WO1994025049A1 - Amidino and guanidino substituted boronic acid inhibitors of trypsin-like enzymes - Google Patents

Amidino and guanidino substituted boronic acid inhibitors of trypsin-like enzymes Download PDF

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WO1994025049A1
WO1994025049A1 PCT/US1994/004058 US9404058W WO9425049A1 WO 1994025049 A1 WO1994025049 A1 WO 1994025049A1 US 9404058 W US9404058 W US 9404058W WO 9425049 A1 WO9425049 A1 WO 9425049A1
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pro
phe
borophe
nhc
alkyl
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PCT/US1994/004058
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French (fr)
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John Matthew Fevig
Charles Adrian Kettner
Sheng-Lian O. Lee
David John Carini
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The Du Pont Merck Pharmaceutical Company
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Priority to AU67038/94A priority Critical patent/AU6703894A/en
Priority to EP94914776A priority patent/EP0696199A4/en
Priority to CA002161216A priority patent/CA2161216A1/en
Priority to JP6524316A priority patent/JPH08509723A/en
Publication of WO1994025049A1 publication Critical patent/WO1994025049A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A61P9/08—Vasodilators for multiple indications
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • 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
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • 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
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • 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
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • 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 relates generally to ⁇ -aminoboronic acids and corresponding peptide analogs in which the alpha substituent is either an aromatic guanidino, isothiouronium, amidino group, halogen, cyano group or an aliphatic amidino, isothiouronium, or formamidino group.
  • Simple boronic acids are inhibitors of serine proteases.
  • Koehler et al. Biochemistry 10: 2477 (1971) reports that 2-phenylethane boronic acid inhibits chymotrypsin at millimolar levels.
  • ⁇ -aminoboronic acids The chemistry of ⁇ -aminoboronic acids was further expanded to the synthesis of peptide analogs containing boronic acid with positive charged sidechains, boroLysine, boroArginine, boroOrnithine, and isothiouronium analogs (EPA 0 293 881, 12/7/88).
  • This series of compounds have provided highly effective inhibitors of thrombin and other trypsin-like enzymes.
  • the boroArginine analogs specifically designed as thrombin inhibitors are highly effective in the
  • this group of compounds is extended to aliphatic amidino and formamidino, to aromatic amidino and guanidino, and to cyano and halogen substituted aromatic boronic acid analogs.
  • boroLysine which contain at least one unnatural amino acid residue.
  • Elgendy et al. Tetrahedron Lett . , 33, 4209-4212 (1992) have described peptides containing ⁇ -aminoboronic acids with aliphatic neutral sidechains which are thrombin inhibitors.
  • Kakkar in (WO 92/07869) has claimed peptide thrombin inhibitors of the general structure, X-Aa 1 -Aa 2 -NH-CH(Y)-Z where Aai and Aa 2 are unnatural amino acid residues.
  • Z is -CN, -COR,
  • aryl is phenyl or napthyl
  • halo F, Cl, Br, I
  • C1-C4-alkyl C1-C4-alkoxy, -NO 2 , -CF 3 , -S(O) r -C1-C4-alkyl, -OH, -NH 2 , -NH(C1-C4-alkyl), -N(C1-C4-alkyl) 2 , -CO 2 R 4 , or
  • R 3 is H, alkyl, aryl, alkylaryl, or an NH 2 -blocking group comprised of 1-20 carbon atoms;
  • R 4 and R 5 are independently
  • aryl is phenyl or napthyl
  • halo F, Cl, Br, I
  • C1-C4-alkyl C1-C7-alkoxy, -NO 2 , -CF 3 , -S(O) r -C1-C4-alkyl, -OH, -NH 2 , -NH (C1-C4-alkyl), -N(C1-C4-alkyl) 2 , -CO 2 R 4 , or
  • A is an amino acid residue or a peptide comprised of 2-20 amino acid residues
  • Y 1 and Y 2 when taken together Y 1 and Y 2 form a d) cyclic boron ester where said chain or ring contains from 2 to 20 carbon atoms and, optionally, 1-3 heteroatoms which can be N, S, or O,
  • n 0 or 1
  • p 0 to 3;
  • q 0 to 4.
  • r is 0 to 2;
  • R 2 is H
  • A is Pro or (D)Phe-Pro
  • Illustrative of the preferred compounds of this invention are the following: ⁇ Ac-(D)Phe-Pro-NH-CH[(CH 2 ) 4 CN]BO 2 -C 10 H 16
  • compositions comprising one or more of the foregoing compounds and methods of using such compositions in the treatment of aberrant proteolysis such as thrombosis in mammals or as reagents used as anticoagulants in the processing of blood to plasma for diagnostic and other commercial purposes.
  • Trp L-tryptophan
  • boronic acid is in the form of the free acid.
  • the pinanediol boronic acid ester and the pinacol boronic acid ester are abbreviated "-C 10 H 16 " and
  • CH NH
  • BSA benzene sulfonic acid
  • THF tetrahydrofuran
  • Boc- t-butoxycarbonyl-
  • Ac- acetyl
  • pNA p-nitro-aniline
  • the compounds of the present invention contain one or more chiral centers and that these stereoisomers may possess distinct physical and biological properties.
  • the present invention comprises all of the stereoisomers or mixtures thereof.
  • the pure enantiomers or diasteromers may be prepared using starting materials with the appropriate stereochemistry, or may be separated from mixtures of undesired stereoisomers by standard
  • NH 2 -blocking group refers to various acyl, thioacyl, alkyl, sulfonyl, phosphoryl, and phosphinyl groups comprised of 1 to 20 carbon atoms. Substitutes on these groups maybe either alkyl, aryl, alkylaryl which may contain the heteroatoms, O, S, and N as a substituent or as inchain component.
  • alkyl, aryl, alkylaryl which may contain the heteroatoms, O, S, and N as a substituent or as inchain component.
  • a number of NH 2 -blocking groups are recognized by those skilled in the art of organic synthesis. By definition, an NH 2 -blocking group may be removable or may remain
  • suitable groups include formyl, acetyl, benzoyl, trifluoroacetyl, and methoxysuccinyl; alkyl and alkylaryl sulfonyl groups, such as n-propylsulfonyl, phenylmethyl and benzylsulfonyl; aromatic urethane protecting groups, such as, benzyloxycarbonyl; and aliphatic urethane protecting groups, such as t-butoxycarbonyl or
  • amino acid residues refers to natural or unnatural amino acids of either D- or L-configuration. Natural amino acids residues are Ala, Arg, Asn, Asp, Aze, Cys, Gln, Glu, Gly, His, Ile, Irg
  • amino acids residues also refers to various amino acids where sidechain functional groups are coupled with appropriate protecting groups known to those skilled in the art.
  • the Peptides Vol 3, 3-88 (1981) discloses numerous suitable protecting groups and is incorporated herein by reference for that purpose.
  • Novel peptide boronic acids containing aliphatic sidechains were prepared by the series of reactions outlined in Scheme I.
  • An example of this product is 1 where the above intermediate is coupled to Ac-(D)Phe-Pro-OH. 1 was converted to the corresponding alkyl cyanide 2. by treatment with
  • the formamidino substituted boronic acid, 5 was prepared by the synthesis of the corresponding alkyl amine such as Ac-(D)Phe-Pro-boroOrn-C 10 H 16 4, Scheme 2 This in turn was prepared by treating 1 with sodium azide followed by hydrogenation (Kettner et al., 1990) The amine, 4, was treated with ethyl formimidate to yield the formamidino compound, 5.
  • N-substituted isothiouronium derivatives and N-substituted guanidines are readily prepared as shown in Scheme 2a.
  • Treatment of bromide 1 with a thiourea produces directly the isothiouronium 21.
  • Alternatively 1 can be converted to the amine 4 as shown in Scheme 2.
  • the amine A then is heated with a formamidinesulfonic acid in the presence of 4-DMAP to afford the guanidine 22 .
  • the required formamidinesulfonic acids can be prepared by oxidation of the corresponding thioureas, see: Walter and Randau, Liebigs Ann . Chem . 722, 98 (1969).
  • the substituted boronic acid, 7, is prepared by treating 4 with dimethyl cyanodithioiminocarbonate or diphenyl cyanodicarbonimiate to yield the S-methyl isourea (6) or O-phenyl isourea, respectively, using a procedure similar to that reported by Barpill et al. J. Hereocycli c Chem . 25, 1698 (1988), Scheme 3. This intermediate is treated with ammonia in either THF or alcohol to yield the desired product.
  • the aromatic substituted cyanides, H were converted to the corresponding amidino compound, 12 , using the same sequence of reactions described for preparation of the aliphatic amidino compound, 3 .
  • Aromatic guanidino inhibitors 20, were prepared from precursor R-boroPhe-C 10 H 16 .
  • scheme 7. The aromatic ring was nitrated by reaction with NO + BF 4- to yield 18. which was reduced to the corresponding amine, 19.
  • the amine is converted to the guanidine by reaction with aminoiminomethane sulfonic acid [Mosher et al.
  • nitrile (Example 1, 0.40 g, 0.70 mmol) was dissolved in 50 mL of a cold solution of saturated HCI in methanol and the solution was stirred overnight at 4°C. The solution was then concentrated under reduced pressure. The residue was dissolved in anhydrous methanol (50 mL), gaseous NH 3 was bubbled through the solution for 1 h, and the solution was heated at 50 °C for 3 h. Solvent was evaporated, the residue was suspended in minimum volume of methanol, and 0.11 g of benzenesulfonic acid (1 eq) was added. Methanol was evaporated and the residue was triturated with hexane to yield the desired product as a pale yellow powder (0.52 g, 99 % yield).
  • Ethyl formimidate ⁇ HCl was prepared by the procedure of Ohme and Schmitz Angew. Chem. Internat . Edit . 6 566 (1967) and Ac-(D)Phe-Pro-boroOrn-C 10 H 16 was prepared by the procedure of Kettner et al. (1990).
  • the formimidate (1.29 g, 11.7 mmol) and 4-N,N-dimethylaminopyridine (1.44 g) were added to a solution of Ac-(D)Phe-Pro-boroOrn-C 10 H 16 ⁇ BSA (2.78 g, 3.92 mmol) dissolved in 40 mL of ethanol.
  • the resulting solution was refluxed for 8 h. After removal of solvent, the residue was purified by chromatography using a column of SephedexTMLH 20 and methanol as a solvent to give pure product (1.28 g, 56 % yield).
  • Boc-Pro-boroOrn-C 10 H 10 ⁇ BSA was also prepared by the procedure described previously (Kettner et al. 1990). This peptide (3.0 g, 6.5 mmol) was dissolved in 25 mL of absolute ethanol, 4-N,N-dimethylaminopyridine (1.6 g, 12.9 mmol) and ethyl formimidate»HCl (1.4 g, 12.9 mmol) were added. The solution was heated on a 85 °C oil bath for 1 h. Solvent was evaporated and the residue was dissolved in methanol and was chromatogramed on a 2.5 X 100 cm column of LH20 in methanol to yield 1.3 g of the desired product.
  • Boc-(P)Phe-Pro-NHCH[(CH 2 ) 3 -NHC(NH)H]B(OH) 2 Boc-(D)Phe-Pro-NHCH[(CH 2 ) 3 - NHC(NH)H]BO 2 -C 10 H 16 .
  • 0.40 BSA, 0.60 HCI (Example 6, 0.16 g, 0.22 mmol) and phenyl boronic acid (0.13g, 1.1 mmol) were placed in mixture of 5 mL of ether and 5 mL of water and was allowed to stir for 4 h at room
  • the first intermediate, Cl-CH[CH 2 -(m-cyanophenyl)]BO 2 -C 10 H 16 was prepared from m-cyanobenzyl bromide and dichloromethyl boronate pinanediol.
  • Zinc dust (1.0 g) in 1 mL of THF was cooled to 0-5°C and a solution of m-cyanobenzyl bromide (1.37 g, 7.0 mmol) in 7 mL of THF was added dropwise (5 sec/drop). The reaction mixture was allowed to stir at 5°C for 2 h.
  • Cl-CH[CH 2 -(m-bromo-phenyl)]BO 2 -C 10 H 16 was prepared making the anion of m-bromobenzyl bromide and coupling it to dichloromethyl boronic acid pinanediol. This intermediate and the corresponding amine were prepared using the procedure described for Example 10. The amine was coupled to Ac-(D)Phe-Pro-OH using the method
  • the above product (0.060 g, 0.080 mmol) was dissolved in 0.5 mL of THF and was allowed to react with 1 equivalent of 30% aqueous ammonia for 30 min at room temperature. Four additional equivalent of ammonia were added and the solution was allowed to stir overnight at room temperature. A large excess of ammonia was added and the reaction mixture was allowed to stir 2 days at room temperature. The reaction mixture was diluted with methylene chloride and was washed with water and
  • ClCH[CH 2 C 6 H 4 -p-CN]BO 2 C 10 H 16 was prepared by making the anion of p-cyanobenzyl bromide and coupling it to dichloromethyl boronate pinanediol. This intermediate and the corresponding amine were prepared using the procedure described for Example 10. NH 2 CH [CH 2 C 6 H 4 -p-CN]BO 2 C 10 H 16 (Example 78) was coupled to Ac-(D)Phe-Pro-OH using the method described in Example 11.
  • Boc-(D)Phe-Pro-boroPhe(mCN)-C 10 H 16 was prepared by reacting Boc-(P)Phe-Pro-OH (0.43 g, 1.2 mmol), H-boroPhe(mCN)-C 10 H 16 ⁇ HCl (0.42 g, 1.2 mmol), N-methylmorpholine (0.26 mL, 2.4 mmol),
  • cyanoborohydride (0.080 g, 1.3 mmol) was added and after 5 min glacial acetic acid (20 ⁇ L) were added. The reaction pH was ⁇ 7. After 5 h, additional acetic acid (20 ⁇ L) were added and the mixture was stirred for 1 h. The reaction mixture was poured into 20 mL of ethyl acetate and the organic phase was washed with 10 mL of saturated aqueous sodium chloride and dried over
  • the chloroform solution was washed with ice-cold 0.1 N hydrochloric acid (2 X 3 mL), ice-cold water (2 X 3 mL), and brine. The resulting organic solution was then dried over anhydrous magnesium sulfate, filtered, and
  • N-Acyl and N-peptide boronic acids which are described in the present invention represent a novel class of potent, reversible inhibitors of trypsin-like enzymes.
  • Trypsin-like enzymes are a group of proteases which hydrolyzed peptide bonds at basic residues
  • enzymes of the blood coagulation and fibrinolytic system required for hemostasis are enzymes of the blood coagulation and fibrinolytic system required for hemostasis. They are Factors II, X, VII, IX, XII, kallikrein, tissue
  • plasminogen activators urokinase-like plasminogen activator, and plasmin.
  • Enzymes of the complement system, acrosin (required for fertilization), pancreatic trypsin are also in this group. Elevated levels of proteolysis by these proteases can result in disease states. For example, consumptive coagulopathy, a condition marked by a decrease in the blood levels of enzymes of both the coagulation system, the fibrinolytic system and accompanying protease inhibitors is often fatal. Intervention by a synthetic inhibitor would clearly be valuable. More specifically, proteolysis by thrombin is required for blood clotting. Inhibition of thrombin results in an effective inhibitor of blood clotting. The importance of an effective inhibitor of thrombin is underscored by the observation that
  • the compounds of this invention When used in the processing of blood products, the compounds of this invention may be mixed with whole blood without the need for any additional
  • the compounds of this invention serve to inhibit blood coagulation thereby facilitating the processing of whole blood into desired cellular
  • the compounds may be removed, if so desired, by membrane ultrafiltration, dialysis, or diafiltration to afford the desired product.
  • membrane ultrafiltration dialysis, or diafiltration to afford the desired product.
  • anticoagulants like heparin allow them to be separated from desired products more easily.
  • v o is the velocity of the control in the absence of inhibitor
  • v s is the velocity in the presence of inhibitor
  • I is the concentration of inhibitor
  • K i is the dissociation constant of enzyme: inhibitor complex
  • S is the concentration of substrate
  • K m is the Michaelis constant
  • Plasma was prepared by diluting blood 1:10 with 3.2% aqueous citric acid and centrifuging. Buffer consisted of 0.10 M Tris, pH 7.4, containing 0.9% sodium chloride, and 2.5 mg/mL bovine serum albumin. Bovine thrombin was obtained from
  • Plasma (200 ⁇ L) and buffer (50 ⁇ L) containing inhibitor were
  • thrombin 50 ⁇ L
  • clotting times were measured. Controls were run under identical conditions except in the absence of inhibitor. The final concentration of thrombin was 4 NIH units/mL.
  • Ki values were measure at 25 °C at pH 7.5.
  • IC50 level of inhibitor required to prolong clotting to the time observed for 2 NIH units/mL thrombin in the absence of inhibitor.
  • Examples 4, 57, and 77 were shown to be effective following i.v. dosing and Examples 4, 56, 57, 60, and 66 effective following oral dosing.
  • oral administration of Examples 31 and 54 resulted in at least a 2-fold elevation in anticoagulant activity in an identical model except activity was measured by increases in thrombin clotting times.

Abstract

This invention relates to Novel α-aminoboronic acid and corresponding peptide analogs of formula (I) are disclosed.

Description

Title
Amidino and Guanidino Substituted Boronic Acid
Inhibitors of Trypsin-Like Enzymes Cross Reference to Related Applications
This application is a continuation-in-part of
Application Serial Number 08/052,835, filed April 27, 1993. Field of the Invention
The present invention relates generally to α-aminoboronic acids and corresponding peptide analogs in which the alpha substituent is either an aromatic guanidino, isothiouronium, amidino group, halogen, cyano group or an aliphatic amidino, isothiouronium, or formamidino group.
Background of the Invention
Simple boronic acids are inhibitors of serine proteases. For example, Koehler et al. Biochemistry 10: 2477 (1971) reports that 2-phenylethane boronic acid inhibits chymotrypsin at millimolar levels. The
synthesis of boronic acid analogs of N-acyl-α-amino acids has yielded more effective inhibitors. AcboroPhe-OH, R-1-acetamido-2-phenylethane boronic acid, inhibits chymotrypsin with a Ki of 4 μM Matteson et al.
J. Am . Chew . Soc . 103: 5241 (1981). More recently, Shenvi, US 4,537,773 (1985) disclosed that boronic acid analogs of α-amino acids, containing a free amino group, were effective inhibitors of aminopeptidases. Shenvi, US 4,499,082 (1985) discloses that peptides containing an α-aminoboronic acid with a neutral side chain were more effective inhibitors of serine proteases exceeding inhibitors disclosed earlier by as much as 3 orders of magnitude in potency. The chemistry of α-aminoboronic acids was further expanded to the synthesis of peptide analogs containing boronic acid with positive charged sidechains, boroLysine, boroArginine, boroOrnithine, and isothiouronium analogs (EPA 0 293 881, 12/7/88). This series of compounds have provided highly effective inhibitors of thrombin and other trypsin-like enzymes. The boroArginine analogs specifically designed as thrombin inhibitors are highly effective in the
inhibition of blood coagulation both in vitro and in vivo . In the present invention, this group of compounds is extended to aliphatic amidino and formamidino, to aromatic amidino and guanidino, and to cyano and halogen substituted aromatic boronic acid analogs.
It should be noted that additional boronic acids have been disclosed. Metternich (EP 0471651) have described peptides containing boroArginine and
boroLysine which contain at least one unnatural amino acid residue. Elgendy et al. Tetrahedron Lett . , 33, 4209-4212 (1992) have described peptides containing α-aminoboronic acids with aliphatic neutral sidechains which are thrombin inhibitors. Kakkar in (WO 92/07869) has claimed peptide thrombin inhibitors of the general structure, X-Aa1-Aa2-NH-CH(Y)-Z where Aai and Aa2 are unnatural amino acid residues. Z is -CN, -COR,
-B(R2)(R3), -P(0)(R)(R), and Y is -[CH2]n-Q or -CH2-Ar-Q where Q = H, amino, amidino, imidazole, guanidino or isothioureido and n=l-5 and where R2 and R3 are the same or different and are selected from the group consisting of OH, OR6, and NR6R7, or R2 and R3 taken together represent the residue of a diol. This specialized group of compounds where Z is -B(R2)(R3) fall within the scope of our present application. It should be noted that this is a narrow subset of Kakkar et al. However, rather specialized chemical transformations are required to prepare these compounds and Kakkar et al. does not make an enabling disclosure. Summary of the Invention
A compound of formula (I)
Figure imgf000005_0002
wherein
R1 is
a) C1-C12-alkyl substituted with -CN, -C(NH)NHR6,
-NHC(NH)H, -NHC(NH)NHR6, -SC(NH)NHR6, -NHC(NH)NHOH,
-NHC(NH)NHCN, -NHC(NH)NHCOR6, or
b) ;
Figure imgf000005_0001
X is
a) halogen (F, Cl, Br, I)
b) -CN,
c) -NO2,
d) -CF3,
e) -NH2
f) -NHC(NH)H,
g) -NHC(NH)NHOH,
h) -NHC(NH)NHCN,
i) -NHC(NH)NHR6,
j) -NHC(NH)NHCOR6,
k) -C(NH)NHR6,
l) -C(NH)NHCOR6,
m) -C(O)NHR2,
n) -CO2R2,
o) -OR2, or
p) -OCF3 q) -SC(NH)NHR6;
R2 is
a) H,
b) C1-C4-alkyl,
c) aryl, wherein aryl is phenyl or napthyl
optionally substituted with one or two substituents selected from the group consisting of halo (F, Cl, Br, I), C1-C4-alkyl, C1-C4-alkoxy, -NO2, -CF3, -S(O)r-C1-C4-alkyl, -OH, -NH2, -NH(C1-C4-alkyl), -N(C1-C4-alkyl)2, -CO2R4, or
d) -C1-C4-alkylaryl, where aryl is defined above; R3 is H, alkyl, aryl, alkylaryl, or an NH2-blocking group comprised of 1-20 carbon atoms;
R4 and R5 are independently
a) H,
b) C1-C4-alkyl, or
c) -CH2-aryl, where aryl is defined above;
R6 is
a) H,
b) C1-C4-alkyl,
c) aryl, wherein aryl is phenyl or napthyl
optionally substituted with one or two substituents selected from the group consisting of halo (F, Cl, Br, I), C1-C4-alkyl, C1-C7-alkoxy, -NO2, -CF3, -S(O)r-C1-C4-alkyl, -OH, -NH2, -NH (C1-C4-alkyl), -N(C1-C4-alkyl)2, -CO2R4, or
d) -C1-C4-alkylaryl, where aryl is defined above;
A is an amino acid residue or a peptide comprised of 2-20 amino acid residues;
Y1 and Y2 are
a) -OH,
b) -F,
c) C1-C8-alkoxy, or
when taken together Y1 and Y2 form a d) cyclic boron ester where said chain or ring contains from 2 to 20 carbon atoms and, optionally, 1-3 heteroatoms which can be N, S, or O,
n is 0 or 1;
p is 0 to 3;
q is 0 to 4;
r is 0 to 2;
and pharmaceutically acceptable salts thereof, with the proviso that when R1 is aliphatic, an R6 substituent on -NHC(NH)NHR6 cannot be H.
Preferred are those compounds of formula (I) where Y1 and Y2 are
a) -OH,
when taken together Y1 and Y2 form a
b) cyclic boron pinacol ester, or
c) cyclic boron pinanediol ester;
R1 is
a) -(CH2)3NHC(NH)H,
b) -(CH2)4C(NH)NH2,
Figure imgf000007_0001
Figure imgf000007_0002
, or
;
Figure imgf000007_0003
R2 is H;
A is Pro or (D)Phe-Pro;
R3 is
a) H,
b) Boc,
c) Z, or
d) Ac, or
e) hydrocinnamoyl
f) C1-C10 alkyl sulfonyl
g) C1-C15 alkylaryl sulfonyl
Illustrative of the preferred compounds of this invention are the following: ● Ac-(D)Phe-Pro-NH-CH[(CH2)4CN]BO2-C10H16
● Ac-(D)Phe-Pro-NHCH[(CH2)4C(NH)NH2]BO2-C10H16 ● Ac-(D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]B(OH)2
● Boc-(D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]B(OH)2.
● Ac-(D)Phe-Pro-boroPhe[m-C(NH)NH2]-C10H16
● Ac-(D)Phe-Pro-boroPhe(m-CH2NH2)-C10H16
● Ac-(D)Phe-Pro-boroPhe(m-Br)-C10H16
● Ac-(D)Phe-Pro-boroArg(CN)-C10H16
● Ac-(D)Phe-Pro-boroPhe(p-CN)-C10H16
● Boc-(D)Phe-Pro-boroPhe-(m-CN)-C10H16
● N,N-(CH3)2-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCl (ISOMER
I)
● Ac-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCl
● Ms-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCl
● Boc-(D)Thiazolylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Boc-(D)3-Pyridylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Ms-(D)3-Pyridylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Boc-(D)2-Pyridylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Boc-(D)2-Thienylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Ms-(D)2-Thienylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Boc-(D)Phe-Aze-boroPhe-(m-CN)-C10H16
● Hydrocinnamoyl-Pro-boroIrg(CH3)-OH●HBr ● Ac-(D)PDe-Pro-boroArg(CH3)-OH●HCl
● PhCH2SO2-(D)Phe-Pro-boroOrn(CH=NH)-OH●HCl
● CH3CH2CH2SO2-(D)Phe-Pro-boroOrn(CH=NH)-OH●HCl ● CH3CH2CH2SO2-(D)Phe-Pro-boroArg(CH3)-OH●HCl ● Ac-(D)Phe-Sar-boroOrn(CH=NH)-OH●HCl
● Boc-(D)Phe-Sar-boroPhe(mCN)-C10H16
● Boc-(D)Phe-Aze-boroOrn(CH=NH)-OH●HCl
● 4-(Phenyl)benzoyl-boroOrn(CH=NH)-C10H10●HCl This invention also provides compositions
comprising one or more of the foregoing compounds and methods of using such compositions in the treatment of aberrant proteolysis such as thrombosis in mammals or as reagents used as anticoagulants in the processing of blood to plasma for diagnostic and other commercial purposes.
Detail Description of the Invention
As used throughout the specifications, the
following abbreviations for amino acid residues or amino acids apply:
Ala = L-alanine
Arg = L-arginine
Asn = L-asparagine
Asp = L-aspartic acid
Aze = azedine-2-carboxlic acid
Cys = L-cysteine
Gln = L-glutamine
Glu = L-glutamic acid
Gly = glycine
His = L-histidine
HomoLys = L-homolysine
Ile = L-isoleucine
Irg = isothiouronium analog of L-Arg Leu = L-leucine
Lys = L-lysine Met = L-methionine
Orn = L-ornithine
Phe = L-phenylalanine
Pro = L-proline
Ser = L-serine
Thr = L-threonine
Trp = L-tryptophan
Tyr = L-tyrosine
Val = L-valine
Sar = L-sarcosine
Phe(4-fluoro) = para-fluorophenylalanine
The "D" prefix for the foregoing abbreviations indicates the amino acid is in the D-configuration .
"D,L" indicates the amino is present in mixture of the
D- and the L-configuration. The prefix "boro" indicates amino acid residues where the carboxyl is replaced by a boronic acid or a boronic acid ester. For example, if R1 is isopropyl and Y1 and Y2 are OH, the C-terminal residue is abbreviated "boroVal-OH" where "-OH"
indicates the boronic acid is in the form of the free acid. The pinanediol boronic acid ester and the pinacol boronic acid ester are abbreviated "-C10H16" and
"-C6H12", respectively. Examples of other useful diols for esterification with the boronic acids are
1,2-ethanediol, 1,3-propanediol, 1,2-propanediol,
2,3-butanediol, 1,2-diisopropylethanediol,
5,6-decanediol, and 1,2-dicyclohexylethanediol. The formamidino modified amino group is abbreviated (CH=NH) . For example, the formamidino analog of -boroOrn-OH {-NH-CH[(CH2)3-NH-CH(NH)H]B(OH)2 } is -boroOrn (CH=NH)-OH.
Analogs containing sidechain substituents are described by indicating the substituent in parenthesis following the name of the parent residue. For example the analog of boroPhenylalanine containing a meta cyano group is -boroPhe (mCN)-. N-alkyl substituents on the guanidino group of boroArg- or on the isothiouronium analogs
(borolrg) are also put in parenthesis in a similar manner. Other abbreviations are: Z, benzyloxycarbonyl;
BSA, benzene sulfonic acid; THF, tetrahydrofuran; Boc-, t-butoxycarbonyl-; Ac-, acetyl; pNA, p-nitro-aniline;
DMAP, 4-N,N-dimethylaminopyridine; Tris,
Tris (hydroxymethyl) aminomethane; MS, mass spectrometry;
FAB/MS, fast atom bombardment mass spectrometry.
LRMS(NH3-CI) and HRMS(NH3-CI) are low and high
resolution mass spectrometry, respectively, using NH3 as an ion source.
It is understood that many of the compounds of the present invention contain one or more chiral centers and that these stereoisomers may possess distinct physical and biological properties. The present invention comprises all of the stereoisomers or mixtures thereof.
If the pure enantiomers or diasteromers are desired, they may be prepared using starting materials with the appropriate stereochemistry, or may be separated from mixtures of undesired stereoisomers by standard
techniques, including chiral chromatography and
recrystalization of diastereomeric salts.
"NH2-blocking group" as used herein, refers to various acyl, thioacyl, alkyl, sulfonyl, phosphoryl, and phosphinyl groups comprised of 1 to 20 carbon atoms. Substitutes on these groups maybe either alkyl, aryl, alkylaryl which may contain the heteroatoms, O, S, and N as a substituent or as inchain component. A number of NH2-blocking groups are recognized by those skilled in the art of organic synthesis. By definition, an NH2-blocking group may be removable or may remain
permanently bound to the NH2. Examples of suitable groups include formyl, acetyl, benzoyl, trifluoroacetyl, and methoxysuccinyl; alkyl and alkylaryl sulfonyl groups, such as n-propylsulfonyl, phenylmethyl and benzylsulfonyl; aromatic urethane protecting groups, such as, benzyloxycarbonyl; and aliphatic urethane protecting groups, such as t-butoxycarbonyl or
adamantyloxycarbonyl. Gross and Meinhoffer, eds., The Peptides, Vol 3; 3-88 (1981), Academic Press, New York, and Greene and Wuts Protective Groups in Organic
Synthesis, 315-405 (1991), J. Wiley and Sons, Inc., New York disclose numerous suitable amine protecting groups and they are incorporated herein by reference for that purpose.
"Amino acid residues" as used herein, refers to natural or unnatural amino acids of either D- or L-configuration. Natural amino acids residues are Ala, Arg, Asn, Asp, Aze, Cys, Gln, Glu, Gly, His, Ile, Irg
Leu, Lys, Met, Orn, Phe, Phe (4-fluoro), Pro, Sar, Ser, Thr, Trp, Tyr, and Val. Roberts and Vellaccio, The Peptides, Vol 5; 341-449 (1983), Academic Press, New York, discloses numerous suitable unnatural amino acids and is incorporated herein by reference for that purpose. Additionally, said reference describes, but does not extensively list, acylic N-alkyl and acyclic α,α-disubstituted amino acids. Included in the scope of the present invention are N-alkyl, aryl, and alkylaryl analogs of both in chain and N-terminal amino acid residues. Similarly, alkyl, aryl, and alkylaryl maybe substituted for the alpha hydrogen. Illustrated below are examples of N-alkyl and alpha alkyl amino acid residues, respectively.
Figure imgf000012_0001
"Amino acids residues" also refers to various amino acids where sidechain functional groups are coupled with appropriate protecting groups known to those skilled in the art. "The Peptides", Vol 3, 3-88 (1981) discloses numerous suitable protecting groups and is incorporated herein by reference for that purpose.
Synthesis
Novel peptide boronic acids containing aliphatic sidechains were prepared by the series of reactions outlined in Scheme I. First, the precursor, NH2-CH[(CH2)nBr]BO2-C10H16. n = 3 or 4, was prepared and coupled with an N-terminal protecting group or with an N-terminal and sidechain protected peptide by the procedure we have described previously [Kettner et al. J. Biol . Chem . 265 18289-18297 (1990)]. An example of this product is 1 where the above intermediate is coupled to Ac-(D)Phe-Pro-OH. 1 was converted to the corresponding alkyl cyanide 2. by treatment with
tetrabutyl ammonium cyanide in THF at 55 °C for 2 hours. This appears to be a general method for introducing the cyano group. In contrast, other common methods of introducing this group can be applied only with limited success. For example, the reaction of Ac-(D)Phe-Pro-NH-CH[(CH2)4-Br]BO2-C10H16 with KCN in N,N-dimethylformamide failed to yield a detectable product. Our data are consistent with the formation of a cyclic product arising from the nucleophilic displacement of the sidechain bromide by the adjacent amide NH.
Treatment of Z-NH-CH[(CH2)4-Br]BO2-C10H16 with NaCN in N,N-dimethylformamide gave the cyano compound, but only in low yield, indicating that cyclization does not occur quite so readily when the urethane protecting group (Z) is present. Typically, 2 was purified by standard techniques such as silica gel chromatography. The corresponding amidine, 3 , was prepared by treating the nitrile with a saturated solution of a mineral acid such as HCl in methanol. Excess solvent and acid were removed by evaporation and the residue was allowed to react with anhydrous ammonia to yield the desired product.
Figure imgf000014_0001
The formamidino substituted boronic acid, 5, was prepared by the synthesis of the corresponding alkyl amine such as Ac-(D)Phe-Pro-boroOrn-C10H16 4, Scheme 2 This in turn was prepared by treating 1 with sodium azide followed by hydrogenation (Kettner et al., 1990) The amine, 4, was treated with ethyl formimidate to yield the formamidino compound, 5.
Scheme 2
Figure imgf000015_0001
N-substituted isothiouronium derivatives and N-substituted guanidines are readily prepared as shown in Scheme 2a. Treatment of bromide 1 with a thiourea produces directly the isothiouronium 21. Alternatively 1 can be converted to the amine 4 as shown in Scheme 2. Employing a method originally described by Kim et al., Tetrahedron Let t . 29, 3183 (1988), the amine A then is heated with a formamidinesulfonic acid in the presence of 4-DMAP to afford the guanidine 22 . The required formamidinesulfonic acids can be prepared by oxidation of the corresponding thioureas, see: Walter and Randau, Liebigs Ann . Chem . 722, 98 (1969).
Scheme 2a
Figure imgf000016_0002
The substituted boronic acid, 7, is prepared by treating 4 with dimethyl cyanodithioiminocarbonate or diphenyl cyanodicarbonimiate to yield the S-methyl isourea (6) or O-phenyl isourea, respectively, using a procedure similar to that reported by Barpill et al. J. Hereocycli c Chem . 25, 1698 (1988), Scheme 3. This intermediate is treated with ammonia in either THF or alcohol to yield the desired product.
Scheme 3
Figure imgf000016_0001
Hydroxyguanidino inhibitors are prepared by
treating 4 with cyanogen bromide or cyanogen chloride followed by hydroxylamine to yield 8, Scheme 4. These are known chemical transformations, Nakahara et. al.
Tetrahedron, 33, 1591 (1977) and Belzecki et al. J.
Chem . Soc . Chem . Commun . , 806 (1970).
Scheme 4.
Figure imgf000017_0001
The preparation of new aromatic boronic acids are shown in Scheme 5. Functionalized benzylic anions containing either a halogen or cyano substituent (the cyano group is shown for illustration) are obtained by treatment with activated Zn metal in THF or other inert solvent and then with CuCN·2LiCl [Berk et al.
Organometallics 9, 3053-3064 (1990)]. Dichloromethyl boronic acid pinanediol was prepared by the method described by Tsai et al. Organometallics 2, 1543-1545 (1983). It was allowed to react with the transmetalated anion to yield 9 . This was the only acceptable method of preparing these functionalized benzylic anions. For example, treatment of p-nitobenzyl chloride with lithium metal using the method of Michel et al. J.
Organometallic Chem . 204, 1-12 (1981) failed to yield an identifiable product. Similarly, treatment of p-cyanobenzyl chloride with lithium naphthalenide in the presence of ZnCl2 using the conditions of Zhu et al . J. Org . Chem . 56, 1445-1453 (1991) did not give the desired product. The α-aminoboronic acid, 10, was obtained by treating 9 with the lithium salt of hexamethyldisilazane and removing the trimethylsilanyl groups by treatment with anhydrous HCl. 10 was coupled to either an N-terminal protecting group or to a peptide using known techniques.
The aromatic substituted cyanides, H, were converted to the corresponding amidino compound, 12 , using the same sequence of reactions described for preparation of the aliphatic amidino compound, 3 .
Scheme 5
Figure imgf000018_0001
11 can be hydrogenated to yield the corresponding aminomethyl group as an aromatic substituent 13, Scheme 6. The corresponding formamidino, cyanoguanidino, hydroxyguanidino and guanidino compounds, 14, 15, 16, and 12, respectively, are prepared by the procedures described for the aliphatic series, Scheme 1.
Figure imgf000019_0001
Aromatic guanidino inhibitors , 20, were prepared from precursor R-boroPhe-C10H16. scheme 7. The aromatic ring was nitrated by reaction with NO+BF4- to yield 18. which was reduced to the corresponding amine, 19. The amine is converted to the guanidine by reaction with aminoiminomethane sulfonic acid [Mosher et al.
Tetrahedral Lett . 29 3183 (1988)] or cyanamide (Kettner et al. 1990). Scheme 7
Figure imgf000020_0001
NMR, proton nuclear magnetic resonance, chemical shifts are reported in δ units, parts per million downfield from the internal tetramethylsilane standard. Elemental analyses were conducted by Galbraith
Laboratories Inc., Knoxville, TN and Microanalysis Inc., Wilmington, DE . FAB/MS samples of free boronic acids did not give consistent results making it difficult to monitor the removal of ester protecting groups by this means. However, the presence of the pinanediol and the pinacol groups are readily observed in NMR spectra. For the pinanediol ester, a methyl group is observed at δ 0.9 and the methyl groups of the pinacol groups are observed as singlet at δ 1.1. Following the removal of pinanediol protecting group, MS were run by treating the sample with ~2 equivalents of pinacol in methanol for 5 minutes and evaporating the solvent. Similarly, MS samples of free boronic acid, obtained by removal of the pinacol, were prepared by treating with pinanediol. In some cases, ethylene glycol was used as a matrix for mass spectroscopy to yield the boronic acid-ethyleneglycol ester (designated EG ester). For the subsequent Example see Table 1 for analytical data.
Example 1
Synthesis of Ac-(D)Phe-Pro-NH-CH[(CH2)4CN]BO2-C10H16
The intermediate, Ac-(D)Phe-Pro-NH-CH[(CH2)4Br]BO2- C10H16, was prepared using the mixed anhydride
procedure. Ac-(D)Phe-Pro-OH (3.04 g, 10 mmol) was dissolved in 50 mL of THF and N-methylmorpholine (1.1 mL, 10 mmol) was added. The solution was cooled to -20°C using a CCI4 dry ice bath and isobutyl
chloroformate (1.30 mL, 10 mmol) was added. After 5 min at -20°C, the mixture was added to NH2-CH[(CH2)4Br]BO2- C10H16●HCl (3.81 g, 10 mmol) which was dissolved in 20 mL of THF and precooled to -20°C. Triethylamine ( 1.39 mL, 10 mmol) was added and the mixture was allowed to stir for 1 h at -20°C and 2 h at room temperature. Insoluble material was removed by filtration and the filtrate was evaporated under a reduced pressure. The residue was dissolved in 50 mL of ethyl acetate and washed
subsequently with 75 mL of 0.2 N HCI, 5% NaHCO3, and saturated aqueous sodium chloride . The organic phase was dried over Na2SO4 and concentrated in vacuo to give Ac-(D)Phe-Pro-NHCH[(CH2)4Br]BO2-C10H16 (6.01 g, 95% yield).
The bromide (1.89 g, 3.0 mmol) and tetra-n-butyl ammonium cyanide (3.2 g, 11.8 mmol, 4 eq) were dissolved in 50 mL of acetonitrile. This solution was heated at 90°C for 3 h and solvent was removed under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate and was washed with three 50 mL portions of saturated aqueous NaCl. The ethyl acetate solution was dried over anhydrous Na2SO4 and evaporated to give 2.5 g of crude product. It was purified by silica gel chromatography using 5% MeOH in CHCI3 as an eluent to yield the desired product (0.50 g, 29% yield).
LRMS (NH3-CI) m/e calcd. for M (C32H45N4O5B) + NH4 +:
594.4. Found: 594. HRMS(NH3-CI) m/e calcd. for M (C32H45N4O5B) + H+ : 577.3561. Found: 577.3555.
Example 2
Synthesis of Ac-(D)Phe-Pro-NHCH[(CH2)4C(NH)NH2]-BO2-C10H16●benzene sulfonic acid
The nitrile, (Example 1, 0.40 g, 0.70 mmol), was dissolved in 50 mL of a cold solution of saturated HCI in methanol and the solution was stirred overnight at 4°C. The solution was then concentrated under reduced pressure. The residue was dissolved in anhydrous methanol (50 mL), gaseous NH3 was bubbled through the solution for 1 h, and the solution was heated at 50 °C for 3 h. Solvent was evaporated, the residue was suspended in minimum volume of methanol, and 0.11 g of benzenesulfonic acid (1 eq) was added. Methanol was evaporated and the residue was triturated with hexane to yield the desired product as a pale yellow powder (0.52 g, 99 % yield).
FABMS: m/e calculated for M (C32H48N5O5B) + H+ :
594.38. Found: 594.14. HRMS(NH3-CI) m/e calcd for M (C32H48N5O5B) + H+: 594.3827. Found: 594.3824. Example 3
Synthesis of Ac-(D)Phe-Pro-NHCH_(CH2)2NHC(NH)H]BO2-C10H16 or Ac-(D)Phe-Pro-boroOrn (CH=NH) -C10H16
Ethyl formimidate●HCl was prepared by the procedure of Ohme and Schmitz Angew. Chem. Internat . Edit . 6 566 (1967) and Ac-(D)Phe-Pro-boroOrn-C10H16 was prepared by the procedure of Kettner et al. (1990). The formimidate (1.29 g, 11.7 mmol) and 4-N,N-dimethylaminopyridine (1.44 g) were added to a solution of Ac-(D)Phe-Pro-boroOrn-C10H16●BSA (2.78 g, 3.92 mmol) dissolved in 40 mL of ethanol. The resulting solution was refluxed for 8 h. After removal of solvent, the residue was purified by chromatography using a column of Sephedex™LH 20 and methanol as a solvent to give pure product (1.28 g, 56 % yield).
HRMS(NH3-CI) m/e calcd. for M (C31H46BN5O5) + H+ : 580.3670. Found: 580.3679. Example 4
Synthesis of Ac- (D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]B(OH)2
The pinanediol protecting group on the boronic acid portion of Ac-(D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]-BO2-C10H16●HCl (Example 3) was removed by
transesterification using the procedure we have
described previously in U.S.Application 08/010731. The pinanediol ester (0.30 g, 0.51 mmol) and phenyl boronic acid (0.31 g, 2.6 mmol) were suspended in 10 mL of a 1:1 mixture of ether and water and was allowed to stir for 2.5 h at room temperature. The phases were separated and the aqueous phase was extensively washed with ether. The aqueous phase was evaporated to yield a solid. This material was triturated with ether to give the desired product as an amorphous white solid, 0.20 g (83 % yield). LRMS (NH3-CI) m/e calcd. for the pinacol ester M (C27H42N5O5B) + H+: 528.3. Found: 528. HRMS (NH3- Cl) m/e calcd. for the pinacol ester M (C27H42N5O5B) + H+: 528.3357. Found: 528.3347.
Example 5
Synthesis of Boc-Pro-NHCH[(CH2)3NHC(NH)H]BO2-C10H16.
Boc-Pro-boroOrn-C10H10●BSA was also prepared by the procedure described previously (Kettner et al. 1990). This peptide (3.0 g, 6.5 mmol) was dissolved in 25 mL of absolute ethanol, 4-N,N-dimethylaminopyridine (1.6 g, 12.9 mmol) and ethyl formimidate»HCl (1.4 g, 12.9 mmol) were added. The solution was heated on a 85 °C oil bath for 1 h. Solvent was evaporated and the residue was dissolved in methanol and was chromatogramed on a 2.5 X 100 cm column of LH20 in methanol to yield 1.3 g of the desired product.
LRMS (NH3-CI) m/e calcd. for M (C25H43N4O5B) + H+ : 491.5. Found: 491.
Example 6
Synthesis of Boc-(D)Phe-Pro-NHCH[[(CH2)3-NHC(NH)H]BO2- C1 0H1 6
The reaction was run using the procedure described for Example 3. Boc-(D)Phe-Pro-boroOrn-C10H16●BSA (3.7 g, 4.78 mmol), 4-N,N-dimethylaminopyridine (1.71 g, 13.8 mmol), and ethyl formimidateΗCl (1.54 g, 13.8 mmol) were dissolved in 50 mL of absolute ethanol and was heated at 85 °C for 7 h. The desired product was obtained by chromatography on a column of LH 20 in a yield of 1.56 g.
HRMS (NH3-CI) m/e calcd for M (C34H52N5O6B) + H+: 638.4089. Found: 638.4082.
Example 7
Synthesis of Boc-(P)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]B(OH)2. Boc-(D)Phe-Pro-NHCH[(CH2)3- NHC(NH)H]BO2-C10H16. 0.40 BSA, 0.60 HCI (Example 6, 0.16 g, 0.22 mmol) and phenyl boronic acid (0.13g, 1.1 mmol) were placed in mixture of 5 mL of ether and 5 mL of water and was allowed to stir for 4 h at room
temperature. The phases were separated and the organic phase was washed with 5 mL of water. The combined aqueous phases were extensively washed with ether. The aqueous phase was evaporated and the residue triturated with ether to yield the desired product as a white solid, 0.10 g. LRMS (NH3-CI) m/e calcd. for the pinacol ester M (C30H48N5O6B) + H+ : 586.4. Found: 586. HRMS (NH3-CI) m/e calcd. for the pinacol ester M
(C30H48N5O6B) + H+: 586.3776. Found: 586.3772.
Example 8
Synthesis of H-(P)Phe-Pro-NHCH[(CH2)2-NHC(NH)H]BO2- C10H16●2HCl
Boc-(D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]BO2-C10H16●0.40 BSA, 0.60 HCI (Example 6, 0.20 g, 0.25 mmol) was
dissolved in 2 mL of 4 N HCI: dioxane and was allowed to stir for 1 h at room temperature. Solvent was
evaporated and the residue was triturated with ether to yield 0.18 g of the desired product.
HRMS (NH3-CI) m/e calcd for M (C29H44N5O4B) + H+: 538.3565. Found: 538.3569.
Example 9
Synthesis of H-(D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]B(OH)2
H-(D)Phe-Pro-NH-CH[(CH2)3-NH-C(NH)H]BO2-C10H1 6●0 . 35 BSA, 0 . 65 HCI (Example 8 , 0 . 10 g, 0 . 16 mmol ) was allowed to react with phenyl boronic acid according to the procedure in Example 4 to yield the desired product , 0 . 053 g . LRMS (NH3-CI ) m/e calcd . for the pinacol ester M (C2 5H40N5O4B) + H+ : 486 .3 . Found: 486. HRMS (NH3-CI) m/e calcd for pinacol ester M (C25H40N5O4B) + H+: 486.3251. Found: 486.3255. Example 10
Synthesis Of H2NCH [CH2C6H4-m-CNlBO2C10H16●HCl or
H-boroPhe (m-CN) -C10H16●HCl
The first intermediate, Cl-CH[CH2-(m-cyanophenyl)]BO2-C10H16, was prepared from m-cyanobenzyl bromide and dichloromethyl boronate pinanediol. Zinc dust (1.0 g) in 1 mL of THF was cooled to 0-5°C and a solution of m-cyanobenzyl bromide (1.37 g, 7.0 mmol) in 7 mL of THF was added dropwise (5 sec/drop). The reaction mixture was allowed to stir at 5°C for 2 h. A mixture consisting of LiBr (1.22 g, 14 mmol), CuCN (0.63 g, 7.0 mmol), and 6 mL of THF was placed in a 50 ml flask and cooled to -40°C; then the benzylic organozinc reagent was added by cannulation. The mixture was allowed to warm to -20°C and stir for 5 min . It was cooled to -78°C and neat dichloromethyl boronic acid pinanediol (1.47 g, 5.6 mmol) was added dropwise. The resulting mixture was stirred at -78°C for 2 h and at room temperature for 2 days. Saturated aqueous NH4CI (20 mL) was added to the mixture and the aqueous
solution was extracted with three 20 ml portions of ether. The combined organic layers was dried over anhydrous MgSO4 and evaporated in vacuo to give crude compound (1.8 g). It was purified by silica gel
chromatography where the column was stepwise eluted with hexane (100 mL) and then 15% ether in hexane (200 mL) to give the desired product 0.53 g (27% yield). LRMS (NH3-CI) m/e calcd. for M (C19H23NO2BCI) +NH4 +: 361.2. Found: 361.1.
To a solution of hexamethyldisilazane (0.21 mL, 0.98 mmol) in 2 mL of THF at -78°C was added n-butyllithium (1.45 M, 0.67 mL, 0.98 mmol). The solution was allowed to slowly warm to room temperature to ensure the anion generation was complete. The resulting solution was then cooled to -78°C and Cl-CH[CH2-(m-cyanophenyl)]BO2-C10H16 (0.33 g, 0.98 mmol) in 2 mL of THF was added. The mixture was allowed to warm to room temperature and to stir overnight. Solvent was
evaporated and 8 mL of hexane was added to give a suspension. HCI in dioxane (4.1 N, 1.5 mL, 6.0 mmol) was added at -78°C. The mixture was slowly warmed to room temperature and stirred for 2 h. Additional hexane (6 mL) was added and crude product was isolated as a precipitate. This product was dissolved in chloroform and insoluble material was removed by filtration. The filtrate was evaporated at a reduced pressure to give an oil (~0.2 g). Final purification was achieved by chromatography on a column of Sephedex™ LH 20 column using methanol as a solvent. H-boroPhe (m-CN)-C10H16●HCl was obtained as an oil (0.12 g, 34% yield). HRMS (NH3-CI) m/e calcd. for M (C19H26BN2O2) + H+ : 325.2087.
Found: 325.2094.
Example 11
Synthesis of Ac-(D)Phe-Pro-boroPhe(m-CN)-C10H16
Ac-(P)Phe-Pro-OH (0.10 g, 0.33 mmol) and N-methylmorpholine (0.037 mL, 0.33 mmol) were allowed to react with isobutyl chloroformate (0.043 mL, 0.33 mmol) in 5 mL of THF at -20°C. After 5 min, H-boroPhe(m-CN)-C10H16●HCl, (Example 10, 0.12 g, 0.33 mmol) dissolved in 3 mL of cold THF and triethylamine (0.046 mL, 0.33 mmol) were added. The mixture was allowed to stir at -20°C for 1 h and to stir at room temperature for an
additional hour. Insoluble material was removed by filtration and solvent was evaporated. The residue was dissolved in ethyl acetate and was washed with 0.20 N HCI, 5 % NaHCO3, and saturated aqueous NaCl. The organic layer was dried over anhydrous Na2SO4 an d was evaporated in vacuo to give 0.2 g of an oil. It was purified by chromatography on a column of Sephedex™ LH 20 yielding 0.13 g of desired product (65% yield). HRMS(NH3-CI) m/e calcd. for M (C35H43BN4O5) + H+:
611.3405. Found: 611.3416.
Example 12
Synthesis of Ac-(D)Phe-Pro-boroPhe[m-C(NH)NH2]-C10H16
Ac- (D)Phe-Pro-boroPhe (m-CN)-C10H16, Example 11, (50 mg) was dissolved in 5 mL of saturated solution of HCI in methanol. The solution was allowed to stir overnight at 4 °C. After removal of solvent, the residue was resuspended in 5 mL of anhydrous methanol, cooled to
0°C, and anhydrous NH3 was bubbled through the solution for 0.5 h. It was heated at 60°C for 6.2 h. Solvent was evaporated and one equivalent of benzene sulfonic acid (13 mg) and 1 mL of methanol were added. Solvent was evaporated under N2 and the product was triturated with ether to give the desired product as a pale brown powder (65 mg, 100% yield). HRMS(NH3-CI) m/e calcd. for M (C35H47BN5O5) + H+ : 628.3670. Found: 628.3688. Example 13
Synthesis of Ac-(D)Phe-Pro-boroPhe(m-CH2NH2)-C10H16
Ac- (P) Phe-Pro-boroPhe (m-CN) -C10H16 was placed in 5 mL of methanol, 10% Pd/C (25 mg) and 0.1N HCI (0.41 mL) were added, and the mixture was stir under H2 at room temperature for 2.5 h. The solution was filtered through Celite and washed with 20 mL of methanol. The filtrate was concentrated under a reduced pressure and the residue was triturated with ether to give pure product as white powder (15.6 mg, 59% yield). HRMS (NH3-Cl) m/e calcd. for M (C35H47N4O5B) + H+ : 615.3718.
Found: 615.3700.
Example 14
Synthesis of Ac-(P)Phe-Pro-boroPhe(m-Br)-C10H16
Cl-CH[CH2-(m-bromo-phenyl)]BO2-C10H16 was prepared making the anion of m-bromobenzyl bromide and coupling it to dichloromethyl boronic acid pinanediol. This intermediate and the corresponding amine were prepared using the procedure described for Example 10. The amine was coupled to Ac-(D)Phe-Pro-OH using the method
described in Example 11.
LRMS(NH3-CI) m/e calcd. for M (C34H43N3O5BrB) + H+: 666.3. Found: 666.2.
Example 15
Synthesis of Ac-(P)Phe-Pro-boroArg(CN)-C10H16
Ac-(D)Phe-Pro-boroOrn-C10H16●HCl (0.15 g, 0.25 mmol), triethylamine (0.035 mL, 0.25 mmol), and diphenyl cyanocarbonimidate (Aldrich, 0.060 g, 0.25 mmol) were heated at a gentle reflux for 5 h in THF and then stirred overnight at room temperature. The sample was diluted with chloroform and washed with water and saturated aqueous NaCl . It was dried over K2CO3 and purified by silica gel chromatgraphy using methanol: chloroform (1:9) as a solvent to yield 80 mg of Ac-(P)Phe-Pro-NH-CH[(CH2)3-NH-C(N-CN)O-Ph]BO2-C10H16.
LRMS(NH3-CI) m/e calcd. for M (C38H49N6O6B) + H+:
697.7. Found: 697.
The above product (0.060 g, 0.080 mmol) was dissolved in 0.5 mL of THF and was allowed to react with 1 equivalent of 30% aqueous ammonia for 30 min at room temperature. Four additional equivalent of ammonia were added and the solution was allowed to stir overnight at room temperature. A large excess of ammonia was added and the reaction mixture was allowed to stir 2 days at room temperature. The reaction mixture was diluted with methylene chloride and was washed with water and
saturated aqueous NaCl. It was dried over K2CO3 and purified by chromatography on a silica gel column using methanol and chloroform (1:9) as a solvent to yield 15 mg of the desired product. LRMS (NH3-CI) m/e calcd. for M (C32H46N7O5B) + H+: 619.5. Found: 620. Example 16
Synthesis of Ac-(D)Phe-Pho-boroPhe(n-CN)-C10H16
ClCH[CH2C6H4-p-CN]BO2C10H16 was prepared by making the anion of p-cyanobenzyl bromide and coupling it to dichloromethyl boronate pinanediol. This intermediate and the corresponding amine were prepared using the procedure described for Example 10. NH2CH [CH2C6H4-p-CN]BO2C10H16 (Example 78) was coupled to Ac-(D)Phe-Pro-OH using the method described in Example 11.
HRMS (NH3-Cl)m/e calcd. for M (C35H43N4O5B) + H+: 611.3405. Found: 611.3408.
Example 17
Synthesis of Boc-(P)Phe-Pro-boroPhe(mCN)-C10H16
Boc-(D)Phe-Pro-boroPhe(mCN)-C10H16 was prepared by reacting Boc-(P)Phe-Pro-OH (0.43 g, 1.2 mmol), H-boroPhe(mCN)-C10H16●HCl (0.42 g, 1.2 mmol), N-methylmorpholine (0.26 mL, 2.4 mmol),
hydroxybenzotriazole●H2O (0.36 g, 2.4 mmol), and dicyclohexylcarbodiimide (0.25 g, 1.2 mmol) in 20 mL of dichloromethane overnight at room temperature. The reaction mixture was filtered and the filtrate was chromatogramed on a 2.5 X 100 cm column of Sephedex LH-20 in methanol to yield 0.36 g of the desired product.
Example 18
Synthesis of H-(D)Phe-Pro-boroPhe(mCN)-C10H16 ●HCl
Boc-(P)Phe-Pro-boroPhe(mCN)-C10H16 (0.21 g) was allowed to react with 2 mL of 4 N HCI dioxane for 2 h at room temperature. Solvent was removed by evaporation and the residue was triturated with ether to yield 0.11 g of the desired product as a white solid. Example 19
Synthesis of H-(D)Phe-Pro-boroPhe(mCN)-OH●HCl H-(P)Phe-Pro-boroPhe(mCN)-C10H16●HCl (0.63 g, 1.0 mmol) was allowed to react with 5 equivalents of
phenylboronic acid using the procedure described for Example 7 to yield 0.46 g of product.
Example 20
Synthesis of N,N Dimethyl-(P)Phe-Pro-boroPhe(mCN)-OH●HCl
H-(D)Phe-Pro-boroPhe(mCN)-OH-HCl (0.20 g, 0.42 mmol), 37% aqueous formaldehyde (0.34 mL, 4.2 mmol) were dissolved in 2 mL of acetonitrile. Sodium
cyanoborohydride (0.080 g, 1.3 mmol) was added and after 5 min glacial acetic acid (20μL) were added. The reaction pH was ~7. After 5 h, additional acetic acid (20 μL) were added and the mixture was stirred for 1 h. The reaction mixture was poured into 20 mL of ethyl acetate and the organic phase was washed with 10 mL of saturated aqueous sodium chloride and dried over
anhydrous sodium sulfate. Evaporation of solvent yielded 0.16 g of an oil which was triturated with ether to give a white solid.
Example 52
Synthesis of Ac-(P)Phe-Pro-NH-H[(CH2)3SC(NH)NHCH3]B(OH)2
The intermediate, Ac-(D)Phe-Pro-NH-CH[(CH2)3Br]BO2C10H16, was prepared using the mixed anhydride procedure of example 1. A solution of this bromide (0.35 g, 0.57 mmol) and 1-methyl-2-thiourea (0.077 g, 0.85 mmol) in 10 mL of absolute ethanol was refluxed for 18 hours. After cooling the solvent was removed under vacuum, and the product was separated from excess thiourea employing chromatography (elution:
methanol) on Sephadex® LH-20 gel to provide 0.31 g (77%) of the isothiouronium product. This boronic acid ester (0.28 g) was then deprotected as described in example 4 to afford 0.13 g (57%) of the desired product. LRMS
(ESI) m/e calcd. for M (C22H34BN5O5S) + H+ : 492. Found: 492. HRMS (NH3-CI) m/e calcd. for ethylene glycol ester M (C24H36BN5O5S) + H+: 518.260847. Found: 518.261656.
Example 54
Synthesis of Ac-(D)Phe-Pro-NH-CH[(CH2)3NHC(NH)NHCH3]- B(OH)2
A solution of Ac-(D)Phe-Pro-boroOrn-BO2C10H16 · HCI [0.50 g, 0.85 mmol, prepared by the procedure of Kettner et al.(1990)], 4-methylaminopyridine (0.21 g, 1.7 mmol), N-methylamino-iminomethanesulfonic acid (0.24 g, 1.7 mmol), and 10 mL of absolute ethanol was refluxed for 18 hours. After cooling the mixture was filtered and the precipitate was washed with chloroform. The combined filtrates were concentrated under vacuum, and the residue was dissolved in 10 mL of chloroform. The chloroform solution was washed with ice-cold 0.1 N hydrochloric acid (2 X 3 mL), ice-cold water (2 X 3 mL), and brine. The resulting organic solution was then dried over anhydrous magnesium sulfate, filtered, and
concentrated. The product was purified employing
chromatography (elution: methanol) on Sephadex® LH-20 gel to provide 0.30 g (55%) of the guanidine. This boronic acid ester was then deprotected as described in example 4 to afford 0.14 g (59%) of the desired product. LRMS (NH3-CI) m/e calcd. for ethylene glycol ester M
(C24H37BN6O5) + H+: 501. Found: 501. HRMS (NH3-CI) m/e calcd. for ethylene glycol ester M (C24H37BN6O5) + H+: 501.299674. Found: 501.300760.
The examples of Table 1 can be prepared by the schemes and procedures described above using the
appropriate starting materials. Table 1.
EX MS Method LRMS LRMS
# Compound CALCD FOUND 1 NH3/CI 594.4 594
Ac-(D)Phe-Pro-NH- (M+NH4)
CH[(CH2)4CN]BO2C10H16
2 NH3/CI 594.4 594
Ac-(D)Phe-Pro-NH-CH[(CH2)4- (M+H)
C(NH)NH2]BO2C10H16●BSA
3 NH3/CI 580.4 580
Ac-(D)Phe-Pro-boroOm(CH=NH)]- (M+H)
C10H16●HCI
4 NH3/CI 528.3 528
Ac-(D)Phe-Pro-boroOm(CH=NH)]-OH●HCI pinacol
ester+H
5 NH3/CI 491.5 491
Boc-Pro-boroOm(CH=NH)-C10H16●HCI (M+H)
6 NH3/CI 638.4 638
Boc-(D)Phe-Pro-boroOrn(CH=NH)]- (M+H)
C10H16●0.5HCI●0.5BSA
7 NH3/CI 586.4 586
Boc-(D)Phe-Pro-boroOrn(CH=NH)]-OH-0.6 pinacol
HCI●0.4 BSA ester+H
8 NH3/CI 538.4 538
H-(D)Phe-Pro-boroOrn(CH=NH)]- (M+H)
C10H16●0.5HCl●0.5BSA
9 NH3/CI 486.3 486
H-(D)Phe-Pro-boroOrn(CH=NH)]-OH-0.65 pinacol
HCI●0.35 BSA ester+H
10
H-boroPhe(mCN)-C10H16●HCI
11 NH3/CI 611.3 611
Ac-(D)Phe-Pro-boroPhe-(m-CN)-C10H16 (M+H)
12 NH3/CI 628.4 628
Ac-(D)Phe-Pro-boroPhe-(m-C(NH)NH2)- (M+H)
C10H16●BSA
13 NH3/CI 615.4 615
Ac-(D)Phe-Pro-boroPhe-(m-CH2NH2)- (M+H)
C10H16●HCI
14 NH3/CI 683.4 683
Ac-(D)Phe-Pro-boroPhe-(m-Br)-C10H16 (M+NH4)
15 NH3/CI 619.5 620
Ac-(D)Phe-Pro-boroArg(CN)-C10H16●HCI (M+H)
16 NH3/CI 628.4 628
Ac-(D)Phe-Pro-boroPhe-(p-CN)-C10H16 (M+NH4)
17 NH3/CI 686.4 686
Boc-(D)Phe-Pro-boroPhe-(m-CN)-C10H16 (M+NH4) 18 NH3/CI 569.3 569
H-(D)Phe-Pro-boroPhe-(m-CN)- (M+H)
C1 0H1 6●HCI
1 9 NH3/CI 461.2 461
H-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCI EG ester+H
20 NH3/CI 489.3 489
N,N-(CH3)2-(D)Phe-Pro-boroPhe-(m-CN)- EG ester+H
OH●HCI (ISOMER I)
21 NH3/CI 615.4 615
Ac-(D)Phe-Pro-boroPhe-(p-CH2NH2)- (M+H)
C1 0H1 6●BSA
22 FAB 628.37 628.44
Ac-(D)Phe-Pro-boroPhe-(p-C(NH)NH2)- (M+H)
C1 0H1 6* BSA
2 3 NH3/CI 520.3 520
Ac-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCI EG
ester+NH4
24 NH3/CI 556.2 556 Ms-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCI EG
ester+NH4
25 NH3/CI 583.4 583.3
N-CH3-(D)Phe-Pro-boroPhe-(m-CN)- (M+H)
C1 0H1 6●HCI
26 NH3/CI 422.3 422
H-Pro-boroPhe-(m-CN)-Cι 0H16*HCI (M+H)
27 NH3/CI 676.4 676.4
Boc-(D)Thiazolylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 0H1 6
28 NH3/CI 670.4 670.4
Boc-(D)3-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 0H1 6
29 NH3/CI 576.3 576
H-(D)Thiazolylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 0H1 6●HCI
30 NH3/CI 570.3 570
H-(D)3-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 0H1 6●HCI
31 NH3/CI 654.3 654
Ms-(D)Thiazolylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 0H1 6
32 NH3/CI 648.3 648
Ms-(D)3-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 0H1 6
33 NH3/CI 700.4 700
N-Boc-N-CH3-(D)Phe-Pro-boroPhe-(m- (M+NH4)
CN)-C1 0H1 6 34 NH3/CI 670.4 670
Boc-(D)2-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H16
35 NH3/CI 481.3 481
Ac-Pro-boroPhe-(m-CN)-C10H16 (M+NH4)
36 NH3/CI 692.4 692
Boc-(D)2-Thienylalanine-Pro-boroPhe-(m- (M+NH4)
CN)-C10H16
37 NH3/CI 570.3 570
H-(D)2-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H16●HCI
38 NH3/CI 575.3 575
H-(D)2-Thienylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H16*HCI
39 NH3/CI 648.3 648
Ms-(D)2-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H16
40 NH3/CI 670.3 670
Ms-(D)2-Thienylalanine-Pro-boroPhe-(m- (M+NH4)
CN)-C10H16
41 NH3/CI 574.3 574
(2-Pyrimidylthio)acetyl-Pro-boroPhe-(m- (M+H)
CN)-C10H16
42 NH3/CI 553.3 553 trans-3-(3-pyridyl)acryl-Pro-boroPhe-(m- (M+H)
CN)-C10H16
43 NH3/CI 573.3 573
(4-Pyridylthio)acetyl-Pro-boroPhe-(m-CN)- (M+H)
C10H16
44 NH3/CI 578.3 578
Succinyl-(D)Phe-Pro-boroPhe-(m-CN)-OH EG
ester+NH4
45 NH3/CI 553.3 555
3-Pyridylpropionyl-Pro-boroPhe-(m-CN)- (M+H)
C10H16
46 NH3/CI 672.4 672
Boc-(D)Phe-Aze-boroPhe-(m-CN)-C10H16 (M+NH4)
47 NH3/CI 555.3 555
H-(D)Phe-Aze-boroPhe-(m-CN)- (M+H)
C10H16●HCI
48 FAB 445.5 445
Hydrocinnamoyl-Pro- EG ester+H
boroOrn(CH=NH)]OH●BSA
49 ESI 461 461
Hydrocinnamoyl-Pro- (M+H)
borolrg(CH2CH=CH2)-OH●HBr
50 ESI 435 435
Hydrocinnamoyl-Pro-borolrg(CH3)-OH● (M+H)
HBr 51 NH3/CI 718 718
Cbz-(D)Phe-Pro-borolrg(CH3)-C1 0H1 6● (M+H)
HBr
52 ESI 492 492
Ac-(D)Phe-Pro-borolrg(CH3)-OH●HBr (M+H)
53 ESI 449 449
Hydrocinnamoyl-Pro-borolrg(CH2CH3)-OH (M+H)
●HBr
54 NH3/CI 501 501
Ac-(D)Phe-Pro-boroArg(CH3)-OH●HCI EG ester+H
55 ESI 418 418
Hydrocinnarnoyl-Pro-boroArg(CH3)-OH● (M+H)
HCI
56 ESI 51 1 51 1
Ms-(D)Phe-Pro-boroArg(CH3)-OH●HCI (M+H)
57 ESI 482 482
Ms-(D)Phe-Pro-boroOrn(CH=NH)-OH●HCI (M+H)
58 ESI 573 573
PhSO2-(D)Phe-Pro-boroArg(CH3)-OH● (M+H)
HCI
59 ESI 544 544
PhSO2-(D)Phe-Pro-boroOrn(CH=NH)-OH● (M+H)
HCI
60 ESI 500 500
Ms-(D)Phe(4-fluoro)-Pro- (M+H)
boroOrn(CH=NH)-OH●HCI
61 ESI 587 587
PhCH2Sθ2-(D)Phe-Pro-boroArg(CH3)-OH (M+H)
●HCI
62 ESI 558 558
PhCH2SO2-(D)Phe-Pro-boroOm(CH=NH)- (M+H)
OH●HCI
63 ESI 510 510
CH3CH2CH2SO2-(D)Phe-Pro- (M+H)
boroOm(CH=NH)-OH●HCI
64 ESI 539 539
CH3CH2CH2SO2-(D)Phe-Pro- (M+H)
boroArg(CH3)-OH●HCI
65 ESI 553 553
CH3(CH2)3SO2-(D)Phe-Pro- (M+H)
boroArg(CH3)-OH●HCI
66 ESI 524 524
CH3(CH2)3SO2-(D)Phe-Pro- (M+H)
boroOrn(CH=NH)-OH●HCI
67
Ac-(D)Phe-Sar-boroOm(CH=NH)-OH●HCI
68
Ms-(D)Phe-Sar-boroOrn(CH=NH)-OH-H 69
Phenethyl-SO2-(D)Phe-Sar- boroOrn(CH=NH)-OH●HCI
70
Boc-(D)Phe-Sar-boroOrn(CH=NH)-OH●HCI
71
N-alpha-[boroOrn(CH=NH)-OH]-(2-trans
benzylcarboxamido)-cyclopentane-1 - carboxamide●HCI
72
H-(D)Phe-Sar-boroOrn(CH=NH)- C1 0H1 6●2HCI
73
Boc-(D)Phe-Sar-boroPhe(m-CN)-C1 0H1 6
74
Boc-(D)Phe-Aze-boroOrn(CH=NH)- OH●HCI
75
H-(D)Phe-Sar-boroPhe(m-CN)- C1 0H1 6●2HCI
76
4-(Phenyl)benzoyl-boroOm(CH=NH)- C1 0H1 6●HCI
77 NH3/CI 620.58 620.
Z-(D)Phe-Pro-boroOrn(CH=NH)-OH●HCI pinacol
ester+H
78
H-boroPhe-(p-CN)-C1 0H1 6●HCI
79 Boc-(D)Phe-Pro- N(CH3)CH[(CH2)3NHC(NH)H]-B(OH)2
80 Boc-(D)Phe-Pro- N(Phenyl)CH[(CH2)3NHC(NH)H]-B(OH)2
81 Boc-(D)Phe-Pro- N(benzyl)CH[(CH2)3NHC(NH)H]-B(OH)2
82 Boc-(D)Phe-Pro- N(CH3)CH[(CH2)3NHC(NH)H]-B(OMe)2
83 Boc-(D)Phe-Pro- N(CH3)CH[(CH2)3NHC(NH)H]-B[N(Me)]2
84 Boc-(D)Phe-Pro- N(CH3)CH[(CH2)3NHC(NH)H]-B(F)2
85 FMoc-(D)Phe-Pro- NHCH[(CH2)3NHC(NH)H]-B(OC1 0H1 6)2 Ac-(D)cyclohexylalanyl-Pro-NHCH[(CH2)3NHC(NH)H]-B(OC10H16)2 Ac-(D)Phe-Gly-NHCH[(CH2)3NHC(NH)H]-
B(OC10H16)2 Ac-(D)Phe-Pro- NHCH[(CH2)3NHC(NOH)NH2]- B(OC10H16)2 Ac- (D)Phe-Pro-boroPhe-(p-Br)-C10H16 Ac- (D)Phe-Pro-boroPhe-(p-NH2)-C10H16 Ac- (D)Phe-Pro-boroPhe-(p- NHC(NH)NH2)-C10H16 Ac- (D)Phe-Pro-boroPhe-(p- CH2NHC(NH)NH2)-C10H16
Ac- (D)Phe-Pro-boroPhe-(m- CH2NHC(NH)NH2)-C10H16
Ac- (D)Phe-Pro-boroPhe-(m- CH2NHC(NH)NHCN)-C10H16 Z-Leu-Ser(Ot-Bu)-Asn-Leu-Ser(Ot-Bu)- Asn-Leu-Ser(Ot-Bu)-Asn-Leu-Ser(Ot-Bu)- Asn-NHCH[(CH2)3NHC(NH)H]- B(OC10H16)2 H-Leu-Ser(Ot-Bu)-Asn-Leu-Ser(Ot-Bu)- Asn-Leu-Ser(Ot-Bu)-Asn-Leu-Ser(Ot-Bu)- Asn-NHCH[(CH2)3NHC(NH)H]- B(OC10H16)2 Z-Leu-Ser-Asn-Leu-Ser-Asn-Leu-Ser-Asn- Leu-Ser-Asn-NHCH[(CH2)3NHC(NH)H]- B(OC10H16)2 H-Leu-Ser-Asn-Leu-Ser-Asn-Leu-Ser-Asn- Leu-Ser-Asn-NHCH[(CH2)3NHC(NH)H]- B(OC10H16)2 Utility
N-Acyl and N-peptide boronic acids which are described in the present invention represent a novel class of potent, reversible inhibitors of trypsin-like enzymes. Trypsin-like enzymes are a group of proteases which hydrolyzed peptide bonds at basic residues
liberating either a C-terminal arginyl or lysyl residue. Among these are enzymes of the blood coagulation and fibrinolytic system required for hemostasis. They are Factors II, X, VII, IX, XII, kallikrein, tissue
plasminogen activators, urokinase-like plasminogen activator, and plasmin. Enzymes of the complement system, acrosin (required for fertilization), pancreatic trypsin are also in this group. Elevated levels of proteolysis by these proteases can result in disease states. For example, consumptive coagulopathy, a condition marked by a decrease in the blood levels of enzymes of both the coagulation system, the fibrinolytic system and accompanying protease inhibitors is often fatal. Intervention by a synthetic inhibitor would clearly be valuable. More specifically, proteolysis by thrombin is required for blood clotting. Inhibition of thrombin results in an effective inhibitor of blood clotting. The importance of an effective inhibitor of thrombin is underscored by the observation that
conventional anticoagulants such as heparin (and its complex with the protein inhibitor, antithrombin III) are ineffective in blocking arterial thrombosis
associated with myocardial infractions and other clotting disorders. However, a low molecular weight thrombin inhibitor, containing a different
functionality, was effective in blocking arterial thrombosis [Hanson and Harker (1988) Proc . Natl . Acad . Sci . U. S.A . 85, 3184-3188]. Therefore, we have chosen to demonstrate utility of compounds in the inhibition of thrombin, both as in buffered solutions and in plasma. Specifically, the compounds have utility as drugs for the treatment of diseases arising from elevated thrombin activity such as myocardial infarction, and as reagents used as anticoagulants in the processing of blood to plasma for diagnostic and other commercial purposes.
When used in the processing of blood products, the compounds of this invention may be mixed with whole blood without the need for any additional
anticoagulants. The compounds of this invention serve to inhibit blood coagulation thereby facilitating the processing of whole blood into desired cellular
components or plasma proteins. Once the processing is complete, the compounds may be removed, if so desired, by membrane ultrafiltration, dialysis, or diafiltration to afford the desired product. The low molecular weight of these compounds relative to conventional
anticoagulants like heparin allow them to be separated from desired products more easily.
Compounds of the present invention are expected to be effective in the control of aberrant proteolysis and a number of accompanying disease states such as
inflammation, pancretitis, and heritary angioedema.
The in vitro effectiveness of compounds of the present invention as inhibitors of the blood coagulation protease thrombin was determined under two different conditions: (1) measurements in buffered solutions using a synthetic substrate; (2) measurement in plasma where the rate of blood clotting is determined. For the former, the chromogenic substrate S2238 (H-(D)Phe-Pip-Arg-PNA, where PIP refers to pipecolic acid; Helena Laboratories, Beaumont, TX) was used following
procedures similar to those described in Kettner et al . J. Biol . Chem . 265 18289-18297 (1990). Here hydrolysis resulted in the release of pNA which was monitored spectrophotometricaly by measuring the increase in absorbance at 405 nm. The Michaelis constant, Km, for substrate hydrolysis was determined at 25 °C in 0.10 M sodium phosphate buffer, pH 7.5, containing 0.20 M NaCl, and 0.5 % PEG 8000 using the method of Lineweaver and Burk.
Values of Ki were determined by allowing thrombin (0.19 nM) to react with substrate (0.20 mM) in the presence of inhibitor. Reactions were allowed to go for 30 minutes and the velocities (rate of absorbance change vs time) were measured in the time frame of 25-30 minutes. The following relationship was used to
calculate Ki values.
Figure imgf000041_0001
where
vo is the velocity of the control in the absence of inhibitor;
vs is the velocity in the presence of inhibitor;
I is the concentration of inhibitor;
Ki is the dissociation constant of enzyme: inhibitor complex;
S is the concentration of substrate;
Km is the Michaelis constant.
Inhibition of blood clotting activity of thrombin in plasma was determined in rabbit plasma. Plasma was prepared by diluting blood 1:10 with 3.2% aqueous citric acid and centrifuging. Buffer consisted of 0.10 M Tris, pH 7.4, containing 0.9% sodium chloride, and 2.5 mg/mL bovine serum albumin. Bovine thrombin was obtained from
Sigma and was diluted to 24 NIH units/mL. Plasma (200 μL) and buffer (50 μL) containing inhibitor were
incubated 3 min at 37 °C in a fibrameter. Reactions were initiated by adding thrombin (50μL) and clotting times measured. Controls were run under identical conditions except in the absence of inhibitor. The final concentration of thrombin was 4 NIH units/mL.
Table 2 - Inhibition of Thrombin.
Ex Ki a
# (nM)
1 750
2 0.26
3 0.38
4 0.28
6 0.085
7 0.040
8 0.18
9 .05
11 3.2
12 2.8
13 4.83
14 10
15 40
16 134
17 0.27
20 0.14
23 0.55
24 0.059
27 0.17
28 0.37
32 0.48
34 0.33
36 0.381
40 0.19
46 0.55
50 <859
54 1 62 0.03
63 0.5
64 0.5
67 8.2
73 81
74 <0.5
76 1 10 a Ki values were measure at 25 °C at pH 7.5.
Another measure of compound effectiveness toward prolonging clotting times can be reported as IC50 (level of inhibitor required to prolong clotting to the time observed for 2 NIH units/mL thrombin in the absence of inhibitor). Representative of data for compounds of the present invention, Examples 3, 7, 9, 11, and 12
increased thrombin clotting times 2-fold at 0.25,
<0.075, 0.10, 0.60, and 0.85 μM, respectively.
The effectiveness of compounds of the present invention as anticoagulants in vivo was demonstrated by the prolongation of the activated partial thromboplastin time of samples of blood taken from conscious dogs or anesthetized rats after either oral or intravenous administration at doses of the compounds from 0.5 to 10 mg/kg. Arterial or venous blood was withdrawn by syringe and mixed with 1/10 volume 3.2% sodium citrate. Plasma was obtained after centrifugation and a standard clinical activated partial thromboplastin time (APTT reagent, Sigma Chemical Co., St. Louis, Mo.) determined at 37°C in a fibrometer. Results from blood samples obtained at various times after dosing showed an effective anticoagulant response which was at least equivalent to doubling of activated partial
thromboplastin time as compared to the value obtained prior to dosing. In this model, Examples 4, 57, and 77 were shown to be effective following i.v. dosing and Examples 4, 56, 57, 60, and 66 effective following oral dosing. Similarly, oral administration of Examples 31 and 54 resulted in at least a 2-fold elevation in anticoagulant activity in an identical model except activity was measured by increases in thrombin clotting times.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Sheng-Lian O. Lee
John Matthew Fevig
Charles Adrian Kettner
David L. Carini
(ii) TITLE OF INVENTION: Amidino and Guanidino Substituted Boronic Acid Inhibitors of Trypsin-Like Enzymes
( i i i ) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: The Du Pont Merck Pharmaceutical
Company
(B) STREET: 1007 Market Street, Legal Department
(C) CITY: Wilmington
(D) STATE: DE
(E) COUNTRY: U.S.
(F) ZIP: 19898
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.50 inch disk
(B) COMPUTER: Apple Macintosh
(C) OPERATING SYSTEM: Apple Macintosh
(D) SOFTWARE: Microsoft Word
( v i ) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/052,835
(B) FILING DATE:
(C) CLASSIFICATION: unknown
(vii) PRIOR APPLICATION DATA: None (vi i i ) ATTORNEY/AGENT INFORMATION:
(A) NAME: Reinert, Norbert, F.
(B) REGISTRATION NUMBER: 1 8,926
(C) REFERENCE/DOCKET NUMBER: DM-6567-A
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 302-892-8867
(B) TELEFAX: 302-892-8536 ( 2 ) INFORMATION FOR SEQ ID NO:1 :
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 2
(B) TYPE: amino acids
(C) TOPOLOGY: linear
( i i ) MOLECULAR TYPE: peptide
(vi) ORIGINAL SOURCE: synthetic
(ix) FEATURE:
(D) OTHER INFORMATION: Example Number 98 at page 36 and within Table 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :
Xaa Xaa Asn Leu Xaa Asn Leu Xaa Asn Leu Xaa Asn
1 5 1 0
(2) INFORMATION FOR SEQ ID NO:2:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 2
(B) TYPE: amino acids
(C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Leu Xaa Asn Leu Xaa Asn Leu Xaa Asn Leu Xaa Asn
1 5 1 0
(3) INFORMATION FOR SEQ ID NO:3:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 2
(B) TYPE: amino acids
(C) TOPOLOGY: linear
( i i ) MOLECULAR TYPE: peptide
(vi) ORIGINAL SOURCE: synthetic
(ix) FEATURE:
(D) OTHER INFORMATION: Example Number 100 at page 36 and within Table 1
( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Xaa Ser Asn Leu Ser Asn Leu Ser Asn Leu Ser Asn 1 5 1 0
( 3 ) INFORMATION FOR SEQ ID NO:4:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 2
(B) TYPE: amino acids
(C) TOPOLOGY: linear
( i i) MOLECULAR TYPE: peptide
(vi) ORIGINAL SOURCE: synthetic
(ix) FEATURE:
(D) OTHER INFORMATION: Example Number 101 at page 36 and within Table 1
(x i) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Leu Ser Asn Leu Ser Asn Leu Ser Asn Leu Ser Asn 1 5 1 0

Claims

What is Claimed is:
A compound of formula (I)
Figure imgf000048_0002
wherein
R1 is
a) C1-C12-alkyl substituted with -CN, -C(NH)NHR6, -NHC(NH)H, -NHC(NH)NHR6, -SC(NH)NHR6, -NHC (NH) NHOH,
-NHC(NH)NHCN, -NHC (NH) NHCOR6, or
b) X
Figure imgf000048_0001
;
X is
a) halogen (F, Cl, Br, I)
b) -CN,
c) -NO2,
d) -CF3,
e) -NH2
f) -NHC(NH)H,
g) -NHC(NH)NHOH,
h) -NHC(NH)NHCN,
i) -NHC(NH)NHR6,
j) -NHC(NH)NHCOR6,
k) -C(NH)NHR6,
l) -C(NH) NHCOR6,
m) -C(O)NHR2,
n) -CO2R2, o) -OR2, or
p) -OCF3
q) -SC(NH)NHR6,;
R2 is
a) H,
b) C1-C4-alkyl,
c) aryl, wherein aryl is phenyl or napthyl
optionally substituted with one or two substituents selected from the group consisting of halo (F, Cl, Br, I), C1-C4-alkyl, C1-C4-alkoxy, -NO2, -CF3,
-S(O)r-C1-C4-alkyl, -OH, -NH2, -NH (C1-C4-alkyl), -N(C1-C4-alkyl)2, -CO2R4, or
d) -C1-C4-alkylaryl, where aryl is defined above;
R3 is H, alkyl, aryl, alkylaryl or an NH2-blocking group comprised of 1-20 carbon atoms;
R4 and R5 are independently
a) H,
b) C1-C4-alkyl, or
c) -CH2-aryl, where aryl is defined above;
R6 is
a) H,
b) C1-C4-alkyl,
c) aryl, wherein aryl is phenyl or napthyl
optionally substituted with one or two substituents selected from the group consisting of halo (F, Cl, Br, I), C1-C4-alkyl, C1-C7-alkoxy, -NO2, -CF3, -S(O)r-C1-C4-alkyl, -OH, -NH2, -NH (C1-C4-alkyl), -N(C1-C4-alkyl)2, -CO2R4, or
d) -C1-C4-alkylaryl, where aryl is defined above; A is an amino acid residue or a peptide comprised of 2- 20 amino acid residues;
Y1 and Y2 are
a) -OH,
b) -F,
c) C1-C8-alkoxy, or when taken together Y1 and Y2 form a
d) cyclic boron ester where said chain or ring contains from 2 to 20 carbon atoms and, optionally, 1-3 heteroatoms which can be N, S, or O,
n is 0 or 1;
p is 0 to 3;
q is 0 to 4;
r is 0 to 2;
and pharmaceutically acceptable salts thereof, with the proviso that when R1 is aliphatic, an R6 subsitiuent on
-NHC(NH)NHR6 cannot be H.
2. A compound of claim 1 where
Y1 and Y2 are
a) -OH,
when taken together Y1 and Y2 form a
b) cyclic boron pinacol ester, or
c) cyclic boron pinanediol ester;
R1 is
a) -(CH2)3NHC(NH)H,
b) -(CH2)4C(NH)NH2,
c)
Figure imgf000050_0001
d)
Figure imgf000050_0002
, or
e)
Figure imgf000051_0002
;
R2 is H;
A is Pro or (D)Phe-Pro;
R3 is
a) H,
b) Boc,
c) Z,
d) Ac,
e) hydrocinnamoyl,
f) C1-C10 alkyl sulfonyl, or
g) C1-C15 alkylaryl sulfonyl,
3. A compound of claim 1 where
is
a)
Figure imgf000051_0001
4. A compound of claim 1 where
R1 is
a) C3-C4-alkyl substituted with -CN, -C(NH)NH2, -NH- C(NH)H.
5. A compound of claim 1 where
R1 is
a)
Figure imgf000052_0001
b)
X
Figure imgf000052_0002
;
X is
a) halogen (Cl, Br)
b) -CN,
c) -C(NH)NH2,
d) -NH2
e) -NHC(NH)NH2;
p is 0 to 1.
6. A compound of claim 1 where R3 is H, alkyl, Ac, Boc, C1-C10 alkyl sulfonyl, C1-C15 alkylaryl sulfonyl, C1-C15 aryl sulfonyl.
7. A compound of claim 1 where n is 0.
8. A compound of claim 1 where [A] is comprised independently of amino acid residues in the D or L configuration selected from the group consisting of Ala, Arg, Asn, Asp, Aze, Cys, Gin, Glu, Gly, His, HomoLys, Ile, Leu, Lys, Met, Orn, Phe, Phe (4-fluoro), Pro, Ser, Thr, Trp, Tyr, and Val.
9. A compound of claim 1 where [A] is comprised of either Pro or (D)Phe-Pro.
10. A compound of claim 1 selected from the group:
Ac-(D)Phe-Pro-NH-CH[(CH2)4CN]BO2-C10H16 ● Ac-(D)Phe-Pro-NHCH[(CH2)4C(NH)NH2]BO2-C10H16● Ac-(D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]B(OH)2
● Boc-(D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]B(OH)2.
● Ac-(D)Phe-Pro-boroPhe[m-C(NH)NH2]-C10H16
● Ac-(D)Phe-Pro-boroPhe(m-CH2NH2)-C10H16
● Ac-(D)Phe-Pro-boroPhe(m-Br)-C10H16
● Ac-(D)Phe-Pro-boroArg(CN)-C10H16
● Ac-(D)Phe-Pro-boroPhe(p-CN)-C10H16
● Boc- (D)Phe-Pro-boroPhe-(m-CN)-C10H16
● N,N-(CH3)2-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCl (ISOMER
I)
● Ac-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCI
● Ms-(D)Phe-Pro-boroPhe-(m-CN)-OH●HCI
● Boc-(D)Thiazolylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Boc-(D)3-Pyridylalanine-Pro-boroPhe-(m-CN)-C10H16● Ms-(D)3-Pyridylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Boc-(D)2-Pyridylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Boc-(D)2-Thienylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Ms-(D)2-Thienylalanine-Pro-boroPhe-(m-CN)-C10H16 ● Boc-(D)Phe-Aze-boroPhe-(m-CN)-C10H16
● Hydrocinnamoyl-Pro-boroIrg(CH3)-OH●HBr
● Ac-(D)Phe-Pro-boroArg(CH3)-OH●HCl
● PhCH2SO2-(D)Phe-Pro-boroOrn(CH=NH)-OH●HCl
● CH3CH2CH2SO2-(D)Phe-Pro-boroOrn(CH=NH)-OH●HCl ● CH3CH2CH2SO2-(D)Phe-Pro-boroArg(CH3)-OH●HCl ● Ac-(D)Phe-Sar-boroOrn(CH=NH)-OH•HCI
● Boc-(D)Phe-Sar-boroPhe(mCN)-C10H16
● Boc-(D)Phe-Aze-boroOrn(CH=NH)-OH●HCl
● 4-(Phenyl)benzoyl-boroOrn(CH=NH)-C10H16●HCl
PCT/US1994/004058 1993-04-27 1994-04-21 Amidino and guanidino substituted boronic acid inhibitors of trypsin-like enzymes WO1994025049A1 (en)

Priority Applications (4)

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AU67038/94A AU6703894A (en) 1993-04-27 1994-04-21 Amidino and guanidino substituted boronic acid inhibitors of trypsin-like enzymes
EP94914776A EP0696199A4 (en) 1993-04-27 1994-04-21 Amidino and guanidino substituted boronic acid inhibitors of trypsin-like enzymes
CA002161216A CA2161216A1 (en) 1993-04-27 1994-04-21 Amidino and guanidino substituted boronic acid inhibitors of tryspin-like enzymes
JP6524316A JPH08509723A (en) 1993-04-27 1994-04-21 Amidino- and guanidino-substituted boronic acid trypsin-like enzyme inhibitors

Applications Claiming Priority (4)

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Cited By (15)

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EP0688788A1 (en) * 1994-06-22 1995-12-27 Adir Et Compagnie Boronic acid peptide derivatives having protease inhibiting activity, process for preparation thereof and pharmaceutical compositions comprising them
EP0724446A1 (en) * 1993-10-07 1996-08-07 The Du Pont Merck Pharmaceutical Company Electrophilic peptide analogs as inhibitors of trypsin-like enzymes
US5561146A (en) * 1994-06-10 1996-10-01 Bristol-Myers Squibb Company Modified guanidino and amidino thrombin inhibitors
FR2739858A1 (en) * 1995-10-11 1997-04-18 Synthelabo Boron-containing sulphonyl and sulphamoyl peptidyl prolinamide(s)
WO2001027138A2 (en) * 1999-10-13 2001-04-19 Tularik Inc. Modulators of srebp processing
US6239150B1 (en) 1995-07-26 2001-05-29 Mitsubishi Chemical Corporation Penicillaminamide derivatives
EP1396269A1 (en) 2002-09-09 2004-03-10 Trigen Limited Boronic acid salts of multivalent metals used in the preparation of a medicament for treating thrombosis
US6933290B2 (en) 2001-05-30 2005-08-23 Novartis Ag 2-{N-(2-amino-3-(heteroaryl or aryl)propionyl)-aminoacyl}-amino}-alkylboronic acid derivatives
US7112572B2 (en) 2002-09-09 2006-09-26 Trigen Limited Multivalent metal salts of boronic acids
US7223745B2 (en) 2003-08-14 2007-05-29 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US7576206B2 (en) 2003-08-14 2009-08-18 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8283367B2 (en) 2005-02-11 2012-10-09 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8541590B2 (en) 2009-12-22 2013-09-24 Cephalon, Inc. Proteasome inhibitors and processes for their preparation, purification and use
US11267803B2 (en) 2016-06-21 2022-03-08 Orion Ophthalmology LLC Carbocyclic prolinamide derivatives
US11377439B2 (en) 2016-06-21 2022-07-05 Orion Ophthalmology LLC Heterocyclic prolinamide derivatives

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RU2354647C2 (en) * 2007-06-28 2009-05-10 Общество С Ограниченной Ответственностью "Бионика" New compounds with thrombin inhibiting function, and based pharmaceutical compositions
RU2353619C2 (en) * 2007-06-28 2009-04-27 Общество С Ограниченной Ответственностью "Бионика" Novel compounds possessing anticoagulant function, based on them pharmaceutical compositions for treatment of thrombotic conditions and plasma-substituting solution for correction of hypercoagulation disorders in hemodilution

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See also references of EP0696199A4 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0724446A1 (en) * 1993-10-07 1996-08-07 The Du Pont Merck Pharmaceutical Company Electrophilic peptide analogs as inhibitors of trypsin-like enzymes
EP0724446A4 (en) * 1993-10-07 1998-01-07 Du Pont Merck Pharma Electrophilic peptide analogs as inhibitors of trypsin-like enzymes
US5561146A (en) * 1994-06-10 1996-10-01 Bristol-Myers Squibb Company Modified guanidino and amidino thrombin inhibitors
EP0688788A1 (en) * 1994-06-22 1995-12-27 Adir Et Compagnie Boronic acid peptide derivatives having protease inhibiting activity, process for preparation thereof and pharmaceutical compositions comprising them
US6239150B1 (en) 1995-07-26 2001-05-29 Mitsubishi Chemical Corporation Penicillaminamide derivatives
FR2739858A1 (en) * 1995-10-11 1997-04-18 Synthelabo Boron-containing sulphonyl and sulphamoyl peptidyl prolinamide(s)
WO2001027138A2 (en) * 1999-10-13 2001-04-19 Tularik Inc. Modulators of srebp processing
WO2001027138A3 (en) * 1999-10-13 2001-11-29 Tularik Inc Modulators of srebp processing
US6649593B1 (en) 1999-10-13 2003-11-18 Tularik Inc. Modulators of SREBP processing
US6933290B2 (en) 2001-05-30 2005-08-23 Novartis Ag 2-{N-(2-amino-3-(heteroaryl or aryl)propionyl)-aminoacyl}-amino}-alkylboronic acid derivatives
US7112572B2 (en) 2002-09-09 2006-09-26 Trigen Limited Multivalent metal salts of boronic acids
US7371729B2 (en) 2002-09-09 2008-05-13 Trigen Limited Boronic acid salts useful in parenteral formulations
EP1396269A1 (en) 2002-09-09 2004-03-10 Trigen Limited Boronic acid salts of multivalent metals used in the preparation of a medicament for treating thrombosis
EP1466917A1 (en) 2002-09-09 2004-10-13 Trigen Limited Method for making peptide boronic acids and acids obtainable thereby
US8058262B2 (en) 2003-08-14 2011-11-15 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US7576206B2 (en) 2003-08-14 2009-08-18 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US7915236B2 (en) 2003-08-14 2011-03-29 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US7223745B2 (en) 2003-08-14 2007-05-29 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8546608B2 (en) 2003-08-14 2013-10-01 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US9233115B2 (en) 2003-08-14 2016-01-12 Millennium Pharmaceuticals Inc. Proteasome inhibitors and methods of using the same
US8283367B2 (en) 2005-02-11 2012-10-09 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8541590B2 (en) 2009-12-22 2013-09-24 Cephalon, Inc. Proteasome inhibitors and processes for their preparation, purification and use
US11267803B2 (en) 2016-06-21 2022-03-08 Orion Ophthalmology LLC Carbocyclic prolinamide derivatives
US11377439B2 (en) 2016-06-21 2022-07-05 Orion Ophthalmology LLC Heterocyclic prolinamide derivatives
US11866422B2 (en) 2016-06-21 2024-01-09 Orion Ophthalmology LLC Carbocyclic prolinamide derivatives

Also Published As

Publication number Publication date
AU6703894A (en) 1994-11-21
IL109319A0 (en) 1994-07-31
EP0696199A4 (en) 1997-06-25
EP0696199A1 (en) 1996-02-14
TW286318B (en) 1996-09-21
JPH08509723A (en) 1996-10-15
CA2161216A1 (en) 1994-11-10

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