WO2015022283A1 - Yap-tead inhibitors - Google Patents

Yap-tead inhibitors Download PDF

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Publication number
WO2015022283A1
WO2015022283A1 PCT/EP2014/067145 EP2014067145W WO2015022283A1 WO 2015022283 A1 WO2015022283 A1 WO 2015022283A1 EP 2014067145 W EP2014067145 W EP 2014067145W WO 2015022283 A1 WO2015022283 A1 WO 2015022283A1
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WO
WIPO (PCT)
Prior art keywords
fmoc
hcy
nle
hcy4
phe
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Application number
PCT/EP2014/067145
Other languages
French (fr)
Inventor
Taishan HU
Zhaohu LIN
Chen LING
Lin Pei
Yiping Rong
Hong Shen
Jason Christopher Wong
Shixiang YAN
Fang Yu
Lingjie Yu
Zhisen ZHANG
Zhenshan Zhang
Zheng Zhou
Original Assignee
F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Application filed by F. Hoffmann-La Roche Ag, Hoffmann-La Roche Inc. filed Critical F. Hoffmann-La Roche Ag
Publication of WO2015022283A1 publication Critical patent/WO2015022283A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring

Definitions

  • the present invention relates to compounds useful for inhibiting the binding of YAP to TEAD. Accordingly, the present invention relates to compounds useful for therapy in a mammal, and in particular to inhibit proliferation of cancer cells that overexpress YAP.
  • Yes-associated protein is a transcriptional co-activator regulated by Hippo pathway.
  • YAP is phosphorylated by the Hippo pathway for cytoplasmic retention, ubiquitination and subsequent proteasomal degradation.
  • organ development, or organ regeneration for instance, after partial hepatectomy
  • the Hippo pathway is inactivated. This allows YAP to translocate into nucleus and interact with transcriptional factors (i.e. TEAD1, 2, 3 and 4) to activate gene expression for cell survival, differentiation and proliferation.
  • transcriptional factors i.e. TEAD1, 2, 3 and 4
  • YAP has been identified as an oncogene in liver cancer by an oncogenomic study (Zender, L.; Spector, M. S.; Xue, W. et al, Cell, 2006, 125, 1253-1267, and Dong, J.; Feldmann, G.; Huang, J. et al. Cell, 2007, 130, 1120- 1133).
  • Defective Hippo pathway and ectopic expression of YAP result in over-grown livers in transgenic mice.
  • YAP promotes tumor growth by overriding the contact-contact inhibition because over-expression of YAP in subcutaneous tumors accelerated tumor growth, and oppositely short hairpin RNA (shRNA)-mediated knockdown of YAP slowed the growth.
  • shRNA short hairpin RNA
  • YAP The functionality of YAP depends on the interaction of YAP with TEADs in nucleus (Zhao, B; Ye, X.; Xu, J. et al. 2008, Genes & Dev. 2008, 1962-1971; and Ota, M.; Sasaki, H. Development 2008, 135, 4059-4069), and YAP proved bind to all 4 TEAD proteins with similar efficiency (Vassilev, A.; Kaneko, K. J.; Shu, H. et al. Genes Dev. 2001, 15, 1229), therefore, disruption of such interaction is believed to abrogate the oncogenic property of YAP.
  • the peptides of invention are designed to block the interaction, and can be further developed into drugs for cancers addicted to YAP over-expression.
  • liver cancer or hepatocellular carcinoma (HCC)
  • HCC hepatocellular carcinoma
  • Objects of the present invention are novel compounds of formula I, their manufacture, medicaments based on a compound in accordance with the invention and their production as well as the use of compounds of formula I for inhibiting the binding of YAP to TEAD. Accordingly, the compounds of formula I are useful for reducing cell proliferation in particular for the treatment of cancer.
  • the compounds according to the present invention may exist in the form of their pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases.
  • Acid- addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like.
  • Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide.
  • the chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin R.J., et al., Organic Process Research & Development 2000, 4, 427-435; or in Ansel, H., et al., In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. Particular are the sodium salts of the compounds of formula I.
  • diastereomeric salts which can be separated by crystallization are formed from the racemic mixtures by reaction with an optically active acid such as e.g. D- or L-tartaric acid, mandelic acid, malic acid, lactic acid or camphorsulfonic acid.
  • an optically active acid such as e.g. D- or L-tartaric acid, mandelic acid, malic acid, lactic acid or camphorsulfonic acid.
  • the present invention provides (i) a compound of the formula I:
  • a 0 is acetyl
  • a 1 is Thr, D-Thr or deleted
  • a 2 is Val
  • a 3 is Pro, 4R-Hyp, 35-Hyp or 4R-Flp;
  • a 4 is Met, Trp, Phe, Phe(2-Cl), Phe(3-Cl), Phe(3-Br), Phe(4-Cl), Cha, Nle, Hie, Ahe, Nal(l) or Nal(2);
  • a 5 is Hey or Hsc
  • a 6 is Leu, Lys or Arg
  • a 7 is Arg
  • A is Lys, His, Ser, Thr, Glu, Asp, Leu, Pro, Nle, Dbu, Orn, D-Lys or alpha-Me-Lys;
  • a 9 is Nle, Cha, Hie, Ahe or Aoc;
  • a 10 is Pro or 4R-Flp
  • a 11 is Ala, Aib, Glu or Hag;
  • a 12 is Ser
  • a 13 is Phe, Phe(2-Cl), Phe(4-F) or Phe(4-Cl);
  • a 14 is Cys or Sec
  • a 15 is Lys, Hag, Ahp or deleted
  • a 16 is Pro, 4R-Hyp, 35-Hyp, 4R-Flp, Tic or deleted;
  • a 17 is Pro, 4R-Hyp, 35-Hyp or deleted
  • a 18 is Glu, Lys, Arg, Phe(4-guanidino) or deleted;
  • a 19 is NH 2 ;
  • X is S or Se;
  • Another embodiment of present invention is (ii) a compound of formula I, wherein A 0 is acetyl;
  • a 1 is Thr, D-Thr or deleted
  • a 2 is Val
  • a 3 is Pro, 4R-Hyp, 35-Hyp or 4R-Flp;
  • a 4 is Met, Trp, Phe, Phe(2-Cl), Phe(3-Cl), Phe(3-Br), Phe(4-Cl), Cha, Nle, Hie, Ahe, Nal(l) or Nal(2);
  • a 5 is Hey or Hsc
  • a 6 is Leu, Lys or Arg
  • a 7 is Arg
  • A is Lys, His, Ser, Thr, Glu, Asp, Leu, Pro, Nle, Dbu, Orn, D-Lys or alpha-Me-Lys;
  • a 9 is Nle, Cha, Hie, Ahe or Aoc;
  • a 10 is Pro or 4R-Flp
  • a 11 is Ala, Aib, Glu or Hag;
  • a 12 is Ser
  • a 13 is Phe, Phe(2-Cl), Phe(4-F) or Phe(4-Cl);
  • a 14 is Cys or Sec
  • a 15 is Lys, Hag or Ahp;
  • a 16 is Pro, 4R-Hyp, 35-Hyp, 4R-Flp or Tic;
  • a 17 is Pro, 4R-Hyp or 35-Hyp;
  • a 18 is Glu, Lys, Arg or Phe(4-guanidino);
  • a 19 is NH 2 ;
  • X is S or Se
  • Another embodiment of present invention is (iii) a compound of formula I or a pharmaceutically acceptable salt thereof, wherein A 5 is Hey and A 14 is Cys, and all re substituents have the significances given before.
  • Another embodiment of present invention is (iv) a compound of formula I or a pharm naacceeuuttiiccaallllyy aacccceeppttaabbllee ssaalltt tthheerreeooff,, wwhheerreeii:n A 5 is Hsc and A 14 is Sec, and all remaining substituents have the significances given before.
  • Another embodiment of present invention is (v) a compound of formula I or a pharmaceutically acceptable salt thereof, wherein A 11 is Ala or Aib, and A 15 is Lys;
  • a 11 is Hag
  • a 15 is Hag or Ahp
  • a 11 and A 15 together with A 12 , A 13 and A 14 , form a
  • a 11 is Glu
  • a 15 is Lys
  • a 11 and A 15 together with A 12 , A 13 and A 14 , form a ring
  • Another embodiment of present invention is (vi) a compound of formula I or a pharm naacceeuuttiiccaallllyy aacccceeppttaabbllee ssaalltt tthheerreeooff,, wwhheerreeii:n
  • a 4 is Met or Phe(3-Cl), and all remaining substituents have the significances given before.
  • Another embodiment of present invention is (vii) a compound of formula I or a pharmaceutically acceptable salt thereof, wherein A is Lys, and all remaining substituents have the significances given before.
  • Another embodiment of present invention is (viii) a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the compound is a peptide truncated after A 14 , or A 16 , or A 17 .
  • Particular compounds of formula I, including their activity data and MS data are summarized in Table 1.
  • PASFCKPPE-NH 2 657.2(M+3H) +
  • Hcy4&Cys 13 E 10&K 14 ⁇ - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-PESFCKPPE-NH 2 ; ⁇ Hcy4&Cys 13, stapled-Hag 1 O&Ahp 14 ⁇ - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-P-Hag- SFC-Ahp-PPE-NH 2 ; ⁇ Hcy4&Cys 13, stapled-Hag 1 O&Hag 14 ⁇ - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-P-Hag- SFC-Hag-PPE-NH 2 ;
  • Hcy4&Cys 13 stapled-Hag 1 O&Hag 14 ⁇ - Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-P-Hag-SFC- Hag-PPE-NH 2 ;
  • Hcy4&Cys 13 stapled-Hag 1 O&Ahp 14 ⁇ - Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-P-Hag-SFC- Ahp-PPE-NH 2 .
  • the compounds of the present invention can be synthesized by any known conventional procedure for the formation of a peptide linkage between amino acids.
  • Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino group of an amino acids or fragment thereof having its carboxy group and other reactive groups protected and the free primary carboxy group of another amino acid or fragment thereof having its amino group or other reactive groups protected.
  • Such conventional procedures for synthesizing the novel compounds of the present invention include, for example, any solid phase peptide synthesis method.
  • the synthesis of the novel compounds can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principle of solid phase methods.
  • Such methods are disclosed in, for example, Merrifield, R. B., J. Amer. Chem. Soc. 85, 2149-2154 (1963); Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980), which are incorporated herein by reference.
  • certain reactive groups on the amino acid for example, the alpha-amino group, a hydroxy group, and/or reactive side chain groups, be protected to prevent a chemical reaction therewith.
  • This may be accomplished, for example, by reacting the tractive group with a protecting group which may later be removed.
  • the alpha amino group of an amino acid or fragment thereof may be protected to prevent a chemical reaction therewith while the carboxy group of that amino acid or fragment thereof reacts with another amino acid or fragment thereof to form a peptide bond.
  • This may be followed by the selective removal of the alpha amino protecting group to allow a subsequent reaction to take place at that site, for example with the carboxy group of another amino acid or fragment thereof.
  • Alpha amino groups may, for example, be protected by a suitable protecting group selected from aromatic urethane-type protecting groups, such as allyloxycarbonyl,
  • Fmoc is used for alpha amino protection.
  • Hydroxy groups of the amino acids may, for example, be protected by a suitable protecting group selected from benzyl (Bzl), 2,6-dichlorobenzyl (2,6 diCl-Bzl), and tert-butyl.
  • a suitable protecting group selected from benzyl (Bzl), 2,6-dichlorobenzyl (2,6 diCl-Bzl), and tert-butyl.
  • t-Bu may, for example, be used.
  • Epsilon-amino groups may, for example, be protected by a suitable protecting group selected from 2-chloro-benzyloxycarbonyl (2-Cl-Z), 2-bromo-benzyloxycarbonyl (2-Br-Z), allycarbonyl and t-butyloxycarbonyl (Boc).
  • Boc may, for example, be used.
  • Beta- and gamma-amide groups may, for example, be protected by a suitable protecting group selected from 4-methyltrityl (Mtt), 2,4,6-trimethoxybenzyl (Tmob), 4,4'- dimethoxydityl (Dod), bis-(4-methoxyphenyl)-methyl and trityl (Trt).
  • Trt may, for example, be used.
  • Indole groups may, for example, be protected by a suitable protecting group selected from formyl (For), mesityl-2-sulfonyl (Mts) and t-butyloxycarbonyl (Boc).
  • Boc may, for example, be used.
  • Imidazole groups may, for example, be protected by a suitable protecting group selected from benzyl (Bzl), t-butyloxycarbonyl (Boc), and trityl (Trt).
  • a suitable protecting group selected from benzyl (Bzl), t-butyloxycarbonyl (Boc), and trityl (Trt).
  • Trt may, for example, be used.
  • Solid phase synthesis may be commenced from C-terminal end of the peptide by coupling a protected alpha-amino acid to a suitable resin.
  • a suitable resin such as a starting material can be prepared by attaching an alpha-amino-protected amino acid by an ester linkage to a p- benzyloxybenzyl alcohol (Wang) resin, or by an amide bond between an Fmoc-linker, such as p-((R,S)-a-(l-(9H-fluoren-9-yl)methoxyformamido)-2,4-dimethyloxybenzyl)- phenoxyacetic acid (Rink linker), and a benzhydrylamine (BHA) resin.
  • Preparation of the hydroxymethyl resin is well known in the art.
  • peptide synthesis is microwave assisted.
  • Microwave assisted peptide synthesis allows for methods to be created that control a reaction at a set temperature for a set amount of time.
  • the amino acids or mimetic are coupled onto the Fmoc-linker-BHA resin using the Fmoc protected form of amino acid or mimetic, with 2-5 equivalents of amino acid and a suitable coupling reagent.
  • the resin may be washed and dried under vacuum.
  • Loading of the amino acid onto the resin may be determined by amino acid analysis of an aliquot of Fmoc-amino acid resin or by determination of Fmoc groups by UV analysis. Any unreacted amino groups may be capped by reacting the resin with acetic anhydride and diisopropylethylamine in methylene chloride.
  • deprotection of alpha amino protecting group and coupling are carried out to extend the peptide.
  • Deprotection of the alpha amino Fmoc protecting groups is realized under basic conditions, and piperidine in DMF (20-40% v/v) may be used for this purpose.
  • Coupling reaction is fulfilled in the presence of activating reagent, such as diisopropylcarbodiimide (DIC), N,N,N',N'-tetramethyl-0-(lH-benzotriazol-l-yl)uronium hexafluorophosphate
  • activating reagent such as diisopropylcarbodiimide (DIC), N,N,N',N'-tetramethyl-0-(lH-benzotriazol-l-yl)uronium hexafluorophosphate
  • HBTU 2-(7-aza-lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate
  • BOP benzotriazol-lyloxy-tri(dimethylamno)phosphonium hexafluorophospate
  • HATU 2-(7-aza-lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate
  • BOP benzotriazol-lyloxy-tri(dimethylamno)phosphonium hexafluorophospate
  • HATU 2-(7-aza-lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate
  • BOP benzotriazol-lyloxy-tri(dimethylamno)phosphonium hexafluor
  • alpha amino group of the final amino acid is deprotected from Fmoc protecting group, and may be capped by acetylation.
  • the peptide- resin is then treated with acidic conditions to release the peptide off the resin. And treatment with a mixture of TFA/H 2 0/TIS/DTT (93:3:3: 1) for 2 hours at room temperature is particular for the purpose.
  • the resin is then filtered off, and the filtrates may be precipitated in cold ethyl ether to give the crude peptide.
  • the disulfide bridge may be realized by variety of methods such those summarized by Annis, I. et al. chapt. 10 disulfide bond formation in peptides in Solid-Phase Peptide
  • the cyclization is realized on resin with iodine as the oxidating reagent, and the diselenide is then released from resin with TFA/H 2 0/TIS, and purified by preparative HPLC.
  • the present invention also relates to a process for the preparation of a compound of formula I:
  • a compound of formula I when manufactured according to the above process is also an object of the invention.
  • the invention also relates to a compound of formula I for use as therapeutically active substance.
  • compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments.
  • compounds of formula I may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
  • physiologically acceptable carriers i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
  • the pH of the formulation depends mainly on the particular use and the concentration of compound, but particularly ranges anywhere from about 3 to about 8.
  • a compound of formula I is formulated in an acetate buffer, at pH 5.
  • the compounds of formula I are sterile.
  • the compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aque
  • compositions are formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of
  • the "effective amount" of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to inhibit YAP interaction with transcriptional factor TEADs. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.
  • the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.001 to 100 mg/kg, with the typical initial range of compound used being 0.1 to 15 mg/kg/day.
  • the compounds of the invention may be administered by any suitable means, including topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral,
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
  • a typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient.
  • Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C, et al., Ansel's Pharmaceutical Dosage Forms and Dru Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005.
  • the formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
  • buffers stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing
  • An example of an aerosol formulation can be prepared by dissolving the compound, for example 5 to 400 mg, of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired.
  • a suitable buffer solution e.g. a phosphate buffer
  • a tonicifier e.g. a salt such sodium chloride
  • the solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants.
  • An embodiment therefore, includes a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or excipient.
  • Another embodiment includes a pharmaceutical composition comprising a compound of Formula I for use in the treatment of a hyperproliferative disease. Another embodiment includes a pharmaceutical composition comprising a compound of Formula I for use in the treatment of cancer.
  • the compounds of the invention inhibit the binding of YAP to transcription factors TEADs in the nucleus. Accordingly, the compounds of the invention are useful for reducing cell proliferation in particular cancer cells. Compounds of the invention are useful for reducing cell proliferation in cells that overexpress YAP. Alternatively, compounds of the invention are useful for reducing cell proliferation in cells in which the Hippo pathway is deregulated, for example, by YAP overexpression.
  • the compounds of the invention are useful for diseases associated with the interaction of YAP with TEAD, or overexpression of YAP.
  • the compounds of the invention are useful for diseases benefit from the inhibition of the interaction of YAP with TEAD.
  • the compounds can be used for the treatment of all cancer types which has Hippo pathway deregulated or fail to undergo apoptosis.
  • the invention relates to the use of compounds for the treatment of solid tumors. More particularly, the invention relates to the use of compounds for the treatment of liver cancer or gastric cancer.
  • the invention relates in particular to the use of compounds for the preparation of a medicament for the treatment of cancer, in particular, solid tumors, more particularly, liver cancer or gastric cancer.
  • Another embodiment includes a method of treating or preventing cancer in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of compounds, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof.
  • Suitable cancers for treatment include bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancer.
  • the compounds of the invention can be used in combination with small-molecule inhibitors such as tyrosine kinase inhibitors, serine/threonine kinase inhibitors, lipid kinase inhibitors, protein-protein inhibitors, etc., cytotoxic agents, radiotherapy, antibodies and cancer vaccines for the treatment of cancer.
  • small-molecule inhibitors such as tyrosine kinase inhibitors, serine/threonine kinase inhibitors, lipid kinase inhibitors, protein-protein inhibitors, etc.
  • cytotoxic agents such as tyrosine kinase inhibitors, serine/threonine kinase inhibitors, lipid kinase inhibitors, protein-protein inhibitors, etc.
  • the microwave assisted reactions were carried out in a Biotage Initiator 2.5.
  • the present representative peptides may be readily synthesized by any solution or solid phase methods for peptide synthesis, which methods are well-known to those skilled in the art.
  • the examples are provided for the purpose of illustration and are by no means to limit the scope of the present invention in any manner.
  • Fmoc amino acids were used with the following side chain protection: Fmoc- Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH, Fmoc-Thr(tBu)-OH.
  • the linear peptide obtained above was dissolved in a mixture of HOAc (2.5 mL) and DMSO (2.5 mL), which was added dropwise to a mixture of DMSO (2.5 mL) and H20 (42.5 mL). The resulting mixture was stirred at room temperature for 2 or 3 days until LCMS indicated the linear peptide was almost consumed, and then lyophilized.
  • the residue was purified by reverse phase preparative HPLC performed on a Waters AutoP purification System (Sample Manager 2767; Pump 2525; Detector: Micromass ZQ and UV 2487 at 214 nm) using Waters SunFireTM Prep-Cig (5 ⁇ , OBDTM 30x100 mm) column.
  • the title peptide was synthesized at 100 ⁇ scale according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Aib-OH.
  • the title peptide was synthesized at 100 ⁇ scale according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Aib-OH.
  • the title peptide was synthesized at 100 ⁇ scale according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Ala-OH.
  • the linear peptide was synthesized at 50 ⁇ scale according to the procedure described for Example 1, using following Fmoc protected amino acids, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)- OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)- OH, Fmoc-Met-OH, Fmoc-Val-OH.
  • Fmoc protected amino acids Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH
  • On-resin diselenide formation and cleavage After the assembly of the sequence, the resin-peptide was transferred to a round flask, and suspended in DCM (20 mL) cooled with ice-water bath. To it was added dropwise a solution of iodine (10 eq., 127 mg) in DCM (20 mL) solution. After addition, the reaction mixture was stirred for another 2 hours. The resin was filtered, washed DCM, and then dried. The cleavage was done by treating the peptide - resin with 10 mL of a mixture of TFA/H20/TIS (94:3:3) for 2 hours at room temperature.
  • the title peptide was synthesized according to the procedure described for Example 67 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)-OH, Fmoc-Val-OH .
  • the title peptide was synthesized according to the procedure described for Example 67 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)-OH, Fmoc-Val-OH, Fmoc-Phe(4-Cl)-OH.
  • the title peptide was synthesized according to the procedure described for Example 67 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Aib-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)-OH, Fmoc-Val-OH, Fmoc-Phe(4-Cl)-OH.
  • the linear peptide was assembled at 50 ⁇ scale on Rink Amide- AM resin according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH with Fmoc-Lys(Alloc)-OH and Fmoc-Glu(OAll)-OH at positions 4 and 8 (numbering from C terminus as the on-resin assembly goes), respectively.
  • On-resin lactam formation After completion of the sequence assembly, the peptide- resin was transferred to a reaction vessel and suspended in DCM (10 mL). To it were added morpholine (1 mL) and catalyst Pd(PPh 3 ) 4 (30 mg). The reaction mixture was flushed with nitrogen and sealed, and then stirred at room temperature for 2 hour to remove the side chain protection of Glu(OAll) and Lys (Alloc) simultaneously. The mixture was filtered, and the resin was washed with DCM (4 mL x3) and DMF (4 mL x 3).
  • On-resin lactamization was then performed in the presence of HATU (0.1 M, 4.0 eq.) and DIEA (0.2 M, 8.0 eq.) in DMF (2.0 mL) twice, each for 1 h. the resin was then filtered off, washed with DMF and DCM, and dried.
  • On-resin ring closing metathesis (RCM): The RCM was performed in a Biotage Initiator 2.5 microwave reactor. To a suspension of resin-peptide in 1,2-dichloroethane (10 mL) was added a solution of lithium chloride in DMF (1 mL, 0.4M), followed by addition of Grubbs catalyst 2nd (30 mg). The reaction mixture was flushed with nitrogen and sealed, then stirred at 100°C for 1 hour under microwave. After cooled to room temperature, the resin was filtered off, washed with DMF and DCM, and dried.
  • RCM On-resin ring closing metathesis
  • Example 78 YAP/TEAD competitive binding assay by Surface Plasmon Resonance (SPR)
  • the biological activity of the peptides of the invention can be determined using the assay described below.
  • YAP protein were dissolved in coupling buffer (10 mg/mL, in 10 mM sodium acetate, pH 4.0) and immobilized onto the CM5 sensor chip of Biacore T100 (GE Healthcare) as the ligand in 3,000 RU with 0.2 M N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) according to the standard primary amine-coupling procedures as briefly described below, the dextran matrix on the sensor chip surface was first activated with a mixture of EDC and NHS to give reactive succinimide esters.
  • coupling buffer 10 mg/mL, in 10 mM sodium acetate, pH 4.0
  • NHS N-hydroxysuccinimide
  • YAP protein in above mentioned buffer was then passed over the surface and immobilized onto the chip by reacting with the esters via its uncharged amino groups or other nucleophilic groups. After that, ethanolamine was injected to deactivate remaining active ester groups on the surface, and remove non-covalently bound ligand.
  • HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20) was used as the running buffer.
  • GST-TEADl 50 nM was pre-incubated at room temperature for 30 minutes with different concentrations of synthetic peptides.
  • reaction mixture was injected over the surfaces of the chip at a flow rate of 30 ⁇ , then regenerated by 10 mM NaOH for 10 s at a flow of 100 ⁇ .
  • Response units were measured at 7 s in the dissociation phase, percent inhibition was calculated from Equation
  • % inhibition 100 x ( RU G ST-TEADI-RU) / (RU G sT-TEADi-RUinhibitor)
  • RU is the specific binding signal for GST-TEADl protein in the presence of the inhibitor
  • RUmhibitor is the binding for inhibitor and the RU GST - TEAD I is the specific signal for the GST-TEADl alone.
  • IC 50 half maximal inhibitory concentration
  • the peptides of the present invention were tested for their capacity to inhibit YAP activity and activation as described herein.
  • the Examples were tested in the above assay and found to have IC 50 of about 0.02 ⁇ to about 2.00 ⁇ .
  • Particular peptides of formula I were found to have IC 50 of about 0.02 ⁇ to about 0.10 ⁇ .
  • a YAP peptide of formula I can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:
  • Example B A YAP peptide of formula I can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:

Abstract

The invention provides novel compounds having the general formula: wherein A0 to A19 and X are as described herein, compositions including the compounds and methods of using the compounds.

Description

YAP-TEAD inhibitors
The present invention relates to compounds useful for inhibiting the binding of YAP to TEAD. Accordingly, the present invention relates to compounds useful for therapy in a mammal, and in particular to inhibit proliferation of cancer cells that overexpress YAP.
FIELD OF THE INVENTION
Yes-associated protein (YAP) is a transcriptional co-activator regulated by Hippo pathway. In well-developed organs or tissues, YAP is phosphorylated by the Hippo pathway for cytoplasmic retention, ubiquitination and subsequent proteasomal degradation. During cell proliferation, organ development, or organ regeneration (for instance, after partial hepatectomy), the Hippo pathway is inactivated. This allows YAP to translocate into nucleus and interact with transcriptional factors (i.e. TEAD1, 2, 3 and 4) to activate gene expression for cell survival, differentiation and proliferation. (Harvey, K. F.; Zhang, X.; Thomas, D. M. Nat. Rev. Cancer 2013, 13, 246-257)
Deregulation of YAP is implicated in cancer. YAP has been identified as an oncogene in liver cancer by an oncogenomic study (Zender, L.; Spector, M. S.; Xue, W. et al, Cell, 2006, 125, 1253-1267, and Dong, J.; Feldmann, G.; Huang, J. et al. Cell, 2007, 130, 1120- 1133). Defective Hippo pathway and ectopic expression of YAP result in over-grown livers in transgenic mice. YAP promotes tumor growth by overriding the contact-contact inhibition because over-expression of YAP in subcutaneous tumors accelerated tumor growth, and oppositely short hairpin RNA (shRNA)-mediated knockdown of YAP slowed the growth. Clinically, YAP gene is amplified and over-expressed in liver cancer tumors. The over- expression is associated with the dismal clinical outcomes of patients because it has been demonstrated that patients whose tumors over-expressed YAP survived shorter comparing to their counterparts without YAP over-expressions.
The functionality of YAP depends on the interaction of YAP with TEADs in nucleus (Zhao, B; Ye, X.; Xu, J. et al. 2008, Genes & Dev. 2008, 1962-1971; and Ota, M.; Sasaki, H. Development 2008, 135, 4059-4069), and YAP proved bind to all 4 TEAD proteins with similar efficiency (Vassilev, A.; Kaneko, K. J.; Shu, H. et al. Genes Dev. 2001, 15, 1229), therefore, disruption of such interaction is believed to abrogate the oncogenic property of YAP. The peptides of invention are designed to block the interaction, and can be further developed into drugs for cancers addicted to YAP over-expression.
Liver cancer, or hepatocellular carcinoma (HCC), is a lethal cancer with very limited treatment options. Surgical resection of tumor can be potentially curative, but most tumors are so advanced at the time of diagnosis that cannot be operated. Sorafenib, a multi-kinase inhibitor, has been approved for treating patients with advanced liver cancer. Despite the benefit of sorafenib in patients' survivals, there are considerable numbers of patients being not responsive towards or intolerable to sorafenib. The incidence rate of liver cancer is exceptionally high in China and Southeast Asia. The number of new cases in Europe and America is also rapidly increasing. It therefore remains imperative to develop new therapeutics for liver cancer treatment.
SUMMARY OF THE INVENTION
Objects of the present invention are novel compounds of formula I, their manufacture, medicaments based on a compound in accordance with the invention and their production as well as the use of compounds of formula I for inhibiting the binding of YAP to TEAD. Accordingly, the compounds of formula I are useful for reducing cell proliferation in particular for the treatment of cancer.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
All peptide sequences mentioned herein are written according to the usual convention whereby the N-termianl amino acid is on the left and the C-termianl amino acids is one the right, unless noted otherwise. A short line between two amino acid residues indicated a peptide bond. Where the amino acid has isomeric forms, it is the L form of the amino acid that is represented unless otherwise expressly indicated.
For convenience in describing this invention, the conventional and nonconventional abbreviations for the various amino acids are used. These abbreviations are familiar to those skilled in the art, but for clarity are listed below: Asp=D=Aspartic Acid; Ala=A= Alanine; Arg=R=Arginine; Asn=N=Asparagine; Gly=G=Glycine; Glu=E=Glutamic acid; Gln=Q=Glutamine; His=H=Histidine;
Ile=I=Isoleucine; Leu=L- Leucine; Lys=K=Lysine; Met=M=Methionine;
Phe=F=Phenylalnine; Pro=P=Proline; Ser=S=Serine; Thr=T=Threonine; Trp=W=Tryptophan; Tyr=Y=Tyrosine; Cys=C=Cysteie; and Val=V=V aline.
Also for convenience, and readily known to one skilled in the art, the following abbreviations or symbols are used to represent the moieties, reagents and the like used in this invention:
Ahp (S)-2-Amino-hept-6-enoic acid
Ahe (S)-2-Amino-heptanoic acid
Aib Alpha-aminoisobutyric acid
Aoc (S)-2-Amino-octanoic acid
Cha Cyclohexyl alanine
Dbu L-2,4-Diaminobutyric acid
Flp Fluoroproline
Gph 4-Guanidinophenylalanine
Hag Homoallylglycine
Hie Homoleucine
Hyp Hydroxylproline
Nal(l) 1-Naphthyl alanine
Nal(2) 2-Naphthylalaline
Nle Norleucine
Orn Ornithine
Phe(2-Cl) 2-Cl-phenylalanine
Phe(3-Br) 3-Br-phenylalanine
Phe(3-Cl) 3-Cl-phenylalanine
Phe(4-Cl) 4-Cl-phenylalanine
Phe(4-F) 4-F-phenylalanine
Sec Selenocysteine
Hsc Homoselenocysteine
Hey Homocysteine
Tic 1 ,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid Trt Trityl
Mtt 4-Methyltrityl
Boc Tert-butoxycarbonyl
Fmoc 9-Fluorenylmethyloxycarbonyl
Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-Sulfonyl tBu tert-Butyl
Acm Acetamidomethyl
Mob Methoxylbenzyl
DMF dimethylformamide
DTT dithiothreitol
TIS trisisopropylsilane
TFA trifluoroacetic acid
HBTU N,N,N',N'-tetramethyl-0-(lH-benzotriazol- l-yl)uronium hexafluorophosphate
HATU 2-(7-aza- IH-benzotriazole- 1-yl)- 1 , 1 ,3,3- tetramethyluronium hexafluorophosphate
HOBt N-hydroxybenzotriazole
HOAt l-hydroxy-7-azabenzotriazole
μL· microliter
μΜ micromoles per liter
calcd calculated
ESI electron spray ionization
HPLC high performance liquid chromatography
g gram
LCMS liquid chromatography mass spectrometry
mg milligram
mmol millimole
min minute
mL milliliter
mM milliliter
mm millimeter
Ac20 acetic anhydride DCM dichloromethane
DIPEA Ν,Ν' -diisopropylethylamine
EDTA ethylenediaminetetraacetic acid
GST-TEAD1 Glutathione S-transferase (GST) fused TEAD1
HEPES 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic
ICso half maximal inhibitory concentration
RU resonance units
TEAD TEA domain family member
The compounds according to the present invention may exist in the form of their pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid- addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin R.J., et al., Organic Process Research & Development 2000, 4, 427-435; or in Ansel, H., et al., In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. Particular are the sodium salts of the compounds of formula I.
Compounds of the general formula I which contain one or several chiral centers can either be present as racemates, diastereomeric mixtures, or optically active single isomers. The racemates can be separated according to known methods into the enantiomers.
Particularly, diastereomeric salts which can be separated by crystallization are formed from the racemic mixtures by reaction with an optically active acid such as e.g. D- or L-tartaric acid, mandelic acid, malic acid, lactic acid or camphorsulfonic acid. INHIBITORS OF YAP-TEAD PROTEIN PROTEIN INTERACTION
The present invention provides (i) a compound of the formula I:
I X— X 1
A°-A1 -A2-A3-A4-A5-A6-A7-A8-A9-A1 °-A -A 2-A13- A14-A15-A16-A17-A18-A19
(I)
wherein
A0 is acetyl;
A1 is Thr, D-Thr or deleted;
A2 is Val;
A3 is Pro, 4R-Hyp, 35-Hyp or 4R-Flp;
A4 is Met, Trp, Phe, Phe(2-Cl), Phe(3-Cl), Phe(3-Br), Phe(4-Cl), Cha, Nle, Hie, Ahe, Nal(l) or Nal(2);
A5 is Hey or Hsc;
A6 is Leu, Lys or Arg;
A7 is Arg;
A is Lys, His, Ser, Thr, Glu, Asp, Leu, Pro, Nle, Dbu, Orn, D-Lys or alpha-Me-Lys;
A9 is Nle, Cha, Hie, Ahe or Aoc;
A10 is Pro or 4R-Flp;
A11 is Ala, Aib, Glu or Hag;
A12 is Ser;
A13 is Phe, Phe(2-Cl), Phe(4-F) or Phe(4-Cl);
A14 is Cys or Sec;
A15 is Lys, Hag, Ahp or deleted;
-A1 1-A12-A13- A14-A15- ' rij ' provided that when A is Glu, and A is Lys, A and A form a ring
-A11-A12-A13- A14-A15- when A11 is Hag, and A15 is Hag or Ahp, A11 and A15 form a ring c~c ;
A16 is Pro, 4R-Hyp, 35-Hyp, 4R-Flp, Tic or deleted;
A17 is Pro, 4R-Hyp, 35-Hyp or deleted;
A 18 is Glu, Lys, Arg, Phe(4-guanidino) or deleted;
A19 is NH2; X is S or Se;
or a pharmaceutically acceptable salt thereof.
Another embodiment of present invention is (ii) a compound of formula I, wherein A0 is acetyl;
A1 is Thr, D-Thr or deleted;
A2 is Val;
A3 is Pro, 4R-Hyp, 35-Hyp or 4R-Flp;
A4 is Met, Trp, Phe, Phe(2-Cl), Phe(3-Cl), Phe(3-Br), Phe(4-Cl), Cha, Nle, Hie, Ahe, Nal(l) or Nal(2);
A5 is Hey or Hsc;
A6 is Leu, Lys or Arg;
A7 is Arg;
A is Lys, His, Ser, Thr, Glu, Asp, Leu, Pro, Nle, Dbu, Orn, D-Lys or alpha-Me-Lys;
A9 is Nle, Cha, Hie, Ahe or Aoc;
A10 is Pro or 4R-Flp;
A11 is Ala, Aib, Glu or Hag;
A12 is Ser;
A13 is Phe, Phe(2-Cl), Phe(4-F) or Phe(4-Cl);
A14 is Cys or Sec;
A15 is Lys, Hag or Ahp;
-A1 1-A12-A13- A14-A15-
'— ril— ' provided that when A is Glu, and A is Lys, A and A form a ring
-A11-A12-A13- A14-A15- when A11 is Hag, and A15 is Hag or Ahp, A11 and A15 form a ring c~c ;
A16 is Pro, 4R-Hyp, 35-Hyp, 4R-Flp or Tic;
A17 is Pro, 4R-Hyp or 35-Hyp;
A 18 is Glu, Lys, Arg or Phe(4-guanidino);
A19 is NH2;
X is S or Se;
or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (iii) a compound of formula I or a pharmaceutically acceptable salt thereof, wherein A5 is Hey and A14 is Cys, and all re substituents have the significances given before.
Another embodiment of present invention is (iv) a compound of formula I or a pharm naacceeuuttiiccaallllyy aacccceeppttaabbllee ssaalltt tthheerreeooff,, wwhheerreeii:n A5 is Hsc and A14 is Sec, and all remaining substituents have the significances given before.
Another embodiment of present invention is (v) a compound of formula I or a pharmaceutically acceptable salt thereof, wherein A11 is Ala or Aib, and A15 is Lys;
or
A11 is Hag, and A15 is Hag or Ahp, and A11 and A15, together with A12, A13 and A14, form a
-A11-A12-A13- A14-A15- c
ring I =c 1
or
A11 is Glu, and A15 is Lys, and A11 and A15, together with A12, A13 and A14, form a ring
-A1 1 -A12- A13- A14-A15-
-N-
; and all remaining substituents have the significances given before.
Another embodiment of present invention is (vi) a compound of formula I or a pharm naacceeuuttiiccaallllyy aacccceeppttaabbllee ssaalltt tthheerreeooff,, wwhheerreeii:n A4 is Met or Phe(3-Cl), and all remaining substituents have the significances given before.
Another embodiment of present invention is (vii) a compound of formula I or a pharmaceutically acceptable salt thereof, wherein A is Lys, and all remaining substituents have the significances given before.
Another embodiment of present invention is (viii) a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the compound is a peptide truncated after A14, or A16, or A17. Particular compounds of formula I, including their activity data and MS data are summarized in Table 1.
Table 1: Sequence, MS data and activity data of particular compounds
Example
No. / ICso
Peptide sequence MS data
Sequence (μΜ) ID
{Hcy5, Cysl4}-Ac-TVPM-Hcy-LRK-Nle- 1036.1(M+2H)+,
1 0.24
PASFCKPPE-NH2 690.9(M+3H)+
{Hcy5, Cysl4}-Ac-(D-Thr)-VPM-Hcy-LRK- 1036.2(M+2H)+,
2 0.16
Nle-PASFCKPPE-NH2 691.1(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 985.8(M+2H)+,
3 0.15
PASFCKPPE-NH2 657.2(M+3H)+
{Hcy4, Cysl3}-Ac-V-(4R-Hyp)-M-Hcy-LRK- 993.3 (M+2H)+,
4 0.07
Nle-PASFCKPPE-NH2 662.7(M+3H)+
{Hcy4, Cysl3}-Ac-V-(3S-Hyp)-M-Hcy-LRK- 993.4 (M+2H)+,
5 0.08
Nle-PASFCKPPE-NH2 662.5(M+3H)+
{Hcy4, Cysl3}-Ac-V-(4R-Flp)-M-Hcy-LRK- 994.5 (M+2H)+,
6 0.08
Nle-PASFCKPPE-NH2 663.2(M+3H)+
{Hcy4, Cysl3}-Ac-V-(4R-Hyp)-Phe(3-Cl)-Hcy- 1018.3 (M+2H)+,
7 0.02
LRK-Nle-PASFCKPPE-NH2 679.4(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Trp-Hcy-LRK-Nle- 1013.5 (M+2H)+,
8 0.19
PASFCKPPE-NH2 675.7 (M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(2-Cl)-Hcy-LRK- 1010.7 (M+2H)+,
9 0.38
Nle-PASFCKPPE-NH2 674.1(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- 1011.4 (M+2H)+,
10 0.03
Nle-PASFCKPPE-NH2 674.1(M+3H)+
{Hcy4, Cysl3}-Ac-VP-Phe(3-Br)-Hcy-LRK- 1032.9 (M+2H)+,
11 0.03
Nle-PASFCKPPE-NH2 688.9(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(4-Cl)-Hcy-LRK- 1011.5 (M+2H)+,
12 0.53
Nle-PASFCKPPE-NH2 673.8(M+3H)+
13 { Hcy4, Cys 13 } - Ac- VP-Cha-Hcy-LRK-Nle- 997.0 (M+2H)+, 0.07 PASFCKPPE-NH2 664.6(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Nle-Hcy-LRK-Nle- 976.4 (M+2H)+,
0.34 PASFCKPPE-NH2 651.2(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Hle-Hcy-LRK-Nle- 983.4 (M+2H)+,
0.42 PASFCKPPE-NH2 655.9(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP- Ahe-Hcy-LRK-Nle- 983.3 (M+2H)+,
0.05 PASFCKPPE-NH2 655.9(M+3H)+
{ Hcy4, Cys 13 } - Ac - VP-Nal( 1 )-Hcy-LRK-Nle- 1018.2 (M+2H)+,
0.21 PASFCKPPE-NH2 679.2(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Nal(2)-Hcy-LRK-Nle- 1018.2 (M+2H)+,
0.52 PASFCKPPE-NH2 679.2(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPF-Hcy-LRK-Nle- 993.6 (M+2H)+,
0.35 PASFCKPPE-NH2 662.6(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-KRK-Nle-P- Aib- 1000.4 (M+2H)+,
0.29 SFCKPPE-NH2 666.8(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-RRK-Nle-P- Aib- 1014.0 (M+2H)+,
0.33 SFCKPPE-NH2 676.3(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-KRK-Nle- 992.9 (M+2H)+,
0.14 PASFCKPPE-NH2 662.2(M+3H)+
{Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(D- 1018.1 (M+2H)+,
0.45 Lys)-Nle-P-Aib-SFCKPPE-NH2 679.6(M+3H)+
{Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(D- 1024.7 (M+2H)+,
0.54 Lys)-Ahe-P-Aib-SFCKPPE-NH2 682.9(M+3H)+
{Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(Me- 1017.3 (M+2H)+,
0.44 K)-Nle-PASFCKPPE-NH2 678.6(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRE- 1011.1 (M+2H)+,
0.26 Nle-PASFCKPPE-NH2 674.7(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRH- 1015.0 (M+2H)+,
0.41 Nle-PASFCKPPE-NH2 677.0(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRL- 1003.2 (M+2H)+,
0.15 Nle-PASFCKPPE-NH2 669.0(M+3H)+ { Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LR-Dbu- 996.3 (M+2H)+,
0.09 Nle-PASFCKPPE-NH2 664.9(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LR-Orn- 1004.1 (M+2H)+,
0.07 Nle-PASFCKPPE-NH2 669.6(M+3H)+
{Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRS- 990.1 (M+2H)+,
0.26 Nle-PASFCKPPE-NH2 660.5(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRT- 997.6 (M+2H)+,
0.38 Nle-PASFCKPPE-NH2 665.2(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRD- 1004.3 (M+2H)+,
0.23 Nle-PASFCKPPE-NH2 669.9(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LR-Nle- 1003.3 (M+2H)+,
0.25 Nle-PASFCKPPE-NH2 669.2(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRP- 995.5 (M+2H)+,
0.21 Nle-PASFCKPPE-NH2 663.9(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LR-Orn- Ahe-P- 992.7 (M+2H)+,
0.24 Aib-SFCKPPE-NH2 662.2(M+3H)+
{Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-KR-Orn- 1011.1 (M+2H)+,
0.06 Nle-PASFCKPPE-NH2 674.6(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LR-Dbu- 1004.3 (M+2H)+,
0.15 Nle-P-Aib-SFCKPPE-NH2 669.6(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LR-Orn- 1010.7 (M+2H)+,
0.11 Nle-P-Aib-SFCKPPE-NH2 674.3(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Cha- 1005.2(M+2H)+,
0.15 PASFCKPPE-NH2 670.6(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK- Ahe- 992.9(M+2H)+,
0.05 PASFCKPPE-NH2 661.9(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Hle- 992.3(M+2H)+,
0.62 PASFCKPPE-NH2 662.0(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK- Aoc- 999.2(M+2H)+,
0.37 PASFCKPPE-NH2 666.6(M+3H)+
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK- Ahe-P- Aib- 999.3(M+2H)+,
0.22 SFCKPPE-NH2 666.6(M+3H)+ { Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- (4R- 994.4(M+2H)+,
0.55 Flp)-ASFCKPPE-NH2 663.3(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- 1017.6(M+2H)+,
0.04 Ahe-PASFCKPPE-NH2 678.8(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP- Ahe-Hcy-LRK- Ahe- 990.5(M+2H)+,
0.07 PASFCKPPE-NH2 660.6(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- 1017.8(M+2H)+,
0.07 Nle-P-Aib-SFCKPPE-NH2 678.8(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- 1024.7(M+2H)+,
0.06 Ahe-P-Aib-SFCKPPE-NH2 683.8(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle-PAS- 1003.3(M+2H)+,
0.17 Phe(4-Cl)-CKPPE-NH2 669.1(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle-PAS- 994.6(M+2H)+,
0.08 Phe(4-F)-CKPPE-NH2 663.6(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle-PAS- 1003.1(M+2H)+,
0.25 Phe(2-Cl)-CKPPE-NH2 669.0(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 994.2(M+2H)+,
0.11 PASFCK-(4R-Hyp)-PE-NH2 662.7(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 993.2(M+2H)+,
0.31 PASFCK-(3S-Hyp)-PE-NH2 663.2(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 1016.6(M+2H)+,
0.11 PASFCK-Tic-PE-NH2 678.0(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 994.5(M+2H)+,
0.26 PASFCK-(4R-Flp)-PE-NH2 663.2(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 993.6(M+2H)+,
0.06 PASFCKP-(4R-Hyp)-E-NH2 662.7(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 993.2(M+2H)+,
0.07 PASFCKP-(3S-Hyp)-E-NH2 662.8(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- 1018.7(M+2H)+,
0.03 Nle-PASFCKP-(4R-Hyp)-E-NH2 679.4(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 985.2(M+2H)+,
0.06 PASFCKPPK-NH2 657.1(M+3H)+ { Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 666.5(M+3H)+,
0.06 PASFCKPPR-NH2 500.0(M+4H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 1023.4(M+2H)+,
0.03 PASFCKPP-Gph-NH2 682.3(M+3H)+
{ Hcy4, Cys 13 } - Ac- VP-Trp-Hcy-LRK-Nle-
783.3(M+2H)+ 0.68 PASFC-NH2
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle-PAS-
768.7(M+2H)+ 0.84 Phe(4-F)-C-NH2
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 920.9(M+2H)+,
0.14 PASFCKPP-NH2 614.5(M+3H)+
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle- 872.9(M+2H)+,
0.48 PASFCKP-NH2 582.1(M+3H)+
{ Hsc4, Sec 13 } - Ac- VPM-Hsc-LRK-Nle-PASF- 1031.7(M+2H)+,
0.27 Sec-KPPE-NH2 688.9(M+3H)+
{ Hsc4, Sec 13 } - Ac- VPM-Hsc-LRK- Ahe-PASF- 1040.6(M+2H)+,
0.07 Sec-KPPE-NH2 693.6(M+3H)+
{ Hsc4, Sec 13 } - Ac- VP- Ahe-Hsc-LRK- Ahe- 1037.6(M+2H)+,
0.05 PASF-Sec-KPPE-NH2 691.8(M+3H)+
{ Hsc4, Sec 13 } -Ac- VP-Phe(3-Cl)-Hsc-LRK-Nle- 1057.9(M+2H)+,
0.07 PASF-Sec-KPPE-NH2 705.3(M+3H)+
{ Hsc4, Sec 13 } -Ac- VP-Phe(3-Cl)-Hsc-LRK-Nle- 1065.5(M+2H)+,
0.18 P-Aib-SF-Sec-KPPE-NH2 710.3(M+3H)+
{ Hcy4&Cys 13 , E 1 O&K 14 } - Ac- VPM-Hcy-LRK- 1005.3(M+2H)+,
0.19 Nle-PESFCKPPE-NH2 670.5(M+3H)+
{ Hcy4&Cys 13, El 0&K14 } -Ac- VP-Phe(3-Cl)- 1037.6(M+2H)+,
0.02 Hcy-LRK-Ahe-PESFCKPPE-NH2 691.9(M+3H)+
{ Hcy4&Cys 13 , stapled-Hag 10& Ahp 14 } - Ac- VP-
1022.3(M+2H)+, Phe(3-Cl)-Hcy-LRK-Ahe-P-Hag-SFC-Ahp-PPE- 0.03
681.8(M+3H)+
NH2
{ Hcy4&Cys 13 , stapled-Hag 1 O&Hag 14 } - Ac- VP-
1014.8 (M+2H)+, Phe(3-Cl)-Hcy-LRK-Ahe-P-Hag-SFC-Hag-PPE- 0.05
677.4(M+3H)+
NH2 { Hcy4&Cys 13 , stapled-Hag 1 O&Hag 14 } - Ac- VP-
1008.3 (M+2H)+,
76 Phe(3-Cl)-Hcy-LRK-Nle-P-Hag-SFC-Hag-PPE- 0.11
672.2(M+3H)+
NH2
{ Hcy4&Cys 13 , stapled-Hag 1 O&Ahp 14 } - Ac- VP-
1015.2 (M+2H)+,
77 Phe(3-Cl)-Hcy-LRK-Nle-P-Hag-SFC-Ahp-PPE- 0.06
677.2(M+3H)+
NH2
More particular compounds of formula I include the following:
{Hcy5, Cysl4}-Ac-(D-Thr)-VPM-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Br)-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Cha-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP- Ahe-Hcy-LRK-Nle-PASFCKPPE-NH2;
{Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(D-Lys)-Nle-P-Aib-SFCKPPE-NH2; {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(Me-K)-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LR-Dbu-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LR-Orn-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-KR-Orn-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Cha-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK- Ahe-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-P- Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-P- Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PAS-Phe(4-F)-CKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCKPPK-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCKPPR-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCKPP-Gph-NH2;
{Hsc4, Secl3}-Ac-VP-Ahe-Hsc-LRK-Ahe-PASF-Sec-KPPE-NH2;
{Hsc4, Secl3}-Ac-VP-Phe(3-Cl)-Hsc-LRK-Nle-P-Aib-SF-Sec-KPPE-NH2;
{ Hcy4&Cys 13 , E 10&K 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-PESFCKPPE-NH2; { Hcy4&Cys 13, stapled-Hag 1 O&Ahp 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-P-Hag- SFC-Ahp-PPE-NH2; { Hcy4&Cys 13, stapled-Hag 1 O&Hag 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-P-Hag- SFC-Hag-PPE-NH2;
{ Hcy4&Cys 13, stapled-Hag 1 O&Hag 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-P-Hag-SFC- Hag-PPE-NH2; and
{ Hcy4&Cys 13, stapled-Hag 1 O&Ahp 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-P-Hag-SFC- Ahp-PPE-NH2.
SYNTHESIS
The compounds of the present invention can be synthesized by any known conventional procedure for the formation of a peptide linkage between amino acids. Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino group of an amino acids or fragment thereof having its carboxy group and other reactive groups protected and the free primary carboxy group of another amino acid or fragment thereof having its amino group or other reactive groups protected.
Such conventional procedures for synthesizing the novel compounds of the present invention include, for example, any solid phase peptide synthesis method. In such a method the synthesis of the novel compounds can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principle of solid phase methods. Such methods are disclosed in, for example, Merrifield, R. B., J. Amer. Chem. Soc. 85, 2149-2154 (1963); Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980), which are incorporated herein by reference.
During the synthesis of peptides, it may be desired that certain reactive groups on the amino acid, for example, the alpha-amino group, a hydroxy group, and/or reactive side chain groups, be protected to prevent a chemical reaction therewith. This may be accomplished, for example, by reacting the tractive group with a protecting group which may later be removed. For example, the alpha amino group of an amino acid or fragment thereof may be protected to prevent a chemical reaction therewith while the carboxy group of that amino acid or fragment thereof reacts with another amino acid or fragment thereof to form a peptide bond. This may be followed by the selective removal of the alpha amino protecting group to allow a subsequent reaction to take place at that site, for example with the carboxy group of another amino acid or fragment thereof. Alpha amino groups may, for example, be protected by a suitable protecting group selected from aromatic urethane-type protecting groups, such as allyloxycarbonyl,
benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p- chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p- biphenyl-isopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and p-methoxy- benzyloxycarbonyl (Moz); and aliphatic urethane-type protecting groups, such as t- butyloxycarbonyl (Boc), diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, and allyloxycarbonyl. In an embodiment, Fmoc is used for alpha amino protection.
Hydroxy groups of the amino acids may, for example, be protected by a suitable protecting group selected from benzyl (Bzl), 2,6-dichlorobenzyl (2,6 diCl-Bzl), and tert-butyl. In an embodiment wherein a hydroxy group of tyrosine, serine, or threonine is intended to be protected, t-Bu may, for example, be used.
Epsilon-amino groups may, for example, be protected by a suitable protecting group selected from 2-chloro-benzyloxycarbonyl (2-Cl-Z), 2-bromo-benzyloxycarbonyl (2-Br-Z), allycarbonyl and t-butyloxycarbonyl (Boc). In an embodiment wherein an epsilon-amino acid group of lysine is intended to be protected, Boc may, for example, be used.
Beta- and gamma-amide groups may, for example, be protected by a suitable protecting group selected from 4-methyltrityl (Mtt), 2,4,6-trimethoxybenzyl (Tmob), 4,4'- dimethoxydityl (Dod), bis-(4-methoxyphenyl)-methyl and trityl (Trt). In an embodiment wherein an amide group of asparagine or glutamine is intended to be protected, Trt may, for example, be used.
Indole groups may, for example, be protected by a suitable protecting group selected from formyl (For), mesityl-2-sulfonyl (Mts) and t-butyloxycarbonyl (Boc). In an embodiment wherein the indole group of tryptophan is intended to be protected, Boc may, for example, be used.
Imidazole groups may, for example, be protected by a suitable protecting group selected from benzyl (Bzl), t-butyloxycarbonyl (Boc), and trityl (Trt). In an embodiment wherein the imidazole group of histidine is intended to be protected, Trt may, for example, be used.
Solid phase synthesis may be commenced from C-terminal end of the peptide by coupling a protected alpha-amino acid to a suitable resin. Such a starting material can be prepared by attaching an alpha-amino-protected amino acid by an ester linkage to a p- benzyloxybenzyl alcohol (Wang) resin, or by an amide bond between an Fmoc-linker, such as p-((R,S)-a-(l-(9H-fluoren-9-yl)methoxyformamido)-2,4-dimethyloxybenzyl)- phenoxyacetic acid (Rink linker), and a benzhydrylamine (BHA) resin. Preparation of the hydroxymethyl resin is well known in the art.
In an embodiment, peptide synthesis is microwave assisted. Microwave assisted peptide synthesis allows for methods to be created that control a reaction at a set temperature for a set amount of time.
Typically, the amino acids or mimetic are coupled onto the Fmoc-linker-BHA resin using the Fmoc protected form of amino acid or mimetic, with 2-5 equivalents of amino acid and a suitable coupling reagent. After coupling, the resin may be washed and dried under vacuum. Loading of the amino acid onto the resin may be determined by amino acid analysis of an aliquot of Fmoc-amino acid resin or by determination of Fmoc groups by UV analysis. Any unreacted amino groups may be capped by reacting the resin with acetic anhydride and diisopropylethylamine in methylene chloride.
After first amino acid attachment on resin, several repetitive cycles including
deprotection of alpha amino protecting group and coupling are carried out to extend the peptide. Deprotection of the alpha amino Fmoc protecting groups is realized under basic conditions, and piperidine in DMF (20-40% v/v) may be used for this purpose. Coupling reaction is fulfilled in the presence of activating reagent, such as diisopropylcarbodiimide (DIC), N,N,N',N'-tetramethyl-0-(lH-benzotriazol-l-yl)uronium hexafluorophosphate
(HBTU), 2-(7-aza-lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate (HATU) and benzotriazol-lyloxy-tri(dimethylamno)phosphonium hexafluorophospate (BOP). HBTU and HATU are particular here. Various additives may be used in the coupling reactions to optimize the reactions, and N-hydroxybenzotriazole (HOBt) and l-hydroxy-7- azabenzotriazole (HOAt) are particular.
After the peptide chain extension, alpha amino group of the final amino acid is deprotected from Fmoc protecting group, and may be capped by acetylation. The peptide- resin is then treated with acidic conditions to release the peptide off the resin. And treatment with a mixture of TFA/H20/TIS/DTT (93:3:3: 1) for 2 hours at room temperature is particular for the purpose. The resin is then filtered off, and the filtrates may be precipitated in cold ethyl ether to give the crude peptide.
The disulfide bridge may be realized by variety of methods such those summarized by Annis, I. et al. chapt. 10 disulfide bond formation in peptides in Solid-Phase Peptide
Synthesis, Volume 289 (Methods in Enzymology) Edited by Fields G. B. et al. Here DMSO is preferred as oxidating reagent (Tarn, J. P. et al. J. Am. Chem. Soc. 1991, 113, 6657). The crude peptide is dissolved in DMSO/AcOH/H20, and stirred at room temperature until the reaction comes to its end as monitored by LCMS. The miture is then lyophilized and submitted to preparative high performance liquid chromatography (HPLC) for purification to give the final product.
In case of diselenide, the cyclization is realized on resin with iodine as the oxidating reagent, and the diselenide is then released from resin with TFA/H20/TIS, and purified by preparative HPLC.
The present invention also relates to a process for the preparation of a compound of formula I:
I X— X 1
A°-A1 -A2-A3-A4-A5-A6-A7-A8-A9-A1 °-A -A 2-A13- A14-A15-A16-A17-A18-A19
(I)
wherein A0 to A19 and X are defined above unless otherwise indicated.
A compound of formula I when manufactured according to the above process is also an object of the invention.
PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION
The invention also relates to a compound of formula I for use as therapeutically active substance.
Another embodiment provides pharmaceutical compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments. In one example, compounds of formula I may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form. The pH of the formulation depends mainly on the particular use and the concentration of compound, but particularly ranges anywhere from about 3 to about 8. In one example, a compound of formula I is formulated in an acetate buffer, at pH 5. In another embodiment, the compounds of formula I are sterile. The compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.
Compositions are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors known to medical practitioners. The "effective amount" of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to inhibit YAP interaction with transcriptional factor TEADs. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.
In one example, the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.001 to 100 mg/kg, with the typical initial range of compound used being 0.1 to 15 mg/kg/day.
The compounds of the invention may be administered by any suitable means, including topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral,
subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
The compounds of the present invention may be administered in any convenient administrative form, e.g., solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
A typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C, et al., Ansel's Pharmaceutical Dosage Forms and Dru Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
An example of an aerosol formulation can be prepared by dissolving the compound, for example 5 to 400 mg, of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired. The solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants.
An embodiment, therefore, includes a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof. In a further embodiment includes a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or excipient.
Another embodiment includes a pharmaceutical composition comprising a compound of Formula I for use in the treatment of a hyperproliferative disease. Another embodiment includes a pharmaceutical composition comprising a compound of Formula I for use in the treatment of cancer.
INDICATIONS AND METHODS OF TREATMENT
The compounds of the invention inhibit the binding of YAP to transcription factors TEADs in the nucleus. Accordingly, the compounds of the invention are useful for reducing cell proliferation in particular cancer cells. Compounds of the invention are useful for reducing cell proliferation in cells that overexpress YAP. Alternatively, compounds of the invention are useful for reducing cell proliferation in cells in which the Hippo pathway is deregulated, for example, by YAP overexpression.
The compounds of the invention are useful for diseases associated with the interaction of YAP with TEAD, or overexpression of YAP.
The compounds of the invention are useful for diseases benefit from the inhibition of the interaction of YAP with TEAD.
More broadly, the compounds can be used for the treatment of all cancer types which has Hippo pathway deregulated or fail to undergo apoptosis. In particular, the invention relates to the use of compounds for the treatment of solid tumors. More particularly, the invention relates to the use of compounds for the treatment of liver cancer or gastric cancer.
The use of compounds for the preparation of medicaments useful in the treatment of cancer is an object of the invention.
The invention relates in particular to the use of compounds for the preparation of a medicament for the treatment of cancer, in particular, solid tumors, more particularly, liver cancer or gastric cancer.
Another embodiment includes a method of treating or preventing cancer in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of compounds, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof. Suitable cancers for treatment include bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancer.
COMBINATION THERAPY
The compounds of the invention can be used in combination with small-molecule inhibitors such as tyrosine kinase inhibitors, serine/threonine kinase inhibitors, lipid kinase inhibitors, protein-protein inhibitors, etc., cytotoxic agents, radiotherapy, antibodies and cancer vaccines for the treatment of cancer.
EXAMPLES
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
General Experimental Conditions
Intermediates and final peptides were purified by preparative HPLC on reversed phase column using X Bridge™ Perp C18 (5 μιη, OBD™ 30 x 100 mm) column or SunFire™ Perp Ci8 (5 μπι, OBD™ 30 x 100 mm) column.
LC/MS spectra were obtained using a MicroMass Plateform LC (Waters™ alliance 2795-ZQ2000). Standard LC/MS conditions were as follows: Acidic condition: A: 0.1% TFA in H2O; B: 0.1% TFA acid in acetonitrile; Basic condition: A: 0.01% NH3 H2O in H2O; B: acetonitrile. Mass spectra (MS): mass ion quoted is the positive mass ion (M+nH)+, n = 1, 2 or 3.
The microwave assisted reactions were carried out in a Biotage Initiator 2.5.
All reactions involving air-sensitive reagents were performed under an argon atmosphere. Reagents and chemicals were used as received from commercial suppliers without further purification unless otherwise noted.
All the amino acids and coupling reagents were purchased from GL Biochem in Shanghai, China, unless indicated otherwise. Fmoc-4R-Flp-OH from Bachem AG, Fmoc- Ahe-OH and Fmoc-Aoc-OH from Watanabe Chemical Industries, LTD., and Fmoc-Ahp-OH is prepared as reported in J. Am. Chem. Soc. 2010, 132, 2904-2906.
Preparative Examples
The present representative peptides may be readily synthesized by any solution or solid phase methods for peptide synthesis, which methods are well-known to those skilled in the art. The examples are provided for the purpose of illustration and are by no means to limit the scope of the present invention in any manner.
Example 1
Preparation of {H -Ac-TVPM-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000023_0001
Synthesis of the linear peptide: The linear peptide was assembled on a Prelude® parallel peptide synthesizer (Protein Technologies, Inc.) at the 50 μιηοΐ scale using Rink Amide-AM resin (0.5 mmol/g) unless otherwise indicated. Deprotection: 20%
piperidine/DMF (2.0 mL) for 3 x 5 min. After-deprotection washes: DMF top wash (2.0 mL) 4 x 30 sec. Coupling: Four-fold excess, 0.2 M AA in DMF (1.0 mL)/ mixture of 0.2M HATU and 0.4 M DIPEA in DMF (1.0 mL) for 2 x 20 min, except for Hey 1 x 45 min. After- coupling washes: DMF bottom wash (2.0 mL, 1 x 30 sec) followed by top wash (2.0 mL) 4 x30 sec. N-terminus acetylation: 0.4 M Ac20 in DMF (1 mL)/0.4 M DIPEA (1 mL) for 1 x 20 min. The Fmoc amino acids were used with the following side chain protection: Fmoc- Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH, Fmoc-Thr(tBu)-OH.
Cleavage of linear peptide: After the sequence was assembled, the resin was washed with DCM and dried, and transferred to a flask. The cleavage was done by treating the peptide-resin with 10 mL of a mixture of TFA/H20/TIS/DTT (93:3:3: 1) for 2 hours at room temperature. The resin was filtered off, and the filtrates were concentrated under reduced pressure and then precipitated in cold ethyl ether to give the crude linear peptide, which was used in the next step without further purification.
Disulfide formation and purification:
The linear peptide obtained above was dissolved in a mixture of HOAc (2.5 mL) and DMSO (2.5 mL), which was added dropwise to a mixture of DMSO (2.5 mL) and H20 (42.5 mL). The resulting mixture was stirred at room temperature for 2 or 3 days until LCMS indicated the linear peptide was almost consumed, and then lyophilized. The residue was purified by reverse phase preparative HPLC performed on a Waters AutoP purification System (Sample Manager 2767; Pump 2525; Detector: Micromass ZQ and UV 2487 at 214 nm) using Waters SunFire™ Prep-Cig (5 μιη, OBD™ 30x100 mm) column. The column was eluted with a linear gradient (30% to 50% of solution B over 30 min) at a flow rate of 40 ml/min (solution A is water containing 0.1% TFA; solution B is acetonitrile containing 0.1% TFA). Fractions were collected and checked by analytical HPLC. Fractions containing pure product were combined and lyophilized to yield 32 mg (26%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for
Figure imgf000024_0001
2070.6 found 1036.1(M+2H)+, 690.9(M+3H)+.
Example 2
Preparation of {Hcy5, Cysl4}-Ac-(D-Thr)-VPM-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000025_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH, Fmoc-D-Thr(tBu)-OH. HPLC purification and lyophilization gave 29 mg (24%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H152N24023S3, 2070.6 found
1036.2(M+2H)+, 691.1(M+3H)+.
Example 3
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000025_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH. HPLC purification and lyophilization gave 15 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H145N23O21S3, 1969.5 found 985.8(M+2H)+,
657.2(M+3H)+. Example 4
Preparation of {Hc 4, Cysl3}-Ac-V-(4R-Hyp)-M-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000026_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH, Fmoc-4R-Hyp(tBu)-OH. HPLC purification and lyophilization gave 16 mg (14%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H145N23O22S3, 1985.5 found 993.3 (M+2H)+, 662.7(M+3H)+.
Example 5
Preparation of {Hc 4, Cysl3}-Ac-V-(3S-Hyp)-M-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000026_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH, Fmoc-3S-Hyp(tBu)-OH.
HPLC purification and lyophilization gave 15 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H145N23O22S3, 1985.5 found 993.4 (M+2H)+, 662.5(M+3H)+.
Example 6
Preparation of {Hc 4, Cysl3}-Ac-V-(4R-Flp)-M-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000027_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH, Fmoc-4R-Flp-OH (Bachem A G). HPLC purification and lyophilization gave 14 mg (12%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for Cg9H144FN23021S3, 1987.5 found 994.5 (M+2H)+, 663.2(M+3H)+.
Example 7
Preparation of {Hcy4, Cysl3}-Ac-V-(4R-Hyp)-Phe(3-Cl)-Hcy-LRK-Nle- PASFCKPPE-NH2
Figure imgf000027_0002
The title peptide was synthesized according to the procedure described for Example 1 Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe- OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-4R-Hyp(tBu)-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 19 mg (16%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H144CLN23022S2, 2035.9 found 1018.3 (M+2H)+, 679.4(M+3H)+.
Example 8
Preparation of Hcy4, Cysl3}-Ac-VP-Trp-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000028_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH. HPLC purification and lyophilization gave 16 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C95H146N24O21S2, 2024.5 found 1013.5 (M+2H)+, 675.7 (M+3H)+.
Example 9
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(2-Cl)-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000028_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe(2-Cl)-OH. HPLC purification and lyophilization gave 53 mg (45%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H144C1N23021S2, 2019.9 found 1010.7 (M+2H)+, 674.1(M+3H)+.
Example 10
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000029_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 52 mg (44%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H144C1N23021S2, 2019.9 found 1011.4 (M+2H)+, 674.1(M+3H)+.
Example 11
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Br)-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000030_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe(3-Br)-OH. HPLC purification and lyophilization gave 54 mg (45%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93Hi44BrN23021S2, 2064.4 found 1032.9 (M+2H)+, 688.9(M+3H)+.
Example 12
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(4-Cl)-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000030_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe(4-Cl)-OH. HPLC purification and lyophilization gave 54 mg (46%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H144C1N23021S2, 2019.9 found 1011.5 (M+2H)+, 673.8(M+3H)+. Example 13
Preparation of {Hc 4, Cysl3}-Ac-VP-Cha-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000031_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Cha-OH. HPLC purification and lyophilization gave 54 mg (46%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H15iN23021S2, 1991.5 found 997.0 (M+2H)+,
664.6(M+3H)+.
Example 14
Preparation of {Hc 4, Cysl3}-Ac-VP-Nle-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000031_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH. HPLC purification and lyophilization gave 30 mg (26%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C9oHi47N23021S2, 1951.4 found 976.4 (M+2H)+, 651.2(M+3H)+. Example 15
Preparation of {Hc 4, Cysl3}-Ac-VP-Hle-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000032_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Hle-OH. HPLC purification and lyophilization gave 29 mg (25%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C9iH149N23021S2, 1965.5 found 983.4 (M+2H)+,
655.9(M+3H)+.
Example 16
Preparation of {Hc 4, Cysl3}-Ac-VP-Ahe-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000032_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Ahe-OH. HPLC purification and lyophilization gave 34 mg (30%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C91H149N23O21S2, 1965.5 found 983.3 (M+2H)+,
655.9(M+3H)+.
Example 17
Preparation of {Hc 4, Cysl3}-Ac-VP-Nal(l)-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000033_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Nal(l)-OH. HPLC purification and lyophilization gave 37 mg (31 ) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C97Hi47N23021S2, 2035.5 found 1018.2 (M+2H)+, 679.2(M+3H)+.
Example 18
Preparation of {Hc 4, Cysl3}-Ac-VP-Nal(2)-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000033_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Nal(2)-OH. HPLC purification and lyophilization gave 39 mg (33%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C97H147N23O21S2, 2035.5 found 1018.2 (M+2H)+, 679.2(M+3H)+.
Example 19
Preparation of {Hc 4, Cysl3}-Ac-VPF-Hcy-LRK-Nle-PASFCKPPE-NH2
Figure imgf000034_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH. HPLC purification and lyophilization gave 15 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H145N2302iS2, 1985.5 found 993.6 (M+2H)+,
662.6(M+3H)+.
Example 20
Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-KRK-Nle-P-Aib-SFCKPPE-NH2
Figure imgf000034_0002
The title peptide was synthesized at 100 μηιοΐ scale according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Aib-OH. HPLC purification and lyophilization gave 69 mg (28%) of the title peptide in TFA salt form as white amorphous powder. LCMS- ESI m/e Calcd for C9oH148N24021S3, 1998.5 found 1000.4 (M+2H)+, 666.8(M+3H)+.
Example 21
Preparation of { Hcy4, Cysl3}-Ac-VPM-Hcy-RRK-Nle-P-Aib-SFCKPPE-NH2
Figure imgf000035_0001
The title peptide was synthesized at 100 μιηοΐ scale according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Aib-OH. HPLC purification and lyophilization gave 52 mg (21%) of the title peptide in TFA salt form as white amorphous powder. LCMS- ESI m/e Calcd for C9oHi48N26021S3, 2026.5 found 1014.0 (M+2H)+, 676.3(M+3H)+.
Example 22
Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-KRK-Nle-PASFCKPPE-NH2
Figure imgf000035_0002
The title peptide was synthesized at 100 μηιοΐ scale according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Ala-OH. HPLC purification and lyophilization gave 54 mg (22%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H146N24021S3, 1984.5 found 992.9
(M+2H)+, 662.2(M+3H)+.
Example 23
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(D-Lys)-Nle-P-Aib- SFCKPPE-NH2
Figure imgf000036_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 8 mg (7%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C94H146C1N23021S2, 2033.9 found 1018.1 (M+2H)+, 679.6(M+3H)+.
Example 24
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(D-Lys)-Ahe-P-Aib- SFCKPPE-NH2
Figure imgf000037_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 15 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C95H148C1N23021S2, 2047.96 found 1024.7 (M+2H)+, 682.9(M+3H)+.
Example 25
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(Me-K)-Nle-PASFCKPPE-
NH2
Figure imgf000037_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-alpha-Methyl-Lys(Boc)-OH, Fmoc- Phe(3-Cl)-OH. HPLC purification and lyophilization gave 17 mg (14%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C94H146C1N23021S2, 2033.9 found 1017.3 (M+2H)+, 678.6(M+3H)+. Example 26
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRE-Nle-PASFCKPPE-NH2
Figure imgf000038_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 30 mg (27%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C92Hi39ClN22023S2, 2020.9 found 1011.1 (M+2H)+, 674.7(M+3H)+.
Example 27
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRH-Nle-PASFCKPPE-NH2
Figure imgf000038_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 27 mg (23%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H139CIN24Q21S2, 2028.9 found 1015.0 (M+2H)+, 677.0(M+3H)+.
Example 28
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRL-Nle-PASFCKPPE-NH2
Figure imgf000039_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 22 mg (20%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93Hi43ClN22021S2, 2004.9 found 1003.2 (M+2H)+, 669.0(M+3H)+.
Example 29
Preparation of {Hc 4, Cys l3}-Ac-VP-Phe(3-Cl)-Hcy-LR-Dbu-Nle-PASFCKPPE-NH2
Figure imgf000039_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Abu(Boc)-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 29 mg (25%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C91H14oClN23021S2, 1991.9 found 996.3 (M+2H)+, 664.9(M+3H)+.
Example 30
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-Orn-Nle-PASFCKPPE-NH2
Figure imgf000040_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 28 mg (24%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C92H142C1N23021S2, 2005.9 found 1004.1 (M+2H)+, 669.6(M+3H)+.
Example 31
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRS-Nle-PASFCKPPE-NH2
Figure imgf000040_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 26 mg (24%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C90H137CI 22O22S2, 1978.8 found 990.1 (M+2H)+, 660.5(M+3H)+.
Example 32
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRT-Nle-PASFCKPPE-NH2
Figure imgf000041_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 22 mg (20%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C91H139C1N22022S2, 1992.8 found 997.6 (M+2H)+, 665.2(M+3H)+.
Example 33
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRD-Nle-PASFCKPPE-NH2
Figure imgf000042_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 25 mg (22%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C91H137C1N22023S2, 2006.8 found 1004.3 (M+2H)+, 669.9(M+3H)+.
Example 34
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-Nle-Nle-PASFCKPPE-NH2
Figure imgf000042_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 14 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H143ClN2202iS2, 2004.9 found 1003.3 (M+2H)+, 669.2(M+3H)+. Example 35
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRP-Nle-PASFCKPPE-NH2
Figure imgf000043_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 16 mg (14%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C92Hi39ClN22021S2, 1998.9 found 995.5 (M+2H)+, 663.9(M+3H)+.
Example 36
Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-LR-Orn-Ahe-P-Aib-SFCKPPE-NH2
Figure imgf000043_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Orn(Boc)-OH, Fmoc-Aib-OH. HPLC purification and lyophilization gave 9 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C9oH147ClN23021S3, 1983.5 found 992.7 (M+2H)+, 662.2(M+3H)+.
Example 37
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-KR-Orn-Nle-PASFCKPPE-NH2
Figure imgf000044_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 18 mg (15%) of the title peptide in TFA salt form as white amorphous powder. LCMS- ESI m/e Calcd for C92H143C1N24021S2, 2020.9 found 1011.1 (M+2H)+, 674.6(M+3H)+.
Example 38
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-Dbu-Nle-P-Aib-SFCKPPE-
NH2
Figure imgf000044_0002
The title peptide was synthesized according to the procedure described for Example 1 g Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Abu(Boc)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Aib-OH. HPLC purification and lyophilization gave 19 mg (16%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C92H142C1N23021S2, 2005.8 found 1004.3 (M+2H)+, 669.6(M+3H)+.
Example 39
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-Orn-Nle-P-Aib-SFCKPPE-
NH2
Figure imgf000045_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Orn(Boc)-OH, Fmoc-Aib-OH. HPLC purification and lyophilization gave 23 mg (19%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H144C1N23021S2, 2019.9 found 1010.7 (M+2H)+, 674.3(M+3H)+.
Example 40
Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-LRK-Cha-PASFCKPPE-NH2
Figure imgf000046_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Cha-OH. HPLC purification and lyophilization gave 21 mg (18%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C92H149N23O21S3, 2009.6 found 1005.2(M+2H)+,
670.6(M+3H)+.
Example 41
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Ahe-PASFCKPPE-NH2
Figure imgf000046_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH. HPLC purification and lyophilization gave 40 mg (34%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C90H147N23O21S3, 1983.5 found 992.9(M+2H)+,
661.9(M+3H)+. Example 42
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Hle-PASFCKPPE-NH2
Figure imgf000047_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Hle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH. HPLC purification and lyophilization gave 33 mg (28%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C90H147N23O21S3, 1983.5 found 992.3(M+2H)+,
662.0(M+3H)+.
Example 43
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Aoc-PASFCKPPE-NH2
Figure imgf000047_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Aoc-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH. HPLC purification and lyophilization gave 29 mg (31 ) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C91H149N23O21S3, 1997.5 found 999.2(M+2H)+,
666.6(M+3H)+.
Example 44
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Ahe-P-Aib-SFCKPPE-NH2
Figure imgf000048_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Aib-OH. HPLC purification and lyophilization gave 26 mg (28%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for CnH^N^O^, 1997.5 found 999.3(M+2H)+, 666.6(M+3H)+.
Example 45
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-(4R-Flp)-ASFCKPPE-NH2
Figure imgf000048_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-4R-Flp-OH. HPLC purification and lyophilization gave 13 mg (11%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H144FN23021S3, 1987.5 found 994.4(M+2H)+, 663.3(M+3H)+.
Example 46
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRK-Ahe-PASFCKPPE -NH2
Figure imgf000049_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH. HPLC purification and lyophilization gave 20 mg (17%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C94H146C1N23021S2, 2033.9 found 1017.6(M+2H)+, 678.8(M+3H)+.
Example 47
Preparation of {Hc 4, Cysl3}-Ac-VP-Ahe-Hcy-LRK-Ahe-PASFCKPPE -NH2
Figure imgf000049_0002
The title peptide was synthesized according to the procedure described for Example 1 g Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH. HPLC purification and lyophilization gave 21 mg (18%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C92H15iN2302iS2, 1979.5 found 990.5(M+2H)+, 660.6(M+3H)+.
Example 48
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRK-Nle-P-Aib-SFCKPPE -
NH2
Figure imgf000050_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Aib-OH. HPLC purification and lyophilization gave 27mg (17%) of the title peptide in TFA salt form as white amorphous powder. LCMS- ESI m/e Calcd for C94Hi46ClN23021S2, 2033.9 found 1017.8(M+2H)+, 678.8(M+3H)+.
Example 49
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LRK-Ahe-P-Aib-SFCKPPE -
NH2
Figure imgf000050_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Aib-OH. HPLC purification and lyophilization gave 29 mg (24%) of the title peptide in TFA salt form as white amorphous powder. LCMS- ESI m/e Calcd for C94H146ClN2302iS2, 2048.0 found 1024.7(M+2H)+, 683.8(M+3H)+.
Example 50
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PAS-Phe(4-Cl)-CKPPE-NH2
Figure imgf000051_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Phe(4-Cl)-OH. HPLC purification and lyophilization gave 16 mg (14%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89Hi44ClN23021S3, 2003.9 found 1003.3(M+2H)+, 669.1(M+3H)+.
Example 51
Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PAS-Phe(4-F)-CKPPE-NH2
Figure imgf000052_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Phe(4-F)-OH. HPLC purification and lyophilization gave 18 mg (16%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for
Figure imgf000052_0002
1987.5 found 994.6(M+2H)+,
663.6(M+3H)+.
Example 52
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PAS-Phe(2-Cl)-CKPPE-NH2
Figure imgf000052_0003
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Phe(2-F)-OH. HPLC purification and lyophilization gave 18 mg (15%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for
Figure imgf000052_0004
2003.9 found 1003.1(M+2H)+, 669.0(M+3H)+. Example 53
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCK-(4R-Hyp)-PE-NH2
Figure imgf000053_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-4R-Hyp(Boc)-OH. HPLC purification and lyophilization gave 14 mg (12%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H145N23022S3, 1985.5 found 994.2(M+2H)+, 662.7(M+3H)+.
Example 54
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCK-(3S-Hyp)-PE-NH2
Figure imgf000053_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-3R-Hyp(Boc)-OH. HPLC purification and lyophilization gave 14 mg (12%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H145N23022S3, 1985.5 found 993.2(M+2H)+, 663.2(M+3H)+.
Example 55
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCK-Tic-PE-NH2
Figure imgf000054_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Tic-OH. HPLC purification and lyophilization gave 19 mg (16%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C^H^N^O^Ss, 2031.5 found
1016.6(M+2H)+, 678.0(M+3H)+.
Example 56
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCK-(4R-Flp)-PE-NH2
Figure imgf000054_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-4R-Flp-OH. HPLC purification and lyophilization gave 15 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for Cg9H144N23021S3, 1987.5 found
994.5(M+2H)+, 663.2(M+3H)+.
Example 57
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCKP-(4R-Hyp)-E-NH2
Figure imgf000055_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-4R-Hyp(Boc)-OH. HPLC purification and lyophilization gave 17 mg (15%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H145N23022S3, 1985.5 found 993.6(M+2H)+, 662.7(M+3H)+.
Example 58
Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCKP-(3S-Hyp)-E-NH2
Figure imgf000055_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-3R-Hyp(Boc)-OH.
HPLC purification and lyophilization gave 17 mg (15%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H145N23022S3, 1985.5 found 993.2(M+2H)+, 662.8(M+3H)+.
Example 59
Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-F)-Hcy-LRK-Nle-PASFCKP-(4R-Hyp)- E-NH2
Figure imgf000056_0001
The title peptide was synthesized according to the procedure described for Example 1 usingFmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-4R-Hyp(Boc)-OH . HPLC purification and lyophilization gave 17 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H144C1N23022S2, 2035.9 found 1018.7(M+2H)+, 679.4(M+3H)+.
Example 60
Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCKPPK-NH2
Figure imgf000057_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH. HPLC purification and lyophilization gave 25 mg (21%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C9oHi5oN24019S3, 1968.5 found 985.2(M+2H)+, 657.1(M+3H)+.
Example 61
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCKPPR-NH2
Figure imgf000057_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH. HPLC purification and lyophilization gave 9 mg (7%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for
Figure imgf000057_0003
1996.6 found 666.5(M+3H)+, 500.0(M+4H+).
Example 62
Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCKPP-Gph-NH2
Figure imgf000058_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Gph(pbf)-OH. HPLC purification and lyophilization gave 33 mg (26%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C94Hi5oN26019S3, 2044.6 found 1023.4(M+2H)+, 682.3(M+3H)+.
Example 63
Preparation of {Hcy4, C sl3}-Ac-VPW-Hcy-LRK-Nle-PASFC-NH2
Figure imgf000058_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH. HPLC purification and lyophilization gave 20 mg (21%) of the title peptide in TFA salt form as white amorphous powder. LCMS- ESI m/e Calcd for C74H1 13N19015S2, 1573.0 found 783.3(M+2H)+.
Example 64 Preparation of {Hcy4, Cysl3}-Ac-VPM-Hcy-LRK-Nle-PAS-Phe(4-F)-C-NH2
Figure imgf000059_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe(4-F)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH. HPLC purification and lyophilization gave 15 mg (16%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for CeeHmFN^OisSs, 1535.9 found 768.7(M+2H)+.
Example 65
Preparation of {Hcy4, C sl3}-Ac-VPM-Hcy-LRK-Nle-PASFCKPP-NH2
Figure imgf000059_0002
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH. HPLC purification and lyophilization gave 17 mg (16%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C84H138N22018S3, 1840.4 found 920.9(M+2H)+, 614.5(M+3H)+.
Example 66 Preparation of {Hcy4 Cysl3}-Ac-VPM-Hcy-LRK-Nle-PASFCKP-NH2
Figure imgf000060_0001
The title peptide was synthesized according to the procedure described for Example 1 using Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc- Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Met-OH. HPLC purification and lyophilization gave 38 mg (36%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C79H131N21O17S3, 1743.3 found 872.9(M+2H)+, 582.1(M+3H)+.
Example 67
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hsc-LRK-Nle-PASF-Sec-KPPE-NH2
Figure imgf000060_0002
The linear peptide was synthesized at 50 μιηοΐ scale according to the procedure described for Example 1, using following Fmoc protected amino acids, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)- OH, Fmoc-Ala-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)- OH, Fmoc-Met-OH, Fmoc-Val-OH.
On-resin diselenide formation and cleavage: After the assembly of the sequence, the resin-peptide was transferred to a round flask, and suspended in DCM (20 mL) cooled with ice-water bath. To it was added dropwise a solution of iodine (10 eq., 127 mg) in DCM (20 mL) solution. After addition, the reaction mixture was stirred for another 2 hours. The resin was filtered, washed DCM, and then dried. The cleavage was done by treating the peptide - resin with 10 mL of a mixture of TFA/H20/TIS (94:3:3) for 2 hours at room temperature. The resin was filtered off, and the filtrates were concentrated under reduced pressure and then precipitated in cold ethyl ether to give the crude diselenide, which was purified according to similar procedure described for example 1. 41mg (34%) of the title diselenide was obtained in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C89H145N2302iSSe2, 2063.3 found 1031.7(M+2H)+, 688.9(M+3H)+.
Example 68
Preparation of {Hc 4, Cysl3}-Ac-VPM-Hsc-LRK-Ahe-PASF-Sec-KPPE-NH2
Figure imgf000061_0001
The title peptide was synthesized according to the procedure described for Example 67 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)-OH, Fmoc-Met-OH, Fmoc-Val-OH . HPLC purification and lyophilization gave 29 mg (24%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C9oH147N2302iSSe2, 2077.3 found
1040.6(M+2H)+, 693.6(M+3H)+.
Example 69
Preparation of {Hcy4, Cysl3}-Ac-VP-Ahe-Hsc-LRK-Ahe-PASF-Sec-KPPE-NH2
Figure imgf000062_0001
The title peptide was synthesized according to the procedure described for Example 67 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)-OH, Fmoc-Val-OH . HPLC purification and lyophilization gave 23 mg (19%) of the title peptide in TFA salt form as white amorphous powder. LCMS- ESI m/e Calcd for C92Hi5iN23021Se2, 2073.3 found 1037.6(M+2H)+, 691.8(M+3H)+.
Example 70
Preparation of {Hc 4, Cysl3}-Ac-VP-Phe(3-Cl)-Hsc-LRK-Nle-PASF-Sec-KPPE-NH2
Figure imgf000062_0002
The title peptide was synthesized according to the procedure described for Example 67 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)-OH, Fmoc-Val-OH, Fmoc-Phe(4-Cl)-OH. HPLC purification and lyophilization gave 16 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C93H144N23021Se2, 2113.7 found 1057.9(M+2H)+, 705.3(M+3H)+.
Example 71 Preparation of {Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hsc-LRK-Nle-P-Aib-SF-Sec-KPPE-
NH2
Figure imgf000063_0001
The title peptide was synthesized according to the procedure described for Example 67 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Sec(Mob)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Aib-OH, Fmoc-Ahe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hsc(Mob)-OH, Fmoc-Val-OH, Fmoc-Phe(4-Cl)-OH. HPLC purification and lyophilization gave 26 mg (21%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C94H146N23021Se2, 2127.7 found 1065.5(M+2H)+, 710.3(M+3H)+.
Example 72
Preparation of {Hcy4 -Ac-VPM-Hcy-LRK-Nle-PESFCKPPE-NH2
Figure imgf000063_0002
The linear peptide was assembled at 50 μιηοΐ scale on Rink Amide- AM resin according to the procedure described for Example 1 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Met-OH, Fmoc-Val-OH with Fmoc-Lys(Alloc)-OH and Fmoc-Glu(OAll)-OH at positions 4 and 8 (numbering from C terminus as the on-resin assembly goes), respectively. On-resin lactam formation: After completion of the sequence assembly, the peptide- resin was transferred to a reaction vessel and suspended in DCM (10 mL). To it were added morpholine (1 mL) and catalyst Pd(PPh3)4 (30 mg). The reaction mixture was flushed with nitrogen and sealed, and then stirred at room temperature for 2 hour to remove the side chain protection of Glu(OAll) and Lys (Alloc) simultaneously. The mixture was filtered, and the resin was washed with DCM (4 mL x3) and DMF (4 mL x 3). On-resin lactamization was then performed in the presence of HATU (0.1 M, 4.0 eq.) and DIEA (0.2 M, 8.0 eq.) in DMF (2.0 mL) twice, each for 1 h. the resin was then filtered off, washed with DMF and DCM, and dried.
The cleavage, disulfide formation and purification followed the procedures described in example 1. And 5 mg (5%) of the final bicyclic peptide was obtained peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C^H^N^C^S^ 2009.5 found 1005.3(M+2H)+, 670.5(M+3H)+.
Example 73
Preparation of {Hcy4&Cysl3, E10&K14}-Ac-VP-Phe(3-Cl)-Hcy-LRK-Ahe- PESFCKPPE-NH2
Figure imgf000064_0001
The title peptide was synthesized according to the procedure described for Example 72 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc- Hcy(Trt)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Val-OH, Fmoc-Lys(Alloc)-OH and Fmoc- Glu(OAll)-OH. HPLC purification and lyophilization gave 6 mg (6%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C96H146C1N23022S2, 2074.0 found 1037.6(M+2H)+, 691.9(M+3H)+. Example 74
Preparation of {Hcy4&Cysl3, stapled-HaglO&Ahpl4}-Ac-VP-Phe(3-Cl)-Hcy-LRK- Ahe-P-Hag-SFC-Ahp-PPE-
Figure imgf000065_0001
Assembly of the linear peptide: The on-resin assembly was performed at 50 umol scale according to the procedure described for Example 1, using following Fmoc protected amino acids, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ahp-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe- OH, Fmoc-Ser(tBu)-OH, Fmoc-Ahe-OH, Fmoc-Hag-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Val-OH.
On-resin ring closing metathesis (RCM): The RCM was performed in a Biotage Initiator 2.5 microwave reactor. To a suspension of resin-peptide in 1,2-dichloroethane (10 mL) was added a solution of lithium chloride in DMF (1 mL, 0.4M), followed by addition of Grubbs catalyst 2nd (30 mg). The reaction mixture was flushed with nitrogen and sealed, then stirred at 100°C for 1 hour under microwave. After cooled to room temperature, the resin was filtered off, washed with DMF and DCM, and dried.
The cleavage, disulfide formation and purification followed the procedures described in example 1. And 8 mg (7%) of the final bicyclic peptide was obtained peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C96H145ClN2202iS2, 2042.9 found 1022.3(M+2H)+, 681.8(M+3H)+.
Example 75
Preparation of {Hcy4&Cysl3, stapled-HaglO&Hagl4}-Ac-VP-Phe(3-Cl)-Hcy-LRK- Ahe-P-Hag-SFC-Hag-PPE-NH2
Figure imgf000066_0001
The title peptide was synthesized according to the procedure described for Example 74 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ahp-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe- OH, Fmoc-Ser(tBu)-OH, Fmoc-Ahe-OH, Fmoc-Hag-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Val-OH. . HPLC purification and lyophilization gave 15 mg (13%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C95H143CI 22O21S2, 2028.9 found 1014.8 (M+2H)+, 677.4(M+3H)+.
Example 76
Preparation of {Hcy4&Cysl3, stapled-HaglO&Hagl4}-Ac-VP-Phe(3-Cl)-Hcy-LRK- Nle-P-Hag-SFC-Hag-PPE-
Figure imgf000066_0002
The title peptide was synthesized according to the procedure described for Example 74 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ahp-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe- OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Hag-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Val-OH. . HPLC
purification and lyophilization gave 6 mg (5%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C94Hi4iClN22021S2, 2014.9 found 1008.3 (M+2H)+, 672.2(M+3H)+. Example 77
Preparation of {Hcy4&Cysl3, stapled-HaglO&Ahpl4}-Ac-VP-Phe(3-Cl)-Hcy-LRK- Nle-P-Hag-SFC-Ahp-PPE-
Figure imgf000067_0001
The title peptide was synthesized according to the procedure described for Example 74 using Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ahp-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe- OH, Fmoc-Ser(tBu)-OH, Fmoc-Nle-OH, Fmoc-Ahp-OH, Fmoc-Hag-OH, Fmoc-Nle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Val- OH. . HPLC purification and lyophilization gave 9 mg (8%) of the title peptide in TFA salt form as white amorphous powder. LCMS-ESI m/e Calcd for C95H143C1N22021S2, 2028.9 found 1015.2 (M+2H)+, 677.2(M+3H)+.
BIOLOGICAL EXAMPLES
Example 78: YAP/TEAD competitive binding assay by Surface Plasmon Resonance (SPR)
The biological activity of the peptides of the invention can be determined using the assay described below.
YAP protein were dissolved in coupling buffer (10 mg/mL, in 10 mM sodium acetate, pH 4.0) and immobilized onto the CM5 sensor chip of Biacore T100 (GE Healthcare) as the ligand in 3,000 RU with 0.2 M N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) according to the standard primary amine-coupling procedures as briefly described below, the dextran matrix on the sensor chip surface was first activated with a mixture of EDC and NHS to give reactive succinimide esters. YAP protein in above mentioned buffer was then passed over the surface and immobilized onto the chip by reacting with the esters via its uncharged amino groups or other nucleophilic groups. After that, ethanolamine was injected to deactivate remaining active ester groups on the surface, and remove non-covalently bound ligand. HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20) was used as the running buffer. GST-TEADl (50 nM) was pre-incubated at room temperature for 30 minutes with different concentrations of synthetic peptides. The reaction mixture was injected over the surfaces of the chip at a flow rate of 30 μΐ^ηιίη, then regenerated by 10 mM NaOH for 10 s at a flow of 100 μΐ^ηιίη. Response units were measured at 7 s in the dissociation phase, percent inhibition was calculated from Equation
% inhibition = 100 x ( RUGST-TEADI-RU) / (RUGsT-TEADi-RUinhibitor)
Where RU is the specific binding signal for GST-TEADl protein in the presence of the inhibitor, RUmhibitor is the binding for inhibitor and the RUGST-TEADI is the specific signal for the GST-TEADl alone.
IC50 (half maximal inhibitory concentration) calculation: Plot curve of % inhibition against tested peptides concentrations with Graphpad Prism 5 software, and the concentration corresponding to 50% inhibition on the curve in IC50.
The peptides of the present invention were tested for their capacity to inhibit YAP activity and activation as described herein. The Examples were tested in the above assay and found to have IC50 of about 0.02 μΜ to about 2.00 μΜ. Particular peptides of formula I were found to have IC50 of about 0.02 μΜ to about 0.10 μΜ.
Results of YAP/TEAD competitive binding assay are given in Table 1.
Example A
A YAP peptide of formula I can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:
Per tablet
Active ingredient 200 mg
Microcrystalline cellulose 155 mg
Corn starch 25 mg
Talc 25 mg
Hydroxypropylmethylcellulose 20 mg
425 mg
Example B A YAP peptide of formula I can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:
Per capsule
Active ingredient 100.0 mg
Corn starch 20.0 mg
Lactose 95.0 mg
Talc 4.5 mg
Magnesium stearate 0.5 mg
220.0 mg

Claims

Claims
1. A compound of formula (I)
I X— X 1
A°-A1 -A2-A3-A4-A5-A6-A7-A8-A9-A1 °-A -A 2-A13- A14-A1 5-A1 6-A1 7-A1 8-A1 9
(I)
wherein
A0 is acetyl;
A1 is Thr, D-Thr or deleted;
A2 is Val;
A3 is Pro, 4R-Hyp, 35-Hyp or 4R-Flp;
A4 is Met, Trp, Phe, Phe(2-Cl), Phe(3-Cl), Phe(3-Br), Phe(4-Cl), Cha, Nle, Hie, Ahe, Nal(l), or Nal(2);
A5 is Hey or Hsc;
A6 is Leu, Lys or Arg;
A7 is Arg;
A is Lys, His, Ser, Thr, Glu, Asp, Leu, Pro, Nle, Dbu, Orn, D-Lys or alpha-Me-Lys;
A9 is Nle, Cha, Hie, Ahe or Aoc;
A10 is Pro or 4R-Flp;
A11 is Ala, Aib, Glu or Hag;
A12 is Ser;
A13 is Phe, Phe(2-Cl), Phe(4-F) or Phe(4-Cl);
A14 is Cys or Sec;
A15 is Lys, Hag, Ahp or deleted;
-A1 1-A12-A13- A14-A15-
'— ril— ' provided that when A is Glu, and A is Lys, A and A form a ring
-A11-A12-A13- A14-A15- when A11 is Hag, and A15 is Hag or Ahp, A11 and A15 form a ring c~c ;
A16 is Pro, 4R-Hyp, 35-Hyp, 4R-Flp, Tic or deleted;
A17 is Pro, 4R-Hyp, 35-Hyp or deleted;
A 18 is Glu, Lys, Arg, Phe(4-guanidino) or deleted; A19 is NH2;
X is S or Se;
or a pharmaceutically acceptable salt thereof.
2. A compound according to claim 1 wherein
A0 is acetyl;
A1 is Thr, D-Thr or deleted;
A2 is Val;
A3 is Pro, 4R-Hyp, 35-Hyp or 4R-Flp;
A4 is Met, Trp, Phe, Phe(2-Cl), Phe(3-Cl), Phe(3-Br), Phe(4-Cl), Cha, Nle, Hie, Ahe, Nal(l) or Nal(2);
A5 is Hey or Hsc;
A6 is Leu, Lys or Arg;
A7 is Arg;
A is Lys, His, Ser, Thr, Glu, Asp, Leu, Pro, Nle, Dbu, Orn, D-Lys or alpha-Me-Lys;
A9 is Nle, Cha, Hie, Ahe or Aoc;
A10 is Pro or 4R-Flp;
A11 is Ala, Aib, Glu or Hag;
A12 is Ser;
A13 is Phe, Phe(2-Cl), Phe(4-F) or Phe(4-Cl);
A14 is Cys or Sec;
A15 is Lys, Hag or Ahp;
-A1 1-A12-A13- A14-A15- ' rij ' provided that when A is Glu, and A is Lys, A and A form a ring
-A11-A12-A13- A14-A15- when A11 is Hag, and A15 is Hag or Ahp, A11 and A15 form a ring c~c ;
A16 is Pro, 4R-Hyp, 35-Hyp, 4R-Flp or Tic;
A17 is Pro, 4R-Hyp or 35-Hyp;
A 18 is Glu, Lys, Arg or Phe(4-guanidino);
A19 is NH2;
X is S or Se;
or a pharmaceutically acceptable salt thereof.
3. A compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein A5 is Hey and A14 is Cys.
4. A compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein A5 is Hsc and A14 is Sec.
5. A compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein A11 is Ala or Aib, and A15 is Lys;
or
A11 is Hag, and A15 is Hag or Ahp, and A11 and A15, together with A12, A13 and A14, form a -A11-A12-A13- A14-A15- or
A11 is Glu, and A15 is Lys, and A11 and A15, together with A12, A13 and A14, form a ring -A1 1 -A12- A13- A14-A15-
6. A compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein A4 is Met or Phe(3-Cl).
7. A compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein A is Lys.
8. A compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein the compound is a peptide truncated after A14, or A16, or A17.
9. A compound according to any one of claims 1 to 4, selected from
{Hcy5, Cysl4}-Ac-TVPM-Hcy-LRK-Nle-PASFCKPPE-NH2;
{Hcy5, Cysl4}-Ac-(D-Thr)-VPM-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- V-(4R-Hyp)-M-Hcy-LRK-Nle-PASFCKPPE-NH2; {Hcy4, Cysl3}-Ac-V-(3S-Hyp)-M-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- V-(4R-Flp)-M-Hcy-LRK-Nle-PASFCKPPE-NH2;
{Hcy4, Cysl3}-Ac-V-(4R-Hyp)-Phe(3-Cl)-Hcy-LRK-Nle-PASFCKPPE-NH2;
{Hcy4, Cysl3 }-Ac-VP-Trp-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } - Ac- VP-Phe(2-Cl)-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Br)-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(4-Cl)-Hcy-LRK-Nle-PASFCKPPE-NH2;
{Hcy4, Cysl3 }-Ac-VP-Cha-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } - Ac- VP-Nle-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Hle-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP- Ahe-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Nal( 1 )-Hcy-LRK-Nle-PASFCKPPE-NH2;
{Hcy4, Cysl3 }-Ac-VP-Nal(2)-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } - Ac- VPF-Hcy-LRK-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-KRK-Nle-P- Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-RRK-Nle-P- Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-KRK-Nle-PASFCKPPE-NH2;
{Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(D-Lys)-Nle-P-Aib-SFCKPPE-NH2;
{Hcy4, Cysl3 }-Ac-VP-Phe(3-Cl)-Hcy-LR-(D-Lys)-Ahe-P-Aib-SFCKPPE-NH2;
{Hcy4, Cysl3}-Ac-VP-Phe(3-Cl)-Hcy-LR-(Me-K)-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRE-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRH-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRL-Nle-PASFCKPPE-NH2;
{Hcy4, Cysl3 }-Ac-VP-Phe(3-Cl)-Hcy-LR-Dbu-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LR-Orn-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRS-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRT-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRD-Nle-PASFCKPPE-NH2;
{Hcy4, Cysl3 }-Ac-VP-Phe(3-Cl)-Hcy-LR-Nle-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LRP-Nle-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LR-Orn- Ahe-P-Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-KR-Orn-Nle-PASFCKPPE-NH2; { Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LR-Dbu-Nle-P-Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } - Ac- VP-Phe(3-Cl)-Hcy-LR-Orn-Nle-P- Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Cha-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK- Ahe-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Hle-PASFCKPPE-NH2;
{Hcy4, Cysl3 }-Ac-VPM-Hcy-LRK-Aoc-PASFCKPPE-NH2;
{Hcy4, Cys 13 }- Ac- VPM-Hcy-LRK- Ahe-P-Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-(4R-Flp)-ASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VP- Ahe-Hcy-LRK-Ahe-PASFCKPPE-NH2;
{ Hcy4, Cys 13 } -Ac-VP-Phe(3-Cl)-Hcy-LRK-Nle-P-Aib-SFCKPPE-NH2;
{Hcy4, Cysl3 }-Ac-VP-Phe(3-Cl)-Hcy-LRK-Ahe-P-Aib-SFCKPPE-NH2;
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle-PAS-Phe(4-Cl)-CKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PAS-Phe(4-F)-CKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PAS-Phe(2-Cl)-CKPPE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCK-(4R-Hyp)-PE-NH2;
{Hcy4, Cysl3 }-Ac-VPM-Hcy-LRK-Nle-PASFCK-(3S-Hyp)-PE-NH2;
{Hcy4, Cys 13 }-Ac-VPM-Hcy-LRK-Nle-PASFCK-Tic-PE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCK-(4R-Flp)-PE-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCKP-(4R-Hyp)-E-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCKP-(3S-Hyp)-E-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-PASFCKP-(4R-Hyp)-E-NH2;
{Hcy4, Cysl3 }-Ac-VPM-Hcy-LRK-Nle-PASFCKPPK-NH2;
{ Hcy4, Cys 13 } - Ac- VPM-Hcy-LRK-Nle-PASFCKPPR-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PASFCKPP-Gph-NH2;
{ Hcy4, Cys 13 } -Ac- VP-Trp-Hcy-LRK-Nle-PASFC-NH2;
{ Hcy4, Cys 13 } -Ac- VPM-Hcy-LRK-Nle-PAS-Phe(4-F)-C-NH2 ;
{Hcy4, Cysl3 }-Ac-VPM-Hcy-LRK-Nle-PASFCKPP-NH2;
{Hcy4, Cys 13 }-Ac-VPM-Hcy-LRK-Nle-PASFCKP-NH2;
{ Hsc4, Sec 13 } - Ac- VPM-Hsc-LRK-Nle-PASF-Sec-KPPE-NH2;
{ Hsc4, Sec 13 } - Ac- VPM-Hsc-LRK- Ahe-PASF-Sec-KPPE-NH2 ;
{ Hsc4, Sec 13 } - Ac- VP- Ahe-Hsc-LRK- Ahe-PASF-Sec-KPPE-NH2;
{ Hsc4, Sec 13 } - Ac- VP-Phe(3-Cl)-Hsc-LRK-Nle-PASF-Sec-KPPE-NH2; {Hsc4, Secl3}-Ac-VP-Phe(3-Cl)-Hsc-LRK-Nle-P-Aib-SF-Sec-KPPE-NH2;
{ Hcy4&Cys 13 , E 10&K 14 } - Ac- VPM-Hcy-LRK-Nle-PESFCKPPE-NH2;
{ Hcy4&Cys 13 , E 1 O&K 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-PESFCKPPE-NH2;
{ Hcy4&Cys 13, stapled-Hag 1 O&Ahp 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-P-Hag-
SFC-Ahp-PPE-NH2;
{ Hcy4&Cys 13, stapled-Hag 1 O&Hag 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK- Ahe-P-Hag- SFC-Hag-PPE-NH2;
{ Hcy4&Cys 13, stapled-Hag 1 O&Hag 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-P-Hag-SFC- Hag-PPE-NH2; and
{ Hcy4&Cys 13, stapled-Hag 1 O&Ahp 14 } - Ac- VP-Phe(3-Cl)-Hcy-LRK-Nle-P-Hag-SFC- Ahp-PPE-NH2.
10. A process for the preparation of a compound of formula I according to any one of
claims 1 to 9
A°-A1 -A2-A3-A4-A5-A6-A7-A8-A9-A1 °-A -A 2-A13- A14-A15-A16-A17-A18-A19
(I)
wherein A0 to A19 and X are defined as in any one of claims 1 to 8.
11. A compound according to any one of claims 1 to 9 for use as therapeutically active substance.
12. A pharmaceutical composition comprising a compound in accordance with any one of claims 1 to 9 and a therapeutically inert carrier.
The use of a compound according to any one of claims 1 to 9 for inhibiting the binding of YAP to TEAD.
The use of a compound according to any one of claims 1 to 9 for diseases associated with the interaction of YAP with TEAD, or overexpression of YAP.
A compound according to any one of claims 1 to 9, when manufactured according to process of claim 10.
16. The invention as hereinbefore described.
PCT/EP2014/067145 2013-08-12 2014-08-11 Yap-tead inhibitors WO2015022283A1 (en)

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CN105524139A (en) * 2014-10-17 2016-04-27 上海宝恒泓康生物技术有限公司 High-activity tumor inhibitor and its preparation method and use
EP3059243A4 (en) * 2013-10-17 2017-11-08 BAO KANG Biomedical Healthcare Inc. Yap protein inhibiting polypeptide and application thereof
WO2021018869A1 (en) 2019-07-29 2021-02-04 Basilea Pharmaceutica International AG 1,2,4-oxadiazol-5-one derivatives for the treatment of cancer
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Title
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ZHISEN ZHANG ET AL: "Structure-Based Design and Synthesis of Potent Cyclic Peptides Inhibiting the YAP-TEAD Protein-Protein Interaction", ACS MEDICINAL CHEMISTRY LETTERS, vol. 5, no. 9, 14 July 2014 (2014-07-14), pages 993 - 998, XP055149232, ISSN: 1948-5875, DOI: 10.1021/ml500160m *

Cited By (10)

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Publication number Priority date Publication date Assignee Title
EP3059243A4 (en) * 2013-10-17 2017-11-08 BAO KANG Biomedical Healthcare Inc. Yap protein inhibiting polypeptide and application thereof
US10526385B2 (en) 2013-10-17 2020-01-07 Bao Kang Biomedical Healthcare Inc YAP protein inhibiting polypeptide and application thereof
CN105524139A (en) * 2014-10-17 2016-04-27 上海宝恒泓康生物技术有限公司 High-activity tumor inhibitor and its preparation method and use
EP3208280A4 (en) * 2014-10-17 2018-10-10 Bao Kang Biomedical Healthcare Inc High activity tumour inhibitor and preparation method and use thereof
US10441628B2 (en) 2014-10-17 2019-10-15 Bao Kang Biomedical Healthcare Inc High activity tumour inhibitor and preparation method and use thereof
CN105524139B (en) * 2014-10-17 2020-04-10 上海宝恒泓康生物技术有限公司 High-activity tumor inhibitor and its preparing process and application
US11548923B2 (en) 2017-01-18 2023-01-10 Fred Hutchinson Cancer Center Peptide compositions and methods of use thereof for disrupting TEAD interactions
WO2021018869A1 (en) 2019-07-29 2021-02-04 Basilea Pharmaceutica International AG 1,2,4-oxadiazol-5-one derivatives for the treatment of cancer
WO2022164835A1 (en) * 2021-01-26 2022-08-04 The United States Government As Represented By The Department Of Veterans Affairs Compositions and methods for inhibiting yap
WO2023057371A1 (en) 2021-10-04 2023-04-13 Basilea Pharmaceutica International Ag, Allschwil 1,2,4-oxadiazol-5-one derivatives for the treatment of cancer

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