HUT77979A - Peptides, process for producing them and pharmaceutical compositions containing them - Google Patents

Description

The present invention relates to parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) compounds, to processes for their preparation, to pharmaceutical compositions containing them and to their use as medicaments.

More particularly, the present invention relates to PTH or PTHrP compounds having potent antagonistic activity at the PTH / PTHrP receptor, in which at least one of the naturally occurring amino acid residues at positions 2 and 10 is replaced by tryptophan or another amino acid residue. having an aromatic or heteroaromatic group in its side chain and optionally substituted at least one of the naturally occurring amino acid residues 3 and 6 with tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain. The compounds will hereinafter be referred to as the compounds of the invention.

A particular embodiment of the present invention relates to PTH or PTHrP compounds wherein the naturally occurring amino acid residue at position 10 is replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic side chain in its side chain and optionally 3 and at position 6, at least one of the naturally occurring amino acid residues is further substituted by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain.

The term PTHrP refers to any naturally occurring form of PTHrP, such as human, bovine, chicken, rat, or mouse PTHrP. For the sake of homogeneity and in accordance with nomenclature conventions, the same numbering system will be used throughout the description herein, whether or not one of the α-amino acid residues of the PTHrP sequence is replaced or omitted according to the invention.

The term PTH refers to any naturally occurring form of PTH, such as human, bovine, chicken, rat, or mouse PTH. For the sake of homogeneity and in accordance with nomenclature conventions, the same numbering system will be used in the following description for avoic acid residues of the PTH sequence, regardless of whether the α-amino acid residue of PTH is substituted or omitted according to the invention.

The term PTHrP compound or PTH compound refers to a peptide comprising an amino acid sequence based on an N-terminal PTHrP fragment or PTH fragment, preferably an amino acid sequence based on a PTHrP fragment or PTH fragment. It begins with any of residues -7 and ends with any of residues 27-38, for example, containing an N-terminal PTHrP fragment or PTH fragment containing 31, 34, 35, 36, 37 or 38 amino acid residues. The terms PTHrP or PTH are thus used to refer to peptides in which one or more amino acid residues of said N-terminal fragment are absent, preferably at the N-terminal. These terms are also used for peptides in which one or more amino acid residues of the naturally occurring sequence are replaced by one or more (natural or non-natural) other amino acid residues in addition to the amino acids 2 and / or 10 of the present invention. and optionally at the 3 and / or 6 position, the compounds are substituted.

The N-terminal fragments 1-38, 1-34 and 1-31 of human PTHrP are shown in Figures 1, 2 and 1, respectively. They have the sequences set forth in SEQ ID NO: 3.

The N-terminal fragments 1-38, 1-34 and 1-31 of human PTH are shown in Figures 4, 5 and 5, respectively. They have the sequences set forth in SEQ ID NO: 6.

The N-terminus of PTHrP or PTH compounds may be a free or protected amino group which may, for example, carry an N-protecting group such as Protective Groups in Organic Synthesis by T. W. Greene, pp. 219-287. pages [J. Wiley & Sons, NY (1981)]; the contents of this book are incorporated herein by reference; preferred protecting groups are R-CO-, RO-CO-, R-O-CH 2 -CO- or R-SO 2 or amino groups which are R '' ', R' '' - NH-CO- or R '' They have a substituent of the formula -NH-CS- as defined below.

The C-terminal group of the compounds of the invention may be COOH, CONH2 or a mono- or disubstituted amide such as -CONRqRj wherein one of R _c and R i is hydrogen and the other is an aliphatic group such as a C 1-6 alkyl group, or both may be aliphatic, or R _c and R j together with the nitrogen atom to which they are attached may form a heterocyclic group such as pyrrolidinyl or piperidinyl.

The PTHrP or PTH compounds of the present invention have potent antagonistic activity at the PTH / PTHrP receptor, e.g., they bind to the PTH / PTHrP receptor, have intrinsic activity (ia) to activate the PTH / PTHrP receptor in a functional bioprobe. less than one (<1), for example in a bioassay having a value of at least 0.3, and antagonizing PTHrP or PTH or a fragment thereof, such as PTHrP (1-34) or PTH ( 1-34) in a functional bioassay with a pA2 of at least 6.5. The compounds of the present invention preferably have an intrinsic activity of 0.03 or less or not detectable in some assays. Exemplary functional bioassays include the osteosarcoma-based adenylate cyclase assay commonly used in the art. This assay allows the in vitro determination of the extent to which a test compound stimulates adenylate cyclase activity or antagonizes the action of PTHrP or PTH or a fragment thereof in the osteosarcoma cells of UMR rats, e.g., rats derived from UMR-106-06. Aurbach (Endocrinology 85: 801-810 (1969)), which will be described below.

The term amino acid refers to naturally occurring or commercially available or non-naturally occurring amino acids or optical isomers thereof. The unnatural amino acid is an amino acid that is not incorporated into a protein under mRNA control, such as β-Nal, fluoro-α-amino acids,

6 such as fluoro-alanine, cyclohexylalanine or trimethylsilylalanine. The term natural amino acids refers to amino acids well known in the art. These can be found, along with their standard abbreviations, in the U.S. Patent Office's Official Gazette, the Tradeiaark Official Gazette, May 15, 1990, page 33, starting with line 46. These amino acids and their abbreviations are incorporated herein by reference.

Natural amino acids are listed below:

THE below alanine D asp aspartic E Glu glutamic acid F Phe phenylalanine G Gly glycine H His histidine I Ile isoleucine K Lys lysine L Leu leucine M Met methionine N Asn asparagine Q Gin glutamine R Arg arginine S Ser serine T Thr threonine V With valine W Trp tryptophan Y Tyr tyrosine

By side chain, an amino acid residue having an aromatic or heteroaromatic group is an amino acid residue whose side chain is, for example, optionally substituted phenylmethyl, 1- or 2-naphthylmethyl, 1- or 2-naphthylmethyl.

2-naphthylethyl, 3- or 4-pyridylmethyl, 3-indolylmethyl or 3-indazolylmethyl; preferably, it is an amino acid residue of the formula -NH-CHR'-CO-, which is further defined below.

A preferred embodiment of the invention relates to compounds of formula I wherein x is 31, 34, 35, 36, 37 or 38, y is 1, 2, 3, 4, 5, 6 or 7,

X ^{2 has the} meaning Val, or independently has any meaning of X ¹⁰ ,

X ³ is Ser, or independently has one of X ¹⁰ ,

^X6 is Gln or has independently one of the meanings of X ^10, X ¹⁰ is Asp or Rio in which X ¹⁰ is Trp or

-NH-CHR'-CO- wherein R 'is a group of formula (a), (b), (c) or (d) wherein n is 1 or 2, m is 1 or 2, o is 0 or 1 .

A is a ring optionally substituted with one or more fluorine, chlorine, nitro, C 1-4 alkyl or C 1-4 alkoxy groups, wherein:

The two alkyl or two alkoxy substituents together may form a ring fused to ring A, the rings B and C being independently substituted in the same manner as given above for ring A, and

Y _a is a direct bond, -CH 2 -, -O, -NH, or -C 1-4 alkyl;

D is the amino acid sequence derived from the N-terminal fragment of PTHrP or PTH, p, q and s are both 1 provided that p is 0 when y> 2, q is 1 when y> 3, and s 0 when y> 6

R is H, R-CO-, RO-CO-, RO-CH ₂ -CO-, R-SO ₂ -,

R ''',R''' - NH-CO-, R '''- NH-CS- or _{NH2 1} -C 6 -alkyl-CO-, wherein R is C ^ -g alkilcsoport7 cu-carboxy-Ci_g alkyl ; ω - [(C 1-4 alkoxy) carbonyl] -C 1-4 alkyl; C ₂ alkenyl; ¢ 5-7 cycloalkyl; C 8 -C 7 cycloalkyl-C 1-4 alkyl; or phenyl, phenyl-Ci_4-alkyl, l-naphthyl, 2-naphthyl, l-naphthyl-Ci_ ₂ alkyl radical or a 2-naphthyl-C ₁ _ ₂ alkyl, each of which is optionally substituted on the ring by one or more fluorine may be substituted by a chlorine atom, a nitro group, a C 1-4 alkyl group or a C 1-4 alkoxy group; heteroaryl; and

R '' 'independently has the same meaning as for R, except

- 9 tJ-carboxy-C 1-8 alkyl and 6J - [(C 1-4 alkoxy) carbonyl] -C 1-8 alkyl; and

R _a is OH or NH ² with the proviso that at least one of X ² and R ¹⁰ has the same meaning as X ¹⁰ .

When the substituents on the A, B, or C rings together form a ring structure, it may be, for example, -O-CH 2 -CH 2 -O-. An example of a polycyclic cycloalkyl group is adamantyl.

The term heteroaryl R refers to a 5-, 6- or 7-membered unsaturated heterocyclic group containing at least one nitrogen atom and optionally an additional heteroatom such as nitrogen, oxygen or sulfur and optionally fused to a benzene ring. The heteroaryl group is preferably an indolyl group, a quinolyl group or an isoquinolyl group.

The compounds of the invention may exist in free form, in the form of a salt or in the form of complexes. Acid addition salts may be formed, for example, with organic acids, polymeric acids and inorganic acids. Such acid addition salt forms include hydrochlorides and acetates. For example, complexes may be formed from the PTHrP or PTH compounds of the present invention by the addition of inorganic materials such as inorganic salts or hydroxides such as calcium and zinc salts and / or by the addition of polymeric organic substances.

In the compounds of the formula I, the following meanings are preferred, either alone or in any combination or subcombination.

• · · «· ·«

1. D is the N-terminal fragment of hPTHrP.

2. D is the N-terminal fragment of hPTH.

3. X ¹⁰ is Trp.

4. X ¹⁰ is -NH-CHR'-CO-, wherein R 'is a group of formula (a), (b) or (c), preferably a group of formula (a) or (c), more preferably (a) ).

5. X ² is Trp or -NH-CHR'-CO-, wherein R 'is a group of formula (a), (b) or (c).

6. X ² and X ¹⁰ are independently Trp or

-NH-CHR'-CO-, wherein R 'is a group of formula (a), (b) or (c).

7. X ² and χΐθ are independently Trp or

-NH-CHR'-CO-, wherein R 'is a group of formula (a), (b) or (c).

8 X ⁶ and X ¹⁰ are independently selected from Trp

-NH-CHR'-CO-, wherein R 'is a group of formula (a), (b) or (c).

9. X ² , X ⁶ and X ¹⁰ are each independently Trp or -NH-CHR'-CO-, wherein R 'is a group of formula (a), (b) or (c).

10. n is 1.

11. y is 3, 4, 5, 6 or 7, preferably 3 or 5.

12. x is 31 or 34.

13. R is RCO, R-SO2, R '' 'or N-Cpg alkylene-CO-.

14. R is C 1-8 alkyl, phenylC 1-4 alkyl, 1- or 2-naphthyl, 1- or 2-naphthyl-C 2-2 alkyl.

15. R is -Carboxy-C1-8-alkyl.

As mentioned above, one or more amino acid residues other than position 2 and / or position 10 may be replaced or absent by a natural or non-natural amino acid residue as defined above. When the compounds of the present invention are hPTHrP derivatives, they may include additional amino acid substitutions, such as at the 3-position, such as Ala, at the 4-position, such as Trp, at the 5-position, e.g. and at position 13 and / or 13, such as Leu, or at position 34, such as D-Ala; the amino acid sequence of these amino acid residues may optionally be shorter at the N-terminus due to the absence of 1-7 amino acid residues. When the compounds of the invention are hPTH derivatives, the amino acid residues may be further substituted, for example, in at least one of the following positions: 8, 16, 18, 33 and 34, such as Leu ⁸ ,

Al ¹⁶ , Gin ¹⁸ , Thr ³³ , Al ³⁴ or the D-isomers thereof.

The invention also relates to a process for the preparation of PTHrP or PTH compounds of the invention. These compounds can be prepared stepwise in solution using the solid phase synthesis method, either by genetic engineering or by a combination of these methods.

For example, the compounds of the invention may be prepared in the following ways:

• · · · · · · · · · · · · · · · · · · · · · · · · · «

- 12 a) removing at least one protecting group present in a PTHrP or PTH compound of the invention, such as a compound of formula I, in a protected form; obsession

b) linking two peptide fragments in protected, partially protected or unprotected form, which form the amino acid sequence of the desired PTHrP or PTH compound, such as the compound of formula (I), and optionally proceeding step a) of the process. , obsession

c) selectively adding a protecting group or substituent to the N-terminal residue of the desired sequence or N-terminal fragment in protected or unprotected form, optionally carrying out the step a) and recovering the resulting free or salt form or complex thereof; PTHrP or PTH compounds.

Process steps a), b) and c) may be carried out analogously to known methods, for example known methods of peptide chemistry, or as described in the Examples below. Where desired, protecting groups may be used in these reactions which are useful as peptide functional groups in peptides but do not participate in the reactions. Protective groups may also be polymeric resins containing functional groups.

When the compounds of the invention contain unnatural or natural residues, they may be prepared by a combination of the stepwise chemical process and the genetic engineering process; consisting of genetically encoded amino acid residues

- 13 peptide sequences (either the whole sequence or a fragment thereof) can be prepared by recombinant techniques and introduced by chemical synthesis of the desired unnatural amino acids or terminal amino substituents and, where desired, the fragments can be combined and the protecting group (s) if present - removable.

In the examples below, all temperatures are given in ° C. The following abbreviations are used.

DMF = dimethylformamide

DMA = N, N-dimethylacetamide-6-sulfonyl

Pmc = 2,2,5,7,8-pentamethylchroman tBu = tert-butyl

PyBOP = Benzotriazol-1-yloxy-tripyrrolidine-phosphonium hexafluorophosphate

Nal (2) = L-3- (2-naphthyl) -alanine

DIEA = N, N-diisopropyl-N-ethylamine

Fmoc = 9-fluorenylmethoxycarbonyl

RT = room temperature

MS = M + as determined by electrospray spectroscopy

EXAMPLE 1 [Trp ^{4 '10]} hPTHrP (1-24) OH

Peptide step by step! is synthesized on a polystyrene-based resin support. The α-amino group is protected by Fmoc and the side chain functional groups are protected as follows: Asp (OtBu), Glu (OtBu), His (Trt), Lys (Boc), Asn (Trt), Gln (Trt), Arg (Pmc), Ser (tBu), Trp (Boc) and Thr (tBu).

• · «· ·· · · ···

- 14 Fmoc-Ala-oxymethyl-4-phenoxymethyl-co (polystyrene-1%-divinylbenzene) was used as starting material for solid-phase peptide syntheses at approximately 0.5 mmol / g. During the cyclic synthesis, the α-amino protecting group is removed, washed, coupled (i.e., the next amino acid residue is attached to the growing peptide chain) and washed. The α-α-Fmoc group was cleaved using 20% piperidine in DMA. The coupling reaction uses four equivalents of Fmoc amino acid and PyBOP reagent, and eight equivalents of DIEA per amino group in DMA. After complete assembly of the peptide chain, the terminal Fmoc protecting group is removed with piperidine in DMA as described above. The peptide is cleaved from the resin support and all side chain protecting groups are simultaneously removed for two hours at room temperature using a reagent containing 5% dodecylmethylsulfide and 5% water in TFA. The resin particles were filtered off, washed with a little TFA, and the product was precipitated from the combined filtrates by addition of 10-20 volumes of diethyl ether, washed with diethyl ether and dried. The product was purified by chromatography on a C-18 broad pore silica gel column using a 2% aqueous phosphoric acid gradient in acetonitrile. The pure compound fractions were combined, filtered through anion exchange resin (Biorad, AG4-X4 acetate form) and lyophilized. The title compound was obtained.

MS: 4146

If everything is done in the same way as in Example 1, but using the appropriate amino acids, the following example4 is the following example.

- 15 compounds can be prepared as follows:

EXAMPLE 2

[Trp ¹⁰ , Leu ¹¹ , Leu ¹³ JhPTHrP (1-34) OH EXAMPLE 3 MS: 4059 [Trp ¹⁰ , Leu ¹¹ JhPTHrP (1-34) OH EXAMPLE 4 MS: 4073 [Trp ^3/10 JhPTHrP (1-34) OH EXAMPLE 5 MS: 4188 [Trp ^2/10 JhPTHrP (1-34) OH EXAMPLE 6 MS: 4176 [Trp ¹⁰ J hPTHrP (2-3 4) OH EXAMPLE 7 MS: 4018.3 [Trp ¹⁰ ] hPTHrP (3-3 4) OH EXAMPLE 8 MS: 3919.2 [Trp ² J hPTHrP (1-3 4) OH EXAMPLE 9 MS: 4104.9 [Trp ¹⁰ ] hPTH (1-34) OH MS: 4189

EXAMPLE 10 [Leu ⁸ , Trp ¹⁰ , Al ¹⁶ , Gin ¹⁸ , Thr ^{3 3} , Al ^{3 34} ] hPTH (1-3 4) OH MS: 4036

EXAMPLE 11 [Leu ⁸ , Trp ¹⁰ , Al ¹⁶ , Gin ¹⁸ , Thr ^{3 3} , Al ^{3 34} ] hPTH (2-3 4) OH MS: 3949

EXAMPLE 12 [Leu ⁸ , Trp ¹⁰ , DLeu ¹¹ , Gin ¹⁸ , Thr ^{3 3} , Al ³⁴ ] hPTH (1-34) OH MS: 4079

EXAMPLE 13 [Phe ² , Trp ¹⁰ JhPTHrP (1-34) -OH MS: 4137.1 • ·· * · ·· * • · * · »*» ·· <t • · · ♦ ··· ·· ··

EXAMPLE 14 [Trp ² , Trp ¹⁰ ] hPTHrP (2-34) -OH

EXAMPLE 15 [Trp ⁶ , Trp ¹⁰ ] hPTHrP (1-34) -OH

EXAMPLE 16 [Trp ⁶ , Trp ¹⁰ ] hPTHrP (6-34) -OH

EXAMPLE 17 [Trp10] hPTHrP (7-34) -OH

EXAMPLE 18 [Trp ³ , Trp ¹⁰ ] hPTHrP (3-34) -OH

EXAMPLE 19 [Trp ¹⁰ ] hPTHrΡ (1-3 4) OH

EXAMPLE 20 [Trp ⁶ , Trp ¹⁰ ] hPTHrP (3-34) NH ₂

EXAMPLE 21 [Phe ⁶ , Trp ¹⁰ ] hPTHrP (1-34) OH

EXAMPLE 22 [Phe ² , Phe ⁶ , Trp ¹⁰ ] hPTHrP (1-34) OH

EXAMPLE 23

MS: 4105.1

MS: 4146.8

MS: 3623.3

MS: 3437.6

MS: 4018.0

MS: 4088.7

MS: 3976.8

MS: 3546.1

MS: 4155.1

^N -3-aminopropionyl [Phe ^6, Nal (2) ^10] hPTHrP (4-34) _NH2 MS: 3932.0

EXAMPLE 24

N ^a Benzyloxycarbonyl [Nal (2) ¹⁰ ] hPTHrP (3-34) -NH ₂ This peptide was prepared as in Example 1, however, starting from approximately 0.4 mmol / g of 4 (2 ', 4'). -dimethoxyphenyl-Fmoc-aminomethyl) phenoxy (polystyrene-1% divinylbenzene); this compound is exemplified

17 µl may be prepared as described in Tetrahedron Letters (1987, 28, 3787-3790). In the final cycle of synthesis, Z-Ser-OH is added to the peptide chain and the peptide is cleaved and purified as described in Example 1.

MS: 3975.3

EXAMPLE 25

^N i1- -acetamide [Trp ^10] hPTHrP (3-3 4) OH

This peptide was prepared in a manner similar to that described in Example 1. At the end of the synthesis, an Fmoc deprotection and acetylation cycle was used, the latter using a 1: 1 by volume mixture of acetic anhydride and DMF. The peptide is cleaved from the resin and purified as described previously.

MS: 3961.1.

The following compounds may be prepared in a manner analogous to that described in Examples 1, 24 or 25.

EXAMPLE 26

^N acetyl [Trp ^10] hPTHrP (2-34) OH MS: 4060.4

EXAMPLE 27

1-Isopropyl- [Leu ⁸ , Trp ¹⁰ , Gin ¹⁸ , Thr ^{3 3} , Al ³ 34] -hPTH (1-34) OH MS: 4091

EXAMPLE 28

N '-2-naphthylacetyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂ MS: 4097.1

EXAMPLE 29

N '-2-naphthylacetyl- [Nal (2) ¹⁰ ] hPTHrP (4-34) NH ₂ MS: 4009.7 ···· ········· · · · · · · · · · · · · · · · · · · ··········

- 18 EXAMPLE 30

^N -2-naphthyl-sulfonyl [Nal (2) ^10] hPTHrP (3-34) _NH2

MS: 4119.2

EXAMPLE 31

N '-2-naphthoxyacetyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂

MS: 4111.5

EXAMPLE 32

^N -2-naphthyl-acetyl- ^[3 Falling, Nal (2) ^10] hPTHrP (3-34) _NH2

MS: 4080.8

EXAMPLE 33

^{N-2-benzyloxycarbonyl [2} Trp, Nal (2) ^10] hPTHrP (2-34) _NH2 MS: 4248.3

EXAMPLE 34

3-Naphth-2-yl-propionyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂

MS: 4110.8

EXAMPLE 35

3-naphth-2-yl-propionyl- [Na 1 (2) ¹⁰ ] hPTHrP (3- 34) NH ₂

MS: 4110.9

EXAMPLE 36

^N -2-naphthyl-acetyl- [Nal (2) ^10, Ala ^34] hPTHrP (3-34) _NH2

MS: 4095.9

EXAMPLE 37

^N -2-naphthyl-acetyl- [Nal (2) ^10] hPTHrP (3-31) _NH2

MS: 3788.0

EXAMPLE 38

^N -3-naphth-2-yl-propionyl- [Nal (2) ^10] hPTHrP (7-34) _NH2

MS: 3630.0

• · · · · · • · · • · • · · · · · • · · · • · · · • · · · · • · · - 19 - 39. Examples ^{of the} N acetyl [Phe ^6, Nal (2) ¹⁰⁾ hPTHrP (6-34) _NH2 MS: 3637.0 EXAMPLE 40 N '-acetyl- [Phe ⁶ , Nal (2) ¹⁰ ) hPTHrP (4-34) NH ₂ MS: 3903.1 EXAMPLE 41 1- (1-Amino-1-cyclopentylcarbonyl) - [Leu ⁸ , Trp ¹⁰ , Gin ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH MS: 4104

The Fmoc-1-aminocyclopentane-1-carboxylic acid used in the preparation of the peptide resin intermediate is, for example, according to the method described by G. Valié et al., Can. J. Chem., 66, 2575-2582 (1988)].

EXAMPLE 42

1-adamantylcarbonyl- [Leu ⁸ , Trp ¹⁰ , Gin ¹⁸ , Thr ^{3 3} , Al ³⁴ ] hPTH (1-34) OH MS: 4155

EXAMPLE 43

1- (3-Indolylcarbonyl) [Leu ⁸ , Trp ¹⁰ , Gin ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH MS: 4150

EXAMPLE 44

1- (3-quinolylcarbonyl- [Leu ⁸ , Trp ¹⁰ , Al ¹⁶ , Gin ¹⁸ ,

Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH MS: 4104

EXAMPLE 45

1- (2-naphthoyl) - [Leu ⁸ , Trp ¹⁰ , Gin ¹⁸ , Thr ^{3 3} , Al ³ 34] hPTH (1-34) OH MS: 4147

- 20 EXAMPLE 46

^Ν α - (2-nafti1) -meti1- [Trp ^10] hPTHrP (2-3 4) -OH Methyl N-naphthoyl (2) -valinátot Lawesson's reagent [SO.

Lawesson et al., Tetrahedron 37: 3635 (1981)], and the resulting N-thiophthaloyl (2) -valinate is reduced as described in FS Guziec et al., Tetrah. Letters 23-26 (1990)]. The methyl ester was hydrolyzed using lithium hydroxide to give N-α- (naphthyl (2) methyl) valine hydrochloride as a crystalline solid, m.p. = 205-210 ° C (with decomposition). This compound was coupled using the PyBOP reagent to the previously prepared and protected [Trp ¹⁰ ] hPTHrP- (3-34) -O-peptide resin from which the α-α-Fmoc group was cleaved. The peptide was cleaved from the resin and purified as described in Example 1.

MS: 4158.2

EXAMPLE 47

^N -2-naphth-2-ylethyl or [Trp ^10] hPTHrP (2-34) OH MS: 4171.9

EXAMPLE 48

^N -2-naphth-2-ylethyl or [Ala ^3, Nal (2) ^10] hPTHrP (3-34) _NH2

MS: 4066.7

EXAMPLE 49

^N -szukcinil- [Phe ^6, Nal (2) ^10] hPTHrP (5-34) _NH2

This peptide was prepared in a similar manner to that described in Example 1, but using 0.63 g of 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxy (polystyrene-1% divinyl) as a starting material. benzene), approximately 0.4 mmol / g

- 21 charges; this compound is described, for example, in Tetrah. 28, 3787-3790 (1987). After coupling of the last amino acid, Fmoc-His (Trt) -OH, the Fmoc group was selectively removed as usual and the peptide resin in dimethylformamide with succinic anhydride (0.5 g) and DIPEA (0.86 ml). ). The peptide was cleaved from the resin and purified in acetate form as described in Example 1 and lyophilized. The title compound was obtained.

MS: 3832.0.

By the procedure described in Example 49, the following compounds were prepared:

EXAMPLE 50

^N -szukcinÍl- [Phe ^6, Nal (2) ^10] hPTHrP (5-31) _NH2 MS: 3522.1

EXAMPLE 51

^N -szukcinil- [Nal (2) ^6, Nal (2) ^10] hPTHrP (5-34) _NH2

MS: 3881.7

EXAMPLE 52

N ^a -Gutaryl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (5-34) NH ₂ MS: 3845.6

EXAMPLE 53

^N -szukcinil- [Phe ^6, Nal (2) ^10, Ala ^34] hPTHrP (5-34) _NH2

MS: 3832.0

EXAMPLE 54

^N -szukcinil- [4-Cl-Phe ^6, Nal (2) ^10] hPTHrP (5-34) _NH2

MS: 3866.5

EXAMPLE 55

Na-succinyl- [4-Cl-Phe ⁵ , 4-Cl-Phe ⁶ , Nal (2) ¹⁰ ] - 22 hPTHrP (5-34) NH ₂ MS: 3910.2

The compounds of the invention, in free form or in the form of their pharmaceutically acceptable salts and complexes, possess valuable pharmacological properties which have been demonstrated in animal experiments. Therefore, the compounds are pharmaceutically useful.

The biological efficacy of the compounds of the invention is determined in vitro by measuring the ability to stimulate (PTH-stimulated (antagonism) synthesis of cyclic AMP) PTH-stimulated (agonism) or inhibitory (UMR-type 106-06) murine osteosarcoma cells according to Marcus and Aurbach, 85 801-810 (1969)]. UMR 106 mice osteosarcoma cells were grown to confluence in DMEM-HAM 'F12 (1: 1) to 10% FCS in 12-well plates. The medium is then replaced with medium containing 2% FCS and 1-5 mCi [3 H] -adenine is added per well. Two hours later, the cells were washed twice with serum-free medium and incubated in 0.1% BSA-DMEM containing 1 mM 3-isobutyl-1-methylxanthine to exclude effects on phosphodiesterases. Test substances were added 20 minutes later either alone or in combination with PTH (antagonist experiment). After 15 minutes, the medium was removed and the reaction was stopped and the cAMP was extracted with 0.5 ml of ice-cold 5% trichloroacetic acid. 0.2 mM unlabeled adenine, adenosine, AMP, ADP, ATP, and cAMP, and 0.4 yCi [14 C] adenosine in vehicle medium to determine yield.

- 23 volumes of 0.5 ml. [3 H] -cAMP was isolated by chromatography on 200-400-well Dowex AG 50W-X4 series resin and alumina, followed by Salomon Y. (Advances in Cyclic Nucleotide Research, Vol. 10, Raven). Press, 35-55. (1979). Results are expressed as a percentage of control solvent and EC5Q values are determined from DRC curves. Antagonist efficacy is calculated from the right-sway of the PTHrP or PTH DRC curves and is expressed as pA2 values. Antagonists are effective compounds of this invention in concentrations from 10 ^-9 to 10 ^"5M. The compounds of Examples 36, 37 and 49 had a pA2 of 10.3 in cells of rat UMR 106-06; 9.7; respectively. 9.3.

The compounds of the present invention also show affinity for binding to PTH receptors, as evidenced by the following data.

Chicken [Tyr ³⁶ ] PTHrP (1-36) amide is iodinated to a specific activity of 2200 Ci / mmol by the lactoperoxidase method (Anawa Lab. AG, Wangen). The monolayers of open kidney cells (OKI) were washed with a 1: 1 mixture of 200 μΐ DMEM and HAM F12 containing 1% BSA and incubated at 16000C with 50,000 cpm [12,5i_Tyr ³⁶ ] chPTHrP (1-3,6) -amide. , 1 μπιοί [Tyr ³⁶ ] in the presence or absence of chPTHrP (1-36) -amide. After incubation, cells are washed with 0.5 ml of medium at 4 ° C, dissolved in 0.5 ml of IN sodium hydroxide solution and radioactivity is determined. Specific binding is defined as the difference between total binding minus non-specific binding. The competition curves with SCTFIT, a non-linear reg · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · <

- 24 Resistance computer programs [Feyen et al., Biochem. Biophys. Gap. Commun. 187: 8-13 (1992)] and data are reported as mean pKg values (n = 2 to 3). The compounds of Examples 36, 37 and 49 have pKp values of 8.3; 7.9; respectively. 8.4.

In addition, the compounds of the invention antagonize the effect of PTH i.v. after infusion, as determined, for example, in rats that had had their parathyroid glands surgically removed. Twenty-four hours after surgical removal of the parathyroid gland, rats are anesthetized, infused with PTH (1-34), and the test compound administered separately through the cervical veins. Urine is taken from the bladder through a cannula inserted through the stomach. Standard methods for measuring urinary phosphate and cAMP, and serum calcium and phosphate. These parameters are used to determine the degree of antagonist activity against in vivo PTH effects. In this assay, the compounds of the invention antagonize PTH effects when administered i.v. infusion at a dose of 1 µg / kg / h to 1 mg / kg / h. The compound of Example 49 completely suppresses PTH-induced phosphaturia, i.e., greater than normal excretion of phosphate in the urine, over a period of 90 minutes. infusion at 190 mg / kg / h and PTH (1-34) i.v. infusion at 4 mg / kg / h.

Accordingly, the compound of the present invention • · * · *

The compounds are useful for the prevention or treatment of any condition associated with increased plasma calcium content caused by excessive release of PTH or PTHrP; such as parathyroid hyperplasia, increased calcium in the blood, for example with or without malignant tumors such as breast cancer, squamous cell carcinomas of the lungs, esophagus, head and neck, and hematologic cancers. The compounds of the present invention are also useful in preventing and treating PTHrP-stimulated tumor growth, tumor invasion and tumor growth, in the treatment of dermatological disorders such as tissue repair therapies such as burns, ulcers and wounds, and in promoting hair growth.

Of course, when treating the symptoms listed, the approximate dose will depend on a number of factors, such as the subject being treated, the route of administration, and the severity of the conditions being treated. However, in general, satisfactory results are obtained in animals at daily doses ranging from about 0.1 mg / kg to about 100 mg / kg. In larger mammals, such as humans, the daily dose is from about 0.1 mg to about 500 mg of the compound of the invention.

The compounds of the invention may be administered in free form or in the form of pharmaceutically acceptable salts or complexes. Such salts and complexes may be prepared in conventional manner and exhibit the same degree of efficacy as the free compounds. Field of the Invention *

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It also encompasses pharmaceutical compositions comprising the compounds of the invention in free base form or in pharmaceutically acceptable salt or complex form with pharmaceutically acceptable diluents or carriers. Such compositions may be prepared in a conventional manner. Unit dosage forms conveniently contain from about 0.025 mg to about 250 mg of the compound of the invention together with a pharmaceutically acceptable diluent or carrier.

The compounds of the invention are administered by any conventional route, for example parenterally, e.g. injectable solutions or suspensions, or nasally or in the form of suppositories. In addition, the compounds of the invention may be administered topically, for example, in the form of a cream, gel or the like, for example, for treating wine or promoting hair growth, as described above.

As stated above, the present invention encompasses:

a) for use as a medicament in the compounds of the present invention or pharmaceutically acceptable salts or complexes thereof;

(b) a method of preventing or treating the above conditions and disorders in individuals in need of such treatment; the method comprising administering to said subject an effective amount of a compound of the invention or a pharmaceutically acceptable salt or complex thereof;

• · ································································································································································································································ We initiates this process, and we will provide you with the

c) the use of the compounds of the present invention or pharmaceutically acceptable salts or complexes thereof for the preparation of a pharmaceutical composition for use in the method of b) above.

The compounds of the present invention may be used as an adjunct or adjunct to other therapies, for example in the treatment of hypercalcaemia (increased calcium content in the blood), in which a bone resorption inhibitor is used, in particular calcitonin or an analogue or derivative thereof, e.g. salmon, eel, or human calcitonin, bisphosphonate, diuretic, or a combination thereof, or tumor therapy, cytostatic therapy, or a combination thereof.

In view of the foregoing, the present invention also encompasses:

d) a method of preventing or treating hypercalcaemia, such as preventing or treating the specific conditions or diseases listed above in an individual in need of such treatment; the method comprising administering to said subject an effective amount of a) a compound of the invention and b) a second drug agent, wherein said second drug agent is the aforementioned pharmaceutical agent.

The compounds of Examples 36, 37 and 49 are preferred for the prevention or treatment of any condition caused by elevated plasma calcium levels caused by excessive release of PTH or PTHrP.

• · · ·

Claims (10)

A patent claim Claims 1. PTH or PTHrP compounds, wherein at least one of the naturally occurring amino acid residues at position 2 and position 10 is replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic side chain and wherein at least one of the naturally occurring amino acid residues in positions 3 and 6 is replaced by tryptophan or another amino acid residue having a side chain aromatic or heteroaromatic group, in free form or in salt or complex form. 2. PTH or PTHrP compounds in which the naturally occurring amino acid residue at position 10 is replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain and optionally the amino acid residues 3 and 6. At least one of the naturally occurring amino acid residues in position 1 is further substituted by tryptophan or another amino acid residue having a side chain aromatic or heteroaromatic group, in free form or in the form of a salt or a complex. Compounds according to claim 1, wherein the side chain of an amino acid residue having an aromatic or heteroaromatic group in its side chain is optionally substituted on a ring. Substituted 3 or 4-pyridylmethyl, 3-indolylmethyl or 3-indazolylmethyl, or a compound of formula (a), (b), (c) or (d) group in which n is 1 or 2, m is 1 or 2, 0 is 0 or 1, THE optionally with one or more fluorine atoms, ring substituted with chloro, nitro, C 1-4 alkyl or C 1-4 alkoxy, wherein two alkyl or two alkoxy substituents together may form a ring fused to ring A, rings B and C being independently substituted as above, for ring, and Y _a is a direct bond, -CH 2 -, -O, -NH, or -C 1-4 alkyl. Compounds of formula (I) according to claim 1, wherein x is 31, 34, 35, 36, 37 or 38, y is 1, 2, 3, 4, 5, 6 or 7, X ^{2 has the} meaning Val, or independently has any meaning of X ¹⁰ , X ³ is Ser, or independently has one of X ¹⁰ , X ^{6 has the} meaning of Gin, or independently has one of the meanings of X ¹⁰ , R ¹⁰ is Asp or X ¹⁰ , where X ¹⁰ is Trp or a · · · · · · · · · · · · · · · · · · · · · · · - 31 -NH-CHR'-CO- wherein R 'is a group of formula (a), (b), (c) or (d) wherein n is 1 or 2, m is 1 or 2, and o is 0 or 1, A is a ring optionally substituted with one or more fluorine, chlorine, nitro, C 1-4 alkyl or C 1-4 alkoxy groups, wherein two alkyl or two alkoxy substituents together may form a ring fused to ring A independently of one another they may be substituted in the same manner as given above for Ring A, and Y _a is a direct bond, -CH 2 -, -O, -NH, or -C 1-4 alkyl; D is the amino acid sequence derived from the N-terminal fragment of PTHrP or PTH, p, q and s are both 1 provided that p is 0 when y> 2, q is 1 when y> 3, and s 0 when y> 6 R is H, R-CO-, RO-CO-, RO-CH ₂ -CO-, R-SO ₂ -, R '·', R '''- NH-CO-, R''' - NH-CS- or NH ₂ C ₁ _ ₆ alkylene C0, where R is C ^ alkyl; ω-carboxy-C 1-8 alkyl; u - [(C 1-4 alkoxy) carbonyl] -C 1-8 alkyl; C ₂ _g alkenyl; C8-7 cycloalkyl; C5-7 cycloalkyl C1-4 alkyl; obsession • · · · • · · · · · · • · · · · · · • · · · · · I ···· ·· ·· w - 32 phenyl, phenyl-Ci_4-alkyl, 1-naphthyl, 2-naphthyl, l-naphthyl-Ci_ ₂ alkyl radical or a 2-naphthyl-C ₁ _ ₂ alkyl, each of which is optionally substituted on the ring by one or more substituted with fluorine, chlorine, nitro, 0-4 alkyl or 0-4 alkoxy; heteroaryl; and R '' 'independently has the same meaning as for R', except for ω-carboxy-C 1 -C 4 alkyl and C 1 -C 4 -alkoxy-carbonyl-C 1 -C 4 alkyl- group report; and R _a is OH or NH ₂ with the proviso that at least one of X ² and R ¹⁰ has the same meaning as X ¹⁰ . The compound of claim 1 or 4, which is derived from the N-terminal fragment of hPTH or hPTHrP. The following compounds according to claim 1: ^N -2-naphthyl-acetyl- [Nal (2) ^10, Ala ^34] hPTHrP (3-34) NH 2, ^N -2-naphthyl-acetyl- [Nal (2) ^10] hPTHrP (3-31) NH2 . N '-succinyl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (5-34) NH ₂ . A process for the preparation of the compounds according to claim 1 in free form or in the form of a salt or a complex, characterized in that: a) removing at least one protecting group present in a PTHrP or PTH compound of the invention, such as a compound of formula I, in a protected form; obsession b) coupling with amide bond protected, partially protected t. Or peptide fragments of unprotected form, such that the amino acid sequence of the desired PTHrP or PTH compound, such as the compound of formula I, is obtained and optionally step a) of the process is performed, or c) selectively adding a protecting group or substituent to the N-terminal residue of the desired sequence or N-terminal fragment in protected or unprotected form, optionally carrying out the step a) and recovering the resulting free or salt form or complex thereof; PTHrP or PTH compounds. The compound of claim 1 for use as a medicament in free form or in physiologically acceptable salt form. A pharmaceutical composition comprising a compound of claim 1 in free form or a physiologically acceptable salt form, together with a pharmaceutically acceptable diluent or carrier. A compound according to claim 1 for use as a medicament in free form or in a physiologically acceptable salt form, together with an additional therapeutic agent, which may be a bone absorption inhibitor and a cytostatic agent.