JPH10502091A - PTH or PTHrP antagonist - Google Patents

Abstract

  (57) [Summary] At least one naturally occurring amino acid residue at positions 2 and 10 has been replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain, optionally at positions 3 and 6 At least one naturally occurring amino acid residue at position 1 may be replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain, an effective antagonist for the PTH / PTHrP receptor Active PTH or PTHrP compounds have, for example, pharmacological activity for the prevention and treatment of conditions associated with increased plasma calcium resulting from the release of excess PTH or PTHrP.

Description

DETAILED DESCRIPTION OF THE INVENTION                   PTH or PTHrP antagonist   The present invention relates to parathyroid hormone (PTH) and parathyroid hormone-related peptide ( (PTHrP) compounds, their production, pharmaceutical preparations containing them and their Regarding the use of   More specifically, the present invention provides for the naturally occurring amino acid residues at positions 2 and 10. At least one of which has tryptophan or an aromatic or heteroaromatic group on its side Substituted with other amino acid residues in the chain, optionally at positions 3 and 6. At least one of the naturally occurring amino acid residues may be tryptophan or aromatic. Or another amino acid residue having a heteroaromatic group in its side chain. PT having effective antagonist activity at the PTH / PTHrP receptor H or PTHrP compounds are provided. This compound is hereinafter referred to as the compound of the present invention. .   In a specific embodiment of the invention, the naturally occurring amino acid residue at position 10 is Ptophan or other amino having an aromatic or heteroaromatic group in its side chain An amino acid substituted at the 3- and 6-positions At least one of the acid residues is tryptophan or an aromatic or heteroaromatic group PTH or may be further substituted with another amino acid residue having Provides a PTHrP compound.   The term “PTHrP” refers to a naturally occurring form of PTHrP, eg, human, Means PTHrP, chicken, rat or mouse. In the following description For consistency and convenience, the α-amino acids of the PTHrP sequence have been replaced according to the invention. Or the amino acid residues in the PTHrP sequence, whether or not And apply the same numbering.   The term “PTH” refers to a naturally occurring form of PTH, eg, human, bovine, chick Means bird, rat or mouse PTH. Consistency and For convenience, the α-amino acids of the PTH sequence have been substituted or removed according to the invention. The same numbering applies to amino acid residues of the PTH sequence, To use.   “PTHrP compound” or “PTH compound” refers to PTHrP or Means a peptide containing an amino acid sequence based on the N-terminal fragment of PTH And preferably begins at any of residues 1-7 and at any of residues 27 to 38 Terminating PTHrP or PTH fragment, eg, 31, 34, 35, 36 N-terminal fragment of PTHrP or PTH containing up to 37 or 38 amino acid residues Lagment is the basis. The term "PTHrP or PTW" therefore refers to N- One or more, preferably N-terminal, amino acid residues of the terminal fragment Should be understood to include peptides in which. This term also refers to the invention Position 2 and / or 10 and optionally position 3 and / or 6 In addition, one or more amino acid residues of a naturally occurring sequence may Or a peptide substituted with more amino acid residues (natural or unnatural). Should be understood to include.   The 1-38, 1-34 and 1-31 N-terminal fragments of human PTHrP are It has the sequence suggested in SEQ ID NO: 1, 2 or 3, respectively.   The 1-38, 1-34 and 1-31 N-terminal fragments of human PTH It has the sequence shown in SEQ ID NO: 4, 5 or 6, respectively.   The N-terminus of a PTHrP or PTH compound may be a free or protected amino group, such as See, for example, "Proactive Groups in Organic Synthesis", TW. Greene, J.M. A with an N-protecting group as disclosed in Willy & Sons NY (1981), 219-287. It is a mino group and is caught. The contents of which are incorporated herein by reference. Preferably, The amino group is represented by R "-CO-, R" -O-CO-, R "-O-CH_Two-CO or R "-SO_TwoOr R '", R'"-NH-CO- or Or an amino group having R "'-NH-CS-.   The C-terminal of the compound of the present invention is COOH, CONH_TwoOr mono- or di- Substituted amides, such as -CONR_cR_d(Where R_cAnd R_dOne is H and The other is an aliphatic residue such as C_1-6Alkyl or both aliphatic residues Or R_cAnd R_dTogether with the nitrogen atom to which they are attached To form a heterocyclic residue, e.g., pyrrolidinyl or piperidinyl). Profit You.   The PTHrP or PTH of the present invention is an effective amino acid for PTH / PTHrP receptor. A functional organism that has a agonist activity, eg, binds to a PTH / PTHrP receptor, In the assay, the intrinsic activity (i.a.) for activation of the PTH / PTHrP receptor, significantly < 1, for example, i.a. of up to 0.3, indicating PTHrP or PTH or their Fragments such as PTHrP (1-34) or PTH (1-34) Antagonize with a pA2 value of at least 6.5 in the physical assay. Preferably a book Compounds of the invention may be 0.03 or lower, or detectable in some assays. Have i.a. Examples of functional bioassays are commonly used in the art Osteosarcoma basic adenylate cyclase assay. This assay is based on the compound to be tested. As described below by Marcus and Aurbach, Endocrinology, 85, 801-810 (1969). According to the method of (1), the rat osteosarcoma cells of the UMR strain, In which adenylate cyclase activity is stimulated, or PTHrP or To the extent that it antagonizes the action of PTH or their fragments, Provides in vitro measurements.   Amino acids are naturally occurring or commercially available or unnatural amino acids Or their optical isomers. Unnatural amino acids, under the direction of mRNA, Amino acids not included in proteins, such as β-Nal and fluoroalanine Fluoro-α-amino acid, cyclohexylalanine or trimethylsilyl-a Lanin.   "Natural amino acids" means those known in the art. They are U.S.P.T.O. Publication, Treadmark Official Gazette, published May 15, 1990, 46 p.33. And standard abbreviations are listed. These amino acids and abbreviations are specifically Specifically incorporated herein by reference. The natural amino acids are listed below:   An amino acid having an aromatic or heteroaromatic group in its side chain has a side chain, for example, Optionally substituted phenyl-methyl, 1- or 2-naphthyl Ru-methyl, 1- or 2-naphthyl-ethyl, 3- or 4-pyridyl-methyl Amino acid residue which is 3-indolyl-methyl or 3-indazolyl-methyl And preferably has the formula -NH-CHR'-CO- as defined below. Amino acid residue.   According to a preferred embodiment of the present invention,     R-[(X^Two)_p, (X^Three)_q, (X)⁶ _s, R^Ten] -D- (yx) R_a              I (In the formula, x is the number of residues selected from 31, 34, 35, 36, 37 or 38; y is the number of residues selected from 1, 2, 3, 4, 5, 6 or 7; X^TwoIs Val or independently X^TenHaving one of the meanings of X^ThreeIs Ser or independently X^TenHaving one of the meanings of X⁶Is Gln or independently X^TenHaving one of the meanings of R^TenIs Asp or X^TenAnd X^TenIs Trp or -NH-CHR'-CO- (Wherein R ′ is a group represented by the formula (a), (b), (c) or (d) here, n is 1 or 2, m is 1 or 2, o is 0 or 1, Ring A is optionally fluoro, chloro, nitro, C_1-4Alkyl and C_1-4A Substituted with one or more substituents selected from the group consisting of And the two alkyl or two alkoxy substituents are fused to ring A to form a ring structure together. Form, Rings B and C can each be independently substituted with those suggested for ring A above. And Y_aIs a direct bond, -CH_Two-, O, NH or N-C_1-6Alkyl) D is the amino acid sequence from PTHrP or the N-terminal fragment of PTH, p , Q and s are each 1 except that when y> 2, p is 0 and y> 3 If q is 0 and y> 6, s is 0; R is H, R "-CO-, R" -O-CO-, R "-O-CH_Two-CO-, R "-S O_Two, R "', R"'-NH-CO-, R "'-NH-CS- or NH_Two-C_1-6Al Kirene-CO- (where R "is C_1-8Alkyl; ω-carboxy-C_1-6Alkyl ; Ω-[(C_1-4Alkoxy) -carbonyl] -C_1-6Alkyl; C_2-8Alkenyl; C_5-7Cycloalkyl; C_5-7Cycloalkyl-C_1-4Alkyl; or each If desired, fluoro, chloro, nitro, C_1-4Alkyl and C_1-4Alkoxy May be ring-substituted with one or more substituents selected from the group consisting of Phenyl, phenyl-C_1-4Alkyl, 1-naphthyl, 2-naphthyl, 1-na Futyl-C_1-2Alkyl or 2-naphthyl-C_1-2Alkyl; heteroaryl; R "'is independently ω-carboxy-C_1-6Alkyl and ω-[(C_1-4Alkoki B) -carbonyl] -C_1-6Other than alkyl, the same meaning as defined for R ″); And R_aIs OH or NH_TwoIn Although, However, X^TwoAnd R^TenAt least one of X^TenHas the same meaning as Is provided.   When the substituents of ring A, B or C together form a ring structure, for example, -O-CH_Two -CH_Two—O—. Examples of polycyclic cycloalkyl include, for example, It is damantill.   Heteroaryl as R "is at least one nitrogen atom and optionally further It has a heteroatom such as N, O or S and is optionally fused to a benzene ring. 5-, 6- or 7-membered unsaturated heterocyclic group which may be substituted. Heteroally Is preferably indolyl, quinolyl or isoquinolyl.   The compounds of the present invention can exist, for example, in free form, in its salt form or in complex form. acid Addition salts may be formed with, for example, organic, polymeric and inorganic acids. With such acid Salted forms include, for example, hydrochlorides and acetates. The complex is, for example, a PT of the present invention. From HrP or PTH, no inorganic substances such as Ca- and Zn-salts Formed by the addition of organic salts or hydroxides and / or the addition of polymerized organic substances Is done.   In the compounds of formula I, the following meanings may be used individually or in any combination Preferred in a combination or sub-combination: 1. D is the N-terminal fragment of hPTHrP. 2. D is the N-terminal fragment of hPTH. 3. X^TenIs Trp. 4. X^TenIs -NH-CHR'-CO- (where R 'is a group represented by the formula (a), (b) or (c), Preferably (a) or (c), and more preferably a group represented by (a)). 5. X^TwoIs Trp or -NH-CHR'-CO- (where R 'is a group represented by the formula (a), (b) or Is a group represented by (c)). 6. X^TwoAnd X^TenIs Trp or -NH-CHR'-CO- (where R ' Is a group represented by formula (a), (b) or (c)). 7. X^ThreeAnd X^TenAre each Trp or -NH-CHR'-CO- (where R 'Is a group represented by the formula (a), (b) or (c)). 8. X⁶And X^TenAre each Trp or -NH-CHR'-CO- (where R ′ is a group represented by formula (a), (b) or (c)). 9. X^Two, X⁶And X^TenIs Trp or -NH-CHR'-CO- (here And R ′ is a group represented by formula (a), (b) or (c)). 10. n is 1. 11. the number of residues in which y is selected from 3, 4, 5, 6 or 7, preferably 3 or 5 . 12. x is the number of residues selected from 31 or 34. 13. R is R "CO-, R" -SO_Two, R "'or H_TwoN-C_1-6Alkylene-CO . 14． R "is C_1-8Alkyl, phenyl C_1-4Alkyl, 1- or 2-naphthyl , 1- or 2-naphthyl-C_1-2Alkyl. 15. R is ω-carboxy-C_1-6Alkyl.   As already mentioned, one or more of the positions other than positions 2 and / or 10 The above amino acid residues are further substituted with the above-mentioned natural or unnatural amino acid residues. Can be removed or removed. When the compound of the present invention is an hPTHrP derivative Further amino acid substitutions, for example at position 3, for example at Ala, at position 4, for example For example, at position Trp, at position 5, for example, optionally substituted Phe, 11 At position and / or position 13, for example Leu, or at position 34, for example D-Ala; the amino acid sequence can optionally include from 1 to 7 amino acids; The shortening at the N-terminus due to the removal of the acid residue. The compound of the present invention is hPTH Ah If the amino acid residue is, for example, from positions 8, 16, 18, 33 and 34, At least one position selected, for example, Leu⁸, Ala¹⁶, Gln¹⁸, Thr³³ , Ala³⁴Or their D-isomers.   The present invention also provides a method for producing the PTHrP or PTH compound of the present invention. . They can be in solution or using solid phase synthesis steps or genetic engineering, or It can be produced in a stepwise manner by a combination of these methods.   The compounds of the present invention are, for example, as follows: a) PTHrP or PTH compounds of the invention in protected form, for example of the formula I Removal of at least one protecting group present on the compound; or b) amide coupling of two peptide fragments in protected, partially protected or unprotected form Where the peptide fragment is the desired PTHrP or PTH compounds, such as those from which the amino acid sequence of a compound of formula I is obtained And then optionally performing step a) of the process; or c) the N-terminus of the desired sequence in protected or unprotected form or the N-terminal fragment thereof A protecting group or substituent is added to the amino group of the terminal residue in a selective manner, and then Performing step a). And the PTHrP thus obtained in free form, in its salt form or in complex form, or Recovery of PTH compounds Can be produced.   Reaction steps a), b) and c) can be carried out, for example, by methods known in the field of peptide chemistry. Similarly, or as described in the examples below. These reactions, if desired In the above, a protecting group suitable for use in a peptide is used as a functional group unrelated to the reaction. Can be used. The term protecting group may also include functionalized polymer resins.   When the compounds of the present invention contain non-natural and natural residues, they will be chemically Can be produced by a combination of methods and genetic engineering; Peptide sequence produced from amino acid residues (complete sequence or fragment ) Can be produced by recombinant techniques and produce the desired unnatural amino acid or terminal amino acid substitution. Substituents can be inserted by chemical synthesis and, if necessary, fragments can be combined. , If protecting group (s) are present, they can be removed.   In all the examples below, the temperature is in ° C., the following abbreviations are used: DMF = dimethylformamide DMA = N, N-dimethylacetamido-6-sulfonyl Pmc = 2,2,5,7,8-pentamethylchroman tBu = tert.butyl PyBOP = benzotriazol-1-yl-oxy-tripyrrolidino-phospho Ium hexafluorophosphate Nal (2) = L-3- (2-naphthyl) -alanine DIEA = N, N-diisopropyl-N-ethylamine Fmoc = 9-fluorenylmethoxycarbonyl RT = room temperature MS = M measured by electron spray (elecrospray) spectroscopy⁺ Example 1 [Trp^4'10] hPTHrP (1-34) OH   The peptide is synthesized in a step-wise manner on a polystyrene-based resin support. Al The far-amino group is protected with Fmoc and the side-chain functionality is protected as follows: Asp (O tBu), Glu (OtBu), His (Try), Lys (Boc), Asn (Trt), Gln (Trt), Ar g (Pmc), Ser (tBu), Trp (Boc) and Thr (tBu).   Deprotection, washing and conjugation of the alpha-amino group (i.e. Peptide step) consisting of repeated cycles of Fmoc-Ala-oxymethyl-4-phenoxyme Use about 0.5 mmol / g of chill-co (polystyrene-1% -divinyl-benzene) . The N-alpha Fmoc group is cleaved using 20% piperidine in DMA. Join In the reaction, 4 equivalents of Fmoc-amino acid and PyBOP-reagent and 8 equivalents Of DIEA in DMA is used per amino group. Complete assembly of peptide chains After completion, the terminal Fmoc-protecting group is removed with piperidine in DMA as described above. You. The peptide is cleaved from the resin support and all side chain protecting groups are removed with 5% dodecyl in TFA. Using a reagent consisting of methyl sulfite and 3% water for 2 hours at RT Than Remove at the same time. The resin particles are collected by filtration, washed with an appropriate amount of TFA, and 10 to 20 volumes. The product is precipitated from the combined filtrates by adding an amount of diethyl ether. Wash with ether and dry. The product was chromatographed on a C-18 wide pore silica column. Purify by chromatography using a gradient of acetonitrile in 2% aqueous phosphoric acid . The fraction containing the pure compound was collected and anion exchange resin (Biorad, AG4-X4 (Acetate form) and lyophilized to give the title compound. MS: 4146   As in Example 1, but using suitable amino acids, the following compounds were prepared: obtain:Example 2: [Trp^Ten, Leu¹¹, Leu¹³] hPTHrP (1-34) OH MS: 4059Example 3 [Trp^Ten, Leu¹¹] hPTHrP (1-34) OH MS: 4073Example 4: [Trp^3'10] hPTHrP (1-34) OH MS: 4188Example 5: [Trp^2'10] hPTHrP (1-34) OH MS: 4176Example 6: [Trp^Ten] hPTHrP (2-34) OH MS: 4018.3Example 7: [Trp^Ten] hPTHrP (3-34) OH MS: 3919.2Example 8: [Trp^Two] hPTHrP (1-34) OH MS: 4104.9Example 9: [Trp^Ten] hPTH (1-34) OH MS: 4189Example 10: [Leu⁸, Trp^Ten, Ala¹⁶, Gln¹⁸, Thr³³, Ala³⁴] hPTH (1-34) OH                                                       MS: 4036Example 11: [Leu⁸, Trp^Ten, Ala¹⁶, Gln¹⁸, Thr³³, Ala³⁴] hPTH (2-34) OH                                                       MS: 3949Example 12: [Leu⁸, Trp^Ten, DLeu¹¹, Gln¹⁸, Thr³³, Ala³⁴] hPTH (1-34) OH                                                       MS: 4079Example 13: [Phe^Two, Trp^Ten] hPTHrP (1-34) -OH MS: 4137.1Example 14: [Trp^Two, Trp^Ten] hPTHrP (2-34) -OH MS: 4105.1Example 15: [Trp⁶, Trp^Ten] hPTHrP (1-34) -OH MS: 4146.8Example 16: [Trp⁶, Trp^Ten] hPTHrP (6-34) -OH MS: 3623.3Example 17: [Trp^Ten] hPTHrP (7-34) -OH MS: 3437.6Example 18: [Trp^Three, Trp^Ten] hPTHrP (3-34) -OH MS: 4018.0Example 19: [Trp^Ten] hPTHrP (1-34) OH MS: 4088.7Example 20: [Trp⁶, Trp^Ten] hPTHrP (3-34) NH_Two       MS: 3966.8Example 21: [Phe⁶, Trp^Ten] hPTHrP (1-34) OH MS: 3546.1Example 22: [Phe^Two, Phe⁶, Trp^Ten] hPTHrP (1-34) OH MS: 4155.1Example 23: N^α-3-aminopropionyl- [Phe⁶, Nal (2)^Ten] hPTHrP (4 -34) NH_Two                                         MS: 3932.0Example 24: N^α-Benzyloxycarbonyl [Nal (2)^Ten] hPTHrP (3-23)- NH_Two   This peptide is described, for example, in Tetrah. Letters, 28: 3787-3790 (1987). 4- (2 ', 4'-dimethoxyphenyl-Fmoc-amino-meth Tyl) -phenoxy-co (polystyrene-1% -divinylbenzene) about 0.4 mmol / Prepared in a similar manner to Example 1 using g as starting material. At the end of the synthesis In the cycle, Z-Ser-OH was added to the peptide chain and Peptide cleavage and purification. MS: 3995.3Example 25: N^α-Acetyl- [Trp^Ten] hPTHrP (3-34) OH   This peptide is used in the same manner as in Example 1. At the end of the synthesis, Fmoc- Final cycle of deprotection and acetylation using 1: 1 mixture of acetic acid and DMF Apply The peptide is cleaved from the resin and purified as described above. MS: 3961.1   The following compounds can be prepared in an analogous manner to Examples 1, 24 or 25.Example 26: N^α-Acetyl- [Trp^Ten] hPTHrP (2-34) OH                                                       MS: 4060.4Example 27: 1-Isocaproyl- [Leu⁸, Trp^Ten, Gln¹⁸, Thr³³, Ala³⁴] hPT HrP (1-34) OH MS: 4091Example 28: N^α-2-Naphthyl-acetyl- [Nal (2)^Ten] hPTHrP (3-34) N H_Two                                                   MS: 4097.1Example 29: N^α-2-Naphthyl-acetyl- [Nal (2)^Ten] hPTHrP (4-34) N H_Two                                                   MS: 4009.7Example 30: N^α-2-Naphthyl-sulfol- [Nal (2)^Ten] hPTHrP (3-34) N H_Two                                                   MS: 419.2Example 31: N^α-2-Naphthoxyacetyl- [Nal (2)^Ten] hPTHrP (3-34) N H_Two                                                   MS: 411.5Example 32: N^α-2-Naphthylacetyl- [Ala^Three, Nal (2)^Ten] hPTHrP (3-3 4) NH_Two                                               MS: 4080.8Example 33: N^α-Benzyloxycarbonyl- [Trp^Two, Nal (2)^Ten] hPTHrP (2-34) NH_Two                                           MS: 4248.3Example 34: 3-Naphth-2-yl-propionyl- [Nal (2)^Ten] hPTHrP (3 -34) NH_Two                                             MS: 410.8Example 35: 3-Naphth-2-yl-propionyl- [Nal (2)^Ten] hPTHrP (3 -34) NH_Two                                             MS: 410.9Example 36: N^α-2-Naphthyl-acetyl- [Nal (2)^Ten, DAla³⁴] hPTHr P (3-34) NH_Two                                         MS: 4095.9Example 37: N^α-2-Naphthyl-acetyl- [Nal (2)^Ten] hPTHrP (3-31) N H_Two                                                   MS: 3788.0Example 38: N^α-3-Naphthyl-2-propionyl- [Nal (2)^Ten] hPTHrP (7-34) NH_Two                                           MS: 3630.0Example 39: N^α-Acetyl- [Phe⁶, Nal (2)^Ten] hPTHrP (6-34) NH_Two                                                       MS: 3637.0Example 40: N^α-Acetyl- [Phe⁶, Nal (2)^Ten] hPTHrP (4-34) NH_Two                                                       MS: 3903.1Example 41: 1- (1-amino-1-cyclopentyl-carbonyl)-[Leu⁸, Tr p^Ten, Gln¹⁸, Thr³³, Ala³⁴] hPTH (1-34) OH MS: 4104   Fmoc-1-aminocyclopen used in production of the present peptide resin intermediate Tan-1-carboxylic acid is described, for example, in G.S. Valle et al., 1988, Can. J. Chem . 66: 2575-2582.Example 42: 1-adamantyl-carbonyl- [Leu⁸, Trp^Ten, Gln¹⁸, Thr³³, A la³⁴] hPTH (1-34) OH MS: 4155Example 43: 1- (3-Indolyl-carbonyl)-[Leu⁸, Trp^Ten, Gln¹⁸, Thr^{Three Three} , Ala³⁴] hPTH (1-34) OH MS: 4150Example 44: 1- (3-quinolyl-carbonyl)-[Leu⁸, Trp^Ten, Ala¹⁶, Gln¹⁸, Thr³³, Ala³⁴] hPTH (1-34) OH MS: 4104Example 45: 1- (2-Naphthoyl)-[Leu⁸, Trp^Ten, Gln¹⁸, Thr³³, Ala³⁴] hP TH (1-34) OH MS: 4147Example 46: N^α-(2-naphthyl) -methyl- [Trp^Ten] hPTHrP (2-34) -OH   N-Naphthoyl (2) -valinate methyl was prepared using Low Wesson's reagent, SO. Law esson et al., Tetrahetdon 37: 3635 (1981). The oil (2-) valinate methyl was converted to FS. Guziec et al., Thetrah. Letter s 23-26 (1990) using the method described for endothiopeptides. You. The methyl ester is hydrolyzed using LiOH to give N-alpha- (naphthyl (2) -Methyl) -valine hydrochloride is obtained as a crystalline solid, mp = 205-210 ° ( Disassembly). This was previously prepared and protected using PyBOP-reagent, from which Removal of the N-alpha Fmoc- group, [Trp^Ten] hTPHrP- (3-34) -O-pepti Bond with resin. The peptide is cleaved from the resin and purified as in Example 1. MS: 4158.2Example 47: N^α-2-naphthyl-2-yl-ethyl- [Trp^Ten] hPTHrP (2-3 4) OH MS: 4171.9.Example 48: N^α-2-naphthyl-2-yl-ethyl- [Ala^Three, Nal (2)^Ten] hP THrP (3-34) OH MS: 4066.7Example 49: N^α-Succinyl- [Phe⁶, Nal (2)^Ten] hPTHrP (5-34) NH_Two   The present peptide is described, for example, in Tetrah. Letters, 23: 3787-3790 (1987) 4- (2 ', 4'-dimethoxyphenyl-Fmoc-amino-methyl)- 0.63 g of phenoxy-co (polystyrene-1% -divinylbenzene), about 0.4 Prepared in a similar manner to Example 1 using mmol / g charge, starting material. Last After coupling of the amino acid, Fmoc-His (Trt) -OH, the Fmoc-group is selectively selected as usual. And the peptide resin was washed with succinic anhydride (0.5 g) and DIPEA (DMP) in DMF. 0.86 ml). The peptide was cleaved from the resin as in Example 1 and purified. Lyophilize in acetate form to give the title compound. MS: 3832.0   The following compounds can be prepared according to the method of Example 49:Example 50: N^α-Succinyl- [Phe⁶, Nal (2)^Ten] hPTHrP (5-31) NH_Two                                                       MS: 3522.1Example 51: N^α-Succinyl- [Nal (2)⁶, Nal (2)^Ten] hPTHrP (5-34) NH_Two                                                      MS: 3881.7Example 52: N^α-Glutaryl- [Phe⁶, Nal (2)^Ten] hPTHrP (5-34) NH_Two                                                       MS: 3845.6Example 53: N^α-Succinyl- [Phe⁶, Nal (2)^Ten, DAla³⁴] hPTHrP (5-34 ) NH_Two                                                MS: 3832.0Example 54: N^α-Succinyl- [4-Cl-Phe⁶, Nal (2)^Ten] hPTHrP (5-34 ) NH_Two                                                MS: 3866.5Example 55: N^α-Succinyl- [4-Cl-Phe^Five, 4-Cl-Phe⁶, Nal (2)^Ten] hPTHrP (5-34) NH_Two                                 MS: 3910.2   The compounds of the present invention in free form or in pharmaceutically acceptable salts and complexes, may be used in animal tests. It exhibits valuable pharmacological properties as suggested by experimentation, and thus indicates a therapy.   The biological activity of the compounds of the present invention is determined by the method of Marcus and Aurbach, Endocrinology. , 85, 801-810 (1969), according to the method of UMR106-06 rat osteosarcoma cells. Promotion (agonism) or inhibition (antagonism) of PTH-stimulated synthesis of click AMP In vitro by measuring the ability of Rat osteosarcoma UMR106 fine Cells were plated in a 12-well plate in DMEM-HAM F12 medium (1: 1) -10% Grow to confluence in FCS. Then, the medium was changed to a medium containing 2% FCS. And 1-5 μCi / well [^ThreeH] -Adenine is added. After 2 hours, the cells are Washed once with serum-free medium, DM containing 1 mM 3-isobutyl-1-methylxanthine Incubate in EM-0.1% BSA to remove phosphodiesterase activity. Leave. Test substances are added 20 minutes later, alone or with PTH (antagonist experiment). Add. After 15 minutes, the medium was removed to stop the reaction, and cAMP was added to ice-cold 5% trichloride. Extraction is performed by adding 0.5 ml of acetic acid. Unlabeled adder for recovery measurements Nin, adenosine, AMP, ADP, ATP and cAMP and 0.4 μCi [^{1 Four} C] -Adenosine containing carrier solution (0.5 ml / well) is added. [^ThreeH] -cAMP To continuous Dowex AG 50W-X4 (200-400 mesh) and alumina chromatography And Salmon Y., Advances in Cyclic Nucleotide Resarc. h, Vol. 10. Count according to Raven Press, 35-55, 1979. The results are %, EC₅₀The value is determined from the DRC curve. PT antagonist effect Calculated from the rightward shift of the DRC curve of HrP or PTH, pA_TwoAs a value obtain. The compound of the present invention comprises 10^-9From 10^-FiveActive as an antagonist at M concentration Sex. The compounds of Examples 36, 37 and 49 were expressed in UMR106-06 cells PA of 10.3, 9.7 and 9.3 respectively_TwoHas a value.   The compounds of the present invention also have a binding affinity for the PTH receptor, for example, Have:   Chicken [Tyr³⁶] PTHrP (1-36) amide was converted to lactoperoxidase method (Ana wa Lab. AG, Wangen) to a specific activity of 2,200 Ci / mmol. Be converted. Opossum kidney cells (OK1) were prepared using DMEM and HA containing 1% BSA. Wash with 200 μl of M F12 (1: 1), [¹²⁵I-Tyr³⁶] chPTHrP (1-36) [Tyr with 50,000 cpm / well³⁶] chPTHrP (1-36) amide Incubate in the presence or absence of 1 μM. After incubation, The cells are washed with 0.5 ml of medium (4 ° C.), lysed with 0.5 ml of 1N NaOH, Measure gender. Specific binding is defined as total binding-non-specific binding. Competition curve Nonlinear regression computer program SCTFIT (Feyen et al., 1992, Biochem. Biophys. Res. Commun. 187: 8-13) and the data were averaged pK_Dvalue( Indicated as n = 2 to 3). The compounds of Examples 36, 37 and 49 were PK of 8.3, 7.9 and 8.4_DHas a value.   In addition, the compounds of the invention have been measured, for example, in thyroid parathyroidectomized rats. Antagonize the effects of PTH after i.v. Thyroid parathyroidectomy Twenty-four hours later, PTH (1-34) and the compound to be tested are added to the anesthetized rat with another cervical static. Inject via the pulse. Urine from the cannulated bladder via the abdominal approach to recover. Phosphate and cAMP in urine and calcium in serum and The content of phosphate is measured using standard methods. Set these parameters to Used to quantify the antagonist effect on the PTH effect ex vivo. Book In the test, the compounds of the invention are dosed from 1 μg / kg / hour to 1 mg / kg / hour. Antagonize the PTH effect when administered by i.v. Example 49 When i.v. was injected at 190 μg / kg / hour, the compound of PTH (1-34) was 4 μg / kg / hour. Injected i.v. at kg / hr, completely inhibits PTH-induced phosphoruria up to 90 minutes I do.   The compounds of the present invention may therefore cause excessive release of PTH or PTHrP. Any condition associated with increased plasma calcium, e.g. metastatic cancer, e.g. bone metastasis For breast cancer with or without breast cancer, squamous cell carcinoma of the lung, cancer of the esophagus, head, neck and blood Useful for the prevention or treatment of associated hyperparathyroidism, hypercalcemia, for example It is. The compounds of the present invention can be used to promote PTHrP-promoted cancer growth and penetration into bone. Prevention or treatment of erosion, treatment of skin diseases, such as tissue repair therapy, e.g., fever It is further useful for treating wounds, ulcers and wounds and for promoting hair growth.   For these indications, the appropriate dose will, of course, be eg the host, the route of administration And depends on the severity of the condition to be treated. However, in general, animals Sufficient results have been obtained with a daily dose of about 0.1 to about 100 mg / kg of animal body weight. You. In large mammals, e.g., humans, the suggested daily doses may be greater than the compound of the present invention. It ranges from about 0.1 to about 500 mg.   The compounds of the present invention may be administered in free form or in a pharmaceutically acceptable salt form or complex. obtain. Such salts and complexes can be prepared by conventional methods and have an activity comparable to that of the free compound. Is shown. The present invention also provides a free base form together with a pharmaceutically acceptable diluent or carrier. Or a pharmaceutical composition comprising a compound of the invention in pharmaceutically acceptable salt or complex form I will provide a. Such compositions may be formulated in a conventional manner. The unit dosage form is preferably Provides about 0.025 to 250 mg of a compound of the present invention to a pharmaceutically acceptable diluent. Or with a carrier.   The compounds of the present invention may be administered by conventional routes, eg, in the form of injectable solutions or suspensions. It may be administered in the form of a nose or suppository, for example parenterally. Alternatively, of the present invention The compound may be in the form of a cream, gel, etc. Alternatively, it can be administered to treat hair growth.   In accordance with the foregoing, the present invention further provides: a) a compound of the present invention or a pharmaceutically acceptable salt thereof for use as a medicament; Or complex; b) a patient-effective amount of a compound of the present invention or a pharmaceutically acceptable salt or complex thereof. Prevention of the above conditions and diseases in a patient in need of treatment, including administering the body Or treatment; c) A compound of the invention for use in the manufacture of a pharmaceutical composition for use in b) above. Or a pharmaceutically acceptable salt or complex thereof; I will provide a.   According to a further aspect of the present invention, the compounds of the present invention may be used for other treatments, e.g. Treatment with bone resorption inhibitors, especially calcitonin or similar Analogs or derivatives such as salmon, eel or human calcitonin, biphosphona In the treatment with a chemotherapy, a diuretic or a combination thereof, or in the treatment of cancer In the case of a cytostatic or adjuvant or adjuvant of a combination thereof Can be used as   In accordance with the foregoing, the present invention provides yet another aspect: d) a patient effective amount of a) a compound of the invention and b) a second drug substance (a second drug substance). Is a therapeutic agent as described above). For example, for preventing or treating the specific condition or disease described above. Prevention or treatment of calcemia I will provide a.   Compounds of Examples 36, 37 and 49 show excess migration of PTH or PTHrP For the prevention or treatment of all conditions associated with increased plasma calcium resulting from separation preferable.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI A61K 38/26 ADT 9051-4C A61K 37/28 ADU ADU 9051-4C ADA (81) Designated countries EP (AT, BE) , CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML) , MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG , MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TT, UA, US (72) Inventor Gombert, Frank Otto Germany Day-79588 Efringen-Kirchen, Am Rebhank 20

Claims (1)

[Claims]   At least one of the naturally occurring amino acid residues at positions 1.2 and 10 is Ptophan or other amino having an aromatic or heteroaromatic group in its side chain An amino acid substituted at the 3- and 6-positions At least one of the acid residues is tryptophan or an aromatic or heteroaromatic group In its free form, which may be further substituted with another amino acid residue having Or PTH or PTHrP compounds in salt or complex form.   2. If the naturally occurring amino acid residue at position 10 is tryptophan or aromatic Or another amino acid residue having a heteroaromatic group in its side chain, If desired, at least one of the naturally occurring amino acid residues at positions 3 and 6 may be Leptophane or other amino acids having aromatic or heteroaromatic groups in their side chains PTH in free form or in salt or complex form, which may be further substituted with a noic acid residue Or a PTHrP compound.   3. An amino acid residue having an aromatic or heteroaromatic group in its side chain is 3- or 4-pyridyl-methyl wherein the chain is optionally ring-substituted, 3- Indolyl-methyl or 3-indazolyl-methyl or formulas (a), (b), ( c) or (d) (In the formula, n is 1 or 2, m is 1 or 2, o is 0 or 1, Ring A is optionally fluoro, chloro, nitro, C_1-4Alkyl and C_1-4A Substituted with one or more substituents selected from the group consisting of Two alkyl or two alkoxy substituents may be taken together to form ring A May form a fused ring structure, Rings B and C may each be independently substituted with those described above for ring A. Is good, and Y_aIs a direct bond, -CH_Two-, O, NH or N-C_1-6Alkyl) The compound according to claim 1, which is an amino acid residue represented by the formula:   4. Formula I     R-[(X^Two)_p, (X^Three)_q, (X)⁶ _s, R^Ten] -D- (yx) R_a              I (In the formula, x is the number of residues selected from 31, 34, 35, 36, 37 or 38; y is the number of residues selected from 1, 2, 3, 4, 5, 6 or 7; X^TwoIs Val or independently X^TenHaving one of the meanings of X^ThreeIs Ser or independently X^TenHaving one of the meanings of X⁶Is Gln or independently X^TenHaving one of the meanings of R^TenIs Asp or X^TenAnd X^TenIs Trp or -NH-CHR'-CO- (Wherein R ′ is a group represented by the formula (a), (b), (c) or (d) here, n is 1 or 2, m is 1 or 2, o is 0 or 1, Ring A is optionally fluoro, chloro, nitro, C_1-4Alkyl and C_1-4A Substituted with one or more substituents selected from the group consisting of Or two alkyl or two alkoxy substituents taken together into ring A May form a fused ring structure, Rings B and C are each independently substituted with those described above for ring A. Is also good, and Y_aIs a direct bond, -CH_Two-, O, NH or N-C_1-6Is alkyl) D is the amino acid sequence from PTHrP or the N-terminal fragment of PTH, p , Q and S are each 1, except that when y> 2, p is 0 and y> 3. If q is 0 and y> 6, S is 0; R is H, R "-CO-, R" -O-CO-, R "-O-CH_Two-CO-, R "-S O_Two, R "', R"'-NH-CO-, R "'-NH-CS- or NH_Two-C_1-6Al Kirene-CO- (where R "is C_1-8Alkyl; ω-carboxy-C_1-6Alkyl ; Ω-[(C_1-4Alkoxy) -carbonyl] -C_1-6Alkyl; C_2-8Alkenyl; C_5-7Cycloalkyl; C_5-7Cycloalkyl-C_1-4Alkyl; or each If desired, fluoro, chloro, nitro, C_1-4Alkyl and C_1-4Alkoxy May be ring-substituted with one or more substituents selected from the group consisting of Phenyl, phenyl-C_1-4Alkyl, 1-naphthyl, 2-naphthyl, 1-na Futyl-C_1-2Alkyl or 2-naphthyl-C_1-2Alkyl; heteroaryl; R "'is independently ω-carboxy-C_1-6Alkyl and ω-[(C_1-4Alkoki B) -carbonyl] -C_1-6Other than alkyl, the same meaning as defined for R ″); And R_aIs OH or NH_TwoIn Although, However, X^TwoAnd R^TenAt least one of X^TenHas the same meaning as The compound according to claim 1, which is a compound represented by the formula:   5. derived from the N-terminal fragment of hPTH or hPTHrP, The compound according to claim 1.   6. N^α-2-Naphthyl-acetyl- [Nal (2)^Ten, DAla³⁴] hPTHrP (3-34 ) NH_Two, N^α-2-Naphthyl-acetyl- [Nal (2)^Ten] hPTHrP (3-31) NH_Two , N^α-Succinyl- [Phe⁶, Nal (2)^Ten] hPTHrP (5-34) NH_TwoA group consisting of The compound according to claim 1, which is selected from:   7. a) PTHrP or PTH compounds of the invention in protected form, such as those of the formula I Removal of at least one protecting group present in the compound being performed; or b) amide coupling of two peptide fragments in protected, partially protected or unprotected form Where the peptide fragment is the desired PTHrP or PTH compounds, such as those from which the amino acid sequence of a compound of formula I is obtained And then optionally performing step a) of the process; or c) the N-terminus of the desired sequence in protected or unprotected form or the N-terminal fragment thereof A protecting group or substituent is added to the amino group of the terminal residue in a selective manner, and then Performing step a). And the PTHrP thus obtained in free form, in its salt form or in complex form, or Recovery of PTH compounds The method for producing a compound according to claim 1, which is in a free form, a salt or a complex form, comprising:   8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof for use as a medicament. Functional salt or complex.   9. A compound or compound according to claim 1 together with a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition comprising a pharmaceutically acceptable salt or complex thereof.   10. Further cure selected from the group consisting of bone resorption inhibitors and cytostatics The compound according to claim 1 or a pharmaceutically acceptable compound thereof for use as a medicament together with a therapeutic drug. Acceptable salts or complexes.   11. An effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof; Comprises administering to the patient a PTH or PTH of the patient in need of treatment, including administering the complex to the patient. Prevention of conditions associated with increased plasma calcium resulting from excessive release of THrP Or treatment, prevention or treatment of cancer growth promoted by PTHrP, Methods of treatment and hair growth promotion.