Deuterium-containing compounds

Abstract

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof (Formula I), wherein each of R<supgt;1< / supgt> - R<supgt;30< / supgt> is independently selected from the group consisting of H and deuterium, and at least one of R<supgt;1< / supgt> - R<supgt;30< / supgt> is deuterium having an abundance level greater than the natural abundance of deuterium. The present invention also provides a pharmaceutical composition containing the compound and the use of the compound.

Description

Technical Field

[0001] The present invention relates to novel deuterated melflufen derivatives having particularly advantageous properties. The novel deuterated melflufen derivatives or their salts can be used for treating or preventing cancer. Background Art

[0002] Melphalan (also known as melphalan flufenamide and L-melphalanyl-4-fluoro-L-phenylalanine ethyl ester) is an anti-tumor agent useful in the treatment of cancer, particularly multiple myeloma. Melphalan is described in WO 01 / 96367 and WO 2014 / 065751. The structure of the hydrochloride salt of melphalan is shown below:

[0003]

[0004] Meflufen is a powerful and highly lipophilic alkylating agent, and it achieves targeted delivery of alkylated metabolites to tumor cells. Compared with other hydrophilic alkylating agents, the high lipophilicity of meflufen allows it to be quickly absorbed into tissues and cells. Once inside the cell, meflufen may bind directly to DNA, or it may be easily hydrolyzed by intracellular peptidases into melphalan, or by intracellular esterases into des-ethylmethfulfen, which also has alkylating properties. The high activity of esterases and peptidases in human tumors allows the rapid formation of meflufen metabolites in these cells, and then the rapid formation allows more meflufen to flow in (Gullbo, J. et al., Journal of Drug Target (J Drug Target) (2003) Vol. 11, pp. 355-363; Wickstrom, M. et al., Biochem Pharmacol (Biochem Pharmacol) (2010) Vol. 79, pp. 2381-1290). Since desethylmelflufen and melphalan are relatively hydrophilic, it is possible to trap these agents inside cells.

[0005] Addition of melflufen to primary cultures of human tumor cells resulted in an activity pattern similar to that of melphalan, but with a 50- to 100-fold higher potency (Wickstrom, M. et al., Invest New Drugs (2008) Vol. 26, pp. 195-204), which was explained by a 10- to 20-fold higher intracellular concentration (Gullbo, J. et al., Journal of Drug Targets, (2003) Vol. 11, pp. 355-363; Wickstrom, M. et al., Biochemical Pharmacology (2010) Vol. 79, pp. 2381-1290). This can be explained by the efficient uptake of melflufen by these cells and the efficient formation of melflufen metabolites.

[0006] Melflufen is usually provided in crystalline form after synthesis. The crystalline form is only soluble in strongly acidic aqueous solutions which are generally unsuitable for manufacturing and pharmaceutical purposes. In previous pharmaceutical formulations, the crystalline form was dissolved in dimethylacetamide (DMA) and glucose solutions. However, this formulation is unstable and prone to the formation of harmful Melflufen dimers. Organic solvents such as DMA may also be harmful to patients and may damage medical devices used for administration. As described in WO 2012 / 146625 and WO 2014 / 065751, lyophilized formulations of Melflufen have been found to have improved stability and solubility in aqueous solutions.

[0007] Intrinsically unstable compounds are difficult to handle and are more likely to form harmful metabolites and impurities. Alkylating agents such as mefluphenazine present further difficulties because they have the potential to form harmful genotoxic metabolites and impurities that may cause off-target effects in patients. Therefore, poorly stable alkylating agents are generally difficult to handle and may have poor pharmacological properties. Therefore, there is a need for mefluphenazine derivatives with improved stability and handling characteristics.

[0008] The present inventors have found that deuterated derivatives of Melflufen have improved properties compared to Melflufen with naturally abundant levels of deuterium. Summary of the invention

[0009] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0010]

[0011] in,

[0012] 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 the is deuterium having an abundance level greater than the naturally occurring abundance of deuterium.

[0013] The present invention further provides a compound of formula (Ia) or a pharmaceutically acceptable salt thereof,

[0014]

[0015] in,

[0016] 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 the is deuterium having an abundance level greater than the naturally occurring abundance of deuterium.

[0017] The present invention further provides a composition comprising deuterated melflufen of formula (I) or (Ia) and an acceptable carrier. The composition may optionally comprise an additional therapeutic agent, such as a protease inhibitor (PI), an immunomodulatory drug (IMiD) or an alkylating agent.

[0018] The present invention further provides a pharmaceutical composition comprising deuterated melflufen of formula (I) or (Ia) and a pharmaceutically acceptable carrier. The pharmaceutical composition may optionally comprise an additional therapeutic agent, such as a protease inhibitor (PI), an immunomodulatory drug (IMiD) or an alkylating agent.

[0019] The present invention further provides a compound or pharmaceutical composition according to the present invention, which is used as a medicament. In addition, a compound or pharmaceutical composition according to the present invention is provided, which is used to treat or prevent cancer, such as multiple myeloma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

[0020] The present invention further provides a method of treating a patient, the method comprising administering a pharmaceutically effective amount of a compound or pharmaceutical composition according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are mean plasma concentrations of Melflufen-d5(III) in combined male and female beagle dogs following 1.25 and 2.5 mg / kg Melflufen-d5(III) infusions.

[0022] Figure 2 The individual and mean (±SD) C values ​​of Melflufen-d5(III) or Melflufen after 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 are shown. max comparison.

[0023] Figure 3Shown are the individual and mean (±SD) AUCs of Melflufen-d5(III) or Melflufen after 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 last comparison.

[0024] Figure 4 Shown are the individual and mean (±SD) C values ​​of desethyl-melflufen following infusion of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs. max comparison.

[0025] Figure 5 Shown are the individual and mean (±SD) AUCs of desethyl-melflufen following infusion of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs last comparison.

[0026] Figure 6a and 6b Shown are mean plasma concentrations of melflufen-d5(III) and its metabolites desethyl-melflufen and melphalan following infusion of 1.25 mg / kg melflufen-d5(III) to male and female beagle dogs (Group 2 combined sexes).

[0027] Figure 7a and 7b Shown are mean plasma concentrations of melflufen-d5(III) and its metabolites desethyl-melflufen and melphalan following infusion of 2.5 mg / kg melflufen-d5(III) to male and female beagle dogs (Group 3 combined sexes).

[0028] Figure 8a and 8b Shown are mean plasma concentrations of melflufen and its metabolites desethyl-melflufen and melphalan following infusion of 2.5 mg / kg melflufen to male and female beagle dogs (Group 4 combined sexes).

[0029] Fig. 9 Figure 2 shows individual and mean (±SD) C values ​​of melphalan following infusion of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs. max comparison.

[0030] Fig.10Shown are the individual and mean (±SD) AUCs of melphalan following infusion of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs last comparison.

[0031] Fig.11 Figure 2 shows the individual and mean (±SD) t of melphalan following infusion of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs. 1 / 2,z comparison.

[0032] Fig.12 Shown are the individual and mean (±SD) AUCs of melphalan following infusion of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) in male and female beagle dogs ∞ comparison. DETAILED DESCRIPTION

[0033] The present invention provides a compound 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 the is deuterium having an abundance level greater than the naturally occurring abundance of deuterium.

[0034] The present invention further provides a compound of formula (Ia) 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 the is deuterium having an abundance level greater than the naturally occurring abundance of deuterium.

[0035] 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 is deuterium. Thus, in 1 mole of naturally occurring hydrogen, 0.156 mmol is deuterium, or 6.022×10 23 Of the naturally occurring hydrogen atoms in a sample, 9.39×10 19 deuterium atom, or one deuterium atom in a sample of 6,413 naturally occurring hydrogen atoms.

[0036] There are 30 carbon-hydrogen (CH) groups in mefluphen and each contains a naturally distributed hydrogen isotope. Therefore, in a sample of mefluphen, the abundance of deuterium at each position is about 0.0156 mol%. Therefore, in 1 mole of mefluphen, there are 4.68 mmol of deuterium, or 6.022×10 23 Among the samples of melflufen molecules, 2.82×10 21 deuterium atoms, or 1 deuterium atom in 214 melflufen molecules.

[0037] In formula (I) or (Ia), R 1 -R 30 Where one or more of the above are indicated herein as "deuterium", the deuterium abundance at the indicated position is greater than the naturally occurring abundance of deuterium. The deuterium abundance level greater than the naturally occurring abundance of deuterium may be at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol%, or 98 mol% deuterium.

[0038] Deuterium is a safe and stable isotope of hydrogen. The energy required to break a carbon-deuterium (CD) bond is higher than the energy required to break a carbon-hydrogen (CH) bond. Therefore, reactions involving the breaking of CD bonds proceed at a slower rate than reactions that break CH bonds. If a CH bond breaks in the rate-determining step of a reaction, then substitution of a CD bond will reduce the reaction rate. This effect is known as the Deuterium Kinetic Isotope Effect (DKIE).

[0039] The effect of deuteration on the pharmacological properties of a drug is unpredictable and must be determined empirically. In some selected cases, deuteration has been shown to improve the pharmacological properties of a drug (see, e.g., WO 2010 / 044981). In other cases, deuteration may not have a clinically relevant effect or may have a negative impact on the pharmacological properties of a drug.

[0040] Deuteration of a drug can reduce the rate at which it is metabolized by enzymes such as cytochromes P450 (CYP), esterases, peptidases, reductases, dehydrogenases, and oxidases, thereby altering its pharmacological properties. Deuteration may also have the effect of altering the metabolic profile of a drug, a phenomenon often referred to as "metabolic switching".

[0041] Compared to non-deuterated drugs, when deuterated drugs bind to metabolic enzymes in different conformations, metabolic switching may occur. This may result in the formation of known metabolites in different proportions, or even the formation of new metabolites (Fischer et al., Curr Opin Drug Discov Devel, 2006, 9 (1), 100-109). It is impossible to predict how an increase in deuterium abundance at a specific position may change the metabolite profile of a drug. It is also impossible to predict whether a changed metabolite profile will improve or be detrimental to the pharmacological properties of a drug.

[0042] 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 infusion, the deuterated melflufen derivatives provide increased systemic exposure of the derivative itself and the active metabolite melphalan compared to an equivalent dose of melflufen. This effect is demonstrated in Example (a) below, and in particular, in Example (a). Figure 3 , 4 , 9 and 10, the figures show the average and individual C after administration of melflufen-d5(III) or melflufen to dogs max and AUC last Melflufen-d5(III) / melflufen or melphalan.

[0043] For the same dose of melflufen-d5 (III) and melflufen, the results of increased exposure of melflufen-d5 (III) and melphalan have extremely significant benefits. As described above, the excellent clinical efficacy of melflufen can be explained by the efficient absorption of melflufen by cells and the effective formation of melflufen metabolites. Therefore, compared with melflufen, derivatives that cause even higher exposure of melflufen derivatives and higher exposure of the active metabolite melphalan are particularly advantageous because they are expected to improve those properties of melflufen at the same time. In addition to the advantages that mean that less compounds need to be prepared, stored and administered, it also allows the administration of lower doses of deuterated melflufen derivatives compared to equivalent doses of melflufen, which reduces the risk of side effects caused by the administration of melflufen; or if the same dose as the dose of melflufen is administered, higher exposure of deuterated melflufen derivatives and melphalan can be achieved, thereby having a better chance of providing clinical benefits to patients without increasing the risk of intolerable toxic side effects.

[0044] Preferred compounds according to the invention are those in which R 1- R 30 Particularly preferred compounds according to the invention are those in which R 1 -R 8 At least one of them is deuterium; R 9 -R 15 At least one of them is deuterium; R16 -R 18 At least one of them is deuterium; R 19 -R 25 At least one of is deuterium; or R 26 -R 30 Those compounds wherein at least one of the

[0045] Other preferred compounds according to the invention are those in which R 1 -R 8 At least two of them are deuterium; R 1 -R 8 At least three of them are deuterium; R 1 -R 8 At least four of them are deuterium; R 1 -R 8 At least five of them are deuterium; R 1 -R 8 At least six of them are deuterium; R 1 -R 8 At least seven of them are deuterium; or R 1 -R 8 Those compounds in which at least eight of the atoms are deuterated.

[0046] Other preferred compounds according to the invention are those in which R 9 -R 15 At least two of them are deuterium; R 9 -R 15 At least three of them are deuterium; R 9 -R 15 At least four of them are deuterium; R 9 -R 15 At least five of them are deuterium; R 9 -R 15 At least six of them are deuterium; or R 9 -R 15 Those compounds wherein at least seven of the atoms are deuterated.

[0047] Other preferred compounds according to the invention are those in which R 16 -R 18 At least two of them are deuterium; or R 16 -R 18 Those compounds wherein at least three of the

[0048] Other preferred compounds according to the invention are those in which R 19 -R 25 At least two of them are deuterium; R 19 -R 25 At least three of them are deuterium; R 19 -R 25 At least four of them are deuterium; R19 -R 25 At least five of them are deuterium; R 19 -R 25 At least six of them are deuterium; or R 19 -R 25 Those compounds wherein at least seven of the atoms are deuterated.

[0049] Other preferred compounds according to the invention are those in which R 26 -R 30 At least two of them are deuterium; R 26 -R 30 At least three of them are deuterium; R 26 -R 30 At least four of them are deuterium; or R 26 -R 30 In a particularly preferred embodiment of the present invention, the compounds according to the present invention are those compounds in which R 26 -R 30 Those compounds wherein five (ie, each) of the

[0050] Other preferred compounds according to the invention are those in which R 1 -R 30 Particularly preferred compounds are those wherein R 1 -R 8 At least one of them is deuterium and R 9 -R 15 , R 16 -R 18 , R 19 -R 25 or R 26 -R 30 A compound in which at least one of R 9 -R 15 At least one of them is deuterium and R 1 -R 8 , R 16 -R 18 , R 19 -R 25 or R 26 -R 30 At least one of them is deuterium; R 16 -R 18 At least one of them is deuterium and R 1 -R 8 , R 9 -R 15 , R 19 -R 25 or R 26 -R 30 At least one of them is deuterium; R19 -R 25 At least one of them 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 them is deuterium and R 1 -R 8 , R 9 -R 15 , R 16 -R 18 or R 19 -R 25 Those compounds wherein at least one of the compounds is deuterated.

[0051] In one embodiment of the present invention, R 1 -R 8 At least two of them are deuterium. For example, the compound according to the present invention can be selected from the following group, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0052]

[0053] In another embodiment of the present invention, R 1 -R 8 At least four of the atoms are deuterium. For example, the compound according to the invention may be selected from the following group, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0054]

[0055] In another embodiment of the present invention, R 1 -R 8 At least eight of the atoms are deuterium. For example, the compounds according to the present invention have the following structure, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0056]

[0057] In another embodiment of the present invention, R 9 -R 15 At least two of them are deuterium, for example, R 9 -R 12At least two of them are deuterium. For example, the compound according to the present invention can be selected from the following group, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0058]

[0059] In another embodiment of the present invention, R 16 -R 18 At least three of them are deuterium. For example, the compound according to the present invention has the following structure, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0060]

[0061] In another embodiment of the present invention, R 19 -R 25 At least one of is deuterium. For example, the compound according to the present invention has the following structure, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0062]

[0063] In another embodiment of the present invention, R 26 -R 30 At least two of them are deuterium. For example, the compound according to the present invention has the following structure, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0064]

[0065] In another embodiment of the present invention, R 26 -R 30 At least three of them are deuterium. For example, the compound according to the present invention has the following structure, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0066]

[0067] In a particularly preferred embodiment of the present invention, R 26 -R 30 Five of them are deuterium (i.e. R 26 -R 30 For example, a compound according to the invention has the following structure, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0068]

[0069] In another embodiment of the present invention, R 9 -R 15 At least one of them is deuterium and R 19 -R 25 At least one of is deuterium. For example, the compound according to the present invention has the following structure, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0070]

[0071] In another embodiment of the present invention, R 9 -R 15 At least one of them is deuterium and R 1 -R 8 or R 26 -R 30 At least one of is deuterium. For example, the compound according to the present invention may be selected from the following group, wherein each of the atoms indicated as deuterium (D) has a deuterium abundance greater than the naturally occurring abundance of deuterium:

[0072]

[0073] The compounds of the present invention can be prepared using methods known to those skilled in the art of organic chemistry and by conventional modifications to the known procedures for preparing meflufen. The procedures for preparing meflufen are described in WO 01 / 96367 and WO 2016 / 180740. The procedures for preparing deuterated compounds are known in the art. See, for example, Sajiki, "New Horizons of Process Chemistry" (2017), Springer, pp. 29-40 and Hanson, "The Organic Chemistry of Isotopic Labelling" (2011), Chapter 3, RSC Publishing.

[0074] The compounds of the present invention can be prepared by synthetic techniques using deuterated reagents. Alternatively, deuterium can be introduced by reducing the reducible part using a deuterated reducing agent. Another alternative method is to use a post-synthetic hydrogen-deuterium exchange reaction using D2 gas in the presence of a metal catalyst (e.g., Pd / C or Pt / C catalyst).

[0075] The compounds of the present invention can be prepared by using a combination of deuterated and non-deuterated reagents. Suitable deuterated reagents are those deuterated reagents in which each deuterium has an abundance level greater than the naturally occurring abundance of deuterium. For example, at least 1mol%, 5mol%, 10mol%, 50mol%, 90mol% or 98mol% deuterium abundance levels. Suitable deuterated reagents include deuterated chloroacetic acid, deuterated chloroethanol, deuterated ethylene oxide, deuterated ethanol, deuterated p-fluoro-phenylalanine, deuterated p-nitro-phenylalanine and deuterated p-amino-phenylalanine. Deuterated reagents can be purchased from commercial suppliers. Alternatively, it can be prepared by a non-deuterated reagent using a hydrogen-deuterium exchange reaction as described above.

[0076] The compounds of the present invention can also be prepared by using a deuterated reducing agent. Suitable deuterated reducing agents include deuterated borane, deuterated borane-Lewis base complexes, borodeuterides, metal deuterides and D2 gas in the presence of a metal catalyst.

[0077] The compounds of the present invention may also be prepared from mefluphenazine using a hydrogen-deuterium exchange reaction.

[0078] Specific methods for preparing compounds according to the invention are described herein in the Examples section.

[0079] For the avoidance of doubt, herein, unless otherwise stated, when the term "deuterated mefluphenazine" is used, it includes salts thereof. Mefluphenazine and its salts, in particular its hydrochloride, are known from, for example, WO 01 / 96367 and WO 2014 / 065751, and the same salts are suitable for use in the present invention.

[0080] Salts of deuterated melflufen suitable for use in the present invention are those in which the counterion is pharmaceutically 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, strong organic carboxylic acids (such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, with halogens, such as saturated or unsaturated dicarboxylic acids, such as hydroxycarboxylic acids, such as amino acids) or with organic sulfonic acids (such as (C1-C4) alkylsulfonic acids or arylsulfonic acids which are unsubstituted or substituted, for example, with halogens). Pharmaceutically acceptable acid addition salts include those formed with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, acetic acid, phosphoric acid, lactic acid, pyruvic acid, trifluoroacetic acid, succinic acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, oxalic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, isethionic acid, ascorbic acid, malic acid, phthalic acid, aspartic acid and glutamic acid, lysine and arginine.

[0081] Preferred salts of deuterated mefluphen 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 mefluphen according to the present invention is a hydrochloride (i.e., an addition salt formed from hydrochloric acid).

[0082] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they react or precipitate or crystallize. These complexes are referred to as "solvates". For example, complexes with water are referred to as "hydrates". The complex can be incorporated into a solvent in stoichiometric or non-stoichiometric amounts. Solvates are described in "Water-Insoluble Drug Formulation", 2nd edition, edited by R. Lui, CRC Press, page 553 and Byrn et al., Pharm Res 12 (7), 1995, 945-954. Before it is made in solution, the deuterated mefluphenazine or its salt of formula (I) and formula (Ia) used in the present invention may be in the form of a solvate. Solvates of deuterated mefluphenazine suitable for use as a medicament are those in which the relevant solvent is pharmaceutically acceptable. For example, a hydrate is a pharmaceutically acceptable solvate.

[0083] Although it is possible to administer the compounds according to the invention alone, it is preferred that they are present in a composition, and in particular, in a pharmaceutical composition. Pharmaceutical compositions include those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion) and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including cutaneous, buccal, sublingual and intraocular) administration, but the most appropriate route may depend, for example, on the condition and disorder of the individual being treated.

[0084] Pharmaceutical compositions of the invention suitable for oral administration may be in the form of discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. Deuterated melflufen may also be in the form of a pill, electuary or paste. Various pharmaceutically acceptable carriers and their formulations are described in standard formulation discussions, such as in EW Martin's Remington's Pharmaceutical Sciences. See also Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supplement. 42: 2S, 1988.

[0085] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that provide the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Preferably, the formulation may be present in unit dose or divided dose containers, such as sealed ampoules and vials. The formulation may be stored in freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as saline or water for injection, immediately before use. Such lyophilized formulations are known from WO2012 / 146625 and WO2014 / 065751 for established meflufenamic compounds. The compounds of the present invention may be formulated in a similar manner, for example in a lyophilized form containing the active ingredient and sucrose, for example in a weight ratio of 1:25 to 1:75 (e.g., 1:50). Ready-to-use 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 may contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents such as 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.

[0086] Pharmaceutical compositions for nasal, aerosol or inhalation administration include solutions in saline which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other solubilizing or dispersing agents as are known in the art.

[0087] Pharmaceutical compositions for rectal administration may be in the form of suppositories with common carriers such as cocoa butter, synthetic glycerides or polyethylene glycols. Such carriers are generally solid at ordinary temperatures but liquefy and / or dissolve in the rectal cavity to release the drug.

[0088] Pharmaceutical compositions for topical administration in the mouth (e.g., buccal or sublingual) include lozenges comprising the active ingredient in a flavored base such as sucrose and acacia or tragacanth and lozenges 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).

[0089] The compounds, compositions and pharmaceutical compositions according to the invention can be used to treat and / or prevent cancer, reduce tumor growth and / or kill tumor cells. Thus, deuterated melflufen can be used to cure patients suffering from cancer diseases and / or prolong their survival. The invention is particularly suitable for treating and / or preventing multiple myeloma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma, especially when the condition relapses or is refractory. The invention is particularly suitable for treating relapsed refractory multiple myeloma.

[0090] The amount of deuterated melflufen required to achieve a therapeutic effect will vary with the specific route of administration and the characteristics of the individual being treated, such as species, age, weight, sex, medical condition, specific disease and its severity, and other relevant medical and physical factors. An ordinarily skilled physician can readily determine and administer the effective amount of deuterated melflufen required to treat or prevent cancer.

[0091] Deuterated melflufen or a salt thereof may be administered daily, every two or three days, weekly, every two weeks, every three weeks or every four weeks or even in a high single dose, depending on the individual and the form of cancer to be treated.

[0092] Preferably, deuterated melflufen or a salt thereof (excluding a majority of any salt) may be administered in an amount of about 15 to 150 mg per administration, for example, 15, 20, 25, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 mg.

[0093] Alternatively, deuterated melflufen or its salt (excluding the majority of any salt) can be administered in a single high dose. The single high dose can be about 150 to 1200 mg, for example about 150 to 800 mg. For example, it can be selected from 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 and 1200 mg. For example, it can be selected from 150, 200, 300, 400, 500, 600, 700 and 800 mg.

[0094] Although deuterated melflufen or its salt can be used as the sole active ingredient of the present invention, it is also possible to use it in combination with one or more other therapeutic agents, and the use of such a combination provides a preferred embodiment of the present invention. Such other therapeutic agents may be medicaments or other pharmaceutically active materials suitable for treating or preventing cancer. Such agents are known in the art. Examples of other therapeutic agents used 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).

[0095] Examples

[0096] Synthesis of the compounds of the present invention

[0097] General Experimental Details

[0098] Unless otherwise stated, all reagents and solvents were purchased from commercial sources and used without further purification.Mefluphen and Mefluphen intermediates can be prepared using the synthetic methods described in WO 2016 / 180740 or in WO 01 / 96367.

[0099] Analytical HPLC / LCMS was performed using an Agilent 1100 series liquid chromatograph / mass selective detector (MSD, single quadrupole) equipped with an electrospray interface and a UV diode array detector. ACE 3 C8 (3.0×50 mm) columns were used with a 10-97% gradient of acetonitrile in 0.1% TFA in water over 3 minutes and at a flow rate of 1 mL / min (condition #1), or Xbridge C18 (3.0×50 mm) columns were used with a 10-97% gradient of acetonitrile in 10 mM ammonium bicarbonate over 3 minutes and at a flow rate of 1 mL / min (condition #2), 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 buffer.

[0100] Example 1-Synthesis of (2S)-2-[[(2S)-2-amino-3-[4-[bis(2-chloroethyl)amino]phenyl]propionyl]amino]-3-(4-fluorophenyl)propionic acid (II)

[0101]

[0102] 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 vacuo. This process was repeated three times. The solution was then evaporated to dryness in vacuo. The crude mixture was used as the starting material for Example 2.

[0103] Example 2 - Synthesis of Meflufen-d5 (III), Meflufen-d6 (IV) and Meflufen-d7 (V)

[0104] Compound II (507 mg, 0.93 mmol) was dissolved in ethanol-d6 (4.86 g, 93.32 mmol) and refluxed. After 2 hours, compound II was almost completely converted into ester. The reaction mixture was cooled to room temperature and then evaporated to dryness in a vacuum to obtain a white solid (495 mg, 95%).

[0105] The NMR of the final compound showed partial deuteration at the ortho position of the aniline functional group. The integration of the double peak at δ 6.78ppm-6.82ppm indicated that the final sample was a heterogeneous mixture of meflufen-d5(III), meflufen-d6(IV) and meflufen-d7(V), wherein the final sample contained about 12.5% ​​meflufen-d5(III). Deuterium-proton exchange is known to occur in deuterated solvents under heating and acidic conditions using protons in the aniline functional group.

[0106] LC-MS (condition 1): t R 2.28 min (purity>97%), m / z [M+H] 505. LC-MS (condition 2): t R 2.63 minutes (purity>98%), m / z [M+H] 505. 1H NMR (400 MHz, 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).

[0107] Example Synthesis of ethyl 3-(2S)-2-[[(2S)-3-[4-[bis(1,1,2,2-tetradeuterium-2-hydroxy-ethyl)amino]phenyl]-2-(tert-butoxycarbonylamino)propionyl]amino]-3-(4-fluorophenyl)propanoate (VI)

[0108]

[0109] (2S)-2-[[(2S)-3-(4-aminophenyl)-2-tert-butoxycarbonylamino)propanoyl]

[0110] [0.29g, 51%) of 4-(4-fluorophenyl)-2-iodo-1-ol (0.17mL, 2.18mmol) was added to the reaction mixture at room temperature. The reaction mixture was stirred for 15 minutes, followed by addition of 1,1,2,2-tetradeuterium-2-iodo-ethanol (0.17mL, 2.18mmol). The reaction mixture was stirred at reflux for one month. After cooling, the reactant was separated from water by DCM and extracted with DCM. The organic phase was concentrated and the product was purified by preparative HPLC to give the title compound (0.29g, 51%).

[0111] Example 4 - Synthesis of Mefluphenazine-d8(VII).

[0112]

[0113] Compound VI (290 mg, 0.51 mmol) was dissolved in DCM, followed by the slow addition of POCl. The reaction mixture was stirred at room temperature overnight. The reaction mixture was separated by water and DCM, and then alkalized by the addition of 1 M NaOH. The title compound was extracted with ether, and the solvent was evaporated in vacuo to give the title compound (96 mg, 37%) as a light yellow foam of about 95% purity. 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).

[0114] 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)

[0115]

[0116] 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 the removal of the solvent using a N2 stream to give the title compound (13.7 mg, 64%). LC-MS (condition 1): t R 1.97 min (purity>95%), m / z [M+H] 478. LC-MS (condition 2): t R 1.72 min (purity>94%), m / z [M+H]478.

[0117] Example 6 - Biological Activity

[0118] Fluorescence microculture cytotoxicity analysis (FMCA) (Larsson, R. et al. -1992: "International Journal of Tumors (Int J Cancer)", 50, 177-185) is used to evaluate compounds. Briefly, 96-well microtiter plates (NUNC, Roskilde, Denmark) were prepared with 20 μl of drug solution at ten times the required concentration and stored at -70 ° C for up to two months. Typically, the substance is first dissolved in anhydrous or acidic ethanol to a concentration of 4.0 to 8.2 mM, and further diluted with sterile water or sterile phosphate-buffered saline. All dilutions were performed directly with water before the experiment to minimize the effects of nitrogen mustard hydrolysis. The final ethanol concentration did not exceed 1% v / v. On the 0th day of the experiment, 180 μL of a cell suspension of sufficient concentration was added to the wells of the thawed plate, six wells served as controls (only cell suspension) and six wells served as blanks (only cell culture medium). After 72 hours of incubation, cells were washed once with PBS and 100 μL of fluorescein diacetate (10 μg / ml) in physiological buffer was added. After another 45 minutes, the generated fluorescence (ex 485 edge; em 528 nm) was measured in a 96-well scanning fluorimeter (Fluoroscan II, Labsystems Oy, Helsinki, Finland). The generated fluorescence is proportional to the number of viable cells, and the data are presented as the viability index (fluorescence in the test wells, expressed as a percentage of the control wells, minus the blank value) and IC 50 (Inhibitory concentration 50%, as calculated by the software GraphPad Prism@ (Graphpad Software Inc., San Diego, CA, USA). Quality criteria for a successful assay include a coefficient of variation of less than 30% in the blank (six wells), control (six wells), and test wells (three), a control signal that exceeds ten times the blank, and finally an initial cell viability of more than 70% (primary human tumor cultures) or 90% (cell lines) as judged by a trypan blue exclusion test.

[0119] Fluorescein diacetate (FDA, Sigma) was dissolved in DMSO to 10 mg / ml and kept frozen in the dark as a stock solution. 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 was used.

[0120] Example 7a: Esterification of fluoro-L-phenylalanine with ethanol (d6) 2 H5) Preparation of ethyl (2S)-2-amino-3-(4-fluorophenyl) propionate hydrochloride

[0121]

[0122] p-Fluoro-L-phenylalanine (1.0 kg, CAS No. 1132-68-9) was slurried in a mixture of ethanol-d6 (2.5 l, CAS No. 1516-08-1) and 1,2-dichloroethane (2.0 l). After the condenser, a scrubber containing NaOH (5M solution) was connected to the outlet of the reactor. To track the degradation of the scrubber fluid, bromothymol blue (1-2 mg) was added.

[0123] The reactor was heated to an internal temperature of 60°C. When the internal temperature reached 60°C, thionyl chloride (600 ml) was added at a slow rate. A very thick precipitate was initially formed. The initially very thick slurry thinned during the course of the reaction. The total time for addition was about 3 hours. The internal temperature was allowed to reach a maximum of 70°C and was controlled by adjusting the mantel temperature accordingly. After complete addition, the mantel temperature was adjusted to maintain the internal temperature between 65-70°C.

[0124] After 3 hours after the addition of thionyl chloride was complete, the conversion to the desired ( 2 H5) ethyl (2S)-2-amino-3-(4-fluorophenyl) propanoate hydrochloride was achieved. After confirming complete conversion (LC-MS analysis, conditions are as follows: ACE 3 C8 (3.0×50 mm) column, 10-90% B gradient, lasting 3 minutes; 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 liters) was added to give the product as a white precipitate. The mixture was stirred to obtain a homogeneous mixture.

[0125] The mixture was then cooled to an internal temperature of 0°C and aged at this temperature for about 30 minutes before filtration. The solid was washed with about 1 liter of tert-butyl methyl ether ( 2 H5) ethyl (2S)-2-amino-3-(4-fluorophenyl)propanoate hydrochloride. And then dried under reduced pressure at a maximum temperature of 30° C. The product is carefully sieved to remove lumps, if any. ( 2 H5) Ethyl (2S)-2-amino-3-(4-fluorophenyl)propanoate hydrochloride was isolated in 92% yield. LC-MS: tR 1.43 min, m / z [M+H] 217.

[0126] Example 7b: 2 H5) Kg-Scale Production of Ethyl (2S)-2-[(2S)-3-{4-[Bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propionamido]-3-(4-fluorophenyl)propanoate (IX)

[0127]

[0128] Melphalan (1.663 kg, 5.45 mol, 1 eq) was added to a mixture of purified water (16.0 kg), NaOH (32%, aqueous solution, 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 eq) and tetrahydrofuran (4.75 kg) was added at 10-15°C. The reaction mixture was stirred at 18-23°C for 4-5 hours until a minimum 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 1.5M 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 distillation in vacuo, and the residue containing (2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propionic acid was dried in vacuo at 20-25° C. for a minimum of 12 hours. HPLC: retention time 11.9 minutes. (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 minutes, flow rate of 1 mL / min, mobile phase A: 1.0 L MQ-water containing 500 μL 85% phosphoric acid, mobile phase B: 1.0 L acetonitrile containing 500 μL 85% phosphoric acid, UV detection at 262 nm).

[0129] The (2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-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, followed by ( 2H5) ethyl (2S)-2-amino-3-(4-fluorophenyl) propanoate hydrochloride (1.377 kg, 5.45 mol, 1.0 equiv), 1-hydroxybenzotriazole, H2O (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 stirred at 18-23°C for 3-4 hours until (2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propanoic acid was achieved. 2 H5) Ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propionamido]-3-(4-fluorophenyl)propanoate minimum 97.0% (HPLC) conversion (HPLC conditions are as follows: sample solvent acetonitrile, Waters, Atlantic T3 (3μ, 4.6×150mm) column, 10-90-10% B gradient over 23 minutes, flow rate of 1 mL / min, mobile phase A: 1.0 L MQ-water containing 500 μL 85% phosphoric acid, mobile phase B: 1.0 L acetonitrile containing 500 μL 85% phosphoric acid, UV detection at 262 nm).

[0130] The pH was adjusted to 3.0-4.0 with 5% KHSO4 (aqueous solution). The organic phase was fixed and the aqueous phase was extracted with dichloromethane (29.0 kg). The first organic phase was washed with 6% NaHCO3. The organic phase was fixed and the remaining aqueous phase was extracted with the second organic phase. The combined organic phases were dried over magnesium sulfate, filtered, and washed with dichloromethane. The dried organic phases were concentrated to 22-26 L by distillation in a vacuum. The concentrated organic phases were 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. The combined organic phases containing ( 2 H5) ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propionamido]-3-(4-fluorophenyl)propanoate (TLC) fraction and evaporate to 26-28 L under reduced pressure. Ethyl acetate (5.8 kg) was added and evaporation was continued to 26-28 L. This step 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}propionamido]-3-(4-fluorophenyl)propanoate. Optionally, ( 2H5) ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propionamido]-3-(4-fluorophenyl)propanoate seed crystals to aid precipitation. Ethyl acetate (5.8 kg) is added again, and the optional seeding step can be repeated. The mixture is evaporated to 19-21 L under reduced pressure and n-heptane (22.1 kg) is added at 35-45°C. The suspension is cooled to -2 to 2°C and stirred for 2-18 hours. The solid is separated by centrifugation, and the filter cake is washed with n-heptane. The solid is dried in vacuo at 30°C to give a white to slightly yellow solid material ( 2 H5) Ethyl (2S)-2-[(2S)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)carbonyl]-amino}propionamido]-3-(4-fluorophenyl)propanoate (2.6 kg, 80%). HPLC: retention time 13.4 minutes.

[0131] Example 7c: Mefluphenazine-d5(III) (( 2 H5) Kg scale production of ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propionamido]-3-(4-fluorophenyl)propionate hydrochloride)

[0132]

[0133] 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}propionamido]-3-(4-fluorophenyl)propanoate (Compound IX) (3.10 kg, 5.14 mol) in 1.3 M HCl acetonitrile 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}propionamido]-3-(4-fluorophenyl)propanoate to ( 2H5) Ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propionamido]-3-(4-fluorophenyl)propanoate hydrochloride with a minimum conversion rate of 99.0% (HPLC) (HPLC conditions are 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 minutes, flow rate of 1 mL / min, mobile phase A: 1.0 L MQ-water containing 500 μL 85% phosphoric acid, mobile phase B: 1.0 L acetonitrile containing 500 μL 85% phosphoric acid, UV detection at 262 nm.).

[0134] The reaction mixture is subjected to fine filtration and diluted with acetonitrile (68.9kg). Then, the jacket temperature of 45°C is used to carry out the distillation under reduced pressure. When the volume of the reaction mixture is 86L, ​​acetonitrile (22.7kg) is added and distillation is continued. When leaving 86L reaction mixture, acetonitrile (22.7kg) is added and distillation is continued. When the volume in the reactor is 86L, ​​acetonitrile (22.7kg) is added and distillation is continued until the volume of 86L is reached in the reactor.

[0135] Tert-butyl methyl ether (68.4 kg) was added at 35-45° C. over a period of 25-45 minutes, followed by cooling to 22-28° C. After stirring at this temperature for 60-120 minutes, the crude ( 2 H5) Ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propionamido]-3-(4-fluorophenyl)propanoate hydrochloride was filtered off and washed with tert-butyl methyl ether (12.5 kg). The crude material was dried in vacuo using a jacket temperature set point of 30° C. in the reactor.

[0136] Acetonitrile (84.0 kg) was added and the resulting suspension was stirred at 48-54° C. for 30-90 minutes, then cooled to 40-45° C. Tert-butyl methyl ether (74.6 kg) was added at 38-45° C. over a period of 40-70 minutes, then cooled to 22-28° C. After stirring at this temperature for 60-120 minutes, the crude ( 2 H5) ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propionamido]-3-(4-fluorophenyl)propanoate hydrochloride was filtered off and washed with tert-butyl methyl ether (14.0 kg). Drying in vacuo at 30-35° C. afforded ( 2H5) Ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propionamido]-3-(4-fluorophenyl)propionate hydrochloride (Mefluphen-d5, (III)) (2.5 kg, 90%). HPLC: retention time 9.0 minutes.

[0137] Biological Testing

[0138] Example (a) In vivo studies in dogs

[0139] A single-dose toxicity comparison study was conducted in dogs to investigate the toxicokinetics of melflufen and melflufen-d5(III) and its metabolites desethyl-melflufen and melphalan following a single intravenous administration of melflufen-d5(III) or melflufen as a 30-minute infusion in dogs.

[0140] (i) Introduction and objectives

[0141] The aim of this study was to compare the acute toxic potential of melflufen-d5(III) and melphalan. The toxicokinetics of melflufen-d5(III), melflufen and its metabolites desethyl-melflufen and melphalan were evaluated in dogs after a single intravenous administration as a 30-min infusion.

[0142] (ii) Materials and methods

[0143] abbreviation

[0144] The following abbreviations are used in this article:

[0145] AUC ∞ The area under the plasma concentration-time curve to infinity

[0146] AUC last The area under the plasma concentration-time curve to reach the last detectable concentration

[0147] C last Finally, the plasma concentration can be detected

[0148] C max Maximum plasma concentration

[0149] CV% Coefficient of variation of the mean, expressed as a percentage

[0150] h hour

[0151] SD Standard deviation

[0152] t 1 / 2,z Apparent terminal half-life

[0153] T lastTime of last detectable concentration

[0154] T max Time of maximum concentration

[0155] %AUC extr Percentage of extrapolated area

[0156] Study Design

[0157] Male and female dogs were given either melflufen-d5(III) or melflufen as a 30-minute infusion according to the following schedule:

[0158]

[0159] The control group was treated with 5% glucose solution.

[0160] Sample collection

[0161] On Day 1, blood samples were collected from a peripheral vein prior to dosing, 15 minutes after the start of the infusion, 30 minutes (just before the end of the infusion), 40 minutes, 1 hour, 2 hours, 4 hours, and 6 hours.

[0162] Blood samples were collected into heparinized collection tubes, placed in an ice-water bath and immediately centrifuged (3 min, 10000 g, +4° C.) The obtained plasma was divided into two aliquots, placed in pre-cooled cryovials and placed in a freezer at -70° C. until analysis.

[0163] Toxicokinetic calculations

[0164] Plasma toxicokinetics of melflufen-d5(III), melflufen and its metabolites desethyl-melflufen and melphalan were analyzed according to standard non-compartmental methods using the Phoenix WinNonlin system (v.6.3, Certara Company, USA).

[0165] After application, the maximum concentration C max and the time to achieve maximum concentration T max The coordinates of the highest plasma concentration during this time course are read as follows. The last detectable concentration C last and the time T of the last detectable concentration last Reported as parameters.

[0166] The area under the plasma concentration versus time curve to reach the last detectable concentration (AUC) was calculated by the linear trapezoidal rule. last .

[0167] When applicable, the following parameters are calculated:

[0168] The terminal half-life t was calculated by linear regression analysis of the natural logarithmic concentration versus time curve according to the following formula: 1 / 2,z :

[0169]

[0170] Where -λ z is the slope of the regression line. 1 / 2,z Estimates.

[0171] By calculating C under the assumption of a single exponential decay last / λ z The portion of the area added to the AUC last , calculate the area under the plasma concentration-time curve (AUC) to reach infinite time ∞ .

[0172] The extrapolated area under the curve (AUC) ∞ The score is calculated as follows:

[0173]

[0174] Individual and descriptive statistics (mean ± SD, CV %) plasma concentrations and toxicokinetic parameters were reported to three significant figures.

[0175] (iii) Results

[0176] Melflufen-d5(III), Melflufen and its metabolites desethyl-melflufen and melphalan were not detected in plasma samples of the control group (Group 1) and in pre-dose samples of treatment groups 2, 3 and 4.

[0177] Systemic exposure parameters for meflufen-d5(III), meflufen and its metabolite desethyl-meflufen, and melphalan were comparable for males and females, therefore descriptive statistics for the combined male and female parameters are also reported.

[0178] Mefluphenazine-d5(III)

[0179] A summary of the toxicokinetic parameters of melflufen-d5(III) is reported in Tables 1 and 2:

[0180] Table 1

[0181]

[0182] Table 2

[0183]

[0184] Flufen-d5(III) infused over 30 minutes at doses of 1.25 mg / kg and 2.5 mg / kg (Groups 2 and 3) reached its maximum plasma concentration in the mid-late infusion and then disappeared within 40 minutes of dosing. Due to insufficient numbers of terminal time points, half-life could not be calculated.

[0185] Exposure to melflufen-d5(III) increased with increasing dose in terms of both peak and area under the curve (2.7-fold vs. 2-fold dose increase calculated based on combined sex parameters).

[0186] Mean + SD plasma concentrations of melflufen-d5(III) after 1.25 and 2.5 mg / kg are shown in Figure 1 middle.

[0187] Melfluphenazine

[0188] A summary of the toxicokinetic parameters of Melflufen is reported in Tables 3 and 4 below:

[0189] Table 3

[0190]

[0191] Table 4

[0192]

[0193] Similar to Melflufen-d5(III), a peak at 15-30 minutes and rapid disappearance in plasma characterized the kinetics of Melflufen at a dose of 2.5 mg / kg (Group 4) infused over 30 minutes.

[0194] Comparison of systemic exposure to melflufen-d5(III) and melflufen

[0195] Figure 2 (C max )and Figure 3 (AUC last ) shows a comparison 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 male and female beagle dogs combined.

[0196] At 2.5 mg / kg, the mean exposure to melflufen was 2-fold lower than the mean exposure to melflufen-d5(III). Figure 2 and 3 As shown in the table, the difference in means is driven in part by the high concentration measured in one male dog treated with melflufen-d5(III). max and AUC last Value Figure 2 and Figure 3It can be seen that compared with mefluphenazine, there is a C max Increase and AUC last A clear trend of increase.

[0197] The inter-animal variability (CV%) in both groups was of the same order of magnitude.

[0198] Desethyl-Mefluphen

[0199] A summary of the toxicokinetic parameters for the metabolite desethyl-melflufen is reported in Tables 5 and 6:

[0200] Table 5

[0201]

[0202] Table 6

[0203]

[0204] After 1.25 and 2.5 mg / kg Melflufen-d5(III) infusions (Groups 2 and 3), the metabolite desethyl-Meflufen appeared in plasma at the first sampling time, reached its maximum concentration 15-30 minutes after dosing (1.25-2.5 mg / kg), and was no longer detectable after 40-60 minutes after dosing. Only at the 2.5 mg / kg dose, a half-life of 5 minutes could be estimated in one animal.

[0205] Relative to a 2-fold increase in the dose of melflufen-d5(III), exposure to desethyl-melflufen increased C max Increased by 3.1 times and AUC last Increased by 3.5 times (calculated based on combined gender parameters).

[0206] Just max and t last In contrast, after administration of Melflufen (Group 4), the plasma profile of desethyl-Meflufen was similar to that observed after administration of Melflufen-d5(III). Only at a dose of 2.5 mg / kg, a half-life of 7 minutes could be estimated in one animal.

[0207] Comparison of systemic exposure to desethyl-melflufen after administration of melflufen-d5(III) or melflufen

[0208] Figure 4 (C max )and Figure 5 (AUC last ) shows a comparison of individual and mean (±SD) systemic exposure parameters of desethyl-melflufen following infusion of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) into combined male and female beagle dogs.

[0209] The mean exposure to desethyl-melflufen was lower after an infusion of 2.5 mg / kg of melflufen than after an infusion of 2.5 mg / kg of melflufen-d5(III). Figure 4 and 5 As shown in the table, the difference in means is mainly driven by the high concentration of desethyl-melflufen measured in one male dog treated with melflufen-d5(III). max Value Figure 4 It can be seen that compared with melflufen, after infusion of melflufen-d5(III), there is a C max Increasing trend.

[0210] Melphalan

[0211] A summary of the toxicokinetic parameters of the metabolite melphalan is reported in Tables 7 and 8:

[0212] Table 7

[0213]

[0214] Table 8

[0215]

[0216] Following 1.25 and 2.5 mg / kg melflufen-d5(III) infusions, the metabolite melphalan appeared in plasma at the first sampling time after each melflufen-d5(III) dose, with an average t of 30 min after dosing. max It reaches its maximum concentration at 4 hours post-dose and can be detected until 4 hours post-dose. The half-life is estimated to be about 40 minutes.

[0217] After melflufen infusion (Group 4), the plasma profile of melphalan was comparable to that formed by melflufen-d5(III). max and t last are similar.

[0218] Exposure to melphalan increased with increasing doses of melflufen-d5(III) in terms of peak and AUC values: A 2-fold dose increase corresponded to a metabolite mean C max A 2.2-fold increase in AUC last and a 2.0-fold increase in AUC∞.

[0219] Melphalan AUC at two ascending doses of melflufen-d5(III) last 48-fold and 35-fold higher exposure than melflufen-d5(III), respectively (mean AUC based on combined sex datalast At two increasing doses of melflufen-d5(III), the AUC of melphalan last Exposures were on average 51.1-fold (range 37-70) and 44.8-fold (range 22-100) higher than those of melflufen-d5(III), respectively (calculated based on individual values ​​for both sexes combined).

[0220] After melflufen infusion, melphalan AUC last Exposures were on average 75-fold higher (range 38-142) than melflufen (calculated based on individual values ​​for both sexes combined).

[0221] Figure 6a (Group 2, logarithmic scale) and 6b (Group 2, non-logarithmic scale) and Figure 7a Mean + SD plasma concentrations of meflufen-d5(III), melphalan, and desethyl-meflufen following infusion of meflufen-d5(III) into dogs (combined sexes) in Group 2 or Group 3 are shown in 7a (Group 3, log scale) and 7b (Group 3, non-log scale). Figure 7a and 7b As shown in Figure , the mean C after infusion of 2.5 mg / kg melflufen-d5(III) max It is 2.73μmol / L.

[0222] Figure 8a Mean + SD plasma concentrations of melflufen, melphalan, and desethyl-melflufen following infusion of melflufen into dogs in Group 4 are shown in 8a (Group 4, logarithmic scale) and 8b (Group 4, non-logarithmic scale). Figure 8a and 8b As shown in Figure , the mean C after infusion of 2.5 mg / kg melflufen max It is 2.23μmol / L.

[0223] Comparison of systemic exposure to melphalan after administration of melflufen-d5(III) or melflufen

[0224] Fig. 9 (C max )and Fig.10 (AUC last ) shows a comparison of individual and mean (±SD) systemic exposure parameters to melphalan following infusion of melflufen-d5(III) (Group 3, 2.5 mg / kg) and melflufen (Group 4, 2.5 mg / kg) into combined male and female beagle dogs. Fig.11 and 12 The following table also shows the 1 / 2,z and AUC ∞ result.

[0225] The mean exposure to melphalan after an infusion of 2.5 mg / kg of melflufen was lower than that after an infusion of 2.5 mg / kg of melflufen-d5(III). max , AUC last and AUC ∞ Value Fig. 9 , 10 As shown in Figure 21, compared with melflufen, after infusion of melflufen-d5(III), there was a C max Increase, AUC last Increase and AUC ∞ Increasing trend. Fig.11 As shown in Figure 2, the mean t of melphalan after infusion of 2.5 mg / kg melflufen-d5(III) 1 / 2,z was lower than that after melflufen infusion, and there was a t of melphalan in some animals after melflufen-d5(III) infusion compared with melflufen. 1 / 2,z Decreasing trend.

[0226] in conclusion

[0227] Following a single 30-minute infusion of melflufen-d5(III) (1.25 and 2.5 mg / kg) or melflufen (2.5 mg / kg), systemic exposure profiles of melflufen-d5(III), melflufen, and its metabolites desethyl-melflufen and melphalan were similar in males and females.

[0228] Following infusion of melflufen-d5(III), melflufen-d5(III) and the metabolite desethyl-melflufen rapidly disappear from the systemic circulation. Desethyl-melflufen exposure is approximately half that of the parent compound.

[0229] The metabolite melphalan is rapidly and extensively formed. After the end of the infusion, melphalan is detected in plasma for up to 4 hours and decays with a terminal half-life of approximately 40 minutes. max ,t last and t 1 / 2,z The results were consistent between doses of melflufen-d5(III). In the combined sexes, the resulting melphalan exposure was approximately 50-fold higher than the melflufen-d5(III) exposure.

[0230] Following escalating dosing of increasing infusions of melflufen-d5(III), systemic exposure increased as expected (melphalan) and slightly more than expected (melflufen-d5(III) and desethyl-melflufen), assuming dosing ratio.

[0231] Comparing equivalent doses of melflufen-d5(III) and melflufen, overall, exposures to melflufen-d5(III) and the active metabolite melphalan were consistently higher following infusion of melflufen-d5(III) compared to melflufen. For equivalent doses of melflufen-d5(III) and melflufen, this increased exposure to melflufen-d5(III) and melphalan was a highly significant benefit.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, in, Each R 26 -R 30 is deuterium having an abundance level of at least 90 mol % deuterium; and R 1 -R 25 Each is H.

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

3. The compound according to claim 1 or 2, wherein the compound has the following structural formula:

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the following structural formula (Ia):

5. The compound according to claim 1, wherein the compound has the following structural formula:

6. The compound according to claim 1, wherein the pharmaceutically acceptable salt of the compound is ( 2 H5) Ethyl (2S)-2-[(2S)-2-amino-3-{4-[bis(2-chloroethyl)amino]phenyl}propionamido]-3-(4-fluorophenyl)propanoate hydrochloride.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 together with a pharmaceutically acceptable carrier, and optionally together with an additional therapeutic agent.

8. The pharmaceutical composition of claim 7, wherein the additional therapeutic agent is a protease inhibitor (PI), an immunomodulatory drug (IMiD), or an alkylating agent.

9. Use of an effective amount of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 or 8 in the preparation of a medicament for treating or preventing cancer, Wherein the cancer is multiple myeloma.

10. A compound having a structural formula selected from the following group: or a pharmaceutically acceptable salt thereof.

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