WO1996003437A1

WO1996003437A1 - PTH OR PTHrP ANTAGONISTS

Info

Publication number: WO1996003437A1
Application number: PCT/EP1995/002993
Authority: WO
Inventors: François Cardinaux; Jean Honoré M. FEYEN; Rainer Gamse; Frank Otto Gombert
Original assignee: Sandoz Ltd.; Sandoz-Patent-Gmbh; Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H.
Priority date: 1994-07-28
Filing date: 1995-07-27
Publication date: 1996-02-08
Also published as: MX9700446A; JPH10502091A; CA2192787A1; NO970356D0; HUT77979A; PL318017A1; AU3167095A; CZ23397A3; BR9508433A; NO970356L; TR199500915A2; EP0773958A1; CO4410206A1; FI970168A; FI970168A0; SK12097A3; IL114736A0

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

PTH or PTHrP antagonists

The present invention relates to parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) compounds, a process for their production, pharmaceutical preparations comprising them and their use as a pharmaceutical.

More particularly the present invention provides a PTH or PTHrP compound 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. The compounds are hereinafter referred to as compounds of the invention.

In a particular embodiment of the invention there is provided a PTH or PTHrP compound in which the amino acid residue naturally occurring in position 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.

The term "PTHrP" refers to any naturally occurring form of PTHrP, e.g. human, bovine, chicken, rat or mouse PTHrP. For consistency and as is conventional, in the following description, the same numbering system will be applied to the amino acid residues of the PTHrP sequence regardless of whether any o-amino acid residue of the PTHrP sequence is replaced or omitted according to the invention.

The term "PTH" refers to any naturally occurring form of PTH, e.g. human, bovine, chicken, rat or mouse PTH. For consistency and as is conventional, in the following description, the same numbering system will be applied to the amino acid residues of the PTH sequence regardless of whether any o-amino acid residue of the PTH sequence is replaced or omitted according to the invention.

By "PTHrP compound" or "PTH compound" is meant a peptide comprising an amino acid sequence based on a N-terminal fragment of PTHrP or PTH respectively, preferably based on a PTHrP or PTH fragment starting with any one of the residues 1-7 and terminating with any one of the residues from 27 to 38 e.g. a N-terminal fragment of PTHrP or PTH comprising up to 31, 34, 35, 36, 37 or 38 amino acid residues.The terms "PTHrP or PTH" will thus be understood as embracing peptides wherein one or more amino acid residues of the said N-terminal fragment is omitted, preferably at the N-terminus. The terms are also to be understood as embracing peptides wherein one or more amino acid residues of the naturally occurring sequence is replaced by one or more other amino acid residues (natural or non natural) in addition to the substitution in position 2 and/or 10 and optionally in 3 and/or 6 according to the invention. The 1-38, 1-34 and 1-31 N-terminal fragments of human PTHrP have the sequences as indicated in SEQ ID No:l, 2 or 3, respectively.

The 1-38, 1-34 and 1-31 N-terminal fragments of human PTH have the sequences as indicated in SEQ ID No: , 5 or 6, respectively.

The N-terminus of the PTHrP or PTH compounds may be a free or a protected amino group, bearing e.g. a N-protecting group as disclosed in "Protective Groups in Organic Synthesis", T. . Greene, J. Wiley & Sons NY (1981), 219-287, the contents of which being herein incorporated by reference, preferably protected by R"-CO-, R"-0-CO-, R"-0-CH₂-CO- or R"-S0₂, or an amino group bearing a radical R' ' ' , R' ' '-NH-CO- or R" ' -NH-CS- such as defined hereunder.

The C-terminus of the compounds of the invention may be COOH, CONH₂ or a mono- or disubstituted amide, e.g. -CONR_cR-i wherein one of R_c and R,, is H and the other is an aliphatic residue, e.g. C_x_₆alkyl, o both are an aliphatic residue, or R_c and R-, together with the nitrogen atom to which they are attached form a heterocyclic residue, e.g. pyrrolidinyl or piperidinyl. PTHrP or PTH compounds in accordance with the invention have potent antagonistic activity at the PTH/PTHrP receptor e.g. bind to the

PTH/PTHrP receptor, have an intrinsic activity (i.a) for activation of the PTH/PTHrP receptor in a functional bioassay significantly <1, e.g. an i.a of at most 0.3, and antagonize PTHrP or PTH or a fragment thereof e.g. PTHrP(1-34) or PTH(1-34) in a functional bioassay with a pA2 value of at least 6.5. Preferably compounds in accordance with the invention have an i.a of 0.03 or lower or even not detectable in some of the assays. Example of a f nctional bioassay is the osteosarcoma-based adenylate cyclase assay employed conventionally in the art. This assay provides an in vitro determination of the extent to which the compound to be tested stimulates adenylate cyclase activity or antagonizes the effect of PTHrP or PTH or a fragment thereof in rat osteosarcoma cells of the UMR lineage, e.g. UMR-106-06 according to the method of Marcus and Aurbach in Endocrinology, ≤_5_, 801-810 (1969) as disclosed hereinafter.

By amino acid is meant a naturally occurring or commercially available or non natural amino acid or an optical isomer thereof. A non natural amino acid is an amino acid which is not incorporated into a protein under mRNA direction, e.g. β-Nal, a fluoro-o-amino acid such as fluoroalanine, cyclohexylalanine or trimethylsilyl-alanine.

"Natural amino acids" refer to those well known in the art. They are listed and standard abbreviations are pr ^τided in the

U.S.P.T.O. publication, Trademark Official G :ette. published

May 15, 1990, p. 33 at 46. These amino acids and abbreviations are specifically incorporated herein by reference.

The natural amino acids are shown below:

A Ala alanine

D Asp aspartic acid

E Glu glutamic acid

F Phe phenylalanine

G Gly glycine H His histidine

I lie isoleucine

K ys lysine

L Leu leucine

M Met methionine N Asn asparagine

Q Gin glutamine

R Arg arginine

S Ser serine

T Thr threonine V Val valine W Trp tryptophane

Y Tyr tyrosine

By amino acid residue bearing an aromatic or heteroaromatic group on its side chain is meant an amino acid residue wherein the side chain is e.g. optionally ring-substituted phenyl-methyl, 1- or 2-naphthyl-methyl, 1- or 2-naphthyl-ethyl, 3- or 4-pyridyl-methyl, 3-indolyl-methyl or 3-indazolyl-methyl; preferably it is an amino acid residue of formula -NH-CHR'-CO- as defined below.

According to a preferred embodiment of the invention, there is provided a compound of formula I

R-[(X²)_P, (X³)_q, (X⁶),,R¹⁰]-D-(y-x)R_a I

wherein x is a residue number selected from 31, 34, 35, 36, 37 or 38, y is a residue number selected from 1, 2, 3, 4, 5, 6 or 7, X² is Val or has independently one of the significances of X¹⁰, X³ is Ser or has independently one of the significances of X¹⁰, X⁶ is Gin or has independently one of the significances of X¹⁰, R¹⁰ is Asp or X¹⁰, X¹⁰ being Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a) , (b) , (c) or (d)

(a) (b)

-(CH₂)fτ ® (CH₂)₀ ^" ©)

(c) (d) wherein n is 1 or 2, m is 1 or 2, o is 0 or 1, ring A is optionally substituted by one or more substituents selected from fluoro, chloro, nitro, C₁.₄alkyl and C_1-4alkoxy, whereby two alkyl or two alkoxy substituents may also form together a ring structure fused to ring A, each of rings B and C independently may be substituted as indicated above for ring A, and

Y_a is a direct bond, -CH₂-, 0, NH or N-C_j.galkyl, D is an amino acid sequence derived from an N-terminal fragment of PTHrP or PTH, each of p, q and s is 1, provided that p is 0 when y > 2, q is 0 when y > 3 and s is 0 when y > 6,

R is H, R"-C0-, R"-0-CO-, R"-0-CH₂-C0-, R"-S0₂-, R ' ' ' , R" ' '-NH-C0-, R" ' '-NH-CS- or NH₂-C₁.₆alkylene-C0- wherein R" is C^aHyl; ω-carboxy-Ci.galkyl; ω-[ (C₁,₄alkoxy) - carbonyll-Cj-ealkyl; C₂__Balkenyl; C_5-7cycloalkyl; C₅.₇cycloalkyl- C₁.₄alkyl; or phenyl, phenyl-C^alkyl, 1-naphthyl, 2-naphthyl, 1-naphthyl-C_halky1 or 2-naphthyl-C₁_₂alkyl each of which being optionally ring substituted by one or more substitutents selected from fluoro, chloro, nitro, C_halky1 and C₁.₄alkoxy; heteroaryl; and R' ' ' has indepedently one of the significances given for R" except the significances of ω-carboxy-Ci.salkyl and ω- [ (C₁,₄alkoxy) -carbonyl]-C_halky1; and R_a is OH or NH₂, with the proviso that at least one of X² and R¹⁰ has the significance of X¹⁰.

When the substitutents of ring A, B or C form together a ring structure, it may be e.g. -0-CH₂-CH₂-0-. Example of polycyclic cycloalkyl is e.g. adamantyl.

By heteroaryl as R" is meant a 5-, 6- or 7-membered unsaturated heterocyclic radical comprising at least one nitrogen atom and optionally further a heteroatom such as N, 0 or S, and optionally condensed with a benzene ring. Heteroaryl is preferably indolyl, quinolyl or isoquinolyl.

The compounds of the invention may exist e.g. in free form, salt form or in the form of complexes thereof. Acid addition salts may be formed with e.g. organic acids, polymeric acids and inorganic acids. Such acid addition salt forms include e.g. the hydro- chlorides and the acetates. Complexes are e.g. formed from the PTHrP or PTH compound of the invention on addition of inorganic substances, e.g. inorganic salts or hydroxides such as Ca- and Zn-salts, and/or an addition of polymeric organic substances.

In the compounds of formula I, the following significances are preferred either individually or in any combination or sub-combination:

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

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

3. X¹⁰ is Trp.

4. X¹⁰ is -NH-CHR'-CO- wherein R' is a radical of formula (a), (b) or (c) , preferably (a) or (c), more preferably (a).

5. X² is Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a) , (b) or (c) ,

6. Each of X² and X¹⁰ is Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a) , (b) or (c) .

7. Each of X³ and X¹⁰ is Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a) , (b) or (c) .

8. Each of X^s and X¹⁰ is Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a) , (b) or (c) .

9. Each of X², X⁶ and X¹⁰ is Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a), (b) or (c) .

10. n is 1.

11 . y is a residue number selected from 3 , 4 , 5 , 6 or 7 , preferably 3 or 5 .

12 . x is a residue number selected from 31 or 34 .

13 . R is R"CO- , R" -S0₂ , R ' ' ' or HzN-Ci-ealkylene-CO- .

14 . R" is Ci.galkyl , phenylC₁,₄alkyl , 1- or 2-naphthyl , 1- or 2-naphthyl-C₁_₂alkyl . 15. R is ω-carboxy-Ci-_ealkyl.

As already mentioned, one or more amino acid residues at positions other than 2 and/or 10 may be further replaced by a natural or unnatural amino acid residue as indicated above or be omitted. When the compounds of the invention are hPTHrP derivatives, they may comprise further amino acid replacement, e.g. in position 3, e.g. Ala, in position 4, e.g. Trp, in position 5, e.g. optionally ring-substituted Phe, in position 11 and/or 13, e.g. Leu, or in position 34, e.g. D-Ala; their amino acid sequence being optionally shortened at the N-terminus by the omission of 1 up to 7 amino acid residues. When the compounds of the invention are hPTH derivatives, amino acid residues may be further replaced e.g. in at least one of the positions selected from 8, 16, 18, 33 and 34, e.g. Leu^B, Ala¹⁶, Gin¹⁸, Thr³³, Ala³⁴ or D-isomers thereof.

The present invention also provides a process for the production of the PTHrP or PTH compounds of the invention. They may be prepared in a stepwise manner either in solution or using the solid phase synthesis process or genetic engineering or by a combination of these methods.

The compounds of the invention may be produced for example as follows by:

a) removing at least one protecting group which is present in a PTHrP or PTH compound of the invention, e.g. a compound of formula I, in protected form; or

b) linking together by an amide bond two peptide fragments in protected, partially protected or unprotected form, the peptide fragments being such that the amino acid sequence of the desired PTHrP or PTH compound, e.g. of formula I, is obtained, and then effecting optionally stage a) of the process, or

c) adding 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 protected or unprotected form and then optionally carrying out step a) , and recovering the PTHrP or PTH compounds thus obtained in free form, in salt form or in complex form.

Process steps a) , b) and c) may be effected in analogy with known methods, e.g. as known in the art of peptide chemistry or as described in the following examples. Where desired, in these reac¬ tions, protecting groups which are suitable for use in peptides may be used for functional groups which do not participate in the reaction. The term protecting group may also include a polymer resin having functional groups.

When the compounds of the invention comprise unnatural and natural residues, they may be produced by a combination of a chemical stepwise process and genetic engineering; the peptide sequence (whether the complete sequence or a fragment) made of genetically encodable amino acid residues may be produced by recombinant techniques and the desired unnatural amino acids or terminal amino substituent may be introduced by chemical synthesis and, where required, the fragments may be combined and the protecting group(s) when present may be removed.

In the following Examples all temperatures are in °C. The following abbreviations are employed.

DMF = dimethylformamide

DMA = N,N-dimethylacetamide-6-sulfonyl

Pmc = 2,2,5,7,8-pentamethylchroman tBu = tert.butyl PyBOP = Benzotriazol-1-yl-oxy-tripyrrolidino- phosphonium hexafluorophosphate

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

DIEA = N,N-diisopropyl-N-ethylamine

Fmoc = 9-fluorenylmethoxycarbonyl RT = room temperature

MS = M^* determined by electrospray spectroscopy

EXAMPLE 1: [Trp⁴-¹⁰]hPTHrP(1-34) OH

The peptide is synthesized in a stepwise manner on a polystyrene based resin support. The alpha-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) .

Fmoc-Ala-oxymethyl-4-phenoxymethyl-co(polystyrene-1%-divinyl- benzene), approx. 0.5 mmol/g, is used as a starting material for the stepwise solid phase synthesis of peptides which consists of repetitive cycles of alpha-amino group deprotection, washing, coupling (i.e., attachment of next amino acid residue to the growing peptide chain) and washing. The N-alpha Fmoc group is cleaved using piperidine, 20% in DMA. In the coupling reaction four equivalents of Fmoc-amino acid and PyBOP-reagent and eight eqivalents of DIEA in DMA are used per amino-group. After complete assembly of the peptide chain the terminal Fmoc- protecting group is removed with piperidine in DMA as before. The peptide is cleaved from the resin support and all side-chain protecting groups are simultanously removed by using a reagent consisting of 5% of dodecylmethylsulfide and 5% water in TFA for two hours at RT. Resin particles are filtered off, washed with some TFA and the product is precipitated from the combined filtrates by the addition of 10 to 20 volumes of diethyl ether, washed with ether and dried. The product is purified by chromatography on a C-18 wide-pore silica column using a gradient of acetonitrile in 2% aqueous phosphoric acid. Fractions containing the pure compound are collected, filtered through an anion-exchange resin (Biorad, AG4-X4 acetate form) and lyophilized to give the title compound.

MS: 4146

In analogy with the procedure of Example 1, but using the appropriate amino-acids the following compounds may be prepared:

[Trp¹⁰, Leu¹¹ , Leu¹³] hPTHrP ( l-34 ) OH M MSS 4059 EXAMPLE 3 : [Trp¹⁰ , Leu¹¹! hPTHrP (1-34 ) OH MS 4073 EXAMPLE 4 [Trp³-¹⁰]hPTHrP ( l-34 ) OH MS 4188

SXAMP B [Trp²-¹⁰] hPTHrP ( l-34 ) OH MS 4176

EXAMPLE [Trp¹⁰] hPTHrP ( 2 -34 ) OH MS 4018 3

EXAMPLE 7 [ Trp¹⁰ ] hPTHr P ( 3 - 34 ) OH MS 3919 2

EXAMP E 6 [Trp²] hPTHrP ( 1-34 ) OH MS 4104 9 [Trp¹⁰] hPTH ( l-34 ) OH MS 4189

EXAMPLE 10 s [Leu⁸ , Trp¹⁰ , Ala¹⁶ , Gin¹⁸ , Thr³³ , Ala³⁴ ] hPTH ( 1-34 ) OH

MS : 4036 EXAMPLE 11; [ Leu⁸ , Trp¹⁰ , Ala¹⁶ , Gin¹⁸ , Thr³³ , Ala³⁴ ] hPTH ( 2 -34 ) OH

MS: 3949

EXAMPLE 12: [Leu⁸,Trp¹⁰,DLeu¹¹,Gin¹⁸,Thr³³,Ala³⁴]hPTH(1-34)OH

MS: 4079

EX MPL 13 [Phe² , Trp¹⁰] hPTHrP ( 1-34 ) -OH MS : 4137 .1

EXΛMP^T-E 1« [Trp² , Trp¹⁰] hPTHrP (2-34 ) -OH MS : 4105 .1

EXAMPLE 15 [Trp⁶ , Trp¹⁰] hPTHrP ( 1-34 ) -OH MS 4146 8

EXAMPLE 16 [ Trp⁶ , Trp¹⁰ ] hPTHr P ( 6 - 34 ) -OH MS 3623 3 [ Trp¹⁰ ] hPTHr P ( 7 - 34 ) -OH MS 3437 6 EXAM LE 18 [ Trp³ , Trp¹⁰ ] hPTHr P ( 3 - 34 ) -OH MS 4018 0 [ Trp¹⁰ ] hPTHr P ( 1 - 34 ) OH MS 4088 7

E AM£L£_20.: [Trp⁶, Trp¹⁰]hPTHrP(3-34)NH₂ MS 3976 8 EXAMPLE 21; [Phe⁶, Trp¹⁰]hPTHrP(l-34)OH MS 3546 1 EXAMPLE 22; [Phe², Phe⁶, Trp¹⁰]hPTHrP(l-34)OH MS 4155 1

EXAMPLE 23; N"-3-aminopropionyl- [Phe⁶ , Nal (2)¹⁰]hPTHrP(4-34)NH₂

MS: 3932.0

EXAMPLE 24; N^«-benzyloxycarbonyl[Nal(2)¹⁰]hPTHrP(3-34)-NH₂

This peptide is prepared in a manner analogous to Example 1 but using 4-(2 ' ,4'-dimethoxyphenyl-Fmoc-amino-methyl)-phenoxy- co(polystyrene-l%-divinylbenzene) , approx. 0.4 mmol/g, which may be prepared, e.g., as described in Tetrah. Letters, 28:3787-3790 (1987) as a starting material. In the last synthesis cycle Z-Ser- OH is added to the peptide chain and the peptide cleaved and purified as in Example 1. MS: 3975.3

EXAMPLE 25; N'-acetyl-[Trp¹⁰]hPTHrP(3-34)OH

This peptide is assembled in analogy with the procedure of Example 1. At the end of the synthesis a final cycle of Fmoc-deprotection and acetylation using a 1:1 mixture of acetic anhydride and DMF is applied. The peptide is cleaved from the resin and purified as before. MS: 3961.1 The following compounds may be prepared in a manner analogous to that of Examples 1, 24 or 25.

EXAMPLE 26; N'-acetyl- [Trp¹⁰]hPTHrP(2-34)OH MS: 4060.4

EXAMPLE 27; 1-Isocaproyl- [Leu⁸,Trp¹⁰,Gin¹⁸,Thr³³,Ala³⁴]- hPTH(l-34)OH MS: 4091

EXAMPLE 28; N*-2-naphthyl-acetyl- [Nal (2 ) ¹⁰] hPTHrP ( 3-34 )NH₂ MS: 4097.1

EXAMPLE 29; N^«-2-naphthyl-acetyl- [Nal (2 ) ¹⁰] hPTHrP (4-34 )NH₂

MS: 4009.7

EXAMPLE 30; N'-2-naphthyl-sulf onyl- [Nal (2 ) ¹⁰] hPTHrP ( 3-34 )NH₂ MS: 4119.2

EXAMPLE 31; N^«-2-naphthoxyacetyl- [Nal (2 ) ¹⁰] hPTHrP ( 3-34 )NH₂ MS: 4111.5

EXAMPLE 32; N"-2-naphthylacetyl- [Ala³ , al (2 ) ¹⁰] hPTHrP (3-34 ) NH₂ MS: 4080.8

EXAMPLE 33; N^«-benzyloxycarbonyl- [Trp² , Nal (2 ) ¹⁰] hPTHrP (2-34 ) H₂ MS: 4248.3

EXAMPLE 34; 3-Naphth-2-yl-proprionyl- [Nal (2 ) ¹⁰] hPTHrP ( 3-34 )NH₂

MS: 4110.8

EXAMPLE 35; 3 -Naphth-2-yl-propionyl- [Nal (2 ) ¹⁰] hPTHrP (3 -34 )NH₂ MS: 4110.9

EXAMPLE 36; N"-2-naphthyl-acetyl- [Nal (2 ) ¹⁰ , DAla³⁴] 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 EXAMPLE 39; N^«-acetyl- [Phe⁶,Nal (2)¹⁰]hPTHrP(6-34)NH₂ MS: 3637.0

EXAMPLE 40; N"-acetyl- [Phe⁶,Nal (2)¹⁰]hPTHrP(4-34)NH₂ MS: 3903.1

E AMPT-E_4J₁: 1- (1-amino-l-cyclopentyl-carbonyl) -

[Leu⁸,Trp¹⁰,Gin¹⁸,Thr³³,Ala³⁴]hPTH(1-34)OH MS: 4104

Fmoc-1-aminocyclopentane-l-carboxylie acid used in the prepara¬ tion of the peptide resin intermediate may be prepared, e.g. as described by G. Valle et al.,1988, in Can.J.Chem.66:2575-2582.

EXAMPLE 42: 1-Adamantyl-carbonyl-[Leu⁸,Trp¹⁰,Gin¹⁸,Thr³³, Ala³⁴]hPTH(l-34)OH MS: 4155

EXAMPLE 43: 1- (3-indolyl-carbonyl)- [Leu⁸,Trp¹⁰,Gin¹⁸,Thr³³, Ala³⁴]hPTH(l-34)OH

MS: 4150

EXAMPLE 44 ; 1- (3-quinolyl-carbonyl- , [Leu⁸ , Trp¹⁰ , Ala¹⁶ 'Gin¹⁸ , Thr³³ , Ala³⁴] hPTH ( 1-34 ) OH MS : 4104

EXAMPLE 45 ; 1- (2-naphthoyl ) - [Leu⁸ , Trp¹⁰, Gin¹⁸ , Thr³³ , Ala³⁴] - hPTH ( l-34 ) OH MS : 4147

BlffinnTlt if ' N^«- (2-naphthyl) -methyl- [Trp¹⁰]hPTHrP(2-34) -OH

Methyl N-naphthoyl (2) -valinate is treated with Lawesson's reagent, S-O. Lawesson et al. , Tetrahedron 37:3635 (1981) and the resulting methyl N-thionaphthoyl (2) -valinate is reduced using the procedure described for endothiopeptides by F.S. Guziec et al., Tetrah. Letters 23-26 (1990). The methyl ester is hydrolyzed using LiOH and N-alpha- (naphtyl (2) -methyl) -valine hydrochloride obtained as a crystalline solid, mp = 205-210° (dec). This is coupled using PyBOP-reagent to previously prepared, protected [Trp¹⁰]hPTHrP- (3-34) -O-peptide resin from which the N-alpha Fmoc- group has been removed. The peptide is cleaved from the resin and purified as in Example 1.

MS: 4158.2

EXAMPLE 47; N*-2-naphth-2 -yl-ethyl- [Trp¹⁰] hPTHrP (2-3 ) OH MS: 4171.9

EXAMPLE 48; N"-2-naphth-2 -yl-ethyl- [Ala³ , Nal (2 ) ¹⁰] hPTHrP (3-34 ) NH₂

MS: 4066.7

EXAMPLE 49; N'-succinyl-[Phe⁶,Nal (2)¹⁰]hPTHrP(5-34)NH₂

This peptide is prepared in a manner analogous to Example 1 but using 4- (2' ,4'-dimethoxyphenyl-Fmoc-amino-methyl)-phenoxy- co(polystyrene-1%-divinylbenzene) , 0.63g, loading approx.

0.4 mmol/g, which may be prepared, e.g., as described in Tetrah. Letters, 28: 3787-3790 (1987) as a starting material. After coupling the last amino acid, Fmoc-His(Trt) -OH, the Fmoc-group is selectively removed as usual and the peptide resin reacted with succinic anhydride (0.5g) and DIPEA (0.86ml) in DMF. The peptide is cleaved from the resin, purified and lyophilized in the acetate form as in Example 1 to give the title compound.

MS: 3832.0

By following the procedure of Example 49, following compounds may be prepared:

EXAMPLE 50; N'-succinyl-[Phe⁶,Nal (2)¹⁰]hPTHrP(5-31)NH₂ MS: 3522.1

EXAMPLE 51; N*-succinyl-[Nal(2)⁶,Nal(2)¹⁰]hPTHrP(5-34)NH₂ MS: 3881.7

EXAMPLE 52; N'-glutaryl- [Phe⁶,Nal (2)¹⁰]hPTHrP(5-34)NH₂

MS: 3845.6

EXAMPLE 53; N*-succinyl- [Phe⁶, Nal (2)¹⁰,DAla³ ]hPTHrP (5-34)NH₂ MS: 3832.0

EXAMPLE 54; N*-succinyl- [4-Cl-Phe⁶ ,Nal (2 ) ¹⁰] hPTHrP (5-34 )NH₂ MS: 3866.5 EXAMPLE 55; N^«-succinyl- [4-Cl-Phe⁵, 4-Cl-Phe⁶, 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 exhibit valuable pharmacological properties as indicated in animal tests and are therefore indicated for therapy.

The biological activity of the compounds of the invention is assessed in vitro by measuring their ability of stimulating (agonism) or inhibiting the PTH-stimulated (antagonism) synthesis of cyclic AMP in UMR 106-06 rat osteosarcoma cells according to the method of Marcus and Aurbach in Endocrinology, £5_, 801-810 (1969) . Rat osteosarcoma UMR 106 cells are grown to confluence in DMEM-HAM's F12 medium (1:1) - 10% FCS in 12 well plates. The medium is then changed to medium with 2% FCS and 1-5 μCi/well [3H]-adenine is added. Two hours later, cells are washed twice with serum-free medium and incubated in DMEM - 0,1% BSA containing 1 mM 3-isobutyl-l-methylxanthine to exclude actions on phospho- diesterases. Test substances are added 20 min later either alone or together with PTH (antagonist experiment) . After 15 min, the medium is removed and the reaction is stopped and cAMP extracted by adding 0.5 ml ice cold 5% trichloroacetic acid. A carrier solution (0.5 ml/well) containing 0.2 mM of unlabelled adenine, adenosine, AMP, ADP, ATP, and cAMP as well as 0.4 μCi [14C]-adenosine for determination of recovery is added. [3H]-cAMP is separated using serial Dowex AG 50W-X4 (200-400 mesh) and alumina chromatography and counted according to Salomon Y. in Advances in Cyclic Nucleotide Research, Vol. 10, Raven Press, 35-55, 1979. Results are calculated in % of solvent control and EC₅₀ values determined from DRC curves. Antagonist potency is calculated from the right ward shift of DRC curves of PTHrP or PTH and is given as pA₂ values. Compounds of the invention are active as antagonists at a concentration of 10^"9 to 10^"5 M. Com¬ pound of Examples 36, 37 and 49 have a pA₂ value in the UMR 106-06 cells of 10.3; 9.7 and 9.3, respectively.

The compounds of the invention also have binding affinity to PTH receptors, e.g. as follows: Chicken [Tyr³⁶]PTHrP(1-36)amide is iodinated to a specific activity of 2,200 Ci/mmol using the lactoperoxidase method (Anawa Lab. AG, Wangen) . Monolayers of opossum kidney cells (OKI) are washed with 200 μl DMEM and HAM's F12 (1:1) containing 1% BSA and incubated at 16°C with 50.000cpm of [¹²⁵I-Tyr³⁶]chPTHrP(1-36)amide per well in the presence or absence of 1 uM [Tyr³⁶]chPTHrP- (l-36)amide. After incubation, cells are washed with 0.5 ml medium (4°C) , lysed with 0.5 ml IN NaOH and radioactivity is determined. Specific binding is defined as total binding minus nonspecific binding. Competition curves are analyzed using

SCTFIT, a non-linear regression computer program (Feyen et al, 1992, Biochem. Biophys. Res. Commun. 187:8-13) and data presented as mean pK_D values (n=2 to 3). Compounds of Examples 36, 37 and 49 have a pK_D value of 8.3; 7.9 and 8.4, respectively.

Furthermore, the compounds of the invention antagonize the effect of PTH after i.v. infusion, e.g. as determined in thyropara- thyroidectomized rats. 24 h after thyroparathyroidectomy, anesthetized rats are infused with PTH(1-34) and the compound to be tested via separate jugular veins. Urine is collected from the urinary bladder which is cannulated via the ventral approach. Phosphate and cAMP content in the urine and calcium and phosphate in serum are measured using standard methodology. These parameters are used to quantify antagonist potencies against PTH effects in vivo. In this test, the compounds of the invention antagonize the PTH effects when administered by i.v. infusion at a dose of from 1 μg/kg/h to 1 mg/kg/h. Compound of Example 49 completely suppresses PTH-induced phosphaturia for up to 90 min when i.v. infused at 190 μg/kg/h, PTH(l-34) being i.v. infused at 4 μg/kg/h.

The compounds of the invention are accordingly useful for preventing or treating all conditions which are associated with increased plasma calcium caused by excessive release of PTH or PTHrP e.g. hyperparathyroidism, hypercalcemia, e.g. associated with malignancies, e.g. breast carcinomas, squamous cell carcinomas of the lung, esophagus and head and neck region and hematological malignancies, with or without bone metastases. The compounds of the invention are furthermore useful for the prevention or treatment of tumour growth, tumour penetration and ingrowth in bones stimulated by PTHrP, for treating dermatological disorders, e.g. tissue repair therapies, for example treatment of burns, ulcerations and wounds, and for hair growth promotion.

For these indications, the appropriate dosage will, of course, vary depending upon, for example, the host, the mode of adminis- tration and the severity of the conditions being treated. Howe¬ ver, in general, satisfactory results in animals are indicated to be obtained at daily dosages from about 0.1 to about 100 mg/kg animal body weight. In larger mammals, for example humans, an indicated daily dosage is in the range from about 0.1 to about 500 mg of the compounds of the invention.

The compounds of the invention may be administered in free form or in pharmaceutically acceptable salt form or complexes . Such salts and complexes may be prepared in conventional manner and exhibit the same order of activity as the free compounds. The present invention also provides a pharmaceutical composition comprising a compound of the invention in free base form or in pharmaceutically acceptable salt form or complex form in associa¬ tion with a pharmaceutically acceptable diluent or carrier. Such compositions may be formulated in conventional manner. Unit dosage forms suitably comprise from about 0.025 to 250 mg of a compound of the invention, together with a pharmaceutical acceptable diluent or carrier therefor.

The compounds of the invention may be administered by any conventional route, for example parenterally e.g. in form of injectable solutions or suspensions, or in a nasal or a sup¬ pository form. The compounds of the invention may alternatively be administered e.g. topically in the form of a cream, gel or the like for example for the treatment of the skin or hair growth as hereinbefore described.

In accordance with the foregoing the present invention further provides:

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

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

c) a compound of the invention or a pharmaceutically acceptable salt or complex thereof for use in the preparation of a phar- maceutical composition for use in the method as in b) above.

According to a further embodiment of the invention, the compounds of the invention may be employed as adjunct or adjuvant to other therapy, e.g. in hypercalcemia to a therapy using a bone resorption inhibitor, in particular a therapy employing a calcitonin or an analogue or derivative thereof, e.g. salmon, eel or human calcitonin, a biphosphonate, a diuretic or any combination thereof, or in case of tumour therapy, a cytostatic agent or any combination thereof.

In accordance with the foregoing the present invention provides in a yet further aspect:

d) a method for preventing or treating hypercalcemia for example for preventing or treating any of the specific conditions or diseases hereinbefore set forth, in a subject in need of such a treatment which method comprises administering to said subject an effective amount of a) a compound of the invention and b) a second drug substance, said second drug substance being a therapeutic agent as indicated above.

Compounds of Examples 36, 37 and 49 are preferred for preventing or treating all conditions which are associated with increase plasma calcium caused by excessive release of PTH or PTHrP.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: SANDOZ LTD.

(B) STREET: Lichtstrasse 35,

(C) CITY: BASLE

(E) COUNTRY: SWITZERLAND

(F) POSTAL CODE (ZIP) : CH-4002

(A) NAME: SANDOZ PATENT GMBH

(B) STREET: Humboldtstrasse 3

(C) CITY: LOERRACH

(E) COUNTRY: GERMANY

(F) POSTAL CODE (ZIP): D-79539

(A) NAME: SANDOZ ERFINDUNGEN VERWALTUNGSGESELLSCHAFT

M.B.H.

(B) STREET: Brunner Strasse 59

(C) CITY: VIENNA

(E) COUNTRY: AUSTRIA

(F) POSTAL CODE (ZIP) : A-1230

(ii) TITLE OF INVENTION: PEPTIDES (iii) NUMBER OF SEQUENCES: 6

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Ala Val Ser Glu His Gin Leu Leu His Asn Lys Gly Lys Ser lie Gin

1 5 10 15

Asn Leu Arg Arg Arg Phe Phe Leu His His Leu lie Ala Glu lie His 20 25 30

Thr Ala Glu He Arg Ala 35 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Ala Val Ser Glu His Gin Leu Leu His Asn Lys Gly Lys Ser He Gin 1 5 10 15

Asn Leu Arg Arg Arg Phe Phe Leu His His Leu He Ala Glu He His 20 25 30

Thr Ala

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Ala Val Ser Glu His Gin Leu Leu His Asn Lys Gly Lys Ser He Gin 1 5 10 15

Asn Leu Arg Arg Arg Phe Phe Leu His His Leu He Ala Glu He 20 25 30

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His 20 25 30

Asn Phe Val Ala Leu Gly 35

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His 20 25 30

Asn Phe

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val 20 25 30

Claims

1. A PTH or PTHrP compound 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,

in free form or in salt or complex form.

2. A PTH or PTHrP compound in which the amino acid residue naturally occurring in position 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,

in free form or in salt or complex form.

3. A compound according to claim 1 wherein the amino acid residue bearing an aromatic or heteroaromatic group on its side chain is an amino acid residue wherein the side chain is optionally ring-substituted 3- or 4-pyridyl-methyl, 3-indolyl-methyl or 3-indazolyl-methyl or a radical of formula (a) , (b) , (c) or (d)

(a) (b)

(c) (d) wherein n is 1 or 2, m is 1 or 2, o is 0 or 1, ring A is optionally substituted by one or more substituents selected from fluoro, chloro, nitro, C_halky1 and Cj.-alkoxy, whereby two alkyl or two alkoxy substituents may also form together a ring structure fused to ring A, each of rings B and C independently may be substituted as indicated above for ring A, and

Y_a is a direct bond, -CH₂-, 0, NH or N-C_1-βalkyl.

4. A compound according to claim 1, which is a compound of formula I

R-[(X²)_P, (X³),, (X⁶)_s,R¹⁰]-D-(y-x)R_a I

wherein x is a residue number selected from 31, 34, 35, 36, 37 or

38, y is a residue number selected from 1, 2, 3, 4, 5, 6 or 7, X² is Val or has independently one of the significances of X¹⁰,

X³ is Ser or has independently one of the significances of

X¹⁰, X⁶ is Gin or has independently one of the significances of X¹⁰, R¹⁰ is Asp or X¹⁰, X¹⁰ being Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a) , (b) , (c) or (d)

(a) (b)

(CH₂)_f .©.

wherein n is 1 or 2, m is 1 or 2, o is 0 or 1, ring A is optionally substituted by one or more substituents selected from fluoro, chloro, nitro, C_halky1 and C^alkoxy, whereby two alkyl or two alkoxy substituents may also form together a ring structure fused to ring A, each of rings B and C independently may be substituted as indicated above for ring A, and Y_a is a direct bond, -CH₂-, 0, NH or N-C.-galkyl, D is an amino acid sequence'derived either from an N-terminal fragment of PTHrP or PTH, each of p, q and s is 1, provided that p is 0 when y > 2, q is 0 when y > 3 and s is 0 when y > 6, R is H, R--C0-, R--0-CO-, R"-0-CH₂-C0-, R"-S0₂-, R' '\ R' ' '-NH-CO-, R' ' '-NH-CS or NH₂-C₁,₆alkylene-CO- wherein R" is Ci.jalkyl; ω-carboxy-Ci.galkyl; ω-[ (C₁,₄alkoxy) - carbonyl]-Cj-salkyl; C₂_₈alkenyl; C₅.₇cycloalkyl;

C_3-7cycloalkyl- C^alkyl; or phenyl, phenyl-C₁-₄alkyl, 1-naphthyl, 2-naphthyl, l-naphthyl-C₁.₂alkyl or 2-naphthyl-C₁.₂alkyl each of which being optionally ring substituted by one or more substitutents selected from fluoro, chloro, nitro, C₁,₄alkyl and C₁.₄alkoxy; heteroaryl; and

R' ' ' has indepedently one of the significances given for R" except the significances of ω-carboxy-Ci.jalkyl and ω-[ (C^alkoxy)-carbonyl]-C_halky!; and R„ is OH or NH₂ , with the proviso that at least one of X² and R¹⁰ has the significance of X¹⁰.

5. A compound according to claim 1 or 4 which is derived from the N-terminal fragment of hPTH or hPTHrP.

6. A compound according to claim 1 which is selected from

N*-2-naphthyl-acetyl-[Nal(2)¹⁰,DAla³⁴]hPTHrP(3-34)NH₂ N*-2-naphthyl-acetyl- [Nal (2)¹⁰]hPTHrP(3-31)NH₂ N^β-succinyl-[Phe ,Nal(2)¹⁰]hPTHrP(5-34)NH₂

7. A process for the production of a compound according to claim 1, in free form or in salt or complex form, which process comprises

8. A compound according to claim 1 in free form or in physiologically acceptable salt form for use as a pharmaceutical.

9. A pharmaceutical composition comprising a compound according to claim 1, in free form or in physiologically acceptable salt form, together with a pharmaceutically acceptable diluent or carrier therefor.

10. A compound according to claim 1 in free form or in physiologically acceptable salt form for use as a pharmaceutical, in association with a further therapeutic agent selected from a bone resorption inhibitor and a cytostatic agent.

11. A method for preventing or treating conditions which are associated with increased plasma calcium caused by excessive release of PTH or PTHrP, for preventing or treating tumor growth stimulated by PTHrP, for treating dermatological disorders and for hair growth promotion, in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a compound according to claim 1 in free form or in a physiologically acceptable salt form.