Patent Grant
6984754
The invention relates to novel aliphatic amino carboxylic and amino phosphoric acids, amino nitrites and amino tetrazoles, their salts, compositions containing such compounds and to the use of such compounds as cellular rescue agents in human and veterinary medicine.
Neurodegenerative disorders (for example, chronic disorders such as Alzheimer's disease, Parkinson'disease, Huntington's disease, Picks's disease, amyotrophic lateral sclerosis and glaucoma as well as acute injuries like stroke, head trauma, Bell's palsy and spinal cord injuries) are now believed to involve apoptotic processes.
Deprenyl, the aliphatic propargylamines, their respective desmethyl analogues and rasagiline have been shown to protect and rescue damaged neurons in several models of degeneration [1-16]. The propargyl group was thought to be a requirement for the protective or rescuing activity of these drugs. Previous studies have examined the N-demethylation and/or depropargylation of these drugs [7, 171].
It has been known for some time that some aliphatic and aromatic acetylenic compounds react with P450 enzymes. One of these reactions results in oxidation of the terminal carbon of the acetylenic functional group to form the corresponding acid [18-20]. The possibility of oxidation of the N-acetylene group of the aliphatic propargylamines to form carboxylic acid metabolites has not been previously addressed. The potential of related acidic compounds (the amino phosphonic acids and amino tetrazoles) and precursors to such compounds (amino nitrites and amino esters) as antiapoptotic agents had also not been previously considered.
The present inventors have found that aliphatic amino carboxylic and amino phosphonic acids, amino nitrites and amino tetrazoles are antiapoptotic agents and may be useful as cellular rescue agents in human and animal treatments.
The present invention relates to a compound of the general formula (I),
wherein:
R1 is (CH2)mCH3 where m is 0 or an integer in the range from 1 to 16, or an alkenyl, alkynyl, alkoxy, alkylthio, or alkyl sulfinyl group having from 2 to 17 carbon atoms. R1 may be unsubstituted or substituted with at least one of the substituents selected from hydroxy, aldehyde, oxo, lower acyloxy, halogen, thio, sulfoxide, sulfone, phenyl, halogen-substituted phenyl, hydroxy-substituted phenyl, cycloalkyl having from 3 to 6 carbon atoms and heterocyclic substituents having between 3 and 6 atoms, of which from 1 to 3 are heteroatoms selected from O, S and/or N,
R2=H, CH3 or CH2CH3,
R3=H or CH3,
R4=H or CH3,
R5=lower alkyl having from 1 to 5 carbon atoms,
n is an integer in the range from 1 to 3,
and X is carboxyl (COOH), carbalkoxy (COOR5), cyano (C≡N), phosphonic acid (PO3H2), phosphonate ester (PO3[R5]2) or 5-tetrazole, and the salts thereof, particularly pharmaceutically acceptable salts thereof.
Compounds of the general formula I in which R1, R2 and R3 differ from one another are chiral. It has been found that the R-enantiomers of some of these classes or sub-classes of compounds (and the S-enantiomers for other classes or subclasses) are useful as cellular rescue agents for the treatment and prevention of diseases in which cell death occurs by apoptosis, such as in many neurodegenerative disorders. For a particular class or subclass of compounds the inactive enantiomer does not prevent apoptosis but can antagonize the antiapoptotic actions of the active enantiomers, and are useful as research tools. The achiral compounds also display cellular rescue properties.
The present invention also relates to the use of compounds of general formula I, as defined above, and salts thereof, as cellular rescue agents for the treatment and prevention of diseases in which cell death occurs by apoptosis including but not limited to, stroke, head trauma, Bell's palsy, spinal cord and other nerve crush injuries, Alzheimer's disease, Parkinson's disease, Pick's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, cardiac myopathies, nephropathy, retinopathy, diabetic complications, glaucoma, as well as idiopathic neuropathies. Accordingly, the present invention provides a method for treating a condition wherein cell death occurs by apoptosis comprising administering an effective amount of a compound of the formula I to an animal in need thereof.
The present invention relates to compounds of the general formula I,
wherein:
R1 is (CH2)mCH3 where m is 0 or an integer in the range from 1 to 16, or an alkenyl, alkynyl, alkoxy, alkylthio, or alkyl sulfinyl group having from 2 to 17 carbon atoms. R1 may be unsubstituted or substituted with at least one of the substituents selected from hydroxy, aldehyde, oxo, lower acyloxy, halogen, thio, sulfoxide, sulfone, phenyl, halogen-substituted phenyl, hydroxy-substituted phenyl, cycloalkyl having from 3 to 6 carbon atoms and heterocyclic substituents having between 3 and 6 atoms, of which from 1 to 3 are heteroatoms selected from O, S and/or N,
R2=H, CH3 or CH2CH3,
R3=H or CH3,
R4=H or CH3,
R5=lower alkyl having from 1 to 5 carbon atoms,
n is an integer in the range from 1 to 3,
and X is carboxyl (COOH), carbalkoxy (COOR5), cyano (C≡N), phosphonic acid (PO3H2), phosphonate ester (PO3[R5]2) or 5-tetrazole, and the salts thereof.
In a preferred embodiment, the present invention provides a compound of the Formula I (as described above) wherein
R1=(CH2)mCH3 where m is 0 or an integer in the range from 1 to 16,
R2=CH3,
R3=H,
R4=H or CH3,
R5=lower alkyl having from 1 to 5 carbon atoms,
n is an integer in the range from 1 to 3,
and X is carboxyl (COOH) or carbalkoxy (COOR5), cyano (C≡N), phosphonic acid (PO3H2), phosphonate ester (PO3[R5]2) or 5-tetrazole, or a pharmaceutically acceptable salt thereof.
Preferred compounds of the invention include:
2-(2-Propylamino)acetic acid;
2-(1-Hexylamino)acetic acid;
(S)-2-(2-Heptylamino)acetic acid;
3-(2-Propylamino)propionic acid;
3-(1-Hexylamino)propionic acid;
(R)-3-(2-Heptylamino)propionic acid;
2-(2-Propylmethylamino)acetic acid;
2-(1-Hexylmethylamino)acetic acid;
(S)-2-(2-Heptylmethylamino)acetic acid;
3-(2-Propylmethylamino)propionic acid;
3-(1-Hexylmethylamino)propionic acid;
(R)-3-(2-Heptylmethylamino)propionic acid;
2-(2-Propylamino)acetonitrile;
(R)-2-(2-Pentylamino)acetonitrile;
2-(1-Hexylamino)acetonitrile;
(S)-2-(2-Heptylamino)acetonitrile;
(R)-3-(2-Heptylamino)propionitrile;
2-(2-Propylmethylamino)acetonitrile;
(R)-2-(2-Pentylmethylamino)acetonitrile;
2-(1-Hexylmethylamino)acetonitrile;
(S)-2-(2-Heptylmethylamino)acetonitrile;
(R)-3-(2-Heptylmethylamino)propionitrile;
2-(2-Propylamino)ethanephosphonic acid;
(R)-2-(2-Pentylamino)ethanephosphonic acid;
(S)-2-(2-Heptylamino)ethanephosphonic acid;
2-(2-Propylmethylamino)ethanephosphonic acid;
(S)-2-(2-Heptylmethylamino)ethanephosphonic acid; and
(R)-2-(2-Heptylamino)ethane-5-tetrazole.
Compounds of formula I which are optically pure enantiomers are novel. Achiral and racemic compounds are novel with the exception of the following exclusions, although their properties as cellular rescue agents are not known.
Particularly preferred are those of the R and S configurations, depending on the subclass of compound. Methods of resolving the racemates are known. Suitable methods include fractional crystallization, derivatization of the racemate followed by stereospecific enzymatic removal of the attached group, and chromatography. It is preferred, however, to make chiral compounds of formula I from chiral reactants, using reactions that do not destroy the stereochemistry. When referring to enantiomers, it is preferred that an enantiomer shall not contain more than 3% of the compound of the opposite configuration. It is particularly preferred that an enantiomer contain less than 1% of the corresponding enantiomer of the opposite configuration.
Some of the inactive enantiomers for a given class or subclass of compounds strongly antagonize the antiapoptotic actions of the active enantiomers, and are useful as research tools.
The invention extends to salts of compounds of formula I. For administration the salts should be pharmaceutically acceptable, but other salts may be useful, for example, in synthesis or for purification.
Methods of Preparing Compounds of the Invention
Compounds of the invention can be prepared in a variety of different ways. One process involves the addition of a primary amine of formula (II)
across the olefinic double bond of α,β-unsaturated carboxylic acid esters (such as methyl acrylate), of vinylphosphonic acids esters or of α,β-unsaturated nitrites (such as acrylonitrile) of formula (III)[21],
H2═CH—X (III)
wherein X is a polarized group such as carboxylic ester, phosphonic ester or nitrile to give the corresponding N-alkylamino propionic esters, ethanephosphonic esters and propionitriles.
It is possible to use an amine of the formula (II) in which R1 differs from R2 in the form of a racemate and to separate the enantiomers subsequently, but it is preferred to use an amine in substantially enantiomerically pure form.
Chiral primary amines (R- or S-forms) were prepared by recrystallization of the tartrates of the racemates from methanol [22], except for (R) and (S)-2-butylamines which were purchased from Aldrich Chemical Co. Enantiomeric purity was determined using gas chromatography with a chiral capillary column and chiral derivatizing agent [23]. In analogy to the above, chiral primary amines can also be added to the C—C double bond of methyl acrylate, diethyl vinylphosphonate or acrylonitrile to give the corresponding chiral methyl N-alkylaminopropionate, chiral diethyl N-alkylaminoethanephosphonate or chiral N-alkylaminopropionitrile.
In one embodiment, an excess of a chiral amine adds to the olefinic bond of acrylonitrile, as depicted in the following scheme.
Excess Amine and Acrylonitrile
where R1=hydrogen, methyl or ethyl and
R2=methyl, ethyl, propyl, butyl, pentyl, hexyl.
In another embodiment an excess of the amine can be added to the olefinic double bond of diethyl vinylphosphonate to give the corresponding diethyl N-alkylaminoethanephosphonate. Hydrochloric acid hydrolysis of the aminophosphonic diester yields the corresponding aminophosphonic acid as the hydrochloride salt.
In yet another embodiment an excess of the amine can be added to the olefinic double bond of methyl acrylate to give the corresponding methyl N-alkylaminopropionate [24]. Hydrolysis of the carboxylate ester with hydrochloric acid produces an amino acid as its hydrochloride salt, in accordance with the following reaction scheme:
Hydrolysis of Esters
A second process to give compounds of the invention which contain only one carbon atom between the nitrogen atom and the functional group involves condensing a primary aliphatic amine of formula (II) with a bromomethyl reactant of formula (IV)
LCH2X (IV)
wherein L is a leaving group, for example a halide, tosyl or mesyl group (bromide is preferred), and X is carboalkoxy (carbomethoxy or carbethoxy is preferred), nitrile or phosphodiethoxy. Again, the amine can be used in racemic or enantiomerically pure form. In one preferred embodiment, two equivalents of the amine are reacted with one equivalent of the bromomethyl analogue of formula (IV) to form the required aminomethanecarboxylate (glycine) ester and the hydrobromide salt of the amine, which can be isolated and reused, in accordance with the following reaction scheme.
Two Equivalents of Amine and One Equivalent of Bromomethyl Ester in Ether
In another preferred embodiment, two equivalents of the amine are reacted with one equivalent of the bromomethyl analogue of formula (IV) to form the required aminomethane nitrile (aminoacetonitrile) and the hydrobromide salt of the amine, which can also be isolated and reused, in accordance with the following reaction scheme.
Two Equivalents of Amine and One Equivalent of Bromoacetonitrile in Ether
Another route to compounds of the invention involves trifluoroacetylation of the amine, followed by reaction with bromomethyl, bromoethyl or bromopropyl analogues of esters of carboxylic acids or nitriles of formula (V).
L(CH2)nX (V)
wherein L is a leaving group, for example a halide, tosyl or mesyl group (bromide is preferred), n is 1, 2 or 3 and X is carboalkoxy (methoxy or ethoxy is preferred), nitrile or phosphodiethoxy.
The amine can be used in racemic or enantiomerically pure form. The amine is reacted with trifluoroacetic anhydride or a trifluoroacetyl halide in an inert organic solvent, for instance a chlorinated hydrocarbon such as methylene dichloride, chloroform or carbon tetrachloride, and a base, for example an organic base such as triethylamine. The N-trifluoroacetylamine is then refluxed with a bromo compound of formula (V), suitably in the presence of a base such as potassium t-butoxide in a polar solvent, for example acetonitrile, and in the presence of a crown ether catalyst, for example 18-crown-6. The product of this reaction is then hydrolyzed, suitably by reaction with a base such as an alkali metal hydroxide in an alcoholic solution.
Tetrazole compounds of the invention were prepared by the addition of azide ion to a nitrile triple bond [25]. Again, the amine can be used in racemic or enantiomerically pure form. In one preferred embodiment azide ion is generated by the slow addition of aluminum chloride to a solution/suspension of sodium azide in tetrahydrofuran at 0° C. To the resulting solution of aluminum azide is added the nitrile (prepared as in the pathways described above) in tetrahydrofuran at room temperature, followed by stirring for 2 hours and then gentle refluxing for several hours, according to the following scheme.
Addition of Azide Ion to Nitrile
N-Methylation of the various secondary amines described above is achieved by reductive methylation using formaldehyde and sodium phosphite [26]. Again, the amine can be used in racemic or enantiomerically pure form. An amino carboxylate ester or amino nitrile (as the hydrochloride salt or free base) is dissolved in aqueous sodium dihydrogen phosphite and reacted with an excess of 37% aqueous formaldehyde at 60° C. for 15 min. The product is isolated by ether extraction after basification of the ice-cold reaction mixture with sodium hydroxide. A preferred embodiment is shown in the following reaction scheme.
N-Methylation of an Amino Ester
Yet another preferred embodiment is shown in the following reaction scheme.
N-Methylation of an Amino Nitrile
Therapeutic Methods of the Invention
As hereinbefore mentioned, the compounds of the formula I (as described above) have many therapeutic applications.
In one aspect, the present invention provides a method for treating or preventing a condition wherein cell death occurs by apoptosis comprising administering an effective amount of a compound of the formula I to an animal in need thereof.
The term “effective amount” as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired result. The term “animal” as used herein includes all members of the animal kingdom including humans.
In another aspect, the present invention provides a use of a compound of the formula I to treat or prevent a condition wherein cell death occurs by apoptosis.
In yet a further aspect, the present invention provides a use of a compound of the formula I to prepare a medicament to treat or prevent a condition wherein cell death occurs by apoptosis.
Conditions wherein cell death occurs by apoptosis includes, but are not limited to, stroke, head trauma, Bell's palsy, spinal cord and other nerve crush injuries, Alzheimer's disease, Parkinson's disease, Pick's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, cardiac myopathies, nephropathy, retinopathy, diabetic complications, glaucoma, as well as idiopathic neuropathies.
Pharmaceutical Compositions
The compounds of the general formula I may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. The compositions containing the compounds of the invention can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
The active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration.
In oral administration, the compounds may be administered as tablets, coated tablets, gelatine capsules, capsules, cachets, and solutions or suspensions to be taken orally. The compounds can also be administered parenterally or through any other suitable administrative route such as intravenous, subcutaneous, depot injections, intramuscular, intrathecal, intraventricular, intra-articular, rectal (suppository, enema), sublingual, buccal, intra-ocular, intra-vitreo, transdermal (skin patch), nasal drops (nebulizer, insufflation), liposomal delivery systems. The daily dosage could likely range from 1 to 100 mg.
Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising a compound of general formula (I) in admixture with a suitable diluent or carrier. The compound may be achiral or a substantially enantiomerically pure R- or S-enantiomer, or a pharmaceutically acceptable salt thereof, in admixture with pharmaceutically acceptable diluents or carriers. The compositions are useful in the treatment or prevention of conditions in which cell death occurs by apoptosis.
In another aspect, the present invention provides a commercial package containing as active ingredient a compound of the formula I, or a pharmaceutically acceptable salt thereof, together with instructions for its use for the treatment or prevention of a condition in which cell death occurs by apoptosis.
In Vitro Protocol for Assessing the Antiapoptotic Capacity of Various Compounds in Cerebellar Granule Cells
The following biological data demonstrate that the compounds of the invention exhibit antiapoptotic properties.
Cultures of cerebellar granule cells (CGC) can be induced into apoptosis by the addition of a high concentration of cytosine arabinoside (AraC) [27]. It has been shown that this is a p53 dependent apoptosis [28]. We have measured the antiapoptotic effect of some amino acids, amino esters, amino phosphonic acids, amino nitriles and amino tetrazoles using this system.
Cultures of CGC were obtained from 6-8 day old Wistar rat pups. Cultures were grown on glass in 35 mm petri dishes for three days and then used for experiments. Aliquots (20 ul) of drug solutions (AraC, anti-apoptotic drugs, drug vehicles) were added to the medium of the culture. 24 Hours later the cultures were fixed with FAM, and stained with bis-benzamide. Normal and apoptotic nuclei were counted to a total of 90-120 cells per culture. The optimum concentration of AraC was found to be 300 uM.
The results are summarized in Tables 1-4.
Detailed Synthetic Procedures
The following non-limiting examples of synthetic procedures are provided.
Methyl 3-(1-hexylamino)propionate hydrochloride [Methyl N-(1-hexyl)-β-alaninate]
To ice-water-cooled 1-hexylamine (7.58 g, 75 mmol) was added dropwise methyl acrylate (4.3 g, 50 mmol). After completion of the addition the temperature was allowed to rise to room temperature and the reaction solution was stirred for 5 hours, then refluxed for 90 minutes. After stirring overnight at 20° C. the product, methyl 3-(1-hexylamino)propionate, was distilled (b.p.=85-88° C./2 mm; lit. b.p. 80-84° C./0.5 mm) as a clear, colorless liquid in a yield of 55%. The hydrochloride salt was prepared by the addition of methanolic HCl (15%) to an ethereal solution of the free base; m.p.=204-205° C. (lit. m.p. 190-192° C.).
Mass spectrum: m/e: 187 (M+); 116 (M—C5H11); 84.
(R)-Methyl 3-(2-heptylamino)propionate hydrochloride [(R)-Methyl N-(2-heptyl)-β-alaninate]
Prepared according to Example 2. The hydrochloride salt was recrystallized from methanol/ether; m.p.=89.5-90.5° C.
Mass spectrum: m/e: 201 (M+); 186 (M—CH3); 130 (M—C5H11)
Methyl 3-(2-propylamino)propionate hydrochloride [Methyl N-(2-propyl)-β-alaninate]
Prepared according to Example 2. The hydrochloride salt was crystallized from methanol/ether; m.p.=108-110° C. (lit. m.p. 107° C.).
Mass spectrum: m/e: 145 (M+); 130 (M—CH3); 98; 72; 56.
(R)-3-(2-Heptylamino)propionic acid hydrochloride [(R)-N-(2-heptyl)-β-alanine]
(R)-3-(2-Heptylamino)propionitrile (75 g, 44.6 mmol) (prepared according to Example 18) was refluxed with concentrated hydrochloric acid (50 mL) for 4 h. After filtration of insoluble ammonium chloride, the aqueous solution was rotary evaporated to dryness. The residue was stirred with dichloromethane (120 mL) for 2 hours and the insoluble ammonium chloride was filtered. The filtrate was concentrated, filtered again and then evaporated to give a colorless oil which crystallized on cooling. The yield of white hygroscopic product was quantitative; m.p.=57-58° C.
Mass spectrum: m/e: 187 (M+); 172 (M—CH3); 116 (M—C5H11); 72.
1H-NMR (D2O, 300 MHz): 0.72 (t, 3H); 1.18 (d, 3H); 1.1-1.3 (m, 6H); 1.42/1.60 (2m, 1H each); 2.67 (t, 2H); 3.17 (m, 3H; αCH & CH2COOH).
The title compound can also be prepared by hydrochloric acid hydrolysis of the methyl ester (prepared according to Example 3).
(S)-3-(2-Heptylamino)propionic acid hydrochloride [(S)-N-(2-heptyl)-β-alanine]
Prepared according to Example 5. The hydrochloride salt, m.p.=56-58° C., is hygroscopic.
Mass spectrum: m/e: 187 (M+); 172 (M—CH3); 128 (M—CH2COOH); 116 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.75 (t, 3H); 1.18 (d, 3H); 1.1-1.3 (m, 6H); 1.45/1.60 (2m, 1H each); 2.68 (t, 2H); 3.20 (m, 3H; αCH & CH2COOH).
3(1-Hexylamino)propionic acid [N-(1-hexyl)-β-alanine]
Methyl 3-(1-hexylamino)propionate hydrochloride (see Example 2 for preparation) was hydrolyzed by refluxing in 2N HCl for 24 hours followed by evaporation to dryness; m.p.=95-97° C. (no lit. value).
Mass spectrum: m/e: 173 (M+); 102 (M—C5H11); 84; 72.
1H-NMR (D2O, 300 MHz): 0.73 (t, 3H); 1.17 (m, 6H); 1.52 (m, 2H); 2.68 (t, 2H); 2.93 (t, 2H); 3.18 (t, 2H).
3-(2-Propylamino)propionic acid hydrochloride [N-(2-propyl)-β-alanine]
Prepared by hydrolysis of the ester (Example 7). The product is a white powder, m.p.=154-155° C. (no lit. value).
Mass spectrum: m/e: 131 (M+); 116 (M—CH3); 98; 56.
1H-NMR (D2O, 300 MHz): 1.20 (d, 6H); 2.70 (t, 2H); 3.19 (t, 2H); 3.32 (m, 1H).
Ethyl 2-(2-propylamino)acetate hydrochloride [Ethyl N-(2-propyl)glycinate]
To a solution of 2-propylamine (4.4 g, 75 mmol) in ether (100 mL) was added ethyl bromoacetate (6.26 g, 37.5 mmol). The solution was stirred at 20° C. for 3 days. The precipitated 2-propylamine hydrobromide was filtered and the filtrate rotary evaporated to give 5.4 g a clear pale yellow liquid (crude yield=99%). The hydrochloride salt was prepared and recrystallized from ethanol/ether; m.p.=120-121° C. (no lit. value).
Mass spectrum: m/e: 145 (M+); 130 (M—CH3); 72 (M—COOC2H5).
2-(2-Propylamino)acetic acid hydrochloride [N-(2-Propyl)glycine]
The sample was prepared from the nitrile (prepared according to Example 25) in 72% yield using a hydrolysis procedure similar to that described in Example 12. The hydrochloride salt was recrystallized from acetone; m.p.=180-184° C. (lit. m.p.203-204.5° C.; 182-183° C.).
Mass spectrum: (CI). 118 (M+1); 102 (M—CH3); 72 (M—CO2H); (EI). 117 (M+); 102 (M—CH3); 72 (M—COOH).
1H-NMR (D2O, 300 MHz): 1.19 (d, 6H); 3.33 (m, 1H); 3.79 (s, 2H).
2-(1-Hexylamino)acetic acid hydrochloride [N-(1-Hexyl)glycine] 1HxActAc
The sample was prepared from the nitrile (procedure analogous to that described in Example 10) in 72% yield using a hydrolysis procedure similar to that described in Example 12. The hydrochloride salt was recrystallized from acetone; m.p.=162-164° C. (lit. m.p. 215-218° C.).
Mass spectrum: m/e: 159 (M+); 114 (M—CO2H); 88 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.72 (t, 3H); 1.20 (m, 6H); 1.55 (m, 2H); 2.96 (t, 2H); 3.77 (s, 2H).
(R)-2-(2-Heptylamino)acetic acid hydrochloride [(R)-N-(2-Heptyl)glycine]
A solution of (R)-2-(2-heptylamino)acetonitrile (1.20 g, 7.79 mmol) (prepared according to Example 23) in concentrated HCl (12 mL) and water (5 mL) was heated for 48 hours at 90° C. After cooling to room temperature, the reaction mixture was concentrated to dryness, filtered, and washed with ethanol to remove NH4Cl. The resulting filtrate was concentrated to give a crude product (1.40 g). A solution of the crude product (0.60 g) in concentrated HCl (20 mL) and water (10 mL) was heated for 24 hours at 90° C. After cooling to room temperature, the reaction mixture was taken to dryness and triturated in ether to give the hydrochloride salt as a crystalline solid (0.60 g, overall 37% yield); m.p.=162-164° C.
Mass spectrum: m/e: (CI). 174 (M+1); 158 (M—CH3); 102 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.74 (t, 3H); 1.18 (d, 3H); 1.1-1.3 (m, 6H); 1.45/1.60 (2m, 1H each); 3.21 (m, 1H); 3.79 (s, 2H).
(S)-2-(2-Heptylamino)acetic acid hydrochloride [(S)-N-(2-Heptyl)glycine]
The product was prepared as described in Example 12 in 72% overall yield by hydrolysis of the nitrile hydrochloride salt; m.p.=161-163° C.
Mass spectrum: m/e: (CI). 174 (M+1); 156 (M—OH); 128 (M—CO2H).
1H-NMR (D2O, 300 MHz): 0.73 (t, 3H); 1.18 (d, m; 9H); 1.47, 1.60 (2m, 1H each); 3.21 (m, 1H); 3.78 (s, 2H).
Methyl 3-(2-propylmethylamino)propionate hydrochloride [Methyl N-(2-propyl)-N-methyl-βalaninate]
To a solution of methyl 3-(2-propylamino)propionate hydrochloride (Example 10) (0.907 g; 5 mmol) in 1N sodium dihydrogen phosphite (25 mL) was added 37% formaldehyde (2.1 mL, 23 mmol). The solution was stirred at 60° C. for 10 min, then cooled in an ice-water bath and basified with 10% sodium hydroxide (10 mL). The resulting solution was saturated with sodium chloride (9 g) and immediately extracted with ether (3×15 mL). The combined filtrates were dried over anhydrous magnesium sulfate and evaporated to dryness to give a clear colorless liquid. The hydrochloride salt, prepared by the addition of methanolic HCl to an ether solution of the free base, precipitated as a viscous oil in 83% yield.
Mass spectrum: m/e: 159 (M+); 144 (M—CH3); 86 (M—CH2COOCH3).
(R)-Methyl 3-(2-heptylmethylamino)propionate hydrochloride [(R)-Methyl N-(2-heptyl)-N-methyl-β-alaninate]
The hydrochloride salt was obtained in 93% yield as a colorless viscous liquid.
Mass spectrum: m/e: 215 (M+); 200 (M—CH3); 144 (M—C5H11).
(R)-3-(2-Heptylmethylamino)propionic acid hydrochloride [N-(2-Heptyl)-N-methyl-β-alanine]
The hydrochloride salt precipitated as a colorless, viscous oil.
Mass spectrum: m/e: 201 (M+); 186 (M—CH3); 130 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.72 (t, 3H); 1.18 (m, 9H); 1.47, 1.59 (2m, 1H each); 2.65 (d, 3H); 2.74 (t, 2H); 3.11 (m, 1H); 3.34 (m, 2H).
(R)-3-(2-Heptylamino)propionitrile hydrochloride
To ice-water-cooled (R)-2-heptylamine (99.4% R) (9.28 g, 80.7 mmol) was added dropwise acrylonitrile (2.85 g, 3.543 mL, 54 mmol). After completion of the addition the temperature was allowed to rise to room temperature and the reaction solution was stirred for 5 hours, then refluxed for 90 minutes. After stirring overnight at 20° C. the product, (R)-3-(2-heptylamino)propionitrile, was distilled as a clear, colorless liquid, b.p.=101-102° C./2 mm, with a yield of 85%. The hydrochloride salt was prepared by the addition of ethanolic HCl (15%) to an ethereal solution of the free base; m.p.=134-135° C.
Mass spectrum: m/e: (CI) 169 (M+H)+; 153 (M—CH3); 97 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.73 (t, 3H); 1.20 (d, m; 9H); 1.45, 1.61 (2m, 1H each); 2.85 (t, 2H); 3.3 (m, 3H).
Elemental Analysis: Calculated: % C=58.66; % H=10.34; % N=13.68.
Found: % C=58.73; % H=10.53; % N=13.47.
The starting material, (R)-2-heptylamine, was resolved from the racemate by repeated recrystallizations of its L-tartrate salt from methanol. Eight recrystallizations using a ratio of volume (of methanol) to weight (of tartrate salt) of 2.4 to 2.6 (increasing as optical purity increased) gave the R-enantiomer with an optical purity of 99.4% R. The optical purity was assessed by derivatization with freshly prepared chiral reagent (S)-N-trifluoroacetylpropyl chloride and then gas chromatography on a chiral column [23].
(S)-3-(2-Heptylamino)propionitrile hydrochloride
(S)-2-Heptylamine as its D-tartrate salt was prepared by recrystallization of the racemate (S-enriched, isolated and prepared from the combined filtrates of the R-enantiomer L-tartrate recrystallizations described in Example 17). The optical purity was 99.4% S. S-2HECN was prepared in 85% yield (b.p. 99-100° C./2 mm) as described for the R-enantiomer in Example 17. The hydrochloride salt was recrystallized from methanol/ether; m.p.=133-134° C.
Mass spectrum: m/e: (CI) 169 (M+H)+; 153 (M—CH3); 128 (M—CH2CN); 97 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.72 (t, 3H); 1.20 (d, m; 9H); 1.45, 1.62 (2m, 1H each); 2.85 (t, 2H); 3.29 (m, 3H).
Elemental Analysis: Calculated: % C=58.66; % H=10.34; % N=13.68.
Found: % C=58.61; % H=10.10; % N=13.42.
3-(2-Propylamino)propionitrile hydrochloride
The free base, b.p.=94° C./30 mm (lit. b.p. 86-87° C./17 mm), was prepared according to the procedure in Example 17 and then converted to the hydrochloride salt which was recrystallized from methanol/ether; m.p.=145.5-146° C. (no lit. value).
Mass spectrum: m/e: 140 (M+); 97 (M—CH3); 72 (M—CH2CN).
1H-NMR (D2O, 300 MHz): 1.20 (d, 6H); 2.85 (t, 2H); 3.29 (t, 2H); 3.36 (m, 1H).
3-(1-Hexylamino)propionitrile hydrochloride
The hydrochloride salt was recrystallized from ethanol/ether; m.p.=188-189° C. (no lit. value).
Mass spectrum: m/e: 154 (M+); 114 (M—CH2CN); 83 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.72 (t, 3H); 1.18 (m, 6H); 1.53 (m, 2H); 2.87 (t, 2H); 2.96 (t, 2H); 3.29 (t, 2H).
3-(3-Pentylamino)propionitrile hydrochloride
The free base, b.p.=84-85° C./2 mm (no lit. value), was converted to the hydrochloride salt, m.p.=118.5-119.5° C. (no lit. value).
Mass spectrum: m/e: 140 (M+); 111 (M—C2H5); 100 (M—CH2CN); 82; 70.
1H-NMR (D2O, 300 MHz): 0.83 (t, 6H); 1.64 (m, 4H); 2.89 (t, 2H); 3.10 (m, 1H); 3.33 (t, 2H).
3-(t-Amylamino)propionitrile hydrochloride
The free base, b.p.=62-63° C./2 mm (no lit. value), was converted to the hydrochloride salt, m.p.=199-200° C. (no lit. value).
Mass spectrum: m/e: 140 (M+, absent); 125 (M—CH3); 111 (M—CH2CH3)
1H-NMR (D2O, 300 MHz): 0.82 (t, 3H); 1.20 (s, 6H); 1.58 (q, 2H); 2.83 (t, 2H); 3.29 (t, 2H).
(R)-2-(2-Heptylamino)acetonitrile hydrochloride
To a solution of (R)-2-heptylamine (1.90 g, 16.5 mmol) in ether (25 mL) were added anhydrous Na2CO3 (1.60 g, 14.9 mmol), and bromoacetonitrile (0.92 mL, 13.2 mmol). The reaction mixture was stirred at room temperature for 24 hours, and for another 14 hours at 80° C. After cooling to room temperature, the reaction mixture was filtered and the filtrate washed with HCl (3 N, 3×10 mL). The aqueous layer was basified with NaOH (6 N) and extracted with ether (3×25 mL). The resulting ethereal solution was dried over Na2SO4, and evaporated to dryness. The residue was fractionated by flash column chromatography (25% EtOAc/hexane, ether) to give a colorless oil (2.10 g, 82%). The hydrochloride salt was prepared by the addition of ethanolic HCl (15%) to an ethereal solution of the free base; m.p.=152-3° C.
Mass spectrum: m/e: (CI). 155 (M+1); 128 (M—CN); (EI) 139 (M—CH3); 83 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.75 (t, 3H); 1.20 (d, 3H); 1.15-1.35 (m, 6H); 1.47/1.62 (2m, 1H each); 3.35 (m, 1H); 4.18 (s, 2H).
Elemental Analysis: Calculated: % C=56.68; % H=10.04; % N=14.69.
Found: % C=56.81; % H=10.20; % N=14.46.
(S)-2-(2-Heptylamino)acetonitrile hydrochloride
The sample was prepared in 96% yield using the above-described procedure (Example 23). The hydrochloride salt was prepared by the addition of ethanolic HCl (15%) to an ethereal solution of the free base and was recrystallized from ethanol/ether. The salt sublimes during melting; m.p.=140° C.
Mass spectrum: m/e: 154 (M+); 139 (M—CH3).
1H-NMR (D2O, 300 MHz): 0.73 (t, 3H); 1.18 (d, m; 9H); 1.43, 1.59 (2m, 1H each); 3.31 (m, 1H); 4.15 (s, 2H).
Elemental Analysis: Calculated: % C=56.68; % H=10.04; % N=14.69.
Found: % C=56.51; % H=9.71; % N=14.79.
2-(2-Propylamino)acetonitrile hydrochloride
The hydrochloride salt was recrystallized from methanol/ether; m.p.=166-167° C. (lit. m.p. 154-156°).
Mass spectrum: m/e: 98 (M+); 83 (M—CH3); 56.
1H-NMR (D2O, 300 MHz): 1.19 (d, 6H); 3.45 (m, 1H); 4.17 (s, 2H).
2-(1-Hexylamino)acetonitrile hydrochloride
The free base of the sample was prepared in 62% yield using the above-described procedure (Example 23). The hydrochloride salt was prepared by the addition of ethanolic HCl (15%) to an ethereal solution of the free base, and was recrystallized from ethanol/ether; m.p.=114-115° C. (lit. m.p. 84-86° C.).
Mass spectrum: m/e: (CI). 141 (M+1)+; 126 (M—CN).
1H-NMR (D2O, 300 MHz): 0.74 (t, 3H); 1.21 (m, 6H); 1.58 (m, 2H); 3.08 (t, 2H); 4.17 (s, 2H).
(R)-3-(2-Heptylmethylamino)propionitrile hydrochloride
(R)-3-(2-Heptylamino)propionitrile (Example 17) (0.85 g, 5.0 mmol) was dissolved in 1N sodium dihydrogen phosphite (NaH2PO3) (25 mL) and 37% formaldehyde (2.1 mL, 23 mmol) was added. Sufficient dioxan was added to give a clear solution (10 mL). The solution was stirred at 60° C. for 15 min during which time it became cloudy. To the cooled solution was then added 20% sodium hydroxide (20 mL) and sodium chloride (9 g). The basic solution was extracted with ether (3×15 mL). The combined extracts were dried over anhydrous magnesium sulfate and then evaporated to give a clear, colorless liquid in quantitative yield. The hydrochloride salt was prepared by adding methanolic HCl to an ether solution of the free base; m.p.=98-98.5° C. Overall yield was 85%.
Mass spectrum: m/e: 182 (M+); 167 (M—CH3); 142 (M—CH2CN); 111 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.77 (t, 3H); 1.20 (d, 3H); 1.15-1.35 (m, 6H); 1.50/1.62 (2m, 1H each); 2.73 (s, 3H); 2.95 (t, 2H); 3.40 (m, 3H; aCH & CH2CN).
Elemental Analysis: Calculated: % C=60.39; % H=10.60; % N=12.80.
Found: % C=59.55; % H=10.29; % N=13.67.
3-(2-Propylmethylamino)propionitrile hydrochloride
The hydrochloride salt was obtained in 90% yield; m.p.=121-121.5° C. (no lit. value) (see Example 27).
Mass spectrum: m/e: 126 (M+); 111 (M—CH3); 86 (M—CH2CN).
1H-NMR (D2O, 300 MHz): 1.23 (d, 6H); 2.73 (s, 3H); 2.97 (t, 2H); 3.52 (br s, 2H); 3.60 (m, 1H).
3-(t-Amylmethylamino)propionitrile hydrochloride
The hydrochloride salt was obtained in 87% yield; m.p.=137-138° C. (no lit. value) (see Example 27).
Mass spectrum: m/e: 154 (M+); 139 (M—CH3); 125 (M—C2H5); 72.
1H-NMR (D2O, 300 MHz): 0.87 (t, 3H); 1.25 (6H); 1.67 (q, 2H); 2.10 (s, 2H); 2.73 (s, 3H); 2.94 (broad t, 2H).
Elemental Analysis: Calculated: % C=56.68; % H=10.04; % N=14.69.
Found: % C=56.62; % H=10.12; % N=14.62.
3-(1-Hexylmethylamino)propionitrile hydrochloride
The hygroscopic hydrochloride salt was obtained in 75% yield; m.p.=77-78.5° C. (no lit. value) (see Example 27).
Mass spectrum: m/e: 168 (M+); 128 (M—CH2CN); 97 (M—C5H11).
1H-NMR (D2O, 300 MHz): 0.72 (t, 3H); 1.20 (m, 6H); 1.60 (m, 2H); 2.79 (s, 3H); 2.95 (t, 2H); 3.09 (t, 2H); 3.42 (t, 2H).
Diethyl 2-(2-propylamino)ethanephosphonate hydrochloride
To ice-cooled 2-propylamine (0.53 mL, 6.2 mmol) was added dropwise diethyl vinylphosphonate (0.4 mL, 3.1 mmol) under N2. The reaction mixture was stirred for 2 hours at 0° C., for 14 hours at room temperature, and for 10 hours at 100° C. After cooling to room temperature, the reaction mixture was concentrated to give a colorless oil (0.89 g). To a solution of the crude product (0.89 g) in ether (40 mL) was added ethanolic HCl (15%), and then stirred for 2 hours at room temperature. The resulting crystallized hydrochloride salt was filtered, washed with ether to give a white solid (0.76 g, overall 95%); m.p.=96-97° C. (no lit. value).
Mass spectrum: m/e: 208 (M—CH3)+; 180 (M—C3H7)+.
2-(2-Propylamino)ethanephosphonic acid hydrochloride
The hydrochloride salt of the product (starting from the diester, Example 31) was prepared in 32% overall yield using the procedure described in Example 37; m.p.=164-166° C. (no lit. value).
Mass spectrum: m/e: (CI). 168 (M+1)+.
1H-NMR (D2O, 300 MHz): 1.16 (d, 6H); 1.84 (m, 2H); 3.08 (q, 2H); 3.27 (m, 1H).
Diethyl 2-(1-hexylamino)ethanephosphonate hydrochloride
The crude product was prepared in 100% yield using the above-described procedure. The hydrochloride salt of the product was prepared by the addition of ethanolic HCl (15%) to an ethereal solution of the product; the salt is a viscous oil.
Mass spectrum: m/e: 265 (M)+, 194 (M—C5H11)+.
2-(1-Hexylamino)ethanephosphonic acid hydrochloride
The hydrochloride salt was prepared by hydrolysis of the diethyl ester using the method described in Example 37; m.p.=145-150° C. (no lit. value).
Mass spectrum: m/e: too involatile
1H-NMR (D2O, 300 MHz): 0.70 (t, 3H); 1.15 (m, 6H); 1.50 (m, 2H); 1.90 (m, 2H); 2.88 (t, 2H); 3.08 (q, 2H).
Elemental Analysis: Calculated: % C=39.11; % H=8.62; % N=5.70.
Found: % C=39.34; % H=8.53; % N=5.89.
(R)-Diethyl 2-(2-heptylamino)ethanephosphonate hydrochloride
The free base was prepared in 93% yield using the above-described procedure. The hydrochloride salt was prepared by the addition of ethanolic HCl (15%) to an ethereal solution of the product; the salt is a viscous oil.
Mass spectrum: m/e: (CI). 280 (M+1)+; 264 (M—CH3)+; 208 (M—C5H11)+.
(S)-Diethyl 2-(2-heptylamino)ethanephosphonate hydrochloride
The free base was prepared in 91% yield using the above-described procedure. The hydrochloride salt was prepared by the addition of ethanolic HCl (15%) to an ethereal solution of the product; the salt is a viscous oil.
Mass spectrum: m/e: (CI). 280 (M+1)+; 208 (M—C5H11)+.
(R)-2-(2-Heptylamino)ethanephosphonic acid hydrochloride
A solution of (R)-diethyl 2-(2-heptylamino)ethanephosphonate hydrochloride (0.19 g, 0.68 mmol) in concentrated HCl (7 mL) was heated at 90° C. for 48 hours. The reaction mixture was then evaporated to dryness and the residue triturated with acetone. The white solid was filtered and air-dried giving an 86% overall yield; m.p.=106-112° C. (no lit. value).
Mass spectrum: m/e: too involatile
1H-NMR (D2O, 300 MHz): 0.73 (t, 3H); 1.25-1.10 (m, 12H); 1.43 (m, 1H); 1.58 (m, 1H); 1.89-1.78 (m, 2H); 3.22-3.05 (m, 3H).
Elemental Analysis: Calculated: % C=41.62; % H=8.93; % N=5.39.
Found: % C=41.68; % H=9.10; % N=5.23.
(S)-2-(2-Heptylamino)ethanephosphonic acid hydrochloride
The hydrochloride salt was prepared in 71% overall yield using the above described procedure; m.p.=106-113° C. (no lit. value).
Mass spectrum: m/e: too involatile
1H-NMR (D2O, 300 MHz): 0.73 (t, 3H); 1.25-1.10 (m, 12H); 1.44 (m, 1H); 1.59 (m, 1H); 1.89-1.78 (m, 2H); 3.22-3.05 (m, 3H).
Elemental Analysis: Calculated: % C=41.62; % H=8.93; % N=5.39.
Found: % C=41.43; % H=9.09; % N=5.33.
(R)-2-(2-Heptylamino)ethane-5-tetrazole hydrochloride
The compound as its hydrochloride salt was prepared in 28% overall yield using the procedure described below in Example 40; the salt is a viscous oil.
Mass spectrum: m/e: (CI). 140 (M—C5H11)+; 128 (M—C2H3N4)+.
1H-NMR (D2O, 300 MHz): 0.71 (t, 3H); 1.18 (m, 9H); 1.44, 1.60 (2m, 1H each); 3.30 (t+m; 3H); 3.45 (m, 2H).
(S)-2-(2-Heptylamino)ethane-5-tetrazole hydrochloride
To an ice-cooled suspension of NaN3 (2.60 g, 40 mmol) in dry THF (20 mL) was added AlCl3 (1.36 g, 10 mmol) in portions under N2, and stirred for 30 minutes. To the resulting suspension was added a solution of (S)-3-(2-heptylamino)propionitrile (1.68 g, 10 mmol) in THF (10 mL), and stirred for 2 hours at 0° C., then gently refluxed for 24 hours. After cooling to room temperature, the reaction mixture was quenched by the careful addition of HCl (3 N, 15 mL), water (5 mL), and the two layers were separated. The lower aqueous layer was extracted with ethyl acetate (3×15 mL). The upper (organic) layer and the organic extracts were combined and dried over Na2SO4, then taken to dryness to give crude product (oil, 1.36 g). The oil was diluted with ether (30 mL) and ethanol (5, mL), stirred for two hours, and filtered to give the hydrochloride salt (0.21 g, 9% overall yield); m.p.=112-113° C. (no lit. value).
Mass spectrum: m/e: (CI). 140 (M—C5H11)+; 128 (M—C2H3N4)+.
1H-NMR (D2O, 300 MHz): 0.72 (t, 3H); 1.20 (d, 3H); 1.15-1.35 (m, 6H); 1.45/1.62 (2m, 1H each); 3.30 (t+m; 3H); 3.43 (m, 2H).
Elemental analysis: Calculated: % C=48.47; % H=8.95; % N=28.27.
Found: % C=48.24; % H=8.85; % N =28.40.
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| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA99/00250 | 3/25/1999 | WO | 00 | 11/5/2001 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO99/48858 | 9/30/1999 | WO | A |
| Number | Name | Date | Kind |
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| 3991208 | Dudzinski et al. | Nov 1976 | A |
| 4548726 | Morris-Sherwood et al. | Oct 1985 | A |
| 4804500 | Miller et al. | Feb 1989 | A |
| 4961873 | Ho et al. | Oct 1990 | A |
| 5565290 | Itakura et al. | Oct 1996 | A |
| 5723239 | Itakura et al. | Mar 1998 | A |
| Number | Date | Country |
|---|---|---|
| 11 39 738 | Nov 1962 | DE |
| 15 67 221 | Apr 1970 | DE |
| 20 52 256 | May 1971 | DE |
| 24 42 239 | Mar 1975 | DE |
| 25 55 769 | Jun 1977 | DE |
| 2 451 913 | Oct 1980 | FR |
| WO 9215551 | Sep 1992 | WO |
| WO 9605286 | Feb 1996 | WO |
| WO 9745115 | Dec 1997 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 60079488 | Mar 1998 | US | |
| 60079489 | Mar 1998 | US |
