a,
`
`Europ‘aisches Patentamt
`European Patent Office
`Office européen des brevets
`
`69 Publication number:
`
`0 393 781
`A2
`
`®
`
`EUROPEAN PATENT APPLICATION
`
`@ Application number: 902009372
`
`@ lnt. Cl.5: C07F 9/12, A61K 31/66
`
`(29 Date of filing: 17.04.90
`
`
`
`20.04.89 IT 2021089
`
`@ Applicant: SIMES, SOCIETA ITALIANA
`MEDICINALI E SINTETICI, S.P.A.
`Via clella Chimica 9
`
`
`
` ® Priority: 20.04.89 IT 2020989
`
`
`
`Date of publication of application:
`24.10.90 Bulletin 90/43
`
`l-36100 Vicenza(IT)
`
`Designated Contracting States:
`AT BE CH DE DK ES FR GB GR IT LI LU NL SE
`
`@ Inventor: Casagrande, Cesare
`Via Campogallo, 21/67
`l-20020 Arese (Milano)(lT)
`Inventor: Santangelo, Francesco
`Via Don Gnocchi, 33
`l-20148 Milano(lT)
`
`
`
`
`
`
`
`l-20131 Milano(lT)
`
`
`
`
`
`Representative: Marchi, Massimo et al
`c/o Marchi & Mittler s.r.l. Viale Lombardia 20
`
`@ Dopamine pro-drug.
`
`@ A monophosphorylated L-dopa ester of formula
`
`R0
`
`R 0
`,1
`
`..*
`
`..
`
`CH2
`
`_
`EH COOR2
`NHR3
`
`'
`
`'
`
`(I)
`
`(wherein the asterisk, R, R1, R2 and R3 have the meanings shown in the description),
`salts thereof with pharmaceutically acceptable acids or bases, a process for the preparation thereof and a
`pharmaceutical composition containing said compound or a salt thereof are described.
`The compound at formula I and the salts thereof are useful in the treatment of Parkinson's disease, renal
`failure. heart tailure and hypertension. -
`
`EP0393781A2
`
`Xerox Copy Centre
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`

`

`EP 0 393 781 A2
`
`DOPAMINE PRO-DRUG
`
`The present invention relates to dopamine pro-drugs and, more particularly, to monophosphorylated L-
`dopa esters having high bioavailability by oral route.
`It is known that dopamine is an endogenous catecholamine endowed with important pharmacological
`effects. However, owing to unfavorable pharmacodynamic profile, dopamine is not therapeutically useful
`when administered to animals.
`
`it is also known that L-dopa, i.e. L-3,4-dihydroxyphenylalanine, is a precursor of dopamine and that L-
`'
`dopa is used in therapy for the treatment of Parkinson's disease. L-dopa, which is pharmacologically inert
`as such, is rapidily absorbed from the small bowel by an active transport system for aromatic aminoacids.
`Since about 95% of orally administered L-dopa is rapidly decarboxylated in the periphery to dopamine
`which does not penetrate the blood-brain barrier, large doses must be taken to allow sufficient accumulation
`of L-dopa in the brain, where its decarboxylation raises the central dopamine concentration required in the
`therapy of Parkinson's disease. Alternatively, the concurrent administration of peripherally acting inhibitors
`of L-dopa decarboxylase can reduce the required dose of L-dopa (Goodman and Gilman's The Pharmaco-
`logical Basis of Therapeutics, 7th Ed., page 475 to 480, Macmillan Publishing Company, New York).
`In
`order to inhibit peripheral decarboxylation,
`it has also been recommended (i) to incorporate L-dopa or a
`derivative thereof capable of being cleaved enzymatically in vivo to yield L-dopa in enteric-coated
`pharmaceutical formulations comprising an effervescent base (UKm,485,676) or (ii) to use some specific
`pro-drugs of L-dopa via oral (US-A-3,891,696; EP-A-0 309 827) or rectal route (US-A—4,663,349).
`in turn, EP‘BP‘O 167 204 teaches improving absorption of catecolamines by phosphorylation of one
`hydroxy phenol group thereof. L-dopa, however, is not a catecolamine and its major drawback is peripheral
`decarboxylation rather than absorption.
`in addition, US-A—3,132,171 discloses that dopadiphosphate is watersoluble and stable in aqueous
`solutions while the pharmacodynamic profile of dopa seems to remain unaffected.
`Finally, US-A—4,618,484 and 4,695,449 disclose mono— and di-phosphorylated derivatives of dope and
`pharmaceutically acceptable salts thereof in the treatment of melanomas wherein the phosphorous is the
`32F isotope.
`We have now surprisingly found that phosphorylation of one hydroxy phenol group of some L-dopa
`esters affords new dopamine pro-drugs which are endowed with a high bioavailability by oral route.‘
`Although the reason for this improvement is still unknown, a decrease in peripheral decarboxylation
`could account for said improved bioavailability.
`It is an object of this invention to provide a monophosphorylated L-dopa ester of formula
`
`R0
`
`R10
`
`-1,
`
`_
`
`CH2
`
`..
`(iIH COOR2
`NHR3
`
`(I)
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`wherein
`
`the asymmetric carbon atom marked with an asterisk has 8 configuration, and either R or R1 is hydrogen
`and the other one is a group of formula
`
`45
`
`50
`
`wherein
`
`ii
`R 0-P-
`
`OH
`
`R4 is hydrogen, phenyl, alkylphenyl or a 01-06 alkyl optionally substituted by one to three groups selected
`from hydroxy, alkoxy, acyloxy, amino, carboxy or alkoxycarbonyl;
`R2 is a straight or branched C1-C4 alkyl, a Cs-Cs cycloalkyl or a phenylalkyl having from 1 to 4 C atoms in
`the alkyl portion optionally substituted by 1 to 3 substituents selected from halogens, C1-C3 alkoxy and 01'
`
`2
`
`

`

`EP 0 393 781 A2
`
`03 alkyl;
`R3 is hydrogen or an acyl group of a natural alpha-aminoacid selected from glycine, alanine, valine, leucine,
`isoleucine, serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, phenylalanine, tyrosine,
`tryptophan, arginine,
`lysine, and .histidine, optionally N-acylated by a C1-C4 acyl, or an acyl group of a
`natural aminoacid ester of formula
`
`-CH-C OR
`CH2)n l
`O
`NHR6
`
`5
`
`0 l
`
`l
`- -(
`C
`
`or
`
`O H
`
`C‘fi”
`NHR6
`
`(CH )
`2 n
`
`COOR
`
`s
`
`wherein
`n is 1 or 2;
`R5 is a C1-C3 alkyl,
`R5 'is hydrogen or a C1-C4 acyl;
`and the salts thereof with pharmaceutically acceptable acids or bases.
`As used herein, the term "natural aminoacid" means those aminoacids wherein the possible asymmet-
`ric carbon atom has 8 configuration.
`Examples of suitable pharmaceutically acceptable acids are hydrochloric, hydrobromic, phosphoric,
`sulfuric,
`lactic, succinic, tartaric, acetic, salicylic, citric, benzoic, p-hydroxybenzoic, naphthalen-Z-sulfonic,
`adipic and pimelic acid.
`,
`Examples of suitable pharmaceutically acceptable bases are sodium, potassium, calcium, magnesium
`and ammonium hydroxide, ethanolamine and tromethanol.
`The compounds of this invention are useful in the treatment of renal failure, Parkinson's disease, heart
`failure and hypertension and may be administered orally.
`Preferred meanings of R4 according to this invention are hydrogen, methyl, ethyl, isopropyl, phenyl and
`benzyi.
`Preferred meanings of R2 are methyl, ethyl,
`phenylethyl and 3-(4-methoxy-phenyl)—ethyl.
`Preferred meanings of Rs are hydrogen or an acyl group of a natural alpha-aminoacid selected from
`glycine, alanine,
`leucine and methionine, or an acyl group of an ester of a natural acidic aminoacid of
`formula
`
`isobutyl, cyclopentyl, cyclohexyl, 2-
`
`isopropyl, butyl,
`
`0 I
`
`I
`-C- (CH2) n‘W'ZH-COOR5
`NHR6
`
`wherein
`n is 2 and
`
`R5 is hydrogen.
`Whenever not differently stated, acyl means the an acyl group of an aliphatic carboxylic acid having
`from 1 to 5 carbon atoms or benzoyl.
`Another object of this invention is to provide a process for the preparation of a compound of formula I
`and the salts thereof, comprising
`(i) phosphorylating an L-dopa ester of formula lla optionally protected at one hydroxy phenol group
`and, when R3 is hydrogen, at the amino group
`
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`EP 0 393 781 A2
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`H0
`
`H
`
`O
`
`-*
`
`..
`
`CH2
`
`_
`(Iii-i COOR2
`NHR
`3
`
`(Ila)
`
`wherein the asterisk, R2 and R3 have the above mentioned meanings,
`with a suitable phosphorylating agent,
`(ii) removing the protective groups, if any, to yield a monophosphorylated L-dopa ester of formula
`
`R0
`
`R10
`
`CH -*CH-CO0R
`2
`|
`NHR3
`
`2
`
`(I)
`
`wherein the asterisk. Ft. R1 and R2 have the above mentioned meanings,
`(iii) optionally reacting a monophosphorylated L-dopa ester of formula I wherein R3 is hydrogen with
`an optionally protected and N-acylated natural alpha-aminoacid or with a natural acidic aminoacid of formula
`
`(III)
`
`(IV)
`
`S
`
`0 H
`
`"CH'COOR
`HO-C-(CH )
`2 n l
`NHRé
`
`. 0
`fl
`HO-C-CH—(CH )
`‘
`2 n
`NHR6
`
`‘COOR
`
`'
`
`5
`
`or
`
`wherein n, R5 and R5 have the above mentioned meanings; and
`(iii) removing the protective groups, if any, to yield a compound of formula I, and
`(iv) if desired, adding a pharmaceutically acceptable acid or base to a compound of formula I to yield
`the corresponding pharmaceutically acceptable salt.
`Step (i)
`is preferably carried out
`in a suitable solvent at a temperature of from -80°C to +100°C.
`Typical examples of suitable solvents are the phosphorylating agent itself and the inert organic solvents
`such as hydrocarbons, halocarbons, ethers, esters, amides, tertiary and heterocyclic amines.
`When phosphorylation releases acidic compounds, step (i) is preferably carried out in the presence of
`suitable acid acceptors such as alkali and earth-alkaline carbonates or bicarbonates or tertiary and
`heterocyclic amines, such as triethylamine and pyridine, which may also act as the solvent.
`Alternatively, in order to neutralize the acidic compounds released during the phosphorylation step, the
`phenol hydroxy group to be phosphorylated can be salified in advance with a base such as sodium hydride,
`sodium methylate or potassium t.butylate.
`,
`Depending on the phosphorylating agent and method which is used, phosphorylation yield compounds
`wherein R4 is hydrogen (hereinafter called phosphoric monoesters) or wherein R4 is different from H
`(hereinafter called phosphoric diesters).
`In the preparation of phosphoric monoesters,’ preferred phosphorylating agents are orthophosphoric,
`pyrophosphoric and polyphosphoric acids, phosphorus pentoxide, chlorophosphoric acids, phosphoryl
`chloride and bromide,
`from which phosphoric monoesters are obtained directly or after an optional
`hydrolysis with water at the end of the phosphorylation reaction; other suitable phosphorylating agents
`having protective groups to be removed at
`the end of the phosphorylation reaction are dibenzyl- or
`diphenyI-phosphochloridate and 2-chloro-2-oxo-1,3,2—benzodioxaphosphate,
`the protective groups being
`
`1O
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`EP 0 393 781 A2
`
`removable by hydrogenolysis and oxidation, respectively, or 4,5-dimethyl-2—(1-imidazolyl)-2-oxo-1,3,2—diox-
`aphosphate; 2-cyanoethylphosphate and dibenzylphosphate require the addition of a suitable condensing
`agent such as N,N-dicyclohexylcarbodiimide.
`Phosphoric diesters can be prepared both by direct phosphorylation or by alkylation of the correspond-
`ing monoesters.
`In case of direct phosphorylation, preferred phosphorylating agents are the phosphodichloridates of
`formula
`
`ll
`R O-P-CL
`
`CL
`
`0 i
`
`l
`R7O-l'3-CL
`0R4
`
`and phosphochloridates of formula
`
`wherein R7 has the same meaning of R4 or is a benzyl group; that at the end of the phosphorylation
`reaction require the removal of
`the chlorine atom or of
`the protective group 0R7 by hydrolysis or
`hydrogenolysis.
`'
`In the case of the alkylation, a phosphoric monoester is reacted with an alcohol of formula
`R4OH
`
`Alternatively, a phosphoric diester, preferably in the form of an alkali or silver salt, carrying benzyl as a
`protective group, can be alkylated with an alkylating agent of formula
`R4X
`wherein
`
`'
`
`X is a halogen atom, an alkylsulphonyloxy or an arylsulphonyloxy group; and then removing by
`hydrogenolysis the benzyl protective group.
`Before step (i), protection of the compound Ila is preferably performed at the amino group, when R3 is
`hydrogen, and also at one of the two phenol hydroxy groups. The latter protection has the aim to direct
`phosphorylation on the free hydroxy group only, so avoiding formation of isomer mixtures.
`In a similar way, the amino group (NH2) of the natural alpha-aminoacid or of compound I" and IV is also
`preferably protected before performing step (iii) or when preparing compound Ila.
`The artisan will recognize that all the protection and deprotection steps optionally performed in the
`process of this invention can be carried out according to conventional techniques of the peptide chemistry.
`The artisan will also recognized that compound He can easily be prepared with the conventional
`techniques of step (iii).
`The process of this invention is substantially similar to that of EP-B1-0 167 204 which is therefore
`incorporated herein by reference.
`'
`When step (i) is carried out without protecting one of the two hydroxy phenol groups of compound Ila, a
`mixture mainly consisting of the two monophosphorylated products, 3- and 4-monophosphate, is obtained.
`The two regioisomers may be separated by chromatography or crystallization.
`The compounds of the present invention are orally adsorbed and generate metabolically dopamine thus
`producing useful pharmacological effects via stimulation of dopaminergic receptors.
`Namely, the compounds of the present invention proved to have a vasodilating effect onto the renal
`district in anesthetized dog at a dose of 0.1-50'mg/kg i.p.
`Mongrels of both sexes anesthetized with sodium pentobarbital (35 mg/kg e.v.) were used.
`Artificial respiration was accomplished by means of a endotracheal tube with a Starling Ideal pump at a
`ventilation frequency of 16-18 cycles/minute, a flow rate of 16-17 ml/kg in order to obtain p02, p002, and
`pH values of arterial blood of 85-100 mmHg, 30—40 mmHg and 7.35-7.45,
`respectively (Radiometer
`Copenhagen BMS 3 MK 2 Blood Microsystem Blood Gas Analyzer). Duodenum was insulated by abdomen
`incision and a polyethylene catheter was inserted for administering the drug. Left renal artery was isolated
`
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`EP 0 393 781 A2
`
`retroperitoneally, an electromagnetic transducer and a pneumatic obturator were positioned around the
`vessel to measure mechanical zero and blood flow, respectively.
`Emodynamic parameters were recorded on a Gould Brush MK 200 Graph Recorder while the pressure
`catheters were connected to a Bell and Howell Pressure Transducers and the electromagnetic flow
`transducer was connected to a Biotronex BL 613 Flowmeter.
`
`Moreover, the compounds of formula l proved to have an antagonist action on the motility depression
`induced by reserpine (4 mg/kg ip) in mouse pretreated 16 hours before with iproniazide (150 mg/kg as) at a
`dose of 50-600 mg/kg i.p. (Wintroub B.V. et at, Am. J. Physiol. 217, 1716, 1969).
`The compounds of this invention are useful in the therapyFParkinson‘s disease and of cardiovascular
`diseases such as heart failure, hypertension and renal failure.
`Another object of the present invention is therefore to provide pharmaceutical compositions containing
`one or more compounds of formula I or their pharmaceutically acceptable salts optionally together with one
`or more excipients suitable for pharmaceutical use.
`The compositions of the present invention may be solid, such as tablets, granules, pills, capsules, or
`liquid, such as solutions, syrups, emulsions and are prepared according to conventional methods.
`They may be administered both enterally and parenterally. The preferred administration way is the oral
`route.
`
`The doses may vary depending on the selected pharmaceutical form and the individual response of the
`patient but usually are in the range of from 100 mg to 5 g per day.
`The following examples are provided in order to better illustrate the present invention without, however,
`limiting it.
`
`Example _1_
`
`Preparation of 3-O-benzyl fl 4-O—benzyl-N-benzyloxycarbonyl-L-dopa w it”
`
`A solution of N-benzyloxycarbonyl-L-dopa ethyl ester (135 g; 0.375 moles), benzyl chloride (94.94 g;
`0.75 moles) and sodium bicarbonate (94.5 g; 1.125 moles) in absolute ethyl alcohol (1.35 I) is refluxed for 8
`hours.
`'
`
`The salts are filtered off and the reaction mixture is kept at 5.. C overnight.
`A solid separates, namely N-benzyloxycarbonyl-S,4-0-dibenzyl-L—dopa ethyl ester which is filtered.
`The solution is evaporated to dryness and the two isomers 3-O-benzylether and 4-O-benzylether are
`separated by chromatography on silica gel column (eluent. CH2CI2; CHsCN = 86:4).
`From the fractions containing the less polar product, N-benzyonycarbonyl-3-O-benzyl-L-dopa ethyl
`ester is obtained, m.p. 74-75° C (isopropyl ether).
`t); 2.97-3.10 (2H, m): 4.07-4.12 (2H, m); 4.57-4.62 (1H,
`1H-NMFl (300 MHz, CDCIa) delta (ppm); 1.23 (3H,
`m); 5.02 (2H, s); 5.11 (2H, d); 6.62 (1H, dd); 6.71 (1H, d); 6.85 (1 H, d); 7.29-7.42 (10H, rn).
`From fractions containing the more polar product, N-benzyloxycarbonyI-4-O-benzyl-L—dopa ethyl ester
`is obtained as chromatographically pure oil (thin layer chromatography, eluent, CHzclzi CHscN = 96:4,
`I2
`vapours detection).
`1H-NMFt (300 MHz, CDGls) delta (ppm): 1.26 (3H, t); 3.03 (2H, d); 4.18 (2H, q); 4.55-4.62 (1 H, m); 5.09 (4H,
`d); 6.57 (1H, dd); 6.7 (1 H, d); 6.82 (1H, d); 7.28-7.42 (10H, m).
`
`Example 2
`
`Preparation of 3-O-benzyl-N-benzyonycarbonyl-L—dopa
`
`NaOH 10N (8.9 ml; 89 mmoles) is added to a solution of 3-O-benzyl-N-benzyloxycarbonyl-L-dopa ethyl
`ester (20 g; 44.5 mmoles), prepared as described in example 1,
`in absolute ethyl alcohol (200 ml) at a
`temperature of 5-10‘ C.
`The reaction mixture is diluted with water (20 ml) and is kept under stirring at room temperature for 9
`hours.
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`EP 0 393 781 A2
`
`Concentrate HCI is added and the sodium chloride which separates is removed by filtration; the solution
`is then concentrated under reduced pressure, diluted with ethyl acetate and washed with water. After drying
`on sodium sulfate, the solvent is evaporated and the oily residue crystallized from a mixture of isopropyl
`ether and petroleum ether. mp. 85 - 88° 0
`Mass spectrum (chemical
`ionization, positive ions,
`adduct).
`
`ionization gas : ammonia) m/e 439 (M+ + 1 + NHa
`
`Example 3
`
`Preparation _o_f 3-O-benzyl-N-benzyloxycarbonyl—L-dopa n.buty| eitel
`
`A solution of 20% tetramethylammonium hydroxyde (5.95 g; 13.1 mmoies) in methyl alcohol is added
`to a solution of 3-O-benzyl-N-benzyloxycarbonyl-L-dopa (5 g; 11.9 mmoies) in methyl alcohol (50 ml). The
`solvent is removed under reduced pressure; the residue is dissolved in dimethylformamide (50 ml) and
`n.butyl
`iodide (4.6 g ; 25 mmoies) is added. After three hours at room temperature under stirring, the
`solution is washed with water and extracted with ethyl ether. The organic layer is separated, washed with
`water, dried on sodium sulfate and evaporated to dryness. The residue is purified by chromatography on
`silica gel column (eluent, CH20I2). The title compound is thus obtained as a chromotographically pure oil
`(thin layer chromatography - eluent, CHZClz: CHSCN = 95:5, l2 vapours detection).
`Mass spectrum (chemical ionization, negative ions, ionization gas : ammonia) m/e 476 (M+ - 1), 388 (M+ -
`C7H7).
`'
`Working in a similar manner the following product has been prepared:
`3-O-benzyi-N-benzyloxycarbonyl-L-dopa 2-phenylethyl ester
`chromotographically pure oil
`(thin layer chromatograW -eluent, CHzclzz CHacN = 95:5,
`detection).
`Mass spectrum (chemical ionization, positive ions, ionization gas : isobutane) m/e 526 (M+ + 1).
`3-O-benzyl-N-benzyloxycarbonyl-L-dopa isopropyl ester
`chromotographically pure oil
`(thin layer chromatofiphy -e|uent, CH2C12: CHaCN = 95:5,
`detection).
`'
`Mass spectrum (chemical ionization, positive ions, ionization gas : isobutane) m/e 464 (M+ + 1).
`
`lz vapours
`
`l2 vapours
`
`3-0-benzyl-N-benzyloxycarbonyl-L—dopa cyclohexyl ester
`
`Thionyl chloride (5,07 g; 42,6 mmoies) is addaed to a solution of 3-O-benzyl-N-benzyloxycarbonyl-L-
`dopa (6 9; 14,2 mmoies) in cyclohexyl alcohol (60 ml) at 0-5° C. After 30 hours at room temperature, the
`reaction mixture is evaporated to dryness and washed as described for the preparation of 3-O-benzyl-N-
`benzyloxycarbonyl-L-dopa n.butyl ester. The product is obtained as a chromotographically pure oil (thin
`layer chromatography - eluent, CH2C|22 CHsCN = 96:4, l2 vapours detection).
`Mass spectrum (chemical ionization, positive ions, ionization gas : isobutane) m/e 504 (M+ + 1).
`
`Example 4
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`Prepartion 9t N-benzyloxycarbonyl-3-benzyloxy-4-dibenzylphosphonyloxy-L-phenylalanine ethyl ester
`
`55
`
`A suspension of 60% NaH in mineral oil (1.8 g; 45 mmoies) is added at 0-50 to a solution of N-
`benzyloxycarbonyl-3-O-benzyl-L-dopa ethyl ester (18.5 g; 41 mmoies), prepared as described in example 1,
`in dimethylformamide (185 ml).
`After one hour at this temperature, a solution of dibenzylphosphochloridate (14.53; 49 mmoies) in
`toluene (145 ml) is added dropwise.
`After further 30 minutes, acetic acid is added (2 ml); the reaction mixture is diluted with water, toluene
`is separated and the aqueous phase is extracted with ethyl ether.
`
`

`

`EP 0 393 781 A2
`
`The combined organic phases are washed with water and dried on Na2804.
`After solvent evaporation, the residue is purified by chromatography on silica gel column (eluting with
`CHzclz with increasing quantities of ethyl acetate up to 10%).
`N-benzyloxycarbonyl-S—benzyloxy-4-dibenzylphosphonyloxy-L-phenylalanine ethyl ester is thus obtained
`in the form of pure oil (thin layer chromatography; eluent, CHZC|2:ethyl acetate = 9:1, l2 vapours detection).
`1H-NMFl (300 MHz. CD013) delta (ppm): 1.21 (3H, t); 3.0-3.11 (2H, m); 4.05-4.18 (2H, m); 4.58-4.66 (1H, m);
`4.95-5.11 (8H, m); 6.64 (1H, dd); 6.77 (1H, d); 7.12 (1H, dd); 7.18-7.39 (21H, m).
`Working in a similar manner the following compounds have been obtained:
`
`N-benzyloxycarbonyl-4-benzyloxy-3-dibenzylphosphonyloxy-L-phenylalanine ethyl ester as a chromatog-
`raphically pure oil (thin layer chromatography - eluent, CHZClzzethyl acetate =-9—:1,
`l2 vapours detection).
`1H-NMR (300 MHz, CDCls) delta (ppm): 1.22 (3H, t); 2.92-3.08 (2H, m); 4.15 (2H, q); 4.55 (1 H, m); 5.01-5.11
`(8H, m); 6.86 (2H. s); 7.88 (1H, s); 7.18-7.42 (21H, m).
`
`chromotog-
`a
`as
`ester
`ethyl
`N-benzyloxycarbonyl-S-benzyony-4-ethylphosphonyloxy-L-phenylalanine
`raphically pure oil (thin layer chromatography - eluent. CHzclzzmethanOINWermcetic acid = 49:15:1r1,
`l2
`vapours detection).
`t); 1.11 (3H, t); 2.72-2.96 (2H, m); 3.78 (2H, m); 3.78
`1H-NMR (300 Mhz, DMSO-ds) delta (ppm): 0.99 (3H,
`(2H, quintet); 4.05 (2H, q); 4.14-4.22 (1 H, m); 4.97-5.02 (2H, m); 6.68 (1H, dd); 6.95 (1H, d); 7.26-7.43 (11H,
`m).
`
`N-benzyloxycarbonyl-3-benzyloxy-4—dibenzylphosphonyloxy-L—phenylalanine n.buty| ester as a chromatog-
`raphically pure oil (thin layer chromotography - eluent, petroleum ether: ethyl acetate—= 65:35,
`l2 vapours
`detection).
`1H-NMR (300 MHz, CDCla) delta (ppm): 0.92 (3H, t); 1.23-1.38 (2H, m); 1.52-1.62 (2H. m); 3.05 (2H, t): 4.00-
`4.15 (2H, m); 4.62 (1H, dd); 4.95 (2H, s); 5.02 (2H, s); 5.06 (2H, s); 5.10 (2H, s); 6.64 (1H,dd); 6.76(1H.d);
`7.12 (1H, dd); 7.19-7.39 (20H, m).
`a
`as
`ester
`2-phenylethyl
`N-benzyloxycarbonyl-3-benzyloxy-4—dibenzylphosphonyloxy-L—phenylalanine
`chromatographically pure oil (thin layer chromotography - eluent, petroleum ether: ethyl acetat = 65:35, l2
`vapours detection).
`a
`as
`ester
`isopropyl
`N-benzyloxycarbonyl-3-benzyloxy-4-dibenzylphosphonyloxy-L-phenylalanine
`chromatographically pure oil (thin layer chromatography - eluent, petroleum ether: ethyl acetate = 65:35, I;
`vapours detection).
`a
`as
`ester
`cyclohexyl
`N-benzyloxycarb'onyl-S-benzyloxy-4-dibenzylphosphonyloxy-L-phenylalanine
`chromatographically pure oil (thin layer chromotography - eluent, petroleum ether: ethyl acetate = 65:35, l2
`vapours detection).
`
`Example 5
`
`Preparation of 3-hydroxy-4-phosphonyloxy-L-phenylalanine M w
`
`A suspension of N-benzyloxycarbonyl-S-benzyloxy-4-dibenzylphosphonyloxy-L-phenylalanine ethyl es-
`ter (23 g; 32.4 mmoles) prepared as described in example 4, 10% palladium on charcoal (2.3 g) in a
`mixture of ethanol (250 ml) and water (50 ml) is kept at a pressure of 3-4 hydrogen atmospheres until
`theoretical absorption.
`The catalyst is filtered off. the solvent is evaporated and the residue is crystallized from a mixture of
`ethyl alcohol :water = 1:2.
`3-hydroxy-4-phosphonyloxy-L—phenylalanine ethyl ester melting at 162-166° C is obtained by filtration.
`1H-NMR (300 MHz, D20) delta (ppm): 1.27 (3H, t); 3.09-3.31 (2H, m); 3.59-3.68 (1H, m); 4.30 (2H. q); 6.78
`(1 H, dd); 6.85 (1H, d); 7.22 (1 H, d).
`Working in a similar manner the following compounds were obtained:
`
`4-hydroxy-3-phosphonyloxy-L—phenylalanine ethyl ester
`m.p. 197-200' c
`—
`‘H-NMR (300 MHz, [320) delta (ppm): 1,27 (3H, t); 3.08-3.32 (2H, m); 4.28 (2H, q); 4.36 (1 H, m); 6.90-6.96
`(2H, m), 7.16 (1 H, s).
`
`
`L-dopa 4-O-ethylphosphate ethyl ester
`having a m.p. higher than 2% (dec.)
`1H-NMFt (300 MHz, D20) delta (ppm): 1.22-1.29 (6H, m); 3.10-3.28 (2H, m); 4.04 (2H, quintet); 4.24-4.35
`
`10
`
`15
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`20
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`30
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`EP 0 393 781 A2
`
`(3H, m); 6.78 (1 H, dd); 6.85 (1 H, d); 7.22 (1H, dd).
`
`3-hydr0xy-4-phosphonyloxy-L-phenylalanine n.buty| ester
`m.p. 184-186 C (from water/ethyl alcohol)
`1H-NMFi (300 MHz, DMSO-ds) delta (ppm) 0.88 (8H, t); 1.29 (2H, sextet); 1.52 (2H, quintet); 2.92 (2H, d);
`4.10 (2H,
`t); 4.19 (1H,
`t); 6.47 (1 H, dd); 6.49 (1H, d); 6.75 (1H, dd). 3-hydroxy-4-phosphonyloxy—L-
`phenylalanine 2-phenylethyl ester
`m.p. 184-190 C (from water/ethyl alcohol)
`t); 4.18-4.31 (2H, m);
`1H-NMR (300 MHz, DMSO-ds) delta (ppm) 2.71-2.82 (2H, m); 2.86 (2H,”t); 3.86 (1H,
`6.34 (1 H, dd); 6.52 (1 H, d); 6.74 (1 H, d); 7.12-7.31 (5H, m). 3-hydroxy-4-phosphonyloxy-L-phenylaianine
`isopropyl ester
`m.p. 165-1-7—5*C (from water/ethyl alcohol)
`1H-NMR (300 MHz, 020) delta (ppm) 1.28 (6H, d); 3.08-3.31 (2H, m); 4.31 (1H, t); 5.11 (1H, quintet); 6.75
`(1H, dd); 6.81 (1H, cl); 7.15 (1H, d).
`3-hydroxy-4-phosphonyloxy-L-phenylalanine cyclohexyl ester
`m.p. 198-200 C (from water/ethyl alcohol)
`—
`1H-NMR (300 MHz, DMSO-ds) delta (ppm) 1.20-1.78 (m, 10H); 2.82-2.97 (2H, m); 4.15 (1H, t); 4.68-4.76
`(1H, m); 6.45 (1H, dd); 6.55 (1 H, d); 6.75 (1H, d).
`
`Example 6
`
`—_.__.____——.._____——_._—_____
`Preparation of N-(N-benzyloxycarbonyl-gamma—L-glutamyl-alfa ethyl ester)—3-hydroxy-4-phosphonyloxy-L-
`
`To a solution of 3-hydroxy-4-phosphonyloxy-L-phenylalanine ethyl ester (4 g; 13.1 mmoles), prepared
`as described in example 5, and NaHCOs
`(1.1 g; 13.1 mmoles)
`in water
`(80 ml). a solution of N-
`benzyloxycarbonyl-L-glutamic-alta-ethyl ester gamma—N-hydroxysuccinimide ester acid (6.39 g; 15.7 moles)
`in ethanol (80 ml).
`'
`After 2 hours, the solution is concentrated to small volume and a saturated solution of N60! is added to
`the residue; the product which solidifies is filtered, washed with a saturated solution of NaCI, with ethyl
`ether, suspended under 'stirring in acetone. The salt is discharged by filtration, the solution is evaporated to
`dryness; ethyl ether is added to the residue and the mixture is filtered. N-(N-benzyioxycarbonyl-gamma-L-
`glutamyI-alta ethyl ester)-3-hydroxy-4-phosphonyloxy-L-phenylalanine ethyl ester sodium salt is thus ob-
`tained and used in next step as it is.
`1H-NMR (300 MHz, DMSO-Dg) delta (ppm); 1.12 (3H, t); 1.18 (3H, t); 1.68-1.96 (2H, m); 2.19 (2H, t); 2.70-
`2.87 (2H, m); 3.98-4.12 (3H, m); 4.28-4.35 (1 H, m); 5.05 (2H, m), 6.46 (1H, dd); 6.55 (1H, d); 6.69 (1 H, d).
`Working in a similar manner the following compounds were obtained:
`N-(N-benzyioxycarbonyl—gamma—L—glutamyl-alfa
`ethyl
`ester)-3-hydroxy-4-ethylphosphonyloxy-L-
`phenylalanine ethyl ester ammonium salt as a chromfiraphically pure oil (thin layer chromatography -
`eiuent, n.butanometic—acid:waterztoluenE acetone = 1:1:1:1:1, U.V. light, l2 vapours detection).
`1H-NMR (300 MHz, DMSO-da) delta (ppm): 1.02-1.18 (9H, m); 1.68-1.96 (1H, m); 2.19 (2H, t); 2.71-2.88 (2H,
`m); 3.45 (1H, m); 3.76 (2H, quintet); 3.96-4.11 (6H, m); 4.28-4.35 (1H, m); 5.02 (2H, d); 6.48 (1H, dd); 6.56
`(1 H, d); 6.66 (1H, dd).
`
`chromatographically pure oil (thin layer chromatography - eiuent, n.butanol:afi5 mwaternolueneiic'etone
`= 1:1:1:1:1, U.V. light, l2 vapours detection).
`
`chromatographically pure oil (thin layer chromatography - eiuent, n.butanol:aEEtiE Hwaternolueneficetone
`= 1:1:1:1:1, U.V. light, l2 vapours detection).
`
`N-(N~benzyloxycarbonyl-L-alanyl)-3-hydroxy-4-phosphonyloxy-L-phenylalanine ethyl ester ammonium salt
`as
`a
`chromatographically
`pure
`oil
`(thin
`layer
`chromatography
`-
`elu—ent,
`n.butanol
`: acfl
`acid:water:toluene:acetone = 1:1:1:1 :1, U.V. light, l2 vapours detection).
`1H-NMFt (300 MHz, D20) delta (ppm): 1.05-1.08 (3H, m); 1.26 (3H, s); 2.80-3.10 (2H, m); 3.65 (1H, Cl); 4.06-
`4.18 (2H, m); 5.12 (2H, s); 6.62-6.74 (2H, m); 7.08 (1H, d); 7.28-7.40 (5H, m).
`
`as
`
`a
`
`chromatographically
`
`pure
`
`oil
`
`(thin
`
`layer
`
`chromatography
`
`-
`
`eiuent,
`
`n.butanol:
`
`acetic
`
`1O
`
`15
`
`20
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`30
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`EP 0 393 781 A2
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`acid:water:toluene:acetone = 1:1:1:1:1, U.V. light, l2 vapours detection).
`
`Example 7
`
`Preparation of N-(gamma—L-glutamyl-alfa-ethyl ester)-3-hydroxy-4-phosphonyloxy-L-phenylalanine ethyl es-
`ter sodium salt
`
`ester)-3-hydroxy-4-
`N-(N-benzyloxycarbonyl-gamma-L-glutamyl-alfa—ethyl
`of
`suspension
`A
`phosphonyloxy-L-phenylalanine ethyl ester sodium salt (7.3 g; 11.8 mmoles), prepared as disclosed in
`example 6, and 10% Pd on charcoal (1.4 g) in 80% ethanol (200 ml) is kept at a pressure of 3-4 hydrogen
`atmospheres until theoretical absorption.
`The catalyst is filtered off, the solvent is evaporated and the residue is suspended in acetone, filtered
`and recrystallized from 95% ethanol obtaining N—(gamma—L-glutamyl-
`alta-ethyl
`ester)-8~hydroxy-4—
`phosphonyloxy—L—phenylalanine ethyl ester sodium salt melting at 139-143° C.
`‘H-NMR (300 MHz, DMSO-da) delta (ppm): 1.26 (3H, t); 1.28 (3H,
`t); 1.88-2.08 (2H, m); 2.3-2.43 (2H, m);
`2.82-2.91 (1H, m); 3.18-3.25 (1H, m); 3.13 (1 H, t); 4.18-4.31 (2H, m); 4.68-4.73 (1H, m); 6.71-6.76 (2H, m);
`7.07 (1H, d).
`
`Working in a simitar manner the following compounds were obtained:
`
`N-(gamma-L-glutamyl-alfa-ethyl ester)-3-hydroxy-4-ethylphosphonyloxy-L-phenylalanine fl estfi sodium
`salt,
`
`m_.p. 90° C (slow decomposition).
`1H—NMR (300 MHz, D20) delta (ppm): 1.20-1.28 (9H, m); 1.85-2.07 (2H, m); 2.38 (2H, t); 2.88-2.96 (1 H, m);
`3.12-3.20 (1H, m); 3.64 (1 H, t); 4.02 (2H, quintet); 4.21 (4H, quintet); 4.63-4.68 (1 H, m); 6.78 (1H, dd); 6.83
`(1 H, d); 7.18 (1 H, dd).
`.
`N-(N-acetyl-L-methionyl)-3-hydroxy-4-phosphonyloxy-L-phenylalanine ethyl
`m.p. 115-120 C
`“'-
`1H-NMR (300 MHz, D20) delta (ppm); 1.22 (3H, t); 1.74-1.98 (2H, m); 1.99 (3H, s); 2.06 (3H, s); 2.32-2.54
`(2H, m); 2.9-3.0 (1 H, m); 3.12-3.25 (1H, m); 4.18 (2H, q); 4.19-4.38 (1H, m); 4.62-4.68 (1H, m); 6.73 (1H, dd);
`6.82 (1H, d); 7.18 (1H, d).
`
`N-glycyI-3-hydroxy-4-phosphonyloxy-L—phenylalanine ethyl ester
`m.p. 224-227 c
`——
`1H—NMR (300 MHz, D20) delta (ppm): 1.29 (3H, t); 2.91-2.94 (1H, m); 3.18-3.23 (1H, m); 3.73 (2H, s); 4.24
`(2H, q); 4.68-4.80 (1 H, m); 6.73-6.79 (2H, m); 7.08 (1 H, cl).
`N-glycyl-4-hydroxy-3-phosphonyloxy-L—phenylalanine ethyl ester
`m.p. 210-213' C
`——
`1H-NMR (300 MHz, D20) delta (ppm). 1.24 (3H, t); 2.92-2.99 (1 H, dd); 3.15-3.22 (1H, dd); 3.80 (2H, s); 4.22
`(2H, d); 4.82 (1 H, m); 6.92 (2H, be); 7.10 (1H, s).
`
`N-(L-alanyl)-3-hydroxy-4-phosphonyloxy-L-phenylalanine ethyl ester
`m.p. 199-203' c
`—
`1H-NMR (300 MHz, D20) delta (ppm): 1.23 (3H, t); 1.49 (3H, t); 2.96-3.04 (1H, dd); 3.16-3.22 (1H, dd); 4.02
`(1 H, q); 4.21 (1 H, q); 4.69 (1 H, m); 6.78 (1 H, dd); 6.85 (1H, d); 7.19 (1 H, d).
`
`N—leucyl~3-hydroxy-4-phosphonyloxy-L-phenylalanine ethyl ester
`m.p. 217-219 c.
`——
`1H-NMR (300 MHz, D20) delta (ppm): 0.94 (3H,t); 1.26 (3H, t); 1.56 (2H, CI); 2.91-2.98 (1H, dd); 3.17-3.25
`(1 H, dd); 3.87 (1 H, t); 4.22 (2H, q); 4.75 (1 H, m); 6.75 (1 H, d); 6.78 (1H, d); 7.08 (1 H, d).
`
`Claims
`
`1. A compound of formula
`
`10
`
`10
`
`15
`
`20
`
`25
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`30
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`35
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`

`EP 0 393 781 A2
`
`R0
`
`R10
`
`CH -*CH-COOR
`2
`I
`NHR
`
`2
`
`(I)
`
`wherein
`
`1O
`
`the asymmetric carbon atom marked with an asterisk has 8 configuration, and either R or R1 is hydrogen
`and the other one is a group of formula
`
`H
`R o~P—
`
`OH
`
`wherein
`
`R4 is hydrogen, phenyl, alkylphenyl or a Cl'CG alkyl optionally substituted by one to three groups selected
`from hydroxy, alkoxy, acyloxy, amino, carboxy or alkoxycarbonyl;
`R2 is a straight or branched C‘l'C4 alkyl, a C3-Cs cycloalkyl or a phenylalkyl having from 1 to 4 C atoms in
`the alkyl portion optionally substituted by 1 to 3 substituents selected from halogens, C1-Ca alkoxy and C1-
`03 alkyl;
`R5 is hydrogen or an acyl group of a natural alpha-aminoacid selected from glycine, alanine, valine, leucine,
`isoleucine, serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, phenylalanine, tyrosine,
`tryptophan, arginine,
`lysine, and histidine, optionally N-acylated by a C1-C4 acyl, or an acyl group of a
`natural aminoacid ester of formula
`
`t
`I
`-C-(CH ) -
`-
`2 n (IZH COORS
`NHR
`6
`
`)
`-
`2 n COORS
`
`0 l
`
`l
`(
`- —CH- CH
`C
`l
`NHRér
`
`-
`
`or
`
`wherein
`
`n is 1 or 2;
`R5 is a 01-03 alkyl;
`R; is hydrogen or a 01-04 acyl;
`and salts thereof with pharmaceutically acceptable acids or bases.
`isopropyl, phenyl or
`2. A compound according to claim 1, wherein R4 is hydrogen, methyl, ethyl,
`benzyl; R2 is methyl, ethyl,
`isopropyl, butyl,
`isobutyl, cyclopentyl, cyclohexyl, 2-phenyl