KR0144864B1 - Novel Peptidase Substrate Analogs - Google Patents

Abstract

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Description

Novel Peptidase Substrate Analogs

The present invention relates to novel analogs of certain peptidase substrates wherein the nitrogen atom of the degradable amide bond is replaced with a difluoromethylene moiety and the carbonyl portion of the adjacent amide bond is replaced with a terminal amine functional group. It has the property of inhibiting renin and is useful for the treatment of hypertension.

In general, the present invention relates to inhibitors of lenin having the general formula:

In the above formula,

R ₁ is an α-amino acid or a peptide, optionally containing a protecting group on its terminal nitrogen atom,

R ₂ is a residue of a characteristic α-amino acid which acts as an inhibitor to the saturated hydrocarbyl moiety or to the active site of the enzyme,

R ₃ is a residue of a characteristic α-amino acid, or saturated hydrocarbyl moiety,

Ra and Rb are activity enhancing moieties.

Before clarifying and / or explaining the scope of the peptidase inhibitor included in Formula 1, it may be convenient to explain some basic concepts related to peptides. Specifically, it will be helpful to read and understand the contents of the present specification and general formulas related to the present specification. For example, all α-amino acids found in proteins except proline have in common a free carboxyl group and a free unsubstituted amino group on the α-carbon atom (since in proline, the α-amino group of proline is substituted, which is actually (alpha) -amino acid or (alpha) -amino group for convenience). In addition, each α-amino acid has a characteristic R-group and the R-group, which is a side chain or residue, is bonded to the α-carbon atom of the α-amino acid. For example, for glycine the R group side chain is hydrogen, for alanine is methyl and for valine isopropyl. Thus, throughout this specification, the R ₂ or R ₃ moiety is a residue for each described α-amino acid. Certain residues of α-amino acids are known in the art.

It is more convenient to define the concept of subtypes related to each of the individual α-amino acids involved in the present invention as well as the domains of the compounds of the present invention encompassed by the general concept of Formula 1, and the various sensory properties of the various α-amino acids. Were classified into several groups. Recognized abbreviations of these groups (D, E, F, G and K) and α-amino acids are shown in Tables I and II, respectively.

Throughout this specification, certain compounds may be defined by the shorthand expression RR [CFRNRaRb]. In the above general formula, the CFRNRaRb moiety in the parenthesis is an α-amino acid in the P′-position, wherein the nitrogen atom is replaced with the CF moiety, the carbonyl moiety is replaced with the NRaRb moiety, and R is And a residue or substituent bonded to the α-carbon atom (not shown) of a P'-amino acid, as modified. Of course, R is an α-amino acid at the P position, the R moiety is an α-amino acid or peptide (up to 4 α-amino acids at maximum) at the P or P to P position, respectively, and its terminal α-amino acid is It may contain an amino protecting group on the nitrogen atom. For example, a compound described as CBZ-His-β-Val-Phe-n-Leu-Leu- [CF-Gly-NHCH] is an amino group in the α-position of the terminal α-amino acid (eg histidine) of the R moiety. Has a carbobenzoxyl (CBZ) protecting group, the P-position α-amino acid is β-Val, the P-position α-amino acid is Phe, the P-position α-amino acid is n-Leu, and the P-position α The amino acid is Leu and the [CFGlyNHCH] portion of the P′-position α-amino acid illustrates the case where the nitrogen atom is replaced by difluoromethylene, and the NHCH is a P′-position α-amino acid (eg, such an example). Is a monomethylamino moiety that replaces the carbonyl moiety of glycine), wherein Ra is methyl and Rb is hydrogen. As in the structure of Formula 1, the same compound can be represented as follows,

Here, of course, His, β-Val, Phe, n-Leu, Leu, Gly moieties represent R residues bonded to the α-carbon atoms of the α-amino acids.

More specifically, the novel compounds having renin inhibitory ability included in the present invention are compounds of the following general formula, their hydrates, isotopes or pharmaceutically acceptable salts.

In the above formula,

R ₁ is P ₂ P ₃ P ₄ P ₅ or P ₂ P ₃ P ₄ P ₅ Pg, Pg is a terminal amino protecting group of the K group, preferably CBZ, Tba or Iva,

이고, 바람직하기로는 n-Val, n-Leu, His, (3-피라졸릴)Ala 또는 (4-피리미디닐)Ala이고,P ₂ is an α-amino acid of C, E or F group, or

Preferably n-Val, n-Leu, His, (3-pyrazolyl) Ala or (4-pyrimidinyl) Ala,

P ₃ is an α-amino acid of the E or F group or is deleted, preferably Nal (1), Phe or 0-methyl tyrosine,

P ₄ is deleted or an α-amino acid of the D, E or F group, preferably Pro, β-Ala or β-Val,

P ₅ is deleted or α-amino acid of group C, E or F, preferably His,

R ₂ is a residue of an α-amino acid of the E or F group, C _1-6 alkyl, benzyl, cyclohexylmethyl, phenethyl or 2-pyridylmethyl, preferably Leu or cyclohexylmethyl,

R ₃ is a residue of an α-amino acid of the E, F or G group, C _1-6 alkyl, benzyl, cyclohexylmethyl, phenethyl or 2-pyridylmethyl, preferably a residue of the E and G groups, most Preferably Gly, Val, n-Val or n-Leu,

Ra is H or a residue of an α-amino acid of the E, F or G group, C _1-6 alkyl, benzyl, cyclohexylmethyl or phenethyl, preferably Gly, Leu, n-Val or Ile,

Rb is H or C _1-6 alkyl, preferably H,

The C, D, E, F, G and K groups consist of:

C: Ser, Thr, Gln, Asn, Cys, His, (3-pyrazolyl) Ala, (4-pyrimidinyl) Ala, and N-methyl derivatives thereof,

D: Pro and Ind,

E: Ala, β-Ala, Leu, Ile, Val, n-Val, β-Val, Met, n-Leu and N-methyl derivatives thereof,

F: Phe, Tyr, 0-methyl tyrosine, (3-pyrazolyl) Ala, (4-pyrimidinyl) Ala, Trp, Nal (1) and their N-methyl derivatives,

G is Gly, Sar,

K: Acetyl (Ac), succinyl (Suc), benzoyl (Bz), t-butyloxycarbonyl (Boc), carbobenzoxy (CBZ), tosyl (Ts), single thread (DNS), isovaleryl ( Iva), methoxy succinyl (MeOSuc), 1-adamantanesulfonyl (AdSO ₂ ), 1-adamantaneacetyl (AdAc), 2-carboxybenzoyl (2-CBZ), phenylacetyl, t-butylacetyl ( Tba), bis [(1-naphthyl) methyl] acetyl (BNMA),

또는

이고, Bz는 플루오로, 클로로, 브로모, 요오드, 트리플루오토메틸, 히드록시, 1 내지 6개의 탄소 원자를 함유하는 알킬, 1 내지 6개의 탄소 원자를 함유하는 알콕시, 카르복시, 알킬카르보닐아미노(여기서, 알킬기는 1내지 6개의 탄소 원자를 함유함), 5-테트라졸로, 및 1 내지 15개의 탄소 원자를 함유하는 아실술폰아미도(단, 아실술폰아미도가 아릴을 함유할 경우, 이 아릴은 플루오로, 클로로, 브로모, 요오도 및 니트로로부터 선택된 것에 의해 더 치환될 수 있음)로 구성되는 군으로부터 독립적으로 선택된 1 내지 3개에 의해 적당하게 치환된 6, 10 또는 12개의 탄소 원자를 함유하는 아릴기이다.Or -A-Rz, where A is

or

And Bz is fluoro, chloro, bromo, iodine, trifluorotomethyl, hydroxy, alkyl containing 1 to 6 carbon atoms, alkoxy containing 1 to 6 carbon atoms, carboxy, alkylcarbonylamino Wherein the alkyl group contains 1 to 6 carbon atoms, 5-tetrazolo, and acylsulfonamido containing 1 to 15 carbon atoms, provided that acylsulfonamido contains aryl, Aryl is 6, 10 or 12 carbon atoms suitably substituted by 1 to 3 independently selected from the group consisting of fluoro, chloro, bromo, iodo and nitro) It is an aryl group containing.

The isotope of the compound of formula (1)

(a) at least one of the α-amino residues of the R ₁ and Q substituents is an abnormal sequence (if a natural sequence is present) or

(b) normal peptidic carbamoyl bonds are modified such as, for example, -CH ₂ NH- (reduced form), -COCH _2- (keto form), -CH (OH) CH _2- (hydroxy form), -CH (NH ₂ ) CH _2- (amino form), -CH ₂ CH _2- , -CH = CH- (trans) (hydrocarbons). Preferably, the compounds of the present invention should not be in isploid form. In the above example where the group K is an -A-Rz moiety, it is preferable that A is -C (= 0)-and Rz is an acylsulfonamido, specifically, an aryl moiety in which the acylsulfonamido is substituted by halogen (Preferably phenyl) and preferred -A-Rz- moieties are 4-[(4-chlorophenyl) sulfonylaminocarbonyl] phenylcarbonyl, 4-[(4-bromophenyl) Sulfonylaminocarbonyl] phenylcarbonyl and 4- [phenylsulfonylaminocarbonyl] phenylcarbonyl (where the parts are abbreviated C1Ф-SAC-Bz-BrФ-SAC-Bz and Ф-SAC-Bz, respectively) .

The compounds of the present invention may be in free form (eg, positive form) or in salt (eg, acid addition salt, alkali metal salt, or anionic (eg ammonium) salt) form. Compounds in free form can be converted to salt form by methods known in the art and vice versa. Examples of salt forms include trifluoroacetate, hydrochloride, sodium, potassium and ammonium forms and the like. Unless otherwise stated, the α-amino acid construct of such peptidase substrate analogs is preferably L-configuration. Of course, when the carbonyl moiety of P ₁ is a reduced type, it can be seen that such a compound is not a hydrate.

및

로 나타낼 수 있다. 마찬가지로, 다른 형태의 유도체(예, N-알킬 유도체)도 또한 유사하게 나타낼 수 있다.In the example where the typical R residue of the α-amino acid contains —OH groups (eg, serine, threonine and tyrosine), it can be seen that such groups can be derived. For example, in each example above the -OH group can be converted to an ether. When converted as above, in an example such as methyl ether, the group may be 0-methyl serine, O-methyl threonine and O-methyl tyrosine, respectively. These methyl ether containing side chains are each

And

It can be represented as. Likewise, other forms of derivatives (eg N-alkyl derivatives) may also be represented similarly.

As used herein, the term alkyl refers to straight, branched and ring forms thereof, specifically, methyl, ethyl, n-butyl, t-butyl, cyclopropyl, n-propyl, pentyl, cyclopentyl, moieties such as n-hexyl, cyclohexyl and cyclohexylmethyl. The term aralkyl includes aryl bonded to C _1-4 alkylene, preferably methyl or ethyl. The term aryl includes both carbocyclic and heterocyclic moieties. Preferred aralkyl and aryl moieties are phenyl, benzyl, naphthylmethyl, phenethyl, 2-pyridylmethyl.

The compound of formula (1) inhibits renin and is therefore used as an antihypertensive agent useful in the treatment of hypertension. Several biochemical parameters of the latent and enzymatic inhibitory properties of the compounds of formula (1) for end use applications can be readily identified by standard biochemical techniques known in the art. The actual dosage range for a particular end use application of a compound of the present invention will of course be determined by the nature of the disease or the nature of the animal or patient being treated by the diagnosing physician. Typical end-use application dosage ranges are from about 0.01 to 10 mg / kg per day for effective treatment, with 0.1 to 10 mg / kg per day being preferred. Preferred compounds are as follows.

5-cyclohexyl-2,2-difluoro-N ^4- [N-isovaleryl-L- (0-methyl) -tyrosyl-Ln-valyl] -3-oxo-1,4-pentanediamine, Hydrochloride,

5-cyclohexyl-2,2-difluoro-N ¹ -isoamyl-N ^4- [N-isovaleryl-L- (0-methyl) -tyrosyl-Ln-valyl] -3-oxo-1 4-pentanediamine, hydrochloride,

1-cyclohexyl-4,4-difluoro-N ^2- [N-isovaleryl-L- (0-methyl) -tyrosyl-Ln-valyl] -3-oxo-2,5-octanediamine, Hydrochloride,

1-cyclohexyl-4,4-difluoro-N ^2- [N-tert-butylacetyl-L- (0-methyl) -tyrosyl-Ln-valyl] -7-methyl-3-oxo-2, 5-octanediamine, hydrochloride,

1-cyclohexyl-4,4-difluoro-N ^2- [N-tert-butylacetyl-L- (0-methyl) tyrosyl-Ln-valyl] -6-methyl-3-oxo-2,5 Heptanediamine, hydrochloride,

5-cyclohexyl-2,2-difluoro-N ^4- [N-isovaleryl-L- (1-napryl) alanine-Ln-valyl] -3-oxo-1,4-pentanediamine, hydrochloride ,

5-cyclohexyl-2,2-difluoro-N ^4- [N- (1-tert-butylsulfonyl-methyl-3-phenyl-propanoyl) -Ln-valyl] -3-oxo-1, 4-pentanediamine, hydrochloride,

5-cyclohexyl-2,2-difluoro-N ^4- [N-isovaleryl-L- (0-methyl) tyrosyl-L- (N-methyl) -n-valyl] -3-oxo- 1,4-pentanediamine, hydrochloride,

5-cyclohexyl-2,2-difluoro-N ^4- [N-isovaleryl-L-phenylalanyl-L- (N-methyl) -n-valyl] -3-oxo-1,4- Pentanediamine, hydrochloride,

5-cyclohexyl-2,2-difluoro-N ¹ -isoamyl-N ^4- [N-isovaleryl-L- (0-methyl) -tyrosyl-L- (N-methyl) -n- Balyl] -3-oxo-1,4-pentanediamine, hydrochloride,

1-cyclohexyl-4,4-difluoro-N ^2- [N-tert-butylacetyl-L- (0-methyl) -tyrosyl-L- (N-methyl) -n-valyl] -6- Methyl-3-oxo-2,5-heptanediamine, hydrochloride.

The scope of the compound is defined within the general aspects and individual subgroups of the present invention and within certain preferred compounds, and methods of preparation are described and illustrated by the following general and specific methods.

In general, the compounds of formula (1) can be prepared using analogous standard chemical reactions known in the art. Important intermediates required for the application of standard peptide coupling techniques can be represented by the following general formula (Va) or (Vb).

In the above formula,

R ₃ ′ is as defined for R ₃ , but not H, and may be a protective form of the specific α-amino acid residues involved,

R ₂ is as defined above,

Pg and Pg 'are the respective protecting groups and are preferably different from one another in order to facilitate selective removal depending on the location, nature and order of reactions required to prepare the final compound from the intermediate, the choice being known in the art and the It carried out according to the theory which a person skilled in the art can understand.

In the case of general formula (1), wherein R ₃ is hydrogen, the process for preparing the required intermediate (Va) is illustrated in Reaction Table A. The intermediate (Vb) required in the case of formula (1) in which R ₃ is not hydrogen is prepared by the method shown in reaction table B.

[Reaction Chart A]

Wherein Pg is an amine protecting group and R ₂ and Ra are as defined above.

In carrying out the steps of Scheme A, it is preferred to start with aldehydes of formula (VI) wherein the protecting group is carbamate and Pg is benzyloxycarbonyl (CBZ). The aldehyde thus protected is condensed with an ester of bromodifluoroacetic acid, preferably ethyl ester, in the presence of zinc. Preferably, this reaction is carried out in anhydrous aprotic solvents such as tetrahydrofuran, ether, dimethoxyethane and the like under nitrogen or argon atmosphere. The reaction mixture is gently heated under reflux conditions, preferably at about 60 ° C. for about 1 to 12 hours. Ester (VII) is converted to amide (VIII) under anhydrous conditions, preferably by treatment with an appropriate amine or ammonia water with a solvent such as anhydrous diethyl ether. This amidation reaction starts at −78 ° C. and is saturated with ammonia, the reaction mixture is slowly warmed to room temperature, and after addition of RaNH ₂ amine, the mixture is refluxed in tetrahydrofuran. The amides thus formed are chemically reduced by reaction with diborane, preferably diborane / dimethylsulfide complex, under nitrogen atmosphere in anhydrous aprotic solvent (e.g. THF) under reflux conditions. This reduction gives the desired amine in the form of an acid (e.g. HCI) salt, and standard reaction conditions (e.g. at room temperature, (BOC)) to protect the amine by adjusting the pH (after normal finishing). ₂ O, tetrahydrofuran) was used to obtain a base with N- protecting group (e.g., Pg 'may be protected suitably as t- butoxycarbonyl alkylcarbonyl).

If the R ₃ 'group is not hydrogen, the method of reaction scheme A is modified to produce the desired intermediate according to reaction scheme B.

[Reaction Chart B]

Wherein R ₃ 'M is an organometallic reagent, preferably lithium or magnesium coupled to the desired R ₃ ' moiety.

The conversion of the ester (VII) to the corresponding R ₃ 'containing ketone with the organometallic reactant occurs by contacting the reactants together under anhydrous conditions at about 0 ° C to -80 ° C in an aprotic solvent (e.g. tetrahydrofuran). . As the reaction temperature is gradually raised to room temperature, the complex is hydrolyzed to yield the desired intermediate ketone (IX), which causes reducing amination reactions known in the art as reported by RF Borch et al. [J. Am. Chem. Soc., 93, 2897 (1971)]. This reductive amination takes place in one to two steps (with isolation of intermediate imines or enamines). For example, the reaction of ammonium acetate with ketone (IX) under slightly acidic conditions in methanol produces an enamine and produces the desired product when reacted with sodium cyanoborohydride. Alternatively, the ketone was treated directly with sodium cyanoborohydride in the presence of ammonium acetate to give the desired amine (as its HCI salt), neutralized in either case, and then protecting the NH ₂ moiety with a suitable protecting group. You can.

If the compound of interest contains both Ra and Rb moieties other than H, such compounds may be prepared by methods known in the art. In this reaction sequence, the protecting group of the (-NRaPg ') moiety is optionally purified by standard amine deprotection methods to obtain an intermediate compound containing the (-NHRa) moiety and, when reacted with the appropriate acyl chloride, an intermediate amide (-NRaCORb). The ') part was obtained and the desired (-NRaR'b) moiety (R'b is Rb rather than H) when reduced to borane, preferably borane dimethylsulfide. This reaction sequence is illustrated as a reaction diagram of the following partial structure, which shows a process for the preparation of compounds wherein Ra is ethyl (Et) and Rb is methyl.

After obtaining the important intermediates (a) and (b) of the general formula (V), the standard α-amino acid or peptide coupling method can be carried out to prepare individual compounds of the general formula (1). In general, one side of the reaction can be represented by the reaction plot C.

[Reaction Chart C]

Among the above, R ₁ , R ₂ , R ₃ , Ra and Pg are as defined above, R ₁ OH is equivalent to RCO ₂ H, Rb is Rb or Pg 'and Pb and Pg are as defined above.

Cleavage of the protecting group may be effective with all standard techniques known in the art, which are illustrated in the specific examples described below. Similarly, after cleaving the Pg protecting group, the coupling method can be carried out according to standard methods known in the art.

Oxidation can be carried out through known Sown Oxidation Procedures or by modified Jones reactions using pyridinium dichromates or chromic anhydride-pyridinium complexes or 1,1,1-triaceoxy-2,1 -You can play as benzoxiodol. Of course, if there are other protecting groups on the residues of the α-amino acid construct, such protecting groups may be removed after oxidation.

Generally, swon oxidation reacts about 2 to 10 equivalents of dimethyl sulfoxide (DMSO) with about 1 to 6 equivalents of trifluoromethylacetic anhydride [(CF ₂ CO) ₂ O] or oxalyl chloride [(COCI) ₂ ]. The reaction was dissolved in an inert solvent (e.g. methylene chloride (CH ₂ Cl ₂ )) and the reaction was about -80 ° C to -50 ° C, under anhydrous conditions and inert atmosphere (e.g. nitrogen or equivalent Functional gas) to produce the sulfonium adduct in situ, to which about 1 equivalent of the appropriate alcohol (VI) is added.

Preferably, the alcohol is dissolved in an inert solvent (eg CH ₂ Cl ₂ ) or a minimum amount of DMSO, the reaction mixture is warmed to about −50 ° C. (for about 10-20 minutes) and then tertiary amine (eg, The reaction is terminated by adding about 3 to 10 equivalents of triethylamine, N-methyl morpholine, etc.).

In general, the modified Jones oxidation method involves reacting pyridinium dichromate in dichloromethane with alcohol (VI) by contacting the reactants together in a water-trapping molecular sieve powder (e.g., grounded 3 sieving molecules). And the contacting is carried out in the presence of glacial acetic acid at about 0 [deg.] C. to 50 [deg.] C., preferably at room temperature, followed by isolation and optionally removal of the amine protecting group.

Alternatively, 1 to 5 equivalents (eg, Sarett reagent) of a chromic anhydride-pyridine complex prepared directly in an inert solvent (eg, CH ₂ Cl ₂ ) in 0 ° C. to 50 ° C., under anhydrous conditions and in an inert atmosphere (Fieser and Fieser's Reagent for Organic Synthesis Vol. 1, page 145, and JACS No. 25, page 422 (1953) by Sarett et al.) Were added to 1 equivalent of alcohol (VI) to react the reactants for about 1 to 15 hours. And then isolated, optionally removing the amine protecting group.

Another method of converting alcohol (VI) to a preferred ketone is the oxidation reaction using periodan (ie 1,1,1-triacetoxy-2,1-benzoxiodol) [J. Org. Chem. 48, 4155 (1983). This oxidation is achieved by contacting about 1 equivalent of alcohol (VI) with about 1-5 equivalents of periodan (preferably 1.5 equivalents), and the reagent is subjected to anhydrous conditions of 0 ° C. to 50 ° C. (preferably room temperature), It is a suspension under an inert atmosphere (preferably nitrogen) and in an inert solvent (e.g. methylene chloride) and the reactants are reacted with each other for about 1 to 48 hours. Any deprotection of the amine protecting group can be carried out as required after isolating the ketone.

The following specific examples illustrate the preparation of compounds of the invention.

Example 1

4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-6-methyl-heptanoic acid, ethyl ester

A mixture of 2.080 g (8.3 mmol) of LN-benzyloxy carbonyl leucine and 2,230 g (11 mmol) ethyl bromodifluoroacetate in dry tetrahydrofuran (15 ml) was activated in dry tetrahydrofuran (10 ml) under nitrogen. It was added dropwise to 0.710 g of reflux suspension of Zinc Wool. The mixture was gently refluxed by adjusting the rate of addition. After the addition was terminated, the solution was stirred for 3 hours at room temperature. The mixture was quenched by addition of 20 ml ethyl acetate, brine and 1M KHSO ₄ (20 ml). The aqueous layer was dried over anhydrous MgSO ₄ , evaporated and purified by flash chromatography (silica gel, ethyl acetate / cyclohexane, 1: 9). 1.130 g of the desired ester were isolated (yield: 36%) (colorless oil).

Rf: 0.57 (ethyl acetate / cyclohexane, 1: 1)

Example 2

4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-6-methyl-heptanamide

Dry ammonia flow at -78 [deg.] C. 0.820 g (2.2 mmol) of 4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-6-methylheptanoic acid, ethyl ester in anhydrous diethyl ether (10 ml) Bubbling through a solution. After saturation, the temperature was raised to room temperature with stirring. Excess ammonia was removed and the solvent was evaporated in vacuo. The residue was dissolved in pentane to afford the desired amide as a solid in quantitative yield.

MS (CI / NH ₃ ): 345 (MH < + >).

Example 3

N ₄ -benzyloxycarbonyl-N ₁ -tert-butoxycarbonyl-2,2-difluoro-3-hydroxy-6-methyl-1,4-heptandiamine

A solution of 1M BH ₃ / (CH ₃ ) ₂ S (1 ml) in dichloromethane was dissolved in 4-chlorobenzyloxycarbonylamino-2,2-difluoro-3-hydroxy- in anhydrous tetrahydrofuran (10 ml) under nitrogen. To a mixture of 0.185 g (0.53 mmol) of 6-methyl-heptanamide was added. This mixture was heated at reflux for 3 hours. After cooling to room temperature, methanol (3 ml) and 1N HCI in diethyl ether (6 ml) were added. The solvent was removed in vacuo. The residue was dissolved in water and the aqueous layer washed with diethyl ether. The pH of the aqueous phase was adjusted to 10. Extraction with diethyl ether gave intermediate amines which were converted directly to its N-Boc protective form [(BOC) ₂ O 1.5 eq: tetrahydrofuran, room temperature]. The desired tert-butylcarbamate was purified by chromatography (silica gel, ethyl acetate / cyclohexane, 1: 1) to give 0.180 g (yield: 79%).

Rf: 0.63 (ethyl acetate / cyclohexane, 1: 1)

Example 4

4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-phenyl-pentanoic acid, ethyl ester

The title compound was prepared from L-N-benzyloxycarbonylphenylalaninal and ethyl bromodifluoroacetate by the method described in Example 1 (yield: 75%).

Rf: 0.5 (ethyl acetate / cyclohexane, 1: 1)

Analysis of C ₂₁ H ₂₃ NO ₅ F ₂

Theoretical: C; 61.91, H; 5.69, N; 3.44

Found: C; 62.19, H; 5.75, N; 3.55

Example 5

4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-phenyl-pentanamide

The title compound was prepared from the ester of Example 4 by the method described in Example 2 (yield: 98%).

Example 6

N ⁴ -benzyloxycarbonyl-N ¹ -tert-butoxycarbonyl-2,2-difluoro-3-hydroxy-5-phenyl-1,4-pentanediamine

The title compound was prepared from the amide of Example 5 by the method described in Example 3 (yield: 64%).

Example 7

N ¹ -tert-butoxycarbonyl-2,2-difluoro-3-hydroxy-5-phenyl-1,4-pentanediamine

N ⁴ -benzyloxycarbonyl-N ¹ -tert-butoxycarbonyl-2,2-difluoro-3-hydroxy-5-phenyl-1,4-pentanediamine (0.464 g, in ethanol (20 ml) 1 mmol) was stirred at room temperature for 5 hours in the presence of 10% palladium (0.020 g) on charcoal under hydrogen atmosphere. The hydrogen atmosphere was then replaced with a nitrogen atmosphere and the catalyst was filtered off. The solvent was removed in vacuo to afford 0.030 g of the desired product (yield: 98%).

Example 8

4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-methyl hexanoic acid, ethyl ester

The title compound was prepared from L-N-benzyloxycarbonylvalinal and ethyl bromodifluoroacetate by the method described in Example 1 (yield: 40%).

Example 9

4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-methyl-hexanamide

The title compound was obtained in quantitative yield from the ester of Example 8 by the method described in Example 2.

Example 10

N ⁴ -benzyloxycarbonyl-N ¹ -tert-butoxycarbonyl-2,2-difluoro-3-hydroxy-5-methyl-1,4-hexanediamine

The title compound was prepared from the amide of Example 9 by the method described in Example 3 (yield: 40%).

Rf: 0.50 (ethyl acetate / cyclohexane, 1: 1)

Example 11

N ¹ -tert-butoxycarbonyl-2,2-difluoro-3-hydroxy-5-methyl-1,4-hexanediamine

The title compound was obtained in quantitative yield from the dicarbamate of Example 10 by the method described in Example 7.

Example 12

Ethyl 4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy butanoate

The title compound was prepared in 33% yield from N-benzyloxycarbonylglycinal, ethyl bromodifluoroacetate and zinc by the method described in Example 1.

Rf: 0.45 (silica gel; ethyl acetate / cyclohexane, 1: 1)

Example 13

4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy butanamide

The title compound was prepared in 95% yield from the ester of Example 12 by the method described in Example 2.

Rf: 0.49 (silica gel; ethyl acetate / cyclohexane, 1: 1)

Example 14

N ⁴ -benzyloxycarbonyl-N ^1- (tert-butoxycarbonyl) -2,2-difluoro-3-hydroxy-1,4-butanediamine

The title compound was prepared in 48% yield from the amide of Example 13 by the method described in Example 3.

Rf: 0.42 (silica gel; ethyl acetate / cyclohexane, 1: 1)

MS (DCI / CI + / NH ₃ ): 392 (MNH ⁺ ₄ , 59); 375 (MH ⁺ , 20); 258 (15); 241 (100).

Example 15

Ethyl 4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy pentanoate

The title compound was prepared in 50% yield from N-benzyloxycarbonylalaninal, ethyl bromodifluoroacetate and zinc by the method described in Example 1.

Rf: 0.49 (silica gel; ethyl acetate / cyclohexane, 1: 1)

Example 16

4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy pentanamide

The title compound was prepared in the yield of 90% from the ester of Example 15 by the method described in Example 2.

Rf: 0.50 (silica gel; ethyl acetate / cyclohexane, 1: 1)

MS (DCI / CI + / NH ₃ ): 392 (MNH ⁺ ₄ , 100); 303 (MH ⁺ , 13); 212 (19); 169 (100).

Example 17

N ⁴ -benzyloxycarbonyl-N ^1- (tert-butoxycarbonyl) -2,2-difluoro-3-hydroxy-1,4-pentanediamine

The title compound was prepared from the amide of Example 16 in a yield of 53% by the method described in Example 3.

Rf: 0.47 (silica gel; ethyl acetate / cyclohexane, 1: 1)

MS (DCI / CI + / NH ₃ ): 406 (MNH ⁺ ₄ , 94); 389 (MH ⁺ , 23); 298 (20); 255 (100).

Example 18

N-benzyloxycarbonyl-3-cyclohexylalanine

To a solution of 3-cyclohexylalanine, hydrochloride (4.75 g, 22.8 mmol) in 2N sodium hydroxide (11.4 ml) benzylchloroformate (3.2 ml, 36 mmol) and 2N sodium hydroxide in tetrahydrofuran (10 ml) at 0 ° C. (11.4 ml) solution was added simultaneously (the pH of the mixture was maintained at about 9-10 due to the addition of 2N sodium hydroxide). The mixture was stirred for 1.5 hours. The solution was washed with diethyl acetate (3 x 20 ml). The aqueous phase was acidified to pH 2 with 3N aqueous hydrochloric acid solution and extracted with ethyl acetate (3 × 50 ml). The mixed organic layer was dried over anhydrous magnesium sulfate. Filter and remove the solvent in vacuo to give 4.80 g of the desired product (yellow oil, 69% yield).

Rf: 0.75 (silica gel; AcOH / BuOH / H ₂ O, 2: 6: 2).

Example 19

2-benzyloxycarbonylamino-3-cyclohexyl-N, O-dimethylpropanehydroxyxamate

To a solution of N-benzyloxycarbonyl-3-cyclohexylalanine (4.60 g, 15 mmol) in anhydrous methylene chloride (60 ml) dicyclohexylcarbodiamide (3.09 g, 15 mmol) and 1-hydroxy at 0 ° C. Benzotriazole hydrate (2.29 g, 15 mmol) was added. After stirring for 0.25 h at 0 ° C., N, O-dimethylhydroxylamine hydrochloride (1.46 g, 15 mmol) and N-methylmorpholine (1.51 g, 15 mmol) were added to the mixture. The temperature was raised to room temperature while the mixture was stirred for 20 hours. The precipitate was filtered off. The solvent was removed in vacuo and the mixture was purified by chromatography (silica gel; ethyl acetate / cyclohexane, 2/8) to afford 3.60 g of the desired hydroxamate (yield: 69%).

Rf: 0.38 (silica gel; ethyl acetate / cyclohexane, 1: 1, UV, I ₂ ).

Example 20

N-benzyloxycarbonyl-3-cyclohexylalanineal

A mixture of 2-benzyloxycarbonylamino-3-cyclohexyl-N, 0-dimethyl propanehydroxyxamate (3.58 g, 10.3 mmol) and lithium aluminum hydride (0.44 g, 11.6 mmol) in anhydrous diethyl ether (100 ml) Was stirred at 0 ° C. for 1 h. 1 M potassium hydrogensulfate (25 ml) was added. The mixture was stirred for 0.5 h and extracted with diethyl ether (2 × 25 ml). The combined organic layers were washed with 2N HCI (3 × 20 ml), water (1 × 20 ml), saturated sodium bicarbonate solution (1 × 20 ml), brine (20 ml) and dried over anhydrous magnesium sulfate. Filtration and the solvent removed in vacuo yielded 2.52 g of the desired aldehyde (85%, yellow oil) and used during the next step without further purification.

Example 21

4-benzyloxycarbonylamino-5-cyclohexyl-2,2-difluoro-3-hydroxy-pentanoic acid, ethyl ester

The title compound was prepared in 37% yield from N-benzyloxycarbonyl-3-cyclohexylalaninal, ethyl bromodifluoroacetate and zinc by the method described in Example 1.

Rf: 0.57 (silica gel; ethyl acetate / cyclohexane, 1: 1)

Example 22

4-benzyloxycarbonylamino-5-cyclohexyl-2,2-difluoro-3-hydroxy-pentanamide

The title compound was prepared in 97% yield from the ester of Example 21 by the method described in Example 2.

Rf: 0.53 (silica gel; ethyl acetate).

MS (DCI / CI + / NH ₃ ): 402 (MNH ⁺ ₄ , 86); 385 (MH <^+> , 13); 294 (23); 169 (40); 126 (100).

Example 23

N ⁴ -benzyloxycarbonyl-N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-3-hydroxy-1,4-pentanediamine

The title compound was prepared from the amide of Example 22 in a yield of 51% by the method described in Example 3.

Rf: 0.59 (silica gel; ethyl acetate / cyclohexane, 1: 1)

Example 24

N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-3-hydroxy-1,4-pentanediamine

The title compound was prepared in 86% yield from the dicarbamate of Example 23 by the method described in Example 7.

Example 25

N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-3-hydroxy-N ^4- [N-isovaleryl-L- (0-methyl) tyrosyl- Ln-valyl] -1,4-pentanediamine

To a stirred solution of 0.325 g (0.86 mmol) of N-isovaleryl-L- (0-methyl) tyrosyl-Ln-valine in dry acetonitrile (8 ml) was added 0.090 g of N-methylmorpholine under nitrogen. The resulting solution was cooled to -20 ° C. Isobutyl chloroformate (0.117 g, 0.86 mmol) was added dropwise to the cold reaction mixture. After 10 minutes, 0.300 g of N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-3-hydroxy-1,4-pentanediamine in dry acetonitrile (3 ml) 0.89 mmol) was added to the cooling mixture. After stirring at −20 ° C. for 4 hours, the temperature of the mixture was raised to room temperature. Stirring was continued for 15 hours at room temperature. The solvent was removed in vacuo and the resulting residue was chromatographed (silica gel; ethyl acetate / cyclohexane, 1: 1) to afford the title compound in 61% yield.

Rf: 0.16 (silica gel; ethyl acetate / cyclohexane, 1: 1)

Analysis of C ₃₆ H ₅₈ N ₄ O ₇ F ₂

Theoretical: C; 62.05, H; 8.39, N; 8.04

Found: C; 62.06, H; 8.30, N; 8.00

Example 26

N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-N ^4- [N-isovaleryl-L- (0-methyl) tyrosyl-Ln-valyl]- 3-oxo-1,4-pentanediamine

A solution of 0.330 g (0.47 mmol) of the alcohol of Example 25 in methylene chloride (5 ml) was added to a mixture of pyridinium dichromate (0.314 g), 3 ′ sieving molecules (0.573 g) and glacial acetic acid (0.010 ml) in methylene chloride (10 ml). Added. Stirring was continued for 15 hours at room temperature. The crude mixture was purified by chromatography (silica gel; ethyl acetate / cyclohexane, 1: 1) to afford the title compound as a white solid, 76% yield.

Rf: 0.67 (silica gel; ethyl acetate)

Analysis of C ₃₆ H ₅₆ N ₄ O ₇ F ₂

Theoretical: C; 62.23, H; 8.12, N; 8.06

Found: C; 62.31, H; 8.06, N; 8.16

Example 27

5-cyclohexyl-2,2-difluoro-N ^4- [N-isovaleryl-L- (0-methyl) tyrosyl-Ln-valyl] -3-oxo-1,4-pentanediamine, hydrochloride

The mixture of 0.207 g (0.3 mmol) of the ketone of Example 26 in a saturated solution of hydrogen chloride in diethyl ether (10 ml) and tetrahydrofuran (1 ml) was stirred at room temperature for 15 hours. This formed a white precipitate. The solvent was removed in vacuo. The residue was dissolved in diethyl ether and evaporated to dryness (3 times). The residue was recrystallized from ethanol / diethyl ether to give 0.128 g (yield 68%) of the title compound.

Rf: 0.65 (silica gel; AcOH / BuOH / H ₂ 0, 2: 6: 2)

Analysis of C ₃₁ H ₄₈ N ₄ O ₅ F ₂ , HCI (1.25 H ₂ O)

Theoretical: C; 56.96, H; 7.94, N; 8.57

Found: C; 56.89, H; 7.99, N; 8.51

Example 28

4-benzyloxycarbonylamino-5-cyclohexyl-2,2-difluoro-3-hydroxy-N-isoamylpentanamide

4-benzyloxycarbonylamino-5-cyclohexyl-2,2-difluoro-3-hydroxy-pentanoic acid, ethyl ester (0.207 g, 0.5 mmol) and isoamylamine in tetrahydrofuran (5 ml) 0.087 g, 1 mmol) was heated at reflux for 15 hours under nitrogen. Diethyl ether was added. The organic solution was washed with 0.1 N HCI and dried over anhydrous magnesium sulfate. Filtration and removal of solvent in vacuo gave a residue, which was purified by chromatography (silica gel; ethyl acetate / cyclohexane, 1: 1). 0.170 g of the desired amide was isolated (yield: 75%).

Example 29

N ⁴ -benzyloxycarbonyl-N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-3-hydroxy-N ¹ -asoamyl-1,4-pentanediamine

The title compound was prepared from the amide of Example 28 by the method described in Example 3.

Example 30

N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-3-hydroxy-N ¹ -isoamyl-1,4-pentanediamine

The title compound was prepared in quantitative yield from the dicarbamate of Example 29 by the method described in Example 7.

Example 31

N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-3-hydroxy-N ¹ -isoamyl-N ^4- [N-isovaleryl-L- (0 -Methyl) tyrosyl-Ln-valyl] -1,4-pentanediamine

The title compound was prepared in a yield of 60% from N-isovaleryl-L- (0-methyl) tyrosyl-L-n-valine and the amine of Example 30 by the method described in Example 25.

Example 32

N ^1- (tert-butoxycarbonyl) -5-cyclohexyl-2,2-difluoro-N ¹ -isoamyl-N ^4- [N-isovaleryl-L- (0-methyl) tyrosyl -Ln-valyl] -3-oxo-1,4-pentanediamine

The title compound was prepared from the alcohol of Example 31 by the method described in Example 26.

Example 33

5-cyclohexyl-2,2-difluoro-N ¹ -isoamyl-N ⁴ [N-isovaleryl-L- (0-methyl) tyrosyl-Ln-valyl] -3-oxo-1,4 -Pentanediamine, hydrochloride

The title compound was prepared from the carbamate of Example 32 by the method described in Example 27.

Example 34

4-benzyloxycarbonylamino-5-cyclohexyl-2,2-difluoro-3-hydroxy-pentanoic acid

A solution of 0.023 g (0.55 mmol) of LiOH, H ₂ O in water (1 ml) was added with 4-benzyloxycarbonylamino-5-cyclohexyl-2,2-di in dimethoxyethane (DME) (3 ml) at 0 ° C. To a mixture of fluoro-3-hydroxy-pentanoic acid, ethyl ester (0.206 g, 0.5 mmol) was added. The temperature was slowly raised to room temperature and the mixture was stirred at room temperature for 15 hours. The mixture was diluted with water (5 ml) and washed with diethyl ether (10 ml). The aqueous layer was acidified to about pH 2.0 with 0.1 N HCI and extracted with diethyl ether (2 × 10 ml). The combined organic layers were washed with brine and dried over anhydrous MgSO ₄ . Filter and remove the solvent in vacuo to afford the desired acid and recrystallize from diethyl ether / pentane.

Example 35

7-benzyloxycarbonylamino-8-cyclohexyl-5,5-difluoro-6-hydroxy-4-octanone

To a rapidly stirred solution of n-propylmagnesium bromide (9 mmol) in anhydrous diethyl ether (20ml) 4-benzyloxycarbonylamino-5-cyclohexyl-2,2-difluoro in anhydrous diethyl ether (10ml) A solution of 0.641 g (2.25 mmol) of rho-3-hydroxy-pentanoic acid was added dropwise. After complete addition of the acid the mixture was refluxed under nitrogen for 2 hours. The crude mixture was poured into a mixture of ice and 0.2 N HCI (50 ml). The layers were separated and the aqueous phase was extracted with diethyl ether (3 × 20 ml). The combined organic layers were washed with saturated sodium bicarbonate solution and brine. The organic layer was dried over anhydrous magnesium sulfate. It was filtered and the solvent was removed in vacuo to give an oil. The residue was purified by chromatography (silica gel, ethyl acetate / cyclohexane, 1: 1) to afford the desired ketone.

Example 36

N ^5- (tert-butoxycarbonyl) -N ² -benzyloxycarbonyl-1-cyclohexyl-4,4-difluoro-3-hydroxy-2,5-octanediamine

7-benzyloxycarbonylamino-8-cyclohexyl-5,5-difluoro-6-hydroxy-4-octanone (0.235 g, 0.46 mmol) in methanol (7 ml), ammonium acetate (0.354 g, 4.6 Mmol) and a mixture of sodium cyanoborohydride (0.020 g, 0.32 mmol) were stirred under nitrogen at room temperature for 20 hours. The mixture was acidified by addition of 1N HCI (4 ml) and the solvent was removed in vacuo. The residue was dissolved in water. The aqueous phase was washed with diethyl ether. The pH of the aqueous phase was adjusted to 10. Extraction with diethyl ether gave the free amine, which was directly converted into N-BOC protected state [(Boc) ₂ O 1.5 eq: tetrahydrofuran, room temperature]. The desired dicarbamate was purified by chromatography (silica gel, ethyl acetate / cyclohexane, 1: 1).

Example 37

N ^5- (tert-butoxycarbonyl) -1-cyclohexyl-4,4-difluoro-3-hydroxy-2,5-octanediamine

The title compound was prepared in quantitative yield from the dicarbamate of Example 36 by the method described in Example 7.

Example 38

N ^5- (tert-butoxycarbonyl) -1-cyclohexyl-4,4-difluoro-3-hydroxy-N2- [N-isovaleryl-L- (0-methyl) tyrosyl-Ln -Valyl] -2,5-octanediamine

The title compound was prepared from N-isovaleryl-L- (0-methyl) tyrosyl-L-n-valine and the amine of Example 37 by the method described in Example 25.

Example 39

N ^5- (tert-butoxycarbonyl) -1-cyclohexyl-4,4-difluoro-N ^2- [N-isovaleryl-L- (0-methyl) tyrosyl-Ln-valyl]- 3-oxo-2,5-octanediamine

The title compound was prepared from the alcohol of Example 38 by the method described in Example 26.

Example 40

1-cyclohexyl-4,4-difluoro-N ^2- [N-isovaleryl-L- (0-methyl) tyrosyl-Ln-valyl] -3-oxo-2,5-octanediamine, hydrochloride

The title compound was prepared from the carbamate of Example 39 by the method described in Example 27.

By following the reference technique and using other known techniques and comparing with known compounds known to be useful in the treatment of the disease, it is possible for a person skilled in the art to practice the invention with suitable materials. Of course, in the end-use application of the compounds of the invention, the compounds may be formulated in sterile solutions or suspensions for oral or parenteral administration in suitable pharmaceutical preparations, for example in the form of tablets, capsules, or elixirs. desirable. The compounds of the present invention can be administered to patients (animals or humans) in need of such treatment several times a day. As noted above, the dosage may vary depending on the severity of the disease, the weight of the patient and various factors as would be known to one skilled in the art.

Typically, the compound is formulated into a pharmaceutical composition as described below.

The required amount of the compound of formula I or a mixture of the compound or physiologically acceptable salt thereof is formulated to allow pharmaceutical implementation in unit dosage form with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, and the like. . The amount of active substance in such compositions or formulations is selected according to the suitable dosage form within the above ranges.

Examples of auxiliaries that can be mixed with tablets, capsules and the like are as follows. Binders such as rubber tragacanth, acacia, corn starch or gelatin, excipients such as microcrystalline cellulose, disintegrating agents such as corn starch, pregelatinized starch, alginic acid, lubricants such as magnesium stearate, sucrose, lactose or saccharin The same sweetener, peppermint, fennel or cherries. When the unit dosage form is a capsule, it may further contain a liquid carrier such as fatty oil in addition to the substance of the above form. Various other materials may be present in the same behavior as the coating or may alter the physical form of the unit dose. For example, tablets may be coated with shellac, sugar or both. Syrups or elixirs may contain active compounds, sucrose as a sweetener, methyl and propyl parabens as preservatives, flavors such as dyes, cherries or orange flavorings.

Sterile compositions for injection comprise conventional pharmaceutical methods by dissolving or suspending the active substance in water for injection or in vehicles such as natural vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil, or in synthetic fatty vehicles such as ethyl oleate. Can be formulated accordingly. If necessary, buffer solutions, preservatives, antioxidants and the like can be mixed.

While the present invention has been described with respect to particular embodiments, it may be further modified and all such modifications, uses or generally fall within the scope of the art known or customary to the art and may be subject to the above-mentioned essentials and furthermore. It is possible to cover the application of the principles of the present invention, including variations of the present invention as covered by one claims.

Claims (2)

A compound of the formula: or a pharmaceutically acceptable salt thereof.

Wherein R ₁ is Iva- (0-methyl) Tyr-nVa1-, R ₂ is cyclohexylmethyl, R ₃ is H, Ra is H and Rb is H. A pharmaceutical composition for treating renin-dependent hypertension, comprising a therapeutically effective amount of a compound of the general formula:

Wherein R ₁ is Iva- (0-methyl) Tyr-nVa1-, R ₂ is cyclohexylmethyl, R ₃ is H, Ra is H and Rb is H.