Deuterium-containing compounds

Abstract

The present invention provides compounds of formula (I), or a pharmaceutically acceptable salt thereof, wherein each R 1 ~R 30 are independently selected from the group consisting of H and deuterium, and R 1 ~R 30 at least one of which is deuterium having an abundance level greater than the naturally occurring abundance of deuterium. The present invention also provides pharmaceutical compositions containing the compounds, and uses of the compounds.

Description

[Technical field]

[0001] The present invention relates to novel deuterated melflufen derivatives having particularly advantageous properties. The novel deuterated melflufen derivatives, or salts thereof, find use in the treatment or prevention of cancer. [Background technology]

[0002] Melflufen (also known as melphalan flufenamide and L-melphalanyl-4-fluoro-L-phenylalanine ethyl ester) is an antitumor agent useful for the treatment of cancer, particularly multiple myeloma. Melflufen is described in WO01 / 96367 and WO2014 / 065751. The structure of the hydrochloride salt of melflufen is shown below. [ka] Melflufen is a potent and highly lipophilic alkylating agent, which achieves targeted delivery of alkylated metabolites to tumor cells. In contrast to other alkylating agents that are hydrophilic, the high lipophilicity of melflufen leads to its rapid uptake into tissues and cells. Once inside the cell, melflufen can directly bind to DNA or be easily hydrolyzed by intracellular peptidases to melphalan, or by intracellular esterases to des-ethyl metoflufen, which also has alkylating properties. The high activity of esterases and peptidases in human tumors leads to the rapid formation of metabolites of melflufen in these cells, which then leads to the influx of more melflufen (Gullbo, J., et al, J Drug Target, (2003) Vol 11, pages 355-363; Wickstrom, M., et al, Biochem Pharmacol (2010) Vol 79, pages 2381 - 1290). Because des-ethylmelflufen and melphalan are relatively hydrophilic, the potential exists for intracellular entrapment of these agents.

[0003] Addition of melflufen to a panel of primary cultures of human tumor cells produces a pattern of activity similar to that of melphalan, but the efficacy is 50-100 times higher (Wickstrom, M., et al, Invest New Drugs (2008) Vol 26, pages 195 - 204), which is explained by the 10-20 times higher intracellular concentration (Gullbo, J., et al, J Drug Target, (2003) Vol 11, pages 355-363;Wickstrom, M., et al, Biochem Pharmacol (2010) Vol 79, pages 2381 - 1290). This can be explained by the highly efficient uptake of melflufen into these cells and the efficient formation of melflufen metabolites.

[0004] Melflufen is generally provided in crystalline form after synthesis. The crystalline form can only be dissolved in strongly acidic aqueous solutions, which are often not suitable for manufacturing and pharmaceutical purposes. In previous pharmaceutical preparations, the crystalline form was dissolved in dimethylacetamide (DMA) and glucose solutions. However, this preparation was unstable and easily formed unwanted melflufen dimers. Organic solvents such as DMA can also be harmful to patients and can damage medical devices used for administration. As described in WO2012 / 146625 and WO2014 / 065751, lyophilized preparations of melflufen have been found to have improved stability and solubility in aqueous solutions.

[0005] Compounds that are inherently unstable are more likely to form undesirable metabolites and impurities that are difficult to handle.Alkylating agents such as melflufen present additional difficulties because they have the potential to form undesirable genotoxic metabolites and impurities that can cause off-target effects in patients.Therefore, alkylating agents with poor stability are often difficult to handle and may have undesirable pharmacological properties.Therefore, there is a need for melflufen derivatives with improved stability and handling properties.

[0006] The present inventors have discovered that deuterated derivatives of melflufen have improved properties compared to melflufen having natural abundance levels of deuterium. Summary of the Invention

[0007] The present invention provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof: [ka] During the ceremony, Each R 1 ~R 30 is independently selected from the group consisting of H and deuterium; R 1 ~R 30 At least one of is deuterium having an abundance level greater than the naturally occurring abundance of deuterium.

[0008] The present invention further provides a compound of formula (Ia), or a pharma- ceutically acceptable salt thereof: [ka] During the ceremony, Each R 1 ~R 30 is independently selected from the group consisting of H and deuterium; R 1 ~R 30 At least one of is deuterium having an abundance level greater than the naturally occurring abundance of deuterium.

[0009] The present invention further provides a composition comprising deuterated melflufen of formula (I) or (Ia) together with an acceptable carrier. The composition may optionally contain an additional therapeutic agent, such as a protease inhibitor (PI), an immunomodulatory agent (IMiD) or an alkylating agent.

[0010] The present invention further provides a pharmaceutical composition comprising deuterated melflufen of formula (I) or (Ia) together with a pharma- ceutically acceptable carrier. The pharmaceutical composition may optionally contain an additional therapeutic agent, such as a protease inhibitor (PI), an immunomodulatory agent (IMiD) or an alkylating agent.

[0011] The present invention further provides a compound or pharmaceutical composition according to the present invention for use as a medicament.Furthermore, the present invention also provides a compound or pharmaceutical composition according to the present invention for use in the treatment or prevention of cancer, such as multiple myeloma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

[0012] The present invention further provides a method of treating a patient comprising administering a pharma- ceutically effective amount of a compound or a pharmaceutical composition according to the invention. [Brief description of the drawings]

[0013] [Figure 1] 1 shows the mean plasma concentrations of melflufen-d5(III) in paired male and female beagle dogs following injections of 1.25 and 2.5 mg / kg of melflufen-d5(III). [Diagram 2] Comparison of individual and mean (±SD) Cmax of melflufen-d5(III) or melflufen following administration of melflufen-d5(III) (Group 3, 2.5 mg / kg) or melflufen (Group 4, 2.5 mg / kg) to male and female beagle dogs is shown. [Diagram 3]Comparison of individual and mean (±SD) AUC last of melflufen-d5(III) or melflufen following administration of melflufen-d5(III) (Group 3, 2.5 mg / kg) or melflufen (Group 4, 2.5 mg / kg) to male and female beagle dogs is shown. [Figure 4] Comparison of individual and mean (±SD) Cmax of desethyl-melflufen following injection of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs is shown. [Diagram 5] 1 shows a comparison of individual and mean (±SD) AUC end of desethyl-melflufen following injection of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) into male and female beagle dogs. [Figure 6a] 1 shows the mean plasma concentrations of melflufen-d5(III) and its metabolites desethyl-melflufen and melphalan following injection of 1.25 mg / kg of melflufen-d5(III) into male and female beagle dogs (sex combined for Group 2). [Figure 6b] 1 shows the mean plasma concentrations of melflufen-d5(III) and its metabolites desethyl-melflufen and melphalan following injection of 1.25 mg / kg of melflufen-d5(III) into male and female beagle dogs (sex combined for Group 2). [Figure 7a] 1 shows the mean plasma concentrations of melflufen-d5(III) and its metabolites desethyl-melflufen and melphalan following injection of 2.5 mg / kg of melflufen-d5(III) into male and female beagle dogs (sex combined in Group 3). [Figure 7b] 1 shows the mean plasma concentrations of melflufen-d5(III) and its metabolites desethyl-melflufen and melphalan following injection of 2.5 mg / kg of melflufen-d5(III) into male and female beagle dogs (sex combined in Group 3). [Figure 8a]1 shows the mean plasma concentrations of melflufen and its metabolites desethyl-melflufen and melphalan following injection of 2.5 mg / kg melflufen into male and female beagle dogs (sex combined in Group 4). [Figure 8b] 1 shows the mean plasma concentrations of melflufen and its metabolites desethyl-melflufen and melphalan following injection of 2.5 mg / kg melflufen into male and female beagle dogs (sex combined in Group 4). [Figure 9] Comparison of individual and mean (±SD) Cmax of melphalan following injection of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs is shown. [Figure 10] 1 shows a comparison of individual and mean (±SD) AUC end of melphalan following injection of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs. [Figure 11] Comparison of individual and mean (±SD) t1 / 2,z of melphalan following injection of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs is shown. [Figure 12] Comparison of individual and mean (±SD) AUC∞ of melphalan following injection of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention provides compounds of formula (I), or a pharma- ceutically acceptable salt thereof, wherein each R 1 ~R 30 is independently selected from the group consisting of H and deuterium; R 1 ~R 30At least one of is deuterium having an abundance level greater than the naturally occurring abundance of deuterium.

[0015] The present invention provides compounds of formula (Ia), or a pharma- ceutically acceptable salt thereof, wherein each R 1 ~R 30 is independently selected from the group consisting of H and deuterium; R 1 ~R 30 At least one of is deuterium having an abundance level greater than the naturally occurring abundance of deuterium.

[0016] The naturally occurring abundance of deuterium is 0.0156 mol%, where mol% is the percentage of the total moles of hydrogen in a sample that are deuterium. Thus, in 1 mole of naturally occurring hydrogen, 0.156 mmol are deuterium, or 6.022 x 10 23 In a sample of naturally occurring hydrogen atoms, 9.39 × 10 19 There are 6,413 atoms of deuterium, or 1 atom of deuterium in a sample of 6,413 naturally occurring hydrogen atoms.

[0017] There are 30 carbon-hydrogen (CH) groups in melflufen, each containing a distribution of naturally occurring hydrogen isotopes. Thus, in a sample of melflufen, the abundance of deuterium at each position is approximately 0.0156 mol%. Thus, in 1 mole of melflufen, there are 4.68 mmol of deuterium, or 6.022 x 10 23 In a sample of melflufen molecules, 2.82 × 10 21 There are 1 deuterium atom in 2 molecules of Melflufen.

[0018] R in formula (I) or (Ia) 1 ~R 30where one or more of is designated herein as "deuterium," the abundance of deuterium at the designated position is greater than the naturally occurring abundance of deuterium. A level of deuterium abundance greater than the naturally occurring abundance of deuterium can be at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol%, or 98 mol% deuterium.

[0019] Deuterium is a safe and stable isotope of hydrogen. The energy required to break a carbon-deuterium (CD) bond is higher than that required to break a carbon-hydrogen (CH) bond. Therefore, reactions involving the breaking of a CD bond proceed at a slower rate than reactions breaking a C-H bond. If a C-H bond is broken in the rate-limiting step of a reaction, its replacement with a C-D bond will decrease the reaction rate. This effect is called the deuterium kinetic isotope effect (DKIE).

[0020] The effect of deuteration on the pharmacological properties of a drug is unpredictable and must be empirically determined. In some selected cases, deuteration has been shown to improve the pharmacological properties of a drug (see, for example, WO2010 / 044981). In other cases, deuteration may have no clinically relevant effect or may have a negative effect on the pharmacological properties of a drug.

[0021] Deuteration of a drug may decrease the rate at which the drug is metabolized by enzymes such as cytochrome P450 (CYP), esterases, peptidases, reductases, dehydrogenases and oxidases, altering its pharmacological properties. It is also possible that deuteration may have the effect of altering the metabolic profile of a drug, a phenomenon often referred to as "metabolic switching."

[0022] Metabolic switching can occur when deuterated drugs bind to metabolic enzymes in a different conformation compared to non-deuterated drugs. This can lead to the formation of different proportions of known metabolites or even the formation of new metabolites (Fischer et al., Curr Opin Drug Discov Devel, 2006, 9(1), 100-109). It is not possible to predict how the increased abundance of deuterium at a particular position may alter the metabolic profile of a drug. It is also not possible to predict whether the altered metabolic profile will improve or be detrimental to the pharmacological properties of the drug.

[0023] The inventors of the present invention have surprisingly found that the deuterated melflufen derivatives according to the present invention have particularly beneficial properties. For example, when administered by injection, the deuterated melflufen derivatives result in increased systemic exposure to the derivative itself, as well as to the active metabolite melphalan, compared to an equivalent dose of melflufen. This effect is shown in Example (a) below, and in particular in the mean and individual Cs after administration of melflufen-d5(III) or melflufen to dogs. 最大 and AUC 最後 Melflufen-d5(III) / melflufen or melphalan are shown in Figures 3, 4, 9 and 10 of Example (a).

[0024] The result of increased exposure to melflufen-d5(III) and melphalan for the same dose of melflufen-d5(III) and melphalan has a very significant benefit. As mentioned above, the excellent clinical efficacy of melflufen can be explained by the very efficient uptake of melflufen into cells and the efficient formation of melflufen metabolites. Therefore, derivatives that lead to even higher exposure of melflufen derivatives and higher exposure of active metabolite melphalan compared to melflufen are particularly advantageous, as they are expected to improve both of these properties of melflufen. In addition to these advantages, which mean that less compound needs to be made, stored and administered, it also allows a lower dose of deuterated melflufen derivatives to be administered compared to an equivalent dose of melflufen, reducing the risk of side effects from the administration of melflufen, or, if the same dose as that of melflufen is administered, a higher exposure to deuterated melflufen derivatives and melphalan can be achieved, leading to a better chance of providing clinical benefits to patients without increasing the risk of intolerable toxic side effects.

[0025] Preferred compounds according to the invention are those comprising R 1 ~R 30 Particularly preferred compounds according to the invention are those in which at least one of R 1 ~R 8 At least one of the groups is deuterium or R 9 ~R 15 At least one of the groups is deuterium or R 16 ~R 18 At least one of the groups is deuterium or R 19 ~R 25 At least one of is deuterium or R 26 ~R 30 At least one of the atoms is deuterium.

[0026] Further preferred compounds according to the invention are those comprising R 1 ~R 8At least two of the nuclei are deuterium or R 1 ~R 8 At least three of the nuclei are deuterium or R 1 ~R 8 At least four of the nuclei are deuterium or R 1 ~R 8 At least five of the nuclei are deuterium or R 1 ~R 8 At least six of the nuclei are deuterium or R 1 ~R 8 At least seven of the 1 to 7 carbon atoms are deuterium or R 1 ~R 8 At least eight of these are deuterium.

[0027] Further preferred compounds according to the invention are those comprising R 9 ~R 15 At least two of the nuclei are deuterium or R 9 ~R 15 At least three of the nuclei are deuterium or R 9 ~R 15 At least four of the nuclei are deuterium or R 9 ~R 15 At least five of the nuclei are deuterium or R 9 ~R 15 At least six of the R 9 ~R 15 At least seven of these are deuterium.

[0028] Further preferred compounds according to the invention are those comprising R 16 ~R 18 At least two of the are deuterium or R 16 ~R 18 At least three of the atoms are deuterium.

[0029] Further preferred compounds according to the invention are those comprising R 19 ~R 25 At least two of the nuclei are deuterium or R 19 ~R 25 At least three of the nuclei are deuterium or R19 ~R 25 At least four of the nuclei are deuterium or R 19 ~R 25 At least five of the nuclei are deuterium or R 19 ~R 25 At least six of the R 19 ~R 25 At least seven of these are deuterium.

[0030] Further preferred compounds according to the invention are those comprising R 26 ~R 30 At least two of the nuclei are deuterium or R 26 ~R 30 At least three of the nuclei are deuterium or R 26 ~R 30 At least four of the are deuterium or R 26 ~R 30 In one particularly preferred embodiment of the invention, the compounds according to the invention are those in which at least five of R 26 ~R 30 Five of them are deuterium.

[0031] Further preferred compounds according to the invention are 1 ~R 30 At least two of R are deuterium. Particularly preferred compounds are 1 ~R 8 At least one of the atoms is deuterium and R 9 ~R 15 , R 16 ~R 18 , R 19 ~R 25 Or R 26 ~R 30 At least one of the groups is deuterium or R 9 ~R 15 At least one of the atoms is deuterium and R 1 ~R 8 , R 16 ~R 18 , R 19 ~R 25 Or R 26~R 30 At least one of the groups is deuterium or R 16 ~R 18 At least one of the atoms is deuterium and R 1 ~R 8 , R 9 ~R 15 , R 19 ~R 25 Or R 26 ~R 30 At least one of the groups is deuterium or R 19 ~R 25 At least one of the atoms is deuterium and R 1 ~R 8 , R 9 ~R 15 , R 16 ~R 18 Or R 26 ~R 30 At least one of is deuterium or R 26 ~R 30 At least one of the atoms is deuterium and R 1 ~R 8 , R 9 ~R 15 , R 16 ~R 18 Or R 19 ~R 25 At least one of the atoms is deuterium.

[0032] In one embodiment of the present invention, R 1 ~R 8 For example, a compound according to the invention may be selected from the following group, where each of the atoms designated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0033] In another embodiment of the present invention, R 1 ~R 8For example, a compound according to the invention may be selected from the following group, where each of the atoms designated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0034] In another embodiment of the present invention, R 1 ~R 8 For example, a compound according to the invention may have the following structure, in which each of the atoms designated as being deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0035] In another embodiment of the present invention, R 9 ~R 15 At least two of the are deuterium, e.g., R 9 ~R 12 For example, a compound according to the invention may be selected from the following group, where each of the atoms designated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0036] In another embodiment of the present invention, R 16 ~R 18 For example, a compound according to the invention may have the following structure, in which each of the atoms designated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0037] In another embodiment of the present invention, R 19 ~R 25 For example, a compound according to the invention may have the following structure, in which each of the atoms designated as being deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0038] In another embodiment of the present invention, R 26 ~R 30 For example, a compound according to the invention may have the following structure, in which each of the atoms designated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0039] In another embodiment of the present invention, R 26 ~R 30 For example, a compound according to the invention may have the following structure, in which each of the atoms designated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0040] In a particularly preferred embodiment of the present invention, R 26 ~R 30 Five of the atoms are deuterium (i.e., R 26 ~R 30 is deuterium.) For example, a compound according to the invention may have the following structure, in which each of the atoms designated as being deuterium (D) has a deuterium abundance that is greater than the naturally occurring abundance of deuterium. [ka]

[0041] In another embodiment of the present invention, R 9 ~R 15 At least one of R is deuterium. 19 ~R 25 For example, a compound according to the invention may have the following structure, in which each of the atoms designated as being deuterium (D) has a deuterium abundance that is greater than the naturally occurring abundance of deuterium. [ka]

[0042] In another embodiment of the present invention, R 9 ~R 15 At least one of R is deuterium. 1 ~R 8 or R 26 ~R 30 For example, a compound according to the invention may be selected from the following group, where each of the atoms designated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium: [ka]

[0043] The compounds of the present invention can be prepared by using methods known to those skilled in the art of organic chemistry and by routine modification of known procedures for preparing melflufen.Procedures for preparing melflufen are described in WO01 / 96367 and WO2016 / 180740.Procedures for preparing deuterated compounds are known in the art.See, for example, Sajiki, New Horizons of Process Chemistry (2017), Springer, pg 29-40 and Hanson, The Organic Chemistry of Isotopic Labelling (2011), Chapter 3, RSC Publishing.

[0044] The compounds of the present invention can be prepared by synthetic techniques using deuterated reagents. Alternatively, deuterium can be introduced by reduction of reducible moieties using deuterated reducing agents. A further alternative approach is the use of post-synthetic hydrogen-deuterium exchange reaction using D2 gas in the presence of metal catalysts, such as Pd / C or Pt / C catalysts.

[0045] The compounds of the present invention can be prepared by using a combination of deuterated and non-deuterated reagents. Suitable deuterated reagents are those in which each deuterium has an abundance level greater than the naturally occurring abundance of deuterium. For example, an abundance level of at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol%, or 98 mol% of deuterium. Suitable deuterated reagents include deuterated chloroacetic acid, deuterated chloroethanol, deuterated ethylene oxide, deuterated ethanol, deuterated para-fluoro-phenylalanine, deuterated para-nitro-phenylalanine, and deuterated para-amino-phenylalanine. Deuterated reagents can be purchased from commercial suppliers. Alternatively, they can be prepared from non-deuterated reagents using hydrogen-deuterium exchange reactions as described above.

[0046] The compounds of the present invention can also be prepared by using deuterated reducing agents. Suitable deuterated reducing agents include borane deuteride, borane deuteride-Lewis base complexes, boro-deuterides, metal deuterides, and D2 gas in the presence of a metal catalyst. 。

[0047] The compounds of the present invention can also be prepared from melflufen using a hydrogen-deuterium exchange reaction.

[0048] Particular methods for preparing compounds according to the invention are described herein in the Examples section.

[0049] For the avoidance of doubt, in this document, the term "deuterated melflufen", when used, includes its salt(s), unless otherwise specified. Melflufen, and its salts, in particular its hydrochloride salt, are known, for example, from WO01 / 96367 and WO2014 / 065751, and the same salts are suitable for use in the present invention.

[0050] The salts of deuterated melflufen suitable for use in the present invention are those in which the counterion is pharma- ceutically acceptable. Suitable salts include those formed with organic or inorganic acids. In particular, suitable salts formed with acids according to the present invention include those formed with mineral acids such as amino acids, strong organic carboxylic acids, such as hydroxycarboxylic acids, such as alkane carboxylic acids of 1 to 4 carbon atoms, unsubstituted or substituted, for example, by halogen, saturated or unsaturated dicarboxylic acids, or organic sulfonic acids, such as (C1-C4)-alkyl or aryl-sulfonic acids, unsubstituted or substituted, for example, by halogen. Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulfuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxalic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, isethionic, ascorbic, malic, phthalic, aspartic, and glutamic acids, lysine, and arginine.

[0051] Preferred salts of deuterated melflufen include acid addition salts such as those formed from hydrochloric acid, hydrobromic acid, acetic acid, p-toluenesulfonic acid, tartaric acid, sulfuric acid, succinic acid, phosphoric acid, oxalic acid, nitric acid, methanesulfonic acid, malic acid, maleic acid, and citric acid. More preferably, the salt of deuterated melflufen according to the present invention is a hydrochloride salt (i.e., an addition salt formed from hydrochloric acid).

[0052] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents with which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates." For example, a complex with water is known as a "hydrate." The complex can incorporate the solvent in stoichiometric or non-stoichiometric amounts. Solvates are described in Water-Insoluble Drug Formulation, 2 nd ed R. Lui CRC Press, page 553 and Byrn et al Pharm Res 12(7), 1995, 945-954. Before being made into solution, the deuterated melflufen of formula (I) and (Ia), or a salt thereof, for use in the present invention may be in the form of a solvate. The solvates of deuterated melflufen suitable for use as a pharmaceutical are those in which the associated solvent is pharma-ceutically acceptable. For example, hydrates are pharma-ceutically acceptable solvates.

[0053] While the compound according to the present invention can be administered alone, it is preferred that it is present in a composition, particularly a pharmaceutical composition.The pharmaceutical composition includes those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous injection, intramuscular, intravascular (bolus or infusion), and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including cutaneous, buccal, sublingual and intraocular) administration, although the most preferred route may depend, for example, on the condition and disorder of the subject being treated.

[0054] The pharmaceutical composition of the present invention suitable for oral administration can be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of active ingredient, as powder or granules, as a solution or suspension in aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion. Deuterated melflufen can also be presented as a bolus, electuary, or paste. Various pharmacologic acceptable carriers and their formulations are described in standard formulation treatises, such as Remington's Pharmaceutical Sciences by EW Martin. See also Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

[0055] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. Preferably, the formulations may be presented in unit-dose or divided-dose containers, such as sealed ampoules and vials. The formulations may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as saline or water for injection, immediately prior to use. Such lyophilized formulations are known from WO2012 / 146625 and WO2014 / 065751 for the established melflufen compound. The compounds of the present invention may be formulated in a similar manner, for example, in lyophilized form containing the active ingredient and sucrose, for example, in a weight ratio of 1:25 to 1:75, for example, 1:50. Extemporaneous injection and infusion solutions and suspensions may be prepared from sterile powders, granules or other dry compositions. Exemplary compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, a suitable non-toxic, parenterally acceptable diluent or solvent, mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agents and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid or Cremaphor.

[0056] Pharmaceutical compositions for nasal, aerosol or inhalation administration include solutions in saline, which can contain, for example, benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and / or other solubilizing or dispersing agents such as those known in the art.

[0057] Pharmaceutical compositions for rectal administration may be presented as a suppository with conventional carriers such as cocoa butter, synthetic glyceride esters, or polyethylene glycols. Such carriers are typically solid at ordinary temperatures, but liquefy and / or melt in the rectal cavity to release the drug.

[0058] Pharmaceutical compositions for topical administration in the oral cavity, e.g., buccal or sublingual administration, include lozenges comprising the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia. Exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).

[0059] The compounds, compositions and pharmaceutical compositions according to the present invention can be used in the treatment and / or prevention of cancer, reducing tumor growth and / or killing tumor cells. Thus, deuterated melflufen can be used to cure and / or prolong the survival of patients suffering from cancer disease. The present invention is particularly useful for the treatment and / or prevention of multiple myeloma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma, especially when the condition is relapsed or refractory. The present invention finds particular use in the treatment of relapsed and refractory multiple myeloma.

[0060] The amount of deuterated melflufen required to achieve a therapeutic effect will vary depending on the specific route of administration and the characteristics of the subject under treatment, such as species, age, weight, sex, medical condition, the specific disease and its severity, and other relevant medical and physical factors.A physician of ordinary skill can easily determine and administer the effective amount of deuterated melflufen required for the treatment or prevention of cancer.

[0061] Deuterated melflufen, or a salt thereof, may be administered daily, every 2 or 3 days, weekly, every 2, 3, or 4 weeks, or even as a high single dose depending on the subject and type of cancer being treated.

[0062] Preferably, deuterated melflufen, or a salt thereof (excluding the mass of any salt) may be administered in an amount of about 15 to 150 mg per dose, for example, 15, 20, 25, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 mg.

[0063] Alternatively, deuterated melflufen, or a salt thereof (excluding the mass of any salt) may be administered in a single high dose. The single high dose may be about 150-1200 mg, for example, about 150-800 mg. For example, it may be selected from 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 and 1200 mg. For example, it may be selected from 150, 200, 300, 400, 500, 600, 700 and 800 mg.

[0064] Deuterated melflufen, or its salt, may be used as the sole active ingredient of the present invention, but may also be used in combination with one or more additional therapeutic agent(s), and the use of such combination provides one preferred embodiment of the present invention. Such additional therapeutic agent may be a drug useful for treating or preventing cancer, or other pharmacologic active material. Such drugs are known in the art. Examples of additional therapeutic agents for use in the present invention include steroids (prednisone and dexamethasone), IMiDs (thalidomide, lenalidomide and pomalidomide), PIs (bortezomib and carfilzomib), histone deacetylase (HDAC) inhibitors (panobinostat) and conventional chemotherapy (alkylating agents (e.g., melphalan, cyclophosphamide) and doxorubicin). EXAMPLES

[0065] Synthesis of Compounds of the Invention General Experimental Details Unless otherwise stated, all reagents and solvents were purchased from commercial suppliers and used without further purification. Melflufen and melflufen intermediates can be prepared using the synthetic methods described in WO2016 / 180740 or WO01 / 96367.

[0066] Analytical HPLC / LCMS was performed using an Agilent 1100 Series Liquid Chromatography / Mass Selective Detector (MSD, single quadrupole) equipped with an electrospray interface and a UV diode array detector. Analysis was performed by two methods using either an ACE 3 C8 (3.0×50 mm) column (condition #1) with a 10-97% gradient of acetonitrile in 0.1% TFA in water over 3 min and a flow rate of 1 ml / min, or an Xbridge C18 (3.0×50 mm) column (condition #2) with a 10-97% gradient of acetonitrile in 10 mM ammonium bicarbonate over 3 min and a flow rate of 1 ml / min, both with UV detection at 305 nm. 1H NMR spectra were recorded on a Bruker 400 MHz instrument at 25° C. Preparative HPLC was performed on a Gilson system equipped with a UV detector using an Xbridge Prep C18 5 μM OBD (19×50 mm) column with acetonitrile and 50 mM ammonium bicarbonate as buffers.

[0067] Example 1 Synthesis of (2S)-2-[[(2S)-2-amino-3-[4-[bis(2-chloroethyl)amino]phenyl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid (II). [ka] Melflufen hydrochloride (500 mg, 0.93 mmol) was suspended in water (10 mL) followed by the addition of concentrated HCl (10 mL). The reaction mixture was stirred at room temperature for 24 hours. Toluene was added to the reaction mixture and the solution was concentrated in vacuum. This process was repeated three times. The solution was then evaporated to dryness in vacuum. The crude mixture was used as starting material for Example 2.

[0068] Example 2 Synthesis of melflufen-d5 (III), melflufen-d6 (IV) and melflufen-d7 (V) [ka] Compound II (507 mg, 0.93 mmol) was dissolved in ethanol-d6 (4.86 g, 93.32 mmol) and refluxed. After 2 h, there was almost complete conversion of compound II to the ester. The reaction mixture was cooled to room temperature and then evaporated to dryness in vacuo to give a white solid (495 mg, 95%).

[0069] NMR of the final compound showed partial deuteration at the ortho position of the aniline functional group. The integral of the doublet at δ 6.78-6.82 ppm suggested that the final sample was a heterogeneous mixture of melflufen-d5(III), melflufen-d6(IV) and melflufen-d7(V), with the final sample containing about 12.5% ​​melflufen-d5(III). Deuterium-proton exchange is known to occur with the proton at the aniline functional group under thermal and acidic conditions in deuterated solvents. LC-MS (Condition #1): R 2.28 min (>97% purity), m / z[M+H]505. LC-MS (condition #2):t R 2.63 min (purity>98%), m / z[M+H]505.1H NMR(400MHz,MeOD):δ / ppm;2.92-2.97(m,1H),3.01-306(m,1H),3.16-3.21(dd,2H),3.67-3.71(m,4H),3.78-3.81(m ,4H),4.02-4.05(m,1H),4.69-4.73(m,1H),6.78-6.82(d,0.25H),7.02-7.07(t,2H),7.19(s,2H),7.24-7.28(m,2H).

[0070] Example 3 Synthesis of ethyl (2S)-2-[[(2S)-3-[4-[bis(1,1,2,2-tetradeuterio-2-hydroxy-ethyl)amino]phenyl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-3-(4-fluorophenyl)propanoate (VI) [ka] Ethyl (2S)-2-[[(2S)-3-(4-aminophenyl)-2-tertbutoxycarbonylamino)propanoyl]amino]-3-(4-fluorophenyl)propanoate (470 mg, 0.99 mmol, see WO2016 / 180740 for suitable synthesis methods) was suspended in acetonitrile. Na2CO3 (210.42 mg, 1.99 mmol) was added to the reaction mixture at room temperature. The reaction mixture was then stirred for 15 minutes before adding 1,1,2,2-tetradeuterio-2-iodo-ethanol (0.17 mL, 2.18 mmol). The reaction mixture was stirred under reflux for 1 month. After cooling, the reaction was partitioned between DCM and water and extracted with DCM. The organic phase was concentrated and the product was purified by preparative HPLC to give the title compound (0.29 g, 51%).

[0071] Example 4 Synthesis of melflufen-d8 (VII). [ka] Compound VI (290 mg, 0.51 mmol) was dissolved in DCM followed by slow addition of POCl3. The reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned over water and DCM and then basified by adding 1M NaOH. The title compound was extracted with diethyl ether and the solvent was evaporated in vacuo to give the title compound as a pale yellow foam of approximately 95% purity (96 mg, 37%). LC-MS (Condition #1):t R 2.27 minutes, m / z[M+H]506. LC-MS (condition #2):t R 2.63 minutes, m / z[M+H]506.1H NMR(400MHz,MeOD):δ / ppm;1.00-1.15(t,3H),2.80-2.84(m,1H),2.91-2.95(m,1H),3.04-3.09(dd,2H),3.90-3.94(m, 1H),4.03-4.08(q,2H),4.58-4.62(m,1H),6.68-6.70(d,2H),6.90-6.94(t,2H),7.07-7.10(d,2H),7.13-7.17(m,2H).

[0072] Example 5 Preparation of (2S)-2-[[(2S)-2-amino-3-[4-[bis(2-chloro-1,1,2,2-tetradeuterioethyl)amino]phenyl]propanoyl]amino]-3-(4-fluorophenyl)propanoate (VIII) [ka] Compound VII (20 mg, 0.04 mmol) was suspended in water (3 mL) followed by the addition of concentrated HCl (3 mL). The reaction mixture was stirred at room temperature overnight. Toluene was added to the reaction mixture and the solution was concentrated in vacuo. This process was repeated three times. The residue was dissolved in acetonitrile / water and transferred to a vial followed by removal of the solvent using a stream of N2 to give the title compound (13.7 mg, 64%). LC-MS (Condition #1):t R 1.97 min (>95% purity), m / z[M+H]478. LC-MS (condition #2):t R 1.72 min (>94% purity), m / z[M+H]478.

[0073] Example 6 Biological activity The Fluorescence Microculture Cytotoxicity Assay (FMCA) (Larsson, R., et al-1992: Int J Cancer, 50,177-185) is used to evaluate the compounds. Briefly, 96-well microtiter plates (NUNC, Roskilde, Denmark) are prepared with 20 μl of drug solution at 10x the desired concentration and stored at -70 °C for up to 2 months. In general, substances are first dissolved in absolute or acidic ethanol to a concentration of 4.0-8.2 mM and further diluted in sterile water or sterile phosphate-buffered saline. All dilutions with water are performed immediately prior to the experiment to minimize the effects of mustard hydrolysis. The final ethanol concentration does not exceed 1% v / v. On day zero of the experiment, 180 μL of cell suspension at the appropriate concentration is added to the wells of the thawed plate, with six wells serving as controls (cell suspension only) and six wells as blanks (cell medium only). After 72 h of incubation, cells are washed once with PBS and 100 μL of fluorescein diacetate (10 μg / ml) in physiological buffer is added. After a further 45 min, the fluorescence generated (ex 485 rim; em 528 nm) is measured in a 96-well scanning fluorometer (Fluoroscan II, Labsystems Oy, Helsinki, Finland). The fluorescence generated is proportional to the number of live cells and the data are expressed as a viability index (fluorescence in test wells in percent of control wells with blank values ​​subtracted) and IC 50 (Presented as inhibitory concentration 50%) as calculated by the software GraphPad Prism @ (Graphpad Software Inc., San Diego, CA, USA). Quality criteria for a successful assay included a coefficient of variation of less than 30% in blank (6 wells), control (6 wells) and test wells (3), respectively, a control signal more than 10-fold that of the blank, and finally an initial cell viability of more than 70% (primary human tumor cultures) or 90% (cell lines), as judged by the trypan blue exclusion test.

[0074] Fluorescein diacetate (FDA, Sigma) is dissolved in DMSO at 10 mg / ml and stored frozen as a stock solution in the dark. Cell growth medium RPMI-1640 (Sigma) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Chemical Co., St. Louis, MO), 2 mM glutamine, 100 μg / ml streptomycin, and 100 μg / ml penicillin is used.

[0075] Example 7a Esterification of p-fluoro-L-phenylalanine with ethanol (d6) 2 H5) Preparation of ethyl (2S)-2-amino-3-(4-fluorophenyl)propanoate hydrochloride [ka] p-Fluoro-L-phenylalanine (1.0 kg, CAS number 1132-68-9) was slurried in ethanol-d6 (2.5 L, CAS number: 1516-08-1) and 1,2-dichloroethane (2.0 L). A scrubber containing NaOH (5 M solution) was connected to the outlet of the reactor, after the condenser. Bromothymol blue (1-2 mg) was added to follow the decomposition of the scrubber liquor.

[0076] The reactor was heated to an internal temperature of 60° C. When the internal temperature reached 60° C., the addition of thionyl chloride (600 ml) was started at a slow rate. Initially, a very thick precipitate formed. The initial very thick slurry thinned over the course of the reaction. The total time for the addition was about 3 hours. The internal temperature was allowed to reach a maximum of 70° C. and was controlled by adjusting the mantle temperature accordingly. After complete addition, the mantle temperature was adjusted to maintain the internal temperature at 65-70° C.

[0077] Desired ( 2H5) Complete conversion to ethyl (2S)-2-amino-3-(4-fluorophenyl)propanoate hydrochloride was achieved 3 hours after the point of complete addition of thionyl chloride. After complete conversion was confirmed (LC-MS analysis with the following conditions: ACE 3 C8 (3.0×50 mm) column with 10-90% B gradient over 3 min, mobile phase A, water 0.1% TFA, mobile phase B, pure acetonitrile, flow rate of 1 mL / min, UV detection at 215-395, 254 and 220 nm), the reaction was cooled (internal temperature about 45° C.) and tert-butyl methyl ether (12.5 L) was added to obtain the product as a white precipitate. The mixture was stirred to obtain a homogeneous mixture.

[0078] The mixture was then cooled to an internal temperature of 0° C. and aged at this temperature for approximately 30 minutes before filtration. 2 H5) Ethyl (2S)-2-amino-3-(4-fluorophenyl)propanoate hydrochloride was washed with about 1 L of tert-butyl methyl ether and then dried under reduced pressure at a maximum temperature of 30° C. The product was carefully sieved to remove any lumps present. 2 The isolated yield of H5) ethyl (2S)-2-amino-3-(4-fluorophenyl)propanoate hydrochloride was 92%. LC-MS: R 1.43 min, m / z[M+H]217.

[0079] Example 7b ( 2 H5) Kg-scale preparation of ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanamido]-3-(4-fluorophenyl)propanoate (IX) [ka] Melphalan (1.663 kg, 5.45 mol, 1 equiv.) was added to a mixture of purified water (16.0 kg), NaOH (32%, aq., 1.04 kg), and tetrahydrofuran (10.0 kg) at 10-15 °C. A mixture of di-tert-butyl dicarbonate (1.308 kg, 5.99 mol, 1.1 equiv.) and tetrahydrofuran (4.75 kg) was added at 10-15 °C. The reaction mixture was stirred at 18-23 °C for 4-5 h until a minimum of 97.0% (HPLC) conversion of melphalan was achieved. The temperature was adjusted to 15-20 °C and, while maintaining this temperature, the pH was adjusted to 2.5-3.0 with 1.5 M HCl. Ethyl acetate (7.34 kg) was added and the phases were separated. The aqueous phase was extracted with ethyl acetate (7.34 kg). The combined organic phases were dried over magnesium sulfate, filtered, and the filter cake was washed with ethyl acetate. The solvent was removed by vacuum distillation, and the residue containing (2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanoic acid was dried in vacuum at 20-25 °C for a minimum of 12 h. HPLC: retention time 11.9 min. (HPLC conditions were as follows: sample solvent acetonitrile:water, 1:1 (v / v), Waters, Atlantic T3 (3μ, 4.6×150 mm) column, 10-90-10% B gradient over 23 min, 1 mL / min flow rate, mobile phase A: 500 μL 85% phosphoric acid in 1.0 L MQ water, mobile phase B: 500 μL 85% phosphoric acid in 1.0 L acetonitrile, with UV detection at 262 nm).

[0080] The (2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(butoxy)carbonyl]-amino}propanoic acid residue was redissolved in dichloromethane (44.0 kg). 4-Methylmorpholine (1.378 kg, 13.63 mol, 2.5 equiv.) was added and ( 2H5) Ethyl (2S)-2-amino-3-(4-fluorophenyl)propanoate hydrochloride (1.377 kg, 5.45 mol, 1.0 equiv.), 1-hydroxybenzotriazole, HO (0.083 kg, 0.54 mol, 0.1 equiv.) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, HCl (1.045 kg, 5.45 mol, 1.0 equiv.). The reaction mixture was cooled to 100° C. for 2 h to obtain the (2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanoic acid (H5). 2 H5) Stir at 18-23 °C for 3-4 h until a minimum of 97.0% (HPLC) conversion to ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanamide]-3-(4-fluorophenyl)propanoate was achieved (HPLC conditions were as follows: sample solvent acetonitrile, Waters, Atlantic T3 (3μ, 4.6×150 mm) column, 10-90-10% B gradient over 23 min, 1 mL / min flow rate, mobile phase A: 500 μL 85% phosphoric acid in 1.0 L MQ water, mobile phase B: 500 μL 85% phosphoric acid in 1.0 L acetonitrile, with UV detection at 262 nm).

[0081] The pH was adjusted to 3.0-4.0 with 5% KHSO4 (aq). The organic phase was settled and the aqueous phase was extracted with dichloromethane (29.0 kg). The first organic phase was washed with 6% NaHCO3. The organic phase was settled and the remaining aqueous phase was back extracted with the second organic phase. The combined organic phase was dried over magnesium sulfate, filtered and washed with dichloromethane. The dried organic phase was concentrated to 22-26 L by distillation in vacuum. The reduced organic phase was applied to column chromatography (silica gel (40-63 pm, 22.4 kg), n-heptane (6.7 kg) and dichloromethane (52.2 kg)). The column was eluted with 6% ethyl acetate / dichloromethane. ( 2H5) The fractions containing ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanamide]-3-(4-fluorophenyl)propanoate (TLC) were combined and evaporated under reduced pressure to 26-28 L. Ethyl acetate (5.8 kg) was added and evaporation continued to 26-28 L. This procedure was repeated. After the addition of ethyl acetate, ( 2 H5) Precipitation of ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanamide]-3-(4-fluorophenyl)propanoate has begun. Optionally, ( 2 Seed crystals of H5) ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanamido]-3-(4-fluorophenyl)propanoate may be added to aid precipitation. Ethyl acetate (5.8 kg) is added again and the optional seeding step may be repeated. The mixture is evaporated under reduced pressure to 19-21 L and n-heptane (22.1 kg) is added at 35-45 °C. The suspension is cooled to -2-2 °C and stirred for 2-18 h. The solid is isolated by centrifugation and the filter cake is washed with n-heptane. The solid is dried in vacuum at 30 °C and ( 2 H5) Ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanamido]-3-(4-fluorophenyl)propanoate (2.6 kg, 80%) was obtained as a white to slightly yellow solid material. HPLC: retention time 13.4 min.

[0082] Example 7c Melflufen-d5 (III) (( 2 H5) Kg scale production of ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propanamido]-3-(4-fluorophenyl)propanoate hydrochloride [ka] (1.3 M HCl in acetonitrile) prepared from hydrogen chloride (1.31 kg, 35.9 mol) and acetonitrile (21.7 kg). 2 H5) A solution of ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanamido]-3-(4-fluorophenyl)propanoate (Compound IX) (3.10 kg, 5.14 mol) was stirred at 29-33° C. for 12-24 hours. ( 2 H5) Ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanamido]-3-(4-fluorophenyl)propanoate, minimum 99.0% (HPLC) 2 H5) Conversion to ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propanamido]-3-(4-fluorophenyl)propanoate hydrochloride was obtained (HPLC conditions were as follows: sample solvent DMSO acetonitrile, 1:9 (v / v), Waters, Atlantic T3 (3μ, 4.6×150 mm) column, 10-90-10% B gradient over 23 min, 1 mL / min flow rate, mobile phase A: 500 μL 85% phosphoric acid in 1.0 L MQ water, mobile phase B: 500 μL 85% phosphoric acid in 1.0 acetonitrile, with UV detection at 262 nm).

[0083] The reaction mixture was subjected to polish filtration and diluted with acetonitrile (68.9 kg). Distillation at reduced pressure was then carried out using a jacket temperature of 45° C. When the volume of the reaction mixture was 86 L, acetonitrile (22.7 kg) was added and distillation was continued. When 86 L of reaction mixture remained, acetonitrile (22.7 kg) was added and distillation was continued. When the volume in the reactor was 86 L, acetonitrile (22.7 kg) was added and distillation was continued until a volume of 86 L in the reactor was reached.

[0084] tert-Butyl methyl ether (68.4 kg) was added over a period of 25-45 min at 35-45° C., followed by cooling to 22-28° C. After stirring for 60-120 min at this temperature, the crude ( 2 H5) Ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propanamide]-3-(4-fluorophenyl)propanoate hydrochloride was filtered off and washed with tert-butyl methyl ether (12.5 kg). The crude material was dried in vacuum in a reactor using a jacket temperature set point of 30° C.

[0085] Acetonitrile (84.0 kg) was added and the resulting suspension was stirred at 48-54 °C for 30-90 min, followed by cooling to 40-45 °C. tert-Butyl methyl ether (74.6 kg) was added over a period of 40-70 min at 38-45 °C, followed by cooling to 22-28 °C. After stirring at this temperature for 60-120 min, the crude ( 2 H5) Ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propanamido]-3-(4-fluorophenyl)propanoate hydrochloride was filtered off and washed with tert-butyl methyl ether (14.0 kg). Drying in vacuum at 30-35° C. yielded ( 2 H5) Ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propanamido]-3-(4-fluorophenyl)propanoate hydrochloride (melflufen-d5, (III)) (2.5 kg, 90%) was obtained as a white to off-white solid. HPLC: retention time 9.0 min.

[0086] Biological testing Example (a) In vivo studies in dogs A comparative single-dose toxicity study in dogs was performed to investigate the toxicokinetics of melflufen and melflufen-d5(III) and their metabolites desethyl-melflufen and melphalan, comparing melflufen-d5(III) and melflufen, after a single intravenous dose of melflufen-d5(III) or melflufen as a 30-minute infusion in dogs.

[0087] (i) Introduction and Objectives This study aimed to compare the potential acute toxicity of melflufen-d5(III) and melflufen.The toxicokinetics of melflufen-d5(III), melflufen and their metabolites desethyl-melflufen and melphalan were evaluated after a single intravenous administration as a 30-minute infusion in dogs.

[0088] (ii) Materials and methods Abbreviation The following abbreviations are used in this document: [Table 1]

[0089] Study design Melflufen-d5(III) or melflufen was given as a 30 minute infusion to male and female dogs according to the following scheme. [Table 2]

[0090] Sample collection Blood samples were collected from a peripheral vein on day 1 pre-dose, 15 min, 30 min (just before the end of the infusion), 40 min, 1 hr, 2 hr, 4 hr, and 6 hr after the start of the infusion.

[0091] Blood samples were collected in heparinized collection tubes, placed in an ice-water bath and immediately centrifuged (3 min, 10000 g, +4° C.). The resulting plasma was divided into two aliquots, placed in pre-cooled cryovials and placed in a 70° C. freezer until analysis.

[0092] Toxicological kinetic calculation Plasma toxicokinetic analysis of melflufen-d5(III), melflufen and its metabolites, desethyl-melflufen and melphalan, was performed according to a standard non-compartmental approach using Phoenix WinNonlin (v. 6.3, Certara Company, USA).

[0093] After administration, the maximum concentration, C 最大 , and T, the time at which the maximum concentration is achieved. 最大 was read as the coordinate of the maximum plasma concentration over time. The last detectable concentration, C 最後 , and T, the time of the last detectable concentration 最後 was reported as a parameter.

[0094] AUC, the area under the plasma concentration versus time curve until the last detectable concentration 最後 was calculated by the linear trapezoidal rule.

[0095] When feasible, the following parameters were calculated: The terminal half-life, t 1 / 2,z was calculated by linear regression analysis of the natural log concentration versus time curve according to the following formula:

number

[0096] AUC, the area under the plasma concentration versus time curve to infinity ∞ , the AUC assuming single exponential decay last Clast / λ z The area was calculated by adding up the parts of the area calculated as:

[0097] AUC, which accounts for the extrapolated area under the curve ∞ The ratio was calculated as follows:

number

[0098] (iii) Results Melflufen-d5(III), melflufen and their metabolites desethyl-melflufen and melphalan were not measured in plasma samples from the control group (Group 1), as well as pre-dose in treatment Groups 2, 3 and 4.

[0099] Systemic exposure parameters for males and females for melflufen-d5(III), melflufen and their metabolites desethyl-melflufen and melphalan were comparable, therefore descriptive statistics for the combined male and female parameters are also reported.

[0100] Melflufen-d5(III) Summary toxicokinetic parameters of melflufen-d5(III) are reported in Tables 1 and 2. [Table 3] [Table 4]

[0101] Melflufen-d5(III) at doses of 1.25 and 2.5 mg / kg infused over 30 minutes (Groups 2 and 3) reached its maximum plasma concentration midway through the infusion and was then eliminated within 40 minutes of the start of dosing. Half-life was not calculated due to an insufficient number of terminal time points.

[0102] Exposure to melflufen-d5(III) increased with dose in terms of peak and area under the curve (2.7-fold for a 2-fold dose increase, calculated for the combined sex parameters).

[0103] The mean +SD plasma concentrations of melflufen-d5(III) after 1.25 and 2.5 mg / kg are shown in FIG.

[0104] Melfulfen Summary melflufen toxicokinetic parameters are reported in Tables 3 and 4 below. [Table 5] [Table 6]

[0105] Comparison between systemic exposure to melflufen-d5(III) and melflufen Comparisons of individual and mean (±SD) systemic exposure parameters of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in combined male and female beagle dogs are shown in Figure 2 (C 最大 ) and Figure 3 (AUC 最後 ) as shown in

[0106] The mean exposure to melflufen was 2-fold lower than that to melflufen-d5(III) at 2.5 mg / kg. As shown in Figures 2 and 3, the difference in the mean values ​​was partially caused by the high concentration measured in one male dog treated with melflufen-d5(III). However, the individual C values ​​for each animal were significantly higher than those for the control dog. 最大 and AUC 最後 As can be seen from Figures 2 and 3 which show values ​​of C 最大 and increased AUC 最後 There is a clear tendency for

[0107] The interanimal variability (CV%) in the two groups was of the same order of magnitude.

[0108] Desethyl-Melflufen Summary toxicokinetic parameters of the metabolite desethyl-melflufen are reported in Tables 5 and 6 . [Table 7] [Table 8]

[0109] After injection of 1.25 and 2.5 mg / kg melflufen-d5(III) (groups 2 and 3), the metabolite desethyl-melflufen appeared in plasma at the first sampling time, reached its maximum concentration at 15-30 min, and was completely undetectable 40-60 min after administration (1.25-2.5 mg / kg). The half-life, which could be estimated in only one animal at the 2.5 mg / kg dose, was 5 min.

[0110] Exposure to desethyl-melflufen increased C 最大 3.1 times and AUC 最後 There was a 3.5-fold increase in the genotype (calculated based on the combined sex parameters).

[0111] After melflufen administration (Group 4), the plasma profile of desethyl-melflufen was 最大 and t 最後 The half-life, which could only be estimated in one animal at the 2.5 mg / kg dose, was 7 minutes.

[0112] Comparison between systemic exposure to desethyl-melflufen following administration of melflufen-d5(III) or melflufen Comparison of individual and mean (±SD) systemic exposure parameters of desethyl-melflufen following injection of melflufen-d5(III) (group 3, 2.5 mg / kg) and melflufen (group 4, 2.5 mg / kg) in combined male and female beagle dogs is shown in Figure 4 (C 最大 ) and Figure 5 (AUC 最後 ) as shown in

[0113] The mean exposure to desethyl-melflufen was lower after injection of melflufen at 2.5 mg / kg than after injection of melflufen-d5(III) at 2.5 mg / kg. As shown in Figures 4 and 5, the difference in the mean values ​​was mainly caused by the high concentration of desethyl-melflufen measured in one male dog treated with melflufen-d5(III). However, the individual C values ​​for each animal were 最大 As can be seen from FIG. 4 which shows the increased C values ​​of desethyl-melflufen following injection of melflufen-d5(III) compared to melflufen. 最大 There is a tendency for

[0114] Melphalan Summary toxicokinetic parameters for the metabolite melphalan are reported in Tables 7 and 8. [Table 9] [Table 10]

[0115] After infusion of 1.25 and 2.5 mg / kg melflufen-d5(III), the metabolite melphalan appeared in plasma at the first sampling time and had a mean t 最大 Its maximum concentration was reached at 100 mg / kg / day and was detectable up to 4 hours after administration after each melphalan-d5(III) dose. The estimated half-life was approximately 40 minutes.

[0116] After melflufen infusion (group 4), the plasma profile of melphalan was similar to that formed by melflufen-d5(III). 最大 and t 最後 was similar.

[0117] Exposure to melphalan increased with the administered dose of melflufen-d5(III) in terms of peak and AUC values: by combining gender, a two-fold dose increase increased the mean C 最大 and a 2.2-fold increase in the AUC of metabolites 最後 and AUC ∞ This corresponded to a 2.0-fold increase in

[0118] Melphalan AUC at two ascending doses of melphalan-d5(III) 最後 were 48- and 35-fold higher than melflufen-d5(III) exposure, respectively (AUC 最後 (calculated with respect to the value) 。 Melphalan AUC at two ascending doses of melphalan-d5(III) 最後 were on average 51.1-fold (range 37–70) and 44.8-fold (range 22–100) higher than melflufen-d5(III) exposure, respectively (calculated for individual values ​​for both sexes combined).

[0119] After melphalan infusion, melphalan AUC 最後 was on average 75-fold (range 38–142) ​​higher than melflufen exposure (calculated for individual values ​​for both sexes combined).

[0120] The mean +SD plasma concentrations of melflufen-d5(III), melphalan and desethyl-melflufen following injection of melflufen-d5(III) in dogs (gender combined) in Groups 2 or 3 are shown in Figures 6a (Group 2, log scale) and 6b (Group 2, non-log scale) and Figures 7a (Group 3, log scale) and 7b (Group 3, non-log scale). As shown in Figures 7a and 7b, the mean C after injection of 2.5 mg / kg melflufen-D5(III) was 0.01 mg / kg. 最大 was 2.73 μmol / L.

[0121] The mean +SD plasma concentrations of melflufen, melphalan and desethyl-melflufen following injection of melflufen in dogs (gender combined) in Group 4 are shown in Figures 8a (Group 4, log scale) and 8b (Group 4, non-log scale). As shown in Figures 8a and 8b, the mean C after injection of 2.5 mg / kg melflufen 最大 was 2.23 μmol / L.

[0122] Comparison between systemic exposure to melphalan following administration of melflufen-d5(III) or melflufen Comparison of individual and mean (±SD) systemic exposure parameters of melphalan following injection of melflufen-d5(III) (group 3, 2.5 mg / kg) and melflufen (group 4, 2.5 mg / kg) in combined male and female beagle dogs is shown in Figure 9 (C 最大 ) and Fig. 10 (AUC 最後 ) is shown in t 1 / 2,z and AUC ∞ The results are also shown in FIG. 11 and FIG.

[0123] The mean exposure to melphalan was lower after injection of melflufen at 2.5 mg / kg than after injection of melflufen-d5(III) at 2.5 mg / kg. 最大 , AUC 最後 and AUC ∞ As can be seen from Figures 9, 10 and 21, which show values ​​of melphalan after infusion of melflufen-d5(III) compared to melflufen,最大 , increased AUC 最後 and increased AUC ∞ As shown in Figure 11, the mean t 1 / 2,z was lower after injection of melflufen-d5(III) at 2.5 mg / kg than after injection of melflufen, indicating a decreased t for melphalan in individual animals after injection of melflufen-d5(III) compared with melflufen. 1 / 2,z There is a tendency for

[0124] conclusion Systemic exposure descriptors for melflufen-d5(III), melflufen and their metabolites desethyl-melflufen and melphalan after a single 30-minute infusion of melflufen-d5(III) (1.25 and 2.5 mg / kg) or melflufen (2.5 mg / kg) were similar in males and females.

[0125] Following infusion of melflufen-d5(III), melflufen-d5(III) and the metabolite desethyl-melflufen were rapidly cleared from the systemic circulation. Exposure to desethyl-melflufen was approximately half that of the parent compound.

[0126] The metabolite melphalan was formed rapidly and extensively; it was detectable in plasma for up to 4 hours after the end of the infusion and was decayed with a terminal half-life of approximately 40 minutes. 最大 , t 最後 and t 1 / 2,z was constant across melflufen-d5(III) doses. In the genders combined, the extent of exposure to formed melphalan was approximately 50-fold higher than melflufen-d5(III) exposure.

[0127] After increasing the titrated dose of melflufen-d5(III) infusion, systemic exposure increased as expected (melphalan) and slightly more than expected assuming dose proportionality (melflufen-d5(III) and desethyl-melflufen).

[0128] When comparing equivalent doses of melflufen-d5(III) and melflufen, the overall exposure to melflufen-d5(III) and the active metabolite melphalan was consistently higher following infusion of melflufen-d5(III) compared to melflufen. This increased exposure to melflufen-d5(III) and melphalan for identical doses of melflufen-d5(III) and melphalan is a highly significant benefit.

Claims

1. A compound of formula (I), or a pharma- ceutically acceptable salt thereof, 【Chemistry 1】 During the ceremony, Each R ２６ R to R are deuterium having an abundance level of at least 90 mol% or 98 mol%; ２５ is H, or a pharma- ceutically acceptable salt thereof.

2. 2. The compound of claim 1, wherein the abundance level is at least 98 mol% deuterium.

3. The structure is of formula (Ia) 【Chemistry 2】 The compound according to claim 1, wherein the compound is represented by the formula:

4. The compound is ２ H ５ 2. The compound of claim 1, which is ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propanamido]-3-(4-fluorophenyl)propanoate hydrochloride.

5. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 4 together with a pharma- ceutically acceptable carrier, and optionally together with an additional therapeutic agent.

6. The pharmaceutical composition of claim 5, wherein the additional therapeutic agent is a protease inhibitor (PI), an immunomodulatory drug (IMiD) or an alkylating agent.

7. A compound according to any one of claims 1 to 4 for use as a medicine.

8. A compound according to any one of claims 1 to 4 for use in the treatment or prevention of cancer.

9. A compound according to any one of claims 1 to 4 for use in the treatment or prevention of multiple myeloma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

10. 7. A composition according to claim 5 or 6 for use as a medicine.

11. 7. A composition according to claim 5 or 6 for use in the treatment or prevention of cancer.

12. 7. The composition of claim 5 or 6 for use in the treatment or prevention of multiple myeloma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

13. 13. Use of a pharma- ceutically effective amount of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for treating a patient.

14. 13. Use of an effective amount of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for treating or preventing cancer.

15. 15. The use according to claim 14, wherein the cancer is any one of multiple myeloma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

16. A compound having a structural formula selected from the group consisting of: 【Chemistry 3】 or a pharma- ceutically acceptable salt thereof.

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