CZ106794A3 - Peptides - Google Patents

Abstract

Di-, tri- and tetrapeptides in which the last alpha -amino acid is based on aspartic acid and attached to a residue A5 which is H; CF3; -Z1-Z2-Y2 wherein each of Z1 and Z2 independently is a direct bond or an alpha -amino acid residue and Y2 is NH2, C1-4 alkylamino, di-(C1-4 alkyl)amino or a heterocyclic radical attached by a nitrogen to Z2; -CH2-X1-Y3 wherein X1 is O or S and Y3 is heteroaryl; -CH2-Y3; substituted phenyl; ring substituted phenoxymethylene or phenylthiomethylene; ring substituted pyridyloxymethylene; or a radical -CH2-X1-CO-Y4 wherein X1 is O or S and Y4 is trialkylmethyl or substituted phenyl or pyridyl, in free form or in salt form, have pharmacological activity, e.g. IL-1 beta release inhibiting properties.

Description

Technical field

F- - “0 1- O r ~ ' > what CV with řo < and O n < OF what > CXX 1 O O σ what O στ ZZ. ! -TO O _____ -

The invention relates to peptides which are pharmaceutically usable, to processes for their preparation, to pharmaceutical compositions containing them and to the use of such compositions as medicaments.

SUMMARY OF THE INVENTION

The present invention provides compounds of formula I

R- / A ₁ -A ₂ / _n -A ₃ -A ₄ -XA ₅ (I) wherein

R represents a hydrogen atom, an amino protecting group or an optionally substituted benzyloxy group,

A 1 is Val, Leu, Ala, Ile or trimethylsilyl-Ala,

A ₂ is Phe or Tyr, n is zero or 1,

A} represents a direct bond, Val, Leu, Ala, Ile, trimethylsilyl-Ala or a divalent radical of formula (a)

wherein ring A is optionally substituted with hydroxy or C 1 -C 4 alkoxy means a direct bond or a bivalent radical of formula (b)

-N-CH-CO (b) wherein

R 1 represents a hydrogen atom or a C 1 -C 4 alkyl group;

Y | represents a residue bonded to the alpha-carbon atom alfaaminokyseliny which is optionally protected -CH ₂ -CH ₂ -N (C ₁ _ ₄ alkyl) _2, imidazol-2-yl-methyl, benzimidazol-2-yl-methyl group , 1H-1,2,4-triazol-3-yl-methyl, pyrazol-3-yl-methyl, indazol-3-yl-methyl or a radical of formula (c) or (d) h ₂ c x ^X 1; n

(C)

(d) in which each of the groups F? R ₂ and R 6 are independently hydrogen, halogen, C 1 -C 4 alkyl, CF 3 or trityl, with at most one of R ₂ and R 6 being hydrogen, and each of R 6 and R 6 are independently hydrogen, C1-C4alkyl, hydroxy, C1-C4alkoxy, CF3, phenyl or halogen, with at most one of R1 and R2 being or together are taken together to form a radical of formula (aa)

1)

NH - CH - CO - N - CH - CO

AND

R.

(aa) 1 and ^R 1 ^Y 1 in which it is as defined above and

R 1 and R 1 together are - (CH ₉ ) _m where m is 2, 3, A or 5, and

X is a divalent radical of formula (ep

(β _χ ) where

R 2 represents a hydrogen atom or a C 1 -C 4 alkyl group and represents a hydrogen atom, CF 3, the radical -CH 2 -X 4 -Y 1, wherein X 2 is O or S and Y 4 is a heteroaryl group, a radical ^or radicals of the general formula formulas (k) to (m)

-CH-X.-CO-Y. 2 1 4 k

(1) or

wherein is tri (C 1-6 alkyl) methyl or

ring B is pyridyl, ring C is phenyl or pyridyl, each of R? and Rg are independently alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, CF ^, halogen, nitro or cyano, and each of the radicals _{R j} R _and R ^ are independently nitro, cyano, CF ^ carbamoyl, 802 ^ 12 ^'- 6H = CH-CN or -CH = CHC0 ₂ Rj2 ^{»kcíe e} -i ^ 12 Nominal group having 1 to 6 carbon atoms, also represents a divalent radical of formula (e ^ )

alkyl alkyl alkyl (e ₂ )

2) X when is hydrogen or is a divalent radical of formula (f)

and represents -Z ₂ -Z-Y2 ~> ^ de each of Z ^ and Z ₂ are independently a direct bond or an alpha-amino acid and Y ₂ represents NH _2, C ^ _ ^ alkylamino, di (C, _ (alkyl) amino or a heterocyclic radical attached by a nitrogen atom to Z ₂ , or radicals of formulas (k) to (o) as defined above, or OR (j), or R (4) - (R 15) represents an alkyl group; C1-C12 optionally substituted by OH or interrupted by oxygen and each of R5 and ^R5 independently of one another hydrogen, C1-C12 alkyl, C1-C7 cycloalkyl or a benzyl group, or

3) X is a divalent radical of formula (g)

(g) and A ₅ is ^-Z 1 ^-Z 2 ^-Y 2 ' ^{as defined} above, or

4) X is a divalent radical of formulas (h) or (j)

and is a radical of formulas (k) to (o), -CH 2 -Y 1 or -CH 2 -X 1 -Y 1 as defined above, with the proviso that only one of A 1 and A 1 may be a direct bond when n is zero, and each of A 1 and A 1 is other than a direct bond when n is 1, and their physiologically hydrolyzable and acceptable esters or amides, in free form, in salt form or in complex form.

Examples of protecting groups in the substituent R are, for example, given in Protective Groups in Organic Synthesis, TW Greene, 3.Wiley and Sons NY (1901), 219-207, and are, for example, an acyl group such as acetyl, methoxysuccinyl, hydroxysuccinyl or a benzoyl group optionally substituted on the phenyl ring, for example, in the para-position by a methoxycarbonyl, methoxy or nitro group, an alkoxycarbonyl group such as a tert. butyloxycarbonyl, arylnethoxycarbonyl, such as 9-fluorenylmethoxycarbonyl or benzyloxycarbonyl, optionally substituted in the para-position by methoxy, nitro, chloro or in the meta-position by phenyl, arylmethyl, such as benzyl, optionally substituted in the ring a methoxy, nitro or chlorine position, or an arylsulfonyl group such as phenylsulfonyl group optionally substituted in the ring in para-polyl by methyl or methoxy, or naphthylsulfonyl group optionally substituted in the ring, for example amino or C 1 -C 4 dialkylamino each alkyl.

When R is a substituted benzyloxy group in the ring, it is preferably a benzyloxy group substituted by a hydroxy or alkoxy group having from 1 to 4 carbon atoms. A preferred R group is unsubstituted benzyloxy.

A preferred halogen atom is a fluorine atom or a chlorine atom.

When A 1 is a substituted radical of formula (a), this radical is preferably substituted with C 1 -C 4 alkoxy, preferably in the para-position to the -C = O group.

Alpha-amino acid means a naturally occurring or commercially available or unnatural alpha-amino acid or its optical isomer. A non-natural alpha-amino acid is an alpha-amino acid that is not incorporated into the codle mRNA rule of the rule, for example, beta-NaI, fluoro-alpha-amino acid,. such as fluoralanine, trimethylsilyl-Ala, or an alpha or nonacid such as

COOH

NH

Wn.

Η _? Ν ch-cooh where is an integer from 1 to 6 and _{n 2} is an integer from 1 to 12

Protecting groups that may be present in Yp are groups that protect the O, S or N functional group in the amino acid side chain of the alpha-amino acid. N-protecting groups are, for example, those mentioned above for R or a C 3 -C 5 alkyl group such as isopropyl, formyl, sugar residue such as 1-deoxy-fructosyl or alpha-glucosyl (1-4) -deoxyfructosyl a C 3 -C 6 dihydroxyalkyl group such as a dihydroxypropyl group, a C 5 -C 7 cycloalkyl group such as a cyclohexyl group or a tropinyl group. O- and S-protecting groups for hydroxyl and thiol functional groups are known and can be, for example, methyl, tert-butyl or benzyl.

When Y 2 is a heterocyclic radical, it may be, for example, a five- or six-membered ring, for example a piperidine or pyrrolidinyl group.

Examples of heteroaryl groups for the substituent include, for example, five-, six- or seven-membered unsaturated heterocyclic radicals containing at least one nitrogen atom and optionally other heteroatoms such as nitrogen, oxygen or sulfur. Preferred Y-j is a heteroaryl group containing from 1 to 4 nitrogen atoms, for example pyridyl, triazolyl, tetrazolyl, triazinedionyl.

In ring B of the radical of formula (o), the nitrogen atom may be in the ortho, meta or para position. If ring C in the radical of formula (k) is pyridyl, then it may be 3-, 4- or 5-pyridyl.

The radicals of formulas (e ^), (62), (f), (g) and (j) are derived from Asp and contain one asymmetric carbon atom and the remainder of formula (h) contains two asymmetric carbon atoms so isomerism occurs. It is to be understood that the present invention encompasses all individual isomeric forms and diastereoisomers, as well as mixtures thereof, for example racemates, unless otherwise indicated.

A radical of formula (ep attached to substituent A _$, which is hydrogen may exist in both cyclic as well as in non-cyclic form, for example as shown by the equation:

It will be understood that when tautomeric forms are present, the present invention includes both lactol and oxo-carboxylic acid forms, i.e. although the compounds of formula I wherein X is a radical of formula (e ^) are preferably referred to only as the form oxo-carboxylic acids, the invention is not to be construed as limited to the particular nomenclature or graphical representation used for these compounds. Similar considerations also apply to starting compounds exhibiting lactol / oxo-carboxylic acid tautomerism as described above.

The same applies to the remainder of the general formula (h), which may also exist in both linear form and cyclic form, as shown by the following equation:

h ⁺ / -h ₂ o js base / water

and for compounds of formula I containing a radical of formula (h) and for the corresponding starting compounds.

By physiologically hydrolyzable and acceptable esters or amides is meant esters and amides which are hydrolysable under physiological conditions to alcohols or amines which are physiologically acceptable per se, i.e. which are non-toxic at the desired dosage levels.

These esters or amides can be prepared by esterification or amidation of a compound of formula kde wherein X is a carboxy-containing residue. These esters include esters with an aliphatic or alicyclic alcohol or a polyol containing 1 to 12 carbon atoms. Amides include amides with aliphatic amines, for example, C 1 -C 4 alkylamines, C 1 -C 4 alkoxyalkylamines and C 1 -C 4 alkoxyalkylamines, such as beta-methoxyethylamine, or aniline.

The compounds of formula I may exist, for example, in free form, in the form of a salt with an acid or in the form of complexes. Acid salts may be formed with, for example, organic acids, polymeric acids, and inorganic acids. Such acid salts are, for example, hydrochlorides and acetates. Salts may also be formed with a carboxyl group present in compounds of Formula I, for example, alkali metal salts such as sodium or potassium, or further substituted or unsubstituted ammonium salts. For example, the complexes are formed from compounds of formula I by addition of an inorganic substance, for example inorganic salts or hydroxides such as Ca- and Zn-salts, and / or the addition of palymeric organic substances.

The following are the meanings of preferred substituents used either singly or in any combination or subcombination that may be included in the compounds of Formula I:

R is an amino protecting group or a benzyloxy group, preferably an amino protecting group. Preferably, R is benzyloxy when n is zero and _{e is a} radical of formula (a).

2. Each of the groups A 1a is other than a direct bond.

3. n is zero.

4. A} is a radical of formula (a) when n / is zero.

5. is a direct bond, Val, Leu, Ala, Ile or trimethylsilyl-Ala.

6. R 6 is a hydrogen atom or a methyl group, preferably a hydrogen atom.

7. Y ₁ is a residue attached to the alpha carbon atom of an alpha amino acid selected from the group consisting of Ala, Leu, His, Phe, Met, Trp, trimethylsilyl-Ala and optionally protected in the side chain with Arg, Orn and Lys, or of formula (c).

8. A ^ and A ^ may also be taken together to form a radical of formula (aa) when n is zero,

9. m is 2 or 3.

10. X is a radical of formula (e ^) and A ^ is other than hydrogen.

11. X is a radical of formula (g).

12. X is a radical of formula (h) or (j).

Is a hydrogen atom or a methyl group, preferably a hydrogen atom.

14. Z is a natural alpha-amino acid residue, preferably an aromatic-heterocyclic alpha-amino acid, especially Pro.

15. Z is a natural alpha-amino acid residue, preferably an aliphatic alpha-amino acid, especially an aliphatic alpha-amino acid containing no other functional groups, most preferably Val.

16. X is a radical of formula (e ^), optionally esterified or amidated, and A ^ is a radical of formula (k), (1) or (o).

17. X is a radical of formula (e ^), (h) or (j), optionally esterified or amidated, and A ₅ is -C ^ -X - ^-Yj, preferably -CH ^ -SYj or -CH ^- Y ^ ·

10. X is a radical of formula (h) or (j), optionally esterified or amidated, and is a radical of formula (k).

19. X is a radical of formula (j), optionally esterified or amidated, and A A is a radical of formula (m).

20. The radical of formula (m) is monosubstituted, preferably in the para position, and more preferably is 4-nitrophenyl.

21. All alpha-amino acid residues present except X have the L configuration. X has a D or L configuration.

22. The asymmetric carbon atom in the radical of formula (h) bearing the OH group has the S configuration. Preferably, the asymmetric carbon atom in the radical (j) bearing the R group has a configuration.

Also preferred are compounds of formula I wherein n is zero, A 1 is a direct bond, Val, Leu, Ala, Ile or trimethylsilyl-Ala, A 1 is as defined above, or and A 1 together form the remainder of the formula ( (aa) as defined above; and

1) X is a radical of formula (e ^) or (e?) As defined above and A ^ is hydrogen, or

2) X is a radical of formula (e ^) or (f) and is a radical of formula (k), (1) or (m), wherein X 1 is 0,

3) X is a radical of formula (g) and A 1 is -Z 2 - ₂ or

4) X is a radical of formula (j) and is a radical of formula (k), (1) or (m) wherein X 1 is O.

The present invention also provides a process for the preparation of the compounds of formula (I), characterized in that:

a) removing at least one protecting group from a compound of formula I in protected form, or adding a protecting group R to the N-terminal group of a compound of formula I, or

(b) converting one compound of formula I into another compound of formula I, or

c) joining together by amide bond two peptide fragments, each containing at least one amino acid in protected or unprotected form and one peptide fragment containing a residue of formulas (e ^) to (j) as defined above, wherein the peptide fragments are such that a protected or unprotected peptide having the sequence of Formula I is obtained, and optionally removing the protecting group or groups from the compound of Formula I in protected form, or

d) for the preparation of compounds of formula I wherein X is a radical of formula (e ^) or (h) and is a radical of formula (k), (1) or (o) or is reacted with a compound of formula III

-A. _X'-O-CH 2 -Z 4 (ΙΠ) where R 1 to A 4 and n have aforesaid meaning "X" is the tek of general formula (e ^) or (h) and Z and it is cleavable

a halogen atom, with the corresponding phenol, thiophenol or HX4-pyridine or with an acid of the formula HX4 -CO-Y4 or a functional derivative thereof or with HX4-Y4, or

e) for the preparation of a compound of formula I

H 0

(I) wherein R 1 to A 8 and n are as defined above and R 6 is an aliphatic or alicyclic radical having 1 to 12 carbon atoms, oxidizes the compound of formula V

H OH

wherein R, A ₁ to A ₅ , on R ₆ are as defined above, and the compound of formula (I) thus obtained is isolated in free or salt form or in complex form.

Processes (a) to (e) may be carried out by methods known in the art.

Removal of the protecting group in step (a) may also include removal of R at the N-terminal group of a compound of formula I. For example, when R is a benzylaxycarbonyl group, this group may be removed by hydrogenation in the presence of catalysts such as palladium.

In accordance with the process of step (b), for example, to prepare a compound of formula I wherein X contains a carboxy group, a compound of formula I wherein X contains an esterified or amidated carboxy group can be hydrolyzed.

This hydrolysis can be carried out by appropriate alkaline or acid hydrolysis, for example in the presence of trifluoroacetic acid.

Further, in accordance with the process of step (b) for preparing a compound of formula I wherein X contains an esterified or amidated carboxy group, a compound of formula I wherein X contains a carboxy or esterified carboxy group may be (trans) esterified or amidated. This ester or amidation formation can be carried out using methods known in the art, for example by converting a carboxy group to a functional reactive group, for example the corresponding carbonyl halide or anhydride, or using a compound of formula I wherein X is a lactone moiety , and reacting such a group with a selected alcohol or amine.

Further, in accordance with the process of step (b) for the preparation of a compound of formula I wherein X is a radical of formula (g), a compound of formula I wherein X is a radical of formula (e ^) or (e ^) is reacted. and is a hydrogen atom with a compound of formula II

H ₂ N-NH-CO-A ₅ (II) wherein A 1 is as defined above. This process can be carried out analogously to known methods used to prepare semicarbazones.

A compound of formula I wherein X is a radical of formula (ep) can also be converted by known methods to a compound of formula I wherein X is a radical of formula (ep, and vice versa).

The process of step (c) may be carried out by methods known in the art of peptide chemistry. A peptide fragment containing a radical of formula (ep to (j)) is also understood to mean a radical containing a protecting group on the -NR 1 - moiety and correspondingly terminating, e.g.

The process of step (d) may be carried out using an OessMartin reagent, for example in the presence of a base or a halogen-precipitating silver salt, or by the Swern oxidation process.

If desired in these reactions, protecting groups can be used for functional groups that do not participate in the reaction.

These may be, for example, amino-protecting groups, carboxy-protecting groups, acetal groups, etc. After the desired reaction, the protecting groups may be removed.

Each of the above processes may be carried out using the starting materials in the form of one or other individual ophthalmic isomer or a mixture thereof / according to the asymmetric carbon atoms present in the radicals X (e) to (j) of X or a precursor thereof. residues. Usually, the S- or R-enantiomers are used as starting materials to give compounds of formula I in which the asymmetric carbon atom in the radicals of formulas (e) to (j) has the S or R configuration.

The starting materials used in steps (d) or (e) of the process of the invention can be prepared in analogy to the process of step (c).

If the preparation of the starting materials is not specifically described, these compounds are known or can be prepared in analogy to known methods used in practice.

The following examples serve to illustrate the present invention in more detail. All temperatures are in ° C.

The following abbreviations are used in the examples:

THF = tetrahydrofuran TFA = trifluoroacetic acid MeOH = methanol EtOAc = ethyl acetate DCC = dicyclohexylcarbodiimide GUEST = hydroxybenzotriazole OF = benzyloxycarbones1 Fmoc = 9-fluorenylmethoxycarbonyl and = amorphous

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Z-Val-Met-Asp (OH) -H

Z-Val-Met-aspartaldehyde beta-tert-butyl ester (1.17 g) was added to 150 ml), TFA (15 ml) and water (1 ml) were added and the reaction mixture was stirred at room temperature for 2 hours. , 5 hours. The solvent was evaporated, toluene was added twice and evaporated again. The crystalline residue was dissolved in water (60 ml) with heating, decanted from the insoluble material and recrystallized at 5 ° C to give a first crop, m.p. 117 ° C. All mother liquors were combined and chromatographed (SiO ₂ , acetone / hexane / EtOAc 60/40 / 0.5) to give a second crop of product and recrystallized after trituration with water.

Example 2

Z-Val-Met-Asp (OH) -H semicarbazone

Example 1 (0.27 g, 0.55 mmol) in MeOH (2 mL) was treated with semicarbazide, HCl (0.6 mL of a 1 molar solution), then 5 drops of pyridine were added. The product crystallized after 10 minutes at room temperature, mp 253-235 ° C.

Example 3

Z-Val-Met-Asp (OH) -H semicarbazonyl-Pro-Val-N (CH 2) ₂

Example 1 (0.48 g, 1 mmol) was dissolved in a mixture of MeOH (2 mL), water (0.5 mL) and 3 drops of pyridine. N- (hydrazinocarbonyl) -Pro-Val-N (CH 2) ₂ (0.3 g, 1 mmol) was dissolved in MeOH (1 mL), water (2 mL) and 3 drops of 2N HCl. The two solutions were mixed and heated to 40-50 ° C for 2 minutes. The product crystallizes under cooling, mp 118-120 ° C

By repeating the procedure of Example 1, the following compounds were prepared using the corresponding starting compounds:

Example 4

Z-Val-Phe-Asp (OH) -H

Example 5

Z-Val-His-Asp (OH) -H By following the procedure of Example 2 or 3, the following compounds of formula IAc / NHA were prepared using the corresponding starting compounds, with _3- and _4- n- ^5.

CIA)

CO ₂ H wherein A 1, A 1 and A 1 are as defined above,

example C. ^A 3 ^A 5 melting point ° C 6 Wall Phe H and 7 Wall His H 150 8 Wall Phe Pro-Val-N _{(CH3) 2} 126-139 9 Wall His Pro-Val-N _{(CH3) 2} 10 Ala-Tyr-Val Phe H 205 Example 11

(3S) -3- (Z-Valyl-phenylalanyl) -amino-5- (2,6-dimethylbenzoyloxy) -4-oxopentanoic acid

(3S, 4RS) -3- (Z-Val-His) amino-4-hydroxy tert-butyl ester

Of 5- (2,6-dimethylbenzoyloxy) pentanoic acid (0.6 mmol) was treated with Dess-Martin reagent (0.77 mmol) in CH 2 Cl 2; (4 mL) for 45 minutes and then filtered. Acetone and 0.5 N NaOH were added and the acetone was evaporated. The remaining crystalline material was washed with water, acidified with 10% tartaric acid and extracted with EtOAc. The combined EtOAc extracts after evaporation gave the tert-butyl ester product as yellow crystals. These were dissolved in (4 mL), TFA (4 mL) was added and the mixture was stirred at room temperature for 15 minutes. Then the mixture is evaporated to dryness and chromatographed (SiO _2> acetone / hexane / EtOAc 60/40/1) to give the desired product as slightly colored crystals.

Example 12

(Z-Valyl-alanyl) - (3R, 4S) -3-Amino-4-hydroxy-5- (2,6-dichloro-benzoyloxy) -pentanoic acid ethyl ester

(Z-valyl-alanyl) - (3R, 4S) -3-amino-4,5-dihydroxypentanoic acid ethyl ester and 4-dimethylaminopyridine were dissolved in pyridine and 2,6-dichlorobenzoyl chloride was added dropwise. The reaction mixture was stirred overnight at room temperature, then ice and water were added and the mixture was extracted with AcOEt. The organic layer was washed with water, then with NaCl solution, dried over Na 2 SO 4, filtered, evaporated and the crude product chromatographed. The title compound is obtained.

The (3R, 4RS) derivative of the title compound was prepared as follows:

Z-Val-Ala-OH (0.26 mmol), DCC (53 mg, 0.26 mmol) and HOBT.F 2 O (39 mg, 0.26 mmol) was dissolved in THF / DMF (2 mL / 2 mL) and stirred for 5 min before (3S, 4RS) -3-Amino-4-hydroxy-5- (2,6-dimethyl-benzoyloxy) -pentanoic acid tert-butyl ester (07 mg, 0.26 mmol) was added. ) in THF (2 mL). The reaction mixture was stirred overnight at room temperature, then evaporated and chromatographed (SiCl 4 / EtOAc / MeOH / NH 95/5 / 0.5) to give the (3R, 4RS) -divative of the title compound.

Example 13

(Z-Valyl-alanyl) - (3R) -3-amino-4-oxo-5- (2,6-dichloro-benzoyloxy) -pentanoic acid ethyl ester

To a solution of oxalyl chloride v was added dropwise dimethylsulfoxide at -50 ° C. After 15 minutes, a solution of the compound of Example 12 in CH 2 Cl 2 was added dropwise. and the reaction mixture was stirred at -40 ° C for 1 hour. Triethylamine is then added and stirring is continued for 3 hours at room temperature. Water was then added and the reaction mixture was extracted with CH ₂ Cl ₂ . The organic layer was washed with NaHCO 3 solution and NaCl solution, dried over Na 2 SO 4, filtered and evaporated. The residue is chromatographed to give the title compound.

By repeating the procedure of Examples 1 to 3 and 11 to 13, the following compounds of formula 18 were prepared using the corresponding starting compounds

(IB)

wherein A 1, A 1, R 2 and R 4 have the meanings given below.

CM O O X O N4 (AT C X XD M-z 4- » O WHAT O 4- » O r— <D. CM O ω 4-> CD «* 4 O r4 X) CD ω C

Γ-Λ

Ό

CD

S-i

Xu

Cl

and-( r-4 1 “4 r-4 <—1 F — f r-4 X >, >. i-4 >> X X O O O O X O O O N N IM N O IM N N C C C C N C C C ω ω ω ω C ω ω i — t cu XI XD XI X ω n XD xd 1 1 1 1 XD 1 1 C 1 CM r- \ 1 ω ω ω x r-4 at. O If_ AT- »-4 X «Μ O C_5 2: LJ O O -w • r4 AND • r4 · »—T • * 4 • «4 • c4 • r4 Ό TZJ CM XD Π XD XD xd XD 1 1 O AND 1 1 1 1 1 M3 NO X NO M3 NO NO NO NO * · » 1 * ·. «* CM CM CL CM CM CM CM CM CM

6-diCl-benzoyl

CM Example R _x / alpha / gc (MeOII) or melting point

CM o

CM

C\

CM o

O

AND-

VO

CM

AND

r — 1 ^ -4 if >> >> O and O N N N C C C here here ω -Ω 10 | YEAH 1 r-H 1 r-4 i — i O what C_3 • fH • «-4 • r-4 Ό -and t O vo 1 VO vC * » F. CM CM CM

CM

O ω in o

JZ -rH here

CL. X <

ra>

i u

>>

FI ra ra>

m in r ** cm cm CM tn ra>

O

CM ro o

o o

i — 4

AND

ra>

σ \

CM vo

C \ o

O

Cv

ra>

o r * \

-diCl-bcnzoy1

VO

CM

CM o

for example Λ-j Λ ₄ R _y R _x / alpha / _Q c (MeOH) or melting point

XXX

Nilo σ \ o <r <r ir \

I CM m lt \

F**\

LT \

CN

C_3 □

Λ- example, Λ R _{4 y} R _x / alpha / ^u ^ c (MeOH) or m.p.

the compounds have the R, S configuration in the aspartyl portion

Example 50

/ alpha / ⁰ * ⁰ = -20.3 c = 1 in MeOH

Example 59

Example 60

O-CO / alpha / ⁰ * ⁰ = -21.5 c = 1.07 MeOH

Example 61

Example 62

By repeating the procedure of Examples 1 to 3 and 12 to 13 using the corresponding starting compounds, the following compounds of formula (IC) were prepared.

(IC) wherein A., R and R are as defined below:

X y

example ^Λ 4 R y R X X 63 Ala H 2,6-diCl-benzoyl Cl \ _ R, S 64 Ala C ₂ ^H 5 - ^C ° - / Oy Cl R 65 Ala tC ₄ ^H 9 2,6-diCl-benzoyl R, S 66 Ala C H _{and the} -0CH 2 3 4 2,6-diCl-benzoyl R, S

Example 67

(3RS) -3- (Z-Valyl-phenylalanyl) amino-4-oxo-6- (p-nitrophenyl) -trans-5-hexenoic acid tert-butyl ester

(3RS) -3- (Z-Valyl-phenylalanyl) amino-4-oxo-pentanoic acid 5-diethylphosphonate (0.2 g, 0.29 mM) was dissolved in THF (15 mL) at 5 ° C. NaH (25 mg, 0.59 mM) was added and the mixture was stirred for 10 minutes. At 0 ° C, p-nitrobenzaldehyde (0.18 g, 1.1 mM) in THF (2 mL) was added and the reaction mixture was stirred at this temperature for 45 minutes. The reaction mixture was then poured into a 5% tartaric acid and extracted with ethyl acetate, the organic phase was dried over Na ₂ S0 ^, evaporated and the product purified by chromatography. The title compound is obtained.

Example 68

Z-Val-Phe-aspartaldehyde dimethyl acetal beta-methyl ester

Z-Val-Phe-Asp (OH) -H (0.8 g) was dissolved in MeOH (25 mL) containing 8% HCl, left at room temperature overnight, then evaporated to dryness and the remaining crystals were washed with ether to give the title compound as white crystals, m.p. 168-171 ° C.

The starting compounds are prepared by the following methods:

Example 69

(3S) -3- (Fluorenylmethoxycarbonyl) amino-5-iodo-4-oxo-butanoic acid tert-butyl ester

To (3S) -3- (fluorenylmethoxycarbonyl) amino-3-carboxybutanoic acid tert-butyl ester (3.5 g, 0.5 mmol) in THF (60 mL) at -10 ° C was added triethylamine (1.3 mL). (9.6 mmol) and then ethyl chloroformate (0.92 mL, 9.6 mmol). After 10 minutes, a solution of diazomethane in ether was slowly added and the reaction mixture was stirred at 0-5 ° C for 45 minutes. HCl (2 N) in ether was added at 5-10 ° C until gas evolution ceased. The reaction mixture was evaporated to dryness, taken up in acetone (50 mL), Nal (4 g) was added and stirred at room temperature for 1 hour. Ether (150 ml) was then added and the reaction mixture was filtered and evaporated. The residue was chromatographed (SiO 2, EtOAc / hexane) to give the title compound as pale yellow crystals.

Example 70

(3S) -3- (Fluorenylmethoxycarbonyl) amino-5- (2,6-dimethylbenzoyloxy) -4-oxo-pentanoic acid tert-butyl ether

(3S) -3- (Fluorenylmethoxycarbonyl) amino31 tert -butyl ester31

5-iodo-4-oxo-butanoic acid (1 g, 1.0 mmol), 2,6-dimethylbenzoic acid (0.5 g, 3.3 mmol) and AgOAc (0.6 g, 3.6 mmol) Dissolve in acetone (25 mL) and heat to boiling for 1 hour. After filtration and evaporation, the crude product is chromatographed (SiO 2, ether / hexane 3/7) to give the title compound.

Example 71

(3S) -3- (Fluorenylmethoxycarbonyl) amino-5- (4-nitrophenoxy) -4-oxo-pentanoic acid tert-butyl ester

(3S) -3- (Fluorenylmethoxycarbonyl) amino-5-iodo-4-oxo-butanoic acid tert-butyl ester (1 g, 1.8 mmol), p-nitrophenol (0.5 g, 3.7 mmol) and (0, 38 g (2.8 mmol) was heated to boiling in acetone (6 mL) for 30 minutes, then Cl 2 Cl 2 was added and the organic phase was washed with 2 N NaHCO 3. The combined organic phases were dried, evaporated and chromatographed (SiO 2, EtOAc / hexane 2/8) to give the title compound as a yellow oil.

Example 72

(Z-Valyl-alanyl) - (3R, 4S) -3-Amino-4,5-dihydroxypentanoic acid ethyl ester

a) Ethyl (3R, 4S) -3-benzylamino-4,5- (isopropylidenedioxy) pentanoate (Y. Yamada, Tetrahedron Letters 1983, 24, 3009) and 10% Pd / C in ethanol are shaken at room temperature in an atmosphere hydrogen for 30 minutes. Filtration and evaporation of the reaction mixture gave ethyl 1- (3R, 4S) -3-amino-4,5- (isopropylidenoxy) pentanoate, which was used without further purification.

b) Z-Val-Ala-OH is dissolved in THF, HOBT.h 2 O and DCJ are added at 5 ° C. The reaction mixture was stirred for 20 minutes at 5 ° C and diisopropylethylamine and ethyl (3R, 4S) -3-amino-4,5- (isopropylidenedioxy) pentanoate in THF were added. The reaction mixture was stirred overnight at room temperature, filtered, evaporated and chromatographed. There was obtained (Z-valyl-alanyl) - (3R, 4S) -3-amino-4,5- (isopropylidenedioxy) pentanoic acid ethyl ester.

c) Example 74b) was dissolved in AcOH / I 2 O (75/25) and stirred at 40 ° C for 4 hours. After evaporation, water was added and the mixture was extracted with AcOEt. The combined extracts were washed with water, NaHCO 3 solution, dried over Na ₂ SO ₄ , filtered and evaporated. The title compound is obtained.

The compounds of formula I, and their physiologically hydrolyzable and acceptable esters and amides, and their pharmaceutically acceptable salts (referred to herein as compounds of the invention) exhibit pharmaceutical activity and are therefore useful as pharmaceuticals.

In particular, the compounds of the invention inhibit IL-1beta secretion, as shown by the following in vitro assay using THP-1 cells and in vivo assay:

a) 900 µl THP-1 cells (0.5 x 10 6 cells) along with 100 U gamma-interferon / 0.9 ml RPMI 1640 medium (containing 2 mM L-glutamine and 5% calf fetal inactivated serum) pipet into 24 wells of culture dishes. 100 µl of test compound is then added. After 3 hours at 37 ° C in an atmosphere of 5% CO ₂ /95% air, 10 µL of lipopolysaccharide (500 µg / ml) was added and incubation continued for a further 40 hours. Appropriate controls (with and without stimulus, solvent) are included in the experiment. The media is then removed and clarified by centrifugation at 1000 g for 10 minutes, then 1.0 ml of 0.01% digitonin is added to the wells to lyse the cells, which are loosened and scraped off with a rubber spatula and kept at 4 ° C. ° C for 10 minutes. Immediately thereafter, lactate dehydrogenase measurement is performed and samples are stored at -20 ° C until further determinations.

These assays are: IL-1β (medium and lysate), IL-6 (medium), TNF-alpha (medium), PGE2 (medium and lysate), lactate dehydrogenase (LDH), and DNA (lysates). IL-1beta, IL-6 and TNF-alpha assays are determined using commercially available ELISA kits (Cistron), PGE2 is measured using standard RIA and DNA is measured fluorometrically using DAPI.

In this assay, the compounds of the invention selectively inhibit the release of IL-1beta at concentrations from about 0.01 to 100 µM. In contrast, levels of IL-6, TNF-alpha, PGE2 and DNA remain substantially unaffected and compounds are non-toxic because LDH release is unchanged. For example, the compounds of Examples 40 and 50 were determined to have an IC 50 (concentration of compound that inhibits 50% release of IL-1beta) or 0.1 µM.

b) LPS-fever

Male Tuttlingen SD rats (150-160 g) were injected with LPS suspension (Sigma, # L-5886, 100 µg / 5 ml glucose solution subcutaneously). Body temperature is measured after 2 hours using a thermistor rectal sensor connected to an ELLAB thermometer. After 4 hours, the test compound is administered orally and after another hour (6 hours after LPS application) the temperature is measured again. The temperature increase measured in the untreated control is calculated as 100% and is expressed as a percentage of the value in the treated group. ED 50 is the dose causing 50% inhibition of the temperature increase determined in control rats. In this assay, the compounds of the invention inhibit LPS-induced temperature increment at a dose ranging from 0.001 to 0.1 mg / kg orally. For example, the compounds of Examples a 50 were determined to have an ED 50 of 0.01 mg / kg per os, and the compound of Example 51 had an ED 50 of 0.05 mg / kg per os.

(c) Carrageenan-induced paw swelling in rats

For each group, 50FA male rats weighing 150-170 g are used. The test compound is administered orally as a suspension in physiological saline and 0.5% tragacanth 1 hour prior to carrageenan injection. Carrageenan (0.1 ml of 1% suspension in physiological saline) is injected by subplantar injection into one hindpaw. The paw swelling is measured with an antiphlogometer according to Kemper and Amelm. A control reading is made immediately after injection and swelling is measured after 3 and 5 hours. The mean of the 3 and 5 hours readings is subtracted after the control measurement and the values obtained in the treated animals are expressed as a percentage of the values obtained from the untreated controls. The value is the dose causing 50 ¾ inhibition of carrageenan-induced swelling after 3 hours. In this assay, the compounds of the invention significantly inhibit carrageenan-induced swelling when administered orally at a dose ranging from 0.02 to 5 mg / kg. For example, it has been determined that the compounds of Examples 40 and 50 have values of εΟ 0.2 ± 3 mg / kg per os.

The compounds of the invention are therefore useful for the treatment of disorders caused by excessive release of IL-1beta, for example in a variety of inflammatory conditions and diseases such as tissue calcium depletion, bone and cartilage degenerative processes such as rheumatoid arthritis and osteo arthritis, inflammatory bowel disease, irritable bowel disease, septic shock, psoriasis, asthma, adult respiratory distress syndrome, type I diabetes, osteoporosis of various origins including, for example, climacteric or postmenopausal osteoporosis, as well as osteoporosis due to age, restraint or trauma, arteriosclerosis and Alzheimer's disease.

For the above uses, the dosage required will, of course, depend upon the mode of administration, the particular condition being treated and the desired effect. However, in general, satisfactory results are obtained with daily dosages ranging from about 0.001 to about 100 mg / kg, preferably from 0.001 to about 10 mg / kg body weight. Suitable daily dosages for larger mammals, for example humans, are in the order of from about 0.1 mg to about 1 g / day, and a single dose, in divided doses of two to four times a day, or in slow release may be conveniently used.

Preferred are the compounds of Examples 50 and 51.

In accordance with the foregoing, the present invention also relates to:

a) A method of treating a condition that is associated with, or caused by, an excess release of IL-1beta, for example, as described above in a subject in need thereof, the method of treatment being characterized by: to be treated, administers an effective amount of a compound of Formula I, a physiologically hydrolyzable and acceptable ester or amide thereof, or a pharmaceutically acceptable salt thereof.

b) A compound of formula I, a physiologically hydrolyzable and acceptable ester or amide thereof, or a pharmaceutically acceptable salt thereof, for use as a medicament, for example, for use as a condition treating agent as set forth above.

The compounds of the invention may be administered by any conventional method, particularly nasally, enterally, preferably orally, e.g. in the form of tablets or capsules, or parenterally, e.g. in the form of injectable solutions or suspensions or in the form of suppositories. µg to 500 mg of a compound of the invention.

The compounds of the invention may be administered in free form or in the form of a pharmaceutically acceptable salt. These salts can be prepared by conventional methods and exhibit the same activity as the free compounds.

Further, the present invention also relates to:

c) A pharmaceutical composition comprising a compound of formula I, a physiologically hydrolyzable and an acceptable ester or amide thereof, or a pharmaceutically acceptable salt thereof, as defined above, together with a pharmaceutically acceptable diluent or carrier. These compositions can be prepared by conventional methods. The compositions may contain up to 99.9% by weight of the active ingredient.

Claims (4)

PATENT CLAIMS Compounds of formula I R / ^A 1-A ₂ / _n A ₃ -A ₄ -XA ₅ (I) wherein R represents a hydrogen atom, an amino protecting group or an optionally substituted benzyloxy group, n represents zero or 1, A 1 is Val, Leu, Ala, Ile or trimethylsilyl-Ala, AND 2 means Phe or Tyr, A} represents a direct bond, Val, Leu, Ala, Ile, trimethylsilyl-Ala or a divalent radical of formula (a) CO (a) wherein ring A is optionally substituted with hydroxy or C 1 -C 4 alkoxy, A ₄ represents a direct bond or a bivalent radical of formula (b) -N-CH-CO- (b) wherein R @ 1 represents a hydrogen atom or a C1 -C4 alkyl group, and Y | represents a residue bonded to the alpha-carbon atom alfaaminokyseliny which is optionally protected, -CH2-CH2-N (C, _ ₄ alkyl) 2, imidazol-2-yl-methyl, benzimidazol-2-y1-methyl, 1H1 , 2,4-triazol-3-ylmethyl, pyrazol-3-ylmethyl, indazol-3-ylmethyl or a radical of formula (c) or (d) (c) wherein each of R ₂ and R 1 are independently hydrogen, halogen, C 1 -C 4 alkyl, CF 3 or trityl, wherein at most one of R ₂ and R 3 is hydrogen, and each of R 4 and R 4 are each independently of one another hydrogen, C1-C4alkyl, hydroxy, C1-C4alkoxy, CF3, phenyl or halogen, where at most one of the groups and R6 is hydrogen, or A 1 and A 2 together form the remainder of formula (aa) -NH-CH-CO-N-CH-COI ^R 1 and ^R 1 ^Y 1 (33) wherein Υ ^ has the meaning given above and ^R 1 and ^R 1 ^{and S} P ^O 1 ^U together form a group - (CH 2). wherein m X xa m 'is 2, 3, 4 or 5, and 1) X represents a divalent radical of formula (e ^) wherein R 8 represents a hydrogen atom or a C 1 -C 4 alkyl group, and A ₅ represents a hydrogen atom, CF 3, the radical -CF 3 -X 4 -Y 1 where X 1 is O or S and Y 1 is a heteroaryl group. or a residue of the formulas (k) to (m) or (m) ‘11 (o) where Y ₄ is a tri (C 1-4) alkylmethyl group or a radical of formula ( ⁷⁾ ring B is pyridyl, ring C is phenyl or pyridyl, each of the radicals and R 8 are independently C 1 -C 4 alkyl, C 1 -C 4 alkoxy , CF 3, halogen, nitro or cyano, and each of R 3, R 4, and R 4 is independently nitro, cyano, CF 3, carbamoyl, ^CO 2 ^R 12 '- ^{ch = ch_cn} or -CH = CHCO ₂ R ₁₂ , wherein R ₁₂ is an alkyl group having 1 to 6 carbon atoms, X is also a divalent radical of formula (e ₂₎ alkyl alkyl alkyl (e ₂₎ 2) X that represents a hydrogen atom, or represents a divalent radical of formula (f) and A ₅ represents a group ^"Z 1 ^'Z 2 ^Y 2' ^{where each of} i ^and 2 i ^are independently of one another a direct bond or an alpha-amino acid and is N 1, R 1, C 1-6 alkylamino, di- (C 1-6 alkyl) amino or a heterocyclic radical attached by a nitrogen atom to Z 2, or radicals of formulas (k) to (o) as defined above, or or NR ^ R ^, where R ^ is an alkyl group having 1 to 12 carbon atoms optionally substituted by OH or interrupted by oxygen and each R14 ³ R 15 and ^are independently of one another hydrogen, alkyl having 1 to 12 carbon atoms, (C 5 -C 7) cycloalkyl or benzyl group; or 3) X is a divalent radical of formula (g) and A 1 is -Z 1 -Z 2 -Y 2 as defined above, or 4) X represents a divalent radical of formulas (h) or (j) (h) (j) and represents a radical of formulas (k) to (o), -CH ₂ -Y 1 or -CH ₂ -X ₁ -Y ₅ , as defined above, with the proviso that only one of A ^ and A ^ can be a direct bond when n is zero, and each of A ^ and A ^ is different than the direct bond when n is 1, and their physiologically hydrolysable and acceptable esters or amides, in free form, in salt form or in the form of complexes. Compounds of formula (I) according to claim 1, wherein n is zero, A 1 is a direct bond, Val, Leu, Ala, Ile or trimethyl silyl-Ala, A 1 is as defined in claim 1 or A 1 and A 1 together together form a residue of formula (aa) as defined in claim 1, and 1) X is a radical of formula (e.) Or (θ?) »D ^E defined in claim 1 and A _$ j _{S and} t _he hydrogen nedo 2) X is a radical of formula (e ^), or (f) and A _$ is a radical of formula (k), (1) or (m) as defined in claim 1, wherein m is 0, 3) X is a radical of formula (g) and A 1 is a group -Z 1 -Z ₂ -Y ₂ as defined in claim 1, or 4) X is a radical of formula (j) and A 1 is a radical of formula (k), (1) or (m) as defined in claim 1, wherein X 1 is O, and their physiologically hydrolyzable and acceptable esters, or amides, in free form, in salt form or in the form of complexes. Compounds of formula I according to claim 1, wherein n is zero and each of A 1 and A 1 is other than a direct bond, and their physiologically hydrolysable and acceptable esters or amides, in free form, in salt form or in the form of complexes. Compounds of formula I according to claims 1 or 3, wherein the residue is attached to the alpha-carbon atom of the alpha-amino acid selected from the group consisting of Ala, Leu, His, Phe, Met, Trp, trimethylsilyl-Ala and optionally in the side chain protected by Arg, Orn and Lys, or a radical of formula (c), and their physiologically hydrolyzable and acceptable esters or amides, in free form, in salt form or in complex form, Compounds of formula I according to claims 1, 3 or 4, wherein X is a radical of formula (e ^), (h) or (j), A5 is other than hydrogen when X is a radical of formula (ep, and their physiologically hydrolyzable and acceptable esters or amides, in free form, in the form of salts or in the form of complexes. 6. (Benzyloxycarbonyl-valyl-alanyl) -3R-3-amino-4-oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid ethyl ester, (Benzyloxycarbonyl-valyl-alanyl) -3R, S-3-amino-4-oxo-ethyl ester 5- (2,6-dichlorobenzoyloxy) pentanoic acid, ethyl ester (benzyloxycarbonyl-valyl-alanyl) -3R-3-amino4-oxo-5- (2,6-dichloropyridyl-4-carbonyloxy) pentanoic acid, (benzyloxycarbonyl- valyl-alalnyl) -3R, S-3-amino-4-oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid, (benzyloxycarbonyl-valyl-alanyl) -3R, S-3-amino-4-oxo-5- ( 2,6-dichlorobenzoyloxy) pentanoic acid, (benzyloxycarbonyl-valyl-alanyl) -3R, S3-amino-4-oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid tert-butyl ester and (benzyloxycarbonyl-valyl-alanyl) - 3S-3-amino-4-oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid in free form, in salt form or in the form of complexes. A process for the preparation of compounds of the formula I and their physiologically hydrolyzable and acceptable esters and amides as defined in claim 1, characterized in that a) removing at least one protecting group from the compound of formula I in protected form or adding a protecting group R as defined in claim 1 to the N-terminal group of a compound of formula I, or (b) converting one compound of formula I into another compound of formula I, or c) join together by amide bond two peptide fragments, each containing at least one amino acid in protected or unprotected form and one peptide fragment containing a residue of formulas (e ^) to (j) as defined in claim 1, wherein the peptide fragments fragments are such that they give a protected or unprotected peptide having the sequence of formula I and, optionally, ^a protecting group očsxrs- n? oo protecting group from a compound of formula I in the form cnráněné or d) for the preparation of compounds of general formula I, wherein X is a radical of general formula (e ^) or (h) and is a radical of general formula (k), (Ϊ) or (o) or -CH 3 -X 1 -Y 2 as are as defined in claim 1, reacting a compound of formula III R / A ₁ -A ₂ / _n -A ₃ -A ₄ -X-CH ₂ -Z _and (III) wherein R, A ^ and A ^ n are as defined in claim 1, X is a radical of formula ( e) or (h) as defined in claim 1, and Z is a leaving group, with the corresponding phenol, thiophenol or HX4-pyridine or an acid of the formula HX4 -CO-Y4 or a functional derivative thereof or with HX ^-Y ^, wherein X ^, Y ^ and Y ^ are as defined in claim 1, or is e) for the preparation of compounds of formula I wherein R, A 1 to A 1 and n are as defined in claim 1, and R is an aliphatic or alicyclic radical containing from 1 to X0 12 carbon atoms, oxidizes a compound of formula V (V) wherein R 1 to A 4 are as defined above, and the resulting compound of formula I, or a physiologically hydrolyzable and acceptable ester or amide thereof, is isolated in free or salt form; or in the form of a complex. A compound according to any one of claims 1 to 6 for use as a medicament. ' A pharmaceutical composition comprising a compound of formula (I) as defined in claim 1, or a physiologically hydrolysable and acceptable ester or amide thereof, in free form or in the form of a physiologically acceptable salt, together with a pharmaceutically acceptable diluent or carrier. A compound of formula I as defined in claim 1, a physiologically hydrolyzable and physiologically acceptable ester or amide thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment of conditions caused by excessive release of IL-1beta.