Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
  • C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention pertains to a number of compounds which can be used in the medicinal field to treat, prophylactically or otherwise, uncontrolled or abnormal proliferation of human tissues. More specifically, the present invention pertains to a number of compounds which act to inhibit the farnesyl transferase enzyme that has been determined to activate ras proteins which in turn activate cellular division and are implicated in cancer and restenosis.
• Ras protein (or p21) has been examined extensively because mutant forms are found in 20% of most types of human cancer and greater than 50% of colon and pancreatic carcinomas (Gibbs J.B., Cell, 65:1 (1991), Cartwright T. , et al. , Chimica Ocr ⁇ i. 10 . :26 (1992)) .
• These mutant ras proteins are deficient in the capability for feedback regulation that is present in native ras and this deficiency is associated with their oncogenic action since the ability to stimulate normal cell division can not be controlled by the normal endogenous regulatory cofactors.
• the recent discovery that the transforming activity of mutant ras is critically dependent on post-translational modifications (Gibbs J., et al.
• blockade of ras dependant processes has the potential to reduce or eliminate the inappropriate tissue proliferation associated with restenosis, particularly in those instances where normal ras expression and/or function is exaggerated by growth stimulatory factors.
• Ras functioning is dependent upon the modification of the proteins in order to associate with the inner face of plasma membranes.
• ras proteins lack conventional transmembrane or hydrophobic sequences and are initially synthesized in a cytosol soluble form.
• Ras protein membrane association is triggered by a series of posttranslational processing steps that are signaled by a carboxyl terminal amino acid consensus sequence that is recognized by protein:farnesyl transferase. This consensus sequence consists of a cysteine residue located four amino acids from the carboxyl terminus, followed by two lipophilic amino acids and the C-terminal residue.
• the sulfhydryl group of the cysteine residue is alkylated by farnesyl pyrophosphate in a reaction that is catalyzed by protein:farnesyl transferase.
• the C-terminal three amino acids are cleaved by an endoprotease and the newly exposed alpha-carboxyl group of the prenylated cysteine is methylated by a methyl transferase.
• the enzymatic processing of ras proteins that begins with farnesylation enables the protein to associate with the cell membrane. Mutational analysis of oncogenic ras proteins indicate that these posttranslational modifications are essential for transforming activity.
• PFTs protein:farnesyl transferases
• farnesyl:protein transferases protein:farnesyl transferases
• the enzyme was charac ⁇ terized as a heterodimer composed of one alpha-subunit (49 kDa) and one beta-subunit (46 kDa) , both of which are required for catalytic activity.
• PCT Application O91/16340 discloses cysteine containing tetrapeptide inhibitors of PFT of the formula CAAX.
• European Patent Application 0461869 discloses cysteine containing tetrapeptide inhibitors of PFT of the formula Cys-Aaa 1 -Aaa 2 -Xaa.
• European Patent Application 0520823 discloses cysteine containing tetrapeptide inhibitors of PFT of the formula Cys-Xaa 1 -dXaa 2 -Xaa 3 .
• European Patent Application 0523873 discloses cysteine containing tetrapeptide inhibitors of PFT of the formula Cys-Xaa 1 -Xaa 2 -Xaa 3 . 2612
• European Patent Application 0528486 discloses cysteine containing tetrapeptide amides inhibitors of PFT of the formula Cys-Xaa 1 -Xaa 2 -Xaa 3 -NRR 1 .
• US 4,022,759 discloses tripeptide antagonists of luteinizing hormone releasing factor of the formula A-R- ⁇ Tyr(benzyl) -Ser(benzyl) -R 2 , wherein one of the definitions of R- j ⁇ is His (benzyl) .
• the present invention is a substituted di- or tripeptide compound of Formula I:
• n 1 or 2;
• A COR 3 , C0 2 R 3 , CONHR 3 , CSR 3 , C(S)OR 3 , C(S)NHR 3 , CF 3 S0 2 , aryl-S0 2 , or alkyl-S0 2 , wherein R 3 is alkyl, (CH 2 ) m -cycloalkyl, (CH 2 ) m -aryl,
• R 1 H, CO-aryl, (CH 2 ) m -aryl, 0(CH 2 ) m -cycloalkyl,
• X one to four substituents, including H, alkyl, CF 3 , F, Cl, Br, I, HO, MeO, N0 2 , NH 2 , N(Me) 2 , 0P0 3 H 2 , or
• R 2 NR(CH 2 ) n C0 2 R 3 , NR(CH 2 ) n CONHR 3 , NR(CH 2 ) n R 3 , NR(CH 2 ) n+1 OR 4 , NR(CH 2 ) n+1 SR 4 , NRCH(COR 5 ) (CH 2 ) n -heteroaryl, NRCH(C0R 5 ) (CH 2 ) n OR 3 ,
• the present invention is also directed to the use of a compound of Formula I, or a pharmaceutically acceptable salt therefrom, to inhibit the activity of a protein:farnesyl transferase enzyme as a method for treating tissue proliferative diseases.
• a further embodiment of the present invention is the use of a pharmaceutical composition including an effective amount of a compound of Formula I as a method for the treatment of cancer. 12612
• a still further embodiment of the present invention is the use of a pharmaceutical composition including an effective amount of a compound of Formula I as a method for the treatment of restenosis.
• a still further embodiment of the present invention is a pharmaceutical composition for administering an effective amount of a compound of Formula I in unit dosage form in the treatment methods mentioned above.
• a final embodiment of the present invention pertains to methods for the preparation of compounds of Formula I by solid phase synthesis, solution phase synthesis, and simultaneous multiple syntheses using a multiple simultaneous synthesis apparatus.
• alkyl means a straight or branched hydrocarbon radical having from 1 to 6 carbon atoms and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, and the like.
• cycloalkyl means a saturated hydrocarbon ring which contains from 3 to 10 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, and the like, unsubstituted or substituted by an alkyl or aryl group.
• heteroaryl means a heteroaromatic ring which is a 2- or 3-thienyl, 2- or 3-furanyl, 2- or 3-pyrrolyl, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl group, unsubstituted or with 1 or 2 substituents from the group of substituents described above for aryl.
• the amino acid or abbreviation is preceded by the appropriate configuration D(R) or DL(RS) . 12612
• Gly-C0 2 Bn is Glycine, benzyl ester
• Ser(OBn) -C0 2 Me is O-Benzyl-serine, methyl ester.
• Gly-CONHBn is Glycine, N-benzyl amide
• Ser(OBn) -CONHEt is O-Benzyl-serine, N-ethyl amide
• Tyr(OBn) -CONHCH 2 CH 2 OBn is O-Benzyl- tyrosine, N- (2- (phenylmethoxy)ethyl) amide.
• optical activity of the amino acid is other than L(S)
• the amino acid or abbreviation is preceded by the appropriate configuration D(R) or DL(RS) . 12612
• optical activity of the amino acid is other than L(S)
• the amino acid or abbreviation is preceded by the appropriate configuration D(R) or DL(RS) . -12-
• Preferred compounds of Formula I consist of compounds of Formula II below:
• R independently H or Me
• Y independently H or Me
• Z independently H or Me
• R 1 (CH 2 ) m -aryl, 0(CH 2 ) m -aryl, OP0 3 H 2 , or CH 2 P0 3 H 2 , wherein m is as defined above;
• R ⁇ NR(CH 2 ) 2 0R 4 , NR(CH 2 ) 2 SR 4 , NRCH(COR 5 ) CH 2 OR 3 ,
• A is CONHR 3
• R 2 is (CH 2 ) 2 0R 4
• at least one of Y and Z is Me.
• the most preferred compounds of Formula I include the following: Cbz-His-Tyr(OBn) -Ser(OBn) -C0 2 Me;
• the compounds of Formula I may be prepared by solid phase peptide synthesis on a peptide synthesizer, for example, an Applied Biosystems 431A peptide synthesizer using activated esters or anhydrides of Boc or Fmoc protected amino acids, acid chlorides, isocyanates, isothiocyanates, etc., on PAM, MBHA, or NH 2 -Rink resins with solution phase modifications to the carboxyl terminus as appropriate.
• Methodology for the solid phase synthesis of peptides is widely known to those skilled in the art thereof (see, for example: J.M. Stewart and J.D. Young in Solid Phase Peptide Synthesis: Pierce Chemical Co.; Rockford, IL (1984); Fields G.B. and Noble R.L.
• the compounds of Formula I may also be prepared by conventional solution peptide synthesis, substituting amines, acid chlorides, isocyanates, etc, for amino acid derivatives where appropriate. Methods for solution phase synthesis of peptides are widely known to those skilled in the art (see, for example, M. Bodanszky, Principles of Peptide Synthesis. Springer-Verlag (1984)) .
• the compounds of Formula I may be prepared by simultaneous multiple solid phase syntheses using an apparatus described by S. H. DeWitt, et al. , Proc. Natl. Acad. Sci. USA. 9 . 0:6909 (1993), and referred to by the trademark, DiversomerTM, both trademark and apparatus being owned in whole by the Warner-Lambert Company.
• the multiple solid phase synthesis apparatus is currently the subject of now abandoned US Serial 07/958,383 filed October 8, 1992 and pending continuation-in-part US Serial 08/012,557 filed February 2, 1993.
• Fmoc-D-His-Tyr(OBn) - C0 2 -CH 2 CH 2 Si (CH 3 ) 3 is linked to 2-Cl-Tr resin using a sterically hindered amine such as DIEA as an HC1 scavenger, the Fmoc protecting group is removed with piperidine, the resulting free amino terminus is acylated with a series of isocyanates, isothiocyanates, activated esters, acid chlorides and the like, the TMS-ethyl ester is cleaved with TBAF, the resulting 12612
• - 19 - free carboxy terminus is activated with a carbodiimide reagent such as EDAC, DCC, or DIC, the activated carboxyl group is reacted with alcohols such as HOBT, NHOS, or pentachlorophenol to give an activated ester, the activated ester is reacted with a series of amines and the resulting array of compounds of Formula I is cleaved from the resin by with hot HOAc or by treatment with TFA at room temperature.
• a carbodiimide reagent such as EDAC, DCC, or DIC
• alcohols such as HOBT, NHOS, or pentachlorophenol
• the activated ester is reacted with a series of amines and the resulting array of compounds of Formula I is cleaved from the resin by with hot HOAc or by treatment with TFA at room temperature.
• RP-HPLC reverse phase-high pressure liquid chromatography
• TLC capillary electrophoresis
• NMR proton nuclear magnetic resonance spectrometry
• amino acid analysis amino acid analysis
• FAB-MS fast atom bombardment mass spectrometry
• ES-MS electrospray mass spectrometry
• the compounds of Formula I are capable of further forming both pharmaceutically acceptable acid addition and/or base salts. All of these forms are within the scope of the present invention.
• Pharmaceutically acceptable acid addition salts of the compounds of Formula I include salts derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, hydrofluoric, phosphorous, and the like, as well as the salts derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
• Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like.
• salts of amino acids such as arginate and the like and gluconate, galacturonate, n-methyl glucamine (see, for example, Berge S.M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science. £6:1-19 (1977)).
• the acid addition salts of said basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner.
• a compound of Formula I can be converted to an acidic salt by treating with an aqueous solution of the desired acid, such that the resulting pH is less than 4.
• the solution can be passed through a C18 cartridge to absorb the compound, washed with copious amounts of water, the compound eluted with a polar organic solvent such as, for example, methanol, acetonitrile, and the like, and isolated by concentrating under reduced pressure followed by lyophilization.
• the free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner or as above.
• the free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention.
• Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
• metals used as cations are sodium, potassium, magnesium, calcium, and the like.
• suitable amines are N,N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge S.M., et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science. 6£: 1 - 19 (1977)).
• the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
• a compound of Formula I can be converted to a base salt by treating with an aqueous solution of the desired base, such that the resulting pH is greater than 9.
• the solution can be passed through a C18 cartridge to absorb the compound, washed with copious amounts of water, the compound eluted with a polar organic solvent such as, for example, methanol, acetonitrile and the 2612
• the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner or as above.
• the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
• Certain of the compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain of the compounds of the present invention possess one or more chiral centers and each center may exist in the R(D) or S(L) configuration. The present invention includes all enantiomeric and epimeric forms as well as the appropriate mixtures thereof.
• the PFT inhibitory activity of compounds of Formula I was assayed in 30 mM potassium phosphate buffer, pH 7.4, containing 7 mM DTT, l. ' 2 mM MgCl 2 , 0.1 mM leupeptin, 0.1 mM pepstatin and 0.2 mM phenylmethylsulfonyl fluoride. Assays were performed in 96 well plates (Wallec) and employed solutions composed of varying concentrations of a compound of Formula I in 100% DMSO.
• radiolabeled farnesyl pyrophosphate [1- 3 H] , specific activity 15-30 Ci/mmol, final concentration 0.12 ⁇ M
• biotinyl) -Ahe-Tyr-Lys-Cys- Val-Ile-Met peptide final concentration 0.1 ⁇ M
• the enzyme reaction was started by addition of 40-fold purified rat brain farnesyl protein transferase. After incubation at 37°C for 30 minutes, the reaction was 12612
• ⁇ 24* terminated by diluting the reaction 2.5-fold with a stop buffer containing 1.5 M magnesium acetate, 0.2 M H 3 P0 4 , 0.5% BSA, and strepavidin beads (Amersham) at a concentration of 1.3 mg/mL. After allowing the plate to settle for 30 minutes at room temperature, radioactivity was quantitated on a microBeta counter (model 1450, Wallec) .
• Compounds of Formula I show IC 50 values of 0.5 nM to 80 ⁇ M (see data table) in this assay and are thus valuable inhibitors of protein: farnesyl transferase enzyme which may be used in the medical treatment of tissue proliferative diseases, including cancer and restenosis.
• the compounds of the present invention can be prepared and administered in a wide variety of oral, rectal and parenteral dosage forms.
• the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally.
• the compounds of the present invention can be administered by inhalation, for example, intranasally.
• the compounds of the present invention can be administered transdermally. It will be obvious to those skilled in the art that the following dosage forms may comprise as the active component, either a compound of Formula I or a corresponding pharmaceutically acceptable salt of a compound of Formula I.
• pharmaceutically acceptable carriers can be either solid or liquid.
• Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
• a solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
• the carrier is a finely divided solid which is in a mixture with the finely divided active component.
• the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
• the powders and tablets preferably contain from 5 or 10 to about 70 percent of the active compound.
• Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
• the term "preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included.
• Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
• a low melting wax such as a mixture of fatty acid glycerides or cocoa butter
• the active component is dispersed homogeneously therein, as by stirring.
• the molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
• Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water propylene glycol solutions.
• liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
• Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents as desired.
• Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
• viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
• solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
• liquid forms include solutions, suspensions, and emulsions.
• These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
• the pharmaceutical preparation is preferably in unit dosage form.
• the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
• the unit dosage form can be a capsules, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
• the quantity of active component in a unit dose preparation may be varied or adjusted from 0.1 mg to 100 mg preferably 0.5 mg to 100 mg according to the particular application and the potency of the active component.
• the composition can, if desired, also contain other compatible therapeutic agents.
• the compounds utilized in the pharmaceutical methods of this invention are administered at the initial dosage of about 0.01 mg/kg to about 20 mg/kg daily.
• a daily dose range of about 0.01 mg/kg to about 10 mg/kg is preferred.
• the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art.
• treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
• the total daily dosage may be divided and administered in portions during the day, if desired.
• Fmoc-NH 2 -Rink resin (0.25 mmol scale) was treated with 20% piperidine in NMP to afford NH 2 -Rink resin.
• Sequential coupling of Fmoc-protected Ser(OBn) and Tyr(OBn) (DCC and HOBT in NMP) and Fmoc deprotection (20% piperidine in NMP) reactions were run using a 4-fold excess of reagents in the coupling steps and traditional resin washing cycles to afford
• Fmoc-Ser(OBn) , Fmoc-Tyr(OBn) , and Cbz-His are sequentially coupled using the deprotection and coupling conditions described in Example 1. Cleavage from the resin is accomplished by treatment with CF 3 S0 2 H for the PAM supported tripeptide or with 50% TFA in DCM for the HMP supported tripeptide. Chromatography as in Example 1 provides Cbz-His-Tyr(OBn) -Ser(OBn) as its TFA salt upon - 31 - lyophilization. See also Example 7 for a solution phase method.
• Step 3 Fmoc-His (Tr) -Tyr(OBn) -CQ 2 CH 2 CH 2 TMS To a solution of HOBT (2.6 g, 19.3 mmol) in DMF
• Step 5 Fmoc-His (2-Cl-Tr Resin) -Tyr(OBn) -CQ 2 CH 2 CH 2 TMS To a suspension of Fmoc-His-Tyr(OBn) - C0 2 CH 2 CH 2 TMS
• the suspension was agitated by shaking for 18 hours, filtered, washed sequentially with a 2:1 mixture of dioxane and 10% citric acid (3x 10 mL) , dioxane:MeOH (3x 10 mL) , dioxane (3x 10 mL) , and CHC1 3 (3x 10 mL) to provide BnNHCO-His (2-Cl-Tr-Resin) -Tyr(OBn) .
• the BnNHCO-His (2-Cl-Tr-Resin) -Tyr(OBn) was suspended in DMF (10 mL) and treated with a carbodiimide coupling reagent such as DIC (0.2 g, 1.6 mmol) and HOBT (0.22 g, 1.6 mmol) .
• a carbodiimide coupling reagent such as DIC (0.2 g, 1.6 mmol) and HOBT (0.22 g, 1.6 mmol) .
• 3-phenoxypropylamine (0.24 g, 1.6 mmol) was added.
• the resulting mixture was shaken 18 hours before filtering the resin and washing with DMF (3x) and CHC1 3 (3x) .
• the resin was suspended in DMF (10 mL) and the carbodiimide/HOBT/3-phenoxypropylamine coupling reaction was repeated. After 18 hours, the resin was filtered and washed with 10 mL each of MeOH (2x
• BnNHCO-His (2-Cl-Tr-Resin) -Tyr(OBn) -CONH(CH 2 ) 3 OPh The highly substiuted dipeptide was cleaved from the resin by treatment with 40% TFA in DCM, shaking for 1 hour at room temperature. The supernate, containing the free dipeptide, was filtered away from the resin and the resin was washed with DCM (6x) . The combined supernate and washings were concentrated in vacuo to provide BnNHCO-His-Tyr(OBn) -CONH(CH 2 ) 3 OPh*TFA.
• Example 4 The method described in Example 4 may be employed in simultaneous multiple syntheses using the Diversomer apparatus described by S.H. DeWitt, et al. , Proc. Natl. Acad. Sci. USA. £0:6909 (1993) .
• Fmoc-D-His (2-C1- Tr Resin) -Tyr(OBn) -C0 2 iPr prepared according to from Example 3 by substituting Fmoc-D-His (Tr) for Fmoc- His (Tr) in Step 3, (100-200 mg) is placed in each of 40 gas dispersion tubes, and the tubes are placed in the Diversomer apparatus.
• the sequential deprotection and coupling reactions described in Example 4 are followed, employing the following acyiating agents and amines in various combinations: Acyiating agents Amines
• BnNHCO-D-His-Tyr CONHOBn ES-MS 647 (m+1) 53.
• BnNHCO-D-His-Tyr (OBn) CONHCH 2 CH 2 CH 2 OPh ES-MS 675 (m+1) 12612
• Step 2 Tyr(OBn) -Ser(OBn) -C0 2 Me*TFA Boc-Tyr(OBn) -Ser(OBn) -C0 2 Me (from Step 1 above,
• Step 3 Cbz-His-Tyr(OBn) -Ser(OBn) -CQ 2 Me To a solution of Cbz-His (1.00 g, 3.47 mmol) in
• Step 2 Cbz-D-His (Tr) -Tyr (OBn) - Ser (OBn) - CC Me
• Example 6 According to Example 6, Step 3, by substituting Cbz-D-His (Tr) for Cbz-His and Tyr(OBn) -Ser(OBn) - C0 2 Me-HCl for Tyr(OBn) -Ser(OBn) -C0 2 Me-TFA, the title compound was obtained as a white solid, mp 78-88°C; FAB-MS 976 (m+1) .
• Step 3 Cbz-D-His-Ty (OBn) -Ser(OBn) -CQ 2 Me
• Example 6 by substituting D-Ser(OBn) -C0 2 Me-TFA for Ser(OBn) -C0 2 Me-TFA in Step 1, the title compound was obtained, mp 168-170°C; FAB-MS 734 (m+1) .
• EXAMPLE 10 N- fof-Methyl-N- fN- f (phenylmethoxy) carbonyl! -L-histidyll - 0- (phenylmethyl) -DL-tyrosyl! -0- (phenylmethyl) -L-serine. methyl ester ⁇ Cbz-His-DL- ( ⁇ -Me)Tyr(OBn) -Ser(OBn) -CQ 2 Me ⁇ According to Example 6, by substituting Boc-DL-
• Example 6 by substituting Boc-Tyr(OBn) -Ser(OBn) -CONHEt for Boc-Tyr(OBn) - Ser(OBn) -C0 2 Me in Step 2, the title compound was obtained, mp 182-188°C; FAB-MS 747 (m+1) .
• Step 1 Cbz-His (1-Me) Benzyl chloroformate (0.24 mL, 1.7 mmol) was added dropwise to a slurry of 1-methyl-L-histidine (0.25 g, 1.5 mmol) in THF (5 mL) and saturated aqueous NaHC0 3 (5 mL) at 0°C. The mixture was allowed to warm to room temperature and stirred overnight. The mixture was concentrated and diluted with H 2 0, washed with ether, and the pH adjusted to 6-7 with IN HC1. The mixture was concentrated, then diluted with CHC1 3 (150 mL) and MeOH (15 mL) , and stirred for 1 hour. The mixture was dried (MgS0 4 ) and concentrated to provide 0.48 g of the title compound which was used without further purification.
• Step 2 Cbz-His (1-Me) -Tyr(OBn) -Ser(OBn) -C0 2 Me
• Example 6 by substituting Boc- homoTyr(OBn) for Boc-Tyr(OBn) in Step 1, and substituting Cbz-D-His for Cbz-His in Step 3, the title compound was obtained; ES-MS 748 (m+1) .
• Step 1 Fmoc-His (Tr) -Tyr(OBn) -Ser(OBn) -C0 2 Me According to Example 13, Step 2, by substituting
• Step 2 His (Tr) -Tyr(OBn) -Ser(OBn) -C0 2 Me Piperidine (4.0 mL) was added to a slurry of
• Step 3 BnNHCO-His (Tr) -Tyr(OBn) -Ser(OBn) -CQ 2 Me
• Benzyl isocyanate (0.053 mL, 0.43 mmol) was added in one portion to a solution of His(Tr) -Tyr(OBn) - Ser(OBn) -C0 2 Me (from Step 2 above, 0.33 g, 0.39 mmol) in EtOAc (5 mL) . The resulting slurry was stirred for 3 hours at room temperature, then concentrated to yield the title compound (0.4 g) , which was used without further purification.
• Step 4 BnNHCO-His-Tyr(OBn) -Ser(OBn) -CO-.Me According to Example 8, by substituting BnNHCO-
• Example 8 by substituting PhCH 2 CH 2 CO-His(Tr) -Tyr(OBn) -Ser(OBn) -C0 2 Me for Cbz-D- His (Tr) -Tyr(OBn) -Ser(OBn) -C0 2 Me, the title compound was obtained, mp 193-196.5°C; ES-MS 732 (m+1) .
• Example 6 According to Example 6, Step 3, by substituting Tyr(OBn) -C0 2 Me-TFA for Tyr(OBn) -Ser(OBn) -C0 2 Me-TFA, the title compound was obtained as a white powder, mp 145-148°C; CI-MS 557 (m+1) .
• Example 7 by substituting Cbz-His- Tyr(OBn) -C0 2 Me for Cbz-His-Tyr(OBn) -Ser(OBn) -C0 2 Me, the title compound was obtained as a white powder, mp 79-92°C; CI-MS 543 (m+1) .
• Step 3 Cbz-His -Tyr(OBn) -CONHCH 2 CH 2 OBn
• Example 6 by substituting 2- (phenyl- methoxy)ethyla ine for Ser(OBn) -TFA and omitting Et 3 N in Step 1 and by substituting Cbz-D-His for Cbz-His in Step 3, the title compound was prepared, mp 161-165°C; FAB-MS 676 (m+1) .
• Example 20 by substituting Boc- ( ⁇ !-Me)Tyr(OBn) for Boc-Tyr(OBn) , the title compound was obtained, mp 66-78°C; ES-MS 690 (m+1) .
• EXAMPLE 23 N- (2-Phenylethyl) -N a - fN- f (phenylmethoxy) carbonyl! - L-histidyl! -0- (phenylmethyl) -L-tyrosinamide ⁇ Cbz-His-Tyr(OBn) -C0NHCH CH 2 Ph) According to Example 19, Step 3, by substituting
• Example 19 by substituting Pyr-C0 2 Me for 2- (phenylmethoxy) ethylamine, the title compound was obtained as a white solid, mp 180-182.5°C (dec) ; FAB-MS 705 (m+1) .
• Boc-Tyr(OBn) -CONH(CH ) 3 OPh 2- (Phenylmethoxy) ethylamine (0.81 g, 5.4 mmol) was added to a premixed solution of EDAC (1.2 g, 6.5 mmol), HOBT (0.87 g, 6.5 mmol), and Boc-Tyr(OBn) -OH (2.0 g, 5.4 mmol) in dry DMF (15 mL) . The resulting mixture was stirred for 18 hours at room temperature.
• Step 4 D-His(Tr) -Tyr(OBn) -CONH(CH 2 ) 3 OPh Fmoc-D-His(Tr) -Tyr(OBn) -CONH(CH 2 ) 3 OPh (from Step 3 above, 1.0 g, 0.99 mmol) in CH 2 C1 2 (5 mL) was treated with piperidine (0.18 g, 2.1 mmol) .
• Step 5 (4-Et0Ph)NHC0-D-His(Tr) -Tyr(OBn) -CONH(CH ) 3 OPh D-His(Tr) -Tyr(OBn) -CONH(CH 2 ) 3 OPh (from Step 4 above, 0.55 g, 0.7 mmol) in CH 2 C1 2 (5 mL) was treated with 4-ethoxyphenyl isocyanate (0.1 g, 0.7 mmol) . The resulting mixture was stirred 1 hour at room temperature. Concentrated in vacuo.
• Step 6 (4-EtOPh)NHCO-D-His-Tyr(OBn) -CONH(CH 2 ) 3 OPh
• 4-EtOPh)NHCO-D-His(Tr) -Tyr(OBn) -CONH(CH 2 ) 3 OPh (from Step 5 above, 0.5 g, 0.52 mmol) in MeOH (5 mL) was treated with Pyridine-HCl (catalytic) . The resulting mixture was stirred at 65°C for 6 hours.
• Example 25 According to Example 25, Step 4, by substituting Fmoc-D-His (Tr) -Tyr(OBn) -CONH(CH 2 ) 3 (2-MeOPh) for Fmoc-D- His (Tr) -Tyr(OBn) -CONH(CH 2 ) 3 OPh, the title compound was obtained as a white foam; ES-MS 798 (m+1) .
• Example 25 by substituting BocTyr(OBn) -CONH(CH 2 ) 2 Ph for BocTyr(OBn) -CONH(CH 2 ) 3 OPh, the title compound was obtained as a white solid; CI-MS 375 (m+1) .
• Step 4 (4-MePh)SQ -D-His(Tr) -Tyr(OBn) -CO (4-Bn- piperazin-l-yl) Fmoc-D-His-Tyr(OBn) -CONH(CH 2 ) 3 OPh, (0.7 g,

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