KR100244418B1 - Thrombin inhibitors - Google Patents

Abstract

The present invention provides novel polyfluorinated alkyl derivatives of certain tripeptides, methods for their preparation, intermediates for their preparation, the use of inhibition of thrombin and pulmonary tryptase, and their use as anticoagulants for the treatment of thrombophlebitis and coronary thrombosis and the treatment of asthma. It is about the end use.

Description

Thrombin inhibitor

The present invention relates to novel polyfluorinated alkyl derivatives of certain tripeptides, methods for their preparation, their preparation intermediates, their use in the inhibition of thrombin and phytase, and as anticoagulants useful for the treatment of thrombophlebitis and coronary thrombosis and the treatment of asthma. It is about end use.

More particularly, the present invention relates to compounds of the general formulas (IA) and (IB), isomers thereof and mixtures thereof, hydrates thereof and pharmaceutically acceptable salts thereof.

Wherein m is 0,1 or 2, n is 0 or 1, the sum of m and n is less than 3 and greater than 0, q is 0 or 1, the sum of both q is 0 or 2, and R ₁ is H or C1-7 alkyl, R ₂ is H or C _1-7 , or R ₁ and R ₂ together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle, and R ₃ is -CF ₃ , -CF ₂ CF ₃ , -CF ₂ (CH ₂ ) _t CH ₃ , -CF ₂ (CH ₂ ) _t COOR ₄ , -CF ₂ CH ₂ ) _t CONHR ₄ , -CF ₂ (CH ₂ ) _t CH ₂ OR ₄ or -CF ₂ (CH ₂ ) vCH = CH ₂ , where t is 2,3 or 4, v is 1,2 or 3, and R ₄ is H or C _1-6 alkyl , A is phenyl or cyclohexyl, B is (CH) ₄ or (CH ₂ ) 4 which together with the two carbon atoms to which it is attached form a C ₆ -cyclic hydrocarbon moiety,

Provided that when R ₁ and R ₂ are both H, R ₃ is not —CF ₃ or —CF ₂ CF ₃ .

Natural amino acids, except glycine, contain chiral carbon atoms. Unless specifically indicated otherwise, preferred compounds are any active amino acid in the L-configuration, with the exception that Phe is in its D-configuration.

The tripeptides of formula (I) may form pharmaceutically acceptable salts with any non-toxic, organic or inorganic acid. Exemplary inorganic acids that form suitable salts include acid metal salts such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and sodium monohydrogen orthophosphotasium hydrogen sulfate. Exemplary organic acids that form suitable salts include mono, di, and tricarboxylic acids. Examples of such acids include acetic acid, trifluoroacetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid and benzoic acid. , Hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, 2-phenoxybenzoic acid, and sulfonic acids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid.

Alkyl groups are linear, branched or cyclic alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, s-pentyl, cyclopentyl, hexyl, ishehexyl, cyclo Hexyl and cyclopentylmethyl. When R ₁ and R ₂ are lower alkyl, one or both are preferably methyl. When R ₁ and R ₂ together with the nitrogen atom to which they are attached form preferred 5- to 6-membered heterocycles, preferred moieties are pyrrolidine and piperidine. Preferred compounds are those wherein at least one of R ₁ and R ₂ is methyl.

In the case of compounds of formulas (IA) and (IB), the P _1- α-amino acid residue (arginine) can be its D- or L-configuration or a mixture thereof, and a P ₂ -α-amino acid residue (proline ) Is preferably its L-configuration, and the α-amino acid residue or substituent on the α-carbon atom of the P ₃ residue (ie .. (CH ₂ ) n ^A residue) or substituent is preferably its D-configuration Preferred residues are Phe and preferred substituents are cyclohexylmethyl residues. In the case of compounds of formula (IB), the P ₃ residue is a so-called "TIC" derivative or "TIC-like" derivative (the expression "TIC" is derived from tetrahydroisoquinoline carboni). In this case, the bicyclic TIC-residue formed of P ₃ nitrogen atom and P ₃ -α-carbon atom has the following general formula.

Wherein (2a) and (2b) represent 1, 2, 3 and 4-tetrahydroisoquinolinyl residues, respectively, and (2'a) and (2'b) represent 1, 2, 3 and 4, respectively. -Decahydroisoquinolinyl residues, and (2C) and (2'C) represent 2, 3-dihydro-1H-isoindolyl and octahydro-1H-isoindolyl residues, respectively. For convenience, these residues can then be classed as TIC-like variants.

Compounds of the present invention can be prepared analogously to methods known in the art. In essence, the synthesis of fluorinated alkyl tripeptides of general formula (I) is carried out on 2-phenyl-5 (4H) oxazolone and trifluoroacetic acid, pentafluoropropionic acid or difluoropentenoic acid (targeted R ₃ residues). According to the modified Dakin-West reaction of anhydrides or acyl halides to obtain the essential polyfluoroalkyl ketone amino acid derivatives used as key intermediates. The reaction is then further advanced to convert these amino acid homologues to the desired peptides of general formula (I). This reaction is illustrated in the following scheme.

Scheme A

Wherein φ is phenyl, A 'is ... (CH ₂ ) n ^A , n is 0 or 1, R is φC (0) NH (CH ₂ ) ₃ , and R' ₁ is an N-protecting group R ' ₂ is H, or C _1-7 alkyl, and R' ₃ is -CF ₃ , -CF ₂ CF ₃ , -CF ₂ (CH ₂ ) _t C (0) NHR ₄ , -CF ₂ (CH ₂ ) _t CH ₂ OR ₄ , or -CF ₂ CH ₂ CH = CH ₂ , and R '' ₃ is -CF ₃ , -CF ₂ CF ₃ , -CF ₂ (CH ₂ ) _t C (O) NHR ₄ ,- CF ₂ (CH ₂ ) CH _t OR ₄ , -CF ₂ (CH ₂ ) vCH = CH ₂ , or CF ₂ (CH ₂ ) COOR ₄ , T is a TIC-like residue as described above, 2a, b and c, and 2'a, b and c, DCC is dicyclohexylcarbodiimide, NMM is N-methylmorpholine, HOB _t is hydroxybenzotriazole (hydrate), and TFA is trifluoro Roacetic acid, TFAA is trifluoroacetic anhydride, and A, R4, t, v, Ia and Ib are as defined above.

Preferred amine protecting groups are Boc or CBz and CBz may also be used, although the preferred protecting group on the arginine residue is represented by the Boc group.

In carrying out the above reaction, N-bis-benzoylated amino acid (3) is cyclized to 5 (4H) -oxazolone (4) in good yield using standard techniques (Ac ₂ O, 30 minutes, 90 。C, oil bath). The solvent is evaporated to give a highly pure compound that can be used without further purification for the next step. The reaction of oxazolone (4) with a suitable polyfluoric acid acyl halide (react in the presence of NEt ₃ ) or anhydride using a modified Dakin-West method is carried out at 40 ° C. (oil bath temperature) for 24 hours under N ₂ atmosphere. ( ¹ H- and ¹⁹ F-NMR-tracking). In the case where all starting material 4 is consumed, C-4-acylated oxazolone 5 is the main product. Residual anhydride and polyfluoric acid formed are removed under vacuum (0.01 torr: 25-70 ° C. oil bath, using acetone / dry ice trap). Alternatively, compound (3) can be converted directly (6) by treatment with R ' ₃ anhydride or R' ₃ acyl halide, in which case the intermediate is not isolated. The oily residue is then mixed with a saturated aqueous solution of freshly prepared oxalic anhydride in tetrahydrofuran. The commercial product oxalic acid is dried for 16 h at 100 ° C. in a drying oven. Two successive sublimations (0.01 Torr, 90 ° C.) yield oxalic anhydride (melting point 104 ° C.), transfer into the flask under N ₂ atmosphere and cover with a septum. Anhydrous tetrahydrofuran is added to the solid phase until the majority (about 4 ml / g) is dissolved and the resulting solution is stirred at room temperature for about 16 hours when gas evolution ceases completely. Conventional finishing operations (ie, washing with EtOAc / H ₂ O, aqueous NaHCO ₃ and brine; drying over MgSO ₄ ) yield a satisfactory yield of the desired fluorinated compound (7) as a mixture in the form of ketones and their hydrates. Get into.

For the desired conversion of α-benzoamide (7), the polyfluoroalkyl ketone functional groups must be temporarily blocked. This can be done by reducing ketone 7 with alcohol 8 (NaBH ₄ : EtOH). The two benzamido functional groups are cleaved by acid hydrolysis to obtain diaminopolyfluoroalkyl alcohol (9). Regioselective protection of the lateral amino groups of the diamino alcohol 9 as trifluoroacetamide 10 is carried out using trifluoroacetic anhydride in trifluoroacetic acid. Guanylation of the ω-amino group with the fully protected arginine homologue (11) is performed using bis-Boc-S-methylisothiourea in triethylamine. The liberation of the flanking amino protective functional groups is carried out using lithium hydroxide in tetrahydrofuran / water to give the compound of formula (12), which is carried out using standard methods known in the art [Nicolaides, E.). , Dewald, H., Westland, R., Lipnik, M. and Posler, J., J. Med. Chem. Vol. 11, 74 (1968)], coupled with a suitable reactant (ie R ' ₁ , R' ₂ N-CH (A ') C (O) ProOH and TicC (O) respectively Obtain fully protected tripeptoxy alcohol homologues of arginine (13a) and (13b), which are oxidized to the corresponding ketones of the alcohol function Compound 12a is residue CF ₂ (CH ₂ ) _t at R ′ ₃ residue Different from the range of compound (2) in that COOR ₄ is added. Thus R ″ ₃ comprises R ′ ₃ and CF ₂ (CH ₂ ) _t COOR ₄ residues and CF ₂ (CH ₂ ) _t COOR ₄ residues Is prepared according to Reactivity B.

Although there are several methods available for the required oxidation, the most preferred method of converting (13a) and (13b) to their corresponding ketones (14a) and (14b) is the Swern oxidation method. . In general, Swarn oxidation (Synthesis, 1981), page 165, contains about 2-10 equivalents of dimethylsulfoxide (DMSO) to about 1-5 equivalents of trifluoroacetic anhydride [(CF ₃ CO) ₂ O] or oxalyl chloride [(COCl) ₂ ]. The reaction is dissolved in an inert solvent, ie methylene chloride (CH ₂ Cl ₂ ), and the reaction is carried out in an inert atmosphere (ie, nitrogen or equivalent gas) under anhydrous conditions at a temperature of about −70 to −30 ° C. To form a sulfonium adduct in the same reactor, to which an appropriate alcohol, i.e., about 1 equivalent of compound (13a) or (13b) is added. Preferably, the alcohol is dissolved in an inert solvent, ie CH ₂ Cl ₂ or a minimum amount of DMSO, and the reaction mixture is warmed to about −20 ° C. (about 10-20 minutes), followed by about 3 to 10 equivalents of tertiary The reaction is completed by adding amines, ie triethylamine, N-methyl morpholine, and the like.

Another method for converting alcohol to the desired ketone is the oxidation using periodinane (ie 1, 1, 1-triacetoxy-2, 1-benzoxiodol) [Dess Martin , J. Org. Chem. 48, 4155 (1983). This oxidation is carried out by contacting about 1 equivalent of alcohol with 1-5 equivalents of periodinan (preferably 1.5 equivalents). The reaction is a suspension in an inert solvent (ie methylene chloride) under an inert atmosphere (preferably nitrogen) under anhydrous conditions of 0-50 ° C. (preferably room temperature) and allowed to react for about 1 to 48 hours. Deprotection of the amine protecting group can be carried out as desired after the ketone is isolated.

Alternatively, the modified Jones oxidation method is carried out by reacting an alcohol with pyridinium dichromate by contacting the reactants together in a powder of molecular sieves (ie crushed 3 A. molecular sieves) that are trapped in water, the contact being approximately 0-50 ° C., preferably at room temperature, in the presence of glacial acetic acid, followed by removal of the amine protecting group after isolation.

Alternatively, 1 to 5 equivalents of chromic anhydride-pyridine complex (ie, Cistert reagent prepared in the same reactor) [Fieser and Fieser, "Reagents for Organic Synthesis", Vol. 1, Vol. 145 Page and Sarrett et al., JACS Vol. 25, page 422 (1953)] in the same reactor in an inert solvent (ie CH ₂ Cl ₂ ) in an inert atmosphere under anhydrous conditions at 0-50 ° C. Prepare and add 1 equivalent of alcohol to this complex and react the reactants for about 1-15 hours and then isolate to remove the amine protecting group.

In the special case where R ₃ is -CF ₂ (CH ₂ ) _t COOR ₄ , the required intermediate [corresponds to the compound of formula (12), provided that the desired R ₃ is -CF ₂ (CH ₂ ) _t COOR ₄ ] The reaction of the following reactivity can be used to prepare the reaction.

Scheme B

Wherein Boc, R ₄ and t are as defined above.

Reactant (17) was prepared using bromo-difluoroethyl acetate (RH Abeles and Ch. P. Govardham, Archives of Biochemistry and Biophysics, Vol. 280, No. 137 (1990)). By reacting with a suitable aldehyde under Reformatsky conditions, it can be prepared with the corresponding difluoro diester alcohol. This alcohol is then mesylated and the mesylated product is removed by reaction with diazabicycloundecane (DBU) and the resulting olefin is reduced by hydrogenation. The mesylation, removal and hydrogenation reactions are described in Hing L. Sham et al., Biochem and Biophys. Res. Comm., Vol. 175, 914 (1991), by standard and known methods and techniques. The intermediate 19 contained in the compound of the structural formula (12a) thus produced is then treated as described in Scheme A.

When preparing a compound wherein R ₃ is -CF ₂ (CH ₂ ) ₂ CH ₃ , the acid anhydride or acyl halide reactant used to prepare the compound of formula (5) is -CF ₂ CH ₂ CH = CH ₂ , and when converting compounds (14a) and (14b), the double bond of the olefin (ie -CF ₂ CH ₂ CH = CH ₂ ) residue may also be reduced. In certain cases where R ₃ is CF ₂ (CF ₂ ) _t CH ₃ , where t is 3 or 4, the corresponding olefin (ie CF ₂ CH ₂ CH═CH—CH ₃ or CF ₂ CH ₂₎ CH = CHCH ₂ CH ₃ ) will be reduced. To prepare these latter olefins, starting acyl halides or anhydrides can be prepared by the methods described in R. W. Lang et al., Tetrahedron Letters, Vol. 29, page 3291 (1988). have.

Furthermore, the method of treatment of the oxidized intermediates [(14a) and (14b)] with the desired compounds of the general formulas (IA) and (IB) depends essentially on the definition of R ₁ , R ₂ and R ″ ₃ residues. In preparing the final compounds wherein R ₁ is H and R ₂ is alkyl, the intermediate used is a compound in which R ′ ₁ is a protecting group and R ′ ₂ is C _1-7 alkyl. The R ′ ₁ protecting group and two protecting groups will be removed by techniques known in the art, such as by reaction with HCl gas / ether or FTA / CH ₂ Cl ₂ , where R ₁ and R ₂ are H, Then R ' ₁ will be a protecting group, R' ₂ will be H, and all three protecting groups (preferably Boc) will be removed as above, if both R ₁ and R ₂ are of a different origin than H in, R is _"1 protecting group (preferably CBz) and, R _'2 is H, such intermediates (14a) are the Suitable aldehyde in the presence of _{H 2 / Pd (OH) 2} / carbon in propanol (i.e., formaldehyde, glutaraldehyde or succinic aldehyde) and N, N- di-alkylation reaction to avoid the purpose pyrrolidine or piperidine derivatives The remaining two Boc-protecting groups on the arginine residue will be cleaved by treatment with HCl / Et ₂ 0 or TFA in CH ₂ Cl ₂ according to standard methods known in the art. ₂ CH ₂ in case it is desired that the CH = CH ₂ moiety) converted to its saturated analogs _{(i.e., -CF 2 (CH 2) 2} CH 3 moiety), the conversion is preferably, using standard hydrogenation techniques Carried out simultaneously with the removal of the amino protecting group.

In the following examples, a method for preparing a compound of formula (I) is shown.

Example I

[L-Prolineamide, N-methyl-D-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3, 3, 3-trifluoro-2-oxopropyl] , Dihydrochloride, hydrate]

[Step A]

[N, N'-bis-benzoylornithine, ornituric acid]

A 250 ml flask equipped with a magnetic stir bar, two dropping funnels and a pH-electrode was charged with 3.37 g (20 mmol) of L-ornithine hydrochloride and 40 ml of 1N NaOH. The stirred solution was cooled to 0 ° C. and a solution of benzoyl chloride (5.0 ml, 44 mmol) and 1N NaOH (44 ml) in diethyl ether (50 ml) was added simultaneously at a rate that kept the pH of the mixture at 12-13. . After the addition was complete the solution was warmed to room temperature and stirred for a further 2 hours. Diethyl ether (200 ml) was added, the phases were separated and the aqueous layer was acidified. (pH 0-1, concentrated HCl). The white precipitate formed was collected, washed with diethyl ether and dried (0.01 Torr) to give 6.7 g (98%) of the title compound.

Melting Point: 184。C

¹ H-NMR (CDCl ₃ , CD ₃ OD 1: 1): δ = 7.8 (m, 4H, benzoyl), 7.5 (m, 6H, benzoyl), 4.6 (m, 1H, CHN), 3.4 (m, 2H , NCH ₂ ), 2. 0-1.5 (m, 4H, CH ₂ CH ₂ ).

[Step B]

[N, N '-[1- (trifluoroacetyl) -1, 4-butanediyl] bis (benzamide), hydrate]

The mixture of ornituric acid (501.5 g, 1.47 mol) and trifluoroacetic acid (TFAA) (400 ml) described in step A was stirred at room temperature for 1 hour. The resulting solution was evaporated (0.1 Torr) and 1 L of TFAA was added. The solution was stirred for 16 h and evaporated to dryness (0.01 Torr). Then 1 L of TFAA was added and the solution was stirred for 4 hours. The solution was redevaporated to dryness (0.1 Torr) followed by further addition of 500 ml of TFAA. The solution was stirred for another 16 h and evaporated to dryness (0.1 Torr). The oily residue was dissolved in 1.6 L of mousse THF and freshly dehydrated oxalic acid (440 g) was added to the solution over 2 hours. 7.5 L of ethyl acetate was added with continuous stirring for 16 hours. The solution was washed once with brine, four times with aqueous sodium bicarbonate solution and once with brine, dried over MgSO 4, and dry drying. 522.8 g (90.5%) of the title compound were obtained.

Rf = 0.33 and 0.27 (eluent: ethyl acetate / petroleum ether 1: 1), ketones and their hydrates

Melting Point: 113-115。C

¹ H-NMR (CDCl ₃ ): δ = 8.0-7.5 (m, 4H, benzoyl), 7.5-7.0 (benzoyl, NH), 6.3 (m, 1H, NH), 5.0 [m, 0.5H, CH (CO ) CF ₃ ], 4.5 [m, 0.5H, CH (OH) ₂ CF ₃ ], 3.4 (m, 2H, NCH ₂ ), 2.2-1.6 (m, 4H, CH ₂ CH ₂ ).

¹⁹ F-NMR (CDCl ₃ ): δ = 86.00 (s, COCF ₃ ), 80.00 [s, CF ₃ C (OH) ₂ ]; Ratio 1: 1, ketones and hydrates

MS (m / s): 393 (MH ⁺ / 30% relative strength).

Elemental analysis for _{C 20 H 19 F 3 N 2} O 3 · H 2 O (410.40)

C H N

Theoretic: 58.53 5.16 6.83

Found: 58.62 5.22 6.64

[Step C]

[N, N '[1- (2, 2, 2-trifluoro-1-hydroxyethyl) -1,4-butanediyl] bis (benzamide)]

NaBH ₄ (45 g, 1.2 mol) was added in portions to a cooled (0 ° C.) stirred solution of trifluoromethylketone (522 g, 1.33 mol) described in Step B in 6.5 L of EtOH. The solution was stirred at 0 ° C. for 10 minutes and then at room temperature for 16 hours. HCl (6N) aqueous solution was added and when the foam stopped, solid sodium carbonate was added until the pH of the solution became basic. AcOEt was added and the phases were separated. The aqueous layer was extracted twice with AcOEt and the mixed organic phases were washed with brine. The organic solution was dried over MgSO ₄ , flash evaporated (20 Torr, 30 ° C.) and left to give an oil that solidified.

Yield 457.6 g (87%).

Melting point: 103 ° C., Rf = 0.7 (AcOEt).

¹ H-NMR (CDC1₃ / CD₃OD 1: 1): δ = 7.9 (m, 4H, benzoyl), 7.4 (m, 6H, benzoyl), 4.4 (m, 1H, CHN), 4.1 (m, 1H, CHCF₃) , 3.3 (m, 2H, NCH₂, 2.0-1.5 (m, 4H, CH₂CH₂).

¹⁹ F-NMR (CDCl ₃ / CD ₃ OD 1: 1) δ = 87.33 and 85.83 (2d, J _HF = 7.5 Hz); Proportion 3: 1.

A small amount of sample (100 mg) was crystallized for CHN analysis.

Elemental Analysis for C ₂₀ H ₂₁ N ₂ O ₃ (394.401)

C H N

Theoretic: 60.76 5.52 7.05

Found: 60.55 5.55 7.01

[Step D]

[3, 6-Diamino-1, 1, 1-trifluoro-2-hexanol, dihydrochloride]

A stirred solution of N, N'-bis-protected diamino alcohol (457 g) described in step C in aqueous HCl 12N solution (5 L) was heated to reflux for 16 h. The solvent was evaporated and the oily residue was dissolved in H ₂ O. The aqueous solution was washed with diethyl ether and evaporated to dryness to give 260 g (87% yield) of trifluoro-diaminoalcohol, hydrochloride as a light colored oil.

¹ H-NMR (D ₂ O): δ = 4.6 (m, 1H, CHCF ₃ ), 3.8 (m, 1H, CHNH), 3.2 (m, 2H, NCH₂), 2.0 (m, 2H, CH ₂ CH₂) .

¹⁹ F-NMR (D ₂ O, C ₆ F ₆ (extract control)): δ = 87.3 and 84.8 (2d, J _HF = 7.5 Hz): ratio 3: 1

[Step E]

[N- [6-[(1,1-dimethylethoxy) carbonyl] amino] -3-amino-2-hydroxy-1, 1, 1-trifluorohexane]

Di-t-butyldicarbonate (217 g, 0.99 mol) was added to the trifluoromethyl-diamino alcohol, dihydrochloride (260 g, 1.0 mol) and NEt₃ described in step D in THF / H ₂ O (3L / 3L). (600 ml, 4.3 mol) was added in portions to a chilled (-5 ° C.) stirring solution. Stirring was continued after complete addition at 0 ° C. for 1 hour and at room temperature for 16 hours. The solution was evaporated to 1/2 of its original volume, acidified (using solid citric acid), washed with diethyl ether, basified (NaOH pellet, pH 12-13) and extracted almost with diethyl ether. The mixed etheric extracts were washed with brine, dried over MgSO ₄ and evaporated (20 Torr, 30 ° C.) to afford ω-protected diamino alcohol.

¹ H-NMR (CDCl ₃ ): δ = 4.8 (m, 1H, CHN), 4.0-3.5 (m, 1H, CHCF₃), 3.2 (m, 2H, NCH₂), 2.5 (m, 3H, D ₂ Exchange with O, OH, NH ₂ ), 1.8-1.3 (m, 4H, 2CH ₂ ), 1.45 (s, 9H, 3CH₃).

¹⁹ F-NMR (CDCl 3): δ = 88.33 and 84.33 (2d, J _HF = 7.5 Hz); Ratio 3: 1.

Aliquots (100 mg) were treated with diethyl ether / methanol (10: 1), the solid phase was collected and dried to give 60 mg of said compound which was analytically pure.

Elemental Analysis for C ₁₁ H ₂₁ F ₃ N ₂ O ₃ (286.301)

C H N

Theoretic: 46.15 7.39 9.78

Found: 46.25 7.62 9.46

[Step F]

[L-Prolineamide, N-methyl-N-[(phenylmethoxy) carbonyl] -D-phenylalanyl-N- [1- [3-[[(1, 1-dimethylethoxy) carbonyl] Amino] propyl] -3,3,3-trifluoro-2-hydroxypropyl

Isobutyl chloroformate (2.46 m1, 18.94 mol) was added to N-methyl-N-phenyl-methyloxycarbonyl-D-phenylalanyl-L-proline (7.06) in dry THF (50 ml) over 10 minutes under N 2 atmosphere. g, 17.22 mol) [S. Bajusz et al., J. Med. Chem. 1990, Vol. 33, p. 1729] and N-methylmorpholine (2.08 ml, 18.94 mol) were added to cooling (-20 ° C.) and stirred solutions. The reaction mixture was stirred at 20 ° C. for 1 hour and the ω-N-protected diaminoalcohol (4.92 g, 17.22 mol) solution described in Step E in THF (anhydrous, 50 ml) was added. The mixture was stirred at 0 ° C. for 1 hour and then held at 4 ° C. for 16 hours. The solvent was evaporated, the residual oil was dissolved in AcOEt / H ₂ O and the layers separated. The organic layer was washed with saturated NaHCO ₃ solution, citric acid and brine, dried over MgSO ₄ and evaporated. The resulting crude tripeptide derivative (15 g, yellow foam) was subjected to flash chromatography on silica gel (eluent: AcOEt / hexane 1: 1) and the mixed product containing fractions were evaporated (20 Torr, 30 ° C: as a cosolvent). CCl ₄ was used twice, then at room temperature 0.01 Torr, 16 hours) to give 11.68 g (62%) of the title compound as a colorless foam. Rf = 0. 24 (AcOEt / hexane 2: 1)

¹H-NMR (90 MHz, CDCl₃): δ = 7.4-7.2 (2m, 10H, 2 aryl), 7.1 (m, 1H, NH), 6.9 (m, 1H, NH), 5.8-5.5 (m, 2H, 2NH), 5.1 (m, 2H, arylCHO₂), 4.8-3.5 [6m, 5H, 4CH, OH (exchanged using D ₂ O)], 3.4-2.6 (m, NCH₃, 2NCH₂, aryl CH₂), 2.2 And 1.9-1.5 (m, 8H, 4CH 2), 1.4 (m, 9H, 3CH 3).

¹⁹ F-NMR (CDCl₃): δ = 87.1-86.8 (m).

[Step G]

[L-Prolineamide, N-[(phenylmethoxy) carbonyl] -N-methyl-D-phenylalanyl-1- [3- (aminopropyl) -3, 3, 3-trifluoro-2- Hydroxypropyl], trifluoroacetate]

A 25 ml solution of TFA in 25 ml of CH ₂ Cl ₂ was added to the protected tripeptides derivative described in step F (7.0 g, 10.32 mol) and the mixture was stirred until foaming stopped (30 minutes, using a bubble counter). The solution was evaporated to dryness (0.01 Torr, 40 hours) to give a yellow foam. The title compound was precipitated from i-propanol / petroleum ether to give 7.05 g (yield: quantitative analysis) of white solid after drying.

Rf = 0.5 (AcOEt / 10% AcOH)

¹ H-NMR (90 MHz, CD ₃ OD): δ = 7.3 (m, 10H, 2 phenyl), 5.1 (m, 2H, CH ₂ O), 4.6-4.45 (m, 1H, CHN-Phe), 4.2 (m, 1H, CHN-Pro), 4.1 (m, 1H, CHN), 3.9 (m, 1H, CHCF ₃ ), 3.1-2.6 (m, 9H, NCH ₃ , 3NCH ₂ ), 2.0-1.3 (m, 8H, 4CH ₂ ).

¹⁹ F-NMR (90 MHz, CD ₃ OD): δ = 88.5 (m, CF ₃ CO ₂ H, CF ₃ ).

Elemental analysis for C ₃₀ H ₃₈ F ₆ N ₄₀ 7H ₂ O (710.66)

C H N

Theoretic: 52.38 5.60 7.49

Found: 52.59 5.60 7.49

[Step H]

[L-Prolineamide, N-[(phenylmethoxy) carbonyl] -N-methyl-D-phenylalanyl-N- [1- [3-[[bis [[(phenylmethoxy) carbonyl] amino ] Methylene] amino] propyl] -3,3,3-trifluoro-2-hydroxypropyl]

N, N'-bis-benzyloxycarbonyl-S-methylisothiourea (3.46 g, 9.7 mol) was added to the trifluoroacetate salt described in Step G (5.57 g, 8 mol) in 50 ml of dry THF under N 2 atmosphere and To NEt 3 (1.67 ml) solution. The solution was stirred at 40 ° C. for 4 days. THF was evaporated and the solid residue was dissolved in Et ₂ O. The ethereal solution was washed with saturated aqueous citric acid solution and brine, dried over MgSO ₄ and the solvent was evaporated. Flash chromatography on solid residue filled with silica gel [230-400 mesh, eluent AcOEt / hexane 1: 2, all residual isothiourea (Rf = 0.8) classified as AcOEt / hexane 1: 1-2: 1] It was applied to a chromatography column and the mixed product containing fractions were evaporated to give 5.08 g (71% yield) of guanidino tripeptide derivative as a colorless foam.

Rf = 0.3 (AcOEt / hexane 2: 1)

¹ H-NMR (360 MHz, CDCl ₃ ): δ = 11.5 (s, broad, 1H, ZNH guanidine), 8.3 (m, 1H NH guanidine), 7.3-7.2 (m, 22H, 4phenyl, 2NH), 5.7 (m, 1H, NH), 5.1 (m, 7H, NH, 3CH ₂ O), 4.8-3.9 (m, 5H, CHN-Phe, CF ₃ CHO, CHN-Pro, CHN, NH), 3.8-2.5 ( m, 9H, NCH ₂ -Pro, NCH ₂ guanidine NCH ₃ , CH ₂ -Phe), 1.8-1.5 (m, 8H, 4CH ₂ ).

¹⁹ F-NMR (360 MHz, ¹ H-decoupled, CDCl ₃ ): δ = 87.49, 87.12, 87.00 and 85.16 (4s).

Elemental analysis for _{C 46 H 51 F 3 N 6} O 9 · 0.5 H 2 O (897.96)

C H N

Theoretic: 61.53 5.84 9.36

Found: 61.53 5.75 9.27

[Step I]

[L-Prolineamide, N-[(phenylmethoxy) carbonyl] -N-methyl-D-phenylalanyl-N- [1- [3-[[bis [[(phenylmethoxy) carbonyl] amino ] -Methylene] amino] propyl] -3, 3, 3-trifluoro-2-oxopropyl], dihydrate]

A 250 ml three-necked flask, equipped with a magnetic stir bar, thermometer and N ₂ inlet, was charged with a solution of oxalylchloride (0.74 ml, 8.4 mmol) in 10 ml of anhydrous CH ₂ Cl ₂ , and dried-ice / acetone bath (-55 ° C., Internal temperature). A solution of DMSO (1.2 ml, 17 mmol) in 200 ml of CH ₂ Cl ₂ was added under an N 2 atmosphere at a rate maintaining an internal temperature of −55 ° C. Stirred continuously for 5 minutes and a portion of the alcohol (4.98 g, 5.6 mmol) solution described in step H in 10 ml of CH ₂ Cl ₂ was added. The mixture was warmed to -25 ° C and stirred at room temperature for 10 minutes. The solution was cooled back down to −55 ° C., 3.9 ml of NEt₃ was slowly added and the ice bath was removed. When the internal temperature reached -20 ° C, a saturated solution of citric acid was added. 250 ml of CH ₂ Cl ₂ were added at room temperature, the phases were separated and the organic layer was washed with H ₂ O, NaHCO 3 saturated solution and brine. The solvent was dried over MgSO ₄ and evaporated to give 5.07 g of yellow oil. Flash chromatography on silica gel (230-400 mesh, eluent AcOEt / hexane 1: 1) and fractions containing the mixed product were evaporated to give 3.71 g (74%) of ketone as a colorless foam.

Rf = 0.5 (AcOEt / hexane 2: 1)

¹ H-NMR (CDCl ₃ ): δ = 11.5 (broad, s, 1H, NH-Z), 8.5 (m, 1H, NH guanidine), 7.3 (m, 20H, 4 phenyl), 5.7 (3m, 1H, NH), 5.1 (m, 6H, 3CH ₂ O), 4.8 (m) and 4.7-4.2 (m, 4H, 4CH), 3.9 (m), 3.7-3.3 (m) and 2.6 (m, 9H, NCH₂- Pro, NCH₂guanidine, NCH₃, aryl CH ₂ ), 2.0-1.5 (m, 8H, 4CH ₂ ).

¹⁹ F-NMR (CDCl ₃ ): δ = 86.08, 85.96, 85.88, and 85.83 (4s).

Elemental analysis for _{C 46 H 49 F 3 N 6} O 9 · H 2 O (913.95)

C H N

Theoretic: 60.45 5.74 9.20

Found: 60.52 5.56 9.16

[Step J]

[L-Prolineamide, N-methyl-D-phenylalanyl-N- [1- [3-[(aminoimino-methyl) amino] propyl] -3, 3, 3-trifluoro-2-oxopropyl ], Dihydrochloride, hydrate]

The ketone solution (2.94 g, 3.3 mmol solution), 15 ml of 1N HCl and 0.5 g of Pd (OH) 2 / C (10%, Perlman catalyst) described in step I in 100 ml of isopropanol were hydrogenated under atmospheric pressure for 4 days. The solvent was evaporated to leave an oily residue, which was dissolved in water. The aqueous phase was washed with ether and lyophilized to give 0.665 g (62% yield) of the title compound as a white powder.

Rf = 0.5, AcOH / BuOH / H ₂ O 1/3/1.

¹ H-NMR (D ₂ O): δ = 7.4 and 7.3 (2 m, phenyl), 4.45, 4.35 and 4.2 [3 m, 2H, NCHCO, CHC (OH) ₂ CF ₃ ], 3.5-3.1 and 2.8 (3 m, 6H, NCH 2 guanidine, NCH ₂ -Pro, CH ₂ -Phe, NCH ₃ ), 2.2-1.4 (m, 8H, 4CH ₂ ), 2.0 (s, CH ₃ CO ₂ H).

¹⁹ F-NMR (D ₂ O): δ-61.6 and -63.4 [2s, ratio 55.45, CF ₃ C (OH) ₂ ].

L-proline-amide, N-methyl-, by substituting the same amount of pentafluoropropionic anhydride (PFPAA) for the TFAA reactant used during step B of this example and subsequently following the methods of steps B to J D-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3, 3, 4, 4, 4-pentafluoro-2-oxobutyl was obtained (Example 6, See step AJ).

Example 2

[L-Prolineamide, ND-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3, 3, 3-trifluoro-2-oxopropyl], hydrofluoride , Acetates, hydrates]

[Step A:]

[L-Prolineamide, N-[(phenylmethoxy) carbonyl] -D-phenylalanyl-N- [1- [3-[(1,1-dimethylethoxy) carbonyl] amino] propyl-3 , 3, 3-trifluoro-2-hydroxypropyl]

Isobutylchloroformate (0.450 ml, 3.46 mmol) was added to phenylmethyloxycarbonyl-D-phenylalanyl-L-proline (ZD-Phe-Pro-OH, 1.27 g, 3.2m mol) and N- under N 2 atmosphere. Methylmorpholine (0.38 ml, 3.46 mmol) was added to cooling (-10 ° C.) and stirred solution. The reaction mixture was stirred at −10 ° C. for 10 minutes and the ω-N-protected diaminoalcohol (0.9 g, 3.19 mmol) solution of step E of Example 1 in tetrahydrofuran (anhydrous, 6 ml) was added. The mixture was stirred at 0 ° C. for 1 hour and then held at + 4 ° C. for 16 hours. AcOEt / H ₂ O (100 ml each) was added and the phases were separated. The organic layer was washed three times with 50 ml each of saturated NaHCO 3 solution, citric acid and brine, dried over MgSO ₄ and evaporated. The resulting crude tripeptide derivative (2.5 g, yellow foam) was subjected to flash chromatography (silica gel, 100 g, eluent: AcOEt / hexane 1: 1) and the mixed product containing fractions were evaporated (20 Torr, 30 ° C .; cosolvent). Twice with CCl ₄ , then 0.01 Torr, room temperature, 16 hours) to give 1.5 g (72%) of the title compound as a colorless foam.

Rf = 0.14 (AcOEt / hexane)

¹ H-NMR (360 MHz, CDCl ₃ ,): δ = 7.4-7.2 (3m, 10H, 2 aryl), 7.1 (m, 1H, NH), 5.8-5.5 (m, 2H, 2NH), AB is 5.10 Concentrated on (J _HA-HB = 10 Hz, 2H, arylCH ₂ O), 4.8-3.5 [6m, 5H, 4CH, OH (exchanged with D ₂ O)], 3.2-2.9 and 2.6 (2m, 6H, 2NCH ₂ , Aryl, CH ₂ ), 2.2 and 1.9-1.5 (m, 8H, 4CH ₂ ), 1.4 (m, 9H, 3CH ₃ ).

¹⁹ F-NMR (CDCl ₃ ): δ = 87.1: 86.8 (2d, J _HF = 7.5 Hz), 86.5 (m), 85.2; _{85.1 (2d, J HF = 7.5Hz} ), 85.0 (m).

MS (CI / NH ₃ ): m / e = 665 (MH ⁺ , 50%).

Elemental analysis for C ₃₃ H ₄₃ F ₃ N ₄ O ₇ · 0.4 CCl ₄ (726.258)

C H N

Theoretic: 54.94 5.97 7.71

Found: 54.74 6.12 7.29

[Step B]

[L-Prolineamide, N-[(phenylmethoxy) carbonyl] -D-phenylalanyl-N- [1-[(3-amino) propyl] -3, 3, 3-trifluoro-2- Hydroxypropyl], trifluoroacetate, hydrate]

TFA 5m1 solution in 5m1 of CH ₂ Cl ₂ was added to the protected tripeptides derivative (1.39 g, 2.09 mmo1) and the mixture was stirred until foaming stopped (20 min, using a bubble counter). The solution was evaporated to dryness (0.01 Torr, 40 h). An aliquot (0.68 g) was obtained from 100% of the resulting yellow foam for the next half and the remainder was treated with i-propanol / hexanes to give 0.73 g of the title compound as a white solid.

Rf = 0.5 (Ac0Et / 10% Ac0H)

¹ H-NMR (360 MHz, CD ₃ OD): δ = 7.3 (m, 10H, 2 phenyl), AB concentrated at 5.1 (J _HA-HB = 23 Hz, 2H, CH ₂ 0), 4.6 (t, J = 7.5 Hz, 0.5H), and 4.45 (dd, J = 7.5 Hz, 0.5H, 1H, CHCF ₃ ), 2.9 and 2.6 (2m, 6H, 3NCH ₂ ), 2.0-1.3 (m, 8H, 4CH ₂ ).

¹⁹ F-NMR (360 MHz, CD ₃ OD): δ = 88.48 (s, CF ₃ CO ₂ H), 88.35, 88.20, 87.21 and 87.18 (4d, J _HF = 7 Hz, CF ₃ ); Proportion 16: 10: 3: 3.

MS (CI / NH ₃ ): m / e = 565 (MH ⁺ , 100% relative intensity).

Elemental analysis for _{C 30 H 36 F 6 N 6} O 7 · 0.5 H 2 O (687.64)

C H N

Theoretic: 52.40 5.42 8.15

Found: 52.30 5.69 7.83

[Step C]

[L-Prolineamide, N-[(phenylmethoxy) carbonyl] -D-phenylalanine-N- [1- [3-[[bis-[[(1,1-dimethylethoxy) carbonyl] amino] Methylene] amino] propyl] -3,3,3-trifluoro-2-hydroxypropyl]

N, N'-bis-Boc-S-methylisothiourea (Bergeron, R, J.) and McManis (McManis, J, S.), J. Org. Chem. 1987, vol. 52, p. 1700] (1.40 g, 4.83 mmol) were added trifluoro acetate salt (12) (0.82 g, 1.2 mmol) and NEt ₃ (0.25 in 4 ml of anhydrous tetrahydrofuran under N ₂ atmosphere. ml, 1.81 mmol). The solution was stirred at 40 ° C. for 4 days. Tetrahydrofuran was evaporated and the solid residue was dissolved in Et ₂ O. The ethereal solution was washed with saturated citric acid solution and brine, dried over MgSO ₄ and the solvent was evaporated. Solid residue was charged with silica gel [30 g, 230-400 mesh, eluent AcOEt / hexane 1: 2, all residual isothiourea (Rf = 0.8) classified as AcOEt / hexane 1: 1-2: 1] The flash chromatography column was applied and the mixed product containing fractions were evaporated to yield 0.44 g (45%) of guanidino tripeptide derivative as expected as a colorless blowing agent. Rf = 0.3 (AcOEt / hexane: 2: 1)

¹ H-NMR (360 MHz, CDCl ₃ ,): δ = 11.5 (s, broad, 1H, BocNH guanidine), 8.3 (m, 1H, NH guanidine), 7.2 (m, 11H, 2phenyl, NH), 6.0 , 5.9, and 5.7 (3m, 1H, NH), AB concentrated at 5.1. (J = 23 Hz, 2H, CH ₂ O), 4.4 (m, 1H, CHN-Phe), 4.3-3.2 (6m, 7H, CHN-Pro, NCH ₂ -Pro, NCH ₂ guanidine A-part, CHCF ₃ , OH), 3.0 (m, 2H, aryl (CH ₂ ), 2.6 (m, 1H, NCH ₂ guanidine B-part), 1.8-1.2 (m, 26H, 6CH ₃ , 4CH ₂ ).

¹⁹ F-NMR (360 MHz, CDCl ₃ ,): δ = 86.95, 86.86, 85.39, and 85.16 (4d, J _HF = 7 Hz); Proportion 5: 5: 1: 1.5.

Elemental analysis for _{C 39 H 43 F 3 N 9} O 6 · H 2 O (824.88)

C H N

Theoretic: 56.79 6.72 10.19

Found: 56.53 6.54 10.05

[Step D]

[L-Prolineamide, N-[(phenylmethoxy) carbonyl] -D-phenylalanyl-N- [1- [3-[[bis-[[(1,1-dimethylethoxy) carbonyl] Amino] methylene] amino] propyl] -3,3,3-trifluoro-2-oxopropyl], hydrate]

A 25 ml three-necked flask, equipped with a magnetic stir bar, thermometer and N ₂ inlet, was charged with a solution of oxalylchloride (0.127 g, 1 mmol) in 1 ml of anhydrous CH ₂ Cl ₂ and dried ice / acetone bath (-55 ° C.). Internal temperature). A solution of DMSO (0.22 g, 2.8 mmol) in 1 ml of CH ₂ Cl ₂ was added at a rate to maintain the internal temperature at −55 ° C. under argon. Stirred continuously for 5 minutes and the alcohol (0.4 g, 0.5 mmol) solution described in step C in ₂ ml CH ₂ Cl _{2 was} added in one portion. The mixture was warmed to -25 ° C and stirred at that temperature for 10 minutes. The solution was cooled back down to −55 ° C., 0.5 ml of NEt ₃ was slowly added and the ice bath was removed. When the internal temperature reached -20 ° C., a saturated solution of citric acid was added. 50 ml of CH ₂ Cl ₂ were added at room temperature, the phases were separated and the organic layer was washed with water, saturated NaHCO ₃ solution and brine. Dry over MgSO ₄ and evaporate the solvent to give 0.34 g of yellow oil. Flash chromatography on silica gel (0.01 g, 230-400 mesh, eluent AcOEt / hexane 1: 1, then 2: 1) and evaporate the mixed product containing fractions to give 0.24 g (60%) of the desired ketone as a colorless foam. )

Rf = 0.5 (AcOEt / hexanes: 2: 1).

¹ H-NMR (CDCl ₃ ): δ = 11.5 (s, broad, 1H, BocNH), 8.5 (m, 1H, NH guanidine), 7.3 (m, 10H, 2phenyl), 5.7 (3m, 1H, NH) , 5.1 (m, 2H, CH ₂ O), 4.8 (m) and 4.7-4.2 (m, 4H, 4CH), 3.9 (m), 3.7-3.3 (m) and 2.6 (m, 4H, NCH-Pro, NCH ₂ guanidine), 3.0 (m, 2H, aryl CH ₂ ), 2.0-1.4 (m, including s at 1.5, 26H, 6CH ₃ , 4CH ₂ ).

¹⁹ F-NMR (CDCl _3, H-decoupled): δ = 86.14 and 85.89 (2d, ratio 1: 2, CF ₃ CO), 80.22 and 79.69 [2s, ratio 1: 1, CF ₃ C (OH) ₂ ].

¹³ C-NMR (CDCl ₃ ): δ = 175.5-174.29 (4s, CONH), 164.1 (2s), 158.8-156.8 (4s) and 153.9 (s, OCON, OCON = C), 136.5 and 136.2 (2s, phenyl ), 130.8-128.0 (8s, phenyl), 125.57, 122.38, and 121.19 (visible part q, J _CF = 107.6 Hz, CF ₃ ), 95.4-94.7 [4s, C (OH) ₂ ], 83.8-83.7 (3s ) And 80.0, 79.9 (2s, C), 68.2-67.8 (3s, CH ₂ O), 61.5-59.4 (2s, NCH-Pro), 55.6-54.4 (species s, NCH-Phe, NCH COCF ₃ ), 47.87 And 47.65 (2s, NCH ₂ -Pro), 41.0-40.7 (2s, NCH ₂ guanidine), 39.11-38.48 (2s, phenyl CH ₂ ), 31.8-24.6 (species s, CH ₂ -Pro, 2-tert.- Butyl, CH ₂ guanidine, CH ₂ -Pro).

Elemental analysis for C ₃₉ H ₅₁ F ₃ N ₆ O ₉ 2H ₂ 0 (840.86)

C H N

Theoretic: 55.71 6.59 9.99

Found: 55.75 6.25 9.78

[Step E]

[L-Prolineamide, ND-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,3-trifluoro-2-oxopropyl], hydrofluoride , Acetate, hydrate]

The reaction flask of the "Sakakibara apparatus" is charged with 0.1 g (0.119 mmol) of the ketone of step D and subjected to liquid HF deprotection of all protecting groups. At the reaction time of 20 minutes, HF was evaporated. Solid residue is AcOH / H₂It was dissolved in 0: 1 mixture and lyophilized. Solid soy sauce is dissolved in water and the mixture is millipore MilliporeFiltered through a filter and freeze-dried again to give 40 mg (57%) of the title compound as a solid.

¹ H-NMR (D ₂ O): δ = 7.4 and 7.3 (2m, phenyl), 4.45, 4.35 and 4.2 [3m, 2H, NCHCO, CHC (OH) ₂ CF ₃ ], 3.5-3.1 and 2.8 (3m, 6H, NCH ₂ guanidine, NCH ₂ -Pro, CH ₂ -Phe), 2.2-1.4 (m, 8H, 4CH ₂ ), 2.0 (s, CH ₃ CO ₂ H).

¹⁹ F-NMR (CD ₂ O): δ = -2.91 (s, CF ₃ CO), -6.16 and -6.34 [2s, ratio 55:45, CF ₃ C (OH) ₂ ], -54.28 (s, F ^- = 1.5 HF / CF ₃ ).

Elemental Analysis for C ₂₁ H ₂₉ F ₃ N ₆ O ₃ 1.5 HF 0.5 CH ₃ CO ₂ H3H ₂ O (584.58)

C H N

Theoretic: 45.20 6.64 14.38

Found: 55.75 6.25 9.78

Substituting the same amount of PFPAA for the TFAA reactant of Example B in Example 1, subsequently performing Steps B, C, D and E of Example 1, using the reaction product in Step A of this Example, and L-prolinamide, N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,4,4,4, -penta, by carrying out steps B, C, D and E of the examples. Fluoro-2-oxobutyl], hydrofluoride and hydrate were obtained.

Example 3

[L-Prolineamide, N, N-dimethyl-D-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,3-trifluoro-2-oxo Propyl], dihydrochloride, hydrate]

[Step A]

[L-Prolineamide, N, N- (dimethyl) amino-D-phenylalanyl-N- [1- [3-[[bis-[[(1,1-dimethylethoxy) carbonyl] amino] methylene ] Amino] propyl] -3,3,3-trifluoro-2-oxopropyl], hydrate]

An aqueous solution of formaldehyde (37%, 24μ1) was added to a suspension of 200 mg of fully protected ketone (product of Step D, Example 2, 0.822 g, 1 mmol) and catalyst pd (OH) ₂ on carbon in isopropanol (10 ml). Was added. The resulting suspension was hydrogenated under atmospheric pressure until hydrogen absorption ceased (about 24 hours). Filtration from the catalyst gave a slightly yellow solution which was evaporated to one third of its original volume. The residue was diluted with 10 ml of water and the solution was lyophilized to give a yellow foam which was used as such in the next step.

[Step B]

[L-Prolineamide, N, N-dimethyl-D-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,3-trifluoro-2-oxo Propyl], dihydrochloride, hydrate]

To a suspension of the prepared ketone (0.72 g, 1 mmol) in anhydrous ether (10 ml) was added a saturated solution of HCl-gas in anhydrous ether (4M, 50 ml). The mixture was stirred for 2 days and the solvent was evaporated to give the title compound (0.60 g) as a slightly yellow foam.

Rf = 0.5, (BuOH / AcOH / H ₂ O: 3/1/1).

The formaldehyde reactant of step A of example 3 was substituted with an equivalent amount of succialdehyde or glutaraldehyde, and the methods of steps A and B were subsequently carried out, respectively, to determine the pyrroly of the dimethyl-D-Phe compound of example 3. Dean and piperidine homologues (i.e. And Corresponding salts).

Similarly, the trifluoroacetic anhydride (TFAA) of step B of Example 1 is replaced with an equivalent amount of pentafluoropropionic anhydride (PFPAA) and treated according to Examples 1-3 to give the corresponding pentafluoropropionyl Homologues [ie compound (a) (b) of (CH ₃ ) ₂ ND-Phe-Pro-Arg-CF ₂ CF ₃ And (c) Salt] was obtained. The nitrogen atom of the indicated pyrrolidine and piperidine residues is the terminal nitrogen atom of D-phenylalanine αamino acid.

Example 4

[L-Prolineamide, N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,4,4,4, -pentafluoro-2-oxobutyl] -1-D- [(1,2,3,4-tetrahydro-3-isoquinolinyl) carbonyl], dihydrochloride, hydrate]

[Step A]

[N- (1,1-dimethylethoxy) carbonyl-D-1,2,3,4-tetrahydro-3-isoquinonol carboxylic acid]

Di-t-butyldicarbonate [(Boc) ₂ O, 6.78 g, 31.0 mmol, 1.1 equiv) and NEt ₃ (4.33 ml, 31.0 mmol 1.1 equiv) were mixed with water (55 ml) and THF (55 ml) To a solution of D-3-TIC (5.00 g, 28.2 mmol) in water. The mixture was stirred at rt for 12 h. After evaporation of THF the aqueous layer was washed twice with Et ₂ O, acidified with saturated citric acid solution and extracted three times with AcOEt. The combined organic layers were washed with brine, dried over MgSO ₄ and evaporated. The residue was dried under reduced pressure (0.01 Torr) to give a protective amino acid as a white foam. Yield 7.32 g (94%).

Melting point: 104-105 ° C., Rf = 0.7 (AcOEt / AcOH = 98: 2).

MS (C ₁₅ H ₁₉ NO _4, 277); (CI / NH ₃ ): m / e = 278 (MH ⁺ , 40%); 239 (90%); 222 (MH ⁺ -56, MH ⁺ H ₂ C = C (CH ₃ ) ₂ , 90%); 178 (MH ⁺ -100, MH ⁺ -Boc, 100%).

¹ H-NMR (CDCl ′ ₃ , 90 MHz): δ = 9.8 (broad s, 1H, COOH); 7.12 (s, 4H, C ₆ H ₄ ); 5.0 (0.5H) and 4.77 (s, 0.5H, NCH); 4.56 (d, J = 9.0 Hz, 2H, NCH ₂ ); 3.12 (m, 2 H, CH ₂ ); 1.4 (broad s, 9H, Boc).

[Step B]

[N- (1,1-Dimethylethoxy) carbonyl-D-1,2,3,4-tetrahydro-3-isoquinolinyl-L-proline-benzyl ester]

DCC (5.38 g, 26.1 mmol, 1.0 equiv) of Boc-D-3-TIC (step A, 7.22 g, 26.1 mmol) and HOBt (3.98 g, 26.1 mmol, 1.0 equiv) in CH ₂ Cl ₂ (200 mL) It was added to cooling and stirring solution (0 ° C.). After the mixture was stirred at 0 ° C. for 30 minutes, L-Pro-benzylester hydrochloride (6.30 g, 26.1 mmol. 1.0 equiv) and NMM (3.10 ml, 28.7 mmol, 1.0 equiv) were added. The solution was stirred for 12 hours at room temperature. After filtration of the DUC, the filtrate was evaporated. The resulting residue was then dissolved in AcOEt and refiltered. The filtrate was washed twice with saturated NaHCO ₃ solution, once with citric acid and then with water and brine, dried over MgSO ₄ and evaporated. After drying at reduced pressure (0.01 Torr), the protected dipeptide was obtained as a white foam.

Yield: 9.69 g (80%)

Melting Point: 93-95 ° C, Rf = 0.5 (Petroleum Ether / AcOEt = 2/1)

MS (C ₂₇ H ₃₂ N ₂ O ₅ = 464): m / e = 465 (MH ⁺ , 100%); 365 (MH ⁺ -100, MH ⁺ -Boc, 40%); 257 [MH ⁺ -208, MH ⁺ -(Boc + OBn), 50%]; 225 (40%); 206 (30%).

¹ H-NMR (CDCl ₃ , 90 MHz): δ = 7.4-7.1 (m, 9H, C ₆ H ₄ , C ₆ H ₅ ); 5.2-4.3 (m, 4H, NCH ₂ -TIC, N-CH-TIC, NCH-Pro); 5.0 (broad s, 2H, OCH ₂ ); 3.5 (m, 2H, NCH ₂ -Pro); 3.0 (m, 2H, CH-TIC); 1.9 (m, 4H, 2CH ₂ -Pro); 1.40 (s, 9 H, Boc).

[Step C]

N- (1,1-dimethylethoxy) carbonyl-D-1,2,3,4-tetrahydro-3-isoquinolinyl-proline]

Palladium hydroxide on carbon (20%, Pd (OH) ₂ C, Perlman catalyst) was converted to BOC-D-3-Tic-L-Pro-benzyl ester (step B) in isopropanol (150 ml) (9.69 g, 20.9 mmol ) Was added to the solution. The mixture was hydrogenated at room temperature for 12 hours at atmospheric pressure while acid precipitated. MeOH (100 ml) was added, the solution was filtered over celite and the filtrate was evaporated. The solid residue was washed with Et ₂ O followed by petroleum ether. Boc-protected dipeptide was obtained by drying under reduced pressure (0.01 Torr) as a white solid. Yield 6.60 g (85%).

Melting point; 216-218 ° C (decomposed)

Rf = 0.75 (BuOH / AcOH / H ₂ O = 3: 3: 1)

MS (C ₂₀ H ₂₆ N ₂ 0 ₅ = 374): (CI / NH ₃ ): m / e = 375 (MH ⁺ , 50%); 275 (MH ⁺ -Boc, 22%), 257 [MH ⁺ -(Box + H ₂ O), 100%]

Elemental analysis for C ₂₀ H ₂₆ N ₂ O ₅ 0.25 H ₂ O (378.9)

C H N

Theoretic: 63.39 7.05 7.39

Found: 63.54 6.94 7.49

α _D ²⁰ : −15.5 (c = 1, MeOH).

¹ H-NMR (CD ₃ OD, 360 MHz): δ = 7.2 (broad s, 4H, C ₆ H ₄ ); 5.2 and 4.9 (2m, 1H, NCH-TIC); 2 AB system centered at 4.58. {[A: 4.75 (J = 16 Hz); B: 4.43 (J = 16 Hz)], 2H, NCH ₂ -TIC)}; 4.3 (m, 1 H, NCH-Pro); 3.9-3.4 (m, 2H, NCH ₂ ); 3.3-2.9 (m, 2H, CH ₂ -TIC); 2.3-1.9 (2m, 4H, 2CH ₂ ); 1.5 (broad s, 9H, Boc).

[Step D:]

[L-Prolineamide, N- [1- [3-[[bis-[[1,1-dimethylethoxy) carbonyl] amino] methylene] amino] porophyll] -3,3,4,4,4 -Pentafluoro-2-hydroxybutyl] -1-[(1,1-dimethylethoxy) carbonyl] -D-[(1,2,3,4-tetrahydro-3-isoquinolinyl) Carbonyl]

DCC (0.255 g, 1.2 mmol, 1.0 equiv) of Boc-D-3-Tic-Pro (0.462 g, 1.2 mmol) and HOBt (0.189 g, 1.2 mmol, 1.0 equiv) in CH ₂ Cl ₂ (15 ml) It was added to cooling and stirring solution (0 ° C.). The mixture was stirred at 0 ° C. for 30 min, at which time amino alcohol 12 (reactivity A) (0.59 g, 1.2 mmol, 1.0 equiv) and NMM (0.15 ml, 1.4 mmol, 1.1 equiv) were added. The solution was stirred at rt for 12 h. The mixture was filtered from the precipitated DCU. The filtrate was washed with a saturated solution of citric acid, KHCO ₃ and brine, dried over MgSO ₄ and evaporated. The resulting residue was flash chromatographed on silica gel (eluent: petroleum ether / AcOEt: 3/2). Tripeptide alcohol homologues were isolated as white solid phase. Yield: 0.62 g (85%)

Melting Point: 102-104 ° C., Rf = 0.3 (Petroleum Ether / AcOEt = 1: 1)

MS (C ₃₈ H ₅₅ N ₆ 0 ₉ F ₅ = 834): (CI / NH ₃ ): m / e = 635 (MH ⁺ -200, MH ⁺ -2 Boc, 100%); 593 [MH ⁺ -242, MH ⁺ -(2 Boc + N = CNH ₂ ), 50%]; 535 (MH ⁺ -300, MH ⁺ -3 Boc, 15%)

¹ H-NMR (CDCl ₃ , 360 MHz): δ = 11.5; 8.4 and 7.5 (broad 3s, 3H, 3NH); 7.2 (m, 4H, C ₆ H ₄ ): 5.1 and 4.8 (broad 2s, 1H, NCH-TIC); 4.80 (d, J = 11.8 Hz, 2H, NCH ₂ -TIC); 4.7-4.4 (m, 2H, NCH-Pro, CF ₂ CH); 4.3-3.7 (m, 3H, NHCH, NCH ₂ -Pro); 3.6-3.5; 3.5-3.3 and 3.1-3.0 (3m, 5H, 2CH ₂ , OH); 2.4-1.6 (m, 8H, 4CH ₂ ); 1.5 (broad s, 27 H, 3 Boc).

¹⁹ F-NMR: (CDCl ₃ , C ₆ F ₆ control extract, 360 MHz); delta = 79.12; 78.94; 78.90 and 78.74 (4s, 3F, CF ₃ , two diastereoisomers and two isomeric cis / trans: ratio 5: 7.5: 1.5: 1); ABX system concentrated at 35.98 [A: 40.57 (J _FA-FB = 280 Hz); B: 31.39 (J _FB-FA = 280 Hz; J _FB-HX = 30 Hz); ABX system concentrated at 35.82 [A: 39.84 (J _FA-FB = 280 Hz); B: 32.30 (J _FB-FA = 280 Hz; J _FB-HX = 30 Hz); ABX system concentrated at 33.95 [A: 39.18 (J _FA-FB = 280 Hz): B: 28.72 (J _FB-FA = 280 Hz; J _FB-HX = 30 Hz)]; ABX system concentrated at 34.41 [A: 39.18 (J _FA-FB = 280 Hz); B: 29.64 (J _FB-FA = 280 Hz; J _FB-HX = 30n Hz); 2F, CF ₂ , two diastereomers and two isomeric cis / trans: ratio: 7.5: 5: 1: 5: 1).

[Step E]

[L-prolineamide, N- [1- [3-[[bis-[[(1,1-dimethylethoxy) -carbonyl] amino] methylene] amino] propyl] -3,3,4,4, 4-pentafluoro-2-oxobutyl] -1-[(1,1-dimethylethoxy) carbonyl] -D- [1,2,3,4-tetrahydro-3-isoquinolinyl) carbon Carbonyl]

A solution of oxalylchloride (0.122 ml, 1.4 mmol, 2.0 equiv) in 3 ml of anhydrous CH ₂ Cl ₂ was cooled to −60 ° C. under N ₂ . Then a solution of dimethylsulfoxide (0.197 ml, 2.8 mmol, 4.0 equiv) in 6 ml of anhydrous CH ₂ Cl ₂ was added dropwise to maintain the internal temperature at −55 ° C. The mixture was stirred for 5 minutes, and then the prepared aminoalcohol (step D, 0.58 g, 0.7 mmol) solution in 5 ml of anhydrous CH ₂ Cl ₂ was added dropwise. After complete addition, the temperature was raised to −20 ° C. for 5 minutes. Then cooled to -55 [deg.] C. again for 0.5 h. NEt ₃ (0.48 ml, 3.5 mmol, 5.0 equiv) was then added to maintain the internal temperature at −55 ° C. After 5 minutes, saturated citric acid solution (2 ml) was added. The mixture was allowed to warm to room temperature and 40 ml of CH ₂ Cl ₂ was added. The phases were separated and the organic phase was washed with water and brine, dried over Na ₂ SO ₄ and evaporated. The resulting residue was subjected to flash chromatography [50 mg of Al ₂ O ₃ (pH: 7.5, pretreated with 3 ml of water; 100 mg of eluent; petroleum ether / CHCl ₃ = 1: 2)]. Product containing fractions were combined and evaporated. The expected compound was obtained as a white foam. Yield: 0.25 g (43%).

Rf: 0.25 (petroleum ether / CHCl ₃ = 1: 2)

MS (C ₃₈ H ₅₃ N ₆ 0 ₉ F ₅ = 832): (CI / NH ₃ ): m / e = 257 (65%); 2257 (100%); 220 (70%).

¹ H-NMR: (CDCl ₃ , 360 MHz): δ = 11.5: 8.4 and 7.9 (broad s, 3H, 3NH); 7.2 (m, 4H, C ₆ H ₄ ): 5.0-4.2 [m, 5H, NCH ₂ -TIC, NCH-TIC, NCH-Pro, (cetone or hydrate)]; 3.8 [broad s, OH of hydrate); 3.7-3.0 (3m, 6H, CH 2 -TIC, NCH ₂ -Pro, NCH ₂ ); 2.4-1.6 (m, 8H, 4CH ₂ ; 1.5 (m, 27H, 3 Boc).

¹⁹ F-NMR (CDCl ₃ , C ₆ F ₆ control extract 360 MHz) δ = 82.81 and 82.65 (2s, 3F, CF ₃ CF ₂ C (OH) ₂ : 80.10 and 80.07 (2s, 3F, CF ₃ CF ₂ CO Ratio 1: 2; 40.58; 40.53 and 40.40 (AB system hardly split, 2F, CF ₃ CF ₂ CO-); 41.05 and 37.92 (AB system hardly split, 2F, CF ₃ CF ₂ C (OH) ₂ ); ratio = 2: 1.

[Step F]

[L-Prolineamide, N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,4,4,4-pentafluoro-2-oxobutyl] -1-D- [ (1,2,3,4-tetrahydro-3-isoquinolinyl) carbonyl], trichlorohydrate]

The protected tripeptide homologue (step E, 0.25 g, 0.3 mmol) was dissolved in 25 ml of anhydrous Et ₂ . A saturated solution of HCl gas in Et ₂ (100 ml) was added and the resulting mixture was stirred for 48 hours at room temperature. The mixture was evaporated to dryness. The oily residue is dissolved in the minimum amount of water and the solution is millipore Filter on filter disc. The filtrate was lyophilized to afford the title compound as a white solid.

Yield: 0.142 g (89%)

Melting point: 158-160 ° C. Decomposition.

Rf = 0.7 (BuOH / AcOH / H ₂ O = 3: 1: 1)

MS: (C ₂₃ H ₂₉ N ₆ O ₃ F ₅ = 532): (CI / NH ₃ ): m / e = 533 (MH ⁺ , 40%); 274 (45%); 257 (100%); 256 (20%); 220 (30%).

C ₂₃ H ₂₉ N ₆ O ₃ F ₅ ; 3HCl; Elemental Analysis for H ₂ O (659.9)

C H N

Theoretic: 41.86 5.19 12.73

Found: 41.41 5.05 12.63

IR: v = 3383 CNH; 1645 (C = C; CONH) cm ^-1 .

¹ H-NMR: (D ₂ O, TSP control extract, 360 MHz); delta = 7.3 (m, 4H, C ₆ H ₄ ); 4.9, 4.8 and 4.7 (3m, 1H, NCH-TC); 4.55 and 4.3 [2m (AB system hardly split), 2H, NCH ₂ -TIC); 4.5 overlap (m, 2H, NCH-Pro, NCH); 3.7 (m, 2H, NCH ₂ -Pro); ABX system concentrated at 3.38 {[A: 3.50 (J _{HA-HX =} 13 Hz; J _HA-HX = 3 Hz); B: 3.15 (J _HB-HA = 13 Hz; J _HA-HX = 3 Hz)], 2H, CH ₂ -TIC}; 3.25 (broad s, 2H, NCH ₂ ); 2.4 and 2.0 (2m (AB system hardly split), 2H, CH-CH ₂ -Pro); 2.1 (m, 2H, CH ₂ -Pro); 2.1 and 1.6 (2m, 4H, 2CH ₂ ).

¹⁹ F-NMR (D ₂ O, CF ₃ COOH, 360 MHz) δ = −3.62 and −3.65 (2s, 3F, CF ₃ ); AB system concentrated at -47.85 [A: -48.20 (J _FA-FB = 280 Hz); B: -47.49 (J _FB-FA = 280 Hz); AB system concentrated at -47.97; [A: -48.62 (J _FA-FA = 280 Hz); B: -47.33 (J _FB-FA = 280 Hz); Ratio = 46:54.

Example 5

[L-Prolineamide, N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,4,, 4-pentafluoro-2-oxobutyl] -1-[(1, 2,3,4-tetrahydro-1-isoquinolinyl) carbonyl], dihydrochloride, hydrate]

[Step A]

[N-[(1,1-dimethylethoxy) carbonyl] -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid]

Di-t-butyldicarbonate (Boc ₂ O, 8.2 g, 37.6 mmol, 1.1 equiv) was prepared from 1,2,3,4-tetrahydroisoquinal acid [Solomon W. in 50 ml of tetrahydrofuran and 50 ml of water. Solomon), J. Chem. SOC., 1974, prepared from isoquinoline-1-carboxylic acid by the method described on page 129, 6.0 g, 33.8 mmol] and added to a stirred solution of NEt ₃ (5.2 ml, 37.6 mmol, 1.1 equiv) I was. The mixture is stirred for 16 hours at room temperature, whereupon tetrahydrofuran is evaporated. The remaining aqueous phase was acidified (solid citric acid) and the protected amino acid was extracted three times using AcOEt. The combined organic layers were washed with brine, dried over MgSO ₄ and evaporated (20 Torr, 30 ° C. and 0.01 Torr, 20 ° C.) to afford 8.0 g (85%) of protected amino acid.

[Step B]

[N-[(1.1-dimethylethoxy) carbonyl]-[(1,2,3,4-tetrahydro-1-isoquinolinyl) carbonyl] -L-proline benzyl ester]

Dicyclohexylcarbodiimide (DCC, 5.38 g, 26 mmol) was treated with protected amino acids (Example 5, Step A; 7.2 g, 26 mmol) and hydroxybenzotriazole hydrate in 100 ml of dichloromethane and 10 ml of tetrahydrofuran. HOBt, 3.98 g, 26 mmol) was added to the stirred and cooled (0 ° C.) solution. After stirring at 0 ° C. for 30 minutes, the mixture was warmed to room temperature for 1 hour and cooled back to 0 ° C. L-Proline benzylester hydrochloride (6.3 g, 26 mmol) and N-methylmorpholine (NMM, 3.2 ml, 26 mmol) were added to the mixture and the resulting mixture was stirred at 0 ° C. for 30 minutes and at room temperature for 16 hours. I was. The mixture was filtered and the solvent was evaporated to give an oil which was dissolved in CH ₂ Cl ₂ . The solution was washed with citric acid, sodium bicarbonate and saturated sodium chloride solution, dried over MgSO ₄ , the solvent was evaporated (20 Torr, 30 ° C. and 0.01 Torr, 20 ° C.), then 10.5 g (84%) of a colorless oil. Got.

Rf = 0.8 (AcOEt: Petroleum Ether = 1: 2)

[Step C]

[N-[(1,1-dimethylethoxy) carbonyl] -1-[(1,2,3,4-tetrahydro-1-isoquinolinyl) carbonyl] -L-proline]

Isopropane was hydrogenated with a protected dipeptide (Example 5, Step B; 9.3 g, 20 mmol) in 150 ml and a 500 mg solution of 10% palladium hydroxide (Pulman catalyst) on carbon for 14 hours at room temperature and atmospheric pressure. Filtration from the catalyst and evaporation of the solvent gave 8.0 g of semisolid oil, which was partially dissolved in methanol / Et ₂ O (1: 1 v / v). The mixture was filtered to give 2.7 g of a colorless oil.

Overall yield (93%).

Rf solid: 0.80 (BuOH / AcOH / H ₂ O = 3/1/1)

Rf oil: 0.80 (BuOH / AcOH / H ₂ O = 3/1/1)

[Step D]

[L-Prolineamide, N- [1- [3-[[bis [[(1,1-dimethylethoxy) carbonyl] amino] methylene] amino] propyl] -3,3,4,4,4- Pentafluoro-2-hydroxybutyl] -N-[(1,1-dimethylethoxy) carbonyl] -1-[(1,2,3,4-tetrahydro-1-isoquinolinyl)- Carbonyl]

Dicyclohexylcarbodiimide (DCC, 0.255 g, 1.2 mmol) was added to the prepared protected dipeptide (step D, 0.374 g, 1.2 mmol) and hydroxybenzotriazole hydrate (HOBt, 0.189 g) in 15 ml of dichloromethane. , 1.2 mmol) was added to the cooling (0 ° C.) and stirred solution. The mixture was plated at 0 ° C. for 30 minutes, at room temperature for 1 hour and cooled back to 0 ° C. Bis-Boc protected arginino homologue (see Example 6, step F; see 0.590 g, 1.2 mmol) and N-methylmorpholine (150 ml, 1.4 mmol, 1.1 equiv) were added. The solution was stirred at rt for 12 h. After the mixture was filtered, the filtrate was washed with a saturated solution of citric acid, KHCO ₃ and brine. The organic solution was dried over MgSO ₄ and evaporated. The residual oil was flash chromatographed on silica gel (10 g, eluent: AcOEt / petroleum ether: 2/3). Tripeptide alcohol homologues were isolated as colorless oils.

Yield: 0.610 g (83%)

Rf: 0.3 (AcOEt / Petroleum ether: 1/1)

[Step E]

[L-Prolineamide, N- [1-3-[[bis [[(1,1-dimethylethoxy) carbonyl] amino] methylene] amino] propyl] -3,3,4,4,4-penta Fluoro-2-oxobutyl] -N-[(1,1-dimethylethoxy) carbonyl] -1-[(1,2,3,4-tetrahydro-1-isoquinolinyl) -carbonyl ]

A solution of oxalylchloride (0.122 ml, 1.4 mmol, 2.0 equiv) in 3 ml of anhydrous CH ₂ Cl ₂ was cooled to −60 ° C. under argon. A solution of DMSO (0.197 ml, 2.8 mmol, 4.0 equiv) in 6 ml of anhydrous CH ₂ Cl ₂ was then added dropwise to maintain the temperature of the solution at -550 ° C. The solution was stirred for 5 minutes and the alcohol (0.58 g, 0.7 mmol) solution prepared in step D in 5 g of anhydrous CH ₂ CL ₂ was added dropwise. After complete addition, the internal temperature was raised to -20 ° C for 5 minutes and the solution was cooled to -55 ° C for 1 hour. Then triethylamine (0.48 ml, was added at a rate that maintains the internal temperature at 3.5 mmol-55 ° C. After 5 minutes, saturated citric acid solution (2 ml) was added The mixture was allowed to warm to room temperature and CH ₂ Cl 40 ml were added _{2. The} phases were separated and the organic phase was washed with water and brine, dried over MgSO ₄ and evaporated The resulting oily residue was subjected to 100 g of Al ₂ O ₃ (pH: 7.5, pretreated with 3 ml of water). Flash chromatography (eluant: petroleum ether / CHCl ₃ : 1/2) The product containing fractions were combined and evaporated to give a colorless foam.

Yield: 0.30 g (50%).

Rf: 0.25 (petroleum ether / CHCl ₃ : 1/2).

[Step F]

[L-Prolineamide, N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,4, 4,4-pentafluoro-2-oxobutyl] -1-[(1 , 2,3,4-tetrahydro-1-1isoquinolinyl) carbonyl], dihydrochloride, hydrate]

The protected tripeptide alcohol (0.30 g, 0.36 mmol) of step F was dissolved in 25 ml of anhydrous Et ₂ O. A saturated solution of HCl gas in Et ₂ O (100 ml) was added and the resulting mixture was stirred for 24 hours at room temperature. The mixture was filtered and the solid residue was further dried (0.01 Torr, 20 ° C., 16 h) to give the title ketone (hydrate form) as a white solid.

Yield: 0.160 g (89%)

Rf: 0.7 (BuOH / H ₂ O / AcOH: 3/1/1)

Example 6

[L-Prolineamide, N-methyl-D-phenylalanyl-N- [1- [3- (aminoiminomethyl) amino] propyl] -3,3,4,4,4-pentafluoro-2- Oxobutyl], dihydrochloride, hydrate]

[Step A]

[N, N '-[1- (pentafluoropropionyl) -1,4-butanediyl] -bis (benzamide), hydrate]

Pentafluoropropionic anhydride (31.8 ml, 50 g, 161 mmol) was added to ornithine 5 (4H) -oxazolone [(14.05 g, 4.3 mmol) under N ₂ atmosphere, Example 1, Step 2, Reaction A, General formula ( To the well stirred powder of 2). The resulting mixture was stirred at 40 ° C for 16 h. At this time an aliquot (about 50 mg) was taken for ¹ H- and ¹⁹ F-NMR spectra (CDCl ₃ ). Disappearing the ¹ H-signal at 4.5 ppm indicates the disappearance of oxazolone, while 42.7; The ¹⁹ F-signal at 46.7 (2S, 2 x CF ₂ ) and 80.0: 80.5 (2S, 2 x CF ₃ ) indicates the formation of 3'-N-4-bispentafluoropropionyl oxazolone. An additional ¹⁹ F-signal showed excess of pentafluoropropionic anhydride (PFPAA) and pentafluoropropionic acid (PFPA) formed. The solvent was then evaporated at 55-60 ° C. (0.5-1 Torr, dry ice-acetone trap) for about 6 hours to give a viscous orange oil. ¹⁹ F-NMR spectra of another aliquot show all PFPAA and PAPA disappeared. 40 ml of a saturated solution of oxalic acid (15.0 g, 150 mmol) in tetrahydrofuran were added, and the resulting orange oil was stirred at 55 ° C. for 6 hours when the foam was completely stopped. The solvent was evaporated (20 Torr, 30 ° C.) and the oily residue was dissolved in AcOEt. This solution was stirred at room temperature for 15 minutes using saturated KHCO ₃ solution (desired hydrolysis of pentafluoropropionamide). The phases were separated and the orange layer was washed with water, 1N HCl and brine, dried over MgSO ₄ and evaporated (20 Torr, 30 ° C .; then 0.1 Torr, 30 ° C.). The resulting orange oil (21.7 g) was flash chromatographed on silica gel (500 g; eluent AcOEt / petroleum ether: 1/3) in two batches of about 11 g. Fractions 11-20 containing N-pentafluoropropionamide A-2 were evaporated to give 7.0 g (27%). Fractions 28-105 containing ketone A-1 were evaporated to give 10.1 g (52%) as a white solid.

Total yield: 79% based on oxazolone.

A-1 ¹ H-NMR (CDCl ₃ ) δ = 8.0-7.85 (m, 5H, aryl, NH), 7.8-7.4 (m, 6H, aryl), 5.5-5.3 (m, 1H, CHCO), 3.9 ( Broad, t, 2H, NCH ₂ ), 2.3-1.8 (m, 4H, 2CH ₂ ).

¹⁹ F-NMR (CDCl ₃ ) δ = 40.33 (d, J = 7.5 Hz, CF ₂ CO), 46.67 (s, CF ₂ CONH), 80.0 (s, 2CF ₃ ).

A-2 ¹ H-NMR (CDCl ₃ ) δ = 8.1-7.8 (m, 4H, aryl), 7.7-7.4 (m, 7H, aryl, NH), 6.6 (m, 1H, NH), 5.3 (m, 1H, CHCO), 3.7 (broad t, 2H, NCH ₂ ), 2.4-1.8 (m, 4H, 2CH ₂ ).

¹⁹ F-NMR (CDCl ₃ / C ₆ F ₆ ) δ = 40.3 (d, J = 7.5 Hz, CF ₂ ), 80.0 (s, CF ₃ ).

MS (CI / NH ₃ ): 443 (MH ⁺ ).

A small sample of A-2 (100 mg) was crystallized from AcOEt / petroleum ether to give 80 mg of the title compound, which was analytically pure.

Elemental Analysis for C ₂₁ H ₁₉ O ₃ N ₂ F ₅ (442.39)

C H N

Theoretic: 57.02 4.33 6.33

Found: 57.14 4.23 6.36

[Step B]

[N, N '-[1- (2,2,3,3,3-pentafluoro-1-hydroxypropyl) -1,4-butanediyl] -bis (benzamide)]

Reduction of two kinds of ketones A-1 and A-2 was carried out in two separate reactions.

1) Reduction of A-1

NaBH ₄ (0.43 g 11 mmol) was added in part to pentafluoroethyl ketone A-1 (10.1 g, 17.1 mmol of cooling (0 ° C.) and stirred solution in EtOH (130 ml) The mixture was allowed to warm to room temperature and further Stir for 1 hour Hydrochloric acid (6N) was added carefully until foaming stopped, the solution was neutralized with Na ₂ CO ₃ and EtOH was evaporated The resulting mixture was redissolved in AcOEt / water, The aqueous layer was extracted twice with AcOEt and the combined organic layers were washed with water and brine.

Dry over MgSO ₄ and evaporate the solvent to afford a white solid which was flash chromatographed on silica gel (300 g, eluent AcOEt / petroleum ether: 1/1, then 4/1). The product containing fractions were evaporated to give 5.88 g (77%) of the desired alcohol as a white solid. R _f = 0.45-0.50 (AcOEt / petroleum ether: 1/1) for diastereoisomers as two severely separated points.

2) reduction of A-2

As described above for A-1, 6.91 g (15.6 mmol) of ketone A-2, 300 mg (7.9 mmol) of NaBH ₄ and 90 ml of EtOH were used to prepare alcohol N, N '-[1- (2,2, 6.05 g (89%) of 3,3,3-pentafluoro-1-hydroxypropyl) -1,4butanediyl] -bis (benzamide) was obtained as a white solid, which was collected from the compound and In comparison.

¹ H-NMR (CDCl ₃ , CD ₃ OD) δ = 7.8-7.5 (m, 4H, aryl), 7.45-7.1 (m, 6H, aryl), 4.5 and 4.2 (2m, 1H, CHOH, ratio 3: 1 ), 3.5 (m, 4H, 2CH ₂ ).

¹⁹ F-NMR (CDC1 ₃ , CD ₃ OD, C ₆ F ₆ ) δ = ABX system concentrated at 36.3 A: 40.3 (J _FA - _FB = 280 Hz, J _FA - _HX = 3 Hz); B: 32.3 (J _FB - _FA = 280 Hz, J _FB - _HX = 30 HZ, CF ₂ ), 79.0 (s, CF ₃ ) = Diastereoisomer 1.

ABX system concentrated at 35.3 A: 36.3, B: 33.3 (with the same coupling constant as above) = diastereoisomer 2; Ratio 3/1.

Elemental Analysis for C ₂₁ H ₂₁ O ₃ N ₂ F ₅ (444.40)

C H N

Theoretic: 56.76 4.76 6.30

Found: 56.94 4.83 6.29

[Step C]

[4,7-Diamino-1,1,1,2,2-pentafluoro-3-heptanol, dihydrochloride]

While advancing the hydrolysis to TLC (BuOH / water / AcOH = 4/1/1), a stirred solution of the above prepared alcohol (step B) (11.78 g, 26.6 mmol) in concentrated aqueous hydrochloric acid solution (240 ml) was stirred under stirring. Heated to reflux. After 16 hours of reaction, the solvent was evaporated and the oily residue was treated twice under these conditions. When complete formation of bisamino alcohol was indicated by TLC, the solution was cooled to room temperature and the solvent was evaporated. The oily residue was dissolved in water and the solution was washed three times with 100 ml of Et ₂ O. The aqueous layer was evaporated to dryness to afford 8.14 g (99%) of the desired diamino alcohol as a brown foam.

¹ H NMR (D ₂ O) δ = 4.6 (m, 1H, CHOHCF ₂ ), 3.1 (m, 2H, NCH ₂ ), 3.6 (m, 1H, CHN), 2.0 (m, 4H, 2CH ₂ )

[Step D]

[4-Trifluoroacetylamino-7-amino-1,1,1,2,2-pentanefluoro-3-heptanol, hydrochloride]

Trifluoroacetic anhydride (3.55 ml, 25 mmol) was added dropwise to a stirred solution of diaminoalcohol (3.09 g, 10 mmol) of step C in 50 ml of trifluoroacetic acid. After stirring for 2 hours at room temperature, 2.5 ml of TFAA was further added to the solution and stirred continuously for 10 hours. The solution was evaporated to dryness to afford a brown oil. Trituration with Et ₂ O / petroleum ether gave a brown solid which was filtered off and washed with petroleum ether. Drying gave the slightly colored solid title compound (3.48 g, 95%), which was pure enough for the next reaction.

¹ H NMR (D ₂ O) δ = 4.6 (m, 2H, 2CH), 3.1 (m, 2H, NCH ₂ ), 2.0-1.7 (m, 4H, 2CH ₂ ).

¹⁹ F-NMR (D ₂ O, ref, CF ₃ CO ₂ H) δ = ABX system concentrated at -49.00, A: -44.00 (J _FA - _FB = 280 Hz); B: -54.00 (J _FB - _FA = 280 Hz) J _FB - _HX = 30 Hz) = isomer 1; ABX concentrated at -49.33, A: 45.00, B: 40.67 (coupling constant as above) = Isomer 2; Ratio 4; One.

MS: (CI / NH ₃ ): 333 (M−H ⁺ ).

[Step E]

[N- [1- [7-bis [(1,1-dimethylethoxy) carbonyl] amino] methylene] amino] -N (4-trifluoroacetylamino) -3-hydroxy-1,1, 1,2,2-pentafluoroheptane]

Bis-Boc-S-methylisothiourea (7.3 g, 25 mmol) was added the hydrochloride salt of step D (5.1 g, 10 mmol) and NEt ₃ (3.5 ml, 25) in anhydrous tetrahydrofuran (100 ml) under N2 atmosphere. mmol) was added to a good stirred solution. The mixture was stirred at 40 ° C. for 60 hours. The solvent was evaporated (placed between the flask and the pump to avoid poisoning the trap with the KMnO ₄ / Na ₂ CO ₃ aqueous solution to methanethiol) and the oily residue was dissolved in AcOEt. This solution was washed with water, citric acid, NaHCO ₃ , and brine saturated solution. Dry over MgSO ₄ and evaporate the solvent to afford an oil (10 g) which was flash chromatographed on silica gel (50 g, eluent AcOEt / petroleum ether: 1/8, then 3/1). The product containing fractions were evaporated to give 2.92 g (51%) of ω-guanidino-γ-amino alcohol protected as a colorless foam.

¹ H-NMR (CDCl ₃ , 360 MHz) δ = 11.20 (s, 1H, NH), 10.31 (d, J = 10 Hz, 1H, NHCOCF ₃ ), 9.73 (broth S, 1H, NHCH ₂ ), 4.45 ( t, J = 9.5 Hz, 1H, CHN), 4.25 d, J = (22 Hz, CHOH), 3.75 (m, 1H, OH) 3.65 and 3.23 (2m, 2H, NCH ₂ ), 2.1 and 1.9 (2m, 4H, 2CH ₂ ), 1.45 and 1.40 (2s, 18H, 2 t-BOC).

¹⁹ F-NMR (CDCl ₃ , Control. C ₆ F ₆ ) δ = ABX system concentrated at 35.50. A: 39.33 (J _FA - _FB = 277 Hz), B: 32.30 (J _FB - _FA = 277 Hz; J _FB - _HX = 22 Hz) = isomer 1; ABX system concentrated at 34.00 A: 40.05 (J _FA - _FB = 277 Hz), B: 27.5 (J _FB - _FA = 277 Hz, J _FB - _HX = 22 Hz) = isomer 2, ratio 4: 1; 78.87 and 79.40 (2s, ratio 4: 1, CF ₃ ); 86.12 and 86.53 (2s, ratio 1: 4 CF ₃ CO)

Elemental analysis for C ₂₀ H ₃₀ F ₈ N ₄ O ₆ 0.5 H ₂ O (574.47)

C H N

Theoretic: 41.17 5.36 9.60

Found: 41.06 5.15 9.57

[Step F]

[N- [1- [7- [bis (1,1-dimethylethoxy) carbonyl] amino] methylene] amino-4-3-hydroxy-1,1,1,2,2, -pentafluoro Heptane]

Freshly prepared aqueous LiOH (1N, 7 ml) solution was added to the stirred solution of prefluoroacetamide of step E in tetrahydrofuran / water (9/1, 50 ml). Stir the solution for 20 hours at room temperature and the starting material disappears (TLC, AcOEt / petroleum ether: 1.5). Tetrahydrofuran was evaporated and the aqueous solution was extracted four times with 50 ml of Et ₂ O. The combined extracts were washed with water and brine and dried over MgSO ₄ . The solvent was evaporated to give 2.18 g (88%) of the aminoalcohol as a white solid.

¹ H-NMR (CDCl ₃ ): δ = 11.5 (m, 1H, NH), 8.4 (m, 1H, NH), 4.1 and 3.9 (2m, 1H, CHOH), 3.5 and 3.2 (2m, 3H, CHN, NCH ₂ ), 2.5 (m, 2H, NH ₂ ), 1.9-1.4 (m, 4H, 2CH ₂ ), 1.50 (s, 18H, 2 t-Boc).

¹⁹ F-NMR (CDCl ₃ ): δ = ABX system concentrated at 37.3; A: 43.67, (J _FA - _FB = 280 Hz; J _FB - _HX = 22 Hz, CF ₂ , isomer 1), 79.0 (s, CF ₃ ) and ABX system concentrated at 39.0 (coupling constants Equals), (CF ₂ isomer 2), 79.5 (s, CF ₃ ); Proportion 4: 1. Elemental Analysis for C ₁₈ H ₃₁ F ₅ N ₄ O ₅ (478.46)

C H N

Theoretic: 45.19 6.53 11.71

Found: 45.13 6.44 11.56

[Step G]

[L-Prolineamide, N-[(1,1-dimethylethoxy) carbonyl] -N-methyl-D-phenylalanyl-N- [1- [3-[[bis [[1,1-dimethyl Ethoxy) carbonyl] amino] methylene] amino] propyl] -3,3,4,4,4-pentafluoro-2-hydroxybutyl]

Dicyclohexylcarbodiimide (0.96 g, 4.7 mmol) was added to Boc-N (methyl) -D-Phe-Pro-OH (1.78 g, 4.7 mmol) and N-hydroxybenzoin CH ₂ C1 ₂ (50 ml). Triazole (0.711 g, 7 mmol) was added to the stirred and cooled (0 ° C.) solution. The mixture was stirred at 0 ° C. for 30 min, at which time the amino alcohol (step F) (2.22 g, 4.64 mmol) and NMM (0.52 ml, 4..7 mmol) prepared above were added. The resulting mixture was further stirred at 0 ° C. for 0.5 h and then warmed to rt. Stir at room temperature for 16 hours. The precipitated DCU was filtered off and the filtrate was washed with citric acid, KHCO ₃ and brine saturated aqueous solution, then dried over MgSO ₄ and the solvent was evaporated to give a viscous oil. Flash chromatography on silica gel (150 g, eluent AcOEt / petroleum ether: 1/1) and the product containing fractions were evaporated to give 3.09 g (70%) of the desired tripeptyl alcohol as a colorless oil.

¹ H-NMR: (CD ₃ OD, 360 MHz): δ = 7.2 (m, 5H, aryl), 5.1-4.9 (2m, 1H, CH-Phe), 4.4-3.9 (m, 3H, CH-Pro, CHOH, CHN), 3.7-3.3 and _{3.2-2.9 (2m, 6H, 2NCH 2} , Phe-CH 2), 2.85-2.7 (5s, 3H, NCH 3), 2.3-1.6 (m, 8H, 4CH 2), 1.5-1.1 (6 s, 27 H, 3 t-Boc).

¹⁹ F-NMR (CD ₃ OD, C ₆ F ₆ control extract, 360 MHz): δ = concentrated ABX system at 43.7. (CF ₂ -Isomer 1 and 2: cis-trans-isomer) and 81.80, 81.55, 81.30 (3s of other isomers, CF ₃ ).

MS (FAB): 837 (MH ⁺ )

[Step H]

[L-Prolineamide, N-[(1,1-dimethylethoxy) carbonyl] -N-methyl-D-phenylalanyl-N- [1- [3-[[bis [[(1,1- Dimethylethoxy) carbonyl] amino] methylene] amino] propyl] -3,3,4,4,4, -pentafluoro-2-oxobutyl]

A 100 ml three-necked flask, equipped with a magnetic stir bar, thermometer and N ₂ inlet, was filled with a solution of oxalylchloride (0.52 ml, 5.7 mmol) in 5 ml of anhydrous CH ₂ C1 ₂ . After cooling the solution at −60 ° C., a solution of dimethylsulfoxide (1.2 ml, 14.3 mmol) in 10 ml of anhydrous CH ₂ C1 ₂ was added via syringe at a rate maintaining the internal temperature at −55 ° C. The mixture was stirred at −55 ° C. for 15 minutes and the alcohol of step F (3.0 g, 3.58 mmol) in 20 ml of anhydrous CH ₂ C1 ₂ was added dropwise. After complete addition, the cooling bath was removed and stirring continued until the internal temperature reached -20 ° C. At this temperature, it is continuously stirred for about 5 minutes and the solution is cooled back to -55 ° C. At this temperature, NEt ₃ (2.5 ml, 17.9 mmol) was added at a rate maintaining the internal temperature of -55 ° C. Finally saturated citro acid solution (10 ml) was added. The mixture was allowed to warm up to room temperature and 200 ml of CH ₂ C1 ₂ was added. The phases were separated and the organic layer was washed with water, saturated NaHCO ₃ solution and brine. Dry over MgSO ₄ and evaporate the solvent to afford a colorless oil (ca. 3 g) which is flash chromatography on silica gel (100 g, eluent AcOEt / petroleum ether: 1/2, then 1/1, then 2/1) I was. The product containing fractions were evaporated to afford 1.0 g (33%) of pure pentafluoroketone as described above as a colorless foam. About 1.6 g of the mixture of ketone and starting alcohol described above in step G was recovered. This can be recycled for another oxidation step.

¹ H-NMR (CDC1 ₃ , 360 MHz): δ = 11.5 (m, 1H, NH), 8.5 (m, 1H, NH), 7.8 (m, 1H, NH), 7.2 (m, 5H, aryl), 5.1-4.3 (m, 3H, α-CH-Phe, α-CH-Pro, α-CHCO), 3.7-3.1 and 3.1-2.6 (2m, 9H, 2NCH ₂ , CH ₂ C ₆ H ₅ , NCH ₃ ) , 2.2-1.6- (m, 8H, 4CH ₂ ), 1.5-1.2 (m, 27H, 9CH ₃ ),

¹⁹ F-NMR (CDC1 ₃ , 360 MHz): δ = 40.33 and 40.19 (2s, CF ₂ CO), ABX system concentrated at 39.0 (CF ₂ ), 80.0 (s, CF ₃ ), 82.7 and 82.9 (2s, CF ₃ ), the ratio 4: 1

[Step I]

[L-Prolineamide, N-methyl-D-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3,4,4,4-pentafluoro-2 Oxobutyl], dihydrochloride, hydrate]

0.9 g (1.07 mmol) of the tripeptide derivative of step H was dissolved in 50 ml of anhydrous Et 2 O. 200 ml of saturated HCl gas / Et ₂ O solution were added and the resulting solution was stirred at room temperature excluding moisture for 48 hours. Petroleum ether (about 100 ml) was added and the precipitate was filtered under N ₂ . The filter residue was dried (0.1 Torr, 40 ° C.) to give 0.6 g of white amorphous powder, which was dissolved in water (20 ml) and the solution was millipore. Filter on filter disc. The filtrate was lyophilized to give 0.5 g (82%) of the title compound as an off-white powder.

¹ H-NMR (D ₂ O): δ = 7.55 (m, 3H, aryl), 7.30 (m, 2, aryl), 4.55 (m, 1H, CH-Phe), 4.38 (m, 1H, CH-Pro ), 4.27 [m, 1H, CHN-C (OH) ₂ ], 3.50 (M, 1H, HCH _A -Pro), 3.37 (m, 1H, C ₆ H ₅ CH _A ), 3.25 (m, 2H, NCH) ₂ -guanidine), 3.15 (m, 1H, C ₆ H ₅ CH _B ), 2.75 (s, broad, 3H, CH ₃ N), 2.73-2.63 (m, 1H, NCH _B -Pro), 2.2-1.4 ( m. 8H, 4CH ₂ ). Approximately 9% impurity may be present at 6.2, 3.7 and 1.2 ppm.

¹⁹ F-NMR (D ₂ O, CF ₃ CO ₂ H control extract) δ = −3.65 and −3.71 (2s, ratio, 45:55, 3F, CF ₃ ), 1 AB system concentrated at −47.80; _{A: -47.44 (J FA - FB} = 281 Hz); B: -48.16 (J _FB - _FA = 281 Hz), AB system concentrated at -48.09; A: -47.64 (J _FA-FB = 281 Hz), -48.53 (J _FB-FA = 281 Hz = CF ₂ ratio 45:55 in _two diastereoisomers, impurity of the remaining alcohol described in step G (about 5%) appears at -6.9 and -54.0, and the unknown structure impurity (9%) appears at -3.3 and -45.5.

Example 7

[L-Prolineamide, N-methyl-D-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3-difluoro-2-oxohexyl], di Hydrochloride, hydrate]

[Step A]

[N- [1- [4- [bis [(1,1-dimethylethoxy) carbonyl] amino] methylene] amino] -1-nitrobutane]

Bis-Bos-S-methylisothiourea (13,8 g 47.6 mmol) was added 4-amino-1-nitrobutane, hydrochloride (2.1 g, 13.6 mmol) in 40 ml of anhydrous dimethylformamide [Keller-Shrlein, Merten , W. Keller-Schierlein, P. Mertens, V. Prelog, and A. Walser, Helv. Chim Acta Vol. 48, Fasc 4 (1965) p. 710]

And 6.6 ml (47.6 mmol) of NEt _{3 to} a stirred solution. The mixture was stirred for 14 h under argon atmosphere. 200 ml of Et ₂ 0 were added and the solution was washed with water and a concentrated solution of citric acid, sodium bicarbonate and sodium chloride. The organic layer was dried over MgSO ₄ and evaporated (20 Torr, 30 ° C.) to give 15 g of a yellow oil. Flash chromatography on silica gel (0.500 g, eluent: Ac0Et / petroleum ether: 1/5) and the product containing fractions were evaporated (20 Torr, 30 ° C and then 0.01 Torr, 20 ° C) to protect spheres as white solids. 3.48 g (71%) of aninodino derivatives were obtained.

R _f = 0.5 (Ac0Et / petroleum ether: 1/4)

Melting Point: 94-96 ℃

[Step B]

[N-1- [bis [(1,1-dimethylethoxy) carbonyl] amino] methylene] -amino-4,4-difluoro-5hydroxy-6-nitro-1-nonene]

Nitro derivatives prepared above (step A, 0.48 g, 1.33 mmol) and 2,2-difluoro-penten-l-al, ethyl hemiacetal (0.283 g, 1.7 mmol) and catalytic amount of potassium carbonate (about 40 mg) The mixture was stirred at 40 ° C for 14 h. The organic layer was dried over MgSO ₄ and the solvent was evaporated (20 Torr, 30 ° C.) to give a colorless oil (0.57 g) which was flash chromatographed on silica gel (20 g, eluent: Ac0Et / petroleum ether: 1 / 5) It was. The product containing fractions were combined and evaporated (20 Torr, 30 ° C. and 0.01 Torr, 20 ° C.) to give 0.332 g (52%) of the desired nitro alcohol as a light yellow solid.

R _f = 0.4 (Ac0Et / petroleum ether: 1/3)

[Step C]

[N-1- [9- [bis [(1,1-dimethylethoxy) carbonyl] amino] methylene] -amino-4,4-difluoro-5-hydroxy-6-aminononan]

0.1 g of freshly prepared Raney-Nickel was added to a stirred solution of nitro alcohol (Step B, 0.33 g, 0.67 mmol) prepared above in i-propanol (40 ml). The mixture was hydrogenated under atmospheric pressure for 15 hours when H ₂ consumption was complete. The mixture was filtered and the solvent was evaporated (20 Torr, 30 ° C. and 0.01 Torr, 20 ° C.) to afford the desired difluoroamino alcohol as a colorless oil. Yield: 302 mg (93%)

[Step D]

[L-Prolineamide, N-[(1,1-dimethylethoxy) carbonyl] -N-methyl-D-phenylalanyl-N- [1- [3-[[bis [[(1,1- Dimethylethoxy) carbonyl] amino] methylene] amino] propyl] -3,3-difluoro-2-hydroxyhexyl]

Example 6, 0.23 g (0.61 mmol) of Boc-N- (methyl) -D-Phe-Pro-0H, 0.093 g (0.61 mmol) in 10 ml CH ₂ Cl ₂ , according to the method as described in step G The reaction was carried out using 0.126 g (0.61 mmol) of DCC, 0.08 g (0.75 mmol) of N-methylmorpholine and 0.23 g (0.61 mmol) of difluoroamino alcohol (Step C) prepared above. Flash chromatography (20 g, eluent: Ac0Et / petroleum ether: 1 / 3-1 / 1) on silica gel afforded 0.24 g (49%) of the protected tripeptide homolog as a colorless oil.

R _f = 0.4-0.6 (Ac0Et / petroleum ether: 1/1)

[Step E]

[L-Prolineamide, N-[(1,1-dimethylethoxy) carbonyl] -N-methyl-D-phenylalanyl-N- [1- [3-[[bis [[(1,1- Dimethylethoxy) carbonyl] amino] methylene] amino] propyl] -3,3-difluoro-2-oxohexane]

The product of step D (0.24 g, 0.29 mmol) was charged with 0.051 ml (0.59 mmol) of oxalyl chloride in 6 ml of anhydrous CH ₂ Cl ₂ , 0.084 ml (1.1 mmol), and according to the method described in Example 5, step H, and 0.2 ml (2 mmol) of NEt ₃ was oxidized to the corresponding ketone. The crude reaction product on silica gel was flash chromatographed (10 g, eluent Ac0Et / petroleum ether: 2/3) and the product containing fractions were evaporated to give 0.16 g (67%) of the desired ketone as colorless oil.

R _f = 0.2-0.3 (2 spots) (Ac0Et / petroleum ether: 2/3)

[Step F]

[L-Prolineamide, N-methyl-D-phenylalanyl-N- [1- [3-[(aminoiminomethyl) amino] propyl] -3,3-difluoro-2-oxohexane], di Hydrochloride, hydrate]

Saturated HCl Gas / Et₂50 ml of 0 solution was added to 0.15 g (0.186 mmol) of the above prepared ketone (step E) and the mixture was stirred for 48 h. The solvent was evaporated and the solid residue dissolved in water. Millipore The solution was filtered on a filter disc and the filtrate was lyophilized to give 0.11 g (100%) of the title mixture as a yellow solid.

R _f = 0.4 and 0.45 (2 diastereoisomers) (BuOH / AcOH / H ₂ O: 3/1/1)

As mentioned above, the compounds of the present invention (IA and IB) have the property of exhibiting host inhibition of thrombin and are thus effective anticoagulants useful for the prevention of both venous and arterial thrombotic diseases.

Formulas (IA) and (IB) using standard in vitro methodology for determining anticoagulant activity as well as thrombin inhibitory activity in human plasma using activated thromboplastin time (aPTT) and thrombin time (TT). It will be found that the compound of is an effective anticoagulant. Anticoagulant data can be expressed as follows.

Anticoagulant Effect in Human Plasma

ID ₂ ^a (μM)

Compound aPTT ^b TT ^b

D-CH ₃ Phe-Pro-Arg-CF ₃ 218 108

D-Phe-Pro-Arg-CF ₃ 148 43

D-Phe-Pro-Arg-CF ₂ CF ₃ 2 1

a amount required to double solidification time

b _a PTT, active partial thromboplastin time TT, thrombin time

In mice, the in vivo antithrombotic effect can be used for evaluation. For example, a FeCl ₃ induced thrombosis model in which D-Phe-Pro-Arg-CF ₂ CF ₃ is administered intravenously at 10 mg / kg per kg body weight followed by continuous infusion at 1 mg / kg per minute In, the compound can prevent infarction in two rats and delay the hardening in the other two of the four rats. Thus, based on in vitro and in vivo standard analytical methods, the compounds of the present invention may be associated with thrombophlebitis at doses in the range of about 2-50 mg per kg of body weight per day (2-20 mg for the preferred compound). It will be found useful in the treatment of arterial thrombosis as well as other types of venous and arterial thrombotic disease states. Of course, the actual dose and frequency will vary depending on the nature and severity of the disease, age, general state of health and other factors known and recognized by diagnostic analysts in the art.

In addition to the above uses of novel compounds of formulas (IA) and (IB) as inhibitors of thrombin, another aspect of the present invention relates to the use of such compounds as effective inhibitors of human lung tryptase useful in the treatment of asthma. will be.

Using the standard in vivo methodology (Journal of Biological Chemistry, Vol. 261, June 5, 7372-7379, page 1986) to determine the inhibitory activity of tryptase, the following results were obtained.

^a rate constant was determined by a progress curve analysis using a nonlinear regression program (ENZFITTER).

Based on this kind of assay, the amount of a compound of the invention in its end use for the treatment of asthma will be about 1-100 mg per treatment, compared to other compounds known to have tryptase inhibitory activity. . Of course, the dosage instructions will depend on the severity of disease penetration, the age and general symptoms of the patient as well as other factors recognized by diagnostic analysts in the art.

Although some of the administered tripeptides of the present invention are effective in passage through the digestive tract following oral administration, the compounds are preferably used via parenteral administration. Subcutaneous, intravenous, intramuscular, or intraperitoneal administration, depot injections, implant preparations and other parenteral methods are preferred methods in which the compounds may be used. In the example of treating asthma, the peptide derivative of the present invention is applied in the form of a spray or dry powdered pharmaceutical formulation, either by using a measured administration erosol or by applying it as an aerosol to the mucosa (ie, nose, throat and bronchial tubes). It can be contained as.

For parenteral administration, the compound is a solution or suspension of the compound in a physiologically acceptable diluent using a pharmaceutical carrier, which may be a sterile liquid, such as water and oil, with or without surfactant and other pharmaceutically acceptable auxiliaries. It can be administered in the injection dose of. Examples of oils that can be used in such oils include petroleum, animal, vegetable or synthetic oils; Examples are peanut oil, soy milk and mineral oil. In general, glycols, such as water, saline, aqueous dextrose and related sugar solutions, ethanol and propylene glycol or polyethylene glycol, are particularly preferred liquid carriers for injectable solutions.

The compounds may be administered in the form of depot injections or implantable preparations which may be formulated in a manner that allows for the sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as a depot injection or implant. Implants can be inert materials such as tetrabiodegradable polymers or synthetic silicones, such as silicones, silicone rubbers made by Dow-Corning Corporation.

In addition, the compounds of the present invention can be administered topically. This can be accomplished by simply preparing a solution of the compound to be administered, using a solvent known to promote transdermal absorption, such as ethanol or dimethyl sulfoxide (DMSO), preferably in the presence or absence of other excipients. Preferably topical administration will be by using reservoirs and porous membrane forms or patches of various solid matrices.

Certain suitable transdermal devices are described in US Pat. Nos. 3,742,951, et al. 3,797,494, 3,996,934 and 4,031,894. Generally, such a device contains a backing member containing one of its mating surfaces, an active ingredient permeable adhesive layer defining the other mating surface and at least one reservoir containing the active ingredient sandwiched between the joining surfaces. do. Alternatively, the active ingredient may be included in a plurality of microcapsules dispersed through the permeable adhesive layer. In one case, the active ingredient is continuously transported through the membrane from the reservoir or microcapsules to the active ingredient permeable adhesive layer in contact with the recipient's skin or mucous membranes. If the active ingredient is absorbed through the skin, control and defined flow of the active ingredient is administered to the recipient. In the case of microcapsules, the encapsulating agent can also act as a membrane.

In another device for transdermal administration of a compound according to the invention, the pharmaceutically effective compound is included in a matrix that is delivered at the desired gradual, constant, controlled rate. The matrix is permeable to release of the compound through diffusion or microporous flow. Release is rate controlled. Such a system without a membrane is described in US Pat. No. 3,921,636. Two or more releases are possible in such a system. Release by diffusion occurs when the matrix is nonporous.

Pharmaceutically effective compounds dissolve and diffuse through the matrix itself. Release by microporous outflow allows the pharmaceutically active compound to be carried through the liquid phase in the pore of the matrix.

Of particular interest are preferred compounds of the general formulas (IA) and (IB) of the present invention which are specified in the following chart.

Wherein CH is cyclohexyl, CHM is cyclohexylmethyl, all substituted for the α-carbon atom of the modified P ₃ -α-amino acid of the tripeptides of the general formula (IA) and Et is ethyl.

Wherein the P ₃ residues 2a, b and c, and 2'a, b and c are as defined above and, when they are combined with the carbonyl residue to which they are attached, modification of the tripeptides of the indicated general formula (IB) Form P ₃ -α-amino acids.

Claims (10)

The following compounds of formula (I), isomers thereof and mixtures thereof, hydrates thereof and pharmaceutically acceptable salts thereof. Where X is or X Is a compound of formula (I) Phosphorus compound of general formula (I) is called compound of (IB). Wherein m is 0, 1 or 2, n is 0 or 1, the sum of m and n is less than 3, greater than 0, q is 0 or 1, the sum of both q is 0 or 2, and R ₁ is H or C _1-7 alkyl, R ₂ is H or C _1-7 alkyl, or R ₁ and R ₂ together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle, R ₃ is -CF ₃ -CF ₂ CF ₃ , -CF ₂ (CH ₂ ) t CH ₃ , -CH ₂ (CH ₂ ) tCOOR _4, -CF ₂ (CH ₂ ) tCONHR ₄ , -CF ₂ (CH ₂ ) tCH ₂ OR ₄ or -CF ₂ (CH ₂ ) vCH = CH ₂ , where t is 2, 3 or 4, v is 1 or 3, R ₄ is H or C _1-6 alkyl, and A is Phenyl or cyclohexyl, B is (CH) ₄ or (CH ₂ ) ₄ which together with the two carbon atoms to which they are attached form a C ₆ -cyclic hydrocarbon residue, provided that both R ₁ and R ₂ are H And R ₃ is not -CF ₃ or -CF ₂ CF ₃ . The compound of claim 1, wherein the R ₃ residue is -CF ₃ , -CF ₂ CF ₃ , -CF ₂ (CH ₂ ) ₂ CH ₃ , -CF ₂ (CH ₂ ) ₂ CO ₂ Et, —CF ₂ (CH ₂ ) ₂ CONHCH ₃ and —CF ₂ (CH ₂ ) _{4 A} compound selected from the group consisting of CONHCH ₃ . The compound of formula (IA) according to claim 2, wherein A is phenyl, n is 1, R 1 is H and R ₂ is C _1-7 alkyl. The compound of claim 3, wherein R ₂ is methyl. The compound of formula (IA) according to claim 2, wherein A is cyclohexyl, R ₁ is H and R ₂ is C _1-7 alkyl. The compound of claim 5, wherein R ₂ is methyl and n is 1. 7. The compound of claim 2 wherein B is (CH ₄ ), each q is 0, m is 2, and n is 0. 4. The compound of claim 2, wherein B is (CH ₄ ), each q is 0, m is 1, and n is 0 or 1. 4. The compound of claim 2, wherein B is (CH ₂ ) ₄ , each q is 1, m is 1 or 2 and n is 0 or 1. 10. The compound of any of claims 4, 7, 8 or 9, wherein R ₃ is -CF ₂ (CH ₂ ) ₂ CH ₃ .