Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C277/00—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
  • C07C277/08—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the present invention relates to a novel process for preparing N-methyl -D-phenylalanyl -N- [1- [3- [ (aminoimino- methyl) amino] propyl] -3 , 3-difluoro-2-oxohexyl] -L-prolinamide and to pharmaceutically acceptable salts thereof.
• N-Methyl -D-phenylalanyl -N- [1- [3- [ (aminoirninomethyl) - amino]propyl] -3 , 3-difluoro-2-oxohexyl] -L-prolinamide dihydrochloride monohydrate (also known as "MDL 75,579DA") is described in U.S. Pat. No. 5,391,705 and is an inhibitor of both thrombin and tryptase.
• the compound is useful in an end-use application as an anticoagulant and for treating thrombophlebitis, coronary thrombosis and in the treatment of asthma.
• the 5-hydroxy-6-amino intermediate is then coupled to a ⁇ - Boc- ⁇ -methyl-D-Phe-L-Pro dipeptide intermediate and the coupled peptide is oxidized using typical oxidizing procedures such as the Swern oxidation, to form N-methyl -D- phenylalanyl -N- [1- [3- [ (aminoirninomethyl) amino] propyl] -3,3- difluoro-2-oxohexyl] -L-prolinamide dihydrochloride monohydrate .
• the bis hydrochloride is then guanylated using a three-step guanylation procedure which involves trifluoroacetic acid anhydride ("TFAA") protection of the internal amine, guanylation of the terminal amine and removal of the TFA-protecting group.
• TFAA trifluoroacetic acid anhydride
• the resulting 5- hydroxy-6-amino intermediate is then coupled to a N-Boc-N- methyl-D-Phe-L-Pro dipeptide intermediate and the coupled peptide is oxidized using typical oxidizing procedures such as the Swern oxidation, to form N-methyl -D-phenylalanyl -N-
• the three-step guanylation process gave poor yields of the final product, MDL 75,579DA, of only about 15%, starting from the ⁇ -9- [l- [3- [bis [ (1, 1-dimethylethoxy) carbonyl] amino]methylene] amino- 6-amino-5-hydroxy-4, 4-difluorononane intermediate. Furthermore, using the Dakin-West, three-step guanylation route, a product purity of only about 92% was obtained.
• the present invention relates to a novel process for preparing N-methyl -D-phenylalanyl -N- [1- [3- [ (aminoimino- methyl) amino]propyl] -3,3-difluoro-2-oxohexyl] -L-prolinamide or a pharmaceutically acceptable salt thereof comprising the steps of :
• the invention further provides a process for preparing N- 9 - [1- [3- [bis (K x -protected) amino]methylene] amino] -6-amino-
• 5-hydroxy-4,4-difluorononane comprising reacting 9,6- diamino-5-hydroxy-4 , 4-difluorononane, or a pharmaceutically acceptable salt thereof, with an appropriate guanylating agent to give N- 9 - [1- [3- [bis (K ⁇ -protected) amino] methylene] - amino] -6-amino-5-hydroxy-4 , 4-difluorononane, wherein K x is an ⁇ -protecting group suitable for protecting the nitrogens of guanyl moieties.
• Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromie, sulphu- ric and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate.
• Illustrative organic acids which form suitable salts include the mono, di and tricarboxylic acids.
• Such acids are, for example, acetic, trifluoroacetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic and sulfonic acids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid.
• Such salts can exist in either the hydrated or substantially anhydrous form.
• Stereoisomers is a general term for all isomers that differ only in the orientation of their atoms in space. It includes isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers or diastereoisomers) .
• the term “enantiomer” refers to two stereoisomers that are non superimposable mirror images of one another.
• chiral center refers to a carbon atom to which four different groups are attached.
• L/D or R/S is used as described in IUPAC-IUB Joint Commission on Biochemical Nomenclature, Eur. J. Biochem. 138, 9-37 (1984) .
• a chiral material may either contain an equal amount of the R and S isomers (or L and D isomers) in which case it is called “racemic” or “a racemate” or it may not contain equal amounts of R and S (or L and D isomers) in which case it is called “optically active” or “nonracemic” .
• N-protecting group suitable for protecting the nitrogens of guanyl moieties is meant to include tert-butyloxycarbonyl (Boc) , carbobenzyloxy (Cbz) , tert- butyldimethylsilyl (TBDMS) , tert-butyldiphenylsilyl (TBDPS) and the like.
• N-protecting group is meant to include any N-protecting group suitable for use in peptide synthethis as described in Greene, "Protective Groups in Organic Chemistry", Chapter 7, John Wiley & Sons, New
• Illustrative examples include, but are not limited to, tert-butyloxycarbonyl (Boc) , carbobenzyloxy (Cbz) , tert-butyldimethylsilyl (TBDMS) , tert- butyldiphenylsilyl (TBDPS) and the like.
• amino acid is meant to include the naturally occurring amino acids which are translated from the genetic code and comprise the building blocks of proteins.
• amino acid also includes, unless specifically stated otherwise, both (L) - and (D) - amino acids, chemically modified amino acids such as amino acid analogs, and naturally occurring amino acids which are not usually incorporated into proteins .
• Abbreviations of amino acid analogs included within the scope of the specification, as well as the amino and carboxy protecting groups are set forth in Table 1.
• step a 9, 6-diamino-5-hydroxy-4,4- difluorononane, or a pharmaceutically acceptable salt thereof, 1 is reacted with an appropriate guanylating agent to form N- 9 - [1- [3- [bis (K ⁇ -protected) amino]methylene] amino] - 6-amino-5-hydroxy-4, 4-difluorononane 2.
• guanylating agent 9, 6-diamino-5-hydroxy-4 , 4-difluorononane, or a pharmaceutically acceptable salt thereof, 1 is contacted with an appropriate guanylating agent .
• Appropriate guanylating agents are well known in the art and are signified by structure la below:
• K refers to an ⁇ -protecting group suitable for protecting the nitrogens of guanyl moieties, including tert-butyloxycarbonyl (Boc) , carbobenzyloxy (Cbz) , tert- butyldi ethylsilyl (TBDMS) , tert-butyldiphenylsilyl (TBDPS) and the like, with tert-butyloxycarbonyl being preferred.
• the substituent LG refers to a suitable leaving group moiety and includes -S-CH 3 , 1-pyrazole and the like.
• guanylating agents include, but are not limited to, bis-Boc-amidinopyrazole, bis-Boc-S-methylisothiourea, bis-Cbz-amidinopyrazole, and the like, with bis-Boc- amidinopyrazole being preferred.
• the reaction is carried out in the presence of a suitable base.
• a suitable base may be utilized to neutralize a salt of the internal amine of 9, 6-diamino-5-hydroxy-4 , 4-difluorononane, or a pharmaceutically acceptable salt thereof, 1, or may be utilized to neutralize the acid liberated when the appropriate guanylating agent, such as bis-Boc- amidinopyrazole, produces acid during the course of the reaction.
• Suitable bases include, but are not limited to, triethylamine, isopropyldiethylamine, N-methyl-morpholine, pyridine, sodium bicarbonate and sodium carbonate.
• the reaction is carried out in a suitable solvent, such as dichloromethane, dimethylformamide, tetrahydrofuran or tetrahydrofuran/water mixtures.
• the guanylating agent is added in a ratio of from about 0.9 to about 1.2 molar equivalents, with from about 0.95 to 1.05 molar equivalents being preferred, for every 1.0 molar equivalent of 9,6- diamino-5-hydroxy-4, 4-difluorononane, or a pharmaceutically acceptable salt thereof, 1.
• the reaction is carried out at a temperature of from about -30°C to about 20°C, with -15°C to 5°C being preferred with -5°C to 0°C being most preferred.
• N- 9 - [1- [3- [bis (K x -protected) amino] ethylene] - amino] -6-amino-5-hydroxy-4 , 4-difluorononane 2 may be isolated from the reaction zone by extraction and evaporation, as is well known in the art.
• N-9- [1- [3- [bis (K x - protected) amino]methylene] amino] -6-amino-5-hydroxy-4,4- difluorononane 2 optionally may be purified by techniques well known in the art, such as chromatography and recrystallization.
• step b N- 9 - [1- [3- [bis (K 1 -protected) - amino]methylene] amino] -6-amino-5-hydroxy-4, 4-difluorononane 2 is coupled with N-K 2 -protected-N-methyl -D-phenylalanyl - L- prolinamide (K 2 - ⁇ -methyl-D-Phe-L-Pro) to provide N-K 2 - protected-N-methyl -D-phenylalanyl -N- [1- [3- [ [bis [ (K x - protected) amino]methylene] amino] propyl] -3 , 3-difluoro-2- hydroxyhexyl] -L-prolinamide 3.
• N- 9 - [1- [3- [bis (K 1 -protected) amino] - methylene] amino] -6-amino-5-hydroxy-4, 4-difluorononane 2 is coupled with K 2 - ⁇ -methyl-D-Phe-L-Pro, wherein K 2 is an N- protecting group, preferably tert-butyloxycarbonyl (Boc) being preferred, using standard solution phase peptide synthesis techniques well known and appreciated by those skilled in the art.
• Standard solution phase peptide synthesis techniques include procedures such as the azide method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexyl- carbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl ester, N- hydroxy-succinic imido ester) method, Woodward reagent K method, carbonyldiimidazole method, phosphorus reagents such as BOP-Cl, or oxidation-reduction methods.
• the carbodiimide coupling is preferred.
• N-Boc-N- methyl-D-Phe-L-Pro (U.S. Pat. No. 5,391,705, issued February 21, 1995) is dissolved in a suitable organic solvent, such as methylene chloride, under an inert atmosphere, such as nitrogen, optionally in the presence of about one equivalent of 1-hydroxybenzotriazole (HOBt) .
• a suitable organic solvent such as methylene chloride
• HOBt 1-hydroxybenzotriazole
• DCC N,N'- dicyclohexylcarbodiimide
• the reaction mixture is allowed to stir for about 1 to 15 hours.
• the coupled product N-K 2 - protected-N-methyl -D-phenylalanyl -N- [1- [3- [ [bis [ (K x - protected)amino]methylene] amino]propyl] -3, 3-difluoro-2- hydroxyhexyl] -L-prolinamide 3, is then isolated and optionally purified by techniques well known in the art such as extractive techniques, precipitation, crystallization and chromatography.
• step c N- K 2 -protected-N-methyl -D- phenylalanyl -N- [1- [3- [ [bis [ (K x -protected) amino]methylene] - amino] ropyl] -3, 3-difluoro-2-hydroxyhexyl] -L-prolinamide 3 is reacted with an appropriate oxidizing agent to provide N-K 2 -protected-N-methyl -D-phenylalanyl -N- [1- [3- [ [bis [ ⁇ K - protected) amino]methylene] amino] propyl] -3 , 3-difluoro-2- oxohexyl] -L-prolinamide 4.
• N-K 2 -protected-N-methyl -D-phenylalanyl - N- [1- [3- [ [bis [ (K- L -protected) amino]methylene] amino] propyl] -3,3- difluoro-2-hydroxyhexyl] -L-prolinamide 3 is reacted with an appropriate oxidizing agent, such as periodinane, a chromic anhydride pyridine complex, pyridinium dichromate, or a dimethyl sulfoxide complex, such as DMSO- (COCl) 2 (Swern conditions) , to provide N-K 2 -protected-N-methyl -D- phenylalanyl -N- [1- [3- [ [bis [ (K ⁇ -protected) amino] methylene] - amino]propyl] -3 , 3-difluoro-2-oxohexyl] -L-prolinamide 4 using standard oxid
• Standard oxidizing techniques include procedures such as the Swern oxidation procedure, Synthesis, 165 (1981) ; the Dess Martin periodinane reaction, Dess Martin, J. Org. Chem. 48, 4155 (1983) ; and the Jones oxidation procedure (see U.S. Pat. No. 5,391,705) ; with the Swern oxidation procedure being most preferred.
• a suitable anhydrous organic solvent such as methylene chloride
• reaction is stirred approximately 30 minutes at a temperature of from about -55°C to about -78°C, an excess of a suitable organic base, such as triethylamine or ⁇ -methylmorpholine, is added and the reaction is allowed to warm to room temperature.
• a suitable organic base such as triethylamine or ⁇ -methylmorpholine
• N- K 2 - protected-N-methyl -D-phenylalanyl -N- [1- [3- [ [bis [ (K x - protected) amino]methylene] amino]propyl] -3,3-difluoro-2- oxohexyl] -L-prolinamide 4 is then isolated and purified by techniques well known to one skilled in the art such as extractive techniques, precipitation, crystallization and chromatography.
• step d N-K 2 -protected-N-methyl -D- phenylalanyl -N- [1- [3- [ [bis [ ( ⁇ -protected) amino] methylene] - amino] propyl] -3 , 3-difluoro-2-oxohexyl] -L-prolinamide 4 is reacted with a suitable deprotecting agent to provide N- methyl -D-phenylalanyl -N- [1- [3- [ (aminoirninomethyl) amino] - propyl] -3, 3-difluoro-2-oxohexyl] -L-prolinamide 5.
• the 9, 6-diamino-5-hydroxy-4 , 4-difluorononane, bis- hydrochloride 1 intermediate required for preparation of the end product 5 can be obtained by using a modified - 17 -
• step a ⁇ -K-L-ornithine 6 is N -protected according to standard N-protecting techniques well-known and appreciated by one skilled in the art to provide N ⁇ -K- N -benzyloxycarbonyl-ornithine 7, wherein K refers to a suitable protecting group as described previously in Scheme A, step a, preferably Cbz.
• ⁇ -Cbz-L-ornithine 6 is N ⁇ -protected using benzoyl chloride and standard Schotten-Baumann conditions. Specifically, a solution of benzoyl chloride in an ethereal solvent, such as diethyl ether, is added concomitantly with sodium hydroxide to a solution of ⁇ -Cbz-L-ornithine 6 in sodium hydroxide, over a period of from about 0.5 to 1 hours, while maintaining the reaction temperature between 0°C to about 5°C. The reaction mixture is then stirred for approximately 4 hours at room temperature, extracted with an ethereal solvent such as diethyl ether and acidified to about pH 1 using concentrated hydrochloric acid solution. Additional water is then added and the mixture is allowed to stand for approximately 48 hours. The solids are collected and purified by techniques well known to one skilled in the art to provide N ⁇ -K-N -benzyloxycarbonyl- ornithine 7.
• step b N ⁇ -K-N -benzyloxycarbonyl- ornithine 7 is cyclized to provide 2-phenyl- [4- (3-K- aminopropyl) ] -5- (4H) -oxazolane 8.
• a slurry of N ⁇ -K-N -benzyloxycarbonyl- ornithine 7 in a suitable organic solvent, such as methylene chloride is contacted with about 0.1 to 1.0 molar equivalents of dicyclohexylcarbodiimide .
• the reaction mixture is then stirred for about 2 to 5 hours and the resulting precipitated dicyclohexylurea by-product is filtered and washed with a suitable organic solvent, such as methylene chloride.
• the filtrate is then concentrated and diluted with a suitable ethereal solvent, such as diethyl ether and the process is optionally repeated.
• step c 2-phenyl- [4- (3-K-aminopropyl) ] -5- (4H) -oxazolane 8 is acylated according to standard acylation techniques to provide the corresponding O-acyl compound of structure 9.
• a suitable tertiary amine such as triethylamine
• 2-phenyl- [4- (3-K- aminopropyl) ] -5- (4H) -oxazolane 8 in a suitable organic solvent, such as tetrahydrofuran or tetrahydrofuran/heptane mixtures, at a temperature range of from about -10°C to about 10°C under an inert atmosphere, preferably nitrogen.
• a solution of ⁇ , ⁇ -difluoropentenoyl chloride, or the corresponding anhydride, in a suitable organic solvent, such as heptane is slowly added to the reaction mixture while maintaining the reaction temperature between about -10°C and 10°C.
• reaction mixture After about 30 to 60 minutes, the reaction mixture is allowed to warm to room temperature and stirred for about another 30 minutes.
• the triethylamine hydrochloride salt is removed by extractive techniques well known in the art, such as filtration, and the filtrate is concentrated to give the corresponding O-acyl compound of structure 9.
• step d the corresponding O-acyl compound of structure 9 is optionally reacted with an acylation catalyst to provide the corresponding C-acyl compound of structure 10.
• the corresponding O-acyl compound of structure 9 is dissolved in a suitable organic solvent, such as tetrahydrofuran and contacted with an acylation catalyst, such as a dialkylaminopyridine, preferably 4- dimethylaminopyridine (DMAP) .
• an acylation catalyst such as a dialkylaminopyridine, preferably 4- dimethylaminopyridine (DMAP) .
• DMAP dimethylaminopyridine
• step e the C-acyl compound of structure 10, is decarboxylated with a decarboxylating agent such as oxalic acid, succinic acid, and the like, with oxalic acid being preferred, to provide NT-9-K-amino-6-benzamido-5-oxo- 4, 4-difluoro-1-nonene 11.
• a decarboxylating agent such as oxalic acid, succinic acid, and the like, with oxalic acid being preferred, to provide NT-9-K-amino-6-benzamido-5-oxo- 4, 4-difluoro-1-nonene 11.
• a solution containing from 1 to 5 molar equivalents of dried oxalic acid in a suitable organic solvent, such as tetrahydrofuran is added to the reaction mixture from Scheme B, step d, containing the C-acyl compound of structure 10, and is left stirring for 16-32 hours.
• the reaction mixture is then concentrated and treated with a suitable acid, such as hydrochloric acid, and extracted with ethyl acetate.
• a suitable base such as sodium carbonate, sodium bicarbonate or sodium hydroxide, optionally washed with brine, dried with a suitable drying agent such as magnesium sulfate and concentrated.
• N -9-K-amino-6- benzamido-5-oxo-4,4-difluoro-1-nonene 11 is then isolated and purified by techniques well known to one skilled in the art such as precipitation and crystallization.
• step f N -9-K-amino-6-benzamido-5-oxo- , 4-difluoro-1-nonene 11, is contacted with an appropriate reducing agent to provide N -9-K-amino-6-benzamido-5- hydroxy-4, 4-difluoro-1-nonene 12.
• reducing agents include but are not limited to lithium tri-t- butyloxyaluminohydride, potassium borohydride, lithium tri- sec-butylborohydride, lithium borohydride, sodium borohydride, and lithium triethylborohydride with sodium borohydride being preferred.
• N -9-K-amino-6-benzamido-5-oxo ⁇ 4, 4- difluoro-1-nonene 11 is contacted with a molar excess of an appropriate reducing agent.
• the reaction is carried out in a suitable solvent.
• suitable solvents for hydride reductions are well known in the art, such as toluene, diethyl ether, methyl t-butyl ether, tetrahydrofuran (THF) and tetrahydrofuran/ethanol mixtures.
• THF tetrahydrofuran
• the reaction is carried out at a temperature in the range of from -78°C to about 10°C.
• the reduced product, N -9-K-amino-6-benzamido- 5-hydroxy-4, 4-difluoro-1-nonene 12 may be isolated from the reaction zone by extraction and then purified by methods well known in the art, such as chromatography and recrystallization.
• step g N -9-K-amino-6-benzamido-5- hydroxy-4 ,4-difluoro-1-nonene 12 is contacted with an appropriate alkylene reducing agent and an appropriate deprotecting agent to provide 9, 6-diamino-5-hydroxy- , 4- difluorononane 1.
• An appropriate alkylene reducing agent includes diborane, diisoalkyl borane, borane/tertiary amine complexes and hydrogen in the presence of a hydrogenation catalyst.
• the most preferred alkylene reducing agent is hydrogen in the presence of a hydrogenation catalyst .
• hydrogenation catalysts include platinum, palladium, rhodium, ruthenium and nickel. Both the metals, as finely dispersed solids or adsorbed on inert supports such as carbon or alumina, and certain soluble complexes of these metals exhibit catalytic activity.
• deprotecting agent includes those described previously in Scheme A, step d.
• Other deprotecting agents, as well as conditions for cleavage of protecting groups are well known in the art and are described in Greene, "Protective Groups in Organic
• N -9-K-amino-6-benzamido-5-hydroxy-4 ,4- difluoro-1-nonene 12 is dissolved in a suitable alcohol, such as isopropanol and a suitable acid such as hydrochloric acid.
• a suitable alcohol such as isopropanol
• a suitable acid such as hydrochloric acid
• the solution is then treated with an alkylene reducing agent, such as palladium dihydroxide adsorbed on an inert carbon support, and shaken under hydrogen gas (40-60 psi) for about 20 to 30 hours.
• the reaction mixture is filtered and concentrated to yield a N ⁇ - benzoyl-difluoro alcohol, hydrochloride.
• N ⁇ -benzoyl- difluoro alcohol, hydrochloride is then contacted with an appropriate deprotecting agent, such as hydrochloric acid and heated to reflux.
• an appropriate deprotecting agent such as hydrochloric acid and heated to reflux.
• the reaction mixture is cooled to room temperature, filtered and the filtrate is extracted with diethyl ether and the layers separated.
• the aqueous layer is treated with activated carbon, heated, filtered and concentrated to provide 9, 6-diamino-5-hydroxy-4,4- difluorononane 1.
• One of ordinary skill in the art can form pharmaceutically acceptable salts of 9, 6-diamino-5- hydroxy-4, 4-difluorononane 1 using techniques and procedures well known in the art.
• the ⁇ , ⁇ -difluoropentenoyl chloride intermediate 8a, required for preparation of the O-acyl compound of structure 9 can be obtained as illustrated in U.S. Pat. No.
• step a 2,2-difluoro-4-pentenoic acid 8c, is prepared by hydrolysis of ethyl ⁇ , ⁇ -difluoropentenoate 8b (U.S. Pat. No. 5,391,705, issued February 21, 1995) by techniques and procedures well known in the art, such as base hydrolysis.
• ethyl ⁇ , ⁇ -difluoropentenoate 8b also known as ethyl 2, 2-difluoro-4-pentenoate (U.S. Pat No. 4,847,401, siiued July 11, 1989)
• ethyl 2, 2-difluoro-4-pentenoate U.S. Pat No. 4,847,401, siiued July 11, 1989
• the reaction mixture is allowed to warm to room temperature for about 2 to 4 hours and then heated at about 40°C to about 55°C for an additional 2 to 4 hours. Ethanol and water are removed from the reaction mixture and 2, 2-difluoro-4-pentenoic acid 8c may be isolated from the reaction zone by extraction and then purified by methods well known in the art.
• step b 2, 2-difluoro-4-pentenoic acid 8c is contacted with a suitable chlorinating agent to yield 2, 2-difluoro-4-pentenoyl chloride 8a.
• An appropriate chlorinating agent is one that converts a hydroxyl group to a chloro group and does not cause the degradation of the starting material or the product .
• Appropriate chlorinating agents include phosphorous trichloride, thionyl chloride, oxalyl chloride and the like.
• 2,2-difluoro-4-pentenoic acid 8c is contacted with about 1.0 to 1.5 molar equivalents of an appropriate chlorinating agent.
• the reaction is carried out in a suitable solvent, such as dichloromethane, toluene or dimethylformamide.
• the reaction is carried out at a temperature of from about 20°C to about 35°C and generally requires about 4 to 24 hours.
• the product, 2,2-difluoro-4- pentenoyl chloride 8a can be isolated by fractional distillation and purified by techniques well known in the art, such as chromatography.
• N-methylated ⁇ -amino acids can be prepared as described in Scheme D and generally in B.S. Pitzele et al . , J. Med. Chem. , 37, 888-896 (1994) , herein incorporated by reference as if fully set forth. All of the substituents, unless otherwise indicated, are previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art.
• step a N-protect step b, carboxyl deprotect
• step a an ⁇ -amino acid of structure 13 wherein X is a suitable ⁇ -carboxyl protecting group, such as a methyl ester or a solid phase resin, is coupled with a suitable protecting group "K", previously defined in Scheme A, step a, in a manner analogous to the procedures described in Scheme A, step b to provide the coupled product .
• X is a suitable ⁇ -carboxyl protecting group, such as a methyl ester or a solid phase resin
• step b the coupled product is deprotected or cleaved from the solid phase under conditions well known in the art to provide the acid of structure 14.
• X is a methyl or ethyl group on structure 13
• the compound is dissolved in a suitable organic solvent, such as ethanol and treated with approximately an equal volume of water.
• a suitable organic solvent such as ethanol
• the resulting acid is then isolated and purified by techniques well known in the art. For example, the organic solvent is removed under vacuum and the remaining aqueous solution is acidified with dilute hydrochloric acid.
• aqueous phase is then extracted with a suitable organic solvent, such ethyl acetate, and the combined organic extracts are dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum.
• a suitable organic solvent such as ethyl acetate
• the residue can then be purified by flash chromatography on silica gel with a suitable eluent, such as methanol/chloroform to provide the acid of structure 14.
• step c the acid 14 is N-methylated to provide the N-protected N-methylated compound of structure 15.
• the acid 14 is dissolved in a suitable organic solvent, such as tetrahydrofuran, cooled to about 0°C and treated with excess methyl iodide. Then 1 to 3 equivalents of sodium hydride is added to the solution which is stirred for about 10 minutes at 0°C and then warmed to room temperature and stirred for 24 to 48 hours.
• a suitable organic solvent such as tetrahydrofuran
• the product is then isolated by techniques well known in the art, such as extractive methods. For example, dilute aqueous hydrochloric acid is added and the reaction is extracted with a suitable organic solvent, such as ethyl acetate. The organic extracts are then combined, washed with 5% sodium thiosulfate, brine, dried over anhydrous magnesium sulfate, filtered through a pad of silica gel and concentrated under vacuum to provide the N-protected, N- methylated compound 15. Alternatively, the N-protected N-methylated compound 15 can be prepared following the procedure described in
• step d the acid 14 is cyclized to provide the oxazolidine described by structure 14a.
• the acid 14 is dissolved in a suitable organic solvent, such as benzene and treated with an excess of paraformaldehyde.
• a suitable organic solvent such as benzene
• paraformaldehyde an organic solvent
• To this is added about 0.2 to 0.4 equivalents of p-toluenesulfonic acid and the reaction is heated at reflux for about 23 hours with continuous removal of water using a Dean-Stark trap.
• the reaction is then allowed to cool to room temperature and the product is isolated and purified by techniques well known in the art.
• the cooled reaction is concentrated under vacuum, the residue taken up in a suitable organic solvent, such as ethyl acetate, rinsed with saturated sodium bicarbonate, the organic phase dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum.
• a suitable organic solvent such as ethyl acetate
• the organic phase dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum.
• the residue is then purified by flash chromatography on silica gel with a suitable eluent, such as ethyl acetate/hexane to provide the oxazolidine 14a.
• step e the oxazolidine 14a is reduced under conditions well known in the art to provide the N- protected N-methylated compound 15.
• the oxazolidine 14a is dissolved in a suitable organic solvent, such as chloroform and treated with excess trifluoroacetic acid.
• a suitable organic solvent such as chloroform
• trifluoroacetic acid To the solution is added an excess of triethylsilane with stirring at room temperature.
• the reaction is allowed to stir for 1 to 7 days and then concentrated under vacuum to provide the N-protected N-methylated compound 15.
• the following examples present typical syntheses as described in Schemes A - D. These examples are understood to be illustrative only and are not intended to limit the scope of the present invention in any way.
• g refers to grams; “mmol” refers to millimoles; “mL” refers to illiliters; “bp” refers to boiling point; “mp” refers to melting point; “°C” refers to degrees Celsius; “mm Hg” refers to millimeters of mercury; “ ⁇ L” refers to microliters; “ ⁇ g” refers to micrograms; and “ ⁇ M” refers to micromolar; “eq” refers to molar equivalents; “cone” refers to concentrated; “sat” refers to saturated.
• step a To a solution of ethyl ⁇ , ⁇ - difluoropentenoate 8b (80 g, 0.49 mol, U.S. Pat. No. 4,847,401, issued July 11, 1989) in H 2 0 (80 mL) is added LiOH (20.9 g, 0.5 mol) over 5 min at 0-5°C. The reaction mixture is allowed to warm to room temperature for 3 h, then heated at 45-50°C for 3 h. Ethanol and H 2 0 are removed (60 torr, 45°C) from the reaction mixture. The resulting orange oil is dried (1 torr, room temperature) for 2 h to obtain an oily solid. The oily solid is diluted in H 2 0 (75 mL) and acidified to pH 1 using cone HCl. The reaction mixture is extracted with diethyl ether (3 X 150 mL) . Cone
• step b Add oxalyl chloride (17.6 mL, 201.8 mmol) to compound 8c (25 g, 183.7 mmol) and dimethylformamide (3 drops) under neat conditions at room temperature. Observe gas evolution for approximately 2 h. After 5 h, treat the reaction mixture with an additional 0.08 eq of oxalyl chloride (1.2 mL) and stir for 18 h. Use fractional distillation (88-91°C) to obtain 24.4 g, 158 mmol of the title compound 8a as an oil in 85% yield. 19F NMR
• step a To a solution of ⁇ -Cbz-L-ornithine 6 (50 g, 187.8 mmol, Advanced ChemTech, Louisville, KY 40228-1075) in 1 N sodium hydroxide (250 mL, 0.25 mol) is added a solution of benzoyl chloride (22 mL, 189.5 mmol) in diethyl ether (200 mL) and 1 N NaOH (313 mL, 0.31 mol) simultaneously over a period of 45 min maintaining the reaction temperature between 0-5°C. Stir the reaction mixture at room temperature for 4 h, extract with diethyl ether (3 X 100 mL) and separate the organic phase.
• ⁇ -Cbz-L-ornithine 6 50 g, 187.8 mmol, Advanced ChemTech, Louisville, KY 40228-1075
• 1 N sodium hydroxide 250 mL, 0.25 mol
• benzoyl chloride 22 mL, 189.5 mmol
• the crude oil is pre-absorbed on silica gel (2 g Si0 2 /l g oil) and purified by flash chromatography using a 10 cm O.D. column containing 6 inches of silica in height and eluting with 3:2 hexane/ethyl acetate to provide 15.4 g, 34.65 mmol of title compound 11 as a light yellow solid in 36 % yield. Total yield of title compound 11 is 37.1 g (87%) .
• Anal. Calcd for C 24 H 2e N 2 0 4 F 2 (444.49) : C, 64.85; H, 5.90; N, 6.30.
• step f To a solution of difluoroketone 11 (38.0 g, 85.5 mmol) in ethanol (500 mL) and tetrahydrofuran (20 mL) is added NaBH 4 (1.66 g, 43.88 mmol) at 0°C. A precipitate forms with 1 h upon warming to room temperature. Dilute the heterogeneous reaction mixture using tetrahyrofuran (55 mL) to give a mostly homogeneous solution and stir for 0.5 h. Concentrate the reaction mixture to a solid, treat with 10% HCl solution (800 mL) and extract with ethyl acetate (400 mL) .
• step g (deprotection): Heat to reflux (110°C) a solution of 9-Amino-6-benzamido-5-hydroxy-4, 4- diflurononane, hydrochloride (26.85 g, 76.5 mmol) in cone aq HCl (250 mL) for 19 h. Benzoic acid crystals develop as the reaction mixture is cooled to room temperature. Remove the crystals by filtration. Extract the filtrate with diethyl ether (250 mL) and separate the layers. Treat the aqueous layer with activated carbon, heat (80°C) , filter through celite, concentrate and dry to provide 21.5 g, 75.9 mmol of title compound 1 as a tan solid in quantitative yield. MS (CI, CH 4 ) m/z (% relative intensity) 211 (M + +l,
• step a Add triethylamine (33 mL, 2 eq) to a solution of diamine bis-hydrochloride 1 (34.4 g, 0.122 mol) in dimethylformamide (250 mL) , added at 0°C. After 5 min, treat the reaction mixture with a solution of bis-Boc amidinopyrazole (36.93 g, 0.119 mol) in dimethylformamide (90 mL) along with simultaneous addition of triethylamine (17 mL, 1 eq) , added at 0°C. Allow the reaction mixture to warm to room temperature and stir overnight .
• step b Add ⁇ , ' -dicyclohexylcarbodiimide (20.76 g, 100.6 mmol) to a solution of ⁇ -Boc- ⁇ -methyl-D- Phe-Pro-OH (37.89, 100.6 mmol) and hydroxybenzotriazole
• step c Add a solution of dimethylsulfoxide (42.5 mL, 0.598 mol) in methylene chloride (700 mL) to a solution of oxalyl chloride (26.1 mL, 0.299 mol) in methylene chloride (350 mL) , added at -55°C. Stir the reaction mixture for 45 min and then treat with a solution of alcohol 3 (80.84 g, 0.100 mol) in methylene chloride (350 mL) over 30 min. Stir the reaction mixture for an additional 15 min, maintaining the temperature between -50°C and -60°C, then treat with diphenylethylamine (157 mL, 0.898 mol) at -55°C over 30 min.
• reaction mixture After stirring for another 1 h at -55°C, the reaction mixture is homogeneous red-colored. Slowly quench the reaction mixture using 10% aq citric acid solution (250 mL) at 0°C. Extract the organic layer, wash with brine (200 mL) , dry (MgS0 4 ) filter and concentrate to provide approximately 80 g of oily foam. Dissolve the foam in a minimum amount of eluent and purify (2 X 40 g) by flash chromatography (5" X 5" Si0 2 column; 3:3:2 Hept/CH 2 Cl 2 /EtOAc) to provide 42.9 g of title compound 4 in
• step d Dissolve ketone 4 (21.6 g, 0.267 mol) in 2 ⁇ HCl/EtOAc (470 mL) , stopper the flask and stir at room temperature for 60 h.
• the reaction mixture is light yellow and under pressure when the flask is vented. Bubble anhydrous HCl into the reaction mixture for 10 min, stopper the flask and stir the mixture for 1 h.
• Liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) may be used to analyze batches of MDL 75,579DA prepared using the process of the present invention.
• WISP 715 autosampler Waters Chromatography
• auxiliary gas (N 2 ) flow 10 mL/min.
• Q3 auxiliary gas
• the voltages to be applied to the conversion dynode and electron multiplier are -15 kV and 1.4 kV, respectively.
• the peak areas of the two main diastereomers of MDL 75,579DA are summed and divided by the total area integrated over the entire analysis, excluding the unretained substances (void peak) . This ratio, based as a percentage, serves as the indication of purity. All purity determinations were performed using peak areas observed in the UV absorption profile. Peak integration was performed using the Peak Pro ⁇ integration software provided by CALS data system (Beckman Instruments, Inc.) . A summary of the purity determinations for three samples prepared using the direct guanylation procedure of the present invention is set forth in Table 2.

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