Process for the preparation of marimastat, marimastat prepared by this process, a pharmaceutical composition comprising the same, and uses thereof

Abstract

The invention relates to a process for the preparation of marimastat. The invention further relates to marimastat prepared by the process, and a pharmaceutical composition comprising said marimastat. The invention also relates to the pharmaceutical composition for use as a medicament, and said marimastat for use as a medicament. The invention further relates to the pharmaceutical composition for use in the prevention and treatment of diseases associated with hyperactivity of extracellular matrix metalloproteinases, and said marimastat for use in the prevention and treatment of diseases associated with hyperactivity of extracellular matrix metalloproteinases.

Claims

1. A process for the preparation of marimastat, characterized in that the process includes the following steps: a) (2S,3R)-2-hydroxy-3-isobutylsuccinic acid is reacted in the environment of 2,2-dimethoxypropane with the addition of p-toluenesulfonic acid, pyridinium p-toluenesulfonate or 10-camphorsulfonic acid at a temperature of 35-55 C. for 15-30 h, then after the reaction is completed, a tertiary amine selected from the group comprising diisopropylethylamine, triethylamine and N-methylmorpholine is added to obtain (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid; b) (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid is reacted with (S)-2-amino-N,3,3-trimethylbutanamide or its hydrochloride in the environment of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate and a tertiary amine selected from the group comprising diisopropylethylamine, triethylamine and N-methylmorpholine in acetonitrile or methylene chloride, then the reaction mixture is subjected to an extraction process to obtain crude (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide, which is then used in step c) or macerated at boiling in methyl tert-butyl ether or isopropyl ether for 1-3 h, and then at room temperature for 15-30 h to obtain (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide; and c) (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide or crude (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide is reacted with a 50% aqueous solution of hydroxylamine in a solvent selected from a lower carboxylic acid ester, a non-polar solvent or a polar solvent, then acetone is added and stirred at boiling for 0.5-1 h, then the reaction mixture is subjected to azeotropic distillation process in order to remove water, and then stirred at room temperature for 15-30 h to obtain marimastat.

2. The process according to claim 1, characterized in that the reaction in step b) is carried out at a temperature from 10 C. to room temperature.

3. The process according to claim 2, characterized in that the reaction in step b) is carried out for 1-24 h.

4. The process according to claim 1, characterized in that the reaction in step c) with the 50% aqueous solution of hydroxylamine is carried out at a temperature from room temperature to boiling point.

5. The process according to claim 4, characterized in that the reaction in step c) with the 50% aqueous solution of hydroxylamine is carried out for 1-30 h.

6. The process according to claim 1, characterized in that the lower carboxylic acid ester is selected from the group comprising ethyl acetate and isopropyl acetate.

7. The process according to claim 1, characterized in that the non-polar solvent is selected from the group comprising 2-methyltetrahydrofuran, 1,2-dimethoxyethane, methylene chloride and chloroform.

8. The process according to claim 1, characterized in that the polar solvent is selected from the group comprising acetonitrile and isopropyl alcohol.

9. The process according to claim 1, characterized in that the tertiary amine in step b) is added in two portions, the first portion before adding (S)-2-amino-N,3,3-trimethylbutanamide or its hydrochloride, the second portion after adding (S)-2-amino-N,3,3-trimethylbutanamide or its hydrochloride.

10. The process according to claim 1, characterized in that the tertiary amine is diisopropylethylamine.

11. The process according to claim 1, characterized in that during the azeotropic distillation process in step c), the volume of distilled solvent is replenished by successive addition of further portions of solvent.

12. Marimastat prepared by the process defined in claim 1.

13. A pharmaceutical composition characterized in that it comprises marimastat defined in claim 12 and a pharmaceutically acceptable carrier.

14. The composition according to claim 13, characterized in that it is in the form of a tablet, modified-release tablet, pill, capsule, powder, granules, pellets, suspension, emulsion, solution, oral liquid forms, e.g. syrup, solution or suspension for injection, solution for infusion, eye drops, ointment, gel, suppository, globule or therapeutic system, e.g. implant, vaginal ring, nanofiber.

15. The pharmaceutical composition defined in claim 13 for use as a medicament.

16. The pharmaceutical composition defined in claim 13 for use in the prevention and treatment of diseases associated with hyperactivity of extracellular matrix metalloproteinases selected from the group comprising post-stroke epilepsy, post-traumatic epilepsy, epilepsy after brain surgery, hypoxic-ischemic encephalopathy, malignant neoplasms, vascular malformations, amyotrophic lateral sclerosis, multiple sclerosis, snake venom poisoning, endometriosis, hemorrhoids, arthritis, nervous system diseases, circulatory system diseases, diabetes and diabetes complications selected from the group comprising diabetic retinopathy and diabetic foot.

17. Marimastat defined in claim 12 for use as a medicament.

18. Marimastat defined in claim 12 for use in the prevention and treatment of diseases associated with hyperactivity of extracellular matrix metalloproteinases selected from the group comprising post-stroke epilepsy, post-traumatic epilepsy, epilepsy after brain surgery, hypoxic-ischemic encephalopathy, malignant neoplasms, vascular malformations, amyotrophic lateral sclerosis, multiple sclerosis, snake venom poisoning, endometriosis, hemorrhoids, arthritis, nervous system diseases, circulatory system diseases, diabetes and diabetes complications selected from the group comprising diabetic retinopathy and diabetic foot.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The subject of the invention is presented in more detail in the examples of implementation and in the drawing, in which:

[0041] FIG. 1 shows the structural formula of marimastat (PKL-021);

[0042] FIG. 2 shows the .sup.1H NMR spectrum of PKL-021;

[0043] FIG. 3 shows the .sup.13C NMR spectrum of PKL-021;

[0044] FIG. 4 shows the IR spectrum (KBr tablet) of PKL-021;

[0045] FIG. 5 shows the HPLC chromatograms of crude PKL-020, PKL-020 (after maceration) and the diastereoisomer PKL-020dias standard;

[0046] FIG. 6 shows the HPLC chromatogram of PKL-021.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of (2S,3R)-2-hydroxy-3-isobutylsuccinic acid (PKL-018)

Step 1: Preparation of diethyl (2R,3R)-2-acetoxy-3-bromosuccinate (PKL-014)

##STR00006##

[0047] Diethyl ()-D-tartrate (PKL-013, 60.0 g, 291.0 mmol) was cooled to 2 C. Then, not exceeding 5 C., 33% HBr in acetic acid (250.0 mL) was added dropwise. The cooling bath was removed and stirred for 4.5 hours. It was then poured into 600 mL of water with ice and extracted with diethyl ether (400+400+200 mL). The combined organic layers were washed with water (3100 mL) and saturated sodium chloride solution (100 mL). Dried over Na.sub.2SO.sub.4. The solvent was evaporated to give 94.0 g of a colorless oil. The crude product was used for the next step.

Step 2: Preparation of diethyl (2R,3R)-2-bromo-3-hydroxysuccinate (PKL-015)

##STR00007##

[0048] The crude acetylbromohydrin (PKL-014, 94.0 g) was dissolved in anhydrous ethanol (940 mL). Then 33% HBr in acetic acid (31 mL) was added and stirred at boiling while slowly distilling off the solvent. 800 mL was distilled (5 hTLCdisappearance of the starting material). After cooling to room temperature, diethyl ether (250 mL) and water (250 mL) were added, and layers were separated. The aqueous layer was extracted with diethyl ether (2250 mL). The combined organic layers were washed with water (2100 mL), a saturated solution of NaHCO.sub.3 (2100 mL) and brine (100 mL), and dried over Na.sub.2SO.sub.4. The solvent was evaporated to give 67.0 g of a colorless oil. The crude product was used for the next step.

Step 3: Preparation of diethyl (2S,3S)-oxirane-2,3-dicarboxylate (PKL-016)

##STR00008##

[0049] Bromohydrin (PKL-015, 67.0 g, 249 mmol) was dissolved in toluene (670 mL). Then, ground K.sub.2CO.sub.3 (138 g, 996 mmol, 4 eq) was addedthe temperature rose to 34 C. The suspension was vigorously stirred on a mechanical stirrer for 2 hours (TLCcomplete disappearance of the starting material). The mixture was filtered on a Schott funnel and K.sub.2CO.sub.3 was washed with toluene. The filtrate was washed with water (5-6100 mL), further with brine (100 mL), and dried over Na.sub.2SO.sub.4. The solvent was evaporated to give 34.9 g of yellow oil (yield: 63%three steps).

[0050] .sup.1H NMR (400 MHz, CDCl.sub.3) 4,26 (m, 4H), 3,66 (t, J=0.70 Hz, 2H), 1,31 (m, 6H);

[0051] .sup.13C NMR (100 Hz, CDCl.sub.3) 166.8, 62.2, 52.0, 14.0.

Step 4: Preparation of diethyl (2S,3R)-2-hydroxy-3-isobutylsuccinate (PKL-017)

##STR00009##

[0052] In a reaction vessel with a mechanical stirrer under Ar atmosphere, CuCN (9.5 g, 106.2 mmol) was suspended in anhydrous THF (115 mL). Cooled to 15 C. and previously prepared 1 M Grignard reagentisobutylmagnesium bromide in THF (101 mL; 101 mmol) was addedtemperature rose to 0 C. The mixture was stirred for 20 minutes at a temperature of 5 C., and then not exceeding 25 C., a solution of PKL-016 (10.0 g, 53.1 mmol) in anhydrous THF (50 mL) was added dropwise. The bath was removed and stirred for 1.5 hours (TLCcomplete disappearance of the starting material). The reaction was quenched with a saturated ammonium chloride solution (170 mL)the temperature rose from 20 to 27 C.stirred for 1.5 hours. It was filtered through Celite. The layers were separated, and the aqueous layer was extracted with diethyl ether (250 mL). The combined organic layers were washed with a saturated sodium chloride solution (100 mL), and dried over Na.sub.2SO.sub.4. The solvent was evaporated to give 11.3 g of yellowish oil (yield: 90%). The crude product was used for the next step without purification. .sup.1H NMR (400 MHz, CDCl.sub.3) 4.24 (m, 3H), 4.13 (qd, J=7.15 Hz, J2=1.46 Hz, 2H), 3.19 (dd, J1=7.78 Hz, J2=1.44 Hz, 1H), 2.92 (ddd, J1=8.34 Hz, J2=6.79 Hz, J3=3.66 Hz, 1H), 1.74 (m, 1H), 1.66 (m, 1H), 1.49 (ddd, J1=12.10 Hz, J2=7.67 Hz, J3=6.19 Hz, 1H), 1.29 (t, J=7.15 Hz, 3H), 1.23 (t, J=7.14 Hz, 3H), 0.92 (ddd, J1=6.22 Hz, J2=4.59 Hz, J3=1.48 Hz, 6H); .sup.13C NMR (100 Hz, CDCl.sub.3) 173.4, 173.0, 71.3, 61.7, 60.8, 46.5, 36.9, 25.7, 22.4, 22.3, 14.13, 14.10.

Step 5: Preparation of (2S,3R)-2-hydroxy-3-isobutylsuccinic acid (PKL-018)

##STR00010##

[0053] The crude hydroxyester (PKL-017, 11.3 g, 45.9 mmol) was dissolved in 1,4-dioxane (68 mL). Then, sodium hydroxide solution (7.3 g, 183.2 mmol) in water (45 mL) was added. The reaction was stirred at room temperature for 21 h (TLCcomplete disappearance of the starting material). It was extracted with diethyl ether (220 mL). The combined organic layers were washed with water (20 mL) and discarded. The combined aqueous layers were acidified with 6M HCl to pH 1. The water was evaporated and the residue was evaporated with acetonitrile. Acetonitrile was added again and filtered through Celite. The acetonitrile was evaporated to give 8.66 g of orange oil (yield: 85.7%two steps).

[0054] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.33 (t, J=1.82 Hz, 1H), 3.09 (m, 1H), 1.72 (m, 3H), 0.94 (t, J=3.60 Hz, 6H);

[0055] .sup.13C NMR (100 Hz, CDCl.sub.3) 178.9, 178.0, 70.4, 46.2, 36.6, 25.5, 22.5, 22.1;

[0056] LC-MS [MH].sup.=188.8; HPLC: 83.9%.

[0057] The obtained PKL-018 was used to prepare marimastat (PKL-021).

Example 2

Preparation of (S)-2-amino-N,3,3-trimethylbutanamide hydrochloride (PKL-012)

##STR00011##

[0058] A solution of t-butyloxycarboamide (PKL-011, 335 g, 1.44 mol) in ethyl acetate (800 mL) was added dropwise to a suspension of PKL-012 sample (8.2 g, previously obtained) in 2M HCl in ethyl acetate (1750 mL, 3.5 mol HCl) at room temperature for 4.5 hours. Formation of white precipitate was observed. Then, the mixture was stirred for additional 22 hours at room temperature to complete crystallization. The crystals were filtered off, washed with ethyl acetate, and dried on air to obtain 251.2 g (243 g after subtraction of 8.2 g of PKL-012, initially added to facilitate the crystallization, 93% yield) of pale yellow crystalline material.

[0059] .sup.1H NMR (400 MHz, D.sub.2O) 3.68 (s, 1H), 2.80 (s, 3H), 1.07 (s, 9H);

[0060] .sup.13C NMR (100 Hz, D.sub.2O) 168.9, 62.0, 32.7, 25.7, 25.6;

[0061] LC-MS [M+H].sup.+=145.1; HPLC: 99.7%.

[0062] The obtained PKL-012 was used to prepare marimastat (PKL-021).

Example 3

Preparation of marimastat (PKL-021)

[0063] An exemplary process of preparing PKL-021 is shown in Scheme 5.

##STR00012##

[0064] PKL-021 and intermediate products: PKL-019 and PKL-020 were characterized by the following methods: NMR, MS, LC-MS, IR, elemental analysis, and the purity was determined using HPLC. .sup.1H and .sup.13C NMR measurements were performed on a Varian 400 MHz spectrometer. LC-MS analyses were performed on a Shimadzu 2020 chromatograph using the electrospray ionization method.

[0065] Compound PKL-021 was further characterized by two-dimensional NMR, IR and elemental analysis. Two-dimensional COSY, HSQC spectra were performed in DMSO-d.sub.6 at room temperature on a Varian 400 MHz spectrometer. IR analysis was performed on a Nicolet iS10 spectrometer in the range of 4000-400 cm.sup.1, with a resolution of 4 cm.sup.1. The PKL-021 sample was prepared in a KBr tablet, approximately 1.5 mg of the compound/200 mg of KBr.

[0066] The purity of PKL-019, PKL-020 and PKL-021 was tested by HPLC on a Shimadzu 3050C 3D chromatograph.

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0067] p-Toluenesulfonic acid (p-TsOH, 18 g, 105 mmol, 0.05 eq) was added to a solution of PKL-018 (465 g of crude PKL-018, 2.05 mol) in 2,2-dimethoxypropane (5 L). The reaction mixture was stirred at 40 C. for 24 hours. The progress of the reaction was monitored by HPLC and TLC (TLC:CHCl.sub.3:CH.sub.3OH:H.sub.2O 6:4:0.3). After the reaction was completed (HPLC:PKL-0183%), diisopropylethylamine (DIPEA, 46 mL, 0.1 eq) was added, and the mixture was concentrated to give a brown oil. The crude product PKL-019 was used in the next step without additional purification.

[0068] Reaction yield: 100%.

[0069] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.45 (1H, d, J=4.9 Hz), 4.11 (1H, q, J=7.1 Hz), 3.0 (1H, ddd, J=8.3 Hz, J=6.3 Hz, 4.8 Hz), 2.02 (1H, s), 1.80-1.75 (2H, m), 1.58 (3H, s), 1.52 (3H, s), 0.95 (3H, d, J=2.2 Hz), 0.93 (3H, J=2.0 Hz);

[0070] LC-MS [MH]=228.8; HPLC: 77.24%.

[0071] Other tertiary amines such as triethylamine (Et.sub.3N) and N-methylmorpholine (NMM) were also tested and a similar result was obtained as with DIPEA. It is preferable to use the same amine that is used in the next step, i.e. coupling PKL-019 with PKL-012.

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0072] The crude PKL-019 (2.05 mol) was dissolved in acetonitrile (5 L). Then, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (929 g, 2.89 mol, 1.4 eq) and diisopropylethylamine (DIPEA, 1.38 L; 7.89 mol, 3.8 eq) were added to the clear solution. Finally, PKL-012 (475 g, 2.63 mol, 1.3 eq) was added portionwise, and the reaction mixture was stirred at room temperature. The progress of the reaction was monitored by TLC method (10% CH.sub.3OH/CH.sub.2Cl.sub.2). The reaction was usually completed within 2 hours. The mixture was diluted with ethyl acetate (5 L) and washed with 5% KHSO.sub.4 (35 L), a saturated solution of NaHCO.sub.3 (35 L), and brine (2.5 L). The organic layer was dried over anhydrous Na.sub.2SO.sub.4. The solvent was evaporated to give 700 g of brown oil (yield of the crude PKL-020:96%).

[0073] The crude product was macerated at boiling in methyl tert-butyl ether (MTBE) (5 L) for 60 min. The mixture was cooled to room temperature and stirred for 17 hours. The crystals were filtered off, washed with MTBE (31.0 L), and dried at room temperature to constant weight to give a white solid of PKL-020 (577 g, 79% yield).

[0074] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.69 (1H, d, J=9.3 Hz), 6.37 (1H, d, J=5.0 Hz), 4.43 (1H, d, J=5.8 Hz), 4.28 (1H, d, J=9.3 Hz), 2.78 (3H, d, J=4.8 Hz), 2.77-2.75 (1H, m), 1.86 (1H, brs), 1.75-1.61 (1H, m), 1.60 (3H, s), 1.50 (3H, s), 1.17 (1H, s), 0.98 (9H, s), 0.90 (6H, t, J=6.1 Hz);

[0075] .sup.13C NMR (100 Hz, CDCl.sub.3) 172, 170.8, 170.6, 110.2, 74.6, 60.6, 49.4, 47.3, 36.8, 34.6, 26.9, 26.7, 26.5, 26.0, 25.8, 25.7, 23.0, 21.9;

[0076] LC-MS [M+H].sup.+=357.2; HPLC: 98.7%.

[0077] Alternatively, the preparation of PKL-020 was performed in methylene chloride as described below.

[0078] The crude PKL-019 (5.26 mmol) was dissolved in methylene chloride (10 mL). Then, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU, 1.86 g, 5.78 mmol) and diisopropylethylamine (DIPEA, 2.7 mL, 15.78 mol) were added to the clear solution. Finally, PKL-012 (950 mg, 5.26 mol) was added portionwise and the reaction mixture was stirred at room temperature. The progress of the reaction was monitored by TLC method (10% CH.sub.3OH/CH.sub.2Cl.sub.2). The reaction was usually completed within 2 hours. The reaction mixture was washed with 5% KHSO.sub.4 (310 mL), saturated solution of NaHCO.sub.3 (310 mL) and brine (5 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4. After evaporating the solvent, 1.81 g of a brown oil was obtained (the crude PKL-020 yield: 97.1%).

[0079] The crude product was macerated at boiling in methyl tert-butyl ether (MTBE, 10 mL) for 60 min. The mixture was cooled to room temperature and stirred for 17 hours. The crystals were filtered off, washed with MTBE (32 mL) and dried at room temperature to constant weight, obtaining 1.49 g of a white solid of PKL-020 (80% yield).

[0080] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.69 (1H, d, J=9.3 Hz), 6.37 (1H, d, J=5.0 Hz), 4.43 (1H, d, J=5.8 Hz), 4.28 (1H, d, J=9.3 Hz), 2.78 (3H, d, J=4.8 Hz), 2.77-2.75 (1H, m), 1.86 (1H, brs), 1.75-1.61 (1H, m), 1.60 (3H, s), 1.50 (3H, s), 1.17 (1H, s), 0.98 (9H, s), 0.90 (6H, t, J=6.1 Hz);

[0081] .sup.13C NMR (100 Hz, CDCl.sub.3) 172, 170.8, 170.6, 110.2, 74.6, 60.6, 49.4, 47.3, 36.8, 34.6, 26.9, 26.7, 26.5, 26.0, 25.8, 25.7, 23.0, 21.9;

[0082] LC-MS [M+H].sup.+=357.2; HPLC: 98.5%.

[0083] In the course of the work, it was observed that, depending on the reaction conditions used, i.e. the type of coupling reagent, the tertiary amine used, the method of its addition and the type of solvent, in addition to the desired product PKL-020, the diasteroisomer PKL-020dias shown below is also formed.

##STR00013##

[0084] A number of different conditions were tested, the results of which are presented in Table 1. Conducting the reaction to obtain PKL-020 at room temperature, using diisopropylethylamine (DIPEA) as a base, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) as a coupling reagent and acetonitrile (ACN) as a solvent, the reaction yield was 96%, and the purity of the obtained crude PKL-020 was 85% with the content of PKL-020dias. 4.4% (item 1, Table 1).

[0085] Good results were also obtained using methylene chloride (DCM) as a solvent in the presence of DIPEA and using TBTU as a coupling reagent (item 2, Table 1). Replacing acetonitrile with methylene chloride resulted in a decrease in the amount of diastereoisomer formed to 1.7%. Two subsequent experiments, i.e. items 3 and 4 in Table 1, prove the beneficial effect of methylene chloride used as a solvent on the ratio of the product PKL-020 to PKL-020dias. The amount of PKL-020dias. formed in both samples was 8.5% vs. 4.9%. By far the weakest result was obtained when triethylamine (Et.sub.3N) was used as a tertiary amine (item 5, Table 1). In this case, the amount of PKL-020dias. formed increased to 32.0%.

[0086] In turn, tests carried out in which 1,1-carbonyldiimidazole (CDI) was used as a coupling reagent showed that the reaction did not proceed at all or the amount of PKL-020dias. formed was above 9% (items 6-8, Table 1).

TABLE-US-00001 TABLE 1 Crude Coupling Yield PKL-020 PKL-020dias. No. Amine Solvent reagent (crude PKL-020) (HPLC) (HPLC) 1 DIPEA ACN TBTU 96% 85% 4.4% 2 DIPEA DCM TBTU 97.1% 76.5% 1.7% 3 NMM ACN TBTU 86.9% 68.5% 8.5% 4 NMM DCM TBTU 102.9% 73.3% 4.9% 5 Et.sub.3N ACN TBTU 88.0% 47.9% 32.0% 6* DIPEA ACN CDI 7 NMM DCM CDI 70.6% 56.1% 9.4% 8 NMM ACN CDI 69.0% 57.5% 9.7% 9 DIPEA DMF TBTU 52.0% 79.1% 4.75% *no reaction product

[0087] In addition, the method of adding the tertiary amine influences the amount of diastereoisomer. Experiments were performed using DIPEA, and it was shown that the active ester formed from PKL-019 and TBTU was easily epimerized in an alkaline environment (item 1, 2, Table 2). According to the above, the lowest amount of diastereoisomer is formed when DIPEA is added in two portions: the first portion of the tertiary amine (1-1.5 eq) is added before the addition of PKL-012 and is used to generate the active ester, the second portion (2-2.8 eq) is added after adding PKL-012, it releases PKL-012 hydrochloride and initiates the amidation reaction (item 3, Table 2).

TABLE-US-00002 TABLE 2 Method of adding reagents to the solution PKL-020:PKL-020dia No. of PKL-019 in MeCN (HPLC) 1 TBTU, DIPEA (3 eq), stirring for 1 h, 83:17 then adding PKL-012 2 TBTU, DIPEA (3 eq), stirring for 3 h, 53:47 then adding PKL-012 3 TBTU, DIPEA (1 eq), stirring for 0,5 h, 98:2 then adding PKL-012, DIPEA (2 eq)

[0088] As a result of the coupling reaction carried out in acetonitrile or methylene chloride, in the presence of DIPEA as a base and TBTU as a coupling reagent, the crude product PKL-020 was obtained, which was isolated by subjecting the reaction mixture to an extraction process. Then, the crude PKL-020 obtained in the above manner was macerated at boiling in methyl tert-butyl ether (MTBE) or isopropyl ether, and then at room temperature. PKL-020 was obtained with a purity of 95-99%, PKL-020dias content below 1% and a yield of 79-85%. Using the maceration process, purification on a chromatographic column was eliminated. Exemplary chromatograms of crude PKL-020, PKL-020 (after maceration) and the diastereoisomer PKL-020dias standard are shown in FIG. 5. HPLC analyzes were performed on a Shimadzu apparatus, using a ReproSil-Pur Basic C8 column, 3 m, 503 mm, UV detector (DAD)/205 nm, samples were dissolved in acetonitrile, a mixture of H.sub.2O:MeOH:ACN:HCOOH was used as a mobile phase.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0089] PKL-020 (560 g, 1.57 mol, 1 eq) was dissolved in ethyl acetate (AcOEt, 5.6 L) at 50 C. and NH.sub.2OH (370 mL, 50% solution in water, 5.97 mol, 3.8 eq) was added. The suspension was stirred at boiling for 1 h (TLC monitoring: 10% CH.sub.3OH/CH.sub.2Cl.sub.2). Then, the temperature of the reaction mixture was decreased to 45 C., and 1.1 L of acetone was added dropwise. The reaction was stirred at boiling for additional 30 minutes. Then, the mixture was subjected to azeotropic distillation from the mixture of AcOEt and acetone to remove water. The volume of distilled solvent was replenished by successive addition of further portions of AcOEt. Then, the reaction mixture was cooled to room temperature and stirred for 17 hours. The crystals were filtered off, washed with AcOEt (21.0 L), acetone (1.0 L), and dried to obtain 445 g of PKL-021 (77% yield) as a white solid.

[0090] LC-MS [MH].sup.=329.95; HPLC: 100%; elemental analysis for C.sub.15H.sub.29N.sub.3O.sub.5, calculated: C 54.36%, H 8.82%, N 12.68%, measured: C54.36%, H8.94%, N12.66%.

[0091] The results of the NMR analysis along with signal assignments, the IR spectrum for compound PKL-021 are presented in FIGS. 2, 3 and 4 and in Tables 3 and 4.

TABLE-US-00003 TABLE 3 .sup.1H and .sup.13C NMR chemical shifts of compound PKL-021 in DMSO-d.sub.6. .sup.13C-NMR .sup.1H-NMR Description of .sup.1H- Number [ppm] [ppm] NMR signals C3; C6; C9 172.7; 171.0; 169.3 C8 71.8 3.70 t, J = 8.00 Hz, 1H C4 60.3 4.15 d, J = 9.51 Hz, 1H C7 48.33 2.67 ddd, J1 = 10.59 Hz, J2 = 8.19 Hz, J3 = 4.09 Hz, 1H C18 37.80 1.43 m, 1H 0.93 m, 1H C13 34.6 C14; C15; C16 27.1 0.87 s, 9H C19 25.8 1.34 m, 1H C1 25.7 2.54 d, J = 4.58 Hz, 3H C20; C21 23.9; 22.2 0.77 dd, J1 = 9.79 Hz, J2 = 6.47 Hz, 6H N10 10.52 s, 1H O11 8.84 s, 1H N2 7.83 q, J = 4.66 Hz, 1H N5 7.45 d, J = 9.54 Hz, 1H O22 5.34 d, J = 7.85 Hz, 1H

TABLE-US-00004 TABLE 4 Characteristic absorption bands of the IR spectrum of compound PKL-021. bands [cm.sup.1] Description 3400-3100 Stretching vibrations of OH bonds and stretching vibrations of NH bonds 2956, 2906 and 2872 Stretching vibrations of CH alkyl bonds 1631 Stretching vibrations of CO bonds of amide groups 1574 and 1524 Bending vibrations of NH bonds 1469 Bending vibrations of CH alkyl bonds 1408 Bending vibrations of OH bonds 1368 Bending vibrations of CH bonds in methyl groups 1312, 1218 and 1145 Stretching vibrations of CN bonds 1067 Stretching vibrations of CO bonds 842-673 Bending vibrations of OH bonds, bending vibrations of NH bonds, stretching vibrations of NO bonds and bending vibrations of CH bonds

[0092] Alternatively, the reaction of PKL-020 with hydroxylamine was also carried out in other solvents, i.e. other lower carboxylic acid esters, e.g. isopropyl acetate, non-polar solvents, e.g. 2-methyltetrahydrofuran, 1,2-dimethoxyethane, methylene chloride, chloroform and polar solvents, e.g. acetonitrile, isopropyl alcohol. The results of the reactions carried out to obtain marimastat (PKL-021) in Step 3 are presented in Table 5. Using the above solvents, PKL-021 was obtained with a yield of over 60% and a purity of over 99%. It is particularly preferred to use ethyl acetate, methylene chloride, 2-methyltetrahydrofuran or chloroform as a solvent in Step 3. Using ethyl acetate, methylene chloride, 2-methyltetrahydrofuran or chloroform, PKL-021 was obtained with a purity of 100%, 99.95%, 99.95% and 99.93%, respectively, and a yield of over 80%.

[0093] The obtained marimastat (PKL-021) did not require an additional purification step, and the amount of individual impurities, including process, degradation, diastereoisomer and enantiomer, was below 0.1%. The exemplary HPLC chromatogram of PKL-021 is shown in FIG. 6.

TABLE-US-00005 TABLE 5 No. solvent purity yield 1 ethyl acetate 100% 77-85%.sup. 2 isopropyl acetate 99.45% 80% 3 1,2-dimethoxyethane 99.70% 72% 4 acetonitrile 99.28% 64% 5 methylene chloride 99.95% 83% 6 2-methyltetrahydrofuran 99.95% 85% 7 isopropanol 99.54% 75% 8 chloroform 99.93% 80%

Example 4

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0094] Step 1 is the same as in Example 3.

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0095] The crude PKL-019 (44.7 mmol) was dissolved in acetonitrile (100 mL) and the resulting solution was cooled to 0-5 C. Then, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (18.6 g; 57.9 mmol; 1.3 eq) and diisopropylethylamine (DIPEA) (9.2 mL; 52.6 mmol, 1.2 eq) were added to the clear solution. Then, PKL-012 (9.5 g; 52.6 mmol; 1.2 eq) and diisopropylethylamine (DIPEA) (18.3 mL; 105.2 mmol, 2.4 eq) were added portionwise. The reaction mixture was stirred at a temperature of 0-5 C. The progress of the reaction was monitored by TLC method (10% CH.sub.3OH/CH.sub.2Cl.sub.2). The reaction was usually completed within 1 hour. The mixture was diluted with ethyl acetate (100 mL) and washed with 5% KHSO.sub.4 (3100 mL), a saturated solution of NaHCO.sub.3 (3100 mL), and brine (50 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4. The purity of the crude PKL-020 was 77%. After filtering off the drying agent, the residue was used for the next step.

[0096] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.69 (1H, d, J=9.3 Hz), 6.37 (1H, d, J=5.0 Hz), 4.43 (1H, d, J=5.8 Hz), 4.28 (1H, d, J=9.3 Hz), 2.78 (3H, d, J=4.8 Hz), 2.77-2.75 (1H, m), 1.86 (1H, brs), 1.75-1.61 (1H, m), 1.60 (3H, s), 1.50 (3H, s), 1.17 (1H, s), 0.98 (9H, s), 0.90 (6H, t, J=6.1 Hz);

[0097] .sup.13C NMR (100 Hz, CDCl.sub.3) 172, 170.8, 170.6, 110.2, 74.6, 60.6, 49.4, 47.3, 36.8, 34.6, 26.9, 26.7, 26.5, 26.0, 25.8, 25.7, 23.0, 21.9;

[0098] LC-MS [M+H].sup.+=357.2; HPLC: 80.5%.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0099] The PKL-020 solution (44.7 mmol in AcOEt) was diluted to a total volume of 200 mL with ethyl acetate and heated to 45 C. Then, NH.sub.2OH (12.2 mL, 50% solution in water, 200 mmol, 4.5 eq) was added. The suspension was stirred at a temperature of 45 C. for 1 h (TLC monitoring: 10% CH.sub.3OH/CH.sub.2Cl.sub.2). Then, 30 mL of acetone were added dropwise. The reaction mixture was stirred at boiling for additional 30 minutes. Then, the reaction mixture was subjected to azeotropic distillation from the mixture of AcOEt and acetone to remove water. The volume of distilled solvent was replenished by successive addition of further portions of AcOEt. Then, the reaction mixture was cooled to room temperature and stirred for 17 hours. The crystals were filtered off, washed with AcOEt (210 mL), acetone (10 mL), and dried to obtain 11.5 g of PKL-021 (78% yield) as a white solid.

[0100] .sup.1H and .sup.13C NMR chemical shifts of compound PKL-021 in DMSO-d.sub.6 are presented in Table 3. Characteristic absorption bands of the IR spectrum of compound PKL-021 are presented in Table 4.

Example 5

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0101] Step 1 is the same as in Example 3.

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0102] The crude PKL-019 (15.8 mmol) was dissolved in acetonitrile (30 mL) and cooled to 10 C. Then, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (5.56 g; 17.35 mmol; 1.1 eq) and diisopropylethylamine (DIPEA) (2.75 mL; 15.76 mmol, 1.0 eq) were added to the clear solution. Then, PKL-012 (2.85 g; 15.77 mmol; 1.0 eq) and diisopropylethylamine (DIPEA) (5.51 mL; 31.54 mmol, 2.0 eq) were added portionwise. The reaction mixture was stirred at 10 C. for 1 hour. The progress of the reaction was monitored by TLC method (10% CH.sub.3OH/CH.sub.2Cl.sub.2). The mixture was diluted with ethyl acetate (30 mL) and washed with 5% KHSO.sub.4 (330 mL), a saturated solution of NaHCO.sub.3 (330 mL), and brine (15 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4. The purity of the crude PKL-020 was 82% (84% yield). The crude product was macerated at boiling in methyl tert-butyl ether (MTBE) (30 mL) for 60 min. The mixture was cooled to room temperature and stirred for 17 hours. The crystals were filtered off, washed with MTBE (310 mL), and dried at room temperature to constant weight to give a white solid of PKL-020 (3.4 g, 61% yield).

[0103] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.69 (1H, d, J=9.3 Hz), 6.37 (1H, d, J=5.0 Hz), 4.43 (1H, d, J=5.8 Hz), 4.28 (1H, d, J=9.3 Hz), 2.78 (3H, d, J=4.8 Hz), 2.77-2.75 (1H, m), 1.86 (1H, brs), 1.75-1.61 (1H, m), 1.60 (3H, s), 1.50 (3H, s), 1.17 (1H, s), 0.98 (9H, s), 0.90 (6H, t, J=6.1 Hz);

[0104] .sup.13C NMR (100 Hz, CDCl.sub.3) 172, 170.8, 170.6, 110.2, 74.6, 60.6, 49.4, 47.3, 36.8, 34.6, 26.9, 26.7, 26.5, 26.0, 25.8, 25.7, 23.0, 21.9;

[0105] LC-MS [M+H].sup.+=357.2; HPLC: 97.1%.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0106] Step 3 is the same as in Example 3.

Example 6

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0107] Step 1 is the same as in Example 3.

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0108] The crude PKL-019 (15.8 mmol) was dissolved in acetonitrile (30 mL). Then, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (5.56 g; 17.35 mmol; 1.1 eq) and 4-methylmorpholine (NMM) (5.2 mL; 47.3 mmol, 3.0 eq) were added to the clear solution. Finally, PKL-012 (475 g; 2.63 mol; 1.3 eq) was added portionwise and the reaction mixture was stirred at room temperature. The progress of the reaction was monitored by TLC method (10% CH.sub.3OH/CH.sub.2Cl.sub.2). The reaction was usually completed within 2 hours. The mixture was diluted with ethyl acetate (30 mL) and washed with 5% KHSO.sub.4 (330 mL), a saturated solution of NaHCO.sub.3 (330 mL), and brine (15 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4. The solvent was evaporated to give 4.9 g of brown solid (yield of the crude PKL-020:86.9%). The crude product was macerated at boiling in methyl tert-butyl ether (MTBE) (30 mL) for 60 min. The mixture was cooled to room temperature and stirred for 17 hours. The crystals were filtered off, washed with MTBE (310 mL), and dried at room temperature to constant weight to give a white solid of PKL-020 (2.74 g, 49% yield).

[0109] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.69 (1H, d, J=9.3 Hz), 6.37 (1H, d, J=5.0 Hz), 4.43 (1H, d, J=5.8 Hz), 4.28 (1H, d, J=9.3 Hz), 2.78 (3H, d, J=4.8 Hz), 2.77-2.75 (1H, m), 1.86 (1H, brs), 1.75-1.61 (1H, m), 1.60 (3H, s), 1.50 (3H, s), 1.17 (1H, s), 0.98 (9H, s), 0.90 (6H, t, J=6.1 Hz);

[0110] .sup.13C NMR (100 Hz, CDCl.sub.3) 172, 170.8, 170.6, 110.2, 74.6, 60.6, 49.4, 47.3, 36.8, 34.6, 26.9, 26.7, 26.5, 26.0, 25.8, 25.7, 23.0, 21.9;

[0111] LC-MS [M+H].sup.+=357.2; HPLC: 96.8%.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0112] Step 3 is the same as in Example 3.

Example 7

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0113] Step 1 is the same as in Example 3.

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0114] Step 2 is the same as in Example 3.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0115] PKL-020 (3 g, 8.42 mmol, 1 eq) was dissolved in 30 mL of methylene chloride at room temperature. Then, NH.sub.2OH (1.98 mL, 50% solution in water, 32 mmol, 3.8 eq) was added. The suspension was stirred at room temperature for 24 h (TLC monitoring: 10% CH.sub.3OH/CH.sub.2Cl.sub.2). Then, 6 mL of acetone was added dropwise. The reaction was stirred at boiling for 30 minutes. Then, the mixture was subjected to azeotropic distillation from the mixture of methylene chloride and acetone to remove water. The volume of distilled solvent was replenished by successive addition of further portions of methylene chloride. Then, the reaction mixture was cooled to room temperature and stirred for 17 hours. The crystals were filtered off, washed with methylene chloride (26 mL), acetone (6 mL), and dried to obtain 2.31 g of PKL-021 (83% yield) as a white solid.

[0116] .sup.1H and .sup.13C NMR chemical shifts of compound PKL-021 in DMSO-d.sub.6 are presented in Table 3. Characteristic absorption bands of the IR spectrum of compound PKL-021 are presented in Table 4.

Example 8

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0117] Step 1 is the same as in Example 3.

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0118] The crude PKL-019 (5.26 mmol) was dissolved in acetonitrile (10 mL). Then, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (1.86 g; 5.78 mmol; 1.1 eq) and triethylamine (NEt.sub.3) (2.2 mL; 15.78 mmol, 3.0 eq) were added to the clear solution. Finally, PKL-012 (1.2 g; 6.84 mol; 1.3 eq) was added portionwise, and the reaction mixture was stirred at room temperature. The progress of the reaction was monitored by TLC method (10% CH.sub.3OH/CH.sub.2Cl.sub.2). The reaction was usually completed within 2 hours. The mixture was diluted with ethyl acetate (10 mL) and washed with 5% KHSO.sub.4 (310 mL), a saturated solution of NaHCO.sub.3 (310 mL), and brine (10 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4. The solvent was evaporated to give 1.65 g of brown solid (yield of the crude PKL-020:88%). The crude product was macerated at boiling in methyl tert-butyl ether (MTBE) (10 mL) for 60 min. The mixture was cooled to room temperature and stirred for 17 hours. The crystals were filtered off, washed with MTBE (33 mL), and dried at room temperature to constant weight to give a white solid of PKL-020 (0.66 g, 35% yield).

[0119] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.69 (1H, d, J=9.3 Hz), 6.37 (1H, d, J=5.0 Hz), 4.43 (1H, d, J=5.8 Hz), 4.28 (1H, d, J=9.3 Hz), 2.78 (3H, d, J=4.8 Hz), 2.77-2.75 (1H, m), 1.86 (1H, brs), 1.75-1.61 (1H, m), 1.60 (3H, s), 1.50 (3H, s), 1.17 (1H, s), 0.98 (9H, s), 0.90 (6H, t, J=6.1 Hz);

[0120] .sup.13C NMR (100 Hz, CDCl.sub.3) 172, 170.8, 170.6, 110.2, 74.6, 60.6, 49.4, 47.3, 36.8, 34.6, 26.9, 26.7, 26.5, 26.0, 25.8, 25.7, 23.0, 21.9;

[0121] LC-MS [M+H].sup.+=357.2; HPLC: 95.1%.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0122] Step 3 is the same as in Example 3.

Example 9

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0123] p-Toluenesulfonic acid (p-TsOH) (36 mg, 0.21 mmol) was added to a solution of PKL-018 (1 g, 5.26 mmol) in 2,2-dimethoxypropane (10 mL). The reaction mixture was stirred at a temperature of 40 C. for 20 hours. The progress of the reaction was monitored by HPLC and TLC methods (TLC:CHCl.sub.3:CH.sub.3OH:H.sub.2O 6:4:0.3). After the reaction was completed (HPLC: PKL-0183%), N-methylmorpholine (NMM) (58 L, 0.53 mmol) was added, and the mixture was concentrated to give a brown oil. The crude product PKL-019 was used in the next step without additional purification.

[0124] Reaction yield: 100%.

[0125] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.45 (1H, d, J=4.9 Hz), 4.11 (1H, q, J=7.1 Hz), 3.0 (1H, ddd, J=8.3 Hz, J=6.3 Hz, 4.8 Hz), 2.02 (1H, s), 1.80-1.75 (2H, m), 1.58 (3H, s), 1.52 (3H, s), 0.95 (3H, d, J=2.2 Hz), 0.93 (3H, J=2.0 Hz);

[0126] LC-MS [MH]=228.8

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0127] Step 2 is the same as in Example 6.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0128] Step 3 is the same as in Example 3.

Example 10

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0129] p-Toluenesulfonic acid (p-TsOH) (36 mg, 0.21 mmol) was added to a solution of PKL-018 (1 g, 5.26 mmol) in 2,2-dimethoxypropane (10 mL). The reaction mixture was stirred at a temperature of 40 C. for 20 hours. The progress of the reaction was monitored by HPLC and TLC methods (TLC: CHCl.sub.3:CH.sub.3OH:H.sub.2O 6:4:0.3). After the reaction was completed (HPLC: PKL-0183%), triethylamine (NEt.sub.3) (74.5 L, 0.53 mmol) was added, and the mixture was concentrated to give a brown oil. The crude product PKL-019 was used in the next step without additional purification.

[0130] Reaction yield: 100%.

[0131] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.45 (1H, d, J=4.9 Hz), 4.11 (1H, q, J=7.1 Hz), 3.0 (1H, ddd, J=8.3 Hz, J=6.3 Hz, 4.8 Hz), 2.02 (1H, s), 1.80-1.75 (2H, m), 1.58 (3H, s), 1.52 (3H, s), 0.95 (3H, d, J=2.2 Hz), 0.93 (3H, J=2.0 Hz);

[0132] LC-MS [MH]=228.8

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0133] Step 2 is the same as in Example 8.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0134] Step 3 is the same as in Example 3.

Example 11

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0135] (1S)-(+)-10-camphorsulfonic acid (49 mg, 0.21 mmol) was added to a solution of PKL-018 (1 g, 5.26 mmol) in 2,2-dimethoxypropane (10 mL). The reaction mixture was stirred at a temperature of 40 C. for 20 hours. The progress of the reaction was monitored by HPLC and TLC methods (TLC: CHCl.sub.3:CH.sub.3OH:H.sub.2O 6:4:0.3). After the reaction was completed (HPLC: PKL-0183%), diisopropylethylamine (DIPEA) (93 L, 0.53 mmol) was added, and the mixture was concentrated to give a yellow oil. The crude product PKL-019 was used in the next step without additional purification.

[0136] Reaction yield: 100%.

[0137] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.45 (1H, d, J=4.9 Hz), 4.11 (1H, q, J=7.1 Hz), 3.0 (1H, ddd, J=8.3 Hz, J=6.3 Hz, 4.8 Hz), 2.02 (1H, s), 1.80-1.75 (2H, m), 1.58 (3H, s), 1.52 (3H, s), 0.95 (3H, d, J=2.2 Hz), 0.93 (3H, J=2.0 Hz);

[0138] LC-MS [MH]=228.8

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0139] Step 2 is the same as in Example 3.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0140] Step 3 is the same as in Example 3.

Example 12

Preparation of marimastat (PKL-021)

Step 1: Preparation of (R)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanoic acid (PKL-019)

[0141] Pyridinium p-toluenesulfonate (PPTS) (13.2 mg, 0.053 mmol) was added to a solution of PKL-018 (0.1 g, 0.526 mmol) in 2,2-dimethoxypropane (1 mL). The reaction mixture was stirred at a temperature of 40 C. for 20 hours. The progress of the reaction was monitored by HPLC and TLC methods (TLC: CHCl.sub.3:CH.sub.3OH:H.sub.2O 6:4:0.3). After the reaction was completed (HPLC:PKL-0183%), diisopropylethylamine (DIPEA) (9.3 L, 0.053 mmol) was added, and the mixture was concentrated to give a yellow oil. The crude product PKL-019 was used in the next step without additional purification.

[0142] Reaction yield: 100%.

[0143] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.45 (1H, d, J=4.9 Hz), 4.11 (1H, q, J=7.1 Hz), 3.0 (1H, ddd, J=8.3 Hz, J=6.3 Hz, 4.8 Hz), 2.02 (1H, s), 1.80-1.75 (2H, m), 1.58 (3H, s), 1.52 (3H, s), 0.95 (3H, d, J=2.2 Hz), 0.93 (3H, J=2.0 Hz);

[0144] LC-MS [MH]=228.8

Step 2: Preparation of (R)-N-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-2-((S)-2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)-4-methylpentanamide (PKL-020)

[0145] Step 2 is the same as in Example 3.

Step 3: Preparation of (2R,3S)-N-1-((S)-3,3-dimethyl-1-methylamino-1-oxobutan-2-yl)-N-4,3-dihydroxy-2-isobutylsuccinamide (PKL-021)

[0146] Step 3 is the same as in Example 3.

Example 13

Preparation of Tablets Comprising marimastat (PKL-021)

[0147] Tablets comprising PKL-021 were produced by wet granulation according to the following steps: [0148] 1. Preparation of granulation liquid [0149] 2. Granulation [0150] 3. Calibration of granulate before drying [0151] 4. Drying [0152] 5. Calibration of granulate after drying [0153] 6. Mixing the granulate with external phase components [0154] 7. Tableting

Step 1: Preparation of Granulation Liquid

[0155] A binder is added to water and stirred until completely dissolved.

Step 2: Granulation

[0156] A mixture of internal phase components is granulated with an aqueous solution of the binder obtained in Step 1 in a high-speed granulator.

Step 3: Calibration of Granulate Before Drying

[0157] Before the drying process, the wet granulate is calibrated using a conical mill or oscillating granulator. The wet granulate can also be leveled manually using a steel sieve with an appropriate mesh size.

Step 4: Drying

[0158] The granulate is dried using a fluid bed dryer or tray dryer at a targeted temperature of approximately 55 C. (40-65 C.) until the required weight loss after drying is achieved (0.2-3.0%).

Step 5: Calibration of Granulate after Drying

[0159] The dried granulate is leveled manually, on a steel sieve or using a conical mill or oscillating granulator.

Step 6: Mixing the Granulate with External Phase Components

[0160] All components of the external phase (except a lubricant) are mixed with the granulate in a low-speed mixer, with the speed adjusted to the size of the mixer tank and its filling. The lubricant is added at the end of the mixing stage.

Step 7: Tableting

[0161] Tableting is performed separately for each dose of tablets comprising PKL-021 (doses are proportional, obtained by tableting to a specific targeted tablet weight).

[0162] Tableting is carried out on a rotary tablet press using appropriate equipment.

Recipe for Tablets Comprising PKL-021

[0163] The concentration of PKL-021 in the product: from 10 to 40%.

[0164] Drug doses: proportional, obtained through targeted tablet weights.

TABLE-US-00006 TABLE 6 Recipe for tablets comprising PKL-021 Amount per 1 tablet Component [% w/w] Internal phase (granulate) PKL-021 (active substance) 10.0-40.0 Binder (as a component of granulation liquid)* 0.5-10.0 Filling agent I 10.0-50.0 Filling agent II 5.0-30.0 Disintegrant 0.0-10.0 Purified water (as a component of granulation liquid)** q.s. External phase Filling agent III (type for direct tableting) 0.0-30.0 Disintegrant 0.0-10.0 Lubricant 0.2-5.0 Total 100.0 *Added in the form of a 4-10% aqueous solution **Removed during the granulate drying process

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