Patent Description:
Melflufen, compound of formula (<NUM>) is a DNA alkylating drug. Melflufen is useful in the treatment of multiple myeloma. Melflufen is marketed in form of HCl salt under the brand name Pepaxto by Oncopeptides.

Melflufen was first disclosed in <CIT>. The application describes a process for preparation of Melflufen starting form L-Melphalan. L-Melphalan is highly toxic compound, use thereof is not suitable for high scale preparation. The process also comprises purification using column chromatography that is also not suitable for high scale preparation. Another process for preparation of Melflufen is described in <CIT>. The applicant discloses that chlorination of compound of formula (<NUM>),
<CHM>
using common chlorination agents SOCl<NUM> or POCl<NUM> led either to significant de-protected side product or require long temperatures and high reaction times. These chlorinating agents are therefore not suitable for high scale production of Melflufen. That was also confirmed as described in Comparative examples <NUM> or <NUM>.

There is therefore a need for improved process for preparation of Melflufen suitable for high scale production.

The present invention relates for preparing Melflufen, compound of formula (<NUM>) or a salt thereof,
<CHM>
the process comprising:.

The presented invention further relates to intermediates used in the process and solid forms thereof.

The salt of compound of formula (<NUM>) can be selected from for example hydrochloride or hydrobromide or methane sulfonate or ethane sulfonate or benzene sulfonate or toluene sulfonate or sulphate or hydrogen sulphate or fluoroacetate such as trifluoroacetate or fluoropropionate, preferably it is hydrochloride. Prot means a protective group that can be selected for example from tert-butyl oxycarbonyl (Boc), methyl oxycarbonyl, ethyl oxycarbonyl, <NUM>- fluorenylmethyl oxycarbonyl (Fmoc), benzyl oxycarbonyl (Cbz), p-methoxybenzyl oxycarbonyl (Moz), <NUM>-adamantyl oxycarbonyl (Adoc), p-bromobenzyl oxycarbonyl, trifluoroacetyl, chloroacetyl, phenylacetyl, benzacetyl, p-toluenesulfonyl (tosyl, Ts), <NUM>-nitrobenzenesulfonyl (Nps), t-butylsulfonyl (Bus), <NUM>- or <NUM>-nitrobenzenesulfonyl (Nosyl), <NUM>,<NUM>-dinitronenzesulfonyl (DNs), and <NUM>-naphthalenesulfonyl, preferably it is tert-butyl oxycarbonyl (Boc).

R<NUM>, R<NUM>, R<NUM>, R<NUM> in compound of formula (<NUM>) can be selected from methyl or ethyl or propyl or isopropyl, preferebly R<NUM>, R<NUM>, R<NUM>, R<NUM> are methyl, i.e. compound of formula (<NUM>) is preferably compound of formula (3A),
<CHM>.

We have surprisingly found that use of compound of formula (<NUM>), preferably compound of formula (3A) provides compound of formula (<NUM>), preferably compound of formula (4A), in high yield and purity. Also the work up of the reaction mixture is easier comparing to work up of the mixture when the SOCl<NUM> or POCl<NUM> is used. Compound of formula (<NUM>), preferably of formula (3A), can be used at low temperature and is suitable for high scale production of Melflufen or a salt thereof.

The step a. can be performed under a protective atmosphere such as nitrogen or argon, in a solvent selected for example from acetonitrile or dimethylformamide or dichloromethane or trichloromethane or an aromatic solvent such as toluene or benzene or an ether such as dimethyl ether or diethyl ether or iso-propyl ether or tetrahydrofurane or <NUM>-methyl tetrahydrofurane or an ester such as methyl acetate or ethyl acetate or isopropyl acetate. Acetonitrile is preferably used.

Compound of formula (<NUM>) is mixed with the solvent. The concentration of compound of formula (<NUM>) in the solvent can be between <NUM>/ml and <NUM>/ml, preferably it is between <NUM>/ml and <NUM>/ml. The mixture is cooled to a temperature between -<NUM> and <NUM>, preferably between <NUM> and <NUM>. To the mixture compound of formula (<NUM>) is added. The molar ratio between compound of formula (<NUM>) and compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably between <NUM>:<NUM> and <NUM>:<NUM>. Compound of formula (<NUM>) can be added in the course of between <NUM> and <NUM> minutes. The mixture is then stirred at the temperature between -<NUM> and <NUM>, preferably between <NUM> and <NUM>, for between <NUM> and <NUM> minutes. The mixture was heated to a temperature between <NUM> and <NUM>, preferably between <NUM> and <NUM> and stirred at this temperature for between <NUM> and <NUM> hours. The reaction progress can be monitored by any suitable analytical technique, e.g. by HPLC or GC. After the reaction is finished the mixture can be optionally cooled to a temperature between -<NUM> and <NUM>. To the mixture water is added to precipitate solid compound of formula (<NUM>). Volume ratio between added water and the solvent used in step a. can be between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is stirred for between <NUM> and <NUM> minutes and solid mass is filtered off. Obtained solid can be optionally dried.

Compound of formula (<NUM>), preferably compound of formula (3A) is commercially available.

Compound of formula (<NUM>) can be transformed to Melflufen, compound of formula (<NUM>) or a salt thereof (step b. ) for example by using an acid such as HCl or HBr or methane sulfonic acid or ethane sulfonic acid or benzene sulfonic acid or toluene sulfonic acid or H<NUM>SO<NUM> or a fluoroacetic acid such as trifluoroacetic acid or fluoropropionic acid, preferably HCl is used. Compound of formula (<NUM>) can be also transformed into Melflufen by using hydrogenation in a presence of a catalyst.

Compound of formula (<NUM>) can be deprotected by any of the process disclosed in the prior art or for example by a process comprising:.

Compound of formula (<NUM>), preferably compound of formula (4A) is mixed with a suitable solvent such as acetonitrile or an alcohol, for example methanol or ethanol or propanol or isopropanol or ethylacetate or tetrahydrofurane or acetone or dichloromethane or trichloromethane or an ether, preferably acetonitrile is used. The concentration of compound of formula (<NUM>), preferably compound of formula (4A), in the solvent can be between <NUM> and <NUM>/ml.

To the mixture an acid such as HCl or HBr or methane sulfonic acid or ethane sulfonic acid or benzene sulfonic acid or toluene sulfonic acid or H<NUM>SO<NUM> or fluoroacetic acid such as trifluoroacetic acid or fluoropropionic acid, preferably HCl, is added. The molar ratio between used acid and the compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:<NUM> and <NUM>:<NUM>. The acid can optionally be used be used in form of a solution in a suitable solvent, for example cyclopentyl methyl ether or an alcohol such as ethanol or propanol or isopropanol or ethylacetate or tetrahydrofurane or acetone. Preferably HCl is be used as a solution in as cyclopentyl methyl ether or ethyl acetate. In comparison with the solvents disclosed in the prior art, cyclopentyl methyl ether or ethyl acetate has following advantages:.

The mixture is then heated to a temperature between <NUM> and <NUM> and stirred at this temperature for between <NUM> and <NUM> hours. The mixture is evaporated to dryness. The rest is dissolved in ethanol at <NUM>-<NUM>. The volume ratio between ethanol and the solvent used in step I to dissolve compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is then cooled to a temperature between <NUM> and <NUM> and stirred at this temperature for between <NUM> and <NUM> minutes to provide a suspension. To the mixture an antisolvent (i.e. a solvent that poorly dissolves Melflufen salt) is added. The volume ratio between ethanol and the antisolvent can be between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is cooled to a temperature between - <NUM> and <NUM> and stirred for between <NUM> and <NUM> hours. Obtained suspension is filtered off and filter cake is washed with the antisolvent and optionally dried.

The process for preparation of compound of formula (<NUM>) by reacting compound of formula (<NUM>) and compound of formula (<NUM>) is a two-step process depicted in following scheme:
<CHM>.

Another embodiment of the presented invention is therefore compound of following formula and use thereof for preparing of Melflufen or a salt thereof:
<CHM>.

Compound of formula (<NUM>) can be prepared by a process comprising:.

Compound of formula (<NUM>) is reacted with a suitable base in a suitable solvent (step I. ) to provide compound of formula (<NUM>) or a salt thereof. Suitable base be selected from a hydroxide such as LiOH or NaOH or KOH or a combination of LiBr and an amine such as triethyl amine or diisopropyl ethyl amine in a presence of water. Suitable solvent can be selected from dimethylformamide or an alcohol such as MeOH or EtOH or dimethylformamide in a mixture with water or the alcohol in a mixture with water. Preferably a mixture of dimethylformamide (DMF) with water or a mixture of the alcohol with water is used. The volume ratio between dimethylformamide and water can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:<NUM> and <NUM>:<NUM>. The volume ration between the alcohol and water can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:<NUM> and <NUM>:<NUM>.

The concentration of compound of formula (<NUM>) in the solvent or solvent mixture can be between <NUM>/ml and <NUM>/ml. The concentration of the base in the solvent can be between <NUM>/ml and <NUM>/ml. The base can be used as a solid or in a form of a solution in a suitable solvent, for example water. The molar ratio between the base and the compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably between <NUM>:<NUM> and <NUM>:<NUM>. Compound of formula (<NUM>) is mixed with the solvent or solvent mixture (solvent1). To the mixture the base is added. The base can be optionally added in parts, for example in <NUM> or <NUM> or <NUM> or <NUM> or <NUM> parts. The mixture is then stirred for between <NUM> and <NUM> hours at <NUM> - <NUM>. The mixture can be optionally cooled to a temperature between <NUM> and <NUM>. pH of the mixture is set to between <NUM> and <NUM>, preferably to <NUM> for example with citric acid (<NUM>% w/w queous solution) or with aqueous solution of HCl. The aqueous solution of the acid can be added in several parts, for example in <NUM> or <NUM> or <NUM> or <NUM> or <NUM> parts, more preferably it is added drop-wise. Obtained reaction mixture can be used in subsequent step for preparation of compound of formula (<NUM>) or compound of formula (<NUM>) can be isolated in a solid form.

Compound of formula (<NUM>) can be isolated by a process comprising adding a solvent (solvent_added) to the mixture. The solvent (solvent_added) can be for example toluene or an alkyl acetate such as ethyl acetate or iso-propyl acetate or <NUM>-methyl tetrahydrofurane or dichloromethane or chloroform or trichloroethylene. The volume ratio between the added solvent (i.e. solvent_added) and the solvent used for mixing with compound of formula (<NUM>) (i.e. solvent1) can be between <NUM>:<NUM> and <NUM>:<NUM>.

In the case when toluene is used as solvent_added, after addition of toluene the mixture is stirred at a temperature between <NUM> and <NUM> for between <NUM> and <NUM> hours to obtain a suspension. The suspension is filtered off and obtained solid is dried to provide a solid form of compound of formula (<NUM>).

In the case of compound of formula (<NUM>), where Prot means tert-butyl oxycarbonyl (Boc), i.e. compound of formula (6A),
<CHM>
the obtained solid of compound of formula (6A), Form B, can be characterized by XRPD pattern having 2θ values <NUM>°, <NUM>° and <NUM>° 2θ (± <NUM> degrees 2θ). The form can be also characterized by XRPD pattern having 2θ values <NUM>°, <NUM>°, <NUM>°, <NUM>° and <NUM>° 2θ (± <NUM> degrees 2θ). The Form B can be further characterized by XRPD 2θ values (± <NUM> degrees 2θ) stated in following table:.

The Form B can be also characterized by XRPD pattern depicted in Figure <NUM>.

In the case when the added solvent (solvent_added) is different from toluene, i.e. selected for example from an alkyl acetate such as ethylacetate or iso-propyl acetate or <NUM>-methyl tetrahydrofurane or dichloromethane or chloroform or trichloroethylene, the solid for can be isolated by using the following process. After addition of the solvent (solvent_added), the mixture is stirred for between <NUM> and <NUM> minutes and the layers are separated. The layer comprising added solvent (solvent_added) is evaporated to dryness. To the rest a second amount of solvent (solvent_added) was added. The volume ratio between solvent (solvent_added) and the solvent used for mixing with compound of formula (<NUM>) (i.e.solvent1) can be between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is then stirred for between <NUM> and <NUM> minutes and the layers are separated. The layer comprising added solvent (solvent_added) is evaporated to dryness to provide compound of formula (<NUM>).

Compound of formula (<NUM>) is commercially available or can be prepared from a compound of formula (<NUM>) by a process disclosed in prior art or by a process comprising reacting compound of formula (<NUM>) with ethylene oxide or with a compound of formula (<NUM>), for example by a process described in Example <NUM>,
<CHM>.

In step II. compound of formula (<NUM>), preferably of compound formula (6A), is reacted with compound of formula (<NUM>) or a salt thereof in a presence of a base and a coupling agent to provide compound of formula (<NUM>), preferably of formula (2A). Compound of formula (<NUM>) is preferably in form of a salt, more preferably in a form of HCl salt. The reaction is performed in a suitable solvent. The suitable solvent can be selected from for example from acetonitrile or ethyl acetate or dimethylsulfoxide or acetone or tetrahydrofurane or <NUM>-methyltetrahydrofurane or dioxane or an alcohol such as methanol or ethanol or propanol or dichloromethane or dimethylformamide or cyclopentyl methyl ester or acetone or water and combination thereof, preferably dimethylformamide in combination with water or acetonitrile is used. In the case a combination of a solvent with water is used, the volume ratio between the solvent and water can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:<NUM> and <NUM>:<NUM>.

As a coupling agent for example propylphosphonic anhydride (T3P), <NUM>-chloro-<NUM>,<NUM>-dimethoxy-<NUM>,<NUM>,<NUM>-triazine (CDMT), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), ethyl-(N',N'- dimethylamino)propylcarbodiimide hydrochloride (EDC); phosphonium-based reagents, for example (benzotriazol-<NUM>-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-<NUM>-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP); aminium-based reagents, for example N,N,N',N'-tetramethyl-O-(<NUM>-benzotriazol-<NUM>-yl)uronium hexafluorophosphate (HBTU), <NUM>-[Bis(dimethylamino)methylene]-<NUM>-<NUM>,<NUM>,<NUM>- triazolo[<NUM>,<NUM>-b]pyridinium <NUM>-oxid hexafluorophosphate (HATU), O-(benzotriazol-<NUM>-yl)- N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), O-(<NUM>-chlorobenzotriazol-<NUM>-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), O-(<NUM>,<NUM>-dihydro-<NUM>-oxo-<NUM>,<NUM>,<NUM>- benzotriazine-<NUM>-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU); immonium aminium-based reagents, for example (<NUM>-benzotriazol-<NUM>-yloxy)-N,N- dimethylmethaniminium hexachloroantimonate (BOMI), <NUM>-(<NUM>-benzotriazol-<NUM>-yloxy)-<NUM>,<NUM>- dihydro-<NUM> -methyl <NUM>-pyrrolium hexachloroantimonate (BDMP) and <NUM>-(<NUM>-azabenzotriazol-<NUM>-yloxy)-<NUM>,<NUM>-dihydro-<NUM>-methyl <NUM>-pyrrolium hexachloroantimonate (AOMP); agents generating acids chlorides, for example thionyl chloride, phosphorus pentachloride, triphosgene, triazines (e.g. cyanuric chlorides, cyanuric fluoride, and derivatives thereof), tetramethylfluoroformamidinium hexafluorophosphate (TFFH), bis(tetramethylene)fluoroformamidinium (BTFFH), and <NUM>,<NUM>-dimethyl-<NUM>-f uoro-<NUM>,<NUM>-dihydro- lH-imidazolium hexafluorophosphate (DFIH); or other coupling reagents, for example <NUM>- (diethylphosphoryloxy)-<NUM>,<NUM>,<NUM>-benzotriazin-<NUM>(<NUM>)-one (DEPBT), carbonyldilmidazole (CDI), and N-Ethoxycarbonyl-<NUM>-ethoxy-l,<NUM>-dihydroquinoline (EEDQ). Preferably propylphosphonic anhydride (T3P) or <NUM>-chloro-<NUM>,<NUM>-dimethoxy-<NUM>,<NUM>,<NUM>-triazine (CDMT) is used, more preferably <NUM>-chloro-<NUM>,<NUM>-dimethoxy-<NUM>,<NUM>,<NUM>-triazine (CDMT) is used.

Suitable base can be selected from N-methyl morpholine or an amine such as trimethyl amine or triethyl amine or diisopropyl ethyl amine (DIPEA) or a carbonate such as sodium carbonate or potassium carbonate or a hydrogen carbonate such as sodium hydrogen carbonate or potassium hydrogen carbonate or a hydroxide such as sodium hydroxide or potassium hydroxide. The base is preferably N-methyl morpholine.

For the preparation of compound of formula (<NUM>), preferably formula (2A), the reaction mixture from the previous step comprising compound of formula (<NUM>), preferably compound of formula (6A), can be used. Compound of formula (<NUM>) can be also prepared from isolated solid compound of formula (<NUM>), preferably compound of formula (6A). In the second case, compound of formula (<NUM>) is mixed with the solvent or a mixture of solvents, preferably with acetonitrile or with a mixture of dimethylformamide with water. The concentration of compound of formula (<NUM>) in the solvent can be between <NUM>/ml and <NUM>/ml, preferably it is between <NUM> and <NUM>/ml. Compound of formula (<NUM>), preferably in form of HCl salt, is added to the mixture or the reaction mixture from the previous step. The molar ratio between compound of formula (<NUM>) and compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>. To the mixture the base is added. The molar ratio between the base and the compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is stirred at <NUM>-<NUM> for between <NUM> and <NUM> minutes. To the mixture the coupling agent is added in the course of between <NUM> and <NUM> minutes. The molar ratio between the coupling agent and the compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is then stirred at <NUM>-<NUM> for between <NUM> and <NUM> hours. The reaction progress can be monitored by any suitable analytical technique, e.g. by HPLC or GC.

Compound of formula (<NUM>) can be also prepared by a process comprising mixing the coupling agent with a solvent or a solvent mixture. The concentration of the coupling agent in the solvent or the solvent mixture can be between <NUM>/ml and <NUM>/ml. The molar ration between the coupling agent and the compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:1and <NUM>:<NUM>. To the mixture the base is added. The molar ratio between the base and the compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is the stirred for between <NUM> and <NUM> minutes. To the mixture compound of formula (<NUM>), preferably in a form of HCl salt is added. The molar ratio between compound of formula (<NUM>) and compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is stirred for between <NUM> and <NUM> minutes. To the mixture compound of formula (<NUM>) is added either in form of isolated solid or in a form of reaction mixture from the previous step. The mixture is then stirred at <NUM>-<NUM> for between <NUM> and <NUM> hours. The reaction progress can be monitored by any suitable analytical technique, e.g. by HPLC or GC.

After the reaction is finished the reaction mixture can be processed either by:.

In the case i. the water immiscible solvent can be selected for example from an alkylacetate, such as methyl acetate or ethyl acetate or propyl acetate or iso-propyl acetate or dichloromethane or chloroform or trichloroethylene, preferably ethyl acetate is used. The volume ratio between water and the water immiscible solvent can be between <NUM>:<NUM> and <NUM>:<NUM>. The volume ratio between added mixture of water and the water immiscible solvent and the solvent used to dissolve compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is then stirred for between <NUM> and <NUM> minutes. The layers are separated and to the organic layer a saturated solution of a hydrogen carbonate such as NaHCO<NUM> or KHCO<NUM> is added. The volume ratio between the organic layer and added saturated solution of the hydrogen carbonate can be between <NUM>:<NUM> and <NUM>:<NUM>. The mixture is stirred for between <NUM> and <NUM> minutes and the layers are separated. The organic layer is dried, for example using MgSO<NUM>. The mixture is then filtrated off and the filtrate is evaporated to dryness to provide compound of formula (<NUM>), preferably compound of formula (2A).

In the case ii. , the volume ratio acetonitrile: water can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably between <NUM>:<NUM> and <NUM>:<NUM>. The volume ratio between the added mixture water and acetonitrile and the solvent or a solvent mixture used to dissolve the compound of formula (<NUM>) can be between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is between <NUM>:<NUM> and <NUM>:<NUM>. The mixture of acetonitrile and water can be added in parts, for example in <NUM> or <NUM> or <NUM> or <NUM> or <NUM> or <NUM> parts, more preferably it is added drop-wise in the course of between <NUM> and <NUM> minutes. The mixture is then concentrated at between <NUM> and <NUM> to approximately <NUM>/<NUM> of the original volume to obtain a suspension. The suspension is cooled to between <NUM> and <NUM> and stirred at this temperature for between <NUM> and <NUM> hours. Obtained solid is filtered off and dried. In the case of compound of formula (<NUM>), where Prot means tert-butyl oxycarbonyl (Boc), i.e. compound of formula (2A),
<CHM>
the obtained solid of compound of formula (2A), Form A, can be characterized by XRPD pattern having 2θ values <NUM>°, <NUM>° and <NUM>° 2θ (± <NUM> degrees 2θ). The form can be also characterized by XRPD pattern having 2θ values <NUM>°, <NUM>°, <NUM>°, <NUM>° and <NUM>° 2θ (± <NUM> degrees 2θ). The Form A can be further characterized by XRPD 2θ values (± <NUM> degrees 2θ) stated in following table:.

The Form A can be also characterized by XRPD pattern depicted in <FIG>.

The invention will be further described with reference to the following examples.

XRPD spectrum of solid compounds was obtained using the following measurement conditions:
Panalytical Empyrean diffractometer with Θ/2Θ geometry (transmition mode), equipped with a PixCell 3D detector;.

<NUM> of methyl (S)-<NUM>-(<NUM>-aminophenyl)-<NUM>-((tert-butoxycarbonyl)amino)propanoate was dissolved in <NUM> of in acetonitrile. <NUM> of sodium iodide, <NUM> of sodium carbonate and <NUM> of <NUM>-bromoethan-<NUM>-ol were added and then the mixture was heated to <NUM>. The mixture was stirred at <NUM> under argon for <NUM> hours. The mixture was cooled to room temperature (<NUM>-<NUM>), filtered and the filter cake was washed with <NUM> of acetonitrile. The mother liquor was evaporated (<NUM>, <NUM> mbar).

The residue was dissolved in <NUM> of ethyl acetate. <NUM> of water was added and the mixture was stirred for <NUM>. The layers were separated. Organic layer was dried and evaporated to give <NUM> of mass. The rest was dissolved in <NUM> of ethyl acetate. The mixture was twice extracted with <NUM> of <NUM>% solution of sodium thiosulphate and then with <NUM> of water. The layers were separated. Organic layer was dried and evaporated to give <NUM> of the product.

<NUM> of compound of formula (5A) was dissolved in <NUM> of methanol. Solution of <NUM> of LiOH·H<NUM>O in <NUM> of water was added. Mixture was stirred for <NUM> hour and <NUM> of LiOH·H<NUM>O was added to reaction mixture. Mixture was stirred for <NUM> hour. Mixture was acidified with <NUM>% (w/w) citric acid to pH <NUM>. To the mixture <NUM> of ethyl acetate was added. Mixture was stirred for <NUM> minutes and layers were separated. Organic layer was evaporated on the rotary evaporator (<NUM>-<NUM> mbar, <NUM>). To the rest <NUM> of ethyl acetate was added and mixture was stirred for <NUM> minutes. Layers were separated. Organic layer was evaporated to dryness (<NUM>-<NUM> mbar) to provide <NUM> of compound of formula (6A) in <NUM> % yield, and <NUM> % purity (HPLC).

<NUM> of compound of formula (6A) was mixed with <NUM> of acetonitrile. To the mixture <NUM> of compound of formula (<NUM>. HCl) was added at <NUM>-<NUM>. <NUM> of N-methyl morpholine was added and the mixture was stirred at <NUM>-<NUM> for <NUM> minutes. <NUM> of AllessanCAP (<NUM>-Propanephosphonic anhydride, <NUM>% solution in acetonitrile) in acetonitrile was added via syringe over <NUM> minutes and resulting solution was stirred at <NUM>-<NUM> for <NUM> hours. To the mixture <NUM> of water and <NUM> of ethyl acetate were added and the mixture was stirred for <NUM> minutes. Layers were separated and organic layer was washed with <NUM> of saturated NaHCO<NUM> solution for <NUM> minutes. Layers were separated. Organic layer was dried with MgSO<NUM>, filtered, and evaporated to dryness (<NUM>-<NUM> mbar, <NUM>) providing <NUM> of compound of formula (2A) in <NUM> % yield and <NUM> % purity (HPLC).

<NUM> of methyl (S)-<NUM>-(<NUM>-(bis(<NUM>-hydroxyethyl)amino)phenyl)-<NUM>-((tert-butoxycarbonyl)amino)propanoate (compound of formula (5A)) was mixed with <NUM> of dimethylformamide (DMF) and <NUM> of water to dissolve the compound of formula (5A). <NUM> of LiOH. H<NUM>O was added and the solution was stirred at <NUM> for <NUM> hours. The solution was cooled down to <NUM> and <NUM> of <NUM>% aq. solution of HCl diluted with <NUM> of water was added dropwise in the course of <NUM> minutes to obtain reaction mixture (RM1).

<NUM> of <NUM>-chloro-<NUM>,<NUM>-dimethoxy-<NUM>,<NUM>,<NUM>-triazine was dissolved in <NUM> of dimethylformamide (DMF) under nitrogen atmosphere. The mixture was cooled down to -<NUM>. <NUM> of <NUM>-Methylmorpholine was added and the mixture was stirred for <NUM> minutes. To the mixture <NUM> of Ethyl (S)-<NUM>-amino-<NUM>-(<NUM>-fluorophenyl)propanoate hydrochloride (compound <NUM>. HCl) was added followed by <NUM> of dimethylformamide. The mixture was stirred for <NUM> minutes. To the mixture previously prepared reaction mixture (RM1) was added in the course of <NUM> minute.

The mixture was stirred for <NUM> hours at <NUM>. To the mixture <NUM> of acetonitrile was added. To the mixture <NUM> of water was added dropwise in the course of <NUM> minutes. The mixture was distilled at <NUM>-<NUM> under vacuum (<NUM> mbar) until the volume of the reaction mixture was reduced to approximately <NUM>. The suspension was cooled down to <NUM> and stirred for <NUM> hour. The product was filtered and washed with <NUM> of water. The solid material was dried on the Buchner funnel for <NUM> hour and then in vacuo for <NUM> hours. <NUM> (<NUM> % yield) of Ethyl (S)-<NUM>-((S)-<NUM>-(<NUM>-(bis(<NUM>-hydroxyethyl)amino)phenyl)-<NUM>-((tert-butoxycarbonyl)amino)pro-panamido)-<NUM>-(<NUM>-fluorophenyl)propanoate was isolated as off-white powder with purity <NUM>% (HPLC IN).

XRPD pattern of obtained solid corresponds to XRPD pattern depicted in <FIG>.

<NUM> of methyl (S)-<NUM>-(<NUM>-(bis(<NUM>-hydroxyethyl)amino)phenyl)-<NUM>-((tert-butoxycarbonyl)amino) propanoate (compound of formula (5A)) was mixed with <NUM> of dimethylformamide and <NUM> of water to dissolve compound of formula (5A). <NUM> of LiOH. H<NUM>O was added and the mixture was stirred at <NUM> for <NUM> hours. The solution was cooled down to <NUM>. A mixture of <NUM> of <NUM>% aq. solution of HCl and <NUM> of water was added dropwise over <NUM> minutes. To the mixture <NUM> of toluene was added dropwise over <NUM> minutes at <NUM>. The mixture was stirred at <NUM> for <NUM> hours. Obtained suspension was filtered off and obtained solid was washed with <NUM> of water. Obtained solid material was dried in the Buchner funnel for <NUM> hour and then in vacuo for <NUM> hours to provide <NUM> (<NUM>% yield) of (S)-<NUM>-(<NUM>-(bis(<NUM>-hydroxyethyl)amino)phenyl)-<NUM>-((tert-butoxycarbonyl)amino)propanoic acid in purity <NUM>% (HPLC IN). XRPD of obtained solid corresponds to XRPD pattern depicted in Figure <NUM>.

<NUM> of <NUM>-chloro-<NUM>,<NUM>-dimethoxy-<NUM>,<NUM>,<NUM>-triazine was dissolved in <NUM> of dimethylformamide under nitrogen atmosphere. The mixture was cooled to -<NUM>. <NUM> of <NUM>-Methylmorpholine was added and the mixture was stirred for <NUM> minutes. <NUM> of Ethyl (S)-<NUM>-amino-<NUM>-(<NUM>-fluorophenyl) propanoate hydrochloride (compound (<NUM>. HCl)) was added followed by <NUM> of dimethylformamide (DMF). The mixture was stirred for <NUM> minutes. <NUM> of (S)-<NUM>-(<NUM>-(bis(<NUM>-hydroxyethyl)amino) phenyl)-<NUM>-((tert-butoxycarbonyl)amino)propanoic acid was added followed by <NUM> of dimethylformamide and <NUM> of water.

The mixture was stirred for <NUM> hours at <NUM>. To the mixture <NUM> of acetonitrile was added. <NUM> of water was added dropwise over <NUM> minutes. The mixture was distilled at <NUM>-<NUM> under vacuum (<NUM> mbar) until the volume of the reaction mixture was reduced to approximately <NUM>. The suspension was cooled down to <NUM> and stirred for <NUM> hour. The solid was filtered and washed with <NUM> of water. The solid material was dried in the Buchner funnel for <NUM> hour and then in vacuo for <NUM> hours. <NUM> (<NUM> % yield) of Ethyl (S)-<NUM>-((S)-<NUM>-(<NUM>-(bis(<NUM>-hydroxyethyl)amino)phenyl)-<NUM>-((tert-butoxycarbonyl)amino)propanamido)-<NUM>-(<NUM>-fluorophenyl)propanoate was isolated with purity <NUM>% (HPLC). XRPD pattern of obtained solid corresponds to XRPD pattern depicted in <FIG>.

<NUM> of compound of formula (4A) was mixed with <NUM> of acetonitrile under argon atmosphere. The mixture was stirred at <NUM>-<NUM> for <NUM> minutes to provide a solution. The mixture was cooled to -<NUM>-<NUM> (ice-bath) and <NUM> of <NUM>-Chloro-N,N,<NUM>-Trimethylpropenylamine (Compound of formula (3A)) was gradually added via syringe over <NUM> minutes keeping reaction temperature below <NUM>. The mixture was stirred at <NUM>-<NUM> for next <NUM> minutes and then at <NUM>-<NUM> for <NUM> hours. To the mixture <NUM> of water was added to provide a suspension. The suspension was then cooled to <NUM>-<NUM> (ice bath) and stirred for <NUM> hour. The suspension was filtered off and filter cake was washed with 2x20 ml of chilled (<NUM>-<NUM>) acetonitrile. Wet product was air dried for <NUM> hours to provide <NUM> of compound of formula (4A) in <NUM>% yield and <NUM>% purity (HPLC).

<NUM> of compound of formula (4A) was mixed with <NUM> of ethanol. <NUM> of hydrogen chloride in cyclopentyl methyl ether (CPME) (<NUM> solution) was added at <NUM>-<NUM>. The mixture was heated up to <NUM> and stirred for <NUM> hours. Mixture was evaporated to dryness (<NUM> - <NUM> mbar, <NUM>) giving an oil. It was dissolved in <NUM> of ethanol and evaporated to dryness (<NUM> - <NUM> mbar, <NUM>). The rest was dissolved in <NUM> of ethanol at <NUM> and then stirred at <NUM>-<NUM> for <NUM> hours giving a suspension. The mixture was cooled to <NUM>-<NUM>, mixed with <NUM> of methyl tert-butyl ether and stirred for <NUM> hours. The solid was filtered off and the filter cake that was washed with 2x10 ml of methyl tert-butyl ether. Obtained solid was air-dried on filter for <NUM> providing <NUM> HCl salt of Melflufen in <NUM>% yield and <NUM>% purity (HPLC).

<NUM> of compound (2A) was added to a reaction vial followed by <NUM> of dichloromethane and <NUM> of SOCl<NUM>. Mixture was stirred at <NUM> for <NUM>. Sample was evaporated in the stream of nitrogen to produce <NUM> of very dark green / black semi-solid containing <NUM> % of Melflufen by assay on external standard. Melflufen was not isolated. Because of low yield and purity of prepared Melflufen, the process is not suitable for high scale production.

<NUM> of compound 2A was added to a reaction vial followed by <NUM> of acetonitrile and <NUM> of POCl<NUM>. Mixture was stirred at <NUM> for <NUM>. Sample was evaporated in the stream of nitrogen to produce <NUM> of a product containing <NUM> % of Melflufen by assay on external standard. Melflufen was not isolated.

Claim 1:
A process for preparing Melflufen, compound of formula (<NUM>) or a salt thereof,
<CHM>
the process comprising:
a. Reacting a compound of formula (<NUM>) with compound of formula (<NUM>) to prepare compound of formula (<NUM>),
<CHM>
<CHM>
Prot means nitrogen protective group;
R<NUM>, R<NUM>, R<NUM>, R<NUM> means C<NUM>-C<NUM> alkyl;
b. Converting compound of formula (<NUM>) into Melflufen, compound of formula (<NUM>) or a salt thereof.