AU653232B2 - Novel thrombin inhibitors - Google Patents

Description

AUSTRALIA

653 Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

PS~ F 92 Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art:

C

Veto.

C

*1

S

C. 6 .00:4 0 Name of Applicant: Merrell Dow Pharmaceuticals Inc.

Actual Inventor(s): Robert James Broersma, Jr.

Bernhard Neises Axel Ganzhorn Celine Tarnus Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: NOVEL THROMBIN INHIBITORS Our Ref 291589 POF Code: 1432/120371 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 6006 1- NOVEL THROMBIN INHIBITORS This invention relates to novel polyfluorinated alkyl derivatives of certain tripeptides, to the methods for their preparation, the intermediates therefore, to their use in inhibiting thrombin and lung tryptase and in their end-use application as anti-coagulants useful in treating thrombophlebitis and coronary thrombosis and in the treatment of asthma.

More specifically this invention relates to the novel 10 compounds of the formulae HN NI- 2

NH

NH

0

IA

N. 2 HN NH 2 2)n R NQ NH NHN CH N N H 2 S0NH fC(H)q :i (H2)m (CH2)n 3 0 0 2 their isomers and mixtures thereof, the hydrates and the pharmaceutically acceptable salts thereof, with the proviso that when R 1 and R 2 are both H, then R 3 is other than -CF 3 or

-CF

2

CF

3 wherein m is zero, one or two, n is zero or one, with the proviso that the sum of m and n is less than three and greater than zero, q is zero or one with the proviso that the sum of both q's is zero or 2, R1 is H or C 1 -7 alkyl,

R

2 is H or C1- 7 alkyl, or R 1 and R 2 taken together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle,

R

3 is -CF 3 CFCF, -CF 2

(CH

2 )tCH 3

-CF

2

(CH

2 )tCOOR 4

-CF

2

(CH

2 )tCONHR 4

-CF

2

(CH

2

CH

2

OR

4 or -CF 2

(CH

2 )vCH=CH 2 with t being 2, 3 or 4, and v is 1, 2 or 3,

R

4 is H or C_-6 alkyl, A is phenyl or cyclohexyl, B is (CH) 4 or (CH 2 4 which, when taken together with the two carbon atoms to which it is attached, forms a Cg- 20 cyclic hydrocarbon moiety.

The natural amino acids, with the exception of glycine, contain a chiral carbon atom. Unless otherwise specifically indicated, the preferred compounds are the optically active 25 amino acids of the L-configuration except, as indicated, it is preferred that Phe be in its D-configuration.

The tripeptides of formula 1 can form pharmaceutically acceptable salts with any non-toxic, organic or inorganic acid. Illustrative inorganic acids which form suitable salts :nclude 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 3 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.

An alkyl group is taken to include straight, branched, or cyclic alkyl groups, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, sec-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl and cyclopentylmethyl. In the instance wherein R 1 and R 2 are lower alkyl, it is preferred that one or both be methyl.

When R 1 and R 2 form a 5-6-membered heterocycle with the nitrogen atom to which they are attached the preferred moieties are pyrrolidine and piperidine. Preferred compounds are those wherein at least one of R1 or R 2 is methyl.

In the instance of compounds of Formula IA and IB, the S 20 Pi-a-amino acid residue (arginine) may be in its D- or L-configuration, or a mixture thereof, the P 2 -a-amino acid residue (proline) preferably is in its L-configuration, and the a-amino acid residue or substituent on the a-carbon atom of the P 3 moiety the *Ii(CH 2 )nA moiety) the residue or substituent preferably is in its D-configuration, and the preferred residue is Phe and the preferred substituent is a cyclohexylmethyl moiety. In the instance of the compounds of Formula IB, the P 3 moiety is what is called a "TIC" derivative or "TIC-like" derivative (the expression "TIC" being derived from tetrahydroisoquinoline carbonyl). In such instances the bicyclic TIC-moiety formed with the P 3 nitrogen atom and the

P

3 -a-carbon atom are of the formulae 4

CH

2

CH

2 2 CH CH 2

C

N-CH HC H H N H

H

(2a) (2b) (2c) CH CH H 2 C CH wherein (2a) and (2b) represent a 1,2,3,4-tetrahydroisoquinolinyl moiety, and represent a 1,2,3,4-decahydroisoquinolinyl moiety, and (2c) and represent 2,3-dihydro-lH- 20 isoindolyl and octahydro-lH-isoindolyl moieties, respectively.

For convenience the moieties hereinafter may also be referred to as TIC-like modifications.

The compounds of this invention may be prepared by procedures analogously known in the art. In essence the synthesis of the fluorinated alkyl tripeptides of Formula I relies on a modified Dakin-West reaction of 2-phenyl-5(4H) oxazolone and the anhydrides or acyl halides of trifluoroacetic acid, pentafluoropropionic acid or difluoropentenoic acid, (depending on the desired R 3 moiety), to yield the requisite polyfluoro alkyl ketone amino acid derivatives for use as key intermediates. Further reactions then allow for the conversion of these amino acid analogs to the desired peptides of Formula I. These reactions are illustrated 1 the following reaction schemes: Reaction Scheme A

R

T

OH

HNo 0 R 1 3 AC 2 3 C 20~ 0or R'1 3 C(O)C1 NEt 3 0 0 R 3 N 0 0 0

(C

2 H4_ HN 0 0

OH

RR

NaBH 4

HII

0 (8) HC 1

A

*4*t

TFAA

3 [TFA] BocHN BocN 7C3 [NEt 3 HClGH 2 *q .1

I

I I

I.

I

0

CF

3

A-HN

LiOR 1K

THF/H

2 0 Ba cHN NBa c

NH

H2 N 4 R.' BocHN NBoc

NH

H

2 N R3

OH

(12) 011) 0 2a) -6 Reaction Scheme A (cont 'd) (1 2a)

A'

Rl 1>ProOH

R'

2 i7~ ~A' DCC/HOBT RPro N*11 1

'-I

R 2 0 T (1 3a) (1 2a) T-,,,,KProOlli 0 3 DCC /IOBT 14MM r<Pr o 0

H

(1 3b) 55 S S 013a) I0 xidation (13b) I xida tion NBoc BocHN NBoc

NH

3 r o -N r KR 1 3 0 ~H 0 Sq

*S

Sb *5 S

S.

S S

S

*5*

S

A'l R r--Pro- R 2 0N (1 4a) Ideprotection and trans formation

'TA

014b) Sdeprotection and transformation lB 7 wherein 0 is phenyl, A' is ie(CH 2 )nA, n is zero or one, R is OC(O)NH(CH 2 3

R'

1 is an N-protecting group,

R'

2 is H, or C 1 -7 alkyl,

R'

3 is -CF 3

-CF

2

CF

3

-CF

2

(CH

2 )tC(O)NHR 4 -CF2(CH 2 )tCH 2 OR4, or -CF2 2

CHC=CH

2 R"3 is -CF 3

-CF

2

CF

3

-CF

2

(CH

2

C(O)NHR

4

-CF

2 (CH2)tCH 2 OR4,

-CF

2

(CH

2 )vCH=CH 2 or CF 2

(CH

2 )tCOOR 4 T is a TIC-like moiety as defined above and depicted as 2a, b and c and 2'a, b and c, DCC is dicyclohexylcarbodiimide, NMM is N-methylmorpholine, HOBtis hydroxybenzotriazole (hydrate), TFA is trifluoroacetic acid, TFAAis trifluoroacetic acid anhydride, and A, R 4 t, v, la and Ib are as previously defined. The preferred amine protecting groups are Boc or CBz and, although the preferred protecting group on the arginine moiety is the Sdepicted Boc group, CBz may also be utilized.

SIn effecting the foregoing reactions, the N-bis-benzoylated amino acid is cyclized by standard techniques to the 5(4H)-oxazolone (Ac 2 0, 30 minutes, 90 0 C oil bath temperature) in good yield. Evaporation of the solvents affords a highly pure compound which can be used without further purification for the next step. Reaction of the oxazolone using a modified Dakin-West procedure, with the appropriate polyfluoroacid acyl halide (with NEt 3 or anhydride, is accomplished at 40 0 C (oil bath temperature) under an atmosphere of N 2 for 24 hours 1 H- and 1 9

F-NMR-

monitoring). When all of the starting material is consumed, the C-4-acylated oxazolones are the main products. Residual anhydride and polyfluorinated acids formed are removed under vacuum (0.01 torr; 25-70 0 C oil bath temperature, acetone/dry ice trap). Alternatively, Compounds 8 may be directly converted to by treatment with the R' 3 anhydrides or R' 3 acyl halides, and in such instances the intermediates are not isolated. The oily residues are then mixed with a saturated solution of freshly prepared anhydrous oxalic acid in tetrahydrofuran. (Commercial oxalic acid is dried for 16 hours at 1000C in a drying oven. Two subsequent sublimations (0.01 Torr, 90°C) afford anhydrous oxalic acid 104°C) which is transferred under an atmosphere of N 2 into a flask and stoppered with a septum.

Anhydrous tetrahydrofuran is added to the solid until most of it has dissolved (about 4 ml/g) and the resulting solutions are stirred at room temperature for about 16 hours, when gas evolution has completely ceased. Work-up EtOAc/H 2 0, aqueous NaHCO 3 brine; drying over MgSO 4 gives in satisfactory yield the desired fluorinated compounds as mixtures of the ketones and their hydrated forms.

*For the intended transformation of the a-benzamides (7) S 20 the polyfluoroalkyl ketone functionality has to be tempo- S rarily masked. This can be achieved by reduction of the ketones (NaBH 4 ;EtOH) to the alcohols The two benzam- S" ido functions are cleaved by acid hydrolysis to produce the diaminopolyfluoroalkyl alcohols Regioselective protection of the lateral amino group of the diamino alcohols as a trifluoroacetamide (10) is effected with trifluoroacetic anhydride in trifluoroacetic acid.

Guanylation of the w-amino group into the fully protected .arginine analogs (11) is effected with bis-Eoc-S-methylisothiourea in triethylamine. Liberation of the lateral-amino protecting function is effected with lithium hydroxide in tetrahydrofuran/water to yield the compounds of Formula (12) which are ready for coupling with the appropriate reactants 2 N-CH(A')C(O)ProOH and the TicC(O)ProOH) according to standard procedures well known in the art 9 [Nicolaides, DeWald, Westland, Lipnik, and Posler, J. Med. Chem. 11, 74 (1968)] to give the completely protected tripeptide alcohol analogs of arginine (13a) and (13b), respectively, which are ready for oxidation of the alcohol function to the corresponding ketones. It is to be noted that compounds (12a) differs in scope from compounds in that it adds the moiety CF 2

(CH

2 )tCOOR 4 to the R13 moiety. Thus, R" 3 embraces the R' 3 and the CF 2

(CH

2 )tCOOR 4 moieties; the latter bieng prepared according to Reaction Scheme B.

Although there are several procedures available for the required oxidation, the most preferred method for conversion of (13a) and (13b) to their corresponding ketones (14a) and (14b), the Swern oxidation procedure is most preferred. In general the Swern oxidation [see Synthesis, (1981), 1G5] is effected by reacting about 2 to 10 equivalents of dimethylsulfoxide (DMSO) with about 1 to 5 equivalents of trifluoroacetic anhydride [(CF 3

CO)

2 01 or oxalyl chloride [(COC1) 2 S 20 said reactants being dissolved in an inert solvent, e.g., Smethylene chloride (CHZCl 2 said reaction being under an inert atmosphere nitrogen or equivalently functioning S- gas) under anhydrous conditions at temperatures of about to -30 0 C to form an insitu sulfonium adduct to which is added about 1 equivalent of the appropriate alcohols, i.e., compounds (13a) or (13b). Preferably, the alcohols are dissolved in an inert solvent, CH 2 C12 or minimum amounts of DMSO, and the reaction mixture is allowed to warm to about -200C (for about 10-20 minutes) and then the reaction is completed by adding about 3 to 10 equivalents of a tertiary amine, triethylamine, N-methyl morpholine, etc.

10 Another alternative process for converting the alcohols to the desired ketones is an oxidation reaction which employs periodinane l,l,l-triacetoxy-2,l-benzoxiodol), [see Dess Martin, J.Org.Chem., 48, 4155, (1983)]. This oxidation is effected by contacting about 1 equivalent of the zlcohols with 1 to 5 equivalents of periodinane (preferably 1.5 equivalents), said reagent being in suspension in an inert solvent methylene chloride) under an inert atmosphere (preferably nitrogen) under anhydrous conditions at 0 C to 50 0 C (preferably room temperature) and allowing the reactants to interact for about 1 to 48 hours.

DLprotection of the amine protecting groups may be effected as desired after the ketones have been isolated.

Alternatively, a modified Jones oxidation procedure may conveniently be effected by reacting the alcohols with pyridinium diuhromate by contacting the reactants together in a water-trapping molecular sieve powder, a grounded 20 3 Angstr6m molecular sieve), wherein said contact is in the S: presence of glacial acetic acid at about 00C to 50 0 C, preferably at room temperature followed by isolation and then removing amine protecting groups.

Alternatively, 1 to 5 equivalents of a chromic anhydridepyridine complex a Sarett reagent prepared insitu) (see Fieser and Fieser "Reagents for Organic Synthesis" Vol.

1, pp. 145 and Sarett, et al., J.A.C.S, 25, 422, (1953)] said complex being prepared insitu in an inert solvent CH2C1 2 in an inert atmosphere under anhydrous conditions at 0 C to 50 0 C to whict complex is added 1 equivalent of the alcohols allowing the reactants to interact for about 1 to hours, followed by isolation and removing amine protecting groups.

11 In the special instance wherein R3is -CF 2

(CH

2 )tCOOR 4 the procedure for preparing necessary intermediates (corresponding to compounds of Formula but wherein the desired R 3 is -CF 2

(CH

2 )tCOOR 4 the reactions of the following reaction scheme may be utilized Reaction Scheme B HClOH 2 N (CE 2 4 N0 (14) Bis-'Boc-S-methylthiourea BocHN.-T, NBoc

NH

(CH

2 4 (16) C S 4* a 54 .554 BocHN--T,,NBoc

NH

20 Ho-- K 2 C0 3 (1)+alkylo C2 (2 t--4 S (17) O 2

N~CF

2

(H

2 tCOOR 4

OH

(18) 54 5S a. 44* (18) H 2 /Raney Nickel BocHN, NBoc

NH

HN

CF

2 (CH) tCOOR 4

OH

(19) wherein Boc, R 4 and t are as previously defined.

12 The reactants (17) may be prepared by reacting bromodifluoroethyl acetate [see R.H. Abeles and Ch. P. Govardham, Archives ofBiochemistry andBiophysics, 280, 137 (1990)] with the appropriate aldehyde, under Reformatsky conditions, to the corresponding difluoro diester alcohols. These alcohols are then mesylated, the mesylated products subjected to elimination by reaction with diazabicycloundecane (DBU) and the so-obtained olfins are reduced by hydrogenation; said mesylation, elimination and hydrogenation being effected by standard and well-known procedures and techniques such as are described in Hing L. Sham et al., Biochem. andBiophys. Res.

Comm., 175, 914 (1991). The so-produced intermediates (19), which are incorporated into the structural formula (12a), are then processed as described in Reaction Scheme A.

In the special instance wherein it is desired to prepare compounds wherein R 3 is -CF 2

(CH

2 2

CH

3 the acid anhydride or acyl halide reactants used to prepare compounds of Formula would bear the moiety -CF 2

CH

2

CH=CH

2 and, when 20 compounds (14a) and (14b) are subjected to transformations, the double bond of the olefins -CF 2

CH

2

CH=CH

2 moiety may also be reduced. In the special instance wherein R 3 is

CF

2

(CH

2 )tCH 3 with t being 3 or 4, the corresponding olefins

CF

2

CH

2

CH=CH-CH

3 or CF 2

CH

2

CH=CHCH

2

CH

3 would be reduced. For the preparation of these latter type olefins, S"the starting acyl halides or anhydrides can be prepared by the methods described by R. W. Lang et al., Tetrahedron Letters, 29, 3291 (1988).

30 Further processing of the oxidized intermediates [(14a) and (14b)] to the desired compounds of Formulae IA and IB depends essentially on the definition of the R 2 and R" 3 moieties. In those instances wherein it is desired to prepare final compounds wherein R, is H and R 2 is an alkyl, then the intermediates to be utilized would be those wherein 13

R'

1 is a protecting group and R' 2 is a C1-7 alkyl. In such instances the R' 1 protecting group and the two protecting groups on the arginine moiety would be removed by techniques well known in the art, such as by reaction with HC1 gas/ether or FTA/CH 2 C1 2 In those instances wherein both R 1 and R 2 are H, then R' 1 would be a protecting group and R' 2 would be H and again all three protecting groups (preferably Boc) would be removed as above. In those instances wherein both R, and R2 are other than H, then R' 1 is a protecting group (preferably CBz) and R' 2 is H and such intermediates (14a) would be reacted with an appropriate aldehyde formaldehyde, glutaraldehyde or succinaldehyde) in the presence of H 2 /Pd(OH) 2 /carbon in isopropanol to form the desired N,N-dialkyled, pyrrolidine or piperidine derivatives. The remaining two Boc-protecting groups on the arginine moiety would be cleaved by treatment with HC1/Et20 or TFA in CH 2 C1 2 according to standard techniques well known in the art. In the instance wherein it is desired to convert the olefinic moiety a -CFCH HCH=CH moiety) to its 20 saturated analog a CF 2

(CH

2 2

CH

3 moiety), the conversion is effected by standard hydrogenation techniques, preferably concurrently with the removal of the aminoprotecting groups.

5 The following examples illustrate the preparation of the compounds of Formula I 14 EXAMPLE 1 L-Prolinamide, N-methyl-D-phenylalanl-N-1-[3-[~(anhinoiminomethyl)aminolpropyl]-3,3,3-trifluoro-2-oxopropyl]-, dihydrochloride, hydrate STEP A: N,N'-bis-Benzoylornithine, ornithuric acid, A 250 ml flask, equipped with a magnetic stirring barr, two dropping funnels, and a pH-electrode was charged~ with 3.37 g (20 mmol) of L-ornithine hydrochloride and 40 ml of N NaOH. The stirred solut,"on was cooled to 0 0 C and solutions of benzoylchloride (5.0 ml, 44 mmol) in diethylether (50 ml) and of N NaOH (44 ml) were added simultaneously at a rate to keep the pH of the mixture at 12-13. Following complete addition the solution was allowed to warm to room temperature and stirred for further two hours. Diethylether (200 ml) was added, phases separated, and the aqueous layer (pH 0-1, HCl conc.). The white precipitate formed collected, washed with diethylether and dried (0.01 Torr) to afford 6.7 g of the title compound. m.p.: 184 0

C.

1 H-NMR (CDCl 3

CD

3 0D 1: 1):8 6=7.8 (in, 4H, benzoyl) 7. 5 (in, 6H, benzoyl), 4.6 (mn, 1H, CHN), 3.4 (mn, 2H, NCH 2 2.0-1t.5 (in, 4H, CH CH 2 *STEP B: N-LN1il(Trifluoroacetyl)-1,4-butaneaiyllbis(benzamide), hydrate, A mixture of the ornithuric acid, as described in Step A, (501.5 g, 1.47 mol) and trifluoroacetic acid (TFAA) (400 ml) was stirred at room temperature for one hour. The 15 resulting solution was evaporated (0.1 Torr) and 1 L of TFAA was added. The solution was stirred for 16 hours and evaporated to dryness (0.01 Torr). Further 1 L of TFAA was added and the solution stirred for 4 hours. Reevaporation of the solution to dryness (0.1 Torr) was followed by addition of further 500 ml of TFAA. The solution was stirred for another 16 hours and evaporated to dryness (0.1 Torr). The oily residue was dissolved in 1.6 L of anhydrous THF and freshly dehydrated oxalic acid (440 g) added to the solution over a period of two hours. Stirring of the mixture was continued for 16 hours, when 7.5 L of ethyl acetate was added. The solution was washed with brine, aqueous sodium bicarbonate and brine, dried over MgSO 4 and evaporated to dryness. Yield of the title compound: 522.8 g Rf 0.33 and 0.27 (eluent: ethyl acetate/petroleum ether ketone and its hydrate.

113-115 0

C.

IH-NMR (CDC1 3 6 8,0-7.5 4H, benzoyl), 7.5-7.0 V e (benzoyl, NH), 6.3 1H, NH), 5.0 0.5H, CH(CO)CF 3 20 2 4.5 0.5H, CH(OH) 2

CF

3 3.4 2H, NCH 2 2.2-1.6 4H,

SCH

2

CH

2 19 F-NMR (CDC1 3 6 86.00 COCF 3 80.00 CF 3

C(OH)

2 ratio 1:1, ketone and hydrate.

MS 393 (MH+/30% rel. intensity).

Analysis calculated for C 20

H

19

F

3

N

2 0 3

H

2 0 (410.40): C: 58.53; H: 5.16; N: 6.83.

Found: C: 58.62; H: 5.22; N: 6.64.

16 STEP C: N,N'[l-(2,2,2-Trifluoro-l-hydroxyethyl)-1,4-butanediyl]bis(benzamide) NaBH 4 (45 g, 1.2 mol) was added portionwise to a cooled (0 0 C) and stirred solution of the trifluoromethyl ketone described in Step B, (522 g, 1.33 mol) in 6.5 L of EtOH. The solution was stirred for 10 minutes at 0°C and then at room temperature for 16 hours. Aqueous HC1 (6N) was added and, when effervescence had stopped, solid sodium carbonate was added until the pH of the solution was basic. AcOEt was added and phases separated. The aqueous layer was extracted twice with AcOEt and the combined organic phases washed with brine. Drying of the organic solution (MgS0 4 and flashevaporation (20 Torr, 300C) afforded an oil which solidified upon standing. Yield 457.6 g 103 0 C, Rf 0.7 (AcOEt).

1 H-NMR (CDC1 3

/CD

3 OD 6 7.9 4H, benzoyl), 7.4 (m, 6H, benzoyl), 4.4 1H, CHN), 4.1 1H, CHCF 3 3.3 (m, 20 2 2H, NCH 2 2.0-1.5 4H, CH 2

CH

2 19 F-NMR (CDC1 3 /CD3OD 1:1) 6 87.33 and 85.83 (2d, JHF Hz); ratio 3:1.

A small sample (100 mg) was allowed to crystallize for CHN analysis.

Analysis calculated for C 20

H

21

F

3

N

2 0 3 (394.401): C: 60.76; H: 5.52; N: 7.05.

Found: C: 60.55; H: 5.55; N: 7.01.

6 I I! 17 STEP D: 3,6-Diamino-l,1,l-trifluoro-2-hexanol, dihydrochloride A stirred solution of the N,N'-bis-protected diaminoalcohol described in Step C (457 g) in aqueous HC1 12N (5 L) was heated for 16 hours to reflux. Solvents were evaporated and the oi.y residue dissolved in H 2 0. The aqueous solution was washed with diethylether and evaporated to dryness to afford 260 g (87% yield) of the trifluoro-diaminoalcohol, hydrochloride as a slightly colored oil.

1 H-NMR (D 2 6 4.6 1H, CHCF 3 3.8 1H, CHNH), 3.2 2H, NCH 2 2.0 2H, CH 2

CH

2 19 F-NMR (D 2 0, C 6F 6 as ext. ref.): 6 87.3 and 84.8 (2d, JHF= 7.5 Hz); ratio 3:1.

STEP E: N-[6-[(1,1-Dimethylethoxy)carbonyl]amino]-3-amino-2-hydroxy- 1,1,1-trifluorohexane SDi-tert-butyldicarbonate (217 g, 0.99 mol) was added portionwise to a cooled (-5 0 C) and well stirred solution of •the trifluoromethyl-diamino alcohol, dihydrochloride described in Step D (260 g, 1.0 mol) and NEt 3 (600 ml, 4.3 mol) in THF/H 2 0 (3 L/3 Stirring was continued after complete addition for 1 hour at 0 C and 16 hours at room temperature. The solution was evaporated to one half of its original volume, acidified (solid citric acid), washed with diethylether, basified (NaOH pellets, pH 12-13), and 30 exhaustively extracted with diethylether. The combined etheral extracts were washed with brine, dried (MgSO 4 and evaporated (20 Torr, 30 0 C) to afford the w-protected diamino alcohol.

18 1 H-NMR (CDCl 3 63 4.8 (in, 1H, CHN), 4.0-3.5 (in, 1H, CHCF 3 3.2 (mn, 2H, NCH 2 2.5 (in, 3H, exchance with D20, OH, NH 2 1.8-1.3 (mn, 4H, 2CH 2 1.45 9H, 3CH 3 1 9 F-NMR (CDC1 3 6 88.33 and 84.33 (2d, JHF= 7.5 Hz); ratio 3:1.

An aliquot (100 ing) was treated with diethylether/methanol the solid collected and dried to afford 60 mg of analytically pure above-described compound.

Analysis calculated for C 1 1

H

2 1

F

3

N

2 0 3 (286.301): C: 46.15; H: 7.39; N: 9.78.

Found: C: 46.25; HI: 7.62; N: 9.46.

STEP

F:

L-Prolinamide, N-methyl-N-[l(phenyliethoxy)carbonyill-D phenylalanyl-N-[l-[3-[ [(l,l-dimethylethoxy)carbonyllaminoLl pi.:opyl 1-3,3, 3-trifluoro-2-hydroxy-propyl I- Isobutylchloroformate (2.46 itl, 18.94 mol) was added 20 under an atmosphere of N 2 ovet a period of 10 minutes to a 9 cooled (-20 0 C) and stirred solution of N-inethyl-N-phenylmethyloxycarbonyl-D-phenylalanyl-L-proline (7.06 g, S 17.22 inol) Bajusz et al., J.Med. Chem., 1990, 33, 1729) and N-methylmorpholine (2.08 ml, 18.94 mol) in anhydrous THF .2 25 (50 ml). The reaction mix~ture was stirred for 1 hour at and a solution of the w-N-protected diaininoalcohol described in Step E,(4.92 g, 17.22 mol) in THF (anhydrous, 50 ml) was added. The mixture was stirred for 1 hour at Q 0 C and then kept for 16 hours at +4 0 C. The solvent was evaporated, the 3 0 residual oil dissolved ini AcOEt/H 2 0, And phases separated.

The organic layer was washed with saturated solutions of NaHCO 3 citric acid, and brine, dried (M960 4 and evaporated. The resulting crude tr !,peptide derivative (15 g, yellow _foam) was applied to flash chromatography on silica gel 19 (eluent: AcOEt/hexane 1:1) and the combined productcontaining fractions were evaporated (20 Torr, 30 0 C; twice with CC1 4 as cosolvent, then 0.01 Torr at room temperature, 16 hours) to afford 11.68 g of the title compound as a colorless foam.

Rf 0.24 (AcOEt/hexane 2:1).

1 H-NMR (90 MHz, CDCl 3 6 7.4-7.2 (2m, 10H, 2'aryl), 7.1 (in, 1H, NH), 6.9 (in, 1H, NH), 5.8-5.5 (mn, 2H, 2NH), 5.1 (m, 2H, arylCH0 2 4.8-3.5 [6m, 5H, 4CH, OH (exchanges with

D

2 3.4-2.6 (mn, NCH 3 2NCH 2 arylCH 2 2.2 and 1.9-1.5 (in,.

8H, 4CH 2 1.4 (in, 9H, 3CH 3 19 F-NMR (CDC1 3 6 87.1-86.8 (in).

STEP G: L-Prolinamide, N-t (2henylinethoxv)carbonyl)-N-inethyl-Dphenylalanyl-l-[3-(aininopropyl 1-3, 3,3-trifluoro-2-hydroxypropyll-, trifluoroacetate 20 A solution of 25 ml of TFA in 25 ml of CH 2 C1 2 was added to the protected tripeptide derivative described in Step F g, 10.32 inol) and the mixture was stirred until effervescence had stopped (30 minutes, bubble counter). The solution was evaporated to dryness (0.01 Torr, 40 hours) to afford a yellow foam. The title compound was precipitated from i.-propanol/petroleum ether to give, after drying, ag" 7.05 g (yield: quantitative) of 'i white solid.

RE 0.5 (AcOEt/l10% AcOH).

1 H-NMR (90 MHz, CD 3 OD) 6 =7.3 (mn, 10H, 2 phenyl), 5.1 (in, sea qO2H, CH 2 4.6-4.45 (in, 1H, CHN-Phe), 4.2 (mn, 1H, CHN-Pro), ~4.1 (in, 1H, CHN), 3.9 (mn, 1H, CHCF),3126(n9HNC, 3NCH 2 2.0-1.3 (mn, 8H, 4CR 2 20 9 F-NMR (90 MHz, CD 3 OD) (S 88.5 (in, CF 3

CO

2 H, CF 3 Analysis calculated for c 3 0 11 38

F

6

N

40 7H 2 0 710.-66): C: 52.38; H: 5.6G; N: 7.49i C: 52.59; H: 5.60; N: 7.409.

STEP i: L-Prolinamide, (phenylinethoxy)carboniyl]-N--ethyl-Dphenylalanyl-N-[l-t3-[ Ebis[[ (phenylrnethoxy)carbonyllamino]methylene]arninolpropyll-3,3,3--trifluoro-2-hydroxypropyl] N,N'-bis-Benzyloxycarbonyl-S--methylisothiourea (3.46 g, 9.7 mol) was added under an atmosphere of N 2 to a solution of the trifluoroacetate salt described in Step G (5.57 g, 8 mol) and NEt 3 (1.67 ml), in 50 ml of anhydrous THF. The solution was stirred at 40 0 C for 4 days. THF was evaporated and the solid residue dissolved in Et 2 O, The ethera.

solution was washed with a saturated solution of citric acid and brine, dried (MgSO 4 and solvents evaporated. The solid residue was applied to a flash chromatography column charged 20 with silica gel (230-400 mesh, eluent AcOEt/hexane 1:2) and when all residual isothiourea (Rf 0.8) had sorted :SosoAcOEt/hexaie 1:1-2:1 and the combined product-containing fractions were evaporated to afford 5.08 g (71% yield) of the guanidino tripeptide derivative as a colorless foam.

Rf 0.3 (AcOEt/hexane 2:1).

IH-NMR (360 M~z, CDC1 3 6S 11.5 broad, 1H, ZNH guanidine), 8.3 (mn, 1H, NH guanidine), 7.3-7.2 (mn, 22H, 4 so, phenyl, 2NH), 5.7 1H, NH), 5.1 (mn, 7H, NH, 3CH 2 4.8- 30 .9 (mn, 5H, CHN-Phe, CF CHO, CHN-Pro, CHN, NH), 3.8-2.5 (at, 9H, NCH 2 -Pro, NCH 2 guanidine NCH,, CH 2 -Phe), 1.8-1.5 (mn, 8H, 4CH 2 21 1 9 F-NMR (360 MHz, 1 H-decoupled, CDC1 3 6 87.49, 87.12, 87.00 and 85.16 (4s).

Analysis calculated for C 4 6

H

51

F

3

N

6 0 9 g0.5 H 2 0 (897.96): C: 61.53; H: 5.84; N: 9.36.

Found: C: 61.53; H: 5.75; N: 9.27.

STEP I: L-Prolinamide, N-[(phenylmethoxy)carbonyl]-N-methyl-Dphenylalanyl-N-[l-[3-[[bis [(phenylmethoxy)carbonyl]amino]- 0 methylene]amino]propyl]-3,3,3-trifluoro-2-oxopropylj, dihydrate A 250 ml three necked flask, equipped with a magnetic stirring barr, thermometer, and an N 2 inlet was charged with a solution of oxalylchloride (0.74 ml, 8.4 mmol) in 10 ml of anhydrous CH 2 C12 and placed in a dry-ice/acetone bath internal temperature). A solution of DMSO (1.2 ml, 17 mmol) in 200 ml of C, 2 C2 was added under an atmosphere of N 2 at a 20 rate to keep the internal temperature at -55 0 C. Stirring was continued for 5 minutes and a solution of the alcohol described in Step H (4.98 g, 5.6 mmol) in 10 ml of CH 2 C1 2 was added in one portion. The mixture was allowed to warm to 0 C and stirred at that temperature for 10 minutes. The solution was cooled again to -55 0 C, 3.9 ml of NEt 3 was added slowly, and the ice bath removed. When the internal Stemperature had reached -20 0 C a saturated solution of citric acid was added. 250 ml of CH 2 C1, were added at room temperature, phases separated and the organic layer washed with H 2 0, a saturated solution of NaHCO 3 and brine. Drying (MgSO 4 and evaporation of the solvents afforded 5.07 g of a yellow oil. Flash chromatography on silica gel (230-400 mesh, eluent AcOEt/hexane 1:1) and evaporation of the 22 comoined product--containing fractions gave 3.71 g of the ketone as a colorless i am.

Rf 0.5 (AcOEt/hexane 2:1).

1 H-NMR (CDC1 3 6 11.5 (broad s, 1H, NH-Z), 8.5 (in, 1H, NH 7.3 (mn, 20H, 4 phenyl), 5.7 (3m, 1H, NH), 5.1 (in, 6H, 3CH 2 4.8 (in) and 4.7-4.2 (in, 4H, 4CH), 3.9 (in) 3.7-3.3 (mn) and 2.6 (in, 9H, NCH 2 -Pro, NGH 2 guanidine, NCH 3 arylCH 2 2.0-1.5 (in, 8H, 4CH 2 19 F-NMR (CDCl 3 6 86.08, 85.96, 85.88 and 85.83 (4s1j.

Analysis f'alculated for C 4 6

H

4 9

F

3

N

6

O

9

.H

2 O (913.95): C: 60.45; H: 5.74; N; 9.20.

Found: C: 60.52; H: 5.56; N: 9.16.

STEP J: L-Prolinamide,N-methyl-D-phenylalanyl-N-f (aininoimin~amethyl)aminolpropyl]-3,3,3-trifluoro-2-oxopropvQ- d rochloride, hlydrate A solution of the ketone described in Step 1 (2.94 gs 3.3 nunol) in 100 of isopropanol, 15 ml of H1-i 1N, and g of Pd(OH) 2 /C Pearlman's catalyst) was hydrogenated under atmospheric pressure for 4 days. Evaporation 25 of the solvent left 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 AcOH/BuOH/H 2 0 1/3/1.

1 H-NMR (D 2 6 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, see*NCH 2 guanidine, NCH 2 -Pro, CH 2 -Phe, NCH 3 1y 2.2-1.4 (mn, 8H, 4CH 2 2.0 CH 3

CO

2

H).

23 1 9 F-NMR (D 6 -6.16 and 34 2s, ratio 55

CF

3

C(OH)

2 ~By substituting the TFAA reactant used in Step B of this example with equivalent quantities of pentafluoropropionic anhydride (PFPAA), and by substantially follwing thie procedures of Steps B to J, there was produced L-Prolinamide, N-wgethyl-D-phenylalanyl-N--(l-(3-( (aminoimninomethyl)aminolpropyll-3,3,4,4,4--pentafluoro-2-oxobutyll- (see Example 6, Steps A to J).

EXAMPLE '2 L-Prolinamide, N-D-phenylalanyl-N-[l-[ 3-Il(amino] iminomethyl) amino ]propyhl 3-trifluoro-2-oxopropdry4ofluoride, acetate, hydrz~te STEP A: L-Prolinamide, N-[(phenvlmethoxy)carbonyl]-D-phenylalanyl-N- :_[l-t3-Ul1,1-dimethylethoxy)carbonyllaminolprcpyl-3,3,3-trifluoro-2-hydroxvypropyl]fees.

Isobutylchloroformate (0.450 ml, 3.,6 mmoj.) was added under an atmosphere of N 2 to a cooled (-10 0 C) and stirred solution Qf phenylraethyloxycarbonyl-D-p.henyla2,anyl-L-proli ne (Z-D-Phe-Pro-OH, 1.27 g, 3.2 mmol) and N-methylmorpiloline (0.38 ml, 3.46 mmol). The reaction mixture was stirred for minutes at -10 0 C and a solution of the w-N-protected diaminoalcohol of Step E of Example 1 (0.9 ge 3.19 nUTmol ja (anhydrous, 6 ml) was added. The mixture was stirred for 1 hour at 0 0 C and then kept for 16 hours at 0 C. AcOEt/H 2 0 (100 ml each) was added and phases separated. The organic layer was washed with saturated soluitions of NaHiC0 3 citric acid, and br'ine (each 3 x50 ml) dried over 24 Ms41and evaporated. The resulting crude tripeptide flerivative (2.5 g, yellow foam) was applied to flasi chromatography (silica gel, 100 g, eluent: AcOEt/hexane 1:1) and thE combined product-containing fractions were evaporated (20 Torr, 30 0 C; twice with CC1 4 as cosolvent, then 0.01 Torr, room temperature, 16 hours) to afford 1.5 g (72%) of the title compound as a colorless foam.

Rf 0.14 (AcOEt/hexane) 1 0 H NMR (360 M~z, CDC1 3 6 7.4-7.2 (3m, 10H, 2 aryl), 7.1 lH, NH), 5.8-5.5 (in, 2H, 2NH), AB centered at 5.10 HA-HB 10H,2,ayC 2 4.8-3.5 r6m, 5H, 4CH, OH (f .w7hanges with D 2 3.2-2.9 and 2.6 (2m, 6H, 2NCH 2 f aryl,

CF.

2 2.2 and 1.9-1.5 (mn, 8H, 4CH 2 1.4 (mn, 9H1, 'jCH 3 )9

I~

1 9F-NMR (CDCl 3 6 87.1; 86.8 (2d, JHF= 7. 5 Hz) 86. 5 (in), 85.2! 85.1 (2d, J HF= 7.5 Hz), 85.0 (mn).

MS (CI/NH 3 m/e= 665 Anal Pis calculated for C 33 1 4 F NO 0-0.4 MC1 (726.258): C 54.94, H1 5.97, N 7.71 C 54.74, H 6.12, N 7.29.

STEP B: L-ProlinainiCe, N-f (ihenylmethoxy)carbonyl]-D-phenylalanyl-N- (3-aiino)propyl)-3,3,3-trifluoro-2-hydroxypropyl]-, trifiluoroacetate, hytdrate A solution of 5 ml TFA inS5 ml of CH 2 C1 2 was added to the protected tripeptide derivative (1.39 g, 2.09 inmol) and the mixture was stirred until effervescence had stopped min, bubble counter). T1he solution was evaporated to dryness (0.01 Torr, 40 hours). An aliquot (0.68 9) was taken from the resulting yellow foam, 100% for the following 25 reaction, and the rest treated with i-propanol/hexane to afford 0.73 g of the title compound as a white solid.

Rf 0.5 (Ac0Et/l0% AcOR).

1 H-NMR (360 MHz, CD 3 0D): '5 7.3 (in, 10H, 2 phenyl), AB's at 5.1 (JHA-HB= 23 Hz, 2H, CH 2 4.6 J= 7.5 Hz, and 4.45 J= 7.5 Hz, 0.5H, 1H, CHCF 3 2.9 and 2.6 (2m, 6H, 2.0-1.3 (in, 8H, 4CH 2 1'F-NMR (360 MHz, CD 3 OD): '5 88.48 CF3CO 2 88.35, 88.20, 87.21, and 87.18 (4d, JHF= 7 Hz, CF 3 ratio 16:10:3:3.

MS (CI/NH 3 m/e= 565 100% rel. intensity).

Anal. calcd. for C 3 0

H

3 6

F

6

N

4 0' 7 0.5 H 2 0 (687.64): C 52.40, H 5.42, N 8.15.

Found: C 52.30, H 5.69, N 7.83.

STEP C: L-Prolinaniide, N,-f (phenylmethoxy)carbonyl]-D-phenylalanine- 3-f bis-f l,1-dimethylethoxy)carbonyllaminolrnethyl- 20enelaminolpropyl]-3,3,3-trifluoro-2-hydroxypropyl]- 'LqN-Bis-Boc-S-methylisothiourea (Bergeron, R.J. and McManis, J. Org. Chem., 1987, 52, 17001 (1.40 g, 4.83 minol) was added under an atmosphere of N 2 to a solution of the tri.fluoroacetate salt (12) (0.82 g, 1.2 minol) and NEt 3 ml, 1.81 mmol) in 4 ml of anhydrous tetrahydrofuran.

The s(-i"tion was stirred at 40 0 C for 4 days. Tetrahydrofuran we- rated and the solid residue dissolved in Et 2 O. The et joJlution was washed with a saturated solution of citric acid and brine, dried (MgSO 4 and solvents evaporated. The solid residue was applied to a flash chromatography column charged with silica gel [30 g, 230-400 mesh, eluent AcOEt/hexane 1:2 and when all residual isothiourea (Rf= 0.8) had sorted AcOEt/hexane 1:1-2:1] and the combined 26 product-containing fractions were evaporated to afford 0.44 g of the expected quanidino tripeptide derivative as a colorless foam.

Rf 0.3 (AcOEt/'hexane: 2:1).

1 H-NMR (360 MHz, CDC1 3 65 11.5 broad, 1H, BocNIH guanidine), 8.3 (in, 1H, NH guanidine), 7.2 (in, 11H, 2 phenyl, NH), 6.0, 5.9, and 5.7 (3m, 1H, NH), AB's centered at 5.1 23 Hz, 2H, CH9Q), 4.4 (in, 1H, CHN-Phe), 4.3-3.2 (6m, 7H, CHN-Pro, NCH 2 -Pro, NCH 2 guanidine A-part, CHCF 3 1 3.0 (mn, 2H, aryl CH 2 2.6 (in, 1H, NC.H 2 guanidine B-part), 1.8-1.2 (in, 26H, 6CH 3 4C,1 2 :1 9 F-NMR (360 MHz, CDCl 3 65 86.95, 86.86, 85.39, and 85.16 (4d, JHF= 7 Hz); ratio 5:5:1:1.5.

Anal. calcd. for C 3 9

H

4 3

F

3

N

9 0 6

-H

2 0 (824.88): C 56.79, H 6.72, N 10.19.

Found C 56.53, H 6.54, N 10.05.

STEP D: V, L-Prolinamide, (Phenvliethoxy)carbonyl]-D-phenylalanyl-N- :*00 El-[3- [fbis-[ t(1,l-dimethylethoxy)carbonyl]amino]methyl- *:009:enejaininolpropylI-3,3,3-trifluoro-2-oxopropylI-, hydrate A 25 ml three necked flask, equipped with a magnetic barr, thermometer, and an N 2 inlet was charged with a solution of oxaly, lchloride (0.127 g, 1 minol) in 1 ml of anhydrous CH 2 Cl 2 anid placed in a dry ice/acetone bath (-550C internal temperature). A solution of DMSO (0.22 g, 2.8 minol) in 1 ml of CH 2 Cl 2 was added under an atmosphere of argo 2 at a rate to keep the internal temperature at -55 0 C. Stirring was continued for 5 minutes and a solution of the alcohol described in Step C (0.4 g, 0.5 nunol) in 2 ml of CH 2 C1 2 was added in one portion. The mixture was allowed to warm to 0 C and stirred at that temperature f~or 10 minutes. The 27 solution was cooled again to -55 0 C, 0.5 ml of NEt 3 was added slowly, and the ice bath removed. When the internal temperature had reached -20 0 C a saturated solution of citric acid was added. 50 ml of CH 2 C12 were added at room temperature, phases separated and the organic layer washed with water, a saturated solution of NaHCO 3 and brine. Drying over MgSO 4 and evaporation of the solvents afforded 0.34 g of a yellow oil. Flash chromatography on silica gel (0.01 g, 230- 400 mesh, eluent AcOEt/hexane 1:1, then 2:1) and evaporation of the combined product-containing fractions gave 0.24 g of the expected ketone as a colorless foam.

Rf 0.5 (AcOEt/hexane: 2:1).

1 H-NMR (CDCl 3 6 11.5 broad, 1H, BocNH), 8.5 1H, NH guanidine), 7.3 10H, 2 phenyl), 5.7 (3m, 1H, NH), 5.1 1 2H, CH 2 4.8 and 4.7-4.2 4H, 4CH), 3.9 (m) 3.7-3.3 and 2.6 4H, NCH 2 -Pro, NCH 2 guanidine), 2H, aryl CH 2 2.0-1.4 including s at 1.5, 26H, 6CH 3 4CH 2 20 19 F-NMR (CDC1 3 H-decoupled): 6 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 (CDC1 3 6 175.5-174.29 (4s, CONH), 164.1 (2s), 158.8-156.8 (4s) and 153.9 OCON, OCON=C), 136.5 and 136.2 (2s, phenyl), 130.8-128,0 (8s, phenyl), 125.57, S. 122.38, and 121.19 (visible part of q, JCF= 107.6 Hz, CF 3 95.4-94.7 [4s, C(OH) 2 83.8-83.7 (3s) and 80.0, 79.9 (2s, 68.2-67.8 (3s, CHO), 61.5-59.4 (2s, NCH-Pro), 55.6-54.4 (several 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 (several s, CH 2 -Pro, 2-tert.-butyl, CH 2 guanidine, CH 2 -Pro).

Anal. calcd. for C 39

H

51

F

3

N

6 0 9 -2H 2 0 (840.86): C 55.71, H 6.59, N 9.99.

Found C 55.75, H 6.25, N 9.78.

28 STEP E: L-Prolinamide, N-D-phenylalanyl-N-[l-[3-[ (aminoiminomethyl)arninolpropyl]-3,3,3-trifluoro-2-oxopropyl]-, hydrofluoride, acetate, hydrate The reaction flask of a "'Sakakibara apparatus" was charged with 0.1 g (0.119 rnmol) of the ketone of Step D and applied to liquid HF deprotection of all protecting groups.

After a reaction time of 20 minutes HF was evaporated, the solid residue dissolved in a mixture-of AcOH/H 2 0 1:1, and lyophilized. The solid residue was dissolved in water, the mixture filtered through a Millipore® filter, and lyophilized again to afford 40 mg of the title compound as a solid.

1 H-NMR (D 2 0) 6 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, :4 NCH 2 guanidine, NCH,-Pro, CH 2 -Phe), 2.2-1.4 (in, 8H, 4CH 2 )1

CH

3

CO

2 H) *0 1 9 F-NMR (CD 0) 6 -2.91 (s,,CF CO) -6.16 and 34 2s, ratio 55:45, CF 3

C(OH)

2 1, -54.28 1.5 HF/CF 3 4 Analysis calculated for C 2 1

H

2 9

F

3

N

6 0 3 *l.5 HFO0.5 CH 3

CO

2 H-3 H 2 0 (584.58): 25C 45.20, H 6.64, N 14.38.

25 Found C 55.75, H 6.25, N 9.78.

By substituting the TFAA reactant of Step B of Example 1 4 4with equivalent amount3 of PFPAA and by following SLCeps B, 304 C, D and E of Example and using the product of that reaction in Step A of this example and by following the teachings of Steps B, C, D and E of this example, there was produced L-prolinamide, (aminoiminoinethyl)amirio]propyl]-3,3,4,4,4-pentafluoro-2-oxobutyl]-, hydrofluoride, hydrate.

29 EXAMPLE 3 L-Prolinanide, N,N-dimethyl-D-phenylalaryl-N-[ (aminoiminomethyl)aminolpropylj-3,3 ,3-trifluoro-2--oxopropyl]-, dihydrochloride, hydrate STEP A: L-Prolinamide, N,N-(dimethyl)amino-D-phenylalanyl-N-[l-[3- [[bis-rr (1,1-dimethylethoxy)carbonyljamino)methyleneb",minolpropyl)-3,3,3-trifluoro-2-oxopropyl]-, hydrate An aqueous solution of formaldehyde 24 pil) was added to a suspension of the fully protected ketone (product of Step D, Example 2, 0.822 g, 1 mmol) in isopropanol (10 ml) and 200 mg of the catalyst Pd(OH) 2 on carbon. The resulting suspension was hydrogenated under atmospheric pressure until hydrogen uptake was stopped 24 hours).

:0.0.0Filtration from the catalyst afforded a slightly yellow solution which was evaporated to one third of its original 20 volume. The residue was diluted with 10 ml water and the solution lyophilized to give a yellow foam which was used as s.uch in the next step.

Step B: L-Prolinamide, N,N-dimethyl-D-phenylalanyl-N-[l-_r3-[(aminoiminomethyl)amino)propyl-3,3,3-trifluoro-2-oxpopyl41dihydrochloride, hydrate To a suspension of the above-prepared ketone (0.72 g, 1 mmol) in anhydrous ether (10 ml) was added a saturated solution of HCl-'gas in anhydrous ether (4 M, 50 ml).

Stirring of the mixture for 2 days and evaporation of the solvents afford the title compound as a slightly yellow foam (0.60 g).

Rf 0.5, (BuOH/AcOH/H 2 0: 3/1/1).

30 By substituting the formaldehyde reactant of Step A of Example 3 with equivalent amounts of succinaldehyde or glutaraldehyde and by following the processes of Steps A and B, there was produced the pyrrolidine and piperidine analogs, respectively, of the dimethyl-D-Phe compound of Example 3, the corresponding salts of C N -D-Phe-Pro-Arg-CF 3 and N -D-Phe-Pro-Arg-CF 3 Similarly, by substituting the trifluoroacetic anhydride (TFAA) of Step B of Example 1 with equivalent amounts of pentafluoropropionic anhydride (PFPAA) and by following the teachings of Examples 1 to 3 there was produced the corresponding pentafluoropropionyl analogs the salts of compounds

(CH

3 2 N-D-Phe-Pro-Arg-CF 2

CF

3 -D-Phe-Pro-Arg-CF 2

CF

3 and N -D-Phe-Pro-Arg-CF 2

CF

3 it being noted that the nitrogen atom of the depicted pyrrolidine and S"piperidine moieties was the terminal nitrogen atom of the D-phenylalanine a-amino acid.

*3 3 31 EXAMPLE 4 L-ProJinamide, N-ll-U 3-l(aiinoiminomethyl)amino]propyl]- 3,3,4,4,4-pentafluoro-2-oxohutyl]-l-D-[ (1,2,3,4-tetrahydro- 3--isocuinolinyl)carbonyl]-, dihydrochloride, hydrate STEP A: N-(1,1--Dimethylethoxy)carbonyl-D-1,2,3,4-tetrahydro-3-isoquinoline carboxylic acid Di-tert-butyldicarbonate (L(Boc) 2 0, 6.78 g, 31.0 mmol, 1.1 eq.] and NEt 3 (4.33 ml, 31.0 mrnol, 1.1 eq) were added to a~ solution of D-3-TIC (5.00 g, 28.2 mmol) in a mixture of water (55 ml) and THF (55 ml). The mixture was stirred at room temperature for 12 hours. After evaporation of THF, the aqueous phase was washed twice with Et 2 O, acidified with a saturated solution of citric acid and extracted three times with AcOEt. The combined organic phases were washed with brine, dried over MgSO 4 and evaporated. The residue was dried at reduced pressure (0.01 Torr) to afford the amino acid as white foam. Yield 7.32 g, 104-105 0 C, Rf =0.7 (AcOEt/A,;Ol 98:2).

MS (C 15

H

1

NO

4 277): (Cl/NH) m/e 278 40%) 239 222 [MH+ 56, M.H+ H 2

C=C(CH

3 2 178 (MH+ 100, 25 MH Boc, 100%).

1 H"NMR: (CDC1 3 90 MiHZ): 6 9.8 (broad s, 1H, COOH); 7.12 4H1, C 6

H

4 5.0 (0.5H) and 4.77 0.5H, NCR); 4.56 (d, J =9.0 liz, 2H!, NCH 2 3.12 (in, 2H, CH 2 1.4 (broad s, 9H, S **Boc).

32 STEP B: N-Cl, l-Dimetrhylethoxy) carbonyl-D-1,2,3, 4-tetrahydro-3-isoquinolinyl-L-proline-benzylester DCC (5.38 g, 26.1 mmol, 1.0 eq.) was added to a cooled and solution of Boc-D-3-TIC (Step A, 7.22 g, 26.1 mxnol) and HOBt (3.98 g, 26.1 mrnol, 1.0 eq.) in CH 2 C1 2 (200 ml). The mixture was stirred at OOW during 30 minutes, and then L-Pro-benzylester hydrochloride (6.30 g, 26.1 minol, eq.) and NMM (3.10 ml, 28.7 iniol, 1.1 eq.) were added.

The solution was stirred at room temperature during 12 hours. After filtration of the DCU, the filtrate was evaporated. The resulting residue was then dissolved in AcOEt and refiltered. The filtrate was washed twice with solutions of NaHCO 3 citric acid, and then once 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 93-95 0 C, Rf 0.5 (pettoleum ether/AcOEt 2/1) 20 M(CHN0= 464): m/e =465 100%); 365 (MH+ 100,

MS(

27

H

32

N

2 5 I4II Boc, 257 [MH+ MH+ (B3oc 225 206 1 H-NMR: (CDCl 3 90 MHZ): 6 =7.4-7.1 (in, 9H, C 6

H

4

C

6

H

5 25 255.2-4.3 4H, NCH 2 -TIC, N-CH-TIC, NCH-Pro); 5.0 (broad s, 2H, OCH 2 3.5 (in, 2H, NCH 2 -Pro); 3.0 (mn, 2H, CH 2 -TIC); 1.9 4H, 2CH 2 -Pro); 1.40 9H, Bc- STEP

C:

N-(1,1-Dimethylethoxy)carbonyl-D-1,2,3,4-tetrahydro-3-isoauinolinyl-proline Palladium hydroxyde on carbon Pd(OH) 2 C, Pearlmnan's catalyst) was added to a solution of Boc-D-3-Tic-L-Probenzy'Lester (Step B) (9.69 g, 20.9 mniol) in isopropanol 33 (150 ml). The mixture was hydrogenated at atmospheric pressure and at room temperature for 12 hours during which the acid precipitates. MeOH (100 ml) was added and the solution was filtered over celite and the filtrate evaporated. The solid residue was washed with Et 2 O and then with petroleum ether. The Boc-protected dipeptide was obtained upon drying at reduc7.d pressure (0.01 Torr) as a white solid. Yield: 6.60 g 216-218*C (decomposed), Rf 0.75 (BuOH/AcOH/H 2 0= 3:1:1) MS (C 2 0

H

26

N

2 0 5 374) 1

CI/NH

3 m/e 375 (MH 4 50%) 275 Boc, 257 [MH+ (Boc H 2 0)0 100%) Analysis calculated for C 2

,H

2 6

N

2 0 5 0.25 H 2 0 (378.9)z C: 63.39; H: 7.05; N: 7.39 Found: C: 63.54; H: 6.94; N: 7.49.

[aID: -<15.5 (c 1, MeOH).

1 H-NMR: (CD OD( 360 MHz): 6=7.2 (broad s, 4H1, C 3 G. 5.

and 4.9 (2 m, 1H, NCH-TIC); 2 AB systems ceintered at 4.58 4.75 (J 16 Hz); B: 4.43 (J 16 Hz)3, 2H1, NCH 2

-TIC));

4.3 (in, 1H1, NCH-Pro); 3.9-3.4 (in, 2H, NCH 2 3.3-2.9 (mn, 211,

CH

2 -TIC); 2.3-1.9 (2 mn, 4H1, 2CH 2 15(ra o) 25 STEP D: L-Prolinamide, (,l-diinethylethoxy)-carbonyllamino]methyleneilaiinolpropyll-3,3,4,4,4-pentafluoro-2- *'.,,hydroxybutyll-l-[ (1,1-dimethylethoxy)carbonyl]-D-[ (1,2,3,4tetrahydro-3-isoguinolinyl)carbonyl- DCC (0.255 g, 1.2 inmol, 1.0 eq., was added to a cooled and stirred solution (0 0 C) of Boc-D-3-Tic-Pro (0.462 g, 1.2 inmol) and HOBt (0.189 g, 1.2 inxol, 1.0 eq.) in CH 2 C1 2 ml). The mixture was stirred at 0 0 C during 30 minutes, when the amino alcohol (12) (Reaction Scheme A) (0.59 g, 34 1.2 rnxol, 1.0 eq.) and NMM1 (0.15 ml, 1.4 mxnol, 1.1 eq.) were added. The solution was stirred at room temperature during 12 hours. The mixture was filtered from the precipitated DCU. The filtrate was washed with saturated solutions of citric acid, KHC0 3 and brine, dried over MgSO 4 and evaporated. The resulting residue was applied to flash chromatography on silica gel (eliient: petroleum ether/AcOEt: 3/2. The tripeptide alcohol analog was isolated as a white solid. Yield: 0.62 g 102-104 0 C, Rf 0.3 (petroleum ether/AcOEt =1:1) MS (C 3 8

H

5 5

N

6 0 9

F

5 834): (Cl/NH 3 m/e 635 (MH+ 200, M 2 Boc, 100%); 593 [MH+ 242, MH+ (2 Boc =NH) 535 (MH+ 300, M.H+ 3 Boc, 1 H-NMR: (CDCl 3 360 MHz): 6 11.5; 8.4 and 7.5 (broad 3s, 3NH); 7.2 (in, 4H, C H 4 5.1 and 4.8 (broad 2s, 1H, NCR- TIC); 4.80 J 11.8 Hz, 2H, NCH 2 -TIC); 4.7-4.4 (mn, 2H, V. 20 NCR-Pro, CF CH); 4.3-3.7 (in, 3H, NHCH, NCH-ro;3635 3.5-3.3 and 3-.1-3.0 (3 m, 5H, 2CR 2 OH); 2.4-1.6 (in, BH, 4CR 2 1. 5 (broad 4, 27 H, 3 Boc) 1 9 F-NMR: (CDCl 3

C

6

F

6 ext. ref., 360 M4Hz): 6 79.12; 78.94; 78.90 and 78.74 (4s, 3F, CF 3 2 diastereoisomers and 2 isomers cis/trans: ratios ABX system centered at 35.98 40.57 (JFA-.FB 280 Hz); B: 31.39 (JFB-FA, 280 Hz; JFI .HX 30 ABX system centered at 3582 3984 (FA.F 280 Hz); B: 32.30 -JBFA 280 Hz; JFB-HX =30 Hz)]I; ABX system centered at 33. 95 39. 18 (JFA-FB 280 Hz) B: 28. 72 (JFB-.FA 280 Hz; JFB-HX 30 Hz)] ABX system centered at 34.41 39.18 (JFA-FB 280 Hz) B: 29.64 (JFB-FA 280 Hz; JFB-HX 30 Hz)]I; 2F, CF 2 2 diastereoisomers and 2 isomers cis/trans: ratios: 7.5:5:1.5:1).

35 Step E: L-Prolinamide, [(1,1-dimethylethoxy)carbonyllamino~methylenelaminollpropyl]-3,3,4,4,4-pentafluoro-2-oxobutyl]-l-j[l ,l-dimethylethoxy)carbonyl ((l,2,3,4-tetrahydro-3-isocruinolinyl)carbonylj- A solution of oxalyle chloride (0.122 ml, 1.4 mi-nol, 2. 0 eq.) in 3 ml of anhydrous CH 2 Cl 2 was cooled under 112 to 0 C. A solution of dimeti-lsulfoxide (0.197 ml, 2.8 mmol, eq.) in 6 ml of anhydrous CH 2 C1 2 was then added dropwise so as to maintain the internal temperature at -55 0 C. The mixture was stirred for 5 minutes and then a solution of the above-prepared aminoalcohol (Step D, 0.58 g, 0.7 mnxol) in ml anhydrous CH Cl 2 was added dropwise. After complete addition, temperature was allowed to raise to -20 0 C during 5 minutes. The solution was then again cooled to -55 0 C for hour. N~t 3 was then added (0.48 ml, 3.5 mmol, 5.0 eq.) jo as to maintain internal temperature at -55 0 C. After minutes a saturated solution of citric acid (2 ml) was The mixture was allowed to warm up to room temprrature and 40 ml of CH 2 Cl 2 was added. Phases were separated and the orilanic phase was washed with water and brine, dried over Na 2

SO

4 and evaporated. The resulting residue was applied to flash chromatography: 50 mg of A1 2 0 3 (pH: 7.5, pretreated with 3 ml of water; 100 mg eluent: petroleum ether/CHCl 3 The product-containing fractions were pooled and evaporated. The expected compound was obtained as a white foam. Yield. 0.25 g Rf: 0.25 (petroleum ether/'CHCl 3 1:2) MS (C 38 53

N

6 0 9

F

5 82:(/N 3 m/e 270 257 00(100%); 220 36 'H-NMR: (CDCl 3 360 MHz) 6 11. 5; 8. 4 an, 7 .9 (broad s, 3H, 3NR); 7.2 (in, 4H, C 6

H

4 5.0-4.2 [im, 5H, NCH 2 -TIC, NCR-TIC, NCR-Pro, (cetone or hydrate)); 3.8 [broad s, OR of 5hydr5,te)]1; 3.7-3.0 (3 m, 6H, CH 2 -TIC, NCH 2 -Pro, NCH 2 2.4-1.6 (mn, 8H, 4CR 2 1.5 27 R, 3 Boc).

19 F-NMR (CDCl 3

C

6

F

6 ext. ref., 360 M~z) 6 82.81 and, 82.65 (2s, 3F, CF 3 CF C(OR) 2 80.10 and 80.07 (2s, 3F, CF CFCO) ratio 1:2; 40.58; 40.53 and 40.40 (AB systems badly resolved, 2F, CF 3

CF

2 41.05 and 37.92 (AD systemts badly resolved, 2F, CF 3

CF

2

C(OR)

2 ratio 2:1.

STEP F- L-Prolinamide, N-[i-f [3-_[__(aminoiininomethyl amino] ro 1 3,3,4,4,4-pentafluoro-2-oxobutyj.I-l-D-( (1,2,3,4-tetrahydro- 3-isoguinolinyl)carbonyl.]-, trichlorohydrate TheproeceA rieptdeanalog (Step E,02 g, .0.3 nunol) was dissolved in 25 ml of anhydrous Et 2 O. A 20 saturated solution of HCl gas in Et 0 (100 ml) was added and the resulting mixture was stirred at tcom. temperature for 48 hours. The mixture was evaporated to dryness. The oily residue was dissolved in a minZmum of water and the solution filtered over a Millipore® filter disk. Tihe filtrate was yophylized to give the title compound as a white solid.

Yield: 0.142 g mn.p.: 158-160 0 C decomposition, Rf =0.7 (BuOH/AcOR/H 2 0= 3:1:1).

MS: (C 2 3

H

2 9

N

6 0 3 Fq 532) (Cl/Nl 3 m/e 533 40%) 274 257 256 220 Analysis calculated for C 2 3

H

29

N

6 0 3 FS; 3 HC1; H2zO (659.9) C: 41.86; Il: 5.19; N: 12.73 Found: C: 41.41; Il: 5.05; N: 12.63.

IR: v 3383 CNR; 1654 CONR) cm- 1 37 1 H-NMR: (D 0, TSP exct. ref., 360 MHz): 6 7.3 4H, C 6 4.9, 4.8 and 4.7 (3 i, 1H, NCH-TIC); 4.55 and 4.3 (2 m (AB systems badly resolved), 2H, NCH 2 -TIC); 4.5 superoos-d (m, 2H, NCH-Pro, NCH); 3.7 2H, NCH 2 -Pro); ABX syqtem centered at 3.38 3.50 (JHAHB 13 Hz; JHA-HX 3 Hz); B: 3.15 (JHB-HA 13 Hz; JHA.HX 3 2H, CH 2 -TIC); 3.25 (broad s, 2H, NCH 2 2.4 and 2.0 (2 in (AB systems badly resolved), 2H, CH-CH,-Pro); 2.1 211, CH 2 -Pro); 2.1 and 1.6 (2 m, 4H, 2CH 2 19 F-NMR (D 2 0,-C 3 COOH, 360 Mfz) 6 -3.62 and -3.65 (2s, 3F, CF31; AB system centered at -47.85 -48.20 (JFA-FB 280 Hz); B: -47.49 (JFB.FA 280 Hz); AB system centered at -47.97; -48.62 (JFA-FB 280 Hz); B: -47.33 (JFB-FA 280 Hz]; ratio 46:54.

too I EXAMPLE L-Proinamide. 1- (sminoinonethvl) amino] propvlb- 313_, 4,4 4-pentnifluoro-2-oxobutvll-4,234-tetrahy dro-lisoguinojnyl)carbrx:yl]-, dihvrdrochlorider hydrate 25 STEP Ati (l,1 Dimethylethoxy)carbonv I ,2,3,4-tetrahydroisog. inoline-i-carboxylic acid o o Di-tert-Butyldicarbonate (Boc 2 O, 8.2 g, 37.6 miol, eq.) was addec to a stirred solutiod1 of l12,3,4-tetrahydroisoquinaldic acid (prepared from isoquinoline-lcarboxylic acid accotding to W. Solomon, J.Chern.,Soc, 1947, 129), 6.0 g, 33.8 mmol] and NEt 3 (5.2 ml, 37.6 mmol, 1.1 eq.) in SO ml of tetrahydrofuran and 50 ml of witer. The 38 mixture TJas stirred at room tem~perature for 16 hours, when tetiahydrofuran was evaporated. The residual aqueous phase was nicid.fied (solid citric acid) and the protected amino acid extracted with AcOEt (three times). The combined organic layers were washed with brine, dried over MgSO 4 and evaporated (20 Torr, 30C =,nd 0.01 Torr, 20 0 C) to afford g of the protected amino acid.

STEP B: (l,l-Dimethylethpxv)carbonyl)-[ (1,2,3,4-tetrahydro-l-isoquinolinyl)carbonyl]-L--proline benzyl ester Dicyclohexylcarbodiimide (DCC, 5.38 g, 26 mraol) was added to a stirred and cooled solution of the protected amino acid (Example 5; Step A; 7.2 g, 26 mxnol) and~ hydroxybenzotriazol hydrate (HOBtr 3.98 gf 26 nunol) in 100 ml o' dichioromethane and 10 ml of tetrahydrofuran.

AfteL stirring for 30 minutes at tiC, the mixture was allowed to warm up to room temperature for 1 hour and cooled to 0 0 C again. L-Prolirie benzylestor hydrochloride (6.3 g, 26 mmol) and N-'methy) *orpholine (NMM, 3. 2 tai, 2G imol) were added to the mixture and the resulting mixture stirred for minutes at OOW and 16 hours at room temperature.

Filtration of the mixture and evaporation of the so~lvents 25 gave an oil which was dissolved in CH 2 C1 2 The solution was washed witht, sat'.jrated solutions of citric acid, voium hydrogen carbonate, and sodium chloride to afford after drying (MgSO 4 and evaporation of zolvents (20 Torr, 301C, and 0 .01 Torr, 20) 10. 5 g of a colorless oil 0.8 (AcOEttpetroleum ether 1:2).

39 STEP C: N-l(1,1-Dimethvlethoxy)carbonl]-I(1,2,3,4-tetrahydro--l-isocquinolinyl)cgarbonyl I-L-proline A solution of the protected dipeptide (Example Step B; 9.3 g, 20 mmol) and 500 mg of 10% palladium hydroxide on carbon (Pearlman's catalyst) in 150 ml of isopropranol was hydrogenated at room temperature and atmospheric pressure for 14 hours. Filtration from the catalyst and evaporation of the solvent gave 8.0 g of a semisolid oil, which was partially dissolved in methanol/Et 2 O (1:1 v/v. Filtration of the mixture gave 2.7 g of a colorless oil. Total yield Rf solid 0.80 (BuOH/AcOH/H 2 0 3/1/1) Rf oil 0.80 (BuOH/AcOH/H 2 0 3/1/1) ~STEP D: L-Frolinamide, N- [bis [U 1, 1-dimethylethoxy) carbon- 20 jjamino]methylenelaminolpro2vll-3,3,4,4,4-pentafluoro-2hvdroxybutyl]-N-[ (l,l-dimethylethoxy)carbonyl-l-[l(1,2,3,4tetrahydro-l-isoguinolinyl )-carbonyl 1- Dicyclohexylcarbodiimide (DCC, 0.255 g, 1.2 mmol) was Se. 25 added to a cool-d (0 0 C) and a stirred solution of the aboveprepared protr-;ted dipeptide (Step D, 0.374 g, 1.2 mmol) and hydroxybenzotriazol hydrat- (HOBt, 0.189 g, 1.2 mmol) in ml of dichloromethane. The mixture was stirred for minutes at 0 0 C and 1 hour at room temperature and cooled again to 0 0 C. The Bis-Boc prot-ected arginino analog (see Example 6, Step F; 0.590 g, 1.2 nunol) and N-inethylmorpholine (150 ml, 1.4 mmol, 1.1 eq.) were added. The solution was stirred at roomn temperature for 12 hours. Filtration of the mixture was followed by washing of the filtrate with 11 saturated solutions o-I citric acid, KHCO 3 and brine. The 40 organic solution was dried over Mg0O 4 and evaporated. The residual oil was applied to flash chromatography on silica gel (10 g, eluent: AcOEt/petroleum ether: The tripeptide alcohol analog was isolated as a colorless oil.

Yield: 0.610 g Rf 0.3 (AcOEt/petroleum ether: 1/1).

STEP E L-Prolinamide, N-[l-[3-[[bis[[(l,l-dimethylethoxy)carbonyl]amino]methyl ene]amino]propyl]-3,3,4,4,4-pentafluoro-2-oxobutyl]-N-[(l,l-dimethylethoxy)carbonyl]-l-[(1,2,3,4-tetrahydro-l-isoquinolinyl)-carbonyl]- A solution of oxalylchloride '0.122 ml, 1.4 mmol, 2.0 eq.) in 3 ml of anhydrous CH 2

CI

2 was cooled under argon to -60 0 C. A solution of DMSO (0.197 ml, 2.8 mmol, 4.0 eq.) in 6 ml of anhydrous CH 2 C1 2 was then added dropwise to maintain temperature of the solution at -550 0 C. The mixture was stirred for 5 minutes and a solution of the alcohol prepared in Step D (0.58 g, 0.7 mmol in 5 g of anhydrous

CH

2 C12 was added dropwise; After complete addition, internal temperature was allowed to raise to -20 0 C (5 minutes) and the solution was cooled to -55 0 C for 1 hour. Triethylamine (0.48 ml, 3.5 mmol) was then added at a rate to maintain 25 internal temperature at -55 0 C. After 5 minutes, a saturated solution of citric acid (2 ml) was added. The mixture was allowed to warm up to room temperature and 40 ml of CH 2 Cl1 was added. Phases were separated and the organic ph. se was washed with water and brine, dried over MgSO 4 and 30 evaporated. The resulting oily residue was flash ch tographed on A1 2 0 3 (pH: 7.5, pretreated with 3 ml wate r0 g eluent: petroleum ether/CHC13: The product-coitai.ing fractions were pooled and evaporated to afford a coloi ess foam. Yield: 0.30 g Rf 0.25 (petroleum ether/CHC13: 1/2).

41 STEP F: L-Prolinamide, N-[1-f3-I(aminoiminomethyl)aminc1.

2 ~y1l- 3,3,4,4,4-pentafluoro-2-oxobutyl]-l-f (1,2,3,4-tet~ahvdro-lisocuinolinyl)carbonyl]-, dihydrochioride, hydrate The protected tripeptide alcohol of Step F (0.30 g, 0.36 mmol) 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 at room temperature for 24 hours. The mixture was filtered and the solid residue further dried (0.01 Torr, 20 0 C, 16 hours) to give the title ketone (hydrate form) as a white solid. Yield: 0.160 g, Rf 0.7 (BuOH/H 2 0/AcOH: 3/1/1).

EXAMPLE 6 L-Prolinamide, N-methyl-D-phenylalanyl-N-[l-[ 3-f (aminoiminomethyl)amino]propv1I-3,3,4,4,4-pentafluoro-2-oxobutylj-I dihydrochloride, _hydrate STEP A: jjnL Jl-(Pentafluoropropionyl)-,4-butanedil]-bis(benz- 5 amide), hydrate Pentafluoropropionic anhydride (31.8 ml, 50 g, 161 mrnol) *was added under an atmosphere of N 2 to a well stirred powder of the ornithine 5(411)-oxazolone (see Example 1, Step 2; 30 Reaction Scheme A, Structure 14.05 g, 4.3 minol). The resulting mixture was stirred at 40*C for 16 hours. At this time an aliquot (about 50 mg) was taken for a 1 H- and 1 9

F-NMR

spectrum (CDCl 3 The disappearance of the 1 H-signal at ppm indicates disappearance of oxazolone, whereas th- 1 9 F-signals at 42.7; 46.7 (2s, 2xCF 2 and 80.0; 80.5 (2s, 42 2xCF 3 indicate the formation of 3'-N-4-bispentafluoropropionyl oxazolone. The additional 19F-signals were assigned to the excess of pentafluoropropionic anhydride (PFPAA) and the pentafluoropropionic acid (PFPA) formed. Solvents were then evaporated at 55-60 0 C (0.5-1 Torr, dry ice-acetone trap) for about 6 hours to give a thick orange oil. A 19

F-NMR

spectrum of another aliquot indicates disappearance of all PFPAA and PFPA. At this time 40 ml of a saturated solution of oxalic acid (15.0 g, 150 mmol) in tetrahydrofuran was added and the resulting orange oil was stirred at 55 0 C for 6 hours when effervescence has totally stopped. The solvent was evaporated (20 Torr, 30 0 C) and the oily residue dissolved in AcOEt. This solution was stirred for 15 minutes at room temperature with a saturated solution of KHCO 3 (intended hydrolysis of pentafluoropropionamide). Phases were separated and the organic layer was washed with water, IN HC1, and brine, dried over MgSO 4 and evaporated (20 Torr, 30 0 C; then 0.1 Torr, 30 0 The resulting orange oil (21.7 g) was subjected to flash chromatography on silica gel 2. 0 (500 g; eluent AcOEt/ petroleum ether: 1/3) in two batches of about 11 g. Fractions 11-20, containing the N-pentafluoropropionamide A-2 were evaporated to give 7.0 g Fractions 28-105 containing the ketone A-i were evaporated to :afford 10.1 g as a white solid.

25 Total yield: 79% based on oxazolone.

A-1 H-NMR (CDC1 3 6 8.0-7.85 5H, aryl, NH), 7.8-7.4 "n 6H, aryl), 5.5-5.3 1H, CHCO), 3,9 (broad, t, 2H,

NCH

2 2.3-1.8 (m,4H, 2CH 2 3.0 19 F-NMR (CDC1 3 6 40.33 J= 7.5 Hz, CF 2 CO), 46.67

CF

2 CONH), 80.0 2CF 3 43 A-2 1 H-NMR (CDC1 3 6 8.1-7.8 4H, aryl), 7.7-7.4 (m, 7H, aryl,NH), 6.6 1H, 5.3 1H, CHCO), 3.7 (broad t, 2H, NCH 2 2.4-1.8 4H, 2CH 2 19F-NMR (CDC1 3

/C

6

F

6 6 40.3 J= 7.5 Hz, CF 2 80.0

CF

3 MS (CI/NH 3 443 A small sample of A-2 (100 mg) was allowed to 1 crystallize from AcOEt/petroleum ether to give 80 mg of analytically pure title compound: Analysis calculated for C 21

H

19 0 3

N

2

F

5 (442.39): C: 57.02; H: 4.33; N: 6.33 Found: C: 57.14; H: 4.23; N: 6.36.

STEP B: N,N'-[l-(2,2,3,3,3-Pentafluoro-l-hydroxypropyl)-1,4-butanediyl]-bis(benzamide) 2The reduction of the two ketones A-1 and A-2 was performed in two separate reactions: 1) Reduction of A-i: NaBH 4 (0.43 g, 11 mmol) was added in one portion to a 25 cooled and stirred solution of the pentafluoroethyl ketone A-1 (10.1 g, 17.1 mmol) in EtOH (130 ml). The mixture was allowed to warm up to room temperature and further stirred for one hour. Hydrochloric acid (6N) was added carefully until effervescence has stopped. The solution was 30 S neutralized with Na 2

CO

3 and EtOH evaporated. The resulting mixture was redissolved in AcOEt/water and phases separated.

The aqueous layer was extracted twice with AcOEt and the combined organic phases washed with water and brine. Drying over MgSO 4 and evaporation of solvents afford a white solid, 44 which was subjected to flash chromatography on silica gel (300 g, eluent AcOEt/petroleun ether: 1/1, then 4/1).

Product-containing fractions were evaporated to give 5.88 g of the desired alcohol as a white solid. Rf 0.45-0.50 (AcOEt/petroleum ether: two badly separated spots for the diastereoisomers.

2) Reduction of A-2: As described above for A-1: 6.91 g (15.6 rnmol) of the ketone A-2, 300 mg (7 9 mmol) NaBH 4 and 90 ml of EtOH afforded 6.05 g of th-e alcohol pentafluoro-l-hydroxyprop ,j-,4-butanediyl]-bis(benzamide) as a white solid, which was in all aspects comparable to the compound obtained above.

1 H-NMR (CDCl 3

,CD

3 OD) 6 7.8-7.5 (in, 4H, aryl), 7.45-7.1 (mn, :00.6*6H, aryl), 4.5 and 4.2 (2m, 1H, CHOHI, ratiAo 3.5 (in, 4H, 2CH 2 19 F-NMR (CDC1 3 CD OD, CF 6 i ABX system centered at 36.3; 4 0. 3 (JFA-FB 8 0 Hz JFA-HX 3 Hz) B: 3 2. 3 (JFB-FA 280 Hz, JFB-HX =30 Hz, CF 2 79.0 CE' 3 diastereoisomer 1.

ABX system centered at 35.3; A: 36.3, B: 33.3 (with equal coupling constants as mentioned above) diastereoisoner 2; too. ratio 3/1.

Analysis calculated for C 21 1fl 2 1 0 3

N

2

F

5 (444.40) .000 C: 56.76; H: 4.76; N: 6.30 30 Found: C: 56.94; H: 4.83; N: 6.29.

45 STEP C: 4,7-Diamino-l,1,1,2,2-pentafluoro-3-heptanol, dihvdrochloride A stirred solution of the above-prepared alcohol (Step B) (11.78 g, 26.6 mmol) in concentrated aqueous hydrochloric acid (240 ml) was heated under stirring to reflux while the progress of hydrolysis was followed by TLC (BuOH/water/AcOH After 16 hours of reaction time solvent was evaporated and the oily residue subjected a second time to the above conditions. When the complete formation of the bisamino alcohol was indicated by TLC, the solution was cooled to room temperature and solvents evaporated. The oily residue was dissolved in water and the solution washed with Et2O (3 x 100 ml). The aqueous layer was evaporated to dryness to afford 8.14 g of the desired diamino alcohol as a brownish foam.

1 H NMR: (D 2 0) 6 4.6 1H, CHOHCF 2 3.1 2H, NCH 2 3.6 1H, CHN), 2.0 4H, 2CH 2 STEP D: 4-Trifluoroacetylamino-7-amino-1,1,1,2,2-pentafluoro-3heptanol, hydrochloride Trifluoroacetic anhydride (3.55 ml, 25 mmol) was added 25 dropwise to a stirred solution of the diamino alcohol of Step C (3.09 g, 10 mmol) in 50 ml trifluoroacetic acid.

After two hours of stirring at room temperature, another ml of TFAA was added to the solution and stirring was continued for 10 hours. The solution was evaporated to dry- 30 ness, giving a brown oil. Trituration with ether afforded a brownish solid, which was filtered and washed with petroleum ether. Drying gave a slightly colored solid of the title compound (3.48 g, which was pure Senough for the following reaction.

46 1 H-NMR (D 2 0) 6 4.6 2H, 2CH), 3.1 2H, NCH 2 2.0-1.7 4H, 2CH 2 19 F-NMR (D 2 0, ref. CF 3

CO

2 H) 6 ABX system centered at -49.00) A: -44.00 (JFAFB 280 Hz); B: -54.00 (JFB-FA 280 Hz) JFB-HX 30 Hz) isomer 1; ABX centered at -49.33, A: 45.00, B: 40.67 (coupling constants as above) isomer 2; ratio 4:1.

MS: (CI/NH 3 333 STEP E: N-[1-[7-bis[(1,1-Dimethylethoxy)carbonyl]-amino]methylene)amino)-N(4-trifluoroacetylamino)-3-hydroxy-l,l,l,2,2pentafluoroheptane Bis-Boc-S-methylisothiourea (7,3 g, 25 mmol) was added under an atmosphere of N 2 to a well stirred solution of the hydrochloride salt of Step D (5.1 g, 10 mmol) and NEt 3 20 (3.5 ml, 25 mmol) in anhydrous tetrahydrofuran (100 ml). The mixture was stirred at 40 0 C for 60 hours. Solvents were evaporated (a trap filled with an aqueous solution of KMnO 4 /Na 2

CO

3 was placed between the flask and the pump to avoid poisoning with methanethiol), and the oily residue 25 dissolved in AcOEt. This solution was washed with water, saturated solutions of citric acid, NaHCO 3 and brine.

Drying over MgSO 4 and evaporation of the solvents afforded s*e. an oil (10 g) which was subjected to flash chromatography on silica gel (50 g, eluent AcOEt/petroleum ether: 1/8, then 30 Product-containing fractions were evaporated ,o afford 2.92 g of the protected w-guanidino-y-amino alcohol as a colorless foam.

47 'H-NMR (CDC1 3 360 MHz) 6 11.20 1H, NH), 10.31 J Hz, 1H, NHCOCF 3 9.73 (broad s, 1H, NHCH 2 4.45 J Hz, 1H, CHN), 4.25 d, J (22 Hz, CHON), 3.75 (mn, 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 tert-Boc).

1 9 F-NMR (CDC1 3 ref. C 6

F

6 S5 ABX system ce~ntered at 35.50; A: 39.33 (JFA-FB 277 Hz), B: 32.30 (JFB-FA 277 Hz; JFB-HX =22 Hz) =isomer 1; ABX system centered at 34.00; A: 40.05 (jFA-FB 277 Hz) B: 27.5 (JBA=277 Hz, JF~X=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, CF3CO) Analysis calculated for C 2 0

H

3

OF

8

N

4 0 6 .0.5 H 2 0 (574.47): C: 41.17; H: 5.36; N: 9.60.

Founl: C: 41.06; H: 5.15; N: 9.57.

STEP F: N-[l-r7-rbis(1,1-Dimethylethoxy)carbonyllaminolmeth-ylenel1amino-4-amino-3-hydroxy-1 2-pentafluoroheptane A freshly prepared aqueous solution of LiOR (1N, 7 ml) was added to a stirred solution of the trifluoroacetamide of Step E g, 5.2 mmcl) in tetrahydrofuran/water (9/1, ml). The solution was stirred at room temperature for ?0 hours, when starting material has disappeared (TLC, AcOEt/petroleum ether: Tetrahydrofuran was evaporated and the aqueous solution extracted with Et 2 O (4 x 50 ml).

The combined extracts were washed with water and brine, and dried over MgSO 4 Evaporation of the solvent afforded 2.18 g a. 30 of the above-described aminoalcohol as a white solid.

1 H-NMR (CDC1 3 6 11.5 (in, 1H, NH), 8.4 1H, NH), 4.1 and 3.9 (2m, 1H, CHOH), 3.5 and 3.2 (2m, 3H, CEN, NCR 2 2.5 (in, 2H, NH 2 4 (in, 4H, 2CH 2 1 1. 50 1811, 2 te rt-Boc).- 48 19 F-NMR (CDC1 3 6 ABX system centered at 37.3; A: 43.67, (JFA-FB 280 Hz; JFB-HX 22 Hz, CF 2 isomer 79.0 (s, CF3) and ABX system centered at 39.0 (coupling constant as above), (CF 2 isomer 79.5 CF 3 ratio 4:1.

Analysis calculated for C 18

H

31

F

5

N

4 0 5 (478.46): C: 45.19; H: 6.53; N: 11.71.

Found: C: 45.13; H: 6.44; N: 11.56.

STEP G: L-Prolinamide, N-[(1,1-dimethylethoxy)carbonyll-N-methyl-Dphenylalanyl-N-fl-[3-f[bisr[(1,1-dimethylethoxy)carbonyl]amino]methylene]aminolpropyl]-3,3,4,4,4-pentafluoro-2hydroxybutyl]- Dicyclohexylcarbodiimide (0.96 g, 4.7 mmol) was added to a stirred and cooled solution of Boc-N(methyl)-D-Phe- Pro-OH (1.78 g, 4.7 mmol) and N-hydroxybenzotriazole (0.711 g, 4.7 mmol) in CH 2 C1l (50 ml). The mixture was stirred for 30 minutes at 0°C, when the above-prepared amino 20 alcohol (Step F) (2.22 g, 4.64 mmol) and NMM (0.52 ml, 4.7 mmol) were added. The resulting mixture was further stirred for 0.5 hour at 0 C and then allowed to warm up to room temperature. Stirring was continued for 16 hours at room temperature. Filtration of the precipitated DCU and 25 washing of the filtrate with saturated solutions of citric acid, KHCO 3 and brine was followed by drying over MgSO 4 and evaporation of the solvent to afford a viscous oil. Flash chromatography on silica gel (150 g, eluent AcOEt/petroleum ether: 1/1) and evaporation of the product-containing 30 fractions afforded 3.09 g of the desired tripeptide alcohol as a colorless oil.

49 'H-NMR: (CD3OD, 360 MHz) 6 7.2 (mn, 5H, aryl), 5.1-4.9 (2m, 1H, CH-Phe), 4.4-3.9 (in, 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 (in, 8H, 4CH 2 1.5-1.1 (6s, 27H, 3 tert-Boc).

1 9 F-NMR (CD 3 OD, C 6

F

6 ext. ref., 360 MHz) 6 about 3 ABX systems centered at 43.7 (CF 2 -isomer 1 and 2; cis-transisomers) and 81.80, 81.55, 81.30 (3s, CF 3 of duff, isomers).

MS (FAB): 837 STEP H: L-Prolinamide, N-f 1-diiethylethoxy)carbonyl 1-N-methyl-Dpenylalanl-N-f-r3-r fbisf [(1,1-dimethylethoxy)carbonyllaminolmethylenelaminoipropyll-3.,3,4,4,4-pentafluoro-2-oxobutyll- C loe* A 100 ml three-necked flask equipped with a magnetic stirring bar, thermomet~er, and a N 2 -inlet was charged with a 2 solution of oxalylchloride (0.52 ml, 5.7 nunol) in 5 ml of too**:anhydrous CH 2 Cl 2 After cooling the solution to -60 0 C, a solution of dimethylsulfox'L.de (1.2 ml, 14.3 inmol) in 10 ml of anhydrous CH 2 C1 2 was addc ?ia a syringe at a rate to keep :internal temperature at -55 0 C. The mixture was stirred for 25 15 minutes at 550C, when the alcohol of Step F (3.0 g, 3.58 mmol) in 20 ml of anhydrous CH 2 Cl 2 was added dropwise.

After complete addition, the cooling bath was removed and S stirring continued until internal temperature reaches -20 0

C.

At this temperature stirring was continued for about 5 minutes and the solution cooled again to -55 0 C. At this *temperature NEt 3 (2.5 ml, 17.9 minol) was added at a rate to keep internal temperature at -55 0 C. Finally a saturated citric acid solution (10 ml) was adde~i. The mixture was allowed to warm up to room temperature and 200 ml of CH 2 Cl 2 was added. Phases were separated d the organic layer 50 washed with water, a saturated olution of NaHCO 3 and brine. Drying over MgSO 4 and evaporation of solvents give a colorless oil (about 3 g) which was subjected to flash chromatography on silica gel (100 g, eluent AcOEt/petroleum ether: 1/2, then 1/1, then Product-containing fractions were evaporated to obtain 1.0 g of the above-described pure pentafluoro ketone as a colorless foam.

About 1.6 g of a mixture of the above-described ketone and starting alconol described in Step G was recovered, which can be recycled for another oxydation.

1 H-NMR (CDCl 3 360 MHz) 6 11.5 (in, 1H, NH), 8.5 (in, 1H, NH), 7.8 (in, 1H, NH), 7.2 (in, 5H, aryl), 5.1-4.3 (mn, 3H, a-CH-Phe, ca-CH-Pro, c-CHCO), 3.7-3.1 and 3.1-2.6 (2m, 9H, 152NCH 2 CH4C 6

H

5

NCH

3 2.2-1.6- 8H, 4CH 2 )f 1.5-1.2 (i, 27H, 9CH 3 1 9 F-NMR (CDCl 3 360 MIHz) 6 40.33 and 40.19 (2s, CF 2 CO), ABX systems centered at 39.0 (CF 2 J I 0. CF') 82.7 and 82.9 (2s, CF 3 ratio 4:1.

STEP I: L-Prolinamide,_N-methyl-D-phenvlalanyvl-N-(l-t3-[ (aininoiminomethyl) amino] propyvll]-3,3, 4,4,4peitafluoro-2-oxobutvl 25dihydrochlorider hydrate 0.9 g (1.07 ininol) of the tripeptide derivative of Step H was dissolved in 50 ml on anhydrous Et 2 O. 200 ml of a saturated HClgas/Et 2 O solution was added and the rew~1ting qOsolution stirred at room temperature for 48 hours (under exclusion of moi-4-sture). Peatroleum ether (about 100 ml) was added anC. the precipitate filtered under N 2 Drying of the filter residue (0.1 Torr, 40 0 C) afforded 0.6 g of a white amorphous powder which was dissolved in water (20 ml) and the solution filtered over a MilliporeO filter disk. The 51 filtrate was then lyophilized to give 0.5 g if the title compound as a %wh),ite fluffy powder.

1 H-NMR (D 2 0) 6 7. 55 (in, 3H, aryl) 7 .30 (in, 2, aryl) 4. (in, 1H, CH-Phe), 4.38 (mn, 1111, CH-Pro-,- 4.27 [in, IR, JEHN-

C(OH)

2 3.50 (mn, 1H, HCHIA-Prco), 3.37 (in, -1H, C 6

H

5 C-TA), 3.25 (mn, 2H, NCH 2 -guanidine), 3.15 (mn, 1H, C 6

H

5 CHB), 2.75 (s, broad, 3H, CH 3 2.73-2.63 (mt, lE, INCH.-Pro), 2.2-1.4 (in, 8H-, 4CZH 2 impurities of about 9% can be seen at 6.2, 3.7 and 1.2 ppm.

1 9 F-NMR (D 2 0, CF 3

CO

2 H ext. ref.) 6 3.65 and -3.71 (2s, ratio, 45:55, 3F, CF 3 2. AB system centered at -47.80; A: -47.44 1 -TF 281 Hz) B: -48.16 (JFBIFA =281 Hz) AB system centered at -48.09; A: -47.64 (JFA.FB 281 Hz), -48.53 (JFB-FA 2821 Hz) CF 2 of the two diastereoisomers, ratio 49:55, impurities of remai~ning alcohol desc, ibed in Step G at -6.9 and -54.0 (about and unknown structure at -3.3 and -45.5 EXAMPLE 7 L-Prolinamide, N-methyl-D-phenylalanyl-N-[l-[3-[ (aminoimino- 25methyl)amino]propyl]-3,3-difluoro-2-oxohexyl]-, dihydrohydrate STEP A: N-tl-[4-[bist (1,1-Diinethvlethoxy)carb-,nyllaiinolmethylene]aoamino]-l-nitrobutane Bis-Boc-S--methy1isothiourea (13.8 g, 47.6 inmol) was added at a time to a stirred solution of 4-amino-1-nitrobutane, hydrochloride [prepared according to W. Keller- Schierlein, P. Mertens, V. Prelog, and A. Walser, Helu. Chim.

52 Acta, 48, Fasc. 4 (1965) 710] (2.1 g, 13.6 mmol) and 6.6 ml (47.6 mmol) of NEt. in 40 ml of anhydrous dimethylformamide.

The mixture stirred under an atmosphere of argon for 14 hours. 200 ml of Et20 were added and the solution washed with water and concentrated solutions of citric acid, sodium bicarbonate, and sodium chloride. The organic layer was dried over MgSO 4 and evaporated (20 Torr, 30 0 C) to afford g of a yellow oil. Flash chromatography on silica gel (0.500 g, eluent: AcOEt/petroleum ether: 1/5) and evaporation of the product-containing fractions (20 Torr, 0 C and then 0.01 Torr, 20 0 C) afforded 3.48 g of the protected guanidino derivative as a whit solid.

Rf 0.5 (AcOEt/petroleum ether: 94--96 0

C.

STEP B: N-l-[9-[bis[(1,1-Dimethylethoxy)carbonyl]amino]methylene]amino-4,4-difluoro-5-hydroxy-6-nitro-l-nonene A mixture of the above-prepared nitroderivative (Step A, 20 0.48 g, 1.33 mmol) and 2,2-difluoro-pentene-l-al, ethyl hemiacetal (0.283 g, 1.7 mmol) and a catalytic amount of potassium carbonate (about 40 mg) was stirred for 14 hours at 40 0 C. AcOEt (50 ml) was added and the resulting mixture washed with water and brine. Drying over MgSO 4 of the organic layer and evaporation of the solvent (20 Torr, 30 0

C)

afforded a colorLess oil (0.57 which was applied to flash chromatography on silica gel (20 g, eluent: AcOEt/ petroleum ether: The product-containing fractions were pooled and evaporated (20 Torr, 30 0 C and 0.01 Torr, 20 0 C) to 30 give 0.332 g of the desired nitroalcohol as a slightly yellow solid.

Rf 0.4 (AcOEt/petroleum ether: 1/3).

53 STEP C: N-1- 9- [bisr 1-Dimethylethoxy) carbonyl 1amino ]methylene]'Iamino-4 ,4-dif:Luoro-5-hydroxy-6-aminononane To a stirred solution of the above-prepared nitroalcohol (Step B, 0.33 g, 0.67 mmcl) in i.-propanol (40 ml) was added 0.1 g of freshly prepared Raney-Nickel. The mixture was applied to hydrogenation under atmospheric pressure for 16 hours when H~2 consumptior completed, Filtration of the mixture and evaporatior. L. solvent (20 Torr, 30 0

C

and 0.01. Torr, 20 0 C) afforde, -esired difluoroamino alcohol as a colorless oil. Yieid: 302 mg STEP D: L-Prolinamide, N-f[ 1-dimethyl ethox1) ca rbonyl I-N-me thy 1-Dphenylala nyl- 1- 3-f [[bis[fi.,-dimethylethoxy)carbonyl]amirolmethylenelaminolpropylbl-3,3-difluoro-2-hVdroxyhexvl]- V. Following the procedure as described for Example 6, Step G: with 0.23 g (0.61 mmol) of Boc-N-(methyl)-D-Phe-Pro- OH, 0.093 a (0.61 minol) of HOBt, 0,126 g (0.61 minol) of DCC, 0.08 g (0.75 mmol) of N-methylmorpholine, and 0.23 g (0.61 mmol) of the above-prepared difluoroamino alcohol (Step in 10 m! of CH 2 C1 2 and flash chromatography of the 25 crude reaction product on silica q~el (20 g, eluent: AcOEt/ petroleum ether: 1/3-1/1) 0.24 g of the protected *tripeptide analog we,,e obtained as a colorless oil Rf 0.4-0.6 (AcOEt/petroleum ether: 1/1).

54 STEP E: L-Prolinamide, N-f (1,1-dimethylethoxy)carbonyl]-N-methyl-D-.

phenylalanyl-N-fl-3-Libis[ Ii(1,l-dimethylethoxy)carbonyvl]amino ]methy'lene Iamino 1prol 3-di fluoro-2-oxohexane- The product of Step D (0.24 g, 0.29 mmol) was oxidized to the corresponding ketone following the procedure described in Example 5, Step H with the following quantities: 0.051 ml (0.59 mmol) of oxalyl chloride, 0.084 ml (1.1 mmol) of DMSO, and 0.2 ml. (2 mmol) of NEt 3 in 6 ml of anhydrous

CH

2 Cl 2 Flash c-iromatography of the crude reaction product (0.19 g) on silica gel (10 g, eluent AcOEt/petroleum ether: 2/3) and evaporation of the product-containing fractions afforded 0.16 g of the desired ketone as a colorless oil.

Rf 0.2-0.3 (two spots) (AcOEt/petroleum ether: 2/3).

STEP F: L-Prolinamide, N-methy1-D-pheny lalany1-N- 3-aminoi-,ino- 20 methyl) amino lpropyl 3-dif lucro-2-oxohexyl]- dihydrochloride, hydrate To 0.15 g (0.186 miol) of the above-prepared ketone ***(Step E) was added 50 ml of a saturated HCl gas/Et 2

O

25 solution and the mixture was stirred for 48 hours. The solvent was evaporated and the solid residue dissolved in water. Filtration of the solutiotL on a MilliporeO filter disk and lyophilization of the filtrate afforded 0.11 g (100%) of the title compound as a yellow solid.

Rf 0.6 and 0.45 (two diastereoisomers) (BuOH/AcOH/H 2 3/l/1).

55 As stated above, the compounds of this invention (IA and IB) possess the property of exhibiting significant inhibition of thrombin indicating that they are effective anti-coagulants useful for the prevention of both venous and arterial thrombotic diseases.

Using standard invitro assay methodology for determining inhibitory activity of thrombin as well as anticoagulant activities in human plasma using activated thromboplastin times (aPTT) and thrombin times it is to be found that the compounds of Formulae IA ind IB are effective anticoagulants. Anticoagulant data may be represented as follows: ANTICOAGULANT EFFECT IN HUMAN PLASMA

ID

2 a (2M) ID2a (M) Compound aPTT b

TT

b 20 D-CH 3 Phe-Pro-Arg-CF 3 218 108 D-Phe-Pro-Arg-CF, 148 43 D-Phe-Pro-Arg-CF 2

CF

3 2 1 aAmount required for doubling the clotting time; 25 baPTT, activated partial thromboplastin time; TT, thrombin time.

Invivo anti-thrombotic effects in rats may also be utilized for evaluation. For example, in the FeCl 3 induced 30 thrombosis model wherein D-Phe-Pro-Arg-JF 2

CF

3 was administered intravenously as a 10 mg/kg of body weight injection, followed by a continuous 1 mg/kg/minute infusion, the compound was capable of preventing occlusion in two rats and prolonging occlusion in the other 2 of the 4 rats. Thus, based upon standard invitro and invivo assay methodology it is 56 to be found that the compounds of this invention, at doses within the range of about 2 to 50 mg per kilogram of body weight (with 2 tc 20 for preferred compounds) per day will be useful in the treatment of thrombophlebitis and coronary thrombosis, as well as other venous and arterial thrombotic disease states. Of course, actual dosage and frequency will vary dependent upon the nature and severity of the condition, age, general health conditions and such other factors well known and appreciated by the skilled attending diagnostician.

In addition to the foregoing use of the novel compounds of Formulae IA and IB as inhibitors of thrombin, another aspect of this invention is the use of these compounds as effective inhibitors of human lung tryptase and, as such~ are useful in the treatment of asthma.

Using standard invitro methodology for determining inhibitory activity of tryptase (Journal of Biological 20 Cheristry, Vol. 261, June 5, pp. 7372-7379, 1986), the following results have been obtained: HUMAN LUNG TRYPTASE 25 30

J

Rate Constantsa kon, M-Is koff, s 1 Ki, M D-Phe-Pro-Arg-CF 3 290 20 1.5 10 4 5.0 x 1.5 x

D-CH

3 -Phe-Pro-Arg-CF 3 200 10 1 5 x 10- 8.0 x 10- 8 6 2.4 x 10 6 D-Phe-Pro-Arg-CF 2

CF

3 1200 40 6.8 x 104 6.0 x10 7 S3.6 x 10 D-3-TIC-Pro-Arg-CF 2

CF

3 4700 200 3.4 x 10-4 7.5 x 10- 8 S 8.3 x 10-1 57 a Rate constants were determined by progress curve analysis using a non-linear regression program (ENZFITTER) Based upon the foregoing type assay, as well as by comparison with other compounds also known to possess tryptase inhibitory activity, it is to be found that the compounds of this invention, in tneir end-use application for the treatment of asthma will be about 1-100 mg per treatment; the dose regimen, of course, being dependent on the severity of the attack, the age and general condition of the patient, as well as other factors well appreciated by the attending diagnostician.

Although it may be possible that some of the administered tripeptides of this invention may survive passage through the gut following oral administration, it is preferred that the compounds be utilized via a non-oral administration. Subcutaneous, intravenous, intramuscular or intraperitoneal administrations, depot injections, implant 20 preparations and such other non-oral methods are the preferred manners by which the compounds may be utilized. In those instances wherein asthma is being treated it is preferred to utilize metered dose aerosols or by application to the mucous membranes, nose, throat and bronchial 25 tubes) in an aerosol can containing a peptide derivative of this invention in the form of a spray or dry powder pharmaceutical formulation.

For a parenteral administration the cmnpounds may be admini-tered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water and oils with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative of oils which can be employed in 58 these preparations are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, ethanol and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

The compounds can be administered in the form of a depot injection or implant preparation which may be formulated in such a manner as to permit a sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants. Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber manufactured by Dow-Corning Corporation.

The compounds of this invention can also be administered 20 topically. This can be accomplished by simply preparing a solution of the compound to be administered, preferably using a solvent known to promote transdermal absorption such as ethanol or dimethyl sulfoxide (DMSO) with or without other excipients. Preferably topical administration will be 25 accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety.

\eo Some suitable transdermal devices are described in U.S.

Pat. Nos. 3,742,951, 3,797,494, 3,996,934, and 4,031,894.

30 These devices generally contain a backing member which defines one of its face surfaces, an active agent permeable adhesive layer defining the other face surface and at least one reservoir containing the active agent interposed between the face surfaces. Alternatively, the active agent may be contained in a plurality of microcapsules distributed 59 throughout the permeable adhesive layer. In either case, the active agent is delivered continuously from the reservoir or microcapsules through a membrane into the active agen, permeable adhesive, which is in contact with the skin or mucosa of the recipient. If the active agent is absorbed through the skin, a controlled and predetermined flow of the active agent is administered to the recipient. In the case of microcapsules, the encapsulating agent may also function as the membrane.

In another device for transdermally administering the compounds in accordance with the present invention, the pharmaceutically active compound is contained in a matrix from which it is delivered in the desired gradual, constant and controlled rate. The matrix is permeable to the release of the compound through diffusion or microporous flow. The release is rate controlling. Such a system, which requires no membrane is described in U.S. Pat. No. 3,921,636. At least two types of release are possible in these systems.

0 20 Release by diffusion occurs when the matrix is non-porous.

The pharmaceutically effective compound dissolves in and diffuses through the matrix itself. Release by microporous flow occurs when the pharmaceutically effective compound is transported through a liquid phase in the pores of the 6* 25 matrix.

*9 •ue 60 The preferred compounds of this invention (IA and IB) which are of particular interest are those compounds specified in the following charts Compounds of Formula S S

S

H

H

CH 3

CH

3

H

H

H

H

H

H

20

H

CH

3

CH

3

CH

3

CH

3

H

H

H

CH

3 CH 3 CH 3

CH

3

CH

3

CH

CH

CH

3

CH

3 CH 3

CH

3

CH

3

CH

3

CH

3 CH 3 CH 3

CH

3

P

3 D-Phe D-Phe D-Phe D-Phe D-Phe D-Phe

D-CHM

D-Phe D-Phe D-Phe

D-CH

D-CH

D-CHM

D-CHM

D-CH

D-CHM

D-CHM

D-CH

P

2 L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Pro L-Prc, L-Pro L-Pro L-Pro P 1 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg

CF

3 C2F

CF

3

C

2

F

CF

2

(CH'

2 2

CH

3

CF

2 (C H2) 2 CH 3 C2FS C F 2 (CH 2 2

CO,

2 Et

CF

2 (CH 2 2 CONHCH 3

CF

2 (C H 2 NH CH 3 C2F C2F

C

2

F

CF

2 (CH 2 2 C0 2 Et C F 2 (C H2)2 CO 2 Et

CF

2

(CH

2 2

CH

3

CF

2

(CH

2 2

CO

2 Et

CF

2

(CH

2 2 CH 3 wherein Cl being the -I is cyclohexyl, substituents on CUM is cyclohexylmethyl, both the a-carbon atom of the modified

P

3 -ct-amino acid of the depicted tripeptide of Formula IA, and Et is ethyl.

61 Compounds of Formula IB

P

3

P

2

P

1 2a L-Pro Arg C 2

F

2b L-Pro Arg C 2

F

2c L-Pro Arg C 2

FS

2'a L-Pro Arg C 2 F 2'b L-Pro Arg C 2 F 2'c L-Pro Arg C2F 2'b L-Pro Arg CF 2

(CH

2 2 CH 3 2'c L-Pro Arg CF 2

(CH

2

),CH

3 2b LPro rg C 2

(CH)

2

C

3 2b L-Pro Arg CF 2

(CH

2 2 CH 3 152c L-Pro Arg CF 2

(CH

2 2

CO

2 E3 152b L-Pro Arg CF 2

(CH

2 2

CO

2 Et 2b L-Pro Arg CF 2 (CH 2 )2CQ 2 Et 2'b L-Pro Arg CF 2

(CH

2 )COEt 2021b L-Pro Arg CF(C 2)3tH 'b LPro rg C 2

(CH

2 )4CH 3 2b L-Pro Arg CF (H)CCH 3 L-Pro Arg CF 2

(CH

2

H

2 3 2b L-Pro Arg CF 2 3

CO

2 Et 2'b L-Pro Arg CF 2

(CH

2 3

CONHCH

3 wherein the P 3 moieties 2a, b and c, and 2'a, b and c are as defined, which, when combined with the carbonyl moiety to which they are attached, form the modified

P

3 -a-amino acid of the depicted tripeptides of Formula lB.

Claims (20)

1. A compound of the formulae HN. NH 2 NH A (CH 2 1 R RNH (DL) 3 R NC (NH, O "2 O O IA HN NH2 NH 'I (H 2 )m (cH 2 )n (D, L 0 0 q 3B their isomers and mixtures thereof, the hydrates and the pharmaceutically acceptable salts thereof, with the proviso that when R 1 and R 2 are both H, then R 3 is other than -CF 3 or -CF 2 CF 3 wherein m is zero, one or two, n is zero or one, with the proviso that the sum of m and n is less than three and greater than zero, q is zero or one with the proviso that the sum of both q's is zero or 2, R 1 is I or C1- 7 alkyl, R 2 is H or C1- 7 alkyl, or R 1 and R 2 taken together with the nitrogen atom to which they are attached form a or 6-membered heterocycle, -63- R 3 is -CF 3 -CF 2 CF 3 -CF 2 (CH 2 )tCH3, -CF 2 (CH 2 )tCOOR 4 -CF,(CH 2 )tCONHR 4 -CF 2 CH 2 )tCH2OR 4 or -CF 2 (CH 2 )vCH=CH 2 with t being 2, 3 or 4, and v is 1, 2 or 3, R 4 is H or C 1 6 alkyl, A is phenyl or cyclohexyl, B is (CH) 4 or (CH 2 4 which, when taken together with the two carbon atoms to which it is attached, forms a C 6 -cyclic hydrocarbon moiety.

2. A compound of Claim 1 wherein the R 3 moiety is selected from the group consisting of -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 CONHCH 3

3. A compound of Claim 2 having the structure of Formula IA wherein A is phenyl, n is one, R 1 is H and R 2 Sis C1-7 alkyl.

4. A compound of Claim 3 wherein R 2 is methyl. A compound of Claim 2 having the structure of Formula IA wherein A is cyclohexyl, R 1 is H and R 2 is C,. 7 alkyl.

6. A compound of Claim 5 wherein R 2 is methyl and n is one.

7. A compound of Claim 2 having the structure of Formula IB wherein B is (CH) 4 each q is zero, m is two, and n is zero. -64-

8. A compound of Claim 2 having the structure of formula IB wherein B is (CH)4, each q is zero, m is one, and n is zero or one.

9. A compound of claim 2 having the structure of formula IB wherein B is (CH 2 4 each q is one, m is one or two and n is zero or one. A compound of any one of claims 4, 7, 8 or 9 wherein R 3 is -CF 2 (CH 2 2 CH 3

11. A process for preparing a compound of the formulae HN NH2 NH (CH 2 NH (DL) nr A IA HNz--NH2 NH I (H (H 2 n (DR N 0 O O IB their isomers and mixtures thereof, the hydrates and the pharmaceutically acceptable salts thereof, with the proviso that when R 1 and R 2 are both H, then R 3 is other than -CF 3 or -CF 2 CF 3 wherein m is zero, one or two, n is zero or one, with the proviso that the sum of m and n is less than three and greater than zero, q is zero or one with the proviso that the sum of both q's is zero or 2, R 1 is H or CI_ 7 alkyl, R 2 is H or C 1 i 7 alkyl, or R 1 and R 2 taken together with the nitrogen atom to which they are attached form a or 6-membered heterocycle, -66- R 3 is -CF 3 -CF 2 CF 3 -CF 2 (CH 2 )tCH 3 -CF 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 with t being 2, 3 or 4, and v is 1, 2 or 3, R 4 is H or C 1 -6 alkyl, A is phenyl or cyclohexyl, B is (CH) 4 or (CH2) 4 which, when taken together wi:h the two carbon atoms to which it is attached, forms a C 6 -cyclic hydrocarbon moiety, which comprises oxidizing a compound of the formulae BocHN NBoc BocHN NBoc NH N A' S" N Pro N R'3 T Pro R 1 3 2 H O O H O (14b) wherein A' is tv(CH 2 )nA, n is zero or one, R' 1 is an N-protecting group, R' 3 is -CF 3 -CF 2 CF 3 -CF 2 (CH 2 )tC(O)NHR 4 -CF 2 (CH 2 )tCH 2 OR 4 or -CF 2 CH 2 CH=CH 2 and T is a TIC-like moiety, followed by removing any protecting group thereof and optionally reducing any olefinic moiety of the R 3 moiety, optionally N-alkylating or N-dialkylating any compound bearing a *[free amino group at the P 3 moiety, and optionally converting the so-produced compounds to their pharmaceutically acceptable salts thereof.

12. iCompounds of formulae IA or IB as defined in claim 1 koen U.SeX eer- eeas medicine.

13. The use of the compounds of formulae IA or IB as r_ defined in claim 1, for the preparation of pharmaceutical S compositions for the inhibition of thrombotic conditions. -67-

14. The use of the compounds of formulae IA or IB as defined in claim 1, for the prer;ration of pharmaceutical compositions for the treatment of thrombophlebitis and coronary thrombosis. The use of the compounds of formulae IA or IB as defined in claim 1, for the preparation of pharmaceutical compositions for the inhibiton of lung tryptase.

16. The use of the compounds of formulae IA or IB as defined in claim 1, for the preparation of pharmaceutical compositions for the treatment of asthma. *G* .9 0

17. A method of inhibiting a thrombotic condition which comprises administering compounds of formulae IA or IB as defined in claim 1.

18. A method of treating thrombophlebitis or coronary thrombosis which comprises administering compounds of formulae IA or IB as defined in claim 1.

19. A method of inhibiting lung tryptase which comprises administering compounds of formulae IA or IB as defined in claim 1.

20. A method of treating asthma which comprises administering compounds of formulae IA or IB as defined in claim 1.

21. A compound of formulae IA or IB as defined in claim 1 substantially as hereinbefore described with reference to any one of the examples.

22. A process for preparing a compound of formulae IA or IB as defined in claim 1 substantially as hereinbefore S" described with reference to any one of the examples. SDATED: 3 June 1992 20 PHILLIPS ORMONDE FITZPATRICK Attorneys for: MERRELL DOW PHARMACEUTICALS INC.

400-3 39 -68- ABSTRACT OF THE INVENTION This invention relates to novel polyfluorinated alkyl derivatives of certain tripeptides, to the methods for their preparation, the intermediates therefore, to their use in inhibiting thrombin and lung tryptase and in their end-use application as anti-coagulants useful in treating thrombo- phlebitis and coronary thrombosis and in the treatment of asthma. e e 4 0 S 0 0 *0*S 0*@0 f&O