CA2192787A1

CA2192787A1 - Pth or pthrp antagonists

Info

Publication number: CA2192787A1
Application number: CA002192787A
Authority: CA
Inventors: Francois Cardinaux; Jean Honore M. Feyen; Rainer Gamse; Frank Otto Gombert
Original assignee: Individual
Current assignee: Novartis AG
Priority date: 1994-07-28
Filing date: 1995-07-27
Publication date: 1996-02-08
Also published as: WO1996003437A1; SK12097A3; IL114736A0; CO4410206A1; PL318017A1; NO970356D0; FI970168A; BR9508433A; FI970168A0; CZ23397A3; NO970356L; MX9700446A; AU3167095A; HUT77979A; EP0773958A1; JPH10502091A; TR199500915A2

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

~ W0 9610343~ 2 1 9 2 7 8 7 PCrlEP95/02993 PTH or PTHrP antagonists The present invention relates to parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) ~ , a process for their production, rh~rr~r~llt;cal preparations comprising them and their use as a rh~rr-~eut;r~1 10 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 ~placed by tryptophane or another amino acid residue bearing an aromatic or het~LuaL~ t;C
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 n aromatic or het~LuaLl ;c group on its side chain. The are hereinafter referred to as compounds of the invention.

In a particular '-'' ' of the invention there is provided a PTH
or PTHrP compound in which the amino acid residue naturally ocr~rr;n~ in position 10 is replaced bY tryptophane or another amino acid residue bearing an aromatic or het~LuaL, t;c group on its side chain, and optionally at least one of the amino acid ~ 25 residues naturally occurring in pocit;rnc 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.r~ human, bovine, chicken, rat or mouse PTHrP. For consistency and as is conventional, in the foll~ ng description, the same numbering system will be applied to the amino acid residues of the ~ PTHrP sequence regardless of whether any ~-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 PTX. For consistency and as is conventional, in the following description, the same numbering system will be applied to the amino 2cid resiaues of the PTX

_ _ _ _ _ _ , .,, . _ . _ . . ... , ... , . . . .. , , . _ _ _ _ _, _ _ W096/03437 2 1 9 2 7 8 7 r~llL~ . ?993 O

sequence reg~rdless of whether aay ~-a~ino ~cid residue of the F~H
sequence is replaced or omitted according to the invention.~

sy "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 tPnmin~;nr, 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 o~ 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 ~ re amino acid residues of the naturally orcnrr;nr~ seguence 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 Arcnr~;ng to the invention.
The 1-38, 1-34 and 1-31 N-terminal fr~,~ ' c of human PTHrP have the seguences as ;n~;ratefl in SEQ ID No:l, 2 or 3, respectively.

The 1-38, 1-34 and 1-31 N-terminal f _ c of human PTH have the sequences as indicated in SEQ ID No:4, 5 or 6, respectively.

The N-terminus o~ the P~HrP or PTH ~ ' may be a free or a protected amino group, bearing e.g. a N-protecting group as ~;crlosp~ in ~Protective Groups in Organic Synthesis-, T.W. Greene, ~ J. Wiley & Sons NY (1981), 219-287, the contents of which being 25 herein incorporated by Lef_Le-ce, preferably protected by Rn-CO-, Rn-O-CO-, R'-O-CH2-CO- or Rn-SO2, or an amino group bearing a radical R''', R'''-NH-CO- or R'''-NH-CS- such as defined heLe~-deL.

The C-terminus of the _ ' of the invention may be COOH, CONH2 or a ~ no- or disubstituted amide, e.g. -CONRCRd wherein one of Rc and Rd is H and the other is an ~l;ph~t;r residue, e.g. Cl6alkyl, or both are an aliphatic residue, or Rc and R~ together with the nitrogen atom to which they are attached form a heterocyclic residue, e.g. pyrrolidinyl or piperidinyl.
PTHrP or PTH _ '~ in a~L~o~ e with the invention have potent ~t~gr~n;ctiC activity at the PTH/PTHrP receptor e.g. bind to the PTH/PTHrP receptor, have an ;ntr;ncir activity ~i.a) for activation of the PTH/PTHrP receptor in a flmr~inn~l bioassay c;rJn;fir~n~ly <l, e.g. an i.a of at most 0.3, and antagonize PTHrP or PTH or a ~ W096l03437 2 1 9 2 7 8 7 r~ ,J

fragment thereof e.g. PTHrP(1-34) or PTH(1-34) in a fllnrtjnnAl bioassay with a pA2 value of at least 6.5. Preferably compounds in r~AnrP with the invention have an i.a of 0.03 or lower or even not ~t~rt~hl e in some of the assays. Example of a flmrt;nnnl 5 bioassay is the osteosarcoma-based adenylate cyclase assay employed conventionally in the art. This assay provides an in vitro ~tPrm;n~t;on of the extent to which the compound to be tested St; lAtPc adenylate cyclase activity or AntAgnn;7~q the effect of PTHrP nr P~h nr a fragment thereof in rat osteosarcoma cells of the UMR lineage, e.g. UMR-106-06 according to the method of Marcus and Aurbach irl ~n~nrr;nnlory, ~, 801-810 (1969) as disclosed hereinafter.

By amino acid is meant a naturally occurring or co~mercially available or non natural amino acid or an optical isomer thereof.
15 A non natural amino acid is an amino acid which is not incorporated into a protein under mRNA direction, e.g. ~-Nal, a fluoro-a-amino acid such as fluoroalanine, cyclohexylalanine or trimethylsilyl-alanine.
~Natural amino acids" refer to those well ~nown in the art. They are listed and standard abbreviations are pr~-.ided in the U.S.P.T.O. publication, Tr~' Offiri~l G.7ette, pllhl;chp~
May 15, 1990, p. 33 at 46. These amino acids and a~breviations are spPr;f;,rA11y incorporated herein by reference.
The natural amino acids are shown below:

~ 25 A Ala alanine D Asp aspartic acid E Glu glutamic acid F Phe phenylalanine G Gly glycine H His histidine I Ile icolP~r;nP
R Lys lysine L Leu leucine M Met h;nn;nP
N Asn asparagine Q Gln g~ nP
R Arg argirine S Ser serine T Thr threonine V Val valine W096/03437 2 1 9 2 7 8 7 PCT~P9S/02993 w Trp try,otopha~e 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 : ' ' of the invention, there is provided a compound of formula I
R_[(x2~pl(x3)i~(x6).~Rlo]-D-(y-x)R.

wherein x is a residue number selected from 31, 34, 35, 36, 37 or 38, y i6 a residue number selected from 1, 2, 3, 4, 5, 6 or 7, X' is Val or has ;nf~ "tly one of the sign;~i c~nr~c of Xl~, X3 is Ser or has in~_L.~..rl_.nly one of the significances of Xl~, X~ is Gln or has ;n~ ly one of the significances of Xl~, Rl~ is Asp or X~~, Xl~ being Trp or -~H-CHR'-CO- wherein R~ is a radical of formula (a), (b), (c) or (d) -(CHz)n -(CH~) (a) -(CH2) j;~-(CHz)m~ Y~~ (CH2)o~>

(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 096/03437 ,~llr~ 93 selected from fluoro, chloro, nitro, C1,alkyl and Cl,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 ;n~pPn~Pntly may be substituted as ;n~;ratP~ aoove for ring A, and Y. is a direct bond, -CHz-, o, NH or N-C16alkyl, D is an amino acid sequence derived ~rom an N-terminal fragment of PTHrP or PTH, each of p, q and s is 1, provided that p is O when y > 2, ~ is O when y > 3 and s is O when y > 6, R is H, R--CO-, R"-O-CO-, R"-O-CH,-CO-, R--SO2-, R''', R'''-~H-CO-, R'''-NH-CS- or NH,-Cl6alkylene-CO-wherein R' is Cl8alkyl; c7-carboxy-Cl6alkyl; c-[(C1~alkoxy)-carbonyl]-Cl6alkyl; C2ealkenyl; Cs7cycloalkyl; Cs7cycloalkyl-C1,alkyl; or phenyl, phenyl-Cl~alkyl, l-naphthyl, 2-naphthyl, l-naphthyl-Cl~alkyl or 2-naphthyl-C12alkyl each of which being optionally ring substituted by one or ~ re substitutents selected from fluoro, chloro, nitro, C1~alkyl and Cl~alkoxy; heteroaryl; and R''' has in,l.L,~ "l1y one of the c;gn;f;r~n~Pc given for R" except the 5j~n;f;rAnrPc of_~-carboxy-Cl6alkyl and ~-[(C1,alkoxy)-carbonyl]-C16alkyl; and R, is OH or NH"
with the proviso that at least one of XZ and R10 has the 5;gn~f;cAnre of Xl~.

_ When the substitutents of ring A, B or C form together a ring ~LLU~LUL~, it may be e.g. -O-CH,-CH2-O-. ~xample of polycyclic cycloalkyl is e.g. adamantyl.

By heteroaryl as R- is meant a 5-, 6- or ~ nc~tllr~
heterocyclic radical comprising at least one nitrogen atom and optionally further a heteroatom such as N, O or 5, and optionAlly ~ 1 with a ~enzene ring. Heteroaryl is preferably indolyl, quinolyl or ;cnquinnlyl.

The ~ _ ' 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 ;nnr~n;r 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 W096/03437 2 1 9 2 7 ~ 7 PCT~P95/02993 ~

sub6tance6, e.g. inorganic 6altc or hydroxides such as Ca- and Zn-salts, and/or an addition of polymeric organic substances.

In the compounds of formula I, the following si~n;fi~An~c are preferred either individually or in any ~'n~tion 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. Xl~ 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' i6 a radical of formula (a), (b) or (c), 6. Each of X~ and XlD is Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a), (b) or (c).

7. Each of X3 and X30 is Trp or -NH-CHR'-CO- wherein R' is a radical of formula (a), (b) or (c).

8. Each of x6 and X10 is Trp or -NH-CHR'-CO- wherein R' is a _ radical of formula (a), (b) or ~c).

9. Each of X2, xc 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 ~.

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

13. R is RRCO-, Rn-SO2, R''' or H2N-Cl6alkylene-CO-.

14. R" is Clnalkyl, phenylC~alkyl, 1- or 2-naphthyl, l- or 2-naphthyl-Cl2alkyl.

W096/03437 ~1 9 2 7 ~ 7 PCT~P9Sl02993 15. R is ~-carboxy-Gltalkyl.

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 ;n~;cAtP~ above or be omitted.
When the I ' of the invention are hPTHrP derivatives, they may comprise further amino acid rPpl A~ ', 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 l up to 7 amino acid residues. When the ~ ds 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.
Leu8, Ala3t, Gln3B, Thr'3, Ala3' or D-isomers thereof.

The present invention also provides a process for the production of the PTHrP or PTH I ' 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 : ''nAt;on of these methods.

The - _ ' 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 25 b) linking together by an amide bond two peptide L _ tc in protected, partially protected or unprotected form, the peptide fl _ ~ being such that the amino acid sequence of the desired PTHrP or PTH compound, e.g. of formula I, is ~htA;nP~, 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 ~), W096/03437 ~l q 2787 ~ ?~93 o and recovering the PTHrP or PTH compounds thus obtained in free form, in salt form or in compleY 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 rlrcrr;hr~ in the following eY~mples. Where desired, in these reac-tions, protecting groups which are suitable for use in peptides may be used for f~ctional groups which do not participate in the reaction. The term protecting group may also include a polymer resin having functional groups.

When the - ulld~ of the invention comprise ~mnr'rllrAl and natural residues, they may be produced by a combination of a chemical stepwise process and genetic engineering; the peptide se~uence (whether the complete seruence or a fragment) made of genetically encodable amino acid residues may be produced by L~ ~nAnt tr~rhr;rl~lrC and the desired unnatural amino acids or terminal amino substituent may be ;~ re~l by chemical synthesis and, where reruired, the LL _ ~C may be combined and the protecting group(s) when present may be removed.

In the following ~Yamples all t~ _ ~LULeS are in ~C. The following abbreviations are employed.

D~F = dimethylformamide DNh = N,N-dimethylAcet~ rl~-6-sulfonyl _ Pmc = 2,2,5,7,8-pentamethylchroman tBu = tert.butyl 25 PyBOP = Benzotriazol-l-yl-oxy-tripyrrolidino-rhr)srhrn i ~ry~ f l u" ~ te Nal(2) = L-3-(2-naphthyl)-alanine DIEA = N,N-diisopropyl-N-ethylamine Fmoc = 9-fluorenylmethoYycarbonyl 30 RT = _ room temperature MS = M~ rlr-t~rm;n~-~ by el~LLu~L~y spectroscopy [Trp~l~jhPTHrP(1-34) OH

The peptide is synthesized in a stepwise manner on a polystyrene based resin support. The alpha- mino 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), ~ WO 96/03437 2 1 9 2 7 8 7 r~ ~ n?993 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 5 repetitive cycles of alpha-amino group deprotection, washing, coupling (i.e., AttA~' t 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 10 e~;ivalents of DIBA in ~A are used per amino-group. After complete assembly of the peptide chain the terminal Fmoc-protecting group is removed with piperidine in 1~ as before. The peptide is cleaved from the resin support and all side-chain protecting groups are simultanously removed by using a reagent 15 consisting of 5P6 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 20 ~hL~ tog~aphy on a C-18 wide-pore silica column using a gradient of acetonitrile in 2# a~aueous phosphoric acid. Fractions rrntA;n;ng the pure compound are collected, filtered through an anion-exchange resin (Biorad, AG4-X4 acetate form) and ly~lrh; l; 7ecl to give the title compound.

25 MS: 4146 In analogy with the p~,.ellu~e of Bxample 1, but using the appropriate amino-acids the frl 11 ~; nq _ '~ may be prepared:

2: [Trp10 Leu11, Leu13]hPTHrP(1-34)OH MS: 4059 ~Y~ PT.-r 3 [Trpl~, Leu11]hPTEIrP(1-34)0H MS: 4073 30 ~Y~T.w 4 [Trp3 1~]hPTHrP(1-34)OH MS: 4188 ll.Y?''PT.W 5 [Trp2 l~]hPTHrP(1-34)0H MS: 4176 .w 6 [Trplo]hpTHrp(2-34)oH MS: 4018.3 .w 7 [Trplo]hpTHrp(3-34)oH MS: 3919.2 rY- .- ~ [Trp2]hPTHrP(1-34)OH MS: 4104.9 35 ~Y~ .W 9 [Trpl~]hPTH(1-34)0H MS: 4189 ' ~ 10: [I.eua Trp10 Ala16,Gln18,Thr33,Ala3~]hPTH(1-34)OH
MS: 4036 W096/03437 2 1 92787 P~ "~ o Leua Trp10,Al216,Gln1B,Thr33,Ala3]hPTH(2-34)OH
MS: 3949 - ,F 1~: [Leu8,Trpl~~3~Leull~Glnla~Thr33~Ala~]hPTH(1-34)0H
~S: 4079 5 ~~~~ ~ 1~: [Phe3,Trpl~hPTHrP~1-34)-OH MS: 4137.1 ~r.~J.F 14 [Trp',Trpl~]hPTHrP(2-34)-OH MS: 4105.1 : [Trp6,Trpl~]hPTHrP(1-34)-OH MS: 4146.8 16: [Trp6,Trpl~]hPTHrP(6-34)-OH MS: 3623.3 ~MrT.F. 17 [Trpl~]hPTHrP(7-34)-OH MS: 3437.6 0 FY~ -F 18: [Trp3,Trpl~]hPTHrP(3-34)-OH MS: 4018.0 : [Trpl~]hPTHrP(1-34)0H MS: 4088.7 20: [Trp6, Trpl~]hPTHrP~3-34)NH2 MS: 3976.8 .~ 21: [Phe5, Trpl~]hPTHrP(1-34)0H MS: 3546.1 .~ 22: [Phe2, Phe6, Trpl~]hPTHrP(1-34)0H MS: 4155.1 EY~'~T.l~ 7'~: N'-3-ami~lopropionyl-[Phe6, Nal~2)l~]hPTHrP(4-34)NH2 MS: 3932.0 .: N -benzylu--y~L~vL,yl[Nal(2)l~]hPTHrP(3-34)-NE~2 This peptide is prepared in a manner analogous to Example 1 but using 4-(2',4'-dimethoxyphenyl-Fmoc-amino-methyl)-phenoxy-CO(PO1Y~LYL~1C 1~-divirylbenzene), approx. 0.4 mmol/g, which may - be prepared, e.g., as ~c~r;hD~ in Tetrah. Letters, 28:3787-3790 (1987) as a st~rting materi~l. In the last synthesis cycle Z-Ser-OH is added to the peptide chain and the peptide cleaved and purified as in Example 1.
25 MS: 3975.3 ~o~-~ 25: N -acetyl-[Trpl~]hPTHrP~3-34)0H

This peptide is assembled in analogy with the pLuce~uL~ of Example 1. At the end of the sy~nthesis a final cycle of Fmoc-deprotection and acetylation using a 1:1 mixture of ~cetic ~nhydride and DMF is applied. The peptide is cleaved from the resin and purified as ~efore.
MS: 3961.1 W096l03437 .~ 7993 The f~ ;n~ ~ may be prepared in a manner analogous to that of Examples 1, 24 or 25.

~Y~ ,v 26 N'-acetyl-[Trpl~]hPTHrP[2-34)0H
MS: 4060.4 5 ~Y~PL~ 27: 1-Isocaproyl-[Leus,Trpl0,Glnl3,Thr3l,Ala3']-hPTH(1-34)OH
~S: 4091 .~ 28: N'-2-naphthyl-acetyl-[Nal(2)1~]hPTHrP(3-34)YH2 MS: 4097.1 10 ~yr ~-~ 2~: N -2-naphthyl-acetyl-[Nal(2)1~]hPTHrP(4-34)NH2 MS: 4009.7 rY- .~ 30: y~-2-naphthyl-sulfonyl-[Nal(2)lo]hpTHrp(3-34)NH2 M~: 4119.2 .~ 31: Y'-2-naphthoxyacetyl-LNal~2)1~]hPTHrP(3-34)NH2 MS: 4111.5 7: y~-2-naphthylccetyl-t~lal,Nal(2)1~]hPTHrP(3-34)yH2 MS: 4080.8 ~ ~ ~ 33:~ N'-benzylu~y~cLL~yl-[Trp2~Nal(2)lo]hpTHrp(2-34)NH2 _ MS: 4248.3 20 ~Y~Y 3~: 3-Naphth-2-yl-proprionyl-~Nal(2)l~]hPTHrP(3-34)NH2 MS: 4110.8 35: 3-Naphth-2-yl-propionyl-[Nal(2)l~]hPTHrP(3-34)NH2 MS: 4110,9 -~L~ 36: y~-2-naphthyl-acetyl-[Nal(2)lo~D~la3']hpTHrp(3-34)NH2 ~S: 4095.9 37: N'-2-naphthyl-acetyl-[Nal(2)l~]hPTHrP(3-31)yH2 MS: 3788.0 38: N~-3-naphth-2-yl-propionyl-[Nal(2)l~]hPTHrP(7-34)yH2 MS: 3630.0 Wo 96/03437 2 1 9 2 7 ~ 7 r~ S~ o 39: N--acetyl-[Phe6,Nal(2)l~]hPT~rP(6-34)NH2 I~S: 3637.0 .~PT.l- 40: N--acetyl-[Phe6,Nal(2)l~]hPTHrP(4-34)NH, NS: 3903.1 5 ~Y~ L~ 41: 1- (l-amino-1-cyclopentyl-carbonyl)-[Leus Trpl~ GlIlls~Thr33~Ala34]hpTH~l-34)oH
NS: 4104 Fmoc-1-aminocyr1 op~nt~n~-1-carbo~ylic acid used irL the prepara-tion of the peptide resin ;nt~ ~Ate may be prepared, e.g. as 10 A-.crr;h~A by G. Valle et al.,1988, in Can.J.Chem.66:2575-2582.

E 1~ ~7: 1-Adamantyl-carbonyl-[Leu3,Trp10,Gln18,Thr33, Ala3']hPTH(1-34)0H
NS: 4155 r---- DL~ 3-indolyl-carbonyl)-[Leu~,Trp10,Gln18,Thr33, Ala3']hPTH(1-34)OH
MS: 4150 : l-(3-,r,~uinolyl-carbonyl-~[Leub~Trplo~Alal6 Gln18, Thr33,Ala3']hPTH(1-34)OH
NS: 4104 - 20 ~ 1-(2-naphthoyl)-[Leus,Trpl0,Glnl8,Thr33,Ala3']-hPTH(1-34)0H
NS: 4147 - 1- 46: N--(2-naphthyl)-methyl-[Trpl~]hPTHrP(2-34)-OH

Methyl N-naphthoyl(2)-valinate is treated with Lawesson s 25 reagent, S-O. Lawesson et al., Tetrahedron 37:3635 (1981) and the resulting methyl N-th;nn~rhthnyl(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 hydrorhlnr;
30 obtained as a crystalline solid, mp = 205-210~ (dec.). This is coupled usirg PyBOP-reagent to previously prepared, protected [Trpl~]hPTHrP-(3-34)-0-peptide resin from which the N-alpha F~noc-group has beerL removed. The peptide is cleaved from the resin and : ~

~ 9~87 W096l03437 ~ l ?~93 purified as in Example 1.

MS: 4158.2 rY~ .~ 47: N~-2-naphth-2-yl-ethyI-[Trplo]hpTHrp(2-34)oH
MS: 4171.9 ~Yr~or.~ 4B: N~-2-naphth-2-yl-ethyl-[Ala3,Nal(2)l~]hPTHrP(3-34)NH2 NS: 4066.7 r~Y~ 49: N'-succinyl-[Phes,Nal(2)1~]hPTHrP(5-34)NH2 This peptide is prepared in a manner analogous to Example l but using 4-(2',4'-dimethoxyphenyl-Fmoc-amino-methyl)-phenoxy-co(polystyrene-l~-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 (0.86m~) in DNF. The peptide is cleaved from the resin, purified and lyoph;l;7e~ in the acetate form as in Example 1 to give the title compound.

NS: 3832.0 By foll, n~ the procedure of Example 49, following _ ' may _ 20 be prepared:

r--~T.ll! 505 N'-succinyl-[Phe6,Nal(2)l~]hPTHrP(5-31)NH~
NS: 3522.1 ~L~ 51: N'-succinyl-[Nal(2) 6, Nal(2)1~]hPTHrP(5-34)NH2 NS: 3881.7 25 ~ 52: N -glutaryl-[Phe6/Nal~2)~~]hPTHrP(5-34)NH2 NS: 3845.6 53: N -succinyl-[Phe6,Nal(2)1~,DAla3']hPTHrP(5-34)NH2 NS: 3832.0 ~~~ S4: N--succinyl-[4-Cl-Phe6,Nal(2)1~]hPTHrP(5-34)NH2 30 NS: 3866.5 - ~q~7~7 W096/03437 P~ 93 O

SS: N~-succinyl-~4-Cl-Phe5, 4-Cl-Phef, Nal~2)1~~-hPTHrP(5-34)NH2 MS: 3910.2 The ~ LLLds of the invention in free form or in the form of p~Arr--P~t;cally acceptable salts and complexes exhibit valuable rh~nr-~nlogical properties as indicated in animal tests and are therefore ;n~l ~A tP~ for therapy.

The biological activity of the - _ ' of the invention is assessed in vitro by measuring their ability of st; lat;n~
~agonism) or in~;hit;ng the PTX-sti lAtP~ (antagonism) synthesis of cyclic AMP in UNR 106-06 rat osteoscLL. cells A~c~r~in7 to the method of ~arcus and Aurbach in FnA~r;n~l~gy, ~, 801-810 (1969). Rat osteo~ 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 uCi/well [3H]-adenine is added. Two hours later, cells ~re washed twice with serum-free medium and ;n~hlted in DM2M - 0,1~ PSA
c~ntA;n;ng 1 mM 3-isobutyl-l-methylYAnt~;np to exclude actions on phospho- diester~ses. Test substances are added 20 min later either ~lone or together with PTX (AntAgrln;ct experiment). After 15 min, the medium is removed and the reaction is stopped and cAMP eYtr~cted by adding 0.5 ml ice cold 5% trichloroacetic acid.
A carrier solution (O.S ml/well~ c~ntA;n;n7 0.2 mM of unlabelled adenine, ~Pn~S;nP~ AMP, ADP, ATP, and cAMP as well as 0.4 ,uCi _ 25 [14C]-~Pn~c;nP for detPnm;nAt;on of recovery is added. [3H]-cAMP
is sepArated using serial Dowex AC 50W-X4 (200-400 mesh) and Alumina ~ILLI tography 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 30 EC50 values flPtPrm;nP~ from DRC curves. Antagonist potency is r~l C~l AtPrl from the right ward shift of DRC curves of PTXrP or PTH and is given as pA2 values. r , ' of the invention are active as antagonists at a c~LLc~LLLLcLtion of 10-' to 10-5 M. Com-pound of Examples 36, 37 and 49 have a PA2 value in the UMR
35 106-06 cells of 10.3; 9.7 and 9.3, respectively. r The ' of the invention also have binding affinity to PTH
receptors, e.g. as follows:

~ W096~03437 2 1 927 87 P~ 7993 Chicken [Tyr35]PTHrP~1-36)amide is iodinated to a specific activity of 2,200 Ci/mmol using the 1~ntn-pprn~;~qe method ~Anawa Lab. AG, Wangen). Monolayers of opossum kidney cells (OKl) are washed with 200 ul DMEM and HAM's F12 (1:1) cnnt~;n;ng 1~ BSA and incubated at 16~C with 50.000cpm of [325I-Tyr36]chPTHrP(1-36)amide per well in the presence or absence of 1 uM [Tyr36]chPTHrP-(1-36)amide. After ;nrllh~t;nn, cells are washed with 0.5 ml medium (4~C), lysed with 0.5 ml lN NaOH and radioactivity is detPrm;nP~ Specific binding is defined as total binding minus 10 nnncper; f; n 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 PXD values (n=2 to 3). C ~-ds of Examples 36, 37 and 49 have a pKD value of 8.3; 7.9 and 8.4, respectively.

Furthermore, the c _ ' of the invention antagonize the effect of PTH after i.v. infusion, e.g. as determined in thyropara-thyro;~rt~ '7P~ rats. 24 h after thyroparathyrn;~Prt~ ~, anesthetized rats are infused with PTH(1-34) and the compound to be tested via separate jugular veins. Urine is nollertP~ from the urinary bladder which ;c nAnn-llAtPd via the ventral approach.
Phosphate and cANP content in the urine and calcium and phosphate in serum are measured using standard hn~nlon~y. These ~aL ~rs are used to guantify nntnrnn;ct potencies against PTH
effects in vivo. In this test, the ~ _ ' of the invention ~nt~nJnn;7e the PTH effects when administered by i.v. infusion at _ a dose of from 1 ,ug/kg/h to 1 mg/kg/h. Compound of Example 49 completely suppresses PTH-induced phnsrh~tllria for up to 90 min when i.v. infused at 190 ug/kg/h, PTH(1-34) being i.v. infused at 4 ug/kg/h.

The _ -c of the invention are accordingly useful for preventing or treating all cnn~;t;nnc which are associated with increased plasma calcium caused by excessive release of PTH or PTHrP e.g. hyperparathyroidism, hypercalcemia, e.g. AcsOr;~
with r-1;n,n~nrjPc, e.g. breast carcinomas, squamous cell carcinomas of the lung, esophagus and head and neck region and hematological r-1;n~n~nr;e5, with or without bone metastases. The ~ o~n~c of the invention are furthermore useful for the prevention or treatment of tumour growth, tumour penetration and ingrowth in bones st; 1~ted by PTHrP, for treating dermatological disorders, e.g. tissue repair therapies, for W096/03437 2 ~ 9 2 7 ~ 7 r~l~ ?~93 O

example treatment of burns, ulcerations and wounds, and for hAir growth promotion.

For these lndications, the appropriate dosage will, of course, vary ~Pp~n~;nr upon, for example, the host, the mode of adminis-tration ano the severity of the conditions being treated. Howe-ver, in general, sat;c~rtnry results in anlmals are ;n~;rAt~A to be obtained at daily dosages from about 0.1 to about 100 mg/kg animal body weight. In larger mammals, for ex_mple humans, an ;n~;rAt~A daily dosage is in the range from about 0.1 to about 500 mg of the ~ ds of the ineention.

The comFounds of the invention may be administered in free form or in phArr~-eutically 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 ~ ' . The lS present invention also provides a i' -r~S;cal comrosition comprising a compound of the invention in free base form or in rhl ~eut;cally acceptable salt form or comFlex form in associa-tion with a phArr--e~ltirAlly acceptable diluent or carriOE. Such compositions may be formulated in conventional manner. ~nit dosage forms suitably comprise from about 0.025 to 250 mg of a compound of the invention, together with a phArr--eut;
acceptable diluent or carrier therefor.

The _ ' of the invention may be administered by any _ conventional route, for example parenterally e.g. in form of injectable 5nlnt;nnc or s~C~nC;nnc, or in a nasal or a sup-pository form. The ~ ' 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 ~s hereinbefore described.

In accordance with the foregoing the present invention further provides:

a) a compound of the invention or a ~ ;r~lly acceptable salt or complex thereof for use as a phArr--~lt;rAl;

b) a method for preventing or treating cnnA;t;nnC and disorders ~s ;nA;r~trA above in a subject in need of such treatment, which method comprises administering to sAid subject an ~ W096/03437 21 q27 ~7 r~ 093 effective amount of a compound of the invention or a ph~rr~~eutically acceptable salt or complex thereof;

c) a compound of the invention or a ph~rr-~fut;cally 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 : -'' t of the invention, the ~
of the invention may be employed as adjunct or adiuvant 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 h;ph~srh~n~te, a diuretic or any com'oination thereof, or in case of tumour therapy, a cytostatic agent or any combination thereof.

In A~ f~- 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 ; n~ t, ed above.
-f __ ~ of Examples 36, 37 and 49 ~re ~ef~lLed forpreventing or treating all conditions which are associated with incre~se plasma calcium caused by excessive release of PTH or PTHrP.

WO 96103437 2 1 9 2 7 ~ 7 F~ r.~ q93 0 SEQUENCE LISTING

~1) GENERAL INFORMATION:
(i) APP_ICANT:
A NAME: SANDOZ LTD.
E STFEET: Lichtstrasse 35, C CITY: BASLE
E COUNTRY: SWITZERLAND
~F POSTAL CODE (Z}P): CH-Ç002 Al NAME: SANDOZ PATENT GM3H
E STREET: ~ l~tqtrAqqe 3 C CITY: LOERRACH
E COUNTRY: GERMANY
~F POSTAL CODE (ZIP): D-79539 ~A) NAME: SANDOZ ~r~'l~WN~N VERWAL.~ scruA~T
N.R.H.
(E) STREET: Erunner Stresse S9 (C) CITY: VIENNA
(E) COUNTRY: AUSTRIA
(F) POSTAL CODE (ZIP): A-123û
(ii) TITLE OF INVENTION: PEPTIDES
(iii) NUh~3ER OF SEQUENCES: 6 (iv) COM'UTE~ READABLE FORM:
Al MEDIUM TYPE: Floppy disk 3 COMPUTER: IHY PC c t;hl~
'CI OP~RATING SYSTEM: PC-DOS/NS-DOS
D SO-TWARE: PatentIn ReleAse #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE r~R~RlcTTr~
A LENGTH: 38 amino acids E TYPE: emino ecid C S~RANnF~n~cc single D TOPOLOGY: ~nXnown (ii) MOLECULE TYPE: protein ~ (iii) ~Yr~~ eL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE ~e~lSIl~N: SEQ ID NO: 1:
Al~ Val Ser Glu His Gl~ Leu Leu His Asn Lys Gly Lys Ser Ile Gln Asn Leu Arg Arg Ar~ Phe Phe Leu His His Leu Ile Ala Glu Ile His Thr Ala Glu Ile Arg Ala W 096/03437 r~l/r~ 7993 (2) INFOR~ATION FOR SEQ ID NO: 2:
(i) SEQUENCE ~A~ A~
A LENGTH: 34 amino acids B TYPE: amino acid C 5~RA~.nN~.cq single :D TOPOLQGY- unknown ~ (ii) MOLECULE TYPE: protein (iii) hY~ln~l'l~: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE ~a~nl~ll~N: SEQ ID NO: 2:
Ala Val Ser Glu His Gln Leu Leu His Asn Lys Gly Lys Ser Ile Gln Asn Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His Thr Ala (2) lN~ ~TO~ FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 31 ~mino ~cids B) TYPE: ~Tino ~cid C) CTRD'~PnN~.q.q: single ID) TOPOLOGY: unknown (ii) NOLECULE TYPE: protein (iii) h-YPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-termin~l -(xi) SEQUENCE Jr~nl~llUN: SEQ ID NO: 3:
Ala Val Ser Glu His Gln Leu Leu His Asn Lys Gly Lys Ser Ile Gln Asn Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE ~DRAI,-~
A) LENGTH: 38 aAlino acids B) TYPE: ~mino -cid C) S~R~RnNE.C.q: single D) TOPOLOGY: ~nknown (ii) MOLECULE TYPE: protein W 096/03~37 2 1 9 2 7 8 7 PCT~EP95/02993 o (iii) ~Y~w~ ~AL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminAl (xi) SEQUENCE ~A~ SEQ ID NO: 4:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Ar~ VA1 Glu Trp Leu Arq Lys Lys Leu Gln Asp VA1 His Asn Phe Val Ala Leu Gly (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE ru~ T~RTcTIcs:
A LENGTH: 34 amino ~cids B TYPE: amino ~cid C S~ nT~sc: single ID TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) ~Yr~ll~ll AL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-termin~l -(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 . 15 Ser Met Glu Ar~ Val Glu Trp Leu Ar~ Dys Lys Leu Gln Asp V21 His Asn Phe (2) INFORXATION FOR SEQ ID NO: 6:
(i) SEQUENCE ~U~ sllc~:
IA) LENGTH: 31 amino ~ids l3) TYPE: amino ~cid C) 5T~ rn~CC sin~le ~D) TOPOLOGY: unXnown ~ii) MOLECULE TYPE: protein (iii) PYPOTUETICAL: NO
(iii) ANTI-SENSF: NO

(v) FRAGMENT TYPE: N-terminAl Wl>96103437 2 ~ 9 2 7 8 7 21 p~ I~r~ ~J~
(xi) SEQUENCE lJ~ lUN: SEQ ID NO: 6:
Ser V~l Ser Glu Ile Gln Leu ~et His Asn Leu Gly Lys His Leu Asn Ser Yet Glu Ar~ Val Glu Trp Leu Ar~ Lys Lys Leu Gln Asp Val -

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) 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 C1-4alkoxy, 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~ is a direct bond, -CH2-, O, NH or N-C1-6alkyl.

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

R-[(X2)p, (X3)q, (X6)s, R10] -D-(y-x)R~ 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 is Ser or has independently one of the significances of X6 is Gln 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) 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 C1-4alkoxy, 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, is a direct bond, -CH2-, O, NH or N-C1-6alkyl, 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 O when y > 2, q is O when y > 3 and s is O when y > 6, R is H, R"-CO-, R"-O-CO-, R"-O-CH2-CO-, R"-SO2-, R''', R'''-NH-CO-, R'''-NH-CS or NH2-C1-6alkylene-CO-wherein R" is C1-~alkyl; .omega.-carboxy-C1-6alkyl; .omega.-[(C1-4alkoxy)-carbonyl]-C1-6alkyl; C2-~alkenyl; C5-7cycloalkyl;
C5-7cycloalkyl- C1-4alkyl; or phenyl, phenyl-C1-4alkyl, 1-naphthyl, 2-naphthyl, 1-naphthyl-C1-2alkyl or 2-naphthyl-C1-2alkyl each of which being optionally ring substituted by one or more substituents selected from fluoro, chloro, nitro, C1-4alkyl and C1-4alkoxy; heteroaryl;
and R''' has independently one of the significances given for R" except the significances of .omega.-carboxy-C1-6alkyl and .omega.-[(C1-4alkoxy)-carbonyl]-C1-6alkyl; 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~-succinyl-[Phe6,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 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.

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.