AU710040B2 - Process for the preparation of N-methyl-D-phenylalanyl-N- (1-(3-((aminoiminomethyl)amino)propyl)-3,3-difluoro-2- oxohexyl)-L-prolinamide - Google Patents

Description

WO'97/35874 WO 9735874PCTIUS97/02869 PROCESS FOR THE PREPARATION OF N-METHYL-D-PHENYLALANYL- N- [(AMINOIMINOMETHYL)AMdINOIPROPYL] -3,3-DIFLUORO- 2 -OXOHEXYL]

-L-PROLINAMIDE

FIELD OF THE INVENTION The present invention relates to a novel process for preparing N-methyl-D-phenylalanyl-N [(aminoiminomethyl)aminolpropyll 3-difluoro-2-oxohexyl] -L-prolinamide and to pharmaceutically acceptable salts thereof.

BACKGROUND OF THE INVENTION N-Methyl-D-phenylalanyN lV-l- [(aminoiminomethyl) amino] propyl] 3-difluoro-2-oxohexyl] -L-prolinamide dihydrochloride monohydrate (also known as "MDL 75,579DAII) 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 thrombophiebitis, coronary thrombosis and in the treatment of asthma. The biochemical and pharmacological effects of N-methyl-D-phenylalanyl-N [(aminoiminomethyl)amino] propyl] 3-difluoro-2-oxohexyll -L-prolinamide dihydrochloride monohydrate are described in R.J. Broersma et al., WO 97/35874 PCT/US97/02869 -2- Thrombosis and Haemostasis 69, 666 (1993) and B. Neises et al., Bioorq. Med. Chem. 3, 1049-1061 (1995).

U.S. Pat. No. 5,391,705 discloses the convergent preparation of N-methyl-D-phenylalanyl-N- [(aminoiminomethyl)amino]propyl]-3,3-difluoro-2-oxohexyl]- L-prolinamide dihydrochloride monohydrate from a nitro intermediate. For example, 4-amino-l-nitrobutane hydrochloride is directly guanylated with bis-Boc-Smethylisothiourea. The guanylated product is then coupled to 2,2-difluoropentene-l-al, ethyl hemiacetal, to form a hydroxy-6-nitro intermediate which is reduced by catalytic hydrogenation to form a 5-hydroxy-6-amino intermediate.

The 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-Dphenylalanyl-N-[1-[3-[(aminoiminomethyl)amino]propyll-3,3difluoro-2-oxohexyl]-L-prolinamide dihydrochloride monohydrate.

Alternatively, B. Neises et al., Bioorg. Med. Chem. 3, 1049-1061 (1995) discloses, and U.S. Pat. No. 5,391,705 suggests, using a modified Dakin-West reaction of 2-phenyl- 3 -benzyloxycarbonylaminopropyl)]-5-(4H)-oxazolane and the anhydrides or acyl halides of 2,2-difluoro-4-pentenoic acid to yield 9,6-diamino-5-hydroxy-4,4-difluorononane, bis hydrochloride. 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. The resulting hydroxy-6-amino intermediate is then coupled to a N-Boc-Nmethyl-D-Phe-L-Pro dipeptide intermediate and the coupled peptide is oxidized using typical oxidizing procedures such WO 97/35874 PCTUS97/028699 -3as the Swern oxidation, to form N-methyl-D-phenylalanyl-N- [1-[3-[(aminoiminomethyl)amino]propyl]-3,3-difluoro-2oxohexyl]-L-prolinamide dihydrochloride monohydrate.

These methods, however, possess some disadvantages.

With regard to the process utilizing an appropriate nitro intermediate, there exist difficulties in the preparation of the 4-guanyl-l-nitrobutane intermediate. For example, when sodium nitrite is added to bromobutylphthalimide, the starting material, high yields of nitrobutylphthalimide, the desired intermediate, are difficult to obtain because of nitrite formation. Also, the nitrite must be hydrolyzed and separated from the desired nitrobutylphthalimide.

Additionally, excess hydrazine is needed to remove the phthalimido group. In order to remove the toxic hydrazine, an excess of the expensive reagent BocO2 is required to be added to the crude mixture and the corresponding carbamates must be removed by chromatography. Besides the difficulty in preparing the appropriate 4-guanyl-1-nitrobutane intermediate, acceptable product purity has been difficult to obtain and numerous chromatographic purifications are required in the synthesis.

As for the Dakin-West route, the three-step guanylation process gave poor yields of the final product, MDL 75,579DA, of only about 15%, starting from the N-9-[1- [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.

However, because of concerns of guanylating the unprotected internal amine the 6-amino group), the art taught and suggested that protection of the internal amine was necessary for selective guanylation of 9,6-diamino-5hydroxy-4,4-difluorononane, bis-hydrochloride; K. Hofmann WO 97/35874 PCTIUS97/02869 -4et al., J. Am. Chem. Soc. 82, 3727-3732 (1960); K.

Narayanan and 0. Griffith, J. Med. Chem. 37, 885-887 (1994); F. Weygard and R. Geiger, Berichte der Deutschen Chemishen Gellellschaft, 89, 647 (1956).

It is an object of the present invention to provide novel methods for the preparation of N-methyl-Dphenylalanyl-N-[1-[3-[(aminoiminomethyl)amino]propyl]-3,3difluoro-2-oxohexyl]-L-prolinamide, or a pharmaceutically acceptable salt thereof that allow for the selective, onestep guanylation of desirable Dakin-West intermediates, as opposed to the difficult to make 4 -guanyl-l-nitrobutane intermediate.

It is a further object of the present invention to provide novel methods for the economical preparation of Nmethyl-D-phenylalanyl-N- [1-[3-[(aminoiminomethyl)amino]propyl]-3,3-difluoro-2-oxohexyl]-L-prolinamide or a pharmaceutically acceptable salt thereof which can be carried out using fewer steps and fewer purifications than are disclosed previously in the art.

It is also a further object of the present invention to provide novel methods for the preparation of [bis (K 1 -protected) 4,4-difluorononane, wherein K, is an N-protecting group suitable for protecting the nitrogens of guanyl moieties, a desirable intermediate useful in the preparation of Nmethyl-D-phenylalanyl-N- [1-[3-[(aminoiminomethyl)amino]propyl-3,3-difluoro-2-oxohexyl]-L-prolinamide or a pharmaceutically acceptable salt thereof.

These and other objects disclosed herein are attained by the following claimed invention.

-4a- Throughout the description and claims of the specification the word comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

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a CAkWINWORDUENNYM\SPECNKI20550 9 7.DOC WO 97/35874 PCTIUS97/02869 SUMMARY OF THE INVENTION The present invention relates to a novel process for preparing N-methyl-D-phenylalanyl-N- (aminoiminomethyl) arinolpropyl] 3-difluoro-2-oxohexyl] -L-prolinamide or a pharmaceutically acceptable salt thereof comprising the steps of: reacting 9,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof, with an appropriate guanylating agent to provide N-9-[l-[3-[bis(Klprotected) amino] methylenel amino] -6-amino-5-hydroxy-4 ,4difluorononane, wherein K, is an N-protecting group suitable for protecting the nitrogens of guanyl moieties; coupling the N-9- [bis (Kl-protected) amino] methylene] amino] -6-amino-5-hydroxy-4, 4-difluorononane with

N-K

2 -protected-N-methyl-D-phenylalanyl-L-prolinamide to provide N- K 2 -protected-NA-methyl D-phenylal anyl [3- [bis (Kj -protected) amino] methylene] amino] propyl]I -3,3 difluoro-2-hydroxyhexyl]-L-prolinamide, wherein K 2 is an Nprotecting group; reacting N-K 2 -protected-N-methyl-D-phenylalanyl-N\- [1- [bis (Kl-protected) amino] methylene] amino] propyl] 3difluoro-2-hydroxyhexyl] -L-prolinamide with an appropriate oxidizing agent to provide N-K 2 -protected-N-methyl-Dphenylalanyl-N- [bis (KI-protected) amino] methylene] amino] propyl] -3,3-difluoro-2-oxohexyl] -L-prolinamide; and deprotecting N- K 2 -protected- N-methyl -D -phenylalanyl -N1- [bis (Kl-protected) amino] methylene] amino] propyl] 3difluoro-2-oxohexyl] -L-prolinamide with a suitable WO 97/35874 PCT/US97/02869 -6deprotecting agent to provide N-methyl-D-phenylalanyl-N- [1- [3-[(aminoiminomethyl)amino]propyl]-3,3-difluoro-2oxohexyl]-L-prolinamide or a pharmaceutically acceptable salt thereof.

The invention further provides a process for preparing [3-[bis(Ki-protected)amino]methylene]amino]-6-amino- 5-hydroxy-4,4-difluorononane comprising reacting 9,6diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof, with an appropriate guanylating agent to give N-9- [bis(K 1 -protected) amino]methylene]amino]-6-amino-5-hydroxy-4,4-difluorononane, wherein K 1 is an N-protecting group suitable for protecting the nitrogens of guanyl moieties.

DETAILED DESCRIPTION OF THE INVENTION As used in this application: a) the designation refers to a bond for which the stereochemistry is not designated.

b) the designation refers to a bond that protrudes forward out of the plane of the page.

c) the designation refers to a bond that protrudes backward out of the plane of the page.

d) the term "pharmaceutically acceptable salt" refers to acid addition salts.

The expression "pharmaceutically acceptable acid addition salts" is intended to apply to any non-toxic organic or inorganic acid addition salt of N-methyl-D- WO 97/35874 PCT/US97/02869 -7phenylalanyl-N- [1-[3-[(aminoiminomethyl)amino]propyl]- 3,3-difluoro-2-oxohexyl]-L-prolinamide or intermediates thereof. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric 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.

Illustrative of 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. The term "chiral center" refers to a carbon atom to which four different groups are attached. The nomenclature 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".

WO 97/35874 PCTIUS97/02869 -8- The term "N-protecting group suitable for protecting the nitrogens of guanyl moieties" is meant to include tert-butyloxycarbonyl (Boc), carbobenzyloxy (Cbz), tertbutyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS) and the like.

The term "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 York (1981). Illustrative examples include, but are not limited to, tert-butyloxycarbonyl (Boc), carbobenzyloxy (Cbz), tert-butyldimethylsilyl (TBDMS), tertbutyldiphenylsilyl (TBDPS) and the like.

As used herein the term "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. The term amino acid also includes, unless specifically stated otherwise, both and 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.

WO 97/35874 PCT/US97/02869 -9- TABLE 1 Amino Acid or Protecting Group Symbol Arginine Arg Phenylalanine Phe Ornithine Orn Proline Pro tert-butyloxycarbonyl Boc carbobenzyloxy Cbz acetyl Ac succinyl Suc phenylacetamidomethyl

PAM

A general synthetic procedure for preparing N-methyl- D-phenylalanyl-N-[1-[3-[(aminoiminomethyl)amino]propyl]- 3,3-difluoro-2-oxohexyl]-L-prolinamide is set forth in Scheme A. In Scheme A, starting materials and reagents unless indicated elsewhere in this application are well known and appreciated by one of ordinary skill in the art.

WO 97/35874 WO 9735874PCTIUS97/02869 SCHEME A

H

2 N 4H CH 3 K-NlNH-K 1

NH

F F step b

CH

3 Coupling step a Guanylation

H

3

C-'

Kjstep c Oxidation step d Deprotection WO 97/35874 PCTIUS97/02869 -11- In Scheme A, step a, 9,6-diamino-5-hydroxy-4,4difluorononane, or a pharmaceutically acceptable salt thereof, 1 is reacted with an appropriate guanylating agent to form N-9- [3-[bis (K 1 -protected)amino]methylene] amino]- 6-amino-5-hydroxy-4,4-difluorononane 2.

For example, 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:

N--K

KI--HN LG la wherein K, refers to an N-protecting group suitable for protecting the nitrogens of guanyl moieties, including tert-butyloxycarbonyl (Boc), carbobenzyloxy (Cbz), tertbutyldimethylsilyl (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.

Appropriate guanylating agents include, but are not limited to, bis-Boc-amidinopyrazole, bis-Boc-S-methylisothiourea, bis-Cbz-amidinopyrazole, and the like, with bis-Bocamidinopyrazole 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- WO 97/35874 PCTIUS97/02869 -12amidinopyrazole, 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. In order to minimize the chance of guanylating the internal amine of 9,6-diamino-5hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof, 1, 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 9,6diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof, 1. The reaction is carried out at a temperature of from about -30 0 C to about 20 0 C, with -15 0

C

to 5 0 C being preferred with -5 0 C to 0°C being most preferred. N-9- [bis(K 1 -protected)amino]methylene]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(Kprotected)amino]methylene]amino]-6-amino-5-hydroxy-4,4difluorononane 2 optionally may be purified by techniques well known in the art, such as chromatography and recrystallization.

In Scheme A, 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-Lprolinamide

(K

2 -N-methyl-D-Phe-L-Pro) to provide N-K 2 protected-N-methyl-D-phenylalanyl-N- WO 97/35874 PCT/US97/02869 -13protected)amino]methylene]amino]propyl]-3,3-difluoro-2hydroxyhexyl]-L-prolinamide 3.

For example, [bis (K 1 -protected) amino]methylene]amino]-6-amino-5-hydroxy-4,4-difluorononane 2 is coupled with K 2 -N-methyl-D-Phe-L-Pro, wherein K 2 is an Nprotecting 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 (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl ester, Nhydroxy-succinic imido ester) method, Woodward reagent K method, carbonyldiimidazole method, phosphorus reagents such as BOP-C1, or oxidation-reduction methods. The carbodiimide coupling is preferred. Some of these methods (especially the carbodiimide method) can be enhanced by adding l-hydroxybenzotriazole. For example, N-Boc-Nmethyl-D-Phe-L-Pro 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 l-hydroxybenzotriazole (HOBt). To this solution is added about one equivalent of N,N'dicyclohexylcarbodiimide (DCC) dissolved in a suitable organic solvent, such as methylene chloride. The reaction mixture is allowed to stir for about 1 to 15 hours. To the mixture is added 1 to 4 equivalents of a suitable base such as N-methylmorpholine or triethylamine and at least one molar equivalent of N-9-[1-[3-[bis(K 1 -protected)amino]methylene]amino]-6-amino-5-hydroxy-4,4-difluorononane WO 97/35874 PCTIS97/02869 -14- 2, dissolved in a suitable organic solvent such as methylene chloride. The reaction mixture is then allowed to stir for about 1 to 15 hours. The coupled product, N-K 2 protected-N-methyl-D-phenylalanyl-N-[1-[3-[[bis[(Kiprotected)amino]methylene]amino]propyl]-3,3-difluoro-2hydroxyhexyl]-L-prolinamide 3, is then isolated and optionally purified by techniques well known in the art such as extractive techniques, precipitation, crystallization and chromatography.

In Scheme A, step c, N-K,-protected-N-methyl-Dphenylalanyl-N- [3-[[bis (K-protected) amino] methylene] amino]propyl]-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- [[bis [(K 1 protected)amino]methylene]amino]propyll-3,3-difluoro-2oxohexyl]-L-prolinamide 4.

For example, N-K 2 -protected-N-methyl-D-phenylalanyl-N-

(K

1 -protected)amino]methylene] amino] propyl]-3,3difluoro-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-(COC1) 2 (Swern conditions), to provide N-K 2 -protected-N-methyl-Dphenylalanyl-N-

(K

1 -protected) amino] methylene] amino]propyl]-3,3-difluoro-2-oxohexyl]-L-prolinamide 4 using standard oxidizing techniques well known and appreciated by those skilled in the art. 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 WO 97/35874 PCT/~S9702869 (1983); and the Jones oxidation procedure (see U.S. Pat.

No. 5,391,705); with the Swern oxidation procedure being most preferred. For example, approximately 1.5 equivalents of oxalyl chloride is dissolved in a suitable anhydrous organic solvent, such as methylene chloride, and cooled to a temperature of from about -55 0 C to about -78 0 C. To this solution is added from about 3 to about 8 equivalents of methyl sulfoxide dropwise, maintaining the temperature at about -55 0 C or below. An equivalent of N-K 2 -protected-Nmethyl-D-phenylalanyl-N- [[bis (K 1 -protected) amino]methylene]amino]propyll-3,3-difluoro-2-hydroxyhexyl]-Lprolinamide 3 is dissolved in a suitable amount of anhydrous organic solvent, such as methylene chloride, and added slowly to the reaction with stirring. After addition is complete the reaction is stirred approximately minutes at a temperature of from about -55"C to about -78 0

C,

an excess of a suitable organic base, such as triethylamine or N-methylmorpholine, is added and the reaction is allowed to warm to room temperature. The oxidized product, N-K 2 protected-N-methyl-D-phenylalanyl-N-[1-[3-[[bis[(K i protected)amino]methylenejamino]propyll-3,3-difluoro-2oxohexyl]-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.

In Scheme A, step d, N-K 2 -protected-N-methyl-Dphenylalanyl-N- (K-protected)amino]methylene]amino]propyl]-3,3-difluoro-2-oxohexyl]-L-prolinamide 4 is reacted with a suitable deprotecting agent to provide Nmethyl-D-phenylalanyl-N-[1-[3-[(aminoiminomethyl)amino]propyl]-3,3-difluoro-2-oxohexyl]-L-prolinamide WO 97/35874 PCTIUS97/02869 -16- For example, N-K 2 -protected-N-methyl-D-phenylalanyl-N- [3-[[bis (K 1 -protected) amino] methylene] amino] propyl]-3,3difluoro-2-oxohexyl]-L-prolinamide 4 is reacted with a suitable deprotecting agent, such as hydrochloric acid in either dioxane or ethyl acetate or trifluoroacetic acid either neat or in dichloromethane. Conditions for cleavage of protecting groups for amino protecting groups are described in Greene, "Protective Groups in Organic Chemistry", Chapter 7, John Wiley Sons, New York (1981).

For example, N-K 2 -protected-N-methyl-D-phenylalanyl-N- [3- [[bis (Kl-protected) amino]methylene]amino]propyl]-3,3difluoro-2-oxohexyl]-L-prolinamide 4 is dissolved in hydrochloric acid/ethylacetate, stirred for about one to several hours. Anhydrous hydrochloric acid is then bubbled into the reaction mixture for about 10-15 minutes, the reaction mixture is stirred for about 20-40 minutes and then concentrated. The residue is then dissolved in water and optionally neutralized with sodium bicarbonate. The deprotected product, N-methyl-D-phenylalanyl-N-[1-[3- [(aminoiminomethyl)amino]propyl]-3,3-difluoro-2-oxohexyl]- L-prolinamide 5 is then isolated and purified by techniques well known to one skilled in the art such as extractive techniques, precipitation, crystallization and chromatography. One of ordinary skill in the art can form pharmaceutically acceptable salts of N-methyl-Dphenylalanyl-N-[1-[3-[(aminoiminomethyl)amino]propyl]-3,3difluoro-2-oxohexyl]-L-prolinamide 5 using techniques and procedures well known in the art.

The 9,6-diamino-5-hydroxy-4,4-difluorononane, bishydrochloride 1 intermediate required for preparation of the end product 5 can be obtained by using a modified WO 97/35874 PCT/US97/02869 -17- Dakin-West reaction as illustrated in U.S. Pat. No.

5,391,705 and set forth in Scheme B. The reagents, starting materials and techniques used in this process are readily available to one of ordinary skill in the art.

WO 97/35874 WO 9735874PCT/US97/02869 -18- SCHEME B

.NHK

step a

OH

Protection 6

NHK

0

OH

N

H

step b Cyclization step c Acylation

NHK

00 9 step d Rearrangement

NHK

step e 0 F step f Decarboxylation I H 0Red uction step g Reduction and Deprotection

NH

2 F

F

H

2 N

CH

3

OH

In Scheme B, step a, 8-K-L-ornithine 6 is Na-protected according to standard N-protecting techniques well-known WO 97/35874 PCTIUS97/02869 -19and appreciated by one skilled in the art to provide Na-K- N -benzyloxycarbonyl-ornithine 7, wherein K refers to a suitable protecting group as described previously in Scheme A, step a, preferably Cbz.

For example, 6-Cbz-L-ornithine 6 is Na-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 6-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 0 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 Na-K-N -benzyloxycarbonylornithine 7.

In Scheme B, step b, Na-K-N -benzyloxycarbonylornithine 7 is cyclized to provide 2-phenyl-[4-(3-Kaminopropyl)]-5-(4H)-oxazolane 8.

For example, a slurry of Na-K-N -benzyloxycarbonylornithine 7 in a suitable organic solvent, such as methylene chloride, is contacted with about 0.1 to 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 WO 97/35874 PCT/US97/02869 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. The cyclized product, 2-phenyl-[4-(3-K-aminopropyl)]-5-(4H)oxazolane 8, is then isolated and purified by techniques well known to one skilled in the art such as precipitation and crystallization.

In Scheme B, 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.

For example, a suitable tertiary amine, such as triethylamine, is added to a solution of 2-phenyl-[4-(3-Kaminopropyl)]-5-(4H)-oxazolane 8 in a suitable organic solvent, such as tetrahydrofuran or tetrahydrofuran/heptane mixtures, at a temperature range of from about -10 0 C to about 10 0 C under an inert atmosphere, preferably nitrogen.

A solution of a,c-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 0 C and 10 0 C. 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.

In Scheme B, step d, the corresponding O-acyl compound of structure 9 is optionally reacted with an acylation WO 97/35874 PCT/LTS97/02869 -21catalyst to provide the corresponding C-acyl compound of structure For example, 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 4dimethylaminopyridine (DMAP). The reaction mixture is then stirred for about 2-4 hours at room temperature to produce the C-acyl compound of structure 10, which is used without further isolation or purification.

In Scheme B, step e, the C-acyl compound of structure is decarboxylated with a decarboxylating agent such as oxalic acid, succinic acid, and the like, with oxalic acid being preferred, to provide N -9-K-amino-6-benzamido-5-oxo- 4,4-difluoro-l-nonene 11.

For example, 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. The layers are separated and the organic layer is washed with an appropriate 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. The decarboxylated product, N-9-K-amino-6benzamido-5-oxo-4,4-difluoro-l-nonene 11, is then isolated and purified by techniques well known to one skilled in the art such as precipitation and crystallization.

WO 97/35874 PCT/US97/02869 -22- In Scheme B, step f, N6-9-K-amino-6-benzamido-5-oxo- 4,4-difluoro-l-nonene 11, is contacted with an appropriate reducing agent to provide N6-9-K-amino-6-benzamido-5hydroxy-4,4-difluoro-l-nonene 12.

As is well known and appreciated in the art, this reduction will give a 5-hydroxy derivative that is a mixture of stereoisomers at the Appropriate reducing agents are well known in the art and include but are not limited to lithium tri-tbutyloxyaluminohydride, potassium borohydride, lithium trisec-butylborohydride, lithium borohydride, sodium borohydride, and lithium triethylborohydride with sodium borohydride being preferred.

For example, N -9-K-amino-6-benzamido-5-oxo-4,4difluoro-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. The reaction is carried out at a temperature in the range of from -78 0 C to about 10 0 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.

In Scheme B, step g, N6-9-K-amino-6-benzamido-5hydroxy-4,4-difluoro-l-nonene 12 is contacted with an WO 97/35874 PCTfUS97/02869 -23appropriate alkylene reducing agent and an appropriate deprotecting agent to provide 9,6-diamino-5-hydroxy-4,4difluorononane 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.

Examples of 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.

An appropriate 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 Chemistry", Chapter 7, John Wiley Sons, New York (1981).

For example, N 9 -K-amino-6-benzamido-5-hydroxy-4,4difluoro-l-nonene 12 is dissolved in a suitable alcohol, such as isopropanol and 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 NIbenzoyl-difluoro alcohol, hydrochloride. The N -benzoyldifluoro alcohol, hydrochloride is then contacted with an appropriate deprotecting agent, such as hydrochloric acid and heated to reflux. The reaction mixture is cooled to WO 97/35874 PCT/US97/02869 -24room 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-4,4difluorononane 1. One of ordinary skill in the art can form pharmaceutically acceptable salts of 9,6-diamino-5hydroxy-4,4-difluorononane 1 using techniques and procedures well known in the art.

The a,-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.

5,391,705 and set forth in Scheme C. The reagents, starting materials and techniques used in this process are readily available to one of ordinary skill in the art.

SCHEME C FF FF F F EtOd step ab C F EHO CI 8b 8c 8a In Scheme C, step a, 2,2-difluoro-4-pentenoic acid 8c, is prepared by hydrolysis of ethyl a,a-difluoropentenoate 8b Pat. No. 5,391,705, issued February 21, 1995) by techniques and procedures well known in the art, such as base hydrolysis.

For example, from about 1.0 to 1.5 molar equivalents of a suitable base, such as a lithium hydroxide, is added to a solution of ethyl a,a-difluoropentenoate 8b, also known as ethyl 2,2-difluoro-4-pentenoate Pat No.

WO 97/35874 PCT/US97/02869 4,847,401, siiued July 11, 1989), in water at a temperature of from about -10°C to about 10 0 C. The reaction mixture is allowed to warm to room temperature for about 2 to 4 hours and then heated at about 40 0 C to about 55 0 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.

In Scheme C, 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.

For example, 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 0 C to about 35 0 C and generally requires about 4 to 24 hours. The product, 2,2-difluoro-4pentenoyl chloride 8a, can be isolated by fractional distillation and purified by techniques well known in the art, such as chromatography.

N-methylated a-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 WO 97/35874 PCT/US97/02869 -26reference 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.

WO 97/35874 PCT/US97/02869 -27- SCHEME D

PO

H2N OX 13 step a, N-protect step b, carboxyl deprotect

O

K-

OH

steV 111mUU1y1a3V= 0 K- OH CH3 step d, cyclization O0

K-

step e, S reduction 14a In Scheme D, step a, an a-amino acid of structure 13 wherein X is a suitable a-carboxyl protecting group, such as a methyl ester or a solid phase resin, is coupled with a suitable protecting group 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.

In Scheme D, 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. For example, wherein X is a methyl or ethyl WO 97/35874 PCTIUS97/02869 -28group on structure 13, the compound is dissolved in a suitable organic solvent, such as ethanol and treated with approximately an equal volume of water. To this solution, with stirring is added 1 to 2 equivalents of lithium hydroxide and the reaction is allowed to stir for 1 to 6 hours. 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. The 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. 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.

In Scheme D, step c, the acid 14 is N-methylated to provide the N-protected N-methylated compound of structure 15. For example, the acid 14 is dissolved in a suitable organic solvent, such as tetrahydrofuran, cooled to about 0 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 OOC and then warmed to room temperature and stirred for 24 to 48 hours.

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, Nmethylated compound WO 97/35874 PCTIS97/02869 -29- Alternatively, the N-protected N-methylated compound can be prepared following the procedure described in Scheme D, steps d and e, from the acid of structure 14.

In Scheme D, step d, the acid 14 is cyclized to provide the oxazolidine described by structure 14a. For example, the acid 14 is dissolved in a suitable organic solvent, such as benzene and treated with an excess of paraformaldehyde. 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.

For example, 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.

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.

In Scheme D, step e, the oxazolidine 14a is reduced under conditions well known in the art to provide the Nprotected N-methylated compound 15. For example, the oxazolidine 14a is dissolved in a suitable organic solvent, such as chloroform and treated with excess 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 WO 97/35874 PCT/US97/02869 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.

As used herein, the following terms have the indicated meanings: refers to grams; "mmol" refers to millimoles; "mL" refers to milliliters; "bp" refers to boiling point; "mp" refers to melting point; refers to degrees Celsius; "mm Hg" refers to millimeters of mercury; refers to microliters; "pg" refers to micrograms; and refers to micromolar; "eq" refers to molar equivalents; "conc" refers to concentrated; "sat" refers to saturated.

EXAMPLE 1 Preparation of 2,2-Difluoro-4-pentenoic acid 8c Scheme C, step a: To a solution of ethyl a,adifluoropentenoate 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 0 C. The reaction mixture is allowed to warm to room temperature for 3 h, then heated at 45-50 0 C for 3 h. Ethanol and H 2 0 are removed torr, 45 0 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 mL) and acidified to pH 1 using conc HC1. The reaction mixture is extracted with diethyl ether (3 X 150 mL). Conc HC1 is added to the aqueous layer until it is pH 1 and the aqueous layer is extracted again with diethyl ether (3 X WO 97/35874 PCTIUS97/02869 -31- 100 mL) The combined organic layers are dried (MgSO 4 concentrated to an oil and distilled (140-170'C) to obtain 58.8 g, 432 mmol of the title compound 8c as an oil in 880yield. NMP. (CDCl 3 6 -106.8 (CFCl 3 as ref) ;13 C NMR (CDCl 3 6 16 8.3 (tI CO 2 H) 12 6. 4 C=C) 12 2. 3 C=C) 115.0 CF 2 38.9 CH,) 'H NMR (CDCl 3 8 2.88 (in, CH 2 2H), 5.28 (in, 1H), 5.32 1H), 5.76 (in, 1H), 8.74 (s, 1H).

EXAMPLE 2 Preparation of 2,2-Difluoro-4-pentenoyl chloride 8a Scheme C, step b: Add oxalyl chloride (17.6 mL, 201.8 inmol) to compound 8c (25 g, 183.7 inmol) 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 0 C) to obtain 24.4 g, 158 minol of the title compound Ba as an oil in 85% yield. 19F NMR (CDCl3) 5 -101.9 1 3 C NMR (CDCl 3 6 167.3 CO 2 H) 125. 5 C=C) 123. 1 C=C) 115. 6 CF 2 38. 3 (t,

CH

2 :H NMR (CDC'l 3 6 2. 90 (mn, 2 H) 5. 33 1H) 5. 36 (s, 1H), 5. 74 (in, 2H) EXAMPLE 3 Preparation of N2-Benzoyl-IN#-benzvloxvcarbonvl-ornithine 7 (K Cbz) Scheme B, step a: To a solution of 8-Cbz-L-ornithine 6 (50 g, 187.8 minol, Advanced ChemTech, Louisville, KY WO 97/35874 PCT/US97/02869 -32- 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 0 C. Stir the reaction mixture at room temperature for 4 h, extract with diethyl ether (3 X 100 mL) and separate the organic phase. Wash the aqueous phase with diethyl ether (3 X 100 mL) and acidify to pH 1 using conc HC1 solution to form white solids. Add additional water (450 mL) and allow the mixture to stand for 48 h. Collect the white solids, wash with water (200 mL) and diethyl ether (200 mL) and dry at 109 0 C (10 torr) overnight to provide 64.04 g, 173.9 mmol of title compound 7 as a white solid in 92% yield. MS-FAB m/z relative intensity) 371 393 100%); 1H NMR (CDC13) 6 7.9 2H, aroyl), 7.7 J 8 Hz, 1H, NH), 7.5-7.2 8H, aroyl, aryl), 6.3 1H, NH), 5.1 (s, 2H, benzyl), 4.7 1H, CH), 3.20 2H, NCH 2 2.1-1.8, 1.8-1.6 (2m, 4H, CH 2 -CH) WO 97/35874 PCT/US97/02869 -33- EXAMPLE 4 Preparation of 2-Phenvl- [4-(3-benzloxvcarbonylaminopropvl)1-5-(4H)-oxazolane 8 (K Cbz) Scheme B, step b: To a slurry of Na-benzamide 7 (25 g, 676.5 mmol) in methylene chloride (250 mL) is added N,N'dicyclohexylcarbodiimide (14.63 g, 70.9 mmol) in four portions. Observe an exotherm (5-10 0 C) while stirring the reaction mixture for 3 h. The precipitated dicyclohexylurea is filtered and washed with methylene chloride (100 mL). Concentrate the filtrate to a yellow oil, dilute with diethyl ether (200 mL), filter and concentrate the filtrate to give a light yellow oil. Dilute the oil with diethyl ether (150 mL) and filter the remaining dicylohexylurea.

Allow the filtrate to crystallize overnight under Argon.

Collect and dry (1 torr) the crystallized product to provide 20.86 g, 59.2 mmol of the title compound 8 as a white solid in 88% yield. MS (CI,CH 4 m/z relative intensity) 353 100%). Anal. Calcd for C 20

H

20

N

2 0 4 (352.30): C, 68.17; H, 5.72; N, 7.95. Found C, 68.02; H, 5.77; N, 7.93; 1H NMR (CDC13) 6 8.0 J 8 Hz, 2H, aroyl), 7.65-7.2 8H, aroyl, aryl), 5.1 (s (with shoulder), 3H, benzyl, NH), 4.4 1H, CH), 3.3 2H,

NCH

2 2.2-2.0 1H) 1.95-1.6 3H, CH 2

-CH

2 EXAMPLE Preparation of N-9Benzvloxvcarbonv l amino 6 benzamido 5 oxo-4,4-difluoro-l-nonene 11 (K Cbz) Scheme B, steps c, d and e: To a solution of azalactone 8 (33.6 g, 95.2 mmol) in tetrahydrofuran (80 mL) and heptane (70 mL), add triethylamine (16 mL, 115 mmol) at WO 97/35874 PCT/US97/02869 -34- 0°C under nitrogen. Slowly add a solution of 2,2-Difluoro- 4-pentenoyl chloride 8a (19.2 g, 124.2 mmol) in heptane mL) to the reaction mixture while maintaining the reaction temperature between 0-3 0 C. After 45 min, allow the reaction mixture to warm to room temperature and stir for another min. Remove a small aliquot, dry for 10 min (5 torr, room temperature) and analyze by HPLC, H NMR, 1F NMR to detect O-acylation of the azlactone. Remove the triethylamine*HCl by filtration and concentrate the filtrate to give a light orange-yellow oil. Analyze a sample of the oil by HPLC, 1H NMR, 1F NMR to confirm formation of the O-acyl intermediate 9. Dissolve the oil in tetrahydrofuran (60 mL), treat with 4-dimethylaminopyridine (1.74 g, 14.2 mmol) and stir for 3 h at room temperature. Remove a small aliquot, dry for min (5 torr, room temperature) and analyze by HPLC, 1H NMR, 1F NMR to confirm the transfer of the O-acyl intermediate 9 to the C-acyl intermediate 10. Add a solution of dried oxalic acid (25.76 g, 286.1 mmol) in tetrahydrofuran mL) to the reaction mixture and stir for 24 h. Remove a small aliquot, filter and dry (1 torr, room temperature) and analyze by 1H NMR and 9F NMR. Concentrate the reaction mixture to 1/4 volume treat with 10% aq HC1 (200 mL), extract with ethyl acetate (150 mL) and separate the layers. Wash the organic layer with sat aq NaHCO 3 (250 mL), brine (250 mL), dry (MgSO 4 and concentrate to provide an orange oil. Dilute the resulting oil with 4:1 hexane/ethyl acetate and allow to stand overnight to provide white solids. Collect and dry (1 torr, room temperature) the product to provide 21.7 g, 48.8 mmol of the title compound 11 as a light yellow solid in 51% yield. Concentrate the filtrate to give an oil. The crude oil is pre-absorbed on silica gel (2 g SiO 2 /1 g oil) and purified by flash chromatography using a 10 cm O.D. column containing 6 WO 97/35874 PUD/US97Y2869 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 MS (CI, CH 4 m/z relative intensity) 445 100%). Anal. Calcd for C 24

H

26

N

2 0 4

F

2 (444.49): C, 64.85; H, 5.90; N, 6.30. Found C, 64,74; H, 5.86; N, 6.20; 9 F NMR (CDC13) 6 -109.3 (dt, J 17, 270 Hz), -103.6 (dt, J 17, 270 Hz), IH NMR (CDC1 3 6 7.8 2H, aroyl), 7.6-7.4 3H, aroyl), 7.3 aryl), 7.1 1H, NH), 5.9-5.1 1H, CH=C), 5.35-5.15 2H, C=CH) 5.1 2H, benzyl), 4.95 1H, CH), 3.3 2H, NCH 2 2.9 2H, CF 2

CH

2 2.1, 1.7 (2m, 4H, CH:-

CH

2 EXAMPLE 6 Preparation of N-9-Benzyloxycarbonvlamino-6-benzamido-5hydroxy-4,4-difluro-l-nonene 12 (K Cbz) Scheme B, step f: To a solution of difluoroketone 11 (38.0 g, 85.5 mmol) in ethanol (500 mL) and tetrahydrofuran 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). Separate the organic phase, dry (MgSO 4 filter and concentrate to provide 38.4 g, 86 mmol of title compound 12 (4/1 diastereomeric mixture) as a gray-white solid in quantitative yield. MS (CI, CH 4 m/z relative intensity) 447 Anal. Calcd for C 2 4H,,N 2 0 4

F

2 (446.51): C, WO 97/35874 PCTIUS97/02869 -36- 64.56; H, 6.32; N, 6.27. Found C, 64.75; H, 6.25; N, 6.17.

19 F NMR (CDC13) 6 (major diastereomer) -106.9 (ddt), -110.5 (ddt), (minor diastereomer) -109.1 H NMR (CDC13) 6 7.8 2H, aroyl), 7.6-7.4 8H, aroyl, aryl), 6.9 1H, NH), 5.9-5.7 1H, CH=C), 5.3 2H, 5.1 2H, benzyl), 4.4 1H, CHN), 4.0, 3.9 (2m, 1H, CHCF 2 3.2 (m, 2H, NCH 2 2.9-2.6 2H, CHF 2

CH

2 1.8 2H) 1.6 (m, 2H, CH2).

EXAMPLE 7 Preparation of 9-Amino-6-benzamido-5-hydroxy-4,4diflurononane, hydrochloride Scheme B, step g (reduction and deprotection): Dissolve a solution of difluoro alcohol 12 (38 g, 85.1 mmol) in isopropanol (800 mL) and 1 N HC1 (200 mL). Treat the solution with 10% Pd(OH) 2 /C (3.81 g) and shake under hydrogen (45 psi) for 28 h. Filter the reaction mixture through celite and concentrate (1 torr, room temperature) to provide 29.9 g, 85.4 mmol, of title compound as an offwhite solid in quantitative yield. MS (CI, CH 4 m/z relative intensity) 315 100%). Anal. Calcd for

C

16

H

2 4

N

2 0 2

F

2 (350.84) C, 54.78; H, 7.18; N, 7.98. Found C, 54.47; H, 7.28; N, 7.83; 9F NMR (CD 3 OD) 5 (major diastereomer) -105.7 (ddt) and -107.9 (minor diastereomer) -107.7 and -110.0 H NMR (CD 3 OD) 8 7.9 2H, aroyl), 7.5 3H, aroyl), 4.4 1H, CHN), 3.9 (dt, J 17 Hz, 5 Hz, 1H, CHCF 2 3.0 2H, NCH 2 2.2-1.4 (2m, 8H, 4CH 2 0.95 J 7Hz, 3H).

WO 97/35874 PCT/US97/02869 -37- EXAMPLE 8 Preparation of 9,6-Diamino-5-hydroxv-4,4-difluorononane, bis-hydrochloride 1 Scheme B, step g (deprotection): Heat to reflux (110 0 C) a solution of 9-Amino-6-benzamido-5-hydroxy-4,4diflurononane, hydrochloride (26.85 g, 76.5 mmol) in conc aq HC1 (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 0 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 F NMR (CD 3 OD) 6 (major diastereomer) -106.4 (dt) and -110.6 (minor diastereomer) -107.6 and -112.6 1H NMR (D20) 6 4.0, 3.9 (2t, J 4 Hz, 1H, CHCF 2 3.5 (m, 1H, CHN), 2.9 2H, NCH 2 2.0-1.5 6H, 3 CH 2 1.3 (m, 2H, CH 2 0.8 J 7 Hz, 3H).

EXAMPLE 9 Preparation of [3-[bis[(1,1-dimethylethoxy)carbonvl]amino]methylenelamino]-6-amino-5-hydroxy-4,4difluorononane 2 (K 1 Boc) Scheme A, 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 WO 97/35874 PCT/US97/02869 -38mL) 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. Add aq citric acid (200 mL) to the reaction mixture and extract the mixture with methylene chloride (2 X 400 mL). Wash the combined organic layers with brine (500 mL) and water (200 mL), dry (Na 2

SO

4 filter and concentrate to provide an oil.

Purify by flash chromatography (silica gel; 7:1 CHC1 3 :MeOH) to give 45.5 g of title compound 2 as a white foam in 83% yield. 19F NMR (CDC13) 6 (major diastereomer) -105.7 (m) and -111.2 1H NMR (CDC13) 6 11.4 1H, NHBoc), 8.3 1H, NH), 3.6 J 5 Hz, 0.5H, CHCF,), 3.4

CHCF

2

NCH

2 2.9 1H, NCH), 2.0-1.0 26H, 2Boc, 4CH 2 0.9, 0.8 (2t, J 7Hz, 3H (4:1 ratio)).

EXAMPLE Preparation of N-[(1,1-dimethylethoxy)carbonvll-N-methyl-Dphenylalanvl-N-[l-[3- [bisf[(1,1-dimethylethoxy)carbonvllamino]methylene aminol propyll -33 -difluoro-2-hvdroxvhexyl L-prolinamide 3 (K Bo Boc K, Scheme A, step b: Add N,N'-dicyclohexylcarbodiimide (20.76 g, 100.6 mmol) to a solution of N-Boc-N-methyl-D- Phe-Pro-OH (37.89, 100.6 mmol) and hydroxybenzotriazole (13.59 g, 100.6 mmol) in methylene chloride (800 mL), added at 0°C. Stir the reaction mixture for 4 h. To the reaction mixture, add amino alcohol 2 (45.5 g, 100.6 mmol) in methylene chloride (200 mL) and N-methylmorpholine (11.1 mL, 100.6 mmol) and stir overnight. The precipitated dicylohexylurea is removed by filtration and the filtrate is concentrated to a yellow oil. Dissolve the oil in ethyl acetate (2 X 250 mL), wash with 10% aq citric acid (2 X 250 mL), brine (250 mL) and H 2 0 (150 mL) Separate the organic WO 97/35874 PCT[US97/02869 -39layer, dry (Na 2

SO

4 filter and concentrate to provide 88 g of crude title product 3 as a light yellow foam in excess of quantitative yield. Crude title product 3 may be carried to the next step without further purification.

9F NMR (CDC13) 8 (major diastereomer) -107.9 and -111.0 1H NMR (CD 3 OD) 6 7.3 5H, C 6 5.1 1H, CH-phe), 4.5-4.0 2H, CH-pro, CH-OH), 3.9-3.3, 3.2 (2m, benzyl, CH 2 -pro, CH 2 -N-gua, CH 3 N, CH-N), 2.2-1.5 12H, 6CH 2 1.5-1.2 (3br s, 27H, 3 Boc), 0.9 (dt, 3H, J 2, 7 Hz, CH 3 EXAMPLE 11 Preparation of N-[(l,l-dimethvlethoxy)carbonyll-N-methyl-Dphenvlalanvl-N-[l-[3-[[bisf[(1l,-dimethvlethoxv)carbonyl]amino]methylenelamino]propyll-3,3-difluoro-2-oxohexyl]-Lprolinamide 4 Boc, K, Boc) Scheme A, 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 0 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 and -60 0 C, then treat with diphenylethylamine (157 mL, 0.898 mol) at -55 0 C over 30 min. After stirring for another 1 h at -55 0 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 WO 97/35874 PCT/US97/02869 with brine (200 mL), dry (MgSO 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 X 5" SiO 2 column; 3:3:2 Hept/CH 2 C1 2 /EtOAc) to provide 42.9 g of title compound 4 in 53% yield. 19 F NMR (CDC13) 6 (major diastereomer) -104.5 and -109.7 IH NMR (CDC1 3 6 11.50 J 5Hz, 1H, NH-Boc), 9.40 J 6Hz, 1H, NH), 7.6, 7.2 (2d, J NH-CHCO (2 isomers)), 5.0, 4.5 (2m, 3H, 3.6-2.7 (m, 9H, 2 CHN, benzyl, CH 3 2.3-1.7 8H, 4 CH2), 1.7-1.1 31H, 3Boc, 2 CH2), 0.95 J 7Hz, CH 3 EXAMPLE 12 Preparation of N-methyl-D-phenvlalanvl-N- [(aminoiminomethvl)amino]propyll-3,3-difluoro-2-oxohexyl L-prolinamide dihydrochloride monohydrate 5 (MDL 75,579DA) Scheme A, step d: Dissolve ketone 4 (21.6 g, 0.267 mol) in 2N 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 HC1 into the reaction mixture for 10 min, stopper the flask and stir the mixture for 1 h.

Concentrate the reaction mixture to a yellow foam, dilute with H 2 0 (100 mL) and methylene chloride (75 mL) and stir vigorously for 20 min. Remove the methylene chloride layer and repeat the methylene chloride wash (75 mL). Filter the aqueous phase through celite and lyophilize to provide 15.2 g of title compound 5 (MDL 75,579DA) as a white solid in 98% yield by LC/MS using a Delta Pack Column (2.1 mm X 150 mm) at 80 0 C. 1F NMR (D 2 0) 6 (major diastereomer) -112.4 (minor diastereomer) -106.2 'H NMR (D 2 0) 8 7.4, 7.3 (2m, 5H, C 6

H

s 4.9 0.2H, CHCOCF 4.1 (dd, J 13 WO 97/35874 PCT/US97/02869 -41- Hz, 3 Hz, 0.8H, CHC(OH) 2

-CF

2 4.55 (dd, J 9Hz, 5Hz, 1H, CH-phe), 4.35 1H, CH-pro), 3.5, 2.6 (2m, 2H, CH 2 -N-pro), 3.25 2H, CH 2 2.70 3H, CHN) 2.2-1.4 12H, 6 CH) 0.95 (2t, 3H, J 7 Hz, CH) EXAMPLE 13 Purity Determinations of MDL 75,579DA Samples By LC/MS Using Electrospray Ionization 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.

A. HPLC Dissolve sample of MDL 75,579DA in initial mobile phase at a concentration of approximately of 1 mg/mL. To reduce the amount of hydrate formed by contact with water, prepare the sample just prior to analysis. Inject a 50 .tL aliquot of this solution using a WISP 715 autosampler (Waters Chromatography), to a Waters Delta Pak narrow bore C18 (2.1 mm x 15 cm) column heated to an apparent temperature of 80 0 C. for analysis.

Prepare mobile phase A as acetonitrile/water (5/95), containing about 0.05% trifluoroacetic acid (TFA), and mobile phase B consisting of acetonitrile/water (95/5), containing about 0.05% TFA. Establish gradient elution at a flow rate of 0.2 mL/min using a Waters 625 HPLC system.

Run a linear gradient from 12 to 57% B for over 45 minutes.

Monitor the UV absorption at 214 nm on-line using a Waters 486 tunable wavelength detector (0.5 AUFS) equipped with a low dead volume micro-flow cell.

WO 97/35874 PCTIUS97/02869 -42- B. Electrospray Mass Spectrometrv Introduce the entire effluent from the HPLC column (0.2 mL/min) into the ESI probe of the Finnigan MAT TSQ 700 triple quadropole mass spectrometer. Roughly situate the ESI syringe needle, holding at 5.5 kV, 2 cm from the stainless steel capillary orifice of the mass spectrometer.

This device, which has an internal diameter of 0.5 mm, separates the ESI spray at atmosphere from the high vacuum conditions of the mass spectrometer. To assist the desolvation process hold the capillary at 200 0 C during the analysis. Accomplish further desolvation by the use of a nitrogen sheath gas (70 psi) introduced coaxial to the ESI needle. In addition, use an auxiliary gas (N 2 flow of mL/min. Acquire mass spectra using Q3 as the scanning quadrupole covering a m/z range of 200 to 1500 in 2 seconds. The voltages to be applied to the conversion dynode and electron multiplier are -15 kV and 1.4 kV, respectively. Accomplish tuning and calibration with a mixture of synthetic peptides using loop injections at a flow rate of 0.2 mL/min.

C. Estimation of Purity 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.).

WO 97/35874 PCTIUS97/02869 -43- 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.

TABLE 2 Purity of Three Batches of MDL 75,579DA Prepared Using the Process of the Invention MDL 75,579DA Batch %Purity (UV 214 nm) C415F-193 96.9 C415F-196 98.3 C415F-198 97.9 mean 97.7 0.7 By using the process of the invention involving a onestep guanylation of 9,6-diamino-5-hydroxy-4,4-difluorononane, bis-hydrochloride, the Dakin-West route known in the art Pat. No. 5,391,705) is shortened by two chemical steps and one chromatography. More dramatically, the overall yield starting from the hydroxy-4,4-difluorononane intermediate is increased from about 15% using the art-known Dakin-West procedure to about 43% using the process of the present invention, while achieving, a highly purified final product of about 98% purity.

Claims (3)

1. A process for preparing N-methyl-D-phenylalanyl- N- [(aminoiminomethyl)aminolpropyll]-3,3-difluoro-2- oxohexyl] -L-prolinamide or a pharmaceutically acceptable salt thereof comprising the steps of: reacting 9,6-diamino-5-hydroxy-4,4-difluorononale, or a pharmaceutically acceptable salt thereof, with an appropriate guanylating agent to provide N\-9-[l-[3-[bis(Kl- protected) amino] methylene] amino] -6-amino-5-hydroxy-4,4- difluorononane, wherein K, is an N-protecting group suitable for protecting the nitrogens of guanyl moieties; coupling the N-9- Ibis (Kl-protected) amino] methylene] amino] -6-amino-5-hydroxy-4, 4-difluorononane with N-K 2 -protected-N-methyl-D-phenylalanyl-L-prolinamide to provide N- K 2 -protected- N-methyl -D -phenyl alanl-N- [3- [I[bis (Kj -protected) amino] methylenel amino] propyl]I 3,3 difluoro-2-hydroxyhexyl]-L-prolinamide, wherein K 2 is an N- protecting group; reacting N-K 2 -protected-N-methyl-D-phenylalanyl-N- Ii- [3 Ibis (K,-protected) amino] methylene] amino] propyl] -3 3 difluoro-2-hydroxyhexyl] -L-prolinamide with an appropriate oxidizing agent to provide N- K 2 -protected- N-methyl -D phenylalanyl-N- [I[bis (Kl-protected) amino] methylene] amino] propyl] -3,3-difluoro-2-oxohexyl] -L-prolinamide; and deprotecting N- K 2 -protected- N-methyl -D -phenyl al anyl-

13- [I[bis [I (i{-protected) amino] methylene] amino] propyl] -3,3- difluoro-2-oxohexyl] -L-prolinamide with a suitable deprotecting agent to provide N-methyl-D-phenylalanYl-N [2- [3-[(aminoiminomethyl)amino]propyl]-3,3-difluoro-2-oxohexyl]-L-prolinamide or a pharmaceutically acceptable salt thereof. 2. A process according to claim 1 wherein the guanylating agent is added in a ratio of about 0.9 to about 1.2 molar equivalents per 1.0 molar equivalent of 9,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof. 3. A process according to claim 1 wherein the guanylating agent is added in a ratio of about 0.95 to about 1.05 molar equivalents per 1.0 molar equivalent of 9,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof. 4. A process according to any one of the preceding claims wherein K, is tert-butyloxycarbonyl. A process according to any one of the preceding claims wherein K 2 is tert-butyloxycarbonyl. 20 6. A process according to any one of the preceding claims wherein the reaction of 9,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof, with an appropriate guanylating agent is carried out at a temperature of from about -30°C to about 200C. 9* 25 7. A process according to any one of the preceding claims wherein the reaction of 9,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically Sacceptable salt thereof, with an appropriate guanylating agent is carried out at a temperature of from about -15oC to about 8. A process according to claim 2 wherein the appropriate guanylating agent is bis-Boc-amidinopyrazole or bis-Boc-S-methylisothiourea. C:\WINWORD\JENNYMISPECNKI\20550-97.DOC -46- 9. A process according to claim 3 wherein the appropriate guanylating agent is bis-Boc-amidinopyrazole or bis-Boc-S-methylisothiourea. A process according to claim 8 wherein the reaction of 9,6- diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof, with an appropriate guanylating agent is carried out at a temperature of from about -15°C to about 11. A process according to any one of the preceding claims wherein the appropriate oxidizing agent is a dimethyl sulfide complex under Swern conditions. 12. A process for preparing 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(Kl-protected)amino]methylene]-amino]-6-amino-5- hydroxy-4,4-difluorononane, wherein K 1 is an N-protecting group suitable for protecting the nitrogens of guanyl moieties. 13. A process according to claim 12 wherein the guanylating agent is added in a ratio of about 0.9 to about 1.2 molar equivalents per 1.0 molar equivalent of 9 ,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof. a

214. A process according to claim 12 wherein the guanylating agent is added in a ratio of about 0.95 to about 1.05 molar equivalents per 1.0 molar 0. equivalent of 9,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof. A process according to any one claims 12 to 14 wherein the reaction of 9,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically C:\WINWORDJENNYM\SPECNKI\20550-97.DOC -47- acceptable salt thereof, with an appropriate guanylating agent is carried out at a temperature of from about -300C to about 200C. 16. A process according to any one of claims 12 to 15 wherein the reaction of 9,6-diamino-5-hydroxy-4,4-difluorononane, or a pharmaceutically acceptable salt thereof, with an appropriate guanylating agent is carried out at a temperature of from about -15°C to about 17. A process according to claim 13 wherein the appropriate guanylating agent is bis-Boc-amidinopyrazole or bis-Boc-S-methylisothiourea. 18. A process according to claim 14 wherein the appropriate guanylating agent is bis-Boc-amidinopyrazole or bis-Boc-S-methylisothiourea. 19. A process according to claim 17 wherein the reaction of 9,6- diamino-5-hydroxy-4,4-difluorononane, bis-hydrochloride with an appropriate guanylating agent is carried out at a temperature of from about -15°C to about S 20 20. A process according to any one of claims 12 to 19 wherein K 1 is tert-butyloxycarbonyl. 21. N-methyl-D-phenylalanyl-N-[1-[3-[(aminoiminomethyl)amino]- •propyl]-3,3-difluoro-2-oxohexyl]-L-prolinamide or a pharmaceutically acceptable 25 salt thereof when made by a process according to any one of claims 1 to 11. 22. N-9-[1-[3-[bis(Kl-protected)amino]methylene]amino]-6-amino-5- hydroxy-4,4-difluorononane when produced by a process according to any one of claims 12 to C:\WINWORD\JENNYM\SPECNKI\20550-97.DOC -48- 23. A process according to claim 1 or claim 12 substantially as herein before described with reference to any of the examples. DATED -:25 May, 1999 PHILLIPS ORMONDE FITZPATRICK Attorneys For: HOECHST MARION ROUSSEL, INC. C:\WrNWORDUENNYM\SPECNKI\20550-97.DOC