred at room temperature for 16h, then evaporated in vacuo, water (100ml) added and washed with EtOAc (2X 100 ml). The pH was adjusted to about 3 by the addition of 1M HCl. The precipitate was removed by filtration and recrystallized (water) to provide the target compound as a yellow powder (0.71g, 47%): melting point 236-238 deg.C (decomposition).¹H NMR(DMSO-D₆)d 1.30(t,3H),4.22(q,2H),4.68(bs,2H)。¹³C NMR(DMSO-D₆)δ14.41,35.27,65.54,107.16,159.14,163.54,168.71,169.15,169.73,183.20,183.34；MS((M+H)⁺) m/z 282. Analysis (C)₁₁H₁₁N₃O₆·2.25H₂O) calcd for C41.06, H4.86, N13.06; found C41.16, H4.46, N12.96.

Pharmacology of

The compounds of the present invention were tested according to the following accepted and trusted test methods.

[³H]AMPA binding assay

In this assay, the substitution from the AMPA receptor is measured³H]AMPA ability to determine drug affinity for AMPA receptors.

The experiments were carried out according to the modification of the methods of Honorere, T. and Nielsen, M. (Neurosci. Lett.1985,54, 27-32). The experiments were performed in the presence of KSCN. This means that only [ 2 ]³H]AMPA high affinity binding sites are labeled.

The membrane preparations used were obtained according to the method in Rancom, R.W. and Stec, J.neurohem.1988, 51, 830-836.

Cortical wedge (cortical wedge) model

The cortical wedge model is an assay that tests rat brain sections in vitro to verify the efficacy of ligands in different glutamate-receptors and to evaluate the pharmacological profile of the ligand (i.e., agonist/antagonist properties). The assay was performed as described by Harrison, N.L. and Simmonds, M.A.Br.J.Pharmacol.1985,84,381-391, modified as Wheatley, P.L.Br.J.Pharmacol.1986,87, 159P.

TABLE 1 cortex wedge model

Compound (I)	Distribution pattern	EC50(μM)	PKi	Receptor subtype
					1	Agonists	1.2		AMPA
2	Agonists	4.8		AMPA
					(S)-2	Agonists	4.4		AMPA
(R)-2	Antagonists		3.28	AMPA
					3	Agonists	40.0		AMPA
8	Agonists	2000		AMPA
					10	Agonists	80		AMPA

11	Partial agonists	325		AMPA
					12	Agonists	40		AMPA
13	Antagonists		3.5	NMDA
					17	Antagonists		3.3	NMDA

Results

The compounds are found to be ligands for Excitatory Amino Acid (EAA) receptors. Some of the compounds were found to be agonists at AMPA receptors, while others were found to be selective AMPA or NMDA receptor antagonists. The compounds show activity in the μ M range.

Formulation examples

The pharmaceutical formulations of the present invention may be prepared by methods conventional in the art.

For example: tablets may be prepared by mixing the active ingredient with conventional adjuvants and/or diluents and subsequently compressing the mixture in a conventional tabletting machine. Examples of adjuvants or diluents include: corn starch, lactose, talc, magnesium stearate, gelatin, lactose, gums, and the like. Any other adjuvants or added coloring agents, fragrances, preservatives and the like may be used provided they are compatible with the active ingredient.

Solutions for injection may be prepared by dissolving the active ingredient and possible additives in a portion of the vehicle, preferably sterile water, adjusting the solution to the desired volume, sterilizing the solution and filling in suitable ampoules or vials. Any suitable additive conventionally used in the art, such as tonicity agents, preservatives, antioxidants, and the like, may be added.

Claims (22)

1. (3-alkoxyisoxazol-4-yl) -substituted 2-aminocarboxylic acid derivatives having the general formula i or ii:orWherein R is₁Is hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, cycloalk (en) yl-C_1-6Alk (en/yn) yl or phenyl-C_1-6Alkane (alkene)A/alkynyl) group, said phenyl group being CF₃Halogen, C_1-6Alkyl or C_1-6Alkoxy is optionally substituted; a is a bond or selected from C_1-6Alkylene radical, C_2-6Alkenylene or C_2-6Spacer groups for alkynylene and cycloalkylene groups; b is-CR_a(NR_bR_c)-COOR₅Group, wherein R_a-R_cAre each hydrogen or C_1-6Alkyl, and R₅Is defined as R₁Or pivaloyloxymethyl, or B is a group of formula III:wherein R is₂、R₃And R₄Are respectively selected from: a) hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, cycloalk (en) yl-C_1-6Alk (en/yn) yl, phenyl-C_1-6Alkyl, thienyl-C_1-6Alkyl, and b) C in which one or more carbon atoms are replaced by N, O and/or S_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, or R₃And R₄Are connected to form C₂-C₆Alkylene radical, C₂-C₆Alkenylene or C₂-C₆Alkynylene, or R₄And R₂Linked to form C optionally mono-or di-substituted by hydroxy or methyl₁-C₃Alkylene radical, C₂-C₃Alkenylene or C₂-C₃Alkynylene, or form CH₂-O-CH₂(ii) a E is COOR₆Wherein R is₆Is defined as R₅Or E is tetrazol-5-yl, 1,2, 4-triazol-3-yl or 1,2, 3-triazol-4-yl; x is O or S; y is O or S.

2. A compound according to claim 1, characterised in that it has formula i.

3. A compound according to claim 1, characterised in that it has formula ii.

4. Any of claims 1-3A compound of which E is COOR₆Group, wherein R₆Is other than hydrogen, preferably C_1-6Alkyl phenyl-C_1-6An alk (en/yn) yl group or a pivaloyloxymethyl group.

5. A compound according to any one of claims 1 to 3, characterized in that E is COOH.

6. A compound according to any one of claims 1 to 3, characterized in that E is tetrazol-5-yl, 1,2, 4-triazol-3-yl or 1,2, 3-triazol-4-yl.

7. A compound according to any one of claims 1 to 6, characterized in that B is of the formula CR_aNR_bR_c-COOR₅A group.

8. A compound of claim 7, characterized in that R_bAnd R_cIs hydrogen and R_aIs hydrogen or C_1-6Alkyl, preferably hydrogen or methyl.

9. A compound according to claim 7 or 8, characterized in that R₅Is hydrogen.

10. A compound according to claim 7 or 8, characterized in that R₅Other than hydrogen, preferably C_1-6Alkyl, phenyl-C_1-6An alk (en/yn) yl group or a pivaloyloxymethyl group.

11. A compound according to any one of claims 1 to 6, characterised in that B is a group of formula III.

12. A compound of claim 11, characterized in that R₂、R₃And R₄Is hydrogen or C_1-6Alkyl, or R₄And R₂Are connected to form C₁-C₃An alkylene group.

13. A compound according to claim 12, characterized in that R₂、R₃And R₄Are each hydrogen.

14. Compounds according to any of claims 1 to 13, characterized in that X is oxygen.

15. Compounds according to any of claims 1-13, characterized in that X is sulfur.

16. Compounds according to any of claims 1-15, characterized in that Y is oxygen.

17. Compounds according to any of claims 1-15, characterized in that Y is sulfur.

18. Compounds according to any of claims 1-17, characterized in that R₁Is C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6Alkynyl, preferably methyl, ethyl, propyl or propargyl.

19. Compounds according to any of claims 1 to 18, characterized in that a is a bond or C₁-C₃Alkylene, preferably methylene.

20. A compound according to claim 2, characterized in that A is a bond or C₁-C₃Alkylene, B being-CH (NH)₂) -COOH or a group of formula III, wherein R₃、R₄And R₂Are each hydrogen, X and Y are both oxygen, and R is₁Is C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6Alkynyl, preferably methyl, ethyl, propyl or propargyl.

21. A pharmaceutical composition, characterised in that it comprises a compound according to any one of claims 1 to 20 together with a pharmaceutically acceptable carrier or diluent.

22. The use of a compound according to any one of claims 1-20 for the preparation of a pharmaceutical composition for the treatment of cerebral ischaemia, huntington's disease, epilepsy, parkinson's disease, alzheimer's disease, schizophrenia, pain, depression or anxiety.