Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a series of PTH and PTHrP analogues that selectively bind to PTH2 receptors and as such may be useful in treating abnormal CNS functions; abnormal pancreatic functions; divergence from normal mineral metabolism and homeostasis; male infertility; regulation of abnormal blood pressure; and hypothalmic disease, to name a few potential uses.
• PTH2 receptor An alternate human parathyroid hormone (PTH) receptor, designated as PTH2 receptor, has been identified in rat and human brain. This receptor is selectively activated by PTH- (1-34) , but not PTH-related protein PTHrP- (1-34) , which has the same calcium-mobilizing activities as PTH- (1-34) . Both PTH and PTHrP share a common G protein- coupled receptor, termed the PTH/PTHrP receptor. The PTH2 receptor is localized predominantly in the brain and pancreas, in contrast to PTH/PTHrP receptor, which is primarily localized in bone and the kidney, the principal target tissue for PTH action. Parathyroid hormone (PTH) is the principal physiological regulator of calcium levels in the blood (Chorev, M.
• PTH-related protein was originally identified as the agent responsible for the paraneoplastic syndrome of humoral hypercalcemia of malignancy (Suva, L.J., et al . , 1987, Science, 237:893-896 and Orloff , J.J., et al . , 1994, Endocrinol . Rev. 15:40-60).
• PTH and PTHrP are products of distinct, yet evolutionary-related genes.
• PTH and PTHrP show sequence similarities only in the N-terminal 13 amino acids, 8 of which are identical (Abou-Samra AB, et al . ,
• PTH has a highly restricted pattern of expression and acts as a classical endocrine hormone
• PTHrP is expressed in a wide variety of normal tissues and functions in a predominantly autocrine/paracrine fashion (Urena, P., et al . , 1993, Endocrinology, 133:617-623; Lee, K. , et al . , 1995, Endocrinology, 136:453-463; and Martin, T.J., et al . , 1995, Miner. Electrolyte Metab., 21:123-128). More recently, PTHrP has been shown to play a fundamental role in embryonic differentiation of bone and cartilage development .
• PTH and PTHrP exert their wide-ranging effects via a common receptor located on the surface of target cells (Juppner, H., et al . , 1988, J. Biol . Chem. , 263:1071-1078; Shigeno, C, et al . , 1988, J. Biol. Chem., 263:18369- 18377) .
• the PTH/PTHrP receptor is a member of a subfamily of G protein-coupled receptor superfamily, which includes the receptors for glucagon, growth hormone-releasing hormone (GHRH) , vasoactive intestinal peptide (VIP) , glucagon-like peptide 1 (GLP-1) , gastric inhibitory polypeptide (GIP) , secretin, pituitary adenylate cyclase- activating polypeptide (PACAP) , calcitonin, and corticotropin-releasing factor (CRF) (Segre, G. , et al . ,
• the PTH/PTHrP receptor recognizes the N-terminal 1-34 regions of both ligands (Schipani, E., et al . , 1993, Endocrinology, 132:2157-2165) and is particularly abundant in classical PTH target tissues such as bone and kidney (Urena, P., et al . , 1993 Endocrinology, 133:35-38) .
• Ligand binding to the PTH/PTHrP receptor can activate at least two signaling pathways; the adenylyl cyclase-cAMP-protein kinase A pathway (Partridge, NC, et al .
• PTH2 receptor An homologous receptor for PTH, designated the PTH2 receptor, has been identified and partially characterized (Behar, V., et al . , 1996, Endocrinology, 137:2748-2757; Gardella, T.J., et al . , 1996, The J. Biol. Chem., 271:19888-19893; Behar, V., et al . , 1996, Endocrinology, 137:4217-4224; and Usdin, T.B., et al . , 1997, Endocrinology, 138:831-834).
• the PTH2 receptor is most similar in sequence to the PTH/PTHrP receptor (51% of the amino acid sequence identify) .
• PTH2 receptor mRNA is not detected in bone or osteosarcoma cell lines, but is expressed in a number of tissues including the exocrine pancreas, lung, heart, vasculature, and epididymis, and is most abundant in the brain (Usdin, T.B., et al . , 1996, Endocrinology, 137:4285- 4297) .
• PTH/PTHrP receptor which binds and is activated by both PTH- (1-34) and PTHrP- (1-34)
• PTH2 receptor binds and is activated only by PTH- (1-34) .
• PTHrP (7-34) was found to recognize PTH2 receptor and weakly activate it.
• His 5 in PTHrP was identified as the "specificity switch" for the PTH2 receptor. Swapping a single amino acid, His 5 from PTHrP, with lie 5 from PTH, resulted in a PTHrP analogue, Ile 5 -PTHrP- (1-34) NH 2 , which acts as a PTH-2 receptor agonist.
• the single amino acid switch converts inactive PTHrP into a potent PTH2 receptor agonist.
• PTHrP binds and activates both receptors, PTH/PTHrP and PTH2 , it is not a selective PTH2 agonist.
• Trp 23 In transient heterologous (with respect to species) expression systems, others have found an additional contribution to hPTH2 receptor selectivity by Trp 23 (Gardella et al . , JBC 1996, 271:19888-19893) .
• Trp 23 Gardella et al . , JBC 1996, 271:19888-19893
• PTH binding leads to PTH2 receptor- mediated activation of both cAMP and [Ca 2+ ] intracellular signaling pathways.
• PTH2 receptor The physiological function of the PTH2 receptor because of its high abundance and distribution in the brain suggests that it may act as a neurotransmitter receptor.
• PTH has been found in the central nervous system (CNS) (Harvey, S., et al . , 1993, J. Endocrinol. 139:353-361), therefore, it is possible that endogenous PTH2 receptor specific ligands, which are distinct from PTH, do exist in the CNS.
• CNS central nervous system
• Usdin reported the isolation of "PTH2 receptor binding activity" from the hypothalamus which was immunologically distinct from PTH.
• PCT Application Number PCT/US97/13360 published as PCT Publication Number WO 98/04591, discloses the use of certain PTHrP analogs which are PTH2 receptor agonists or antagonists .
• the compounds of the present invention are not only selective toward a receptor subtype but also signal specifically through the stimulation of [Ca +2 ] x transients. Therefore, the compounds of the present invention are receptor subtype and signaling pathway selective.
• this invention provides a PTH analogue or a truncated PTH analogue or a pharmaceutically acceptable salt thereof that selectively binds to the PTH2 receptor.
• a preferred PTH analogue or a truncated PTH analogue or a pharmaceutically acceptable salt thereof is where the analogue is a selective PTH2 receptor agonist.
• Another preferred PTH analogue or a truncated PTH analogue or a pharmaceutically acceptable salt thereof is where the analogue is a selective PTH2 receptor antagonist.
• a more preferred PTH analogue that selectively binds to the PTH2 receptor is an analogue of formula (I) , (R 1 R 2 )-A 1 -A 2 -A 3 -A 4 -A 5 -A 6 -A 7 -A 8 -A 9 -A 10 -A 11 -A 1 -A 13 -A 14 -A 15 -A 16 -A 17 -A 18 -
• a 2 is a lipophilic amino acid
• a 3 is a hydrophilic or a lipophilic amino acid
• a 4 is a hydrophilic amino acid
• a 5 is a hydrophilic or a lipophilic amino acid
• a 6 is a hydrophilic amino acid or is deleted
• a 7 is a hydrophilic or a lipophilic amino acid or is deleted
• a 8 is a lipophilic amino acid or is deleted
• a 9 is a hydrophilic amino acid or is deleted;
• a 10 is a hydrophilic amino acid or is deleted;
• a 11 is a hydrophilic or a lipophilic amino acid or is deleted
• a 12 is a hydrophilic or a lipophilic amino acid or is deleted;
• a 13 is a hydrophilic amino acid;
• a 14 is a hydrophilic amino acid or is deleted
• a 15 is a lipophilic amino acid or is deleted
• a 16 is a hydrophilic or a lipophilic amino acid or is deleted
• a 17 is a hydrophilic or a lipophilic amino acid or is deleted
• a 18 is a lipophilic amino acid or is deleted;
• a 19 is a hydrophilic or a lipophilic amino acid or is deleted;
• a 20 is a hydrophilic amino acid or is deleted;
• a 21 is a hydrophilic or a lipophilic amino acid or is deleted;
• a 22 is a lipophilic or a hydrophilic amino acid or is deleted
• a 23 is a hydrophilic or a lipophilic amino acid
• a 24 is a hydrophilic or a lipophilic amino acid
• a 25 is a hydrophilic amino acid
• a 26 is a hydrophilic amino acid
• a 27 is a lipophilic or a hydrophilic amino acid
• a 28 is a lipophilic amino acid
• a 29 is a lipophilic or a hydrophilic amino acid
• a 30 is a hydrophilic or a lipophilic amino acid
• a 31 is a lipophilic or a hydrophilic amino acid or is deleted
• a 32 is a hydrophilic amino acid or is deleted;
• a 33 is a hydrophilic amino acid or is deleted;
• a 34 is a lipophilic amino acid or is deleted;
• a 35 is a lipophilic amino acid or is deleted;
• a 36 is a lipophilic or a hydrophilic amino acid or is deleted;
• a 37 is a lipophilic amino acid or is deleted;
• a 38 is a lipophilic or a hydrophilic amino acid or is deleted;
• R 1 and R 2 are each independently selected from the group consisting of H, (C 1-30 ) alkyl , (C 2-30 ) alkenyl , phenyl- (C. 30 ) alkyl, naphthyl (C- ⁇ o ) alkyl , hydroxy(C 1 _ 30 ) alkyl, hydroxy (C 2 . 30 ) alkenyl , hydroxy-phenyl (C x .
• R 3 is OH , NH 2 , (Ci. j oJ alkoxy or NH-Y-CH 2 - Z , where Y is a ( C ⁇ _ 30 ) hydrocarbon moiety and Z is C0 2 H or CONH 2 ; provided that the compound is not PTH (1-34) R 3 , PTH (1-35) R 3 ,
• PTH(1-36)R 3 PTH(1-37)R 3 , or PTH(1-38)R 3 .
• PTH analogues that selectively binds to the PTH2 receptor is an analogue of formula (II) ,
• a 1 is Ser, Ala, Dap, Thr, Aib or is deleted;
• a 2 is Val, Leu, lie, Phe, Nle, /3-Nal, Aib, p-X-Phe, Ace,
• a 3 is Ser, Thr, Aib or is deleted;
• a 4 is Glu, Asp or is deleted;
• a 5 is Leu, Val, Nle, lie, Cha, /3-Nal, Trp, Pal, Ace, Phe, p-
• a 6 is Gin, a hydrophilic amino acid or is deleted
• a 7 is Leu, Val, Nle, lie, Cha, /3-Nal, Trp, Pal, Ace, Phe, p- X-Phe, a lipophilic amino acid, or is deleted;
• a 8 is Met, Nva, Leu, Val, lie, Cha, Ace, Nle, p-X-Phe, Phe, j ⁇ -Nal, Bpa, a lipophilic amino acid or is deleted;
• a 9 is His, a hydrophilic amino acid or is deleted
• a 10 is Asn, a hydrophilic amino acid or is deleted;
• a 11 is Leu, Val, Nle, lie, Cha, /3-Nal, Trp, Pal, Ace, Phe, p-X-Phe, a hydrophilic amino acid or is deleted;
• a 12 is Gly, Ace, Aib, or is deleted
• a 13 is Lys, Arg or HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) ;
• a 14 is His or is deleted;
• a 15 is Leu, Val, Nle, lie, Cha, /3-Nal, Trp, Pal, Ace, Phe, p-X-Phe or is deleted;
• a 16 is Ser, Asn, Ala, Aib or is deleted
• a 17 is Ser, Thr, Aib or is deleted
• a 18 is Met, Nva, Leu, Val, He, Nle, p-X-Phe, Phe, /3-Nal, Ace, Cha, Aib or is deleted;
• a 19 is Glu, Aib or is deleted
• a 20 is Arg, Lys, HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) or is deleted;
• a 21 is Val, Leu, He, Phe, Nle, /3-Nal, Aib, p-X-Phe, Ace, Cha, Met or is deleted;
• a 22 is Ace, Aib, Glu or is deleted;
• a 23 is Trp, Ace, Phe, p-X-Phe, Aib, /3-Nal or Cha;
• a 24 is Leu, Ace, He, Val, Phe, /3-Nal, Nle, Aib, p-X-Phe or Cha;
• a 25 is Arg, Lys or HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) ;
• a 26 is Arg, Lys or HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) ;
• a 27 is Lys, Aib, Leu, hArg, Gin, Ace, Arg, Cha, Nle, He, Val, Phe, 3-Nal, or p-X-Phe, where the Lys is optionally substituted on the e -amino group by an acyl group;
• a 28 is Leu, Ace, Cha, He, Val, Phe, Nle, /3-Nal, Aib or p-X- Phe;
• a 29 is Gin, Ace or Aib;
• a 30 is Asp, Lys, Arg or is deleted;
• a 31 is Val, Leu, Nle, Ace, Cha, Phe, He, j ⁇ -Nal Aib, p-X-Phe or is deleted;
• a 32 is His or is deleted;
• a 33 is Asn or is deleted;
• a 34 is Phe, Tyr, Amp, Aib, /3-Nal, Cha, Nle, Leu, He, Ace, p-X-Phe or is deleted;
• a 35 is Val, Leu, Nle, Ace, Cha, Phe, He, j ⁇ -Nal Aib, p-X-Phe or is deleted;
• a 36 is Ala, Val, Aib, Ace, Nva, Abu or is deleted;
• a 37 is Leu, Val, Nle, He, Cha, j ⁇ -Nal, Trp, Pal, Ace, Phe, p-X-Phe, a lipophilic amino acid, or is deleted;
• a 38 is Gly, Ace, Aib, or is deleted; where X for each occurrence is independently selected from the group consisting of OH, a halo and CH 3 ;
• R 1 and R 2 are each independently selected from the group consisting of H, (C ⁇ Q ) alkyl, (C 2 . 30 ) alkenyl , phenyl- (C 1-3 o) alkyl, naphthyl (C 1-30 ) alkyl, hydroxy (C x _ 30 ) alkyl , hydroxy (C 2 _ 30 ) alkenyl , hydroxy-phenyl (C x.
• E 1 is (C 1 _ 30 ) alkyl , (C 2 _ 30 ) alkenyl, phenyl (C 1-30 ) alkyl , naphthyl (C ⁇ 30 ) alkyl, hydroxy alkyl , hydroxy (C 2 _ 30 ) alkenyl , hydroxy-phenyl ( C 1 _ 30 ) alkyl or hydroxy-naphthyl ( C x _
• R 3 is OH, NH 2 , (C 1 . 30 )alkoxy or NH-Y-CH 2 -Z, where Y is a (C J ⁇ Q ) hydrocarbon moiety and Z is C0 2 H or CONH 2 ; n for each occurrence is independently an integer from 1 to 5 ; and
• R 4 for each occurrence is independently (C x -
• this invention provides a PTHrP analogue that selectively binds to the PTH2 receptor of the formula (IV) ,
• a 3 is Ser, Aib, Thr or is deleted
• a 4 is Glu, Asp or is deleted
• a 5 is His, He, Ace, Val, Nle, Phe, Leu, p-X-Phe, /3-Nal,
• a 6 is Gin, a hydrophilic amino acid or is deleted;
• a 7 is Leu, Val, Cha, Nle, /3-Nal, Trp, Pal, Ace, Phe, p-X- Phe, Aib, a lipophilic amino acid or is deleted;
• a 8 is Leu, Met, Ace, Cha, Aib, Nle, Phe, He, Val, /3-Nal, p- X-Phe, a lipophilic amino acid or is deleted;
• a 9 is His, a hydrophilic amino acid or is deleted;
• a 10 is Asp, Asn, a hydrophilic amino acid or is deleted;
• a 11 is Lys, Arg, Leu, Cha, Aib, p-X-Phe, He, Val, Nle, Ace, Phe, /3-Nal, HN-CH ( (CH 2 ) n NH-R 4 ) -C (O) , a lipophilic D-amino acid, a hydrophilic amino acid or is deleted;
• a 12 is Gly, Ace, Aib or is deleted;
• a 13 is Lys, Arg, HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) or is deleted;
• a 14 is Ser, His or is deleted;
• a 15 is He, Ace, Cha, Leu, Phe, Nle, /3-Nal, Trp, p-X-Phe,
• a 16 is Gin, Aib or is deleted
• a 17 is Asp, Aib or is deleted;
• a 18 is Leu, Aib, Ace, Cha, Phe, He, Nle, /3-Nal, Val, p-X- Phe or is deleted;
• a 19 is Arg, Lys, Aib, HN-CH ( (CH 2 ) n NH-R 4 ) -C (O) or is deleted;
• a 20 is Arg, Lys, HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) or is deleted;
• a 21 is Arg, Lys, HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) or is deleted;
• a 22 is Phe, Glu, Aib, Ace, p-X-Phe, /3-Nal, Val, Leu, He,
• a 23 is Phe, Leu, Lys, Ace, Cha, j ⁇ -Nal, Aib, Nle, He, p-X- Phe, Val or Trp;
• a 24 is Leu, Lys, Ace, Nle, He, Val, Phe, /3-Nal, Aib, p-X- Phe, Arg or Cha;
• a 25 is His, Lys, Aib, Ace, Arg or Glu;
• a 26 is His, Aib, Ace, Arg or Lys
• a 27 is Leu, Lys, Ace, Arg, He, Val, Phe, Aib, Nle, /3-Nal, p-X-Phe or Cha;
• a 28 is He, Leu, Lys, Ace, Cha, Val, Phe, p-X-Phe, Nle, ⁇ -
• a 29 is Ala, Glu, Ace, Aib or is deleted
• a 30 is Glu, Leu, Nle, Cha, Aib, Ace, Lys, Arg or is deleted;
• a 31 is He, Leu, Cha, Lys, Ace, Phe, Val, Nle, /3-Nal, Arg or is deleted;
• a 32 is His or is deleted
• a 33 is Thr, Ser or is deleted
• a 34 is Ala, Phe, Tyr, Cha, Val, He, Leu, Nle, j ⁇ -Nal, Aib,
• a 35 is Glu, Asp or is deleted
• a 36 is He, Ace, Cha, Leu, Phe, Nle, /3-Nal, Trp, p-X-Phe,
• a 37 is Arg, Lys, HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) or is deleted;
• a 38 is Ala, Phe, Tyr, Cha, Val, He, Leu, Nle, /3-Nal, Aib, Ace or is deleted;
• R 1 and R 2 are each independently selected from the group consisting of H, (C ⁇ Q ) alkyl , (C 2 _ 30 ) alkenyl , phenyl- (C ⁇ . 30 ) alkyl, naphthyl (C- ⁇ Q ) alkyl , hydroxy(C x _ 30 ) alkyl , hydroxy (C 2 _ 30 ) alkenyl , hydroxy-phenyl (C x _
• R 3 is OH, NH 2 , (Ci. ao Jalkoxy or NH-Y-CH 2 -Z, where Y is a (C 1-30 ) hydrocarbon moiety and Z is C0 2 H or CONH 2 ; n for each occurrence is independently an integer from 1 to 5 ; and
• R 4 for each occurrence is independently (C 1 -
• this invention provides a method of selectively binding the PTH2 receptor which comprises administering to a patient in need thereof an effective amount of a PTH analogue or a truncated PTH analogue or a pharmaceutically acceptable salt thereof that selectively binds to a PTH2 receptor.
• this invention provides a method of selectively eliciting an agonist response from the PTH2 receptor which comprises administering to a patient in need thereof an effective amount of a PTH analogue or a truncated PTH analogue or a pharmaceutically acceptable salt thereof which is a selective PTH2 receptor agonist.
• this invention provides a method of selectively eliciting an antagonist response from the
• PTH2 receptor which comprises administering to a patient in need thereof an effective amount of a PTH analogue or a truncated PTH analogue or a pharmaceutically acceptable salt thereof which is a selective PTH2 receptor antagonist.
• this invention provides a compound of the formula (III) , (R*R 2 ) -A 1 -A 2 -A 3 -A 4 -A 5 -A 6 -A 77 -A 8 -A 9 - -A 10 - -A 11 - -A 12 - -A 13 - -A 14 - -A 15 - -A 16 - -A 17 - -A 18 -
• a 1 is Ser, Ala, Dap, Thr, Aib or is deleted;
• a 2 is Val, Leu, He, Phe, Nle, /3-Nal, Aib, p-X-Phe, Ace,
• a 3 is Ser, Thr, Aib or is deleted;
• a 4 is Glu, Asp or is deleted;
• a 5 is Leu, Val, Nle, He, Cha, /3-Nal, Trp, Pal, Ace, Phe, p-
• a 6 is Gin, a hydrophilic amino acid or is deleted
• a 7 is Leu, Val, Nle, He, Cha, /3-Nal, Trp, Pal, Ace, Phe, p- X-Phe, a lipophilic amino acid, or is deleted;
• a 8 is Met, Nva, Leu, Val, He, Cha, Ace, Nle, p-X-Phe, Phe,
• a 9 is His, a hydrophilic amino acid or is deleted
• a 10 is Asn, a hydrophilic amino acid or is deleted;
• a 11 is Leu, Val, Nle, He, Cha, j ⁇ -Nal, Trp, Pal, Ace, Phe, p-X-Phe, a hydrophilic amino acid or is deleted;
• a 12 is Gly, Ace, Aib, or is deleted
• a 13 is Lys, Arg or HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) ;
• a 14 is His or is deleted;
• a 15 is Leu, Val, Nle, He, Cha, /3-Nal, Trp, Pal, Ace, Phe, p-X-Phe or is deleted;
• a 16 is Ser, Asn, Ala, Aib or is deleted
• a 17 is Ser, Thr, Aib or is deleted
• a 18 is Met, Nva, Leu, Val, He, Nle, p-X-Phe, Phe, /3-Nal, Ace, Cha, Aib or is deleted;
• a 19 is Glu, Aib or is deleted
• a 20 is Arg, Lys, HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) or is deleted;
• a 21 is Val, Leu, He, Phe, Nle, /3-Nal, Aib, p-X-Phe, Ace,
• a 22 is Ace, Aib, Glu or is deleted
• a 23 is Trp, Ace, Phe, p-X-Phe, Aib, j ⁇ -Nal or Cha;
• a 24 is Leu, Ace, He, Val, Phe, /3-Nal, Nle, Aib, p-X-Phe or
• a 25 is Arg, Lys or HN-CH ( (CH 2 ) n NH-R 4 ) -C (O)
• a 26 is Arg, Lys or HN-CH ( (CH 2 ) n NH-R 4 ) -C (O) ;
• a 27 is Lys, Aib, Leu, hArg, Gin, Ace, Arg, Cha, Nle, He, Val, Phe, /3-Nal, or p-X-Phe, where the Lys is optionally substituted on the e -amino group by an acyl group;
• a 28 is Leu, Ace, Cha, He, Val, Phe, Nle, /3-Nal, Aib or p-X- Phe;
• a 29 is Gin, Ace or Aib;
• a 30 is Asp, Lys, Arg or is deleted;
• a 31 is Val, Leu, Nle, Ace, Cha, Phe, He, /3-Nal Aib, p-X-Phe or is deleted;
• a 32 is His or is deleted;
• a 33 is Asn or is deleted;
• a 34 is Phe, Tyr, Amp, Aib, /3-Nal, Cha, Nle, Leu, He, Ace, p-X-Phe or is deleted;
• a 35 is Val, Leu, Nle, Ace, Cha, Phe, He, /3-Nal Aib, p-X-Phe or is deleted;
• a 36 is Ala, Val, Aib, Ace, Nva, Abu or is deleted;
• a 37 is Leu, Val, Nle, He, Cha, j ⁇ -Nal, Trp, Pal, Ace, Phe, p-X-Phe, a lipophilic amino acid, or is deleted;
• a 38 is Gly, Ace, Aib, or is deleted; where X for each occurrence is independently selected from the group consisting of OH, a halo and CH 3 ; R 1 and R 2 are each independently selected from the group consisting of H, (C ⁇ Q ) alkyl , (C 2 .
• R 1 or R 2 is COE 1 where E 1 is (C 1-30 ) alkyl ,
• R 3 is OH, NH 2 , (C ⁇ ) alkoxy or NH-Y-CH 2 -Z, where Y is a (C 1 _ 30 ) hydrocarbon moiety and Z is C0 2 H or CONH 2 ; n for each occurrence is independently an integer from 1 to 5 ; and R 4 for each occurrence is independently (C x -
• a preferred group of compounds of formula (III) are the compounds listed as Examples 1-73, shown hereinbelow.
• this invention provides a compound of formula (V) , (R 1 R 2 )-A 1 -A 2 -A 3 -A 4 -A 5 -A 6 -A 7 -A 8 -A 9 -A 10 -A 11 -A 12 -A 13 -A 14 -A 15 -A 16 -A 17 -A 18 - A 19 -A 20 -A 21 -A 22 -A 23 -A 24 -A 25 -A 26 -A 27 -A 28 -A 29 -A 30 -A 31 -A 32 -A 33 - A 34 - A 35 - A 36 -
• a 3 is Ser, Aib, Thr or is deleted
• a 4 is Glu, Asp or is deleted
• a 5 is His, He, Ace, Val, Nle, Phe, Leu, p-X-Phe, /3-Nal,
• a 6 is Gin, a hydrophilic amino acid or is deleted;
• a 7 is Leu, Val, Cha, Nle, j ⁇ -Nal, Trp, Pal, Ace, Phe, p-X- Phe, Aib, a lipophilic amino acid or is deleted;
• a 8 is Leu, Met, Ace, Cha, Aib, Nle, Phe, He, Val, /3-Nal, p- X-Phe, a lipophilic amino acid or is deleted;
• a 9 is His, a hydrophilic amino acid or is deleted;
• a 10 is Asp, Asn, a hydrophilic amino acid or is deleted;
• a 11 is Lys, Arg, Leu, Cha, Aib, p-X-Phe, He, Val, Nle, Ace,
• a 12 is Gly, Ace, Aib or is deleted;
• a 13 is Lys, Arg, HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) or is deleted;
• a 14 is Ser, His or is deleted
• a 15 is He, Ace, Cha, Leu, Phe, Nle, /3-Nal, Trp, p-X-Phe,
• a 16 is Gin, Aib or is deleted;
• a 17 is Asp, Aib or is deleted;
• a 18 is Leu, Aib, Ace, Cha, Phe, He, Nle, /3-Nal, Val, p-X- Phe or is deleted;
• a 19 is Arg, Lys, Aib, HN-CH ( (CH 2 ) n NH-R 4 ) -C (O) or is deleted;
• a 20 is Arg, Lys, HN-CH ( (CH 2 ) n NH-R 4 ) -C (0) or is deleted;
• a 21 is Arg, Lys, HN-CH ( (CH 2 ) n NH-R 4 ) -C (O) or is deleted;
• a 22 is Phe, Glu, Aib, Ace, p-X-Phe, /3-Nal, Val, Leu, He,
• a 23 is Phe, Leu, Lys, Ace, Cha, /3-Nal, Aib, Nle, He, p-X- Phe, Val or Trp;
• a 24 is Leu, Lys, Ace, Nle, He, Val, Phe, /3-Nal, Aib, p-X- Phe, Arg or Cha;
• a 25 is His, Lys, Aib, Ace, Arg or Glu;
• a 26 is His, Aib, Ace, Arg or Lys
• a 27 is Leu, Lys, Ace, Arg, He, Val, Phe, Aib, Nle, jS-Nal, p-X-Phe or Cha;
• a 28 is He, Leu, Lys, Ace, Cha, Val, Phe, p-X-Phe, Nle, j ⁇ - Nal, Aib or is deleted;
• a 29 is Ala, Glu, Ace, Aib or is deleted
• a 30 is Glu, Leu, Nle, Cha, Aib, Ace, Lys, Arg or is deleted;
• a 31 is He, Leu, Cha, Lys, Ace, Phe, Val, Nle, /3-Nal, Arg or is deleted;
• a 32 is His or is deleted
• a 33 is Thr, Ser or is deleted
• a 34 is Ala, Phe, Tyr, Cha, Val, He, Leu, Nle, /3-Nal, Aib, Ace or is deleted;
• a 35 is Glu, Asp or is deleted
• a 36 is He, Ace, Cha, Leu, Phe, Nle, /3-Nal, Trp, p-X-Phe,
• a 37 is Arg , Lys , HN-CH ( (CH 2 ) n NH-R 4 ) -C (O) or is deleted ;
• a 38 is Ala , Phe , Tyr, Cha , Val , He , Leu, Nle , j ⁇ -Nal , Aib ,
• R 1 and R 2 are each independently selected from the group consisting of H, (C ⁇ Q ) alkyl , (C 2 _ 30 ) alkenyl , phenyl- (C x . 30 ) alkyl, naphthyl (C ⁇ Q ) alkyl , hydroxy (C x .
• R 1 or R 2 is COE 1 where E 1 is (C- ⁇ Q ) alkyl , (C 2 _ 30 ) alkenyl, phenyl (C 1 _ 30 ) alkyl , naphthyl (C x _
• R 3 is OH, NH 2 , (C 1 _ 30 )alkoxy or NH-Y-CH 2 -Z, where Y is a (C ⁇ . 30 ) hydrocarbon moiety and Z is C0 2 H or CONH 2 ; n for each occurrence is independently an integer from 1 to 5 ; and
• R 4 for each occurrence is independently (C ⁇ -
• a preferred group of compounds of formula (V) are the compounds listed as Examples 74-86, shown hereinbelow.
• this invention provides a method of selectively binding the PTH2 receptor which comprises administering to a patient in need thereof an analogue of formula (I), (II) or (III) or a pharmaceutically acceptable salt thereof.
• this invention provides a method of selectively binding the PTH2 receptor which comprises administering to a patient in need thereof a compound of formula (III) or (V) or a pharmaceutically acceptable salt thereof.
• a compound of formula (III) or (V) or a pharmaceutically acceptable salt thereof is where the compound is selected from Examples 1-73 or Examples 74-86.
• this invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising an analogue of formula (I) , (II) or (III) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
• this invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising a compound of formula (III) or (V) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
• a pharmaceutical composition comprising a compound selected from Examples 1-73 or Examples 74-86.
• this invention is directed to a method of treating a medical disorder that results from altered or excessive action of the PTH2 receptor, which comprises administering to a patient in need thereof an effective amount of a PTH analogue or a truncated PTH analogue or a pharmaceutically acceptable salt thereof that selectively binds to the PTH2 receptor, sufficient to inhibit the activation of the PTH2 receptor of said patient.
• a preferred method of the immediately foregoing method is where said medical disorder is abnormal CNS functions, abnormal pancreatic functions, divergence from normal mineral metabolism and homeostasis, male infertility, abnormal blood pressure or a hypothalmic disease.
• this invention provides a method of treating a medical disorder that results from altered or excessive action of the PTH2 receptor, which comprises administering to a patient in need thereof an effective amount of an analogue of formula (I), (II) or (III), sufficient to inhibit the activation of the PTH2 receptor of said patient.
• a preferred method of the immediately foregoing method is where said medical disorder is abnormal CNS functions, abnormal pancreatic functions, divergence from normal mineral metabolism and homeostasis, male infertility, abnormal blood pressure or a hypothalmic disease .
• this invention is directed to a method of treating a medical disorder that results from altered or excessive action of the PTH2 receptor, which comprises administering to a patient in need thereof an effective amount of a compound of formula (III) or (V) , sufficient to inhibit the activation of the PTH2 receptor of said patient.
• a preferred method of the immediately foregoing method is where said medical disorder is abnormal CNS functions, abnormal pancreatic functions, divergence from normal mineral metabolism and homeostasis, male infertility, abnormal blood pressure or a hypothalmic disease.
• Preferred of each of the foregoing methods is where the compound is selected from Examples 1-73 or Examples 74-86.
• N-terminal amino acid all abbreviations (e.g. Ala or A of amino acids in this disclosure stand for the structure of -NH-CH (R) -CO- , wherein R is the side chain of an amino acid (e.g., CH 3 for Ala) .
• R X R 2 the side chain of an amino acid
• R 1 and R 2 are as defined above.
• Bpa is p-benzoylphenylalanine .
• ⁇ -amino acids ⁇ - (2 -naphthyl ) alanine, norleucine, oi , j ⁇ -diaminopropionic acid, cyclohexylalanine, norvaline, 4- amino-phenylalanine, ⁇ - (3 -pyridinyl) alanine and ce- aminoisobutyric acid, respectively.
• Ace is an amino acid selected from the group of 1-amino- 1- cyclopropanecarboxylic acid; 1-amino-l- cyclobutanecarboxylic acid; 1-amino-l- cyclopentanecarboxylic acid; 1-amino-l- cyclohexanecarboxylic acid; 1-amino-l- cycloheptanecarboxylic acid; 1-amino-l- cyc looc t anecarboxyl i c acid; and 1-amino-l- cyclononanecarboxylic acid.
• hydroxyalkyl, hydroxyphenylalkyl , and hydroxynaphthylalkyl may contain 1-4 hydroxy substituents .
• (C 1 . 12 ) hydrocarbon moiety is an alkyl group, an alkenyl group or an alkynyl group .
• hydrophilic amino acid is an amino acid having at least one hydrophilic functional group in addition to those required for peptide bond formation, such as: Arg, Asp, Asn, Glu, Gin, Gly, His, Lys, Orn (ornithine) , Ser, Thr, /3-Ala, Ala, Aad ( ⁇ -aminoadipic acid) , /3-Aad (/3-aminoadipic acid) , Apm ( ⁇ -aminopimolic acid) , Cit (citrulline) , Gla ( ⁇ -carboxy-glutamic acid) , hArg (homo-Arg) , hCit (homo-Cit) , hSer (homo-Ser) , Dba (a , ⁇ -diamino-butyric acid), Dpa ( ⁇ , /3-diaminopropionic acid), Amp (p-amino-phenylalanine) ,
• a lipophilic amino acid is an uncharged, aliphatic or aromatic amino acid, such as: Val, Leu, He, Pro, Cys, Phe, Met, Trp, Tyr, Cha, /3-Nal, Aib, Ace, Ala, Abu ( ⁇ -aminobutyric acid), Nle, Nva (norvaline) , Bpa (p-benzoyl-phenylalanine) , hPhe (homo-Phe) , hPro (homo- Pro) , 1-Nal (/3- (1 -naphthyl) alanine) , 2-Nal (/3(2- naphthyl) alanine) , Oic (octahydroindode-2-carboxylic acid) , Tic (1, 2 , 3 , 4-tetrahydroisoquinoline-3-carboxylic acid) , Pen (penicillamine) , Phg (phenylglycine)
• Physiologically active truncated homologue or analogue of PTH refers to a polypeptide having a sequence comprising less than the full complement of amino acids found in PTH.
• a peptide of this invention is also denoted herein by another format, e.g., [D-Nle 8 ] hPTH (1-34) NH 2 , with the substituted amino acids from the natural sequence placed between the set of brackets (e.g., D-Nle 8 for Met 8 in hPTH) .
• the abbreviation hPTH stands for human PTH, and hPTHrP for human PTHrP.
• the numbers between the parentheses refer to the number of amino acids present in the peptide (e.g., hPTH(l-34) is amino acids 1 through 34 of the peptide sequence for human PTH) .
• hPTH(l-34) and hPTHrP(l-34) are listed in Nissenson, et al . , Receptor, 3:193 (1993).
• the designation "NH 2 " in PTH(1-34)NH 2 indicates that the C-terminus of the peptide is amidated.
• PTH (1-34) means that the C-terminus is the free acid.
• the peptides of this invention can be prepared by standard solid phase peptide synthesis. See, e.g., Stewart, J.M. , et al . , Solid Phase Synthesis (Pierce Chemical Co., 2d ed. 1984).
• the substituents R 1 and R 2 of the above generic formula may be attached to the free amine of the N-terminal amino acid by standard methods known in the art.
• alkyl groups e.g., (C x _ 12 ) alkyl , may be attached using reductive alkylation.
• Hydroxyalkyl groups e.g., (C 1-12 ) hydroxyalkyl
• Acyl groups e.g., COE 1
• the coupling should be performed with an additional 3 molar equivalents of HOBT.
• the synthesis of the peptide starts with BocHN-Y-CH 2 -COOH which is coupled to the resin.
• the synthesis of the peptide starts with Boc-HN-Y-CH 2 -COOH which is coupled to PAM resin.
• R 3 is OH the first amino acid is coupled to PAM resin.
• the compounds of this invention can be tested for binding to the human PTH2 (hPTH2) receptor for the ability to stimulate adenylyl cyclase and/or intracellular calcium transients by the assay described below.
• hPTH2 human PTH2
• Tissue culture media and sera were purchased from Life Technologies (Grand Island, NY) , and all tissue culture plastics were obtained from Corning (Corning, NY) .
• Adenosine and 3 -isobutyl-1-methyl xanthine (IBMX) were purchased from Research Biochemicals
• Fura-2 acetoxylmethyl ester was obtained from Molecular Probes (Eugene, OR) , and hPTHrP was purchased from Bachem (Torrance, CA) .
• [ 3 H] -Adenine was purchased from New England Nuclear (Boston, MA) .
• Na 125 I was obtained from Amersham Corp. (Arlington Heights, IL) . All other analytical grade reagents were purchased from Sigma (St. Louis, MO) .
• HEK-293/C- 21 cells (160,000 receptors/cell) and stably transfected HEK-293/C- 21 cells (Beth Israel Deaconess Medical Center-Division of Bone and Mineral Metabolism, Boston, MA) , which express the hPTH/PTHrP receptor, are maintained in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 95% air/5% C0 2 . The medium is changed every 2 days before confluency and every day after confluency. The cells are sub-cultured 1:10 once a week.
• Receptor binding assay Ligand binding is performed using Saos-2/B-10, HEK/C-21 cells or HEK/BP-16 cells using HPLC-purified [ 125 I] [Nle 8 - 18 , Tyr 34 ] bPTH- (1-34) NH 2 ( 125 I -PTH) as radioligand. Saos-2 cells are maintained for four days until they reach confluence.
• the medium is replaced with 5% FBS in RPMI 1640 medium and incubated for about 2 hrs at room temperature with 10 x 10 4 cpm mono- 125 I- [Nle 8,18 , Tyr 34 (3- 125 I) ] bPTH (1-34)NH 2 in the presence of competing peptides of the invention at various concentrations between 10 _11 M to 10 " 4 M.
• the cells are washed four times with ice-cold PBS and lysed with 0.1 M NaOH, and the radioactivity associated with the cells is counted in a scintillation counter.
• the binding assay is conducted with various peptides of the invention, and the Kd value (half maximal inhibition of binding of mono- 15 I- [Nle 8 - 18 , Tyr 34 (3- 125 I) ] bPTH (1-34) NH 2 ) for each peptide is calculated.
• Adenylyl cyclase assay is performed in Saos-2/B-10 cells, HEK/C21 cells, and HEK/BP- 16 cells.
• the ability of the peptides of the invention to induce a biological response in Saos-2/B-10 cells is measured. More specifically, any stimulation of the adenylate cyclase is determined by measuring the level of synthesis of cAMP (adenosine 3 ' , 5 ' -monophosphate) as described previously in Rodan, et al . , J. Clin. Invest. 72: 1511 (1983) and Goldman, et al .
• cAMP adenosine 3 ' , 5 ' -monophosphate
• the cells are treated with 1 mM IBMX [isobutylmethyl-xanthine, Sigma, St. Louis, MO] in fresh medium for 15 in, and a peptide to be tested is added to the medium to incubate for about 5 min.
• the reaction is stopped by the addition of 1.2 M trichloroacetic acid (TCA) (Sigma, St. Louis, MO) followed by sample neutralization with 4 N KOH.
• cAMP is isolated by the two-column chromatographic method (Salmon, et al . , 1974, Anal. Biochem. 58, 541) .
• the radioactivity is counted in a scintillation counter (Liquid Scintillation Counter 2200CA, PACKARD, Downers Grove, IL) .
• Measurements of [Ca 2+ ] 1 Measurements of intracellular Ca 2+ ( [Ca 2+ ] ) are performed in Saos-2/B-10 cells, HEK/C-21 cells and HEK/BP-16 cells. For measurement of [Ca 2+ ] 1 , cells are harvested from 150-cm 2 flasks using
• K is the product K d (F 0 /F s ), where K d is the effective dissociation constant (224 nM) , F 0 is the intensity of the 380-nm excitation signal in the absence of calcium, and F s is the intensity of the 380-nm excitation signal at saturating calcium concentrations.
• salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid) , and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids) .
• organic acids e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic or pamoic acid
• inorganic acids e.g., hydrochloric acid, sulfuric acid, or phosphoric acid
• polymeric acids e.g., tannic acid, carboxy
• a therapeutically effective amount of a peptide of this invention and a pharmaceutically acceptable carrier substance e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle
• a pharmaceutically acceptable carrier substance e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle
• a therapeutic composition e.g., a pill, tablet, capsule, or liquid
• administration e.g., orally, intravenously, transdermally, pulmonarily, vaginally, subcutaneously, nasally, iontophoretically, or by intratracheally
• the pill, tablet or capsule that is to be administered orally can be coated with a substance for protecting the active composition from the gastric acid or intestinal enzymes in the stomach for a period of time sufficient to allow it to pass undigested into the small intestine.
• the therapeutic composition can also be in the form of a biodegradable or nonbiodegradable sustained release formulation for subcutaneous or intramuscular administration. See, e.g., U.S. Patents 3,773,919 and 4,767,628 and PCT Application No. WO 94/15587.
• Continuous administration can also be achieved using an implantable or external pump (e.g., INFUSAIDTM pump) .
• the administration can also be conducted intermittently, e.g., single daily injection, or continuously at a low dose, e.g., sustained release formulation.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
• Preparations according to this invention for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, or emulsions.
• non-aqueous solvents or vehicles are propylene glycol , polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
• Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use .
• compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.
• compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
• a compound of this invention can be administered in a sustained release composition such as those described in the following patents.
• U.S. Patent No. 5,672,659 teaches sustained release compositions comprising a bioactive agent and a polyester.
• the dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained.
• the selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Generally, dosage levels of between 0.0001 to 10 mg/kg of body weight daily are administered.
• a preferred dosage range is 0.001 to 0.5 mg/kg of body weight daily which can be administered as a single dose or divided into multiple doses.
• the compounds of the instant invention are illustrated by the following examples, but are not limited to the details thereof.
• EXAMPLE 1 rcha 7 ' 11 , D-Nle 8 . Nle 18 , Tyr 34 1 hPTH (1-34) NH, The peptide [Cha 7 - 11 , D-Nle 8 , Nle 18 , Tyr 34 ] hPTH (1-34) NH 2 was synthesized on an Applied Biosystems (Foster City, CA) model 430A peptide synthesizer which was modified to do accelerated Boc-chemistry solid phase peptide synthesis. See Schnoize, et al . , Int. J. Peptide Protein Res., 90:180 (1992) .
• Boc-Asn (Xanthyl)
• Boc-Arg (Tos) -OH
• Boc-Glu (OcHex) -OH
• Boc-His (DNP) -OH
• Boc-Cha- OH Boc-D-Nle-OH
• Boc-Nle-OH Boc-Nle-OH
• Boc-Val-OH Boc-Leu-OH
• Boc-Trp (formyl) -OH and Boc-Tyr (Br-Z) -OH
• the resin was treated with a solution of 20% mercaptoethanol/10% DIEA in DMF for 2 x 30 min. to remove the DNP group on the His side chain.
• the resin was washed with DMF.
• the N-terminal Boc group was then removed by treatment with 100% TFA for 2 x 2 min.
• the resin was washed with DMF and was treated with ethanolamine :H 2 0:DMF/15 : 15 : 70 for 2 x 30 min. to remove the formyl protecting group on Trp residue.
• the partially-deprotected peptide-resin was washed with DMF and DCM and dried in vacuo .
• the final cleavage was done by stirring the peptide-resin in 10 mL of HF containing 1 mL of anisole and dithiothreitol (24 mg) at about 0°C for about 75 min. HF was removed by a flow of nitrogen. The residue was washed with ether (6 x 10 mL) and extracted with 4N HOAc (6 x 10 mL) .
• the peptide mixture in the aqueous extract was purified on a reverse-phase preparative high pressure liquid chromatography (HPLC) using a reverse phase VYDACTM C 18 column (Nest Group, Southborough, MA) .
• Fractions were collected and checked on analytical HPLC. Those containing pure product were combined and lyophilized to dryness . 114 mg of a white solid was obtained. Purity was >98% based on analytical HPLC analysis. Electro-spray mass spectrometer analysis gave the molecular weight at 4176.4 (in agreement with the calculated molecular weight of 4176.9).
• EXAMPLE 2 TD-Nle 8 .
• Nle 18 , Tyr 34 l hPTH (1-34)NH, Boc-protected amino acids, N-hydroxybenzotriazole (HOBt) , N,N' -dicyclohexylcarbodilmide (DCC) and p- methylbenzhydrylamine resin were purchased from Applied Biosystems (Foster City, CA) .
• Boc- (3-Iodo) Tyrosine [0- (3- BrBz) ] was purchased from Peninsula Laboratories (Belmont, CA) .
• B&J brand dichloromethane, N-methylpyrrolidone (NMP) and acetonitrile were obtained from Baxter (McGraw Park, IL) .
• N- ⁇ -Boc-amino derivatives were used in the course of the automated solid-phase peptide synthesis: Arg (N°-tosyl ) , Asp(O-cHex), Glu(O-Bzl), His (N n -Bom) , Lys (N 6 -2-Cl-Z) , Ser(0- Bzl) , Thr(O-Bzl), and Tyr(2-Br-Z) . Synthesis started at a 0.5 mmol scale and was split into two halves after the incorporation of Glu 22 .
• the "Low-HF” step included mixing the suspension of the resin-bound peptide in a mixture (20 mL/g of resin-bound peptide) containing (% vol) 60% dimethylsulfide, 5% p-thiocresol , 5% p-cresol, 5% ethane dithiol, and 25% HF for about 2 hours at about 0°C. After removal of the volatile reagent under vacuum and washing the resin-bound peptide consecutively with petroleum-ether and ether it was returned to the reaction vessel for the "High-HF" step.
• the resin-bound peptide was resuspended in a mixture (20 mL/g of resin-bound peptide) containing (% vol) 5% butane dithiol, 5% p-cresol, and 90% HF for about 1 hour at about 0°C . After removing the reagents as previously described the crude peptide was dissolved in 50% (v/v) acetic acid and the solution was diluted with water and lyophilized. The peptide was purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) (PrepPak VYDAC ® C18, 300A cartridge, 15 ⁇ m, 5.5x35 cm).
• RP-HPLC preparative reverse-phase high performance liquid chromatography
• the solvent system employed included a two solvent system: A: 0.1% (v/v) TFA in water and B: 0.1% (v/v) TFA in acetonitrile, generating the following linear gradient: 0-15% B in A in the first 10 min followed by 15-45% B in A in the next 120 min at a flow-rate of 70 mL/min and monitored at 220 nm.
• Fractions were analyzed on an analytical RP-HPLC system (VYDAC® (C18, 300A, 5 ⁇ m, 4.6x150cm) employing a linear gradient of 20-50% B in A for 30 min at a flow rate of 1 ml/min and monitored at 220 nm, the retention time is 18.24 minutes.
• Example 3-4 were synthesized substantially according to the procedure of Example 1 using the appropriate, protected amino acids and Example 5 was synthesized substantially according to Example 2 using the appropriate, protected amino acids.
• Examples 6 to 86 can be synthesized substantially according to the procedure of Example 1 using the appropriate, protected amino acids.
• Example 6 D-Nle 8 , Nle 18 , Tyr>PTH(1-34)NH 2
• Example 7 D-Nle 8 ]hPTH(1-34)NH 2
• Example 8 D-Leu 8 , Nle 18 , Tyr 34 ]hPTH(1-34)NH 2
• Example 9 D-Cha 8 , Nle 18 , Tyr 34 ]hPTH(1-34)NH 2
• Example 10 D-Phe 8 , Nle 18 , Tyr 34 ]hPTH(1-34)NH 2
• Example 1 D-Nal 8 ,Nle 18 , Tyr 34 ]hPTH(1-34)NH 2
• Example 12 D-Abu 8 , Nle 18 , Tyr 34 ]hPTH(1-34)NH 2
• Example 13 D-Met 8 ]hPTH(1-34)NH 2
• Example 14 Cha 7 ' 11 , D-Met 8 ]hPTH(1-34)NH 2
• Example 15 D-lle 8 ]hPTH(1-34)NH 2
• Example 16 Cha 7 ' 1
• Example 80 [ lie 5 , des-Leu 18 , Trp 23 ]hPTHrP(1-34)NH 2
• Example 82 [lie 5 , D-Leu 8 , Glu 22 ' 25 , Leu 23 ' 28 ' 31 , Lys 26 ' 30 , Aib 29 ]hPTHrP(1-34)NH 2
• Example 84 [lie 5 , D-Leu 8 , Glu 22 ' 25 ' 29 , Leu 23 ' 28 ' 31 , Lys 26 ' 30 ]hPTHrP(1-34)NH 2
• Example 85 [lie 5 , D-Leu 8 , Glu 22 ' 25 ' 29 , Trp 23 , Lys 26 ' 30 , Leu 28 ' 31 ]hPTHrP(1-34)NH 2

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