Patent Description:
Melphalan, having formula (I)
<CHM>
is a chemotherapy drug belonging to the class of nitrogen mustard alkylating agents. It is able to alkylate the DNA guanine bases and to form stable bonds between the two DNA helices, thus inhibiting DNA replication. Melphalan can be used for the treatment of different neoplasias, such as multiple myeloma, ovarian carcinoma and amyloidosis. Melphalan is also used in the pediatric field for the therapy of retinoblastoma.

<CIT>, relates to a process for the manufacture of Melphalan which comprises heating a compound of formula:
<CHM>
wherein R<NUM> is hydrogen or -COOC<NUM>H<NUM>, R<NUM> is hydrogen and R<NUM> is a -CHO or -CH<NUM>CO group and R<NUM> and R<NUM> together represent a group of formula
<CHM>
with a chlorinating agent selected from the group consisting of phosphorus oxychloride (POCl<NUM>) and thionyl chloride (SOCl<NUM>).

The compound of general formula:
<CHM>
is prepared by reaction of a compound of formula:
<CHM>
with ethylene oxide.

In particular, <CIT> exemplifies a method for the synthesis of Melphalan which comprises:.

<CIT>, relates to an improvement of the process disclosed in <CIT>; the improvement consists in the optical resolution of N-acetyl-p-nitro-D,L-phenylalanine with brucine or p-nitro-N-phthaloyl-D,L-phenylalanine with cinchonidine to respectively obtain N-acetyl-p-nitro-L-phenylalanine and p-nitro-N-phthaloyl-L-phenylalanine that are submitted to synthetic steps similar to those disclosed in <CIT>.

<CIT>, discloses a process for the preparation of Melphalan starting from (S)-<NUM>-nitro-phenylalanine ethyl ester, which is protected at the amino group as tert-butoxy carbamate; after the protection reaction, the process is continued in the same way as disclosed in <CIT>, with the sole difference that the concentration of hydrochloric acid in the final deprotection reaction of the amino and carboxy moieties is in the range <NUM> - <NUM> instead of <NUM>.

<CIT>, discloses a process comprising the hydroxyethylation of the amino group on the phenyl ring of a protected p-aminophenylalanine of formula:
<CHM>.

Thus, the known processes for the synthesis of Melphalan require a hydroxyethylation step of p-amino-L-phenylalanine, either in a protected or a free form. Such step is carried out using ethylene oxide as alkylating agent; however, ethylene oxide is a gas, which presents safety concerns, especially when used on industrial scale.

Therefore, the need is still felt to provide a process for the synthesis of Melphalan that does not require the use of ethylene oxide.

<NPL> discloses the reaction of ethylene sulfate with various aniline derivatives and other amines to produce mono-alkylated amine compounds.

The Applicant has found out that Melphalan (I) can be advantageously synthesized in optically pure L-form through a process comprising the regiospecific bis sulfate-alkylation of the aromatic amino group of a <NUM>-amino-L-phenylalanine.

Thus, in a first aspect, the invention relates to a process for the synthesis of Melphalan (I):
<CHM>
said process comprising the reaction of a <NUM>-amino-L-phenylalanine protected at the carboxy and amino aminoacidic groups with an agent able to convert the aromatic amino group into a group of formula: -N(CH<NUM>CH<NUM>OS(O)nO-)<NUM>, wherein n is <NUM> or <NUM>, followed by conversion of the -N(CH<NUM>CH<NUM>OS(O)nO-)<NUM> group into a -N(CH<NUM>CH<NUM>Cl)<NUM> group.

In greater detail, the process of the invention comprises the following steps:.

Preferably, R<NUM> is straight or branched lower alkyl group, more preferably straight or branched C<NUM>-C<NUM> alkyl, more preferably ethyl. In one embodiment, R<NUM> is hydrogen and R<NUM> is a group of formula R<NUM>-C(O)-, wherein R<NUM> is hydrogen or straight or branched lower alkyl, preferably straight or branched C<NUM>-C<NUM> alkyl. In another embodiment, R<NUM> and R<NUM>, together with the nitrogen atom they are bound to, represent a group of formula:
<CHM>.

In a preferred embodiment of the invention, R<NUM> is ethyl and R<NUM> and R<NUM>, together with a nitrogen atom they are bound to, form a group of formula (VI) as defined above.

In a preferred embodiment, a protected <NUM>-amino-L-phenylalanine of formula (II) is reacted with <NUM>,<NUM>,<NUM>-dioxathiolane <NUM>,<NUM>-dioxide (IIIa).

A protected <NUM>-amino-L-phenylalanine (II) can be obtained according to methods known in the art. In particular, a compound of formula (IIa):
<CHM>
can be obtained as disclosed in <NPL> with regard to the nitration and esterification reactions and in <NPL> with regard to the protection of the glycinic nitrogen.

Step a) can be carried out in an organic solvent selected from straight or branched C<NUM>-C<NUM> hydrocarbons, straight or branched halogenated C<NUM>-C<NUM> hydrocarbons, linear or cyclic C<NUM>-C<NUM> ethers or C<NUM>-C<NUM> esters, C<NUM>-C<NUM> nitriles, dimethylformamide (DMF), dimethylacetamide (DMA) and dimethylsulfoxide (DMSO) at temperatures ranging from <NUM> to <NUM>. Conveniently, the reaction is carried out in dichloromethane (DCM) or acetonitrile (ACN) at room temperature (<NUM>). At the end of the reaction, the solvent is distilled off and the residue is taken up with an alcohol, preferably with ethanol, thereby obtaining a chemically and optically pure compound (IV).

The compounds of general formula (IV) and their use as intermediates for the synthesis of Melphalan represent a further aspect of the invention, wherein R<NUM> is ethyl, R<NUM> and R<NUM>, together with the nitrogen atom they are bound to, represent a group of formula (VI) as defined above, X is -OS(O)nO- and n, M+ and M<NUM>+ are defined above.

A particularly preferred compound of formula (IV) is compound (IVa), wherein R<NUM> is ethyl, R<NUM> and R<NUM>, together with the nitrogen atom they are bound to, represent a group of formula (VI) as defined above, X is -OS(O)<NUM>O- and M+ and M<NUM>+ are as defined above:
<CHM>.

Step b) can be carried out in two different ways.

In a first embodiment [hereinafter "embodiment A"], a compound of formula (IV), preferably the protected <NUM>-(bis-(<NUM>-ethylsulfate)-amino-L-phenylalanine (IVa), is first reacted with an inorganic chloride source, such as LiCl, NaCl, KCl, CaCl<NUM>, MgCl<NUM> or BaCl<NUM>, preferably CaCl<NUM>, MgCl<NUM> or BaCl<NUM>, even more preferably BaCl<NUM> or CaCl<NUM>, thereby obtaining protected <NUM>-(bis-(<NUM>-chloroethyl)-amino-L-phenylalanine of formula (V) as defined above, optionally followed by crystallization in a suitable solvent. The reaction with the inorganic chlorine source is carried out in an organic solvent/water mixture, wherein the organic solvent is typically selected from ethyl acetate, dimethylformamide and mixtures of water with tetrahydrofuran, methyltetrahydrofuran, acetonitrile or alcohols at a temperature typically ranging from <NUM>° C to <NUM> preferably at room temperature.

Thereafter, step c) is carried out by treating protected <NUM>-(bis-(<NUM>-chloroethyl)-amino-L-phenylalanine (V) with an acid at pH < <NUM> in water at a temperature ranging from <NUM> to <NUM>, preferably at <NUM>, to provide an acidic aqueous solution comprising Melphalan (I) bis-hydrochloride salt. The acid can be inorganic or organic and is typically selected from hydrochloric, sulfuric, citric, methane sulfonic, toluene sulfonic, acetic, perchloric acid or mixtures thereof. Finally, Melphalan can be crystallized by increasing the pH of the aqueous acidic solution; preferably, Melphalan is crystallized as hydrochloride salt (mono hydrochloride salt) by increasing the pH to a value ranging from <NUM> to about <NUM>, preferably from <NUM> to <NUM>; pH increase can be achieved using an organic or inorganic base, typically an organic amine or ammonia; according to a preferred embodiment, the base is ammonia.

In a second embodiment [herein after "embodiment B"] step b) is carried out as follows. First, a compound of formula (IV), preferably a protected <NUM>-(bis-(<NUM>-ethylsulfate)-amino-L-phenylalanine (IVa) is desulfated using absolute ethanol and sulfuric acid, thereby obtaining protected <NUM>-(bis-(<NUM>-hydroxyethyl)-amino-L-phenylalanine of formula (VII):
<CHM>.

The resulting protected <NUM>-(bis-(<NUM>-hydroxyethyl)-amino-L-phenylalanine (VII) is then reacted with POCl<NUM> or with SOCl<NUM> to provide a compound of formula (V), which is submitted to step c), as illustrated above. The reaction with POCl<NUM> or with SOCl<NUM> is carried out in an organic solvent, which may be selected from toluene and halogenated hydrocarbons, preferably dichloromethylene or trichloromethylene and at a temperature ranging from room temperature to <NUM>. Conveniently, the solvent is toluene and the reaction temperature is room temperature.

The process of the invention is thus advantageous as it avoids the use of ethylene oxide. With specific regard to the use of <NUM>,<NUM>,<NUM>-dioxathiolane <NUM>,<NUM>-dioxide (IIIa), a first advantage is due to the use of <NUM>,<NUM>,<NUM>-dioxathiolane <NUM>,<NUM>-dioxide (IIIa) to perform the bis-alkylation of protected <NUM>-amino-L-phenylalanine (II). Indeed, <NUM>,<NUM>,<NUM>-dioxathiolane <NUM>,<NUM>-dioxide (IIIa) can be used in a considerably lower amount with respect to ethylene oxide (about <NUM> eq with respect to the protected <NUM>-amino-L-phenylalanine (II) vs about <NUM> eq ethylene oxide to perform the same reaction). <NUM>,<NUM>,<NUM>-Dioxathiolane <NUM>,<NUM>-dioxide (IIIa) can be used in a stoichiometric amount with respect to protected <NUM>-amino-L-phenylalanine (II) and is completely degraded in the reaction. It will also be appreciated that <NUM>,<NUM>,<NUM>-dioxathiolane <NUM>,<NUM>-dioxide (IIIa) is cheaper than ethylene oxide and is not included in the list of toxic gases. Moreover, carrying out the alkylation with <NUM>,<NUM>,<NUM>-dioxathiolane <NUM>,<NUM>-dioxide (IIIa) envisages the use of an organic solvent, which does not promote the hydrolysis of the protective groups on the amino and carboxyl groups of the amino acid moiety.

A second advantage is specifically associated to step b), embodiment A. Indeed, the use of an inorganic chloride as chlorinating agent to perform the chlorination of <NUM>-(bis-(<NUM>-ethylsulfate)-amino-L-phenylalanine (IVa) avoids using SOCl<NUM> or POCl<NUM> and, as a consequence, the release of hydrochloric acid and the need to quench the reaction with organic bases, which often leads to the production of toxic gases like SO<NUM>. When inorganic chlorides are used, the waste products of the chlorination reaction are inorganic sulfates, whose toxicity is low; moreover, inorganic sulfates are poorly soluble and can be removed from the reaction mixture by simple filtration.

The invention is hereinafter illustrated in greater detail in the following experimental section.

Commercially available reagents and solvents with the following purity degree were used.

L-Phenylalanine monohydrate (<NUM>, <NUM> mmol) was dissolved in H<NUM>SO<NUM> <NUM>% v/v (<NUM>) and cooled to <NUM>; the resulting solution was added dropwise and under stirring with a mixture (<NUM>, <NUM>/<NUM> vol/vol) of concentrated HNO<NUM> and concentrated H<NUM>SO<NUM> (the mixture was prepared in advance and cooled to room temperature). The resulting reaction solution was stirred for <NUM> hrs at room temperature, then the pH was adjusted to <NUM>-<NUM> with <NUM>% NaOH and the resulting precipitate was collected by filtration. The wet precipitate was recrystallized from <NUM> water to provide <NUM>-nitro-L-phenylalanine monohydrate as a pale yellow powder (<NUM>, <NUM>%).

<NUM>-Nitro-L-phenylalanine (<NUM>, <NUM> mmol) was suspended in <NUM> absolute ethanol ad cooled to <NUM> and the resulting mixture was stirred for <NUM>. Thionyl chloride (<NUM>, <NUM>, <NUM> eq) was slowly dropped in the reaction mixture under stirring, keeping the internal temperature under <NUM>. After completion of the addition, the resulting mixture was heated at <NUM> for <NUM> hrs, then concentrated to <NUM> and slowly added with ethyl ether (<NUM>) at room temperature to provide a white precipitate that was collected by filtration and dried (<NUM>, <NUM>%).

<NUM>-Nitro-L-phenylalanine ethyl ester hydrochloride (<NUM>, <NUM> mmol) was suspended in <NUM> acetonitrile under stirring at room temperature, then anhydrous Cs<NUM>CO<NUM> (<NUM> eq, <NUM>) was added to the suspension. A solution of N-carbethoxyphthalimide (<NUM>, <NUM> eq) in <NUM> acetonitrile was added during <NUM> and the resulting reaction mixture was heated at <NUM> for <NUM> hrs. The reaction mixture was filtered on a sintered glass filter and the cake was washed with <NUM> of acetonitrile. The mother liquors were concentrated to dryness and the yellow oily residue was crystallized by adding methanol (<NUM> volumes/dry residue), cooling the resulting suspension to <NUM> for an hour and filtering. The wet product was dried at <NUM> under max vacuum for <NUM> hrs (yield: <NUM>, <NUM>%).

<NUM>-Nitro-L-phenylalanine ethyl ester hydrochloride (<NUM>, <NUM> mmol) was suspended in <NUM> of <NUM>-Methyl-THF under stirring at room temperature, then KHCO<NUM> aqueous solution (<NUM>% w/V; <NUM>, <NUM>) was added to the suspension under stirring.

The phases were left to separate and the aqueous one eliminated. N-carbethoxyphthalimide (<NUM>, <NUM> eq) was added, and the resulting reaction mixture was heated at <NUM> for <NUM> hrs. After <NUM> hrs, the temperature was decreased at <NUM>±<NUM>, and Triethylamine (<NUM>; <NUM> mmol) was added to the reaction mixture (stirred for <NUM> hrs). The reaction mixture was washed with <NUM> HCl (<NUM>) and brine (<NUM>). The obtained solution was directly used for the preparation of N-Phthalimido-<NUM>-amino-L-phenylalanine ethyl ester (IIa) - Example <NUM>.

N-phthalimido-<NUM>-nitro-L-phenylalanine ethyl ester (<NUM>, <NUM> mmol) was dissolved in ethyl acetate (<NUM>) under stirring at room temperature. When dissolution was complete, <NUM>% Pd on activated charcoal (<NUM>, <NUM>% mol) was added and the mixture was stirred for <NUM> hrs under hydrogen atmosphere (<NUM> bar) at room temperature. At the reaction completion the catalyst was filtered off and the solution was concentrated to dryness. The resulting crude (<NUM>, <NUM>%) was used without other purification for the further synthetic steps.

N-phthalimido-<NUM>-nitro-L-phenylalanine ethyl ester (Solution obtained in Example <NUM>), was kept under stirring at room temperature. An ammonium formate/Phosphoric acid buffer (Ammonium formate:<NUM>; Phosphoric acid <NUM>%: <NUM> in <NUM> of water ) was added to the mixture, together with <NUM>% Pd on activated charcoal (<NUM>). The mixture was stirred for <NUM>. 5hrs at <NUM>. At the reaction completion the catalyst was filtered off and the phases separated. The organic one was concentrated to dryness and the crude recrystallised from isopropanol (<NUM> Volumes/weight -<NUM>). Yield <NUM>%; <NUM>.

N-Phthalimido-<NUM>-amino-L-phenylalanine ethyl ester (IIa) (<NUM>, <NUM> mmol) was suspended in <NUM> dichloromethane (DCM) or acetonitrile (ACN), then sodium hydrogencarbonate (<NUM>; <NUM> mmol) was added to the suspension at room temperature (RT) and in one portion. <NUM>,<NUM>,<NUM>-Dioxathiolane <NUM>,<NUM>-dioxide (IIIa) (<NUM>; <NUM> mmol) was dissolved in DCM (<NUM>) and slowly added to the reaction mixture (<NUM>); the resulting reaction mixture was stirred at RT for 18hrs. Upon completion of the reaction, DCM was distilled off under vacuum and replaced with absolute ethanol (<NUM>). The solid precipitate was recovered by filtration under nitrogen atmosphere and dried at <NUM> for <NUM> hrs (yield: <NUM>; <NUM>%).

N-phthalimido-<NUM>-(bis-<NUM>-ethylsulfate)-amino-L-phenylalanine (<NUM>; <NUM>. 6mmol) was suspended in ethyl acetate previously saturated with water at RT (<NUM>), then added with dimethylformamide (<NUM>). The mixture was stirred at RT for <NUM>, then barium chloride dihydrate (<NUM>; <NUM> mmol) was added in two consecutive portions (<NUM>. The reaction was stirred at RT for <NUM> hrs and then filtered on a sintered glass filter to remove solid sulfates. The ethyl acetic organic phase was washed twice with brine (<NUM> x <NUM>) and dried under vacuum till dryness. Yield: <NUM>; <NUM>%.

N-phthalimido-<NUM>-(bis-<NUM>-ethylsulfate)-amino-L-phenylalanine (<NUM>; <NUM>. 6mmol) was suspended in acetonitrile (<NUM>). Anhydrous calcium chloride was added, and the mixture was stirred at <NUM> for 13hrs and then filtered on a sintered glass filter to remove solid sulfates. The solvent was removed under vacuum. Yield: <NUM>; <NUM>%.

N-phthalimido-<NUM>-(bis-<NUM>-ethylsulfate)-amino-L-phenylalanine (<NUM>; <NUM> mmol) was suspended in absolute ethanol (<NUM>) at RT. Concentrated H<NUM>SO<NUM> (<NUM>; <NUM> mmol) was slowly dropped in the reaction mixture under stirring. When the addition was completed, the mixture was heated at <NUM> for an hour. Ethanol was removed by distillation under vacuum and the oily residue was dissolved in methylene chloride (<NUM>). The resulting organic solution was washed twice with a sodium hydrogencarbonate solution <NUM>% w/vol, dried over magnesium sulfate end evaporated under vacuum till dryness. The oily residue was recrystallized with ethyl acetate (yield <NUM>; <NUM>%).

N-phthalimido-<NUM>-(bis-<NUM>-hydroxyethyl) amino-L-phenylalanine ethyl ester (Va) (<NUM>, <NUM> mmol) was dissolved in toluene (<NUM>). Freshly distilled POCl<NUM> (<NUM>; <NUM>, <NUM> mmol) was slowly added in the reaction mixture at RT (<NUM>). The reaction was heated at <NUM> for <NUM>, then the solvent was removed under vacuum (yield: <NUM>; <NUM>%).

Claim 1:
A process for the synthesis of Melphalan (I):
<CHM>
said process comprising the reaction of a <NUM>-amino-L-phenylalanine protected at the carboxy and amino aminoacidic groups with an agent able to convert the aromatic amino group into a group of formula: -N(CH<NUM>CH<NUM>OS(O)nO-)<NUM>, wherein n is <NUM> or <NUM> followed by conversion of the -N(CH<NUM>CH<NUM>OS(O)nO-)<NUM> group into a -N(CH<NUM>CH<NUM>Cl)<NUM> group.