SK12097A3 - Parathormone derivatives and parathormone correlated peptide, preparation method thereof and pharmaceutical composition containing them - Google Patents

Abstract

PTH or PTHrP compounds having potent antagonistic activity at the PTH/PTHrP receptor in which at least one of the amino acid residues naturally occurring in positions 2 and 10 is replaced by tryptophane or another amino acid residue bearing an aromatic or heteroaromatic group on its side chain, and optionally at least one of the amino acid residues naturally occurring in positions 3 and 6 is further replaced by tryptophane or another amino acid residue bearing an aromatic or heteroaromatic group on its side chain, having pharmacological activity, e.g. prevention or treatment of conditions which are associated with increased plasma calcium caused by excessive release of PTH or PTHrP.

Description

Parathyroid hormone derivatives and a parathyroid hormone related peptide, process for their preparation and pharmaceutical compositions containing them.

Technical field

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

SUMMARY OF THE INVENTION

In particular, the invention provides derivatives of parathyroid hormone and a parathyroid hormone related peptide which exhibit potent PTH / PTHrP receptor antagonist activity in which at least one of the amino acid residues naturally occurring at positions 2 and 10 is replaced with tryptophan or another amino acid residue thereof. and optionally wherein at least one of the amino acid residues naturally occurring at positions 3 and 6 is replaced by tryptophan or another amino acid residue that contains an aromatic or heteroaromatic group in its side chain. These compounds are hereinafter referred to as compounds of the invention.

According to a particular embodiment, the invention provides parathyroid hormone derivatives wherein the amino acid residue naturally occurring at position 10 is replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain and wherein

2, further optionally, at least one of the amino acid residues naturally occurring at positions 3 and 6 is replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain.

The term parathyroid hormone related peptide (PTHrP) refers to any naturally occurring form of a parathyroid hormone related peptide, for example, a human, bovine, chicken, rat or murine parathyroid hormone related peptide. If applicable, the same amino acid residue numbering system of the parathyroid-related peptide sequence will be used in the following description, regardless of whether any α-amino acid residue of the parathyroid-related peptide sequence is replaced or deleted according to the invention.

The terms parathyroid related peptide derivative (PTHrP derivative) or parathyroid hormone derivative (PTH derivative) refer to a peptide comprising an amino acid sequence based on the N-terminal fragment of a parathyroid hormone peptide, or an N-terminal fragment of a parathyroid hormone, preferably a parathormone related peptide fragment or fragment. it starts with any of residues 1 to 7 and ends with any of residues 27 to 38, for example the N-terminal fragment of a parathyroid-related peptide or a parathyroid hormone containing up to 31, 34, 35, 36, 37 or 38 amino acid residues. Thus, it is understood that the terms parathyroid-related peptide or parathyroid hormone include peptides in which one or more amino acid residues of said N-terminal fragment are deleted, preferably at the N-terminus. It is to be understood that these terms also include peptides in which, in addition to substitution at position 2 and / or 10 and optionally 3 and / or 6 of the invention, one or more amino acid residues of a naturally occurring sequence is replaced by one

- 3 or more other amino acid residues (natural or unnatural). The N-terminal fragments 1-38, 1-34 and 1-31 of the human parathyroid hormone-related peptide have the sequences shown in SEQ ID NOs: 1, 2 or 3.

The N-terminal fragments 1-38, 1-34, and 1-31 of the human parathyroid hormone have the sequences shown in SEQ ID NOs: 4, 5 or 6.

The N-terminus of a parathyroid hormone derivative or a parathyroid hormone related peptide may be a free or protected amino group carrying, for example, an N-protecting group as described in Protective Groups in Organic Synthesis, TW Green, J. Wiley Sons NY (1981), 219-287 and preferably is protected by R-CO-, RO-CO-, RO-CH 2 -CO- or R-SO 2 or may be an amino group bearing the radical R ''',R' ^Z '-NH-CO- or ^RZZZ -NH-CS as defined below.

The C-terminus of the compounds of the invention may be COOH, CONH ₂ or a mono- or disubstituted amide group -CONR _c R _{d (} where one of R _{c and} R _d is hydrogen and the other is an aliphatic radical such as an alkyl group containing 1 to 6 or both R _c and R _d represent aliphatic radicals R _c and R _d together with the nitrogen atoms to which they are attached form a heterocyclic radical, for example pyrrolidinyl or piperidinyl.

The parathyroid hormone-related or parathyroid hormone-related peptide derivatives of the invention exhibit potent PTH / PTHrP receptor antagonist activity, e.g., bind to a PTH / THrP receptor, exhibit intrinsic activity (ia) in functional bioassay for PTH / PTHrP receptor activation substantially below 1, such as ia maximum 0.3 and antagonize a parathyroid hormone related or parathyroid hormone

-4 (1-34) or parathyroid hormone (—-34) in a functional bioassay with a pA2 value of at least 6.5. Preferred compounds of the invention exhibit an intrinsic activity value of 0.03 or less or even undetectable in some assays. An example of a functional bioassay is the adenylate cyclase assay based on osteosarcoma, which takes place. commonly used in the field. This assay determines the in vitro extent to which a test compound stimulates adenylate cyclase activity or antagonizes the action of a parathyroid hormone or parathyroid hormone related peptide or fragment thereof in rat osteosarcoma cells of the UMR line, e.g., UMR-106-06 according to the method described by Marcus and Aurbach in Endocrinology, 85, 801-810 (1969) as described below.

By amino acid is meant a naturally occurring (natural), commercially available or non-naturally occurring (unnatural) amino acid, or an optical isomer thereof. Unnatural amino acids are amino acids that are not incorporated into the mRNK listing proteins, for example, β-Nal, a fluoro-α-amino acid such as fluoroalanine, cyclohexylalanine or trimethylsilylalanine.

Natural amino acids are well known in the art. A list of these, together with standard abbreviations, is given in U.S.P.T.O., Trademark Official Gazette, May 15, 1990, p. 33 in 46.

Natural amino acids are listed below:

A Ala alanine D Asp aspartic acid E Glu glutamic acid F Phe phenylalanine G Gly glycine

H His histidine I Ile isoleucine The Lyz lysine L Leu leucine M Met methionine N same time asparagine Q gin glutamine R Arg arginine WITH Ser serine T Thr threonine IN wall valine W Trp tryptophan Y Tyr tyrosine

By amino acid residue having an aromatic or heteroaromatic group in its side chain is meant an amino acid residue whose side chain is optionally substituted on the ring, for example. phenylmethyl, 1- or 2-naphthylmethyl, 1- or 2-naphthylethyl, 3- or 4-pyridylmethyl, 3indolylmethyl or 3-indazolylmethyl, preferably being an amino acid residue of the formula -NH-CHR'-CO- as defined below.

According to a preferred embodiment, the invention provides a compound of formula I

R [(X ²⁾ p, ^(X3) q, (X ^{6), R10]} -D- (y-x) R (I) wherein x is the residue selected from the group consisting of the numbers 31,

34, 35, 36, 37 and 38

-6 y is the remainder number selected from the group consisting of 1,

2 3 4 5 6 7

X ² is wall or independently One of významov referred to the X ^10, X ³ for It is Ser ¹⁰ of X, or has independently one of významov listed X ^p is gin or independently One of významov

referred to the X ^10,

R ¹⁰ is Asp or X ¹⁰ , wherein

^X10 is Trp or -NH-CHR'-CO-, wherein R 'is a), (b), (c) or (d)

where n my value 1 or 2 m my value 1 or 2 about my value 0 or 1

- ring 7 is optionally substituted with one or more substituents selected from the group consisting of fluorine, chlorine, nitro, C 1 -C 4 alkyl and C 1 -C 4 alkoxy wherein the two alkyl or two alkoxy substituents may together also to form a ring structure fused to ring A, each of rings B and C may be independently substituted as described above for ring A, and

_And Y is a direct link, -CH ₂ -, O, NH, or N-alkyl of 1 to 6 carbon atoms,

D represents an amino acid sequence derived from a N-terminal fragment of a parathyroid-related peptide, each of p, cj and s. is 1, with p being 0 if y is greater than 2, p being 0 if y is greater than 3, and s being 0 if y is greater than 6,

R is hydrogen, R '' -CO-, R '' -O-CO-, R '' -OCH ₂ -CO-, R-SO ₂ -, R ''',R''' - NH- CO-, R'-NH-CS or NH ₂ -alkylene-CO- having 1 to 6 carbon atoms in the alkylene moiety, wherein

R '' represents an alkyl group having 1 to 8 carbon atoms, a ω-carboxyalkyl group having 1 to 6 carbon atoms in the alkyl moiety, a ω - [(alkoxy) carbonyl] alkyl group having 1 to 4 carbon atoms in the alkoxy moiety and 1 to 6 carbon atoms in the alkyl moiety, alkenyl

- a C8-C8 group, a C7-C7 cycloalkyl group, or a phenyl group, a C1-C4 phenylalkyl group, a 1-naphthyl group, a 2-naphthyl group, a 1- naphthylalkyl having 1 to 2 carbon atoms in the alkyl moiety or 2-naphthylalkyl having 1 to 2 carbon atoms in the alkyl moiety, any of which is optionally substituted on the ring by one or more substituents selected from fluorine, chlorine, nitro, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy, or heteroaryl,

R '' 'independently has one of the meanings given for R' 'with the exception of a C 1 -C 6 -carboxyalkyl group and a C 1 -C 4 -alkoxy (C 1 -C 4) alkoxy group and 1 to 6 carbon atoms in the alkyl moiety; and

R _a is hydroxy or amino, provided that at least one of X and R is X ¹⁰ .

When the substituents of ring A, B or C together form a ring structure, it may be, for example, -O-CH2-CH2-O-. An example of a polycyclic cycloalkyl group is, for example, adamantyl.

Heteroaryl in the meaning of the symbol R '' means a 5-, 6- or 7-membered unsaturated heterocyclic radical containing at least one nitrogen atom and optionally another

A heteroatom, such as nitrogen, oxygen or sulfur, fused to a benzene ring. A preferred group is indolyl, quinolyl or a group.

and optionally is heteroaryl isoquinolyl

The compounds of the invention may exist, for example, in free form, in salt form, or in the form of their complexes. Acid addition salts may be formed with, for example, organic acids, polymeric acids, and inorganic acids. Such acid addition salts include, for example, hydrochlorides and acetates. For example, complexes are formed from a parathyroid hormone-related peptide derivative by addition of inorganic substances, for example inorganic salts or hydroxides such as calcium or zinc salts, and / or the addition of polymeric organic substances.

In the compounds of formula I, the following meanings of the individual substituents are preferred. either individually or in any combination or sub-combination:

represents the N-terminal fragment of hPTHrP;

represents the N-terminal fragment of hPTH;

represents Trp;

represents -NH-CHR'-CO-, wherein R 'is a radical of formula (a), (b) or (c), preferably (a) or (c), even more preferably a radical of formula (a);

X ^z is Trp or the group -NH-CHR'-CO-, wherein R 'is a radical of general formula

- formula (a), (b) or (c);

each X ² and X ¹⁰ is Trp or -NH-CHR'-CO- wherein R is a radical of formula (a), (b) or (c);

X ³ and X ¹⁰ are each Trp or -NH-CHR'-CO-, wherein R 'is a radical of formula (a), (b) or (c);

X ⁵ and X ¹⁰ are each Trp or -NH-CHR'-CO- wherein R 'is a radical of formula (a), (b) or (c);

each X ^2, X ⁶ and X ¹⁰ is Trp alebo.skupinu -NH-CHR'-CO- wherein R 'is a radical of formula (a), (b) or (c);

is 1;

is a residue number selected from the group consisting of the numbers 3, 4, 5, 6 and 7, preferably the number 3 or 5;

is a residue number selected from the group consisting of numbers 31 and 34;

R''CO-, R''-SO ₂ , R'''or H ₂ N-alkylene-CO- having 1 to 6 carbon atoms in the alkylene moiety;

x

-1114. R 'is C 1 -C 8 alkyl, C 1 -C 4 phenylalkyl, 1- or 2-naphthyl or 1 or 2-naphthyl C 1 -C 2 alkyl;

15. R represents a ω-carboxyalkyl group having 1 to carbon atoms in the alkyl moiety.

As mentioned above, one or more amino acid residues located at positions other than 2 and / or 10 may be further replaced by a natural or unnatural amino acid residue as mentioned above or may be deleted. When the compounds of the invention are derivatives of hPTHrP, additional amino acid substitutions may be made therein, e.g. at the 3-position, e.g. Ala, at the 4-position, e.g. Trp, at the 5-position, e.g. optionally substituted on the Phe at position 11 and / or 13 , for example Leu, or at position 34, for example D-Ala; and their amino acid sequence may optionally be truncated at the N-terminus by deleting 1 to 7 amino acid residues. When the compounds of the invention are derivatives of hPTH, the amino acid residues may further be replaced, for example, in at least one of positions 8, 16, 18, 33 and 34, e.g. Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ , or their D isomers.

The invention also provides a process for preparing parathyroid hormone derivatives or a parathyroid hormone related peptide of the invention. They may be prepared sequentially either in solution or by solid phase synthesis or by genetic engineering methods or a combination of these methods.

The compounds of the invention can be prepared, for example, by:

a) removes at least one protecting group that is present in a parathyroid hormone or parathyroid hormone-related peptide derivative according to the invention, for example a compound of formula I, in protected form, or

b) joining two peptide fragments in a protected, partially protected or unprotected form to each other by an amide bond, the peptide fragments being such that the amino acid sequence of the desired parathyroid hormone related peptide derivative, e.g. of formula I, is obtained, optionally followed by step a) way or that

c) introducing a protecting group or substituent in a selective manner to the amino group of the N-terminal residue of the desired sequence or N-terminal fragment thereof in a protected or unprotected form, optionally carrying out process step a) and isolating the parathyroid hormone-related peptide or parathyroid hormone derivatives in a free, salt or complex form.

Process steps a), b) and c) can be carried out analogously to known methods, for example as known in the art of peptide chemistry or as described in the following examples. If desired, protecting groups that are suitable for use in peptides for non-participating functional groups can be used in these reactions. The term protecting group may also mean a polymer resin containing functional groups.

If the compounds of the invention contain unnatural and natural residues, they can be prepared by combining a chemical multi-step process and genetic engineering. peptide

The 13 sequence (either a complete sequence or fragment) constructed from genetically encoded amino acid residues can be produced by genetic engineering and the desired unnatural amino acids or terminal amino substituent can be incorporated by chemical synthesis and, if desired, the fragments can be joined and deleted. protecting group. or protecting groups, if present.

The following abbreviations are used in the following examples:

DM F dimethylformamide, DMA N, N-dimethylacetamide pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl, Bu t. butyl, PyBOP benzotriazol-yloxytripyrolidinofosfónium- hexafluorophosphate, Nal (1) L-3- (2-naphthyl) alanine, DIEA N, N-diisopropyl-N-ethylamine a Fmoc 9-fluorenylmethoxycarbonyl

Where mass spectrometry data is reported, it is M ⁺ determined by electrospray spectrometry.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 ^{[4-Trp 10]} hPTHrP (1-34) OH

The peptide is synthesized in multiple stages on a polystyrene-based resin, the α-amino group is protected with a 9-fluorophenylmethoxycarbonyl group, and the side-chain functional groups are protected as follows:

-14Asp (OtBu), Glu (OtBu), His (Trt), Lys (Boe), Asn (Trt), Gin (Trt), Arg (Pmc), Ser (tBu), Trp (Boe), and Thr (tBu) .

Fmoc-Ala-oxymethyl-4-phenoxymethyl-co (polystyrene-1% -vinylbenzene) (approximately 0.5 mmol / g) is used as a starting material for multi-step peptide synthesis. on a solid phase, which consists of repeated cycles of deprotection of the α-amino group, washing, coupling (i.e., attaching an additional amino acid residue to the growing peptide chain) and washing. The Ν-α-9-fluorenylmethoxycarbonyl group is cleaved using 20% piperidine in N, N-dimethylacetamide. In the condensation reaction, 4 equivalents of 9-fluorenylmethoxycarbonylamino acid and benzotriazol-1-yloxytripyrrolidine phosphonium hexafluorophosphate and 8 equivalents of N, N-diisopropyl-N-ethylamine in N, N-dimethylacetamide are used per amino group. Upon completion of the assembly of the peptide chain, the terminal 9-fluorenylmethoxycarbonyl protecting group is removed with piperidine in N, N-dimethylacetamide as described above. The peptide is cleaved from the resin used as the carrier and all side-chain protecting groups are simultaneously removed using a reagent consisting of 5% dodecylmethylsulfide and 5% water in trifluoroacetic acid, which is left to react for two hours at room temperature. The resin particles are filtered off, washed with trifluoroacetic acid and the product precipitated from the combined filtrates giving 10 to 20 volumes of diethyl ether. The precipitate was washed with ether and dried. The product is purified by chromatography on a large pore C-18-silica gel column using a gradient of acetonitrile in 2% aqueous phosphoric acid. Fractions containing pure compound were collected, filtered through anion exchange resin (Biorad, AG4-X4 acetate form) and lyophilized to afford the title compound.

Mass spectrometry: 4146.

By analogous procedure to that described in Example 1, but using the appropriate amino acids, the following compounds can be prepared:

Example 2 [Trp ¹⁰ , Leu ¹¹ , Leu ¹³ ] hPTHrP (1-34) OH

Mass spectrometry: 4059

Example 3 [Trp ¹⁰ , Leu ¹¹ ] hPTHrP (1-34) OH

Mass spectrometry: 4073

Example 4 [Trp ^3.10 ] hPTHrP (1-34) OH

Mass spectrometry: 4188

Example 5 [Trp ^{2 '10 l]} hPTHrP (1-34) OH

MS 4176

Example 6 [Trp ¹⁰ ] hPTHrP (2-34) OH

Mass spectrometry: 4018.3

Example 7 [Trp ¹⁰ ] hPTHrP (3-34) OH

Mass spectrometry: 3919.2

Example 8 [Trp ² ] hPTHrP (1-34) OH

Mass spectrometry: 4104.9

Example 16 [Trp ¹⁰ ] hPTHrP (1-34) OH

Mass spectrometry: 4189

Example 10 [Leu ⁸ , Trp ¹⁰ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTHrP (1-34) OH Mass Spectrometry: 4036

Example 11 [Leu ⁸ , Trp ¹⁰ , Ala ¹⁵ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTHrP (2-34) OH Mass spectrometry: 3949

Example 12 [Leu ⁸ , Trp ¹⁰ , Dleu ¹¹ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTHrP (1-34) OH Mass Spectrometry: 4079

Example 13 [Phe ² , Trp ¹⁰ ] hPTHrP (1-34) OH

Mass spectrometry: 4137.1

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

Mass spectrometry: 4105.1

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

Mass spectrometry: 4146.8

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

Mass spectrometry: 3623.3

Example 17

-17 [Trp ¹⁰ ] hPTHrP (7-34) OH

Mass spectrometry: 3437.6

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

MS: 4018.0

Example 19 [Trp ¹⁰ ] hPTHrP (1-34) OH

Mass spectrometry: 4088.7

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

MS 3976.8

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

Mass spectrometry: 3546.1

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

Mass spectrometry: 4155.1

Example 23

N ^and -3-aminopropionyl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (4-34) NH ₂

Mass spectrometry: 3932.0

Example 24

N ^and -benzyloxykarbonyl- [Nal (2) ^10] hPTHrP (3-34) ₂ NH

This peptide was prepared in an analogous manner to Example 1, but using 4- (2 ', 4'-dimethoxyphenyl-9-fluorenylmethoxycarbonylaminomethyl) -phenoxy-co (poly-18-styrene-1% -dinylbenzene) as the starting material (approximately 0, 2, 4'-dimethoxyphenyl) 4 mmol / g), which may be prepared, for example, as described in Tetrah.Letters, 28: 3787-3790 (1987). In the last synthesis cycle, Z-Ser-OH (benzyloxycarbonyl-Ser-OH) is added to the peptide chain, and the peptide is cleaved and purified as described in Example 1.

Mass spectrometry: 3975.3

Example 25

N ^and -2-acetyl- [Trp ¹⁰ ] hPTHrP (3-34) OH

This peptide is assembled in a manner analogous to Example 1. At the end of the synthesis, a final cycle of 9-fluorenylmethoxycarbonyl protecting group removal and acetylation is performed using a 1: 1 mixture of acetic anhydride and dimethylformamide. The peptide is cleaved from the resin and purified as above. .

Mass spectrometry: 3961.1

The following compounds can be prepared in an analogous manner 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-Isocaproyl- [Leu ⁸ , Trp ¹⁰ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTHrP (1-34) OH

MS: 4091

Example 28

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

Mass spectrometry: 4097.1

Example 29

-19N ^and -2-naphthylacetyl- [Nal (2) ¹⁰ ] hPTHrP (4-34) NH ₂ Mass spectrometry: 4009.7

Example 30

N ^and -2-naphthylsulfonyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂ Mass spectrometry: 4119.2

Example 31 '

N '2-naphthoxyacetyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂ Mass spectrometry: 411.5

Example 32

N ^and -2-naphthylacetyl- [Ala ³ , Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂ Mass spectrometry: 4080.8

Example 33

N '-benzyloxycarbonyl- [Trp ³ , Nal (2) ¹⁰ ] hPTHrP (2-34) NH ₂ Mass spectrometry: 4248.3

Example 34

3-Naphth-2-yl-propionyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂ Mass spectrometry: 4110.8

Example 35

3-Naphth-2-yl-propionyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂ Mass spectrometry: 4110.9

Example 36

N ^and -2-naphthylacetylethyl- [Nal (2) ¹⁰ , DAla ³⁴ ] hPTHrP (3-34) NH ₂ Mass spectrometry: 4095.9

Example 37

N 2 -2-naphthylacetyl- [Nal (2) ¹⁰ ] hPTHrP (3-31) NH ₂

-20Mass spectrometry: 3788.0

Example 38

N ^and -3-naphth-2-ylpropionyl- [Nal (2) ¹⁰ ] hPTHrP (7-34) NH ₂

Mass spectrometry: 3630.0

Example 39

N '-acetyl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (6-34) NH ₂

Mass spectrometry: 3637.0

Example 40

N ^α -acetyl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (4-34) NH ₂

Mass spectrometry: 3903.1

Example 41

1- (1-Amino-1-cyclopentylcarbonyl) [Leu ⁸ , Trp ¹⁰ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTHrp (1-34) OH

Mass spectrometry: 4104

The 9-fluorenylmethoxycarbonyl-1-aminocyclopentane-1-carboxylic acid used to prepare the intermediate peptide resin can be prepared, for example, as described by G. Valle et al., 1988, Can. J. Chem. 66: 2575–2582.

Example 42 1-adamantylcarbonyl- [Leu ⁸ , Trp ¹⁰ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH Mass Spectrometry: 4155

Example 43

1- (3-indolylcarbonyl) - [Leu ⁸ , Trp ¹⁰ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH

Mass spectrometry: 4150. Example 44

-21 1- (3-quinolylcarbonyl) - [Leu ⁸ , Trp ¹⁰ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH

Mass spectrometry: 4104

Example 45

1- (2-naphthyl) - [Leu ⁸ , Trp ¹⁰ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH

Mass spectrometry: 4147

Example 46

^And N - (2-naphthyl) methyl- [Trp ^10] hPTHrP (2-34) OH

Methyl N-2-naphthoyl valinate is treated with Lawesson's reagent, SO. Lawesson et al., Tetrahedron 37: 3635 (1981) and the methyl N-2-thionaphthyl-valinate formed is reduced using the procedure described for the endothiopeptides FS Guziec et al., Tetrah. Letters 23-26 (1990). The methyl ester is hydrolyzed using lithium hydroxide to give N - (- (2-naphthylmethyl) valine hydrochloride) as a crystalline solid melting at 205-210 ° C with decomposition. This compound was condensed with the previously prepared [Trp ¹⁰ ] hPTHrP (3-34) -O-peptide resin from which the Na-9-fluorenylmethoxycarbonyl group was removed using benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate. The peptide is cleaved from the resin and purified as in Example 1.

Mass spectrometry: 4158.2

Example 47

N ^and -2-naphth-2-ylethyl- [Trp ¹⁰ ] hPTHrP (2-34) OH

Mass spectrometry: 4171.9

Example 48

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

-22 Mass Spectrometry: 4066.7

Example 49

N ^and succinyl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (5-34) NH ₂

This peptide was prepared in an analogous manner to Example 1, but using 0.63 g of 4- (2 ') starting material.

4'-dimethoxyphenyl-9-fluorenylmethoxycarbonylaminomethyl) phenoxy (polystyrene-1% -divinylbenzene) (about 0.4 mmol / g), which can be prepared, for example, as described in Tetrah. Letters, 28: 3787-3790 (1987). After addition of the last amino acid, 9-fluorenylmethoxycarbonyl-His (Trt) -OH, the 9-fluorenylmethoxycarbonyl group is selectively cleaved by conventional means and the peptide resin is reacted with 0.5 g of succinic anhydride and 0.86 ml of N, N-diisopropyl-N-ethylamine in dimethylformamide. The peptide is cleaved from the resin, purified and lyophilized in the acetate form as in Example 1 to give the title compound.

Mass spectrometry: 3832.0

The following compounds can be prepared as described in Example 49:

Example 50

N ^and succinyl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (5-31) NH ₂

Mass spectrometry: 3522.1

Example 51

N ^and succinyl- [Nal (2) ⁶ , Nal (2) ¹⁰ ] hPTHrP (5-34) NH ₂

Mass Spectrometry: 3881.7

Example 52

N '-glutaryl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (5-34) NH ₂

Mass spectrometry: 3845.6

-23Example 53

N ^and succinyl- [Phe ⁶ , NaI (2) ¹⁰ , DAla ³⁴ ] hPTHrP (5-34) NH ₂

Mass spectrometry: 3832.0

Example 54

N ^and succinyl- [4-Cl-Phe ⁶ , Nal (2) ¹⁰ ] hPTHrp (5-34) NH ₂

Mass spectrometry: 3866.5

Example 55

N ^and -sukcinyl- [4-Cl-Phe ^6, 4-Cl-Phe ^6, Nal (2) ^10] hPTHrP (5-34) _NH2 MS: 3910.2

The compounds of the invention in free form or in the form of pharmaceutically acceptable salts and complexes possess valuable pharmacological properties as confirmed in animal tests and are suitable for therapy.

The biological activity of the compounds of the invention is assessed in vitro by measuring their ability to stimulate (agonism) or inhibit parathyroid hormone-stimulated (antagonism) synthesis of cyclic AMP in rat osteosarcoma UMR 10606 cells, according to the Marcus and Aurbach method described in Endocrinology, 85, 801-810 (1969). . Rat Osteosarcoma UMR 106 cells are grown to a continuous growth phase in a 1: 1 mixture of DMEM and HAM-12 media with 10% calf fetal serum (FCS) on a twelve-well plate. Subsequently, the medium is changed to medium with 2% calf fetal serum and 1-5 µ 5 [ ³ H] adenine is added per well. Two hours later, cells are washed twice with serum-free medium and incubated in DMEM medium with 0.1% bovine serum albumin (BSA) containing 1 mM 3-isobutyl-1-methylxanthine to eliminate phosphodiesterase action. 20 minutes later test substances are added either alone or together with parathyroid hormone (antagonism test). After 15 minutes

-24 The medium is removed, the reaction is stopped and the cyclic AMP, ADP, ATP and AMP as well as 0.4 μθί [ ¹⁴ C] -adenosine are extracted to determine the yield. [ ³ H] -cAMP is separated using Dowex AG 50W-X4 chromatography (pore size 0.074-0.037) and alumina chromatography, and the amount is determined as described by Salomon Y. in Advances in Cyclic Nucleotide Research, Volume 10, Raven Press, 35-55, 1979. Results are expressed as a percentage of the solvent control and EC50 values are determined from DCR curves. The antagonist potency is calculated from the left to right-directed shift of the DCR curves for the parathyroid hormone-related peptide or parathyroid hormone and is expressed as pA2 values. The compounds are effective as antagonists at concentrations of 10 ^{"9-10" 5} M. The compounds of Examples 36, 37 and 49 show the UMR 106-06 cells of pA2 values of 10.3, 9.7 respectively 9.3.

The compounds of the invention also exhibit binding affinity for PTH receptors, as determined, for example, as follows:

Chicken [Tyr ³⁶ ] PTHrP (1-36) amide is iodinated to a specific activity of 2,200 Ci / mmol using the lactoperoxidase method (Anawa Lab.AG, Wangen). The opossum kidney cell layers (OKI) are washed with 200 μΐ of a 1: 1 mixture of DMEM and HAM F12 containing 1% bovine serum albumin (BSA) and incubated at 16 ° C with 50,000 cpm (pulse counts from). minute [ ¹²⁵ I-Tyr ³⁶ ] chPTHrP (1-36) amide (ch stands for chicken PTHrP) per well in the presence or absence of 1 μΜ [Tyr ³⁶ ] chPTHrP- (1-36) amide. The cells are washed with 0.5 ml of 4 ° C medium, lysed with 0.5 ml of 1N sodium hydroxide and radioactivity is determined.The specific binding is defined as total binding minus non-specific binding The competition curves are analyzed using a non-linear regression computer program SCTFIT ( Feyen et al., 1992, Biochem. Biophys. Res. Commun. 187: 8-13) and data are expressed as

-25 average pK _D values (n = 2 to 3). The compounds of Examples 36, 37 and 49 have a pK _{D of} 8.3, 7.9 and 8.4, respectively.

In addition, the compounds of the invention antagonize parathyroid hormone action after intravenous infusion, for example as determined in thyroparathyroidectomized rats · 24 hours after tyroparathyroidectomy, anesthetized rats receive PTH (1-34) infusion by isolated jugular veins and the compound to be tested. Urine is collected from the bladder into which a cannula is inserted from the ventral side. Urine and phosphate and cAMP phosphate and serum and phosphate levels are measured by standard methods. These parameters are used to quantify the potency of antagonists against parathyroid hormone activity in vivo. In this test, the compounds of the invention antagonize the action of parathyroid hormone when administered by intravenous infusion at a dose of from 1 µg / kg / h to 1 mg / kg / h. The compound of Example 49 completely suppressed parathyroid hormone induced phosphaturia for up to 90 minutes when administered by intravenous infusion at 190 at a PTH (1-34) intravenous infusion at 4 μς / kg / h.

Accordingly, the compounds of the invention are useful for the prevention or treatment of all conditions associated with increased plasma calcium due to excessive release of parathyroid hormone or parathyroid hormone related peptide, e.g., hyperparathyroidism, hypercalcemia, e.g., associated with cancer, such as cancer flat cell lung, smuggler malignant tumors, head and neck region, and hematologic malignancies with or without bone metastasis. The compounds of the invention are further suitable for the prevention or treatment of tumor growth, penetration and tumor growth into parathyroid-related peptide-stimulated bones, and for the treatment of dermatological disorders, for example tissue repair therapies, for example, treating burns, ulcers and wounds and promoting hair growth.

The compounds of the invention may be administered in free form, in the form of pharmaceutically acceptable salts or complexes. Such salts and complexes may be prepared in conventional manner and exhibit the same order of activity as the free compounds. The invention also provides pharmaceutical compositions. comprising a compound of the invention in free base or pharmaceutically acceptable salt or complex form, and in combination with a pharmaceutically acceptable diluent or carrier. Such compositions may be formulated in conventional manner. Dosage unit forms conveniently contain from about 0.025 to 250 mg of a compound of the invention, together with a pharmaceutically acceptable diluent or carrier.

The compounds of the invention may be administered by any conventional means, for example parenterally, for example in the form of injectable solutions or in the form of suspensions or in the nasal or suppository form. Alternatively, the compounds of the invention may be administered, for example, topically in the form of a cream, gel, and the like to treat the skin or promote hair growth as described above.

Accordingly, the invention further includes:

a) a compound of the invention or a pharmaceutically acceptable salt or complex thereof for use as a medicament;

b) a method of preventing or treating the conditions and disorders as set forth above in a patient in need thereof, which method comprises administering to said patient an effective amount of a compound of the invention or a pharmaceutically acceptable salt or complex thereof; and

-27c) a compound of the invention or a pharmaceutically acceptable salt or complex thereof for use in the preparation of a pharmaceutical composition for use in the method described in b) above.

According to another. In an embodiment of the invention, the compounds of the invention may be used as an adjunct or adjuvant in other therapy, for example in the case of hypercalcemia in therapy using a bone resorption inhibitor, in particular in therapy using calcitonin salmon, eel or human calcitonin, bisphosphonate, diuretic or any combination thereof; in the case of tumor therapy, a cytostatic agent or any combination thereof.

In accordance with the above, the invention includes as yet another embodiment:

d) a method of preventing or treating hypercalcemia, for example the prevention or treatment of any of the specific conditions or diseases mentioned above, in a patient in need thereof, which method comprises administering to said patient an effective amount of a) a compound of the invention; the active agent is a therapeutic agent as mentioned above.

The compounds of Examples 36, 37 and 49 are useful for the prevention or treatment of all conditions associated with increased plasma calcium due to excessive release of parathyroid hormone or parathyroid hormone related peptide.

Sequence list

SEQ ID NO. 1:

-28 (i) sequence characteristics:

(A) length: 38 amino acids (B) type: amino acid (C) number of chains: single-stranded (D); unknown

(Ii) Molecule type: protein (Iii) hypothetical: it's not (Iii) anti-sense: it's not (IN) fragment type : N - terminal (Xi) SEQ ID NO

Ala wall Ser Glu His gin Leu Leu His same time Lys Gly Lys Ser Ile gin 1. 5 10 15 same time Leu Arg Arg Arg Phe Phe Leu His His Leu Ala Ala Glu Ile His 20 25 30 Thr Ala Glu Ile Arg Ala 35

SEQ ID NO. 2:

(i) sequence characteristics:

(A) length: 34 amino acids (B) type: amino acid (C) number of chains: single chain (D) topology: unknown (ii) type of molecule: protein (iii) hypothetical: not (iii) anti-sense: not ( v) fragment type: N-terminal (xi) representation of SEQ ID NO. 2:

Ala wall Ser Glu His Gin Leu 1 5 same time Leu Arg Arg 20 Arg Phe Phe Thr Ala

Leu His same time 10 Lys Gly Lys Ser Ile 15 gin Leu His 25 His Leu Ile Ala Glu 30 Ile His

Sequence Information SEQ

ID # 3:

(i) sequence characteristics:

(A) length: 31 amino acids (B) type: amino acid (C) number of chains: single chain (D) topology: unknown

(Ii) Molecule type: protein (Iii) hypothetical: not (Iii) anti - sense: no (in) fragment type: N - terminal (Xi) SEQ ID NO. 3: Ala Val Ser Glu His Gin Leu His Asn Lys Gly Lys Ser ile Gin 1 5 10 15 Asn Leu Arg Arg Arg Phe Phe Leu His His Leu White Ala Glu Ile 20 25 30 SEQ ID NO. 4: (I) sequence characteristics: (A) length: 48 amino acids (B) type: amino acid (C) number of strings: single chain (D) topology: unknown (Ii) Molecule type: protein (Iii) hypothetical: not (Iii) anti - sense: no

-30 (v) fragment type: N-terminal (xi) representation of SEQ ID NO. 4:

Ser 1 wall Ser Glu Ile 5 gin Leu Met Kis Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg 20 wall Glu Trp Leu Arg Lys Lys Leu gin Asp 30 Val His same time Phe wall 35 Ala Leu Gly

SEQ ID NO. 5:

(I) sequence characteristics: (A) length: 34 amino acids (B) type: amino acid (C) number of strings: single chain 1 (D) topology: unknown (Ii) Molecule type: protein (Iii) hypothetical: not (Iii) anti - sense: no (IN) fragment type: N - terminal (Xi) SEQ ID NO. 5:

Ser 1 wall Ser Glu Ile 5 Gin Leu Met His same time 10 Leu Gly Lys His Leu 15 same time Ser Met Glu Arg 20 wall Glu Trp Leu Arg 25 Lys Lys Leu gin asd 30 ' wall His

Asn Phe

SEQ ID NO. 6:

(i) sequence characteristics:

(A) length: 31 amino acids (B) type: amino acid (C) number of chains: single chain (D) topology: unknown (ii) type of molecule: protein (iii) hypothetical: not (iii) anti-sense: not

-31 (V) (xi) fragment type: N-terminal representation of SEQ ID NO. 6:

Ser Val Ser Glu White Gin Leu Met His Asn Leu Gly Lys His Leu Asn 15 10 15

Ser Met Glu Arg Val Glu Trp Leu 25 30

Claims (11)

PATENT CLAIMS A derivative of a parathyroid hormone or a parathyroid hormone-related peptide in which at least one of the amino acid residues naturally occurring 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 further comprising at least one of the amino acid residues naturally occurring at positions 3 and 6, replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain, in free form or in the form of a salt or complex. 2. A derivative of a parathyroid hormone or a parathyroid hormone related peptide in which the amino acid residue naturally occurring at position 10 is replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain and optionally further comprising at least one of the amino acid residues, naturally occurring at positions 3 and 6, replaced by tryptophan or another amino acid residue that contains in its side chain an aromatic or heteroaromatic group, in free form or in the form of a salt or complex. The parathyroid hormone or parathyroid hormone-related derivative according to claim 1, wherein the amino acid residue containing an aromatic or heteroaromatic group in its side chain is an amino acid residue whose side chain is optionally substituted on the ring with a 3-33 or 4-pyridylmethyl group, 3- indolylmethyl or 3-indazolylmethyl, or a radical of formula (a), (b), (c) or (d) - < ^CH 2> n (C) (d) where n my value 1 or 2 m my value 1 or 2 about my 0 or 1 circle A is a possibly substituted substituents selected from the group consisting of fluorine, chlorine, nitro, C 1 -C 4 alkyl and C 1 -C 4 alkoxy, wherein the two alkyl or two alkoxy substituents may also together form a ring structure fused to ring A, each of rings B and C may be independently substituted as described above for ring A, and -34Y _and represents a direct bond, -CH ₂ -, an oxygen atom, an NH group or an N-alkyl group having 1 to 6 carbon atoms. The parathyroid hormone or parathyroid hormone-related peptide of claim 1, which is a compound of formula I R - [(X ² ) P, (X ³ ) q, (X ⁶ ) s; R ¹⁰ ] -D- (yx) R a (I) wherein x is a remnant number selected from a group comprising numbers 31, 34 35 36, 37 and 38 y is a number of the rest selected from a group comprising numbers 1, 2 3, 4, 5, 6 and 7, x ² represents Val or my independently one of významov referred to the X ^10, X ³ is Ser or independently has one of the meanings specified for X ¹⁰ , X ⁶ is Gln or has independently one of the meanings of X ^10, R ¹⁰ is Asp or X ¹⁰ , wherein X ¹⁰ represents Trp or the -NH-CHR'-CO- moiety, wherein R 'is a radical of formula (a), (b), (c) -35or (d) where n my value 1 or 2, m my value 1 or 2, 0 my value 0 Or ring A is optionally substituted with one or more substituents selected from the group consisting of atom, fluorine, chlorine, nitro, alkyl of 1 to 4 carbon atoms and alkoxy of 1 to 4 carbon atoms, wherein the two alkyl or two alkoxy substituents may also together to form a ring structure fused to ring A, each of rings B and C may be independently substituted as described above for ring A, and _And Y is a direct bond, - CH ₂ -, hydrogen -36 oxygen, NH or N-C 1 -C 6 -alkyl, D represents an amino acid sequence derived from a N-terminal fragment of a parathyroid hormone-related or parathyroid hormone peptide, each of p and pass having a value of 1, p having a value of 0 if y is greater than 2, p having a value of 0 if y is greater than 3 s a value of 0 if y is greater than 6, R is H, R '' - CO-, R ^Z -O-CO-, RO-CH ₂ -CO-, R-SO ₂ -, R ', R'-NH-CO-, R '' -NH-CS- or NH ₂ -alkylene-CO- having 1 to 6 carbon atoms in the alkylene moiety, wherein R '' represents an alkyl group having 1 to 8 carbon atoms, a ω-carboxyalkyl group having 1 to 6 carbon atoms in the alkyl moiety, a ω - [(alkoxy) carbonyl] alkyl group having 1 to 4 carbon atoms in the alkoxy moiety and 1 to 6 C 2 -C 8 alkenyl, C 5 -C 7 cycloalkyl, C 5 -C 7 cycloalkylalkyl and C 1 -C 4 alkyl, or phenyl, phenylalkyl (C 1 -C 4) alkyl, 1-naphthyl, 2-naphthyl, 1-naphthyl (C 1 -C 2) alkyl, or 2-naphthyl (C 1 -C 2) alkyl group, -37 wherein any of these groups is optionally substituted on the ring by one or more substituents selected from the group consisting of fluorine, chlorine, nitro, C 1 -C 4 alkyl and C 1 -C 4 alkoxy or heteroaryl, R 'independently has one of the meanings given for R' 'except for the ω-carboxyalkyl group having 1 to 6 carbon atoms in the alkyl moiety and the ω - [(alkoxy) carbonyl] alkyl group having 1 to 4 carbon atoms in the alkoxy group and (1-6C) alkyl moieties; and R _a is hydroxy or amino, with the proviso that at least one of X ² and R ¹⁰ is X ¹⁰ . The parathyroid hormone or parathyroid hormone related peptide of claim 1 or 4, which is derived from the N-terminal fragment of human parathyroid hormone, hPTH, or the human parathyroid hormone related peptide, hPTHrP. The parathyroid hormone or parathyroid hormone related peptide of claim 1, selected from the group consisting of: N '-2-naphthylacetyl- [Nal (2) ¹⁰ , DAla ³⁴ ] hPTHrP (3-34) NH 2, N' -2-naphthylacetyl- [Nal (2) ¹⁰ ] hPTHrP (3-31) NH 2, and N ^and -succinyl- [Phe ⁶ , NaI (2) ¹⁰ ] hPTHrP (5-34) NH ₂ . A process for the preparation of a parathyroid hormone derivative or a parathyroid hormone-related peptide according to claim 1 in free or salt or complex form, characterized by: -38 that is a) removes at least one protecting group that is present in a parathyroid hormone-related or parathyroid hormone-related peptide derivative according to the invention, for example a compound of formula I, in protected form, or b) joining two peptide fragments in protected form, partially protected or unprotected form to each other by an amide bond, the peptide fragments being such that the amino acid sequence of the desired parathyroid hormone or parathyroid hormone related peptide derivative is obtained, for example of formula I, optionally converted step a) of the process, or that c) introduces a protecting group or substituent in a selective manner to the amino group of the N-terminal residue of the desired sequence or N-terminal fragment thereof in the protected or unprotected process a. on the what possibly place step a) and isolate do as follows obtained derivatives peptide related parathyroid hormone or parathyroid hormone in free in form in the form of a salt or in the form of a complex. The parathyroid hormone or parathyroid hormone related peptide of claim 1 in free form or in the form of a physiologically acceptable salt for use as a medicament. 9. A pharmaceutical composition comprising a parathyroid hormone derivative or a parathyroid hormone related peptide of claim 1 in free form; or In the form of a physiologically acceptable salt together with a pharmaceutically acceptable diluent or carrier. The parathyroid hormone or parathyroid hormone-related derivative according to claim 1 in free or physiologically acceptable salt form for use as a medicament together with another therapeutic agent selected from the group consisting of bone resorption inhibitors and cytostatic agents. A method for preventing or treating conditions associated with increased plasma calcium due to excessive release of parathyroid hormone or parathyroid hormone related peptide, for preventing or treating tumor growth stimulated by a parathyroid hormone related peptide for treating dermatological disorders and promoting hair growth in a patient who such treatment is in need of administering to said patient an effective amount of a parathyroid hormone derivative or a parathyroid hormone related peptide of claim 1 in free or physiologically acceptable salt form.