EP1135023A1 - Aminobenzisoxazole compounds and libraries thereof - Google Patents

Aminobenzisoxazole compounds and libraries thereof

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Publication number
EP1135023A1
EP1135023A1 EP99958891A EP99958891A EP1135023A1 EP 1135023 A1 EP1135023 A1 EP 1135023A1 EP 99958891 A EP99958891 A EP 99958891A EP 99958891 A EP99958891 A EP 99958891A EP 1135023 A1 EP1135023 A1 EP 1135023A1
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EP
European Patent Office
Prior art keywords
substituted
alkyl
formula
compounds
unsubstituted
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99958891A
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German (de)
French (fr)
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EP1135023A4 (en
Inventor
Michael Robert Wiley
Savatore Donato Lepore
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Eli Lilly and Co
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Eli Lilly and Co
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Publication of EP1135023A1 publication Critical patent/EP1135023A1/en
Publication of EP1135023A4 publication Critical patent/EP1135023A4/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/20—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin

Definitions

  • This invention relates to novel aminobenzisoxazole compounds, libraries thereof, and methods of screening such a library for useful therapeutic compounds.
  • Certain aminobenzisoxazole compounds of the present invention have demonstrated activity as thrombin inhibitors. Other therapeutic activities are expected, depending on the substituents attached to the aminobenzisoxazole system.
  • combinatorial chemistry is the parallel, simultaneous preparation of multiple derivatives of a central, fixed moiety often referred to as the "scaffold,” “core” or “backbone”.
  • the number of compounds which may be produced to establish a library is limited only by the number of reagents available to form the derivatives.
  • Combinatorial chemistry may be used to generate libraries which are mixtures of individual compounds and complete identification of the individual compounds are postponed until after positive screening.
  • "parallel array synthesis" whereby individual reaction products are simultaneously synthesized but each reaction is carried out in a separate vessel is generally preferred.
  • a compound library may be prepared, stored and assayed in standard microtiter plates such as the plates often used in the biological sciences.
  • the scaffold is typically attached to a solid support, e.g., a polymer, which is insoluble in the reaction solvent. When the entire reaction sequence has been completed, the library compounds are detached from the support.
  • combinatorial libraries can be prepared in solution phase. It is also possible to tether the reactants to a support with the scaffold in solution. In all cases, the net effect is to produce a library of many derivatives of the scaffold each in its own small reaction vessel.
  • HTS high throughput screening
  • the present invention provides combinatorial libraries of aminobenzisoxazole compounds of the formula (I):
  • a 1 , A 2 and A 3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A 1 , A 2 and A 3 is not a bond;
  • R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is 0, 1, 2, 3, or 4.
  • the present invention provides a library of 4-amidino-3-hydroxylphenyl compounds of the formula (lb):
  • a ⁇ A 2 and A 3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A 1 , A 2 and A 3 is not a bond;
  • R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and
  • c is O, 1, 2, 3 or 4.
  • the present invention also provides libraries of aminobenzisoxazole compounds of the formula (III):
  • X-CO- is D-prolinyl, D-homoprolinyl, R 2 -(CH2) -NH-CH 2 -C(O)-,
  • # denotes a chiral center that is (L);
  • R 2 is -COOR !4 , -SO 2 (C ⁇ -C 4 alkyl), -SO 3 H, -P(O)(ORl4) 2 or tetrazol-5-yl;
  • R 3 is hydrogen or (C ⁇ -C 4 )alkyl;
  • R4 is carboxy or methylsulfonyl;
  • R 5 is NHR 6 , NHCOR 6 or NHCOOR 6 ;
  • R 6 is (C ⁇ -C ⁇ o)alkyl, (C 3 -C 8 )cycloalkyl or a (C 3 -C 8 )cycloalkyl-(C ⁇ -C 6 )alkyl group containing 4-10 carbons;
  • R 7 is (C 3 -C 8 )cycloalkyl, (C ⁇ -Cg)alkyl,
  • R 8 is -OH, (C ⁇ -C 4 )alkoxy, or -NH-R 12 ;
  • R 9 is hydrogen or (C 1 -C 4 )alkyl;
  • R 10 is hydrogen, (C ⁇ -C 4 )alkyl, (C ⁇ -C 4 )alkoxy, hydroxy, halo or (C 1 -C 4 ) alkylsulfonylamino;
  • R 11 is (C ⁇ -C )alkyl, (C ⁇ -C )fluoroalkyl bearing one to five fluoros, -(CH 2 ) d -R 2 , or unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
  • R 12 is hydrogen, (C ⁇ -C 4 )alkyl, R ⁇ SO 2 -, R n OC(O)-, R n C(O)-, R l3 C(O)- or -
  • R 13 is -COOR 14 or tetrazol-5-yl
  • each R is independently hydrogen or (Cj-C4)alkyl
  • R is (C ⁇ -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, or -(CH 2 ) c -L-(CH2) b -R 17 ;
  • R 16 is (C ⁇ -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, or -(CH2) c -L-(CH2) b - R 17 ;
  • L is a bond, -O-, -S-, or -NH-;
  • R 17 is (C ⁇ -C 4 )alkyl, (C 3 -C 8 )cycloalkyl, -COOH, -CONH 2 , or Ar, where Ar is unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
  • R is -CH 2 -, -O-, -S-, or -NH-;
  • R is a bond or, when taken with R and the three adjoining carbon atoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom of which may be -O-, -S-, or -NH-; each a, independently, is 0, 1 or 2; each b, independently, is 0, 1, 2 or 3; each c, independently, is 0, 1, 2, 3 or 4; and each d, independently, is 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), (lb), or (III), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier, diluent or excipient.
  • the aminobenzisoxazole compounds of formula (III) are useful whenever inhibition of thrombin is useful, for example, for the prophylaxis and treatment of thromboembolic diseases such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process.
  • thromboembolic diseases such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process.
  • the process for making libraries of compounds of the formulae (I), (lb), and (III) of the invention may be carried out in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means.
  • the libraries of compounds (I), (lb) and (III) according to the present invention may be screened for biological activity.
  • the library to be screened is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibody; and the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined.
  • a biological substance usually a protein such as a receptor, enzyme, membrane binding protein or antibody; and the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined.
  • this will comprise determining whether the biological substance is bound to one or more of the members of the library.
  • binding may be determined by attaching a label to the biological substance.
  • Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels.
  • Alcohols include groups of the formula -OH.
  • Alkyl refers to a substituted or unsubstituted, straight, branched or cyclic hydrocarbon chain containing of from 1 to 20 carbon atoms.
  • Preferred alkyl groups are lower alkyl groups, i.e., alkyl groups containing from 1 to 6 carbon atoms.
  • Preferred cycloalkyls have from 3 to 10, preferably 3-6, carbon atoms in their ring structure.
  • Suitable examples of unsubstituted alkyl groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso-butyl, tert-butyl, sec-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like.
  • Alkylene refers to an unbranched or branched carbon chain containing 1-20, preferably 1-10, carbon atoms. Branched carbon chains include both linear and cyclic structures.
  • alkenyl refers to a substituted or unsubstituted, straight, branched or cyclic, unsaturated hydrocarbon chain that contains at least one double bond and 2 to 20, preferably 2 to 6, carbon atoms.
  • cycloalkenyl groups contain having five to eight carbon atoms and at least one double bond.
  • Examples of cycloalkenyl groups include cyclohexadienyl, cyclohexenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cycloheptadienyl, cyclooctatrienyl and the like.
  • Alkoxy refers to a substituted or unsubstituted, -O-alkyl group. Exemplary unsubstituted alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, t-butoxy, and the like.
  • Alkynyl refers to a substituted or unsubstituted, straight, branched or cyclic unsaturated hydrocarbon chain containing at least one triple bond and 2 to 20, preferably 2 to 6, carbon atoms.
  • “Amine” refers to a unsubstituted or substituted amino (-NH 2 ) group.
  • the amine can be primary (-NH 2 ), secondary (-NHR) or tertiary (-NR 2 ), depending on the number of substituents (R).
  • substituted amino groups include methylamino, dimethylamino, ethylamino, diethylamino, 2-propylamino, 1- propylamino, di(n-propyl)amino, di(iso-propyl)amino, methyl-n-propylamino, t- butylamino, and the like.
  • amino acid refers to a compound that contains an amino group and a carboxylic acid group. Typically, the phrase refers to ⁇ -amino acids, although other amino acids (such as ⁇ - and (-amino acids) can be used.
  • the amino acids can either be "D,” “L,” or a racemic mixture.
  • Naturally encoded amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn.
  • Synthetic amino acids can include ⁇ -amino acids containing aryl or hetaryl groups attached as side chains.
  • Examples of synthetic amino acids include norvaline, sarcosin, n-leucine, 1-naphthylalanine, 2- indolinecarboxylic acid and the like.
  • Synthetic amino acids also include ⁇ , ⁇ - disubstituted amino acids such aminoisobutyric acid (AIB) and those described by Scott et al, Tetrahedron Lett. 1997, 38(21 ⁇ :3695; inco ⁇ orated herein by reference.
  • AIB aminoisobutyric acid
  • amino protected amino acid refers to an amino acid which includes an amino-protecting group bonded to the nitrogen atom to prevent reaction.
  • amino acid side chain refers to the group attached to the ⁇ -carbon of an ⁇ - amino acid. Suitable amino acid side chains include the ⁇ -side chain of the naturally encoded amino acids. Alternatively, any side chain of a synthetic amino acid can be used, including lower alkyl, alkyloxy, alkylamino, alkylthio, and the like.
  • Aprotic solvent refers to polar solvents of moderately high dielectric constant which do not contain an acidic hydrogen.
  • Examples of common aprotic solvents are dimethyl sulfoxide (DMSO), dimethylformamide (DMF), sulfolane, tetrahydrofuran (THF), diethyl ether (Et 2 O), methyl-t-butyl ether, or 1,2-dimethoxyethane.
  • Aryl refers to any monovalent aromatic carbocyclic group of 6 to 10 carbon atoms. Preferred aryl groups include phenyl and naphthyl.
  • Assay kit as used in accordance with the present invention refers to an assemblage of at least two cooperative elements, namely (1) a well plate apparatus and (2) biological assay materials.
  • Bio assay materials are materials necessary to conduct a biological evaluation of the efficacy of any library compound in a screen relevant to a selected disease state.
  • “Diverse library” means a library where the substituents on the combinatorial library scaffold are highly variable in constituent atoms, molecular weight, and structure and the library, considered in its entirety, is not a collection of closely related homologues or analogues (compare to "directed library”).
  • Halogen refers to fluorine, chlorine, iodine or bromine. The preferred halogen is fluorine or chlorine.
  • Heterocyclic (Het or heterocyclic) refers to a stable, saturated, partially unsaturated, or aromatic group containing 5 to 10, preferably 5 or 6, ring atoms. The ring can be substituted 1 or more times with a substituent. The ring can be mono-, bi- or polycyclic. The heterocyclic group consists of carbon atoms and from 1 to 3 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
  • heterocyclic groups include acridine, benzathiazoline, benzimidazole, benzofuran, benzothiapene, benzthiazole, benzothiophenyl, carbazole, cinnoline, furan, imidazole, lH-indazole, indole, isoindole, isoquinoline, isothiazole, mo ⁇ holine, oxazole (i.e.
  • 1,2,3-oxadiazole phenazine, phenothiazine, phenoxazine, phthalazine, piperazine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinazoline, quinoline, quinoxaline, thiazole, 1,3,4-thiadiazole, thiophene, 1,3,5-triazines, triazole (i.e. 1,2,3-triazole), and the like. It will be appreciated that many of the above heterocycles may exist in tautomeric forms. All such forms are included within the scope of this invention.
  • Inert atmosphere refers to reaction conditions in which the mixture is covered with a layer of inert gas such as nitrogen or argon.
  • Library or “Combinatorial library” means a large number of chemical derivatives used in screening for biological activity or other activity. In general, a library will have greater than 20 members, preferably the library will have at least 50 members, more preferably the library will have at least 96 members and most preferably the library will have at least 1000 members.
  • Library compound is an individual reaction product (usually a single compound) in a combinatorial library.
  • Lead compound means a compound in a selected combinatorial library for which the assay kit has revealed significant activity relevant to a selected disease state.
  • Nitriles include groups of the formula -C ⁇ N.
  • Organic solvent includes solvents containing carbon, such as halogenated hydrocarbons, ether, toluene, xylene, benzene, and tetrahydrofuran.
  • Peptide refers to a chain of amino acids connect from carboxy terminus to amino terminus via peptide bonds. Typically, the chain can be a mixture of various amino acids. Chains of ⁇ -amino acids are preferred, although other amino acids (such as ⁇ - and (-amino acids) can be used. The amino acids within the chain can either be “D,” “L,” or a racemic mixture. The chain can be of any length, including 2 amino acids (dipeptide), 3 amino acids (tripeptide), or more. The terms “oligopeptide” and “polypeptide” should be understood to be included within this term. The average length can vary between 2 to 100 amino acids, preferably 2-20, more preferably 2-10, most preferably 2-5.
  • “Pharmaceutically acceptable salt” and “salts thereof means organic or inorganic salts of the pharmaceutically important molecule.
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
  • the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically important organic molecule may have more than one charged atom in its structure. Situations where multiple charged atoms are part of the molecule may have multiple counterions.
  • the molecule of a pharmaceutically acceptable salt may contain one or more than one charged atoms and may also contain, one or more than one counterion. The desired charge distribution is determined according to methods of drug administration.
  • Protecting group means a group used to protect a heteroatom such as oxygen, nitrogen, sulfur or phosphorus from chemical reaction.
  • a O-protecting group is used to protect an oxygen heteroatom, such as in a hydroxy group, from reaction.
  • O-protecting groups include Boc, t-butyl ether, benzyl ethers, and the like.
  • N-protecting groups examples include acetyl (Ac), 1-adamantanesulphonyl (AdSO 2 ), 1-adamantaneacetyl (AdAc), benzoyl (Bz), t- butoxycarbonyl (Boc), carbobenzoxy (Cbz), 2-carboxybenzoyl (2-Cbz), dansyl (DNS), isovaleryl (Iva), Fmoc, methoxysuccinyl (MeOSuc), nitropiperonyl, pyrenylethoxycarbonyl, nitroveratryl (NV), nitrobenzyl, succinyl (Sue), tosyl (Ts), and such amino protecting groups which are functionally equivalent thereto.
  • Protecting groups are well known in the art, see for example Protective Groups in Organic Synthesis, Peter G. M. Wuts (Editor), Theodora W. Greene, 3rd ed. (April 1999), Vch Pub.; Protective Groups in Organic Synthesis, Theodora W. Greene, Peter G. Wuts (Contributor), 2nd ed., (March 1991) John Wiley & Sons.
  • Preferred protecting groups include, but are not limited to, the "Boc" protecting group, trialkyl silyl groups such as TBS (tert-butyldimethylsilyl, Si(CH 3 ) 2 C(CH 3 ) 3 ), MEM (2-methoxyethoxymethyl), MOM (methoxymethyl), SEM (2-(trimethylsilyl)ethoxymethyl), and THP (tetrahydropyrany 1) .
  • TBS tert-butyldimethylsilyl, Si(CH 3 ) 2 C(CH 3 ) 3
  • MEM 2-methoxyethoxymethyl
  • MOM methoxymethyl
  • SEM 2-(trimethylsilyl)ethoxymethyl
  • THP tetrahydropyrany 1
  • Protic solvent refers to a solvent containing hydrogen that is attached to oxygen, and hence is appreciably acidic.
  • Common protic solvents include such solvents as water, methanol (MeOH), ethanol (EtOH), 2-propanol (PrOH), and 1-butanol (n- BuOH).
  • Protic acid refers to an acid having an acidic hydrogen.
  • Preferred protic acids include acetic acid, hydrochloric acid (HC1), formic acid, perchloric acid, sulfuric acid (H 2 SO 4 ), and phosphoric acid (H 2 PO 4 ) in an aqueous medium.
  • the most preferred protic acids are hydrochloric acid, sulfuric acid, and formic acid.
  • “Scaffold” as used in accordance with the present invention refers to the invariable region of the compounds that are members of the combinatorial library.
  • Solid support broadly refers to any structure which is capable of supporting the chemical compound and is substantially inert to the chemical reactions conducted on the surface.
  • Exemplary solid supports include, but are not limited to, metals, resins, polymers, gels, glass beads, silica gels, ceramic supports and other solid and semi-solid compositions.
  • substantially pure is intended to mean at least about 90 mole percent, more preferably at least about 95 mole percent, and most preferably at least about 98 mole percent of the desired enantiomer or stereoisomer is present compared to other possible configurations.
  • Substituted means that the moiety contains at least one, preferably 1-3 substituent(s).
  • ⁇ - Amino acids can be mono-substituted at the backbone nitrogen atom, substituted at the ⁇ -carbon (to form a ⁇ , ⁇ -disubstituted ⁇ -carbon) or substituted 1-3 times at one or more side chain atoms.
  • Suitable substituents include hydrogen (H) and hydroxyl (-OH), amino (-NR 2 ), oxy (-O-), carbonyl
  • substituents can optionally be further substituted with 1-3 substituents.
  • substituted substituents include carboxamide, alkylmercapto, alkylsulphonyl, alkylamino, dialkylamino, carboxylate, alkoxycarbonyl, alkylaryl, aralkyl, alkylheterocyclic, (C l -C4)fluoroalkyl groups (such as trifluoromethyl or 2,2,2- trifluoroethyl) and the like.
  • Thiols include compounds of the formula -SH or -SR where R is a substituent, preferably alkyl.
  • exemplary thiols include methanethiol, ethanethiol, propanethiol, and the like.
  • a 1 , A 2 and A 3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A 1 , A 2 and A 3 is not a bond;
  • R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is O, 1, 2, 3, or 4.
  • Compounds of formula (I) may be made by using methods including those described in Appendix A. Briefly, the compounds may be synthesized (for example, as shown for a compound of formula (III) in which the residue is attached to the 6- position of the group of the formula (II) and in which X-CO- is R 7 -(CH 2 ) a -CH(NHR 12 )- CO-) by:
  • R 1 , R 11 , and R m are hydrogen or a substituent; Hal is a halogen; NP is a protected amino group; and c is 0, 1, 2, 3 or 4; with a compound of the formula (V):
  • R 1 to R IV and NP are as defined above;
  • P' is a protecting group; and each a, independently, is 0, 1 or 2; to form a N- protected first intermediate;
  • the temperature for the cyclization and displacement procedure may be between about 0° and 85° C but is preferably between about 30° and 70° and is most preferably at about 55 ° C .
  • the solvents suitable for the cyclization and displacement procedure include protic and aprotic solvent mixtures, aqueous and anhydrous solvent mixtures.
  • a preferred solvent is TFA
  • a more preferred solvent mixture is TFA:H2O
  • a most preferred solvent mixture is TFA: 5 N HCl/H 2 O.
  • the ratio of this TFA:5 N HCl/ ⁇ 2 O mixture may vary between about 1 :1 to about 99:1 TFA: 5 N HCl/H 2 O, but preferably is between about 80:1 to 1:1 TFA:5 N HCl H 2 O, and is most preferably at about 4:1 TFA:5 N HCl/H 2 O.
  • the time for the cyclization and displacement reaction may vary, but generally is between about 1 minute and 4 days but preferably between about 1 hour and 20 hours.
  • aminobenzisoxazole compounds of the invention having a basic group may be isolated in the form of an acid addition salt.
  • a salt of the compound of formula (I) formed with an acid such as one of those mentioned above is useful as a pharmaceutically acceptable salt for administration of the antithrombotic agent and for preparation of a formulation of the agent.
  • Other acid addition salts may be prepared and used in the isolation and purification of the compound.
  • optically active isomers and diastereomers of the compounds of formula (I) are also considered part of this invention.
  • Such optically active isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. This resolution can be carried out by derivatization with a chiral reagent followed by chromatography or by repeated crystallization. Removal of the chiral auxiliary by standard methods affords substantially optically pure isomers of the compounds of the present invention or their precursors. Further details regarding resolutions can be obtained in Jacques, et al., Enantiomers, Racemates, and Resolutions, John Wiley & Sons, 1981.
  • the present invention provides a library of 4-amidino-3-hydroxylphenyl compounds of the formula (lb): R-A 3 -A 2 -A'-NH-(CH 2 ) C -R lb wherein R lb is a substituted or unsubstituted group of the formula (lib):
  • a 2 and A 3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A 1 , A 2 and A 3 is not a bond;
  • R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is O, 1, 2, 3 or 4.
  • 4-Amidino-3-hydroxylphenyl compounds of the formula (lb) can be formed by ring opening catalytic hydrogenation of aminobenzisoxazole compounds of the formula (I) as exemplified in the following examples.
  • X-CO- is D-prolinyl, D-homoprolinyl, R 2 -(CH2) -NH-CH 2 -C(O)-,
  • R 2 is -COOR 14 , -SO 2 (C ⁇ -C 4 alkyl), -SO 3 H, -P(O)(OR 14 )2 or tetrazol-5-yl;
  • R 3 is hydrogen or (C ⁇ -C 4 )alkyl;
  • R4 is carboxy or methylsulfonyl
  • R 5 is NHR 6 , NHCOR 6 or NHCOOR 6 ;
  • R is (C ⁇ -C ⁇ o)alkyl, (C 3 -C 8 )cycloalkyl or a (C 3 -C 8 )cycloalkyl-(C ⁇ -C 6 )alkyl group containing 4-10 carbons;
  • R 7 is (C 3 -C 8 )cycloalkyl, (C ⁇ -C 8 )alkyl,
  • R 8 is -OH, (C i -C 4 )alkoxy, or -NH-R 12 ;
  • R 9 is hydrogen or (C ⁇ -C 4 )alkyl
  • R 10 is hydrogen, (C ⁇ -C 4 )alkyl, (C ⁇ -G 4 )alkoxy, hydroxy, halo or (C 1 -C 4 ) alkylsulfonylamino;
  • R 11 is (C ⁇ -C 4 )alkyl, (C ⁇ -C 4 )fluoroalkyl bearing one to five fluoros, -(CH 2 ) d -R 2 , or unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
  • R 12 is hydrogen, (C ⁇ -C 4 )alkyl, R n SO 2 -, R n OC(O)-, R n C(O)-, R i C(O)- or - (CH 2 ) d -R2;
  • R 13 is -COOR 14 or tetrazol-5-yl
  • each R is independently hydrogen or (C 1 -C4)alkyl; -Y-CO- is
  • R " is (C ⁇ -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, or -(CH2) c -L-(CH 2 ) b -R 17 ;
  • R 16 is (C ⁇ -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, or -(CH2) c -L-(CH2)b- R 1? ;
  • L is a bond, -O-, -S-, or -NH-;
  • R 17 is (C ⁇ -C 4 )alkyl, (C 3 -C 8 )cycloalkyl, -COOH, -CONH 2 , or Ar, where Ar is unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
  • R is -CH 2 -, -O-, -S-, or -NH-;
  • R is a bond or, when taken with R and the three adjoining carbon atoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom of which may be -O-, -S-, or -NH-; each a, independently, is 0, 1 or 2; each b, independently, is 0, 1, 2 or 3; each c, independently, is 0, 1, 2, 3 or 4; and each d, independently, is 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
  • This library is particularly useful for screening for compounds with antithrombin activity.
  • R 12 is, e.g., methyl, are referred to as the N ⁇ -methyl-phenylglycyl group and abbreviated MePhg.
  • Substituted compounds wherein R 10 is other than hydrogen are referred to by the type and position of the substituent group, e.g., 3'-chlorophenylglycyl or Phg(3-Cl).
  • R 12 is, e.g., methyl, are referred to as the N ⁇ -methyl-phenylalanyl group and abbreviated MePhe.
  • Substituted compounds wherein R 10 is other than hydrogen are referred to by the type and position of the substituent group, e.g., 3'-chlorophenylalanyl or Phe(3-Cl).
  • azetidine-2-carbonyl prolinyl, or homoprolinyl
  • Azt Pro or hPro
  • the group represents a saturated bicyclic system of the 4,5; 5,5; 6,5; 7,5; or 8,5 type.
  • the stereochemistry at 3a is cis to the carbonyl; the other bridgehead bond may be either cis or trans except that the 4,5 and
  • variable ring which includes the three carbon atoms shown, is a saturated carbocyclic system of 4-8 atoms. All of the ring atoms may be carbon, or one of the ring atoms may be a hetero atom selected from -O-, -S-, and -NH-.
  • This definition includes the moiety derived from octahydroindole-2-carboxylic acid, as represented by The various cis and trans forms of this moiety are contemplated by this invention.
  • the preferred isomer derived from [2S- (2 ⁇ ,3a ⁇ ,7a ⁇ )]-octahydroindole-2-carboxylic acid is abbreviated "Ohi" and is represented by
  • the asterisks in Y denote a chiral center that corresponds to (L) in the natural amino acids.
  • the asterisk in X denotes a chiral center that is (D) or (DL); the # in X denotes a chiral center that is (L).
  • Preferred compounds of the formula (III) (1-24) are shown in the examples.
  • X-CO- is D-prolinyl, D-homoprolinyl, R 2 -(CH2) -NH-CH 2 -C(O)-,
  • R 2 is -COOR 14 , -SO 2 (C ⁇ -C 4 alkyl), -SO 3 H, -P(O)(OR 1 )2 or tetrazol-5-yl;
  • R 3 is hydrogen or (C ⁇ -C 4 )alkyl;
  • R 4 is carboxy or methylsulfonyl
  • R 5 is NHR6, NHCOR6 or NHCOOR 6 ;
  • R 6 is (C ⁇ -C ⁇ o)alkyl, (C 3 -C 8 )cycloalkyl or a (C 3 -C 8 )cycloalkyl-(C ⁇ -C 6 )alkyl group containing 4-10 carbons;
  • R 7 is (C 3 -C 8 )cycloalkyl, (C j -C 8 )alkyl,
  • R 8 is -OH, (C ⁇ -C 4 )alkoxy, or -NH-R 12 ;
  • R 9 is hydrogen or (C ⁇ -C 4 )alkyl
  • R 10 is hydrogen, (C ⁇ -C 4 )alkyl, (C ⁇ -C 4 )alkoxy, hydroxy, halo or (C 1 -C 4 ) alkylsulfonylamino;
  • R 11 is (C ⁇ -C 4 )alkyl, (C ⁇ -C 4 )fluoroalkyl bearing one to five fluoros, -(CH 2 ) d -R 2 , or unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
  • R 12 is hydrogen, (C ⁇ -C 4 )alkyl, R u SO 2 -, R n OC(O)-, R u C(O)-, R 13 C(O)- or - (CH 2 ) d -R2;
  • R 13 is -COOR 14 or tetrazol-5-yl
  • each R is independently hydrogen or (C 1 -C4)alkyl; -Y-CO- is
  • R is (C ⁇ -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, or -(CH2) c -L-(CH2) b -R 17 ;
  • R 16 is (C ⁇ -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, or -(CH2) c -L-(CH2) b - R 17 ;
  • L is a bond, -O-, -S-, or -NH-;
  • R 17 is (C ⁇ -C 4 )alkyl, (C 3 -C 8 )cycloalkyl, -COOH, -CONH 2 , or Ar, where Ar is unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
  • R is -CH 2 -, -O-, -S-, or -NH-;
  • R is a bond or, when taken with R and the three adjoining carbon atoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom of which may be -O-, -S-, or -NH-; each a, independently, is 0, 1 or 2; each b, independently, is 0, 1, 2 or 3; each c, independently, is 0, 1, 2, 3 or 4; and each d, independently, is 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
  • 4-Amidino-3-hydroxylphenyl compounds of the formula (Illb) can be formed by ring opening catalytic hydrogenation of aminobenzisoxazole compounds of the formula (III).
  • U.S. 5,863,929 discloses that certain compounds of the formula (Illb) inhibit thrombin.
  • the present invention encompasses compounds of formula (I), (lb), (III) or (Illb) as a mixture of diastereomers, as well as in the form of an individual diastereomer, and that the present invention encompasses compounds of formula (I), (lb), (III) or (Illb) as a mixture of enantiomers, as well as in the form of an individual enantiomer, any of which mixtures or form possesses inhibitory properties against thrombin, it being well known in the art how to prepare or isolate particular forms and how to determine inhibitory properties against thrombin by standard tests including those described below.
  • compounds of formula (I), (lb), (III) or (Illb) may exhibit polymo ⁇ hism or may form a solvate with water or an organic solvent.
  • the present invention also encompasses any such polymo ⁇ hic form, any solvate or any mixture thereof.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), (lb), (III), or a novel compound of formula (Illb), or a pharmaceutically acceptable salt or solvate thereof, in association with a pharmaceutically acceptable carrier, diluent or excipient.
  • This invention also provides pharmaceutical compositions for use in therapeutic methods.
  • Pharmaceutical compositions of the invention comprise an effective amount of a compound of formula (I), (lb), (III) or a novel compound of formula (Illb) in association with a pharmaceutically acceptable carrier, excipient or diluent.
  • the compound is formulated in gelatin capsules or tablets which may contain excipients such as binders, lubricants, disintegration agents and the like.
  • a pharmaceutically acceptable diluent e.g. physiological saline (0.9 percent), 5 percent dextrose, Ringer's solution and the like.
  • the compound can be formulated in unit dosage formulations comprising a dose between about 0.1 mg and about 1000 mg.
  • the compound is in the form of a pharmaceutically acceptable salt such as for example the sulfate salt, acetate salt or a phosphate salt.
  • An example of a unit dosage formulation comprises 5 mg of a compound of the present invention as a pharmaceutically acceptable salt in a 10 ml sterile glass ampoule.
  • Another example of a unit dosage formulation comprises about 10 mg of a compound of the present invention as a pharmaceutically acceptable salt in 20 ml of isotonic saline contained in a sterile ampoule.
  • the compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal.
  • the compounds of the present invention are preferably formulated prior to administration.
  • the active ingredient in such formulations comprises from 0.1 percent to 99.9 percent by weight of the formulation.
  • pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the present pharmaceutical compositions are prepared by known procedures using well known and readily available ingredients.
  • the compositions of this invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
  • the active ingredient will usually be admixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container.
  • the carrier when it serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient.
  • the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
  • An aminobenzisoxazole compound of formula (III) or a novel compound of formula (Illb) is useful whenever inhibition of thrombin is useful, for example, for the prophylaxis and treatment of thromboembolic diseases such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process.
  • thromboembolic diseases such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process.
  • the present invention further provides a method of inhibiting thrombin comprising administering to a mammal in need of treatment, a thrombin inhibiting dose of a compound of formula (III) or a novel compound of formula (Illb).
  • the present invention also provides a method of inhibiting thrombosis in a mammal comprising administering to a mammal in need of treatment, an antithrombotic dose of a compound of formula (III) or a novel compound of formula (Illb).
  • the compounds of the invention are believed to selectively inhibit thrombin over other proteinases and nonenzyme proteins involved in blood coagulation without appreciable interference with the body's natural clot lysing ability (the compounds have a low inhibitory effect on fibrinolysis). Also, they generally exhibit increased selectivity for thrombin compound to the prior amidinophenyl compounds. Further, such selectivity is believed to permit use with thrombolytic agents without substantial interference with thrombolysis and fibrinolysis.
  • the invention in one of its aspects provides a method of inhibiting thrombin in mammals comprising administering to a mammal in need of treatment an effective (thrombin inhibiting) dose of a compound of formula (III) or a novel compound of formula (Illb).
  • the invention provides a method of treating a thromboembolic disorder comprising administering to a mammal in need of treatment an effective (thromboembolic disorder therapeutic and/or prophylactic amount) dose of a compound of formula (III) or a novel compound of formula (Illb).
  • the invention in another of its aspects provides a method of inhibiting coagulation in a mammal comprising administering to a mammal in need of treatment an effective (coagulation inhibiting) dose of a compound of formula (III) or a novel compound of formula (Illb).
  • the thrombin inhibition, coagulation inhibition and thromboembolic disorder treatment contemplated by the present method includes both medical therapeutic and/or prophylactic treatment as appropriate.
  • the invention relates to treatment, in a human or other mammal, of conditions where inhibition of thrombin is required.
  • the compounds of the invention are expected to be useful in mammals, including man, in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility are in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues.
  • disorders in which the compounds have a potential utility, in treatment and/or prophylaxis include venous thrombosis and pulmonary embolism, arterial thrombosis, such as in myocardial ischemia, myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis.
  • the compounds have expected utility in the treatment or prophylaxis of atherosclerotic disorders (diseases) such as coronary arterial disease, cerebral arterial disease and peripheral arterial disease.
  • the compounds are expected to be useful together with thrombolytics in myocardial infarction.
  • the compounds have expected utility in prophylaxis for reocclusion after thrombolysis, percutaneous transluminal angioplasty (PTCA) and coronary bypass operations. Further, the compounds have expected utility in prevention of rethrombosis after microsurgery. Further, the compounds are expected to be useful in anticoagulant treatment in connection with artificial organs and cardiac valves. Further, the compounds have expected utility in anticoagulant treatment in hemodialysis and disseminated intravascular coagulation. A further expected utility is in rinsing of catheters and mechanical devices used in patients in vivo, and as an anticoagulant for preservation of blood, plasma and other blood products in vitro.
  • the compounds have expected utility in other diseases where blood coagulation could be a fundamental contributing process or a source of secondary pathology, such as cancer, including metastasis, inflammatory diseases, including arthritis, and diabetes.
  • the anticoagulant compound is administered orally, parenterally e.g. by intravenous infusion (iv), intramuscular injection (im) or subcutaneously (sc).
  • the specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the rate of administration, the route of administration, and the condition being treated.
  • a typical daily dose for each of the above utilities is between about 0.01 mg/kg and about 1000 mg/kg.
  • the dose regimen may vary e.g. for prophylactic use a single daily dose may be administered or multiple doses such as 3 or 5 times daily may be appropriate.
  • a compound of the invention is administered by iv infusion at a rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferably between about 0.1 mg/kg/h and about 5 mg/kg/h.
  • the method of this invention also is practiced in conjunction with a clot lysing agent e.g. tissue plasminogen activator (t-PA), modified t-PA, streptokinase or urokinase.
  • a clot lysing agent e.g. tissue plasminogen activator (t-PA), modified t-PA, streptokinase or urokinase.
  • a clot lysing agent is usually employed.
  • a compound of the invention can be administered prior to or along with the lysing agent or subsequent to its use, and preferably further is administered along with aspirin to prevent the reoccurrence of clot formation.
  • the method of this invention is also practiced in conjunction with a platelet glycoprotein receptor (Ilb/IIIa) antagonist, that inhibits platelet aggregation.
  • a compound of the invention can be administered prior to or along with the Ilb/IIIa antagonist or subsequent to its use to prevent the occurrence or re
  • a compound of the invention can be administered prior to or along with aspirin or subsequent to its use to prevent the occurrence or reoccurrence of clot formation.
  • a compound of the present invention is administered in conjunction with a clot lysing agent and aspirin.
  • the process for making libraries of compounds of the formulae (I), (lb), (III), and (Illb) of the invention may be carried out in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means.
  • the process of the invention is preferably carried out in containers adapted for parallel array synthesis.
  • parallel array synthesis individual reaction products are prepared in each of multiple reaction zones.
  • the reaction zones are physically separated from one another in a reaction vessel.
  • a preferred parallel synthesis embodiment of the present invention is a diverse compound library in the form of a plurality of wellplates, each wellplate having wells containing a separate reaction product (library compound).
  • the library compounds are conveniently identified by their wellplate number and "x" column and "y” row coordinates.
  • the process of making the library of phenyl ether compounds may be conveniently carried out in a conventional wellplate apparatus. It is particularly advantageous to carry out the method of the invention in a standard wellplate apparatus such as a plastic 96 well microtiter plate, or FLEXCHEM TM 96 well Synthesis Assembly available from Robbins Scientific.
  • the wellplate apparatus is in the form of a rigid or semi-rigid plate.
  • the plate has a common surface containing openings of a plurality of reservoirs arranged in rows and columns.
  • a standard form of wellplate apparatus is a rectangular plastic plate having 8 rows and 12 columns (total 96) of liquid retaining depressions, or reservoirs, on its surface.
  • a wellplate apparatus may optionally have other elements or structure such as a top or cover (e.g., plastic or foil), a bottom in a form such as a plate or reservoir, clamping means to secure the wellplate and prevent loss of its contained compounds.
  • the amount of solid bound compound introduced into each reaction zone will depend on the desired amount of each library compound that is needed for conducting biological assays, archival storage and other related needs. Typically, the desired amount of individual reaction product is from 1 microgram to 50 milligrams.
  • the amount of solid bound compound in each reaction zone is represented by the symbol "(n)", where (n) represents the equivalents of compound.
  • the reactant is used in excess.
  • the method of the invention contemplates solution phase reactions where a stoichiometric excess of the coupling reactant is used.
  • the amount of coupling reactant used to ensure an excess is defined as at least 1.1 (n) and preferably a larger excess in the range of from 1.25(n) to 5(n), where the variable (n) is as previously defined.
  • the 1.1 multiplier is used to ensure at least a 10% stoichiometric excess of coupling agent is present to drive the reaction to completion.
  • the reaction zone is maintained at a temperature and for a time sufficient to permit reaction of solid bound compound with the coupling reactant, that is, to complete consumption of the solid bound compound and form an amount of compound necessary to conduct biological assays to determine the efficacy of the prepared library compounds.
  • reaction times for a single step of the reaction are generally from about 0.1 seconds to about 24 hours, with reaction times of 1 second to 60 minutes being most often used.
  • the temperature of the reaction may be any temperature between the freezing point and the boiling point of the liquid reaction medium, but is generally between about -10°C and about 60°C, with 10°C to 40°C being preferred and ambient temperatures (about 20°C-30°C) being most preferred.
  • the reactions may be conducted at subatmospheric pressure or superatmospheric pressure (viz., about 60 Kg./m 2 and about 2100 Kg./m 2 absolute), but ambient atmospheric pressure (about 10330 Kg./m , absolute) is most often used.
  • the completion of the coupling and deprotecting steps may be determined by a number of conventional techniques, including, but not limited to, chromatography (preferably, thin layer chromatography).
  • the purification of the library compound dissolved in the solvent phase of the reaction may be done by any conventional chemical or physical method.
  • the last purification step of the process may optionally be supplemented by a solvent removal step in which the solute library compound is removed from its solvent by conventional processes known in the art; such as solvent evaporation, distillation, salting out, solvent extraction, and etc.
  • the libraries of compounds (I), (lb) and (III) according to the present invention may be screened for biological activity.
  • the library to be screened is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibodies wherein the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined.
  • a biological substance usually a protein such as a receptor, enzyme, membrane binding protein or antibodies wherein the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined.
  • this will comprise determining whether the biological substance is bound to one or more of the members of the library.
  • binding may be determined by attaching a label to the biological substance.
  • Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels.
  • the degree of binding affinity may be determined by quantitating the amount or intensity of the bound label.
  • various biologically active compounds may be selected by identifying which compounds bind the particular biological substance most effectively.
  • Illustrative additional assays include, but are not limited to, in vitro assays such as enzymatic inhibition, receptor - ligand binding, protein - protein interaction, and protein - DNA interaction; cell based, functional assays such as transcriptional regulation, signal transduction / second messenger, and viral infectivity; add, incubate & read assays such as scintillation proximity assays (SPA), fluorescence polarization assay, fluorescence correlation spectroscopy, colorimetric biosensors, cellular reporter assays using reporter genes such as luciferase, green fluorescent protein, ⁇ -lactamase, and the like; and electrical cell impedance sensor assays.
  • in vitro assays such as enzymatic inhibition, receptor - ligand binding, protein - protein interaction, and protein - DNA interaction
  • cell based, functional assays such as transcriptional regulation, signal transduction / second messenger, and viral infectivity
  • incubate & read assays such as
  • APTT assay Activated partial thromboplastin time.
  • DIBAL diisobutylaluminum hydride
  • DIPEA diisopropylethyl amine
  • Fmoc 9-fluorenylmethoxycarbonyl.
  • HC1 hydrochloric acid
  • HCl aq aqueous HC1.
  • HOBT 1-hydroxybenzotriazole hydrate
  • hPro homo-proline
  • HTS technology high throughput screening technology
  • i-PrOH isopropanol
  • IR Infrared Spectrum
  • PT assay prothrombin time assay.
  • RPHPLC Reversed Phase High Performance Liquid
  • Si ⁇ 2 silica gel
  • TBS t-butyldimethylsilyl.
  • TEA triethylamine
  • TFA trifluoroacetic acid
  • THF tetrahydrofuran
  • TLC thin layer chromatography
  • TMS trimethyl silyl.
  • Ts tosyl (p-toluenesulfonyl)
  • the reaction vessel was rotated at 55 °C in a Robbins oven for 12 h, and allowed to cool for 1 h.
  • the resin was then rinsed with 2 x 2 mL of CH 2 C1 2 , 2 x 2 mL of MeOH, 2 2 mL of H 2 O, and 4 x 2 mL of MeOH.
  • the resin was dried in a 35 °C vacuum oven for 12 h.
  • the resin was then suspended in 25% TFA/CH C1 2 (3 mL) and rotated for 2 h.
  • the resin was again rinsed with 2 2 mL of CH 2 C1 2 and 4 x 2 mL of MeOH.
  • the resin was dried in a 35 °C vacuum oven for 12 h.
  • the resin was then suspended in 25% TFA/CH 2 C1 2 (3 mL) and rotated for 2 h.
  • the resin was again rinsed with 2 x 2 mL of CH 2- Cl 2 and 4 2 mL of MeOH.
  • the resin was dried in a 35 °C vacuum oven for 12 h.
  • the resin was then suspended in 10% TFA/CH 2 C1 2 (3 L) and rotated for 20 min.
  • the resin was rinsed with 2 x 2 mL of CH 2 C1 2 ,2 x 2 mL of 10% Et 3 N/CH 2 Cl 2 , 4 x 2 mL of CH 2 C1 2 .
  • the resin was then suspended in CH 2 C1 2 (2 mL) followed by addition of ethanesulfonyl chloride (403 ⁇ L, 4.28 mmol) and pyridine (346 ⁇ L, 4.28 mmol).
  • the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H 2 O, 2 2 mL of MeOH, 2 x 2 mL of CH 2 C1 2 and 2 x 2 mL of MeOH.
  • the resin was dried in a 35 °C vacuum oven for 12 h.
  • the resin was then suspended in TFA (2 mL) and 5 N HCl aq (0.5 mL) and the vessel was rotated for 2 h in a 55 °C oven.
  • the TFA/HCl aq was collected and the resin was rinsed with 2 2 mL of CH 2- Cl 2 .
  • the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H 2 O, 2 x 2 mL of MeOH, 2 x 2 mL of CH 2 C1 2 and 2 x 2 mL of MeOH.
  • the resin was dried in a 35°C vacuum oven for 12 h.
  • the resin was then suspended in 25% TFA/CH 2 C1 2 (3 mL) and rotated for 2 h.
  • the resin was again rinsed with 2 x 2 mL of CH 2 C1 2 and 4 x 2 mL of MeOH.
  • the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H 2 O, 2 x 2 mL of MeOH, 2 x 2 mL of CH 2 C1 2 and 2 x 2 mL of MeOH.
  • the resin was dried in a 35°C vacuum oven for 12 h.
  • the resin was then suspended in 25% TFA/CH 2 /C1 2 (3 mL) and rotated for 2 h.
  • the resin was again rinsed with 2 x 2 mL of CH 2 C1 2 and 4 x 2 mL of MeOH.
  • the resin was then suspended in DMF (2 mL) followed by addition of t-butylbromoacetate (compound C)(35 ⁇ L, 0.214 mmol) and DIPEA (112 ⁇ L, 0.642 mmol). After rotating the reaction vessel for another 12 h, the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H 2 O, 2 x 2 mL of MeOH, 2 x 2 mL of CH 2 C1 2 and 2 x 2 mL of MeOH. The resin was dried in a 35°C vacuum oven for 12 h.
  • dipeptide 9b Formation of dipeptide 9b.
  • the 3 -aminobenzisoxazole 9 (1.49 g, 2.8 mmol) was added to 250 mL round bottom flask followed by the addition of 5%> Pd(C) (310 mg, 0.145 mmol Pd).
  • the flask was slowly evacuated and released under a nitrogen atmosphere.
  • EtOH 50 mL
  • 0.1 N HCl aq 50 mL
  • the flask was again evacuated and released under an atmosphere of hydrogen (balloon). The reaction was allowed to stir vigorously for 30 min and removed from the hydrogen atmosphere.
  • Active ingredient means an aminobenzisoxazole compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.
  • Hard gelatin capsules are prepared using the following ingredients:
  • Formulation 2 A tablet is prepared using the ingredients below:
  • the components are blended and compressed to form tablets each weighing 665 mg.
  • Formulation 3 An aerosol solution is prepared containing the following components:
  • Propellant 22 (Chlorodifluoromethane) 70.00
  • the active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -30 °C and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container.
  • Formulation 4 Tablets, each containing 60 mg of active ingredient, are made as follows:
  • the active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly.
  • the aqueous solution containing polyvinylpyrrolidone is mixed with the resultant powder, and the mixture then is passed through a No. 14 mesh U.S. sieve.
  • the granules so produced are dried at 50 °C and passed through a No. 18 mesh U.S. Sieve.
  • the sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
  • Capsules each containing 80 mg of active ingredient, are made as follows:
  • the active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities.
  • Formulation 6 Suppositories, each containing 225 mg of active ingredient, are made as follows:
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
  • Formulation 7 Suspensions, each containing 50 mg of active ingredient per 5 ml dose, are made as follows:
  • the active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste.
  • the benzoic acid solution, flavor and color are diluted with a portion of the water and added, with stirring. Sufficient water is then added to produce the required volume.
  • Formulation 8 An intravenous formulation may be prepared as follows: Active ingredient 100 mg
  • the solution of the above ingredients generally is administered intravenously to a subject at a rate of 1 ml per minute.
  • the compounds provided by the invention selectively inhibit the action of thrombin in mammals.
  • the inhibition of thrombin is demonstrated by in vitro inhibition of the amidase activity of thrombin as measured in an assay in which thrombin hydrolyzes the chromogenic substrate, N-benzoyl-L-phenylalanyl-L-valyl-L- arginyl-p-nitroanilide, N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide.
  • the assay is carried out by mixing 50 ⁇ l buffer (0.03M Tris, 0.15M NaCl, pH 7.4) with 25 ⁇ l of human thrombin solution (purified human thrombin, Enzyme Research Laboratories, South Bend, Indiana, at 8 NIH units/ml) and 25 ⁇ l of test compound in a solvent (50%) aqueous methanol (v:v)). Then 150 ⁇ l of an aqueous solution of the chromogenic substate (at 0.25 mg/ml) are added and the rates of hydrolysis of the substrate are measured by monitoring the reactions at 405 nm for the release of p-nitroaniline. Standard curves are constructed by plotting free thrombin concentration against hydrolysis rate.
  • the hydrolysis rates observed with test compounds are then converted to "free thrombin" values in the respective assays by use of the standard curves.
  • the bound thrombin (bound to test compound) is calculated by subtracting the amount of free thrombin observed in each assay from the known initial amount of thrombin used in the assay.
  • the amount of free inhibitor in each assay is calculated by subtracting the number of moles of bound thrombin from the number of moles of added inhibitor (test compound).
  • the Kass value is the hypothetical equilibrium constant for the reaction between thrombin and the test compound (I).
  • Kass [Thrombin-I ] [ (Thrombin) x (I) ] Kass is calculated for a range of concentrations of test compounds and the mean value reported in units of liter per mole.
  • a thrombin inhibiting compound of formula (I) of the instant invention exhibits a Kass of 0.1 X 10 ⁇ L/mole or much greater.
  • N-Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide for factor Xa
  • N-Cbz-D-Arg-Gly-Arg-p- nitroanilide for factor IXa assay as the factor Xa substrate
  • Pyroglutamyl-Pro-Arg-p- nitroanilide for Factor XIa and for aPC
  • H-D-Pro-Phe-Arg-p-nitroanilide for factor Xlla
  • Pyroglutamyl-Gly-Arg-p-nitroanilide for urokinase
  • Bovine trypsin is purchased from Worthington Biochemicals, Freehold, New Jersey, and human plasma kallikrein from Kabi Vitrum, Swiss, Sweden.
  • Chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide for plasma kallikrein is purchased from Kabi Vitrum, Swiss, Sweden.
  • N-Benzoyl-Phe-Val-Arg-p-nitroanilide, the substrate for human thrombin and for trypsin is synthesized according to procedures described above for the compounds of the present invention, using known methods of peptide coupling from commercially available reactants, or purchased from Midwest Biotech, Fishers, Indiana.
  • plasmin Human plasmin is purchased from Boehringer Mannheim, Indianapolis, Indiana; nt-PA is purchased as single chain activity reference from American Diagnostica, Greenwich, Connecticut; modified-t-PA6 (mt-PA6) is prepared at Eli Lilly and Company by procedure known in the art (See, Burck, et al., J. Biol. Chem., 265, 5120-5177 (1990). Plasmin chromogenic substrate H-D-Val-Leu-Lys-p- nitroanilide and tissue plasminogen activator (t-PA) substrate H-D-Ile-Pro-Arg-p- nitroanilide are purchased from Kabi Vitrum, Sweden.
  • Thrombin inhibitors preferably should spare fibrinolysis induced by urokinase, tissue plasminogen activator (t-PA) and steptokinase.
  • fibrinolytic system sparing can be studied by the use of human plasma clots and their lysis by the respective fibrinolytic plasminogen activators.
  • Dog plasma is obtained from conscious mixed-breed hounds (either sex Hazelton-LRE, Kalamazoo, Michigan, U.S.A.) by venipuncture into 3.8 percent citrate.
  • Fibrinogen is prepared from fresh dog plasma and human fibrinogen is prepared from in-date ACD human blood at the fraction 1-2 according to previous procedures and specifications. Smith, Biochem. J Cincinnati 185, 1-11 (1980); and Smith, et al., Biochemistry. 11, 2958-2967, (1972).
  • Human fibrinogen (98 percent pure/plasmin free) is from American Diagnostica, Greenwich, Connecticut. Radiolabeling of fibrinogen 1-2 preparations is performed as previously reported. Smith, et al., Biochemistry.
  • Urokinase is purchased form Leo Pharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase is purchased from Hoechst-Roussel Pharmaceuticals, Somerville, New Jersey.
  • thrombin inhibitors 25 ul of supernate is added into 1.0 ml volume of 0.03 M tris/0.15 M NaCl buffer for gamma counting. Counting controls 100 percent lysis are obtained by omitting thrombin (and substituting buffer). The thrombin inhibitors are evaluated for possible interference with fibrinolysis by including the compounds in the overlay solutions at 1, 5, and 10 ug/ml concentrations. Rough approximations of IC50 values are estimated by linear extrapolations from data points to a value which would represent 50 percent of lysis for that particular concentration of fibrinolytic agent.
  • Dog plasma and rat plasma are obtained from conscious mixed-breed hounds (either sex, hazelton-LRE, Kalamazoo, Michigan, U.S.A.) or from anesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, Indiana, U.S.A.) by venipuncture into 3.8 percent citrate.
  • Fibrinogen is prepared from in-date ACD human blood as the fraction 1-2 according to previous procedures and specifications. Smith, Biochem. J.. 185. 1-11 (1980); and Smith, et al., Biochemistry. 11, 2958-2967 (1972).
  • Human fibrinogen is also purchased as 98 percent pure/plasmin free from American Diagnostica, Greenwich, Connecticut.
  • Coagulation reagents ACTIN, Thromboplastin, and Human plasma are from Baxter Healthcare Corp., Dade Division, Miami, Florida.
  • Bovine thrombin from Parke-Davis (Detroit, Michigan) is used for coagulation assays in plasma.
  • Coagulation assay procedures are as previously described. Smith, et al., Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulation instrument (American LABor, Inc.) is used for all coagulation assay measurements.
  • the thrombin time (TT) is measured by adding 0.05 ml saline and 0.05 ml thrombin (10 NTH units/ml) to 0.05 ml test plasma.
  • the activated partial thromboplastin time (APTT) is measured by incubation of 0.05 ml test plasma with 0.05 ml Actin reagent for 120 seconds followed by 0.05 ml CaCl2 (0.02 M).
  • the prothrombin time is measured by adding 0.05 ml saline and 0.05 ml Thromboplastin-C reagent to 0.05 ml test plasma.
  • the compounds of formula I are added to human or animal plasma over a wide range of concentrations to determine prolongation effects on the TT, APTT and PT assays. Linear extrapolations are performed to estimate the concentrations required to double the clotting time for each assay.
  • mice Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc., Indianapolis, IN) are anesthetized with xylazine (20 mg/kg, s.c.) and ketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37 °C). The jugular vein(s) is cannulated to allow for infusions.
  • the left jugular vein and right carotid artery are cannulated with 20 cm lengths of polyethylene PE 60 tubing.
  • Blood is circulated through the shunt for 15 min before the thread is carefully removed and weighed. The weight of a wet thread is subtracted from the total weight of the thread and thrombus (see J.R. Smith, Br J Pharmacol. 77:29, 1982).
  • the carotid arteries are isolated via a midline ventral cervical incision.
  • a thermocouple is placed under each artery and vessel temperature is recorded continuously on a strip chart recorder.
  • a cuff of tubing (0.058 ID x 0.077 OD x 4 mm, Baxter Med. Grade Silicone), cut longitudinally, is placed around each carotid directly above the thermocouple.
  • FeCl 3 hexahydrate is dissolved in water and the concentration (20 percent) is expressed in terms of the actual weight of FeCl 3 only.
  • 2.85 ⁇ l is pipetted into the cuff to bathe the artery above the thermocouple probe.
  • Arterial occlusion is indicated by a rapid drop in temperature. The time to occlusion is reported in minutes and represents the elapsed time between application of FeCl 3 and the rapid drop in vessel temperature (see K.D. Kurz, Thromb. Res., 60:269,1990).
  • peptide thrombin inhibitors inhibit thrombin and at higher concentration may inhibit, other serine proteases, such as plasmin and tissue plasminogen activator.
  • the rate of spontaneous thrombolysis is determined by implanting a labeled whole blood clot into the pulmonary circulation. Rat blood (1 ml) is mixed rapidly with bovine thrombin (4 IU, Parke Davis) and 125 I human Fibrogen (5 ⁇ Ci, ICN), immediately drawn into silastic tubing and incubated at 37 °C for 1 hour.
  • the aged thrombus is expelled from the tubing, cut into 1 cm segments, washed 3X in normal saline and each segment is counted in a gamma counter.
  • a segment with known counts is aspirated into a catheter that is subsequently implanted into the jugular vein.
  • the catheter tip is advanced to the vicinity of the right atrium and the clot is expelled to float into the pulmonary circulation.
  • One hour after implant, the heart and lungs are harvested and counted separately.
  • Thrombolysis is expressed as a percentage where:
  • % Thrombolysis (injected cpm - lung cpm) x 100 injected cpm
  • Plasma thrombin time (TT) and activated partial thromboplastin time (APTT) are measured with a fibrometer. Blood is sampled from a jugular catheter and collected in syringe containing sodium citrate (3.8 percent, 1 part to 9 parts blood). To measure TT, rat plasma (0.1 ml) is mixed with saline (0.1 ml) and bovine thrombin (0.1 ml, 30 U/ml in TRIS buffer; Parke Davis) at 37 °C.
  • APTT For APTT, plasma (0.1 ml) and APTT solution (0.1 ml, Organon Teknika) are incubated for 5 minutes (37 °C) and CaCl 2 (0.1 ml, 0.025M) is added to start coagulation. Assays are done in duplicate and averaged.
  • a measure of bioactivity plasma thrombin time (TT) serves as a substitute for the assay of parent compound on the assumption that increments in TT resulted from thrombin inhibition by parent only.
  • the time course of the effect of the thrombin inhibitor upon TT is determined after i.v bolus administration to anesthetized rats and after oral treatment of fasted conscious rats. Due to limitations of blood volume and the number of points required to determine the time course from time of treatment to the time when the response returns to pretreatment values, two populations of rats are used. Each sample population represents alternating sequential time points. The average TT over the time course is used to calculate area under the curve (AUC).
  • AUC area under the curve
  • the index of bioavailability is calculated by the formula shown below and is expressed as percent relative activity.
  • the area under the curve (AUC) of the plasma TT time course is determined and adjusted for the dose. This index of bioavailability is termed "% Relative Activity” and is calculated as
  • Compound solutions are prepared fresh daily in normal saline and are injected as a bolus or are infused starting 15 minutes before and continuing throughout the experimental perturbation which is 15 minutes in the arteriovenous shunt model and 60 minutes in the FeCb model of arterial injury and in the spontaneous thrombolysis model.
  • Bolus injection volume is 1 ml/kg for i.v., and 5 ml/kg for p.o. and infusion volume is 3 ml/hr.
  • Results are expressed as means +/- SEM. One-way analysis of variance is used to detect statistically significant differences and then Dunnett's test is applied to determine which means are different. Significance level for rejection of the null hypothesis of equal means is PO.05.
  • Pharmacokinetic model Test compound is formulated immediately prior to dosing by dissolving in sterile 0.9 percent saline to a 5 mg/ml preparation. Dogs are given a single 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5 ml) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1,2,3,4 and 6 hours after dosing. Samples are collected in citrated Vacutainer tubes and kept on ice prior to reduction to plasma by centrifligation. Plasma samples are analyzed by HPLC-MS.
  • Plasma concentration of test compound is recorded and used to calculate the pharmacokinetic parameters: elimination rate constant, Ke; total clearance, Clt; volume of distribution, VD; time of maximum plasma test compound concentration, Tmax; maximum concentration of test compound of Tmax, Cmax; plasma half-life, t ⁇ .5; and area under the curve, A.U.C.; fraction of test compound absorbed, F. Canine Model of Coronary Artery Thrombosis
  • Surgical preparation and instrumentation of the dogs are as described in Jackson, et al., Circulation, 82, 930-940 (1990).
  • Mixed-breed hounds (aged 6-7 months, either sex, Hazelton-LRE, Kalamazoo, MI, U.S.A.) are anesthetized with sodium pentobarbital (30 mg/kg intravenously, i.v.), intubated, and ventilated with room air. Tidal volume and respiratory rates are adjusted to maintain blood PO 2 , PCO 2 , and pH within normal limits.
  • Subdermal needle electrodes are inserted for the recording of a lead II ECG.
  • the left jugular vein and common carotid artery are isolated through a left mediolateral neck incision.
  • a left thoracotomy is performed at the fifth intercostal space, and the heart is suspended in a pericardial cradle.
  • a 1- to 2-cm segment of the left circumflex coronary artery (LCX) is isolated proximal to the first major diagonal ventricular branch.
  • a 26- gauge needle-tipped wire anodal electrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm long is inserted into the LCX and placed in contact with the intimal surface of the artery (confirmed at the end of the experiment).
  • the stimulating circuit is completed by placing the cathode in a subcutaneous (s.c.) site.
  • An adjustable plastic occluder is placed around the LCX, over the region of the electrode.
  • a precalibrated electromagnetic flow probe (Carolina Medical Electronics, King, NC, U.S.A.) is placed around the LCX proximal to the anode for measurement of coronary blood flow (CBF).
  • CBF coronary blood flow
  • the occluder is adjusted to produce a 40-50 percent inhibition of the hyperemic blood flow response observed after 10-s mechanical occlusion of the LCX.
  • All hemodynamic and ECG measurements are recorded and analyzed with a data acquisition system (model M3000, Modular Instruments, Malvern, PA. U.S.A.). Thrombus Formation and Compound Administration Regimens
  • Electrolytic injury of the intima of the LCX is produced by applying 100- ⁇ A direct current (DC) to the anode. The current is maintained for 60 min and then discontinued whether the vessel has occluded or not. Thrombus formation proceeds spontaneously until the LCX is totally occluded (determined as zero CBF and an increase in the S-T segment).
  • Compound administration is started after the occluding thrombus is allowed to age for 1 hour.
  • a 2-hour infusion of the compounds of the present invention at doses of 0.5 and 1 mg/kg/hour is begun simultaneously with an infusion of thrombolytic agent (e.g. tissue plasminogen activator, streptokinase, APSAC). Reperfusion is followed for 3 hour after administration of test compound.
  • Reocclusion of coronary arteries after successful thrombolysis is defined as zero CBF which persisted for at least 30 minutes.
  • Hematology and template bleeding time determinations Whole blood cell counts, hemoglobin, and hematocrit values are determined on a 40- ⁇ l sample of citrated (3.8 percent) blood (1 part citrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900, Sequoia-Turner. Mount View, CA, U.S.A.). Gingival template bleeding times are determined with a Simplate II bleeding time device (Organon Teknika Durham, N.C., U.S.A.). The device is used to make 2 horizontal incisions in the gingiva of either the upper or lower left jaw of the dog.
  • Each incision is 3 mm wide x 2 mm deep.
  • the incisions are made, and a stopwatch is used to determine how long bleeding occurs.
  • a cotton swab is used to soak up the blood as it oozes from the incision.
  • Template bleeding time is the time from incision to stoppage of bleeding. Bleeding times are taken just before administration of test compound (0 min), 60 min into infusion, at conclusion of administration of the test compound (120 min), and at the end of the experiment.
  • solid support is intended to have a relatively broad meaning including, but not limited to, a resin, a polymer, a gel, glass beads, silica gel, a ceramic solid support or other solid composition.
  • solid support bound oxime means a solid support that at least has one oxime moiety chemically attached thereto.
  • this compound may be represented by the formula (IX):
  • g represents a solid support, such as defined above.
  • solid support bound member means a solid support that has at least one functional moiety chemically attached thereto.
  • this may be represented by the formula (III):
  • oxime resin means a solid support where the functional moiety is an oxime, and the solid support is a resin.
  • Kaiser resin means an oxime functionalized polystyrene resin, an example of that is defined by E.T.Kaiser in a 1980 publication (Degrado, W.F.;Kaiser. E.T.; J.Org. Chem.,1980, 45, 1295).
  • a preferred resin is an oxime functionalized polystyrene, such as an oxime-polystyrene resin derived from p- nitrobenzophenone polystyrene resin according to the following formula.
  • halo means a member selected from the group consisting of fluoro, chloro, bromo and iodo.
  • Alkyl is the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl group, and that groups may include one or more double or triple bonds.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone, and more preferably 20 or fewer and most preferred 10 or fewer.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3-6 carbons in the ring structure.
  • alkyl substituents include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso-butyl, tert- butyl, sec-butyl, cyclobutyl, pentyl, hexyl, cyclohexyl, etc.
  • "lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure.
  • the aliphatic cyclic groups can be single or polycyclic containing between about 3 to 12 carbons per ring, but preferably between 3 and 9 carbons per ring.
  • Haloalkyl and “alkylhalo” both refer to mono- or poly- halogen radical substituted alkyl radicals, with the alkyl radicals having the analogous length and possible substitution as described above. Typically, these terms refer to groups of the formula X n -(CX'X") m -, where n and m are each independently an integer > 1, and X, X' and X" are each independently hydrogen or halogen (so long as at least one of X, X' and X" are halogen).
  • Hydroxyalkyl and “alkylhydroxide” and “alkyl alcohol” all refer to a mono or poly hydroxide radical substituted alkyl radical, with the alkyl radicals having the analogous length and possible substitution as described above.
  • Alkyloxyalkyl ether and “alkyloxyaryl ether” both refer to ether functional radicals of either the dialkyl radical or the alkyl, aryl radical configuration, with the alkyl radicals and the aryl radicals having the analogous length and possible substitution to the alkyl and aryl radicals defined herein.
  • Alkenyl and “alkynyl” refer to unsaturated aliphatic substituents analogous in length and possible substitution to the alkyl radicals described above, but that contain at least one double or triple bond, respectively.
  • Amino means an amino radical substituted with up to 2 alkyl radicals as defined above or with 1 alkyl radical and a hydrogen radical, or with two or more hydrogen radicals or with the substitution required to complete the nitrogen's valence requirements.
  • Aryl as used herein includes 5-10 membered aromatic monocyclic or fused polycyclic moieties that may include from zero to four heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthyline, benzathiazoline, benzothiapene, benzofurans, indole, quinoline, etc.
  • the aryl group can be substituted at one or more positions with halo, alkyl, alkoxy, alkoxy carbonyl, haloalkyl, cyano, amino sulfonyl, aryl, sulfonyl, aminocarbonyl, carboxy, acylamino, alkyl sulfonyl, amino and substituted or unsubstituted substituents.
  • heteroaryl is a mono-, bi- or tricyclic, - N-, -O- or -S- heteroaryl substituent, such as benzofuran, benzothiophene, furan, imidazole, indole, isothiazole, oxazole, piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinoline, thiazole and thiophene.
  • a "library” means a large number of chemical derivatives used in screening for biological activity or other activity.
  • a library will have greater than 20 members, preferably the library will have at least 50 members, more preferably the library will have at least 96 members and most preferably the library will have at least 1000 members.
  • chemically derivatized means the chemical manipulation such as addition to, oxidation of, substitution for, reduction of, or cyclization of the selected R group or R groups of the intermediate. Chemical derivatization also means the manipulation of two or more groups of the intermediate such that additional aryl or alkyl rings are formed and that rings may be fused or unfused to the intermediate ring, and that new ring may be substituted with further chemically derivatizable substituents.
  • pharmaceutically acceptable salt and “salts thereof means organic or inorganic salts of the pharmaceutically important molecule.
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
  • the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically important organic molecule may have more than one charged atom in its structure. Situations where multiple charged atoms are part of the molecule may have multiple counterions.
  • the molecule of a pharmaceutically acceptable salt may contain one or more than one charged atoms and may also contain, one or more than one counterion.
  • the desired charge distribution is determined according to methods of drug administration.
  • Examples of pharmaceutically acceptable salts are well known in the art but, without limiting the scope of the present invention, exemplary presentations can be found in the Physicia 's Desk Reference, The Merck Index, The Pharmacopoeia and Goodman & Gilman's 77ze Pharmacological Basis of Therapeutics.
  • TFA means trifluoro acetic acid
  • HC1 means hydrochloric acid
  • THF means tetrahydrofuran
  • DMF means dimethylformamide
  • DIPEA diisopropylethyl amine
  • TMS means a trimethyl silyl radical
  • TBS means a t-butyldimethyl silyl radical.
  • leaving group means halo, oxo, thioxo radicals and activated alcohols such as a p-toluene sulfonyl activated alcohols and other groups that are susceptible to displacement and replacement by a nucleophile under selected conditions of temperature, solvent and time.
  • shaffold means a common chemical structure found within a library of organic compounds. Similarly, within a combinatorial chemical library the scaffold forms the basis for a diverse series of chemical derivatization, additions and subtractions. Importantly, regardless of the extent of the chemical derivatization performed on the scaffold, the product is within the scope of the combinatorial library. All other acronyms and abbreviations have the corresponding meaning as published in journals relative to the art of organic chemistry.
  • a general method for making polycyclic heterocyles according to the first aspect of the invention involves the following method.
  • each of R 1 , R 2 , R 3 and R 4 is a stable moiety independently selected from the group consisting of halo, haloalkyl, cyano, nitro, R a -Q-, R a -Q-alkyl, R a -Q-alkenyl, R a -Q- alkynyl, R a -Q-arylalkyl and R a -Q-aryl;
  • R a is hydrogen, alkyl, aryl or arylalkyl
  • R b is hydrogen, alkyl, aryl or arylalkyl, where R a and R b may together with the nitrogen to that they are attached form a ring, R 5 is selected from the group consisting of halo, nitro, and haloalkyl.
  • each of R 1 , R 2 , R 3 and R 4 may be substituted one to three times with a substituent selected from the group consisting of alkyl, alkenyl, alkynyl, halo, hydroxy, alkoxy, alkylthio, sulfonyl, aryl, heteroaryl and where the substituents of the moieties substituted can themselves be substituted with one to three further substituents, if desired.
  • a substituent selected from the group consisting of alkyl, alkenyl, alkynyl, halo, hydroxy, alkoxy, alkylthio, sulfonyl, aryl, heteroaryl and where the substituents of the moieties substituted can themselves be substituted with one to three further substituents, if desired.
  • Particularly one of R 1 , R 2 , R 3 or R 4 is an haloalkyl, more particularly either R 1 , R 3 or R 4 is a CF 3 radical and most particularly
  • R 1 , R 2 , R 3 or R 4 is an hydroxyalkyl, more particularly one of R 1 , R 2 , R 3 or R 4 is methoxy and most particularly R 3 is a methoxy radical.
  • R 1 , R 2 , R 3 or R 4 is halo, cyano or nitro.
  • R 3 or R 4 is bromo, and most particularly R 3 is bromo.
  • R 3 is most particularly a cyano radical.
  • R 3 is most particularly a nitro radical.
  • R 5 is selected from fluoro, chloro or nitro. If a 5,6 polycycle is desired, then R 5 is a halo. Also, R 5 is between to Cj 2 haloalkyl, particularly R 5 is between Cj to C 6 haloalkyl and more particularly R 5 is a halomethyl and most particularly R 5 is bromomethyl.
  • R 6 of formula (I) is selected from the group consisting of cyano and a radical of formula (II) where L is selected from the group consisting of -O-, -S- and -NH-.
  • R 6 is the radical of formula (II)
  • L is preferably -O- or -S- and most preferably -O-.
  • R 7 of formula (II) is selected from the group consisting of alkyl, aryl, arylalkyl, alkyloxyalkyl, aryloxyalkyl, alkylamino, dialkylamino, arylamino, alkoxy carbonyl, amino, alkoxy, hydroxy and heteroaryl.
  • R 7 is preferably aryl and most preferably R 7 is phenyl.
  • R 5 is halomethyl and R 6 is cyano.
  • R 5 is halo
  • R 6 is formula (II)
  • L is oxygen and R 7 is a phenyl radical.
  • R 5 is halo and R 6 is cyano.
  • g is a solid support selected from the group consisting of a resin, a polymer, a gel, glass beads, silica gel, a ceramic solid support or other solid composition. More preferably the solid support is a resin and most preferably the solid support is a polystyrene resin.
  • h is selected from the group consisting of alkyl and aryl.
  • h is aryl, more preferably h is a substituted aryl and most preferably h is p- nitrophenyl.
  • R is selected from the group consisting of -NH-, -O- and -S-, most preferably R is -O-.
  • Suitable conditions for this reaction include having a suitable solvent mixture, a suitable temperature and reacting for a suitable period of time.
  • a suitable solvent mixture is preferably of basic pH.
  • the base is an aikoxide, more preferably the base is an alkoxide of fewer than 5 carbons and most preferably the base is K + OBu ⁇
  • a suitable solvent is either protic or aprotic, preferably the solvent is aprotic and even more preferably the solvent is aprotic and anhydrous and most preferably the solvent is THF.
  • the reaction temperature is preferably between about 0° C and 85° C but more preferably between about 30° C and about 70° C and most preferably at about 55° C.
  • the reaction time is preferably between about 1 minute and 2 days, more preferably between about 1 hour and 1 day and most preferably between about 8 hours and 14 hours.
  • the compounds (I) and (III) react forming an intermediate (IV) where n > 0, and g, h, R 1 , R 2 , R 3 , R 4 , R 6 and R 13 are as defined above.
  • This intermediate (IV) is optionally chemically derivatized prior to the cyclization and displacement procedure.to form a corresponding intermediate (IV):
  • R 1 , R 2 , R 3 and R 4 substituents are each independently the same as substituents R 1 , R 2 , R 3 and R 4 respectively if not derivatized or are each independently the chemically derivatized substituents respectively.
  • Examples of chemical derivatization reactions include, but are not limited to, the following general derivatizations procedures.
  • the BOC protected nitrogen of the intermediate compound of Example ix is deprotected and reacted with acetic anhydride.
  • the TBS (t-butyldimethyl silyl) protected alcohol of the intermediate compound of Example x is deprotected with tetrabutylamonium fluoride and reacted with a chlorophenol under Mitsunobu conditions to give the p-chlorophenyl ether derivatized intermediate.
  • the methylene ester radical at R 3 of the intermediate of Example xv is subjected to ester hydrolysis/amidation providing the p-chlorophenyl methylaminocarbonyl methylenamide substituent.
  • R 1 , R 2 , R 3 and R 4 substituents are each independently the same as substituents R 1 , R 2 , R 3 and R 4 respectively if not derivatized or are each independently the chemically derivatized substituents respectively.
  • each of R 1' , R 2 , R 3' and R may be correspondingly the same as R 1 , R 2 , R 3 and R 4 or may represent the result of the optional chemical derivatization of the corresponding substituent, prior to cyclization and displacement.
  • chemical derivatization includes other ring formation or ring closure reactions such as where any two of R 1 , R , R and R 4 together form an aryl or an alkyl ring of between about 5 and 14 atoms.
  • R is amino, hydroxy or the same as R above.
  • the temperature for the cyclization and displacement procedure may be between about 0° and 85° C but is preferably between about 30° and 70° and is most preferably at about 55° C.
  • the solvents suitable for the cyclization and displacement procedure include protic and aprotic solvent mixtures, aqueous and anhydrous solvent mixtures.
  • a preferred solvent is TFA
  • a more preferred solvent mixture is TFA:H 2 O
  • a most preferred solvent mixture is TFA:5 N HCl/H 2 O.
  • the ratio of this TFA:5 N HCl/H 2 O mixture may vary between about 1 :1 to about 99:1 TFA:5 N HCl H 2 O, but preferably is between about 80:1 to 1 :1 TFA:5 N HCl/H 2 O, and is most preferably at about 4:1 TFA:5 N HCl H 2 O.
  • the time for the cyclization and displacement reaction may vary, but generally is between about 1 minute and 4 days but preferably between about 1 hour and 20 hours.
  • a second aspect of the invention is directed to a solid support bound intermediate (IV) above.
  • the intermediate (IV) can be derivatized before the cyclization and displacement procedure to thereby provide further options for the groups R 1 , R 2 , R 3 and R 4 in the final product.
  • the preferred, yet optional, derivatizations of the intermediate compound (IV), and preferred conditions whereby optional derivatizations occur are as described above.
  • a third aspect of the invention is directed to a library of polycyclic heterocycle compounds where the library contains a plurality of diverse compounds (V):
  • each of R 1 , R 2 , R 3 and R 4 is a stable moiety independently selected from the group consisting of halo, haloalkyl, cyano, nitro, R a -Q-, R a -Q-alkyl, R a -Q-alkenyl, R a -Q- alkynyl, R a -Q-arylalkyl and R a -O-aryl;
  • R a is hydrogen, alkyl, aryl or arylalkyl;
  • Q is a single bond, -O-, -NR b -, -CO-, -NR b -CO-, -CO-NR b -, -CO-O-, -O-CO-, S(O)bond
  • R b is hydrogen, alkyl, aryl or arylalkyl, where R a and R b may together with the nitrogen to that they are attached form a ring, and n > 0;
  • R 7 is selected from the group consisting of alkyl, aryl, arylalkyl, alkyloxy alkyl, aryloxyalkyl, alkylamino, dialkylamino, arylamino, alkoxy carbonyl, amino, alkoxy, hydroxy and heteroaryl; and
  • R 13 is selected from the group consisting of -NH-, -O- and -S-.
  • Preferred substituents for R 1 , R 2 , R 3 , R 4 , R 7 , and values for n are as described above, in the included examples and the appended claims.
  • a fourth aspect according to the present invention preferably produces a library of compounds where the compounds comprise a diverse chemical library according to the general methods discussed above. All of the compounds in such a library have a common scaffold, e.g., compound (V).
  • a combinatorial library according to the present invention, diversity is introduced via the R 1 , R 2 , R 3 , R 4 , R 7 , and R 13 substituents as discussed more fully above. These substituents are selected to allow the creation of a chemically diverse library that, as one goal, maximizes the exploration of molecular spatial properties. Such maximization increases the likelihood of creating compounds that will be biologically active against selected targets.
  • a fifth aspect of the invention is directed to an assay kit for the identification of biologically active compounds, the kit comprising assay materials and a well plate apparatus where each well in the apparatus contains a compound of the library described above.
  • the fourth and fifth aspects of the solid support mediated method of the invention may be carried out by way of parallel synthesis in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means.
  • the solid support mediated method of the invention is preferably carried out in containers adaptable to parallel array syntheses.
  • parallel array synthesis individual reaction products are prepared in each of multiple reaction zones. The reaction zones are physically separated from one another in a reaction vessel. Compounds can be added to the reaction vessel by multiple delivery apparatus, automated or robotic apparatus, any of that may be either manually or computer controlled.
  • a preferred parallel synthesis embodiment of the present invention is a diverse polycyclic heterocycle compound library in the form of a plurality of wellplates, each wellplate having wells containing a separate reaction product (library compound).
  • libraries compound a separate reaction product
  • their wellplate number and "x" column and "y" wellplate row coordinates conveniently identify the library compounds.
  • the process of making the library of polycyclic heterocycle compounds may be conveniently carried out in a conventional wellplate apparatus. It is particularly advantageous to carry out the method of the invention in a standard wellplate apparatus such as a plastic 96 well microtiter plate.
  • the wellplate apparatus is in the form of a rigid or semi-rigid plate, the plate having a common surface containing openings of a plurality of reservoirs arranged in rows and columns.
  • a standard form of wellplate apparatus is a rectangular plastic plate having 8 rows and 12 columns (total 96) of liquid retaining depressions, or reservoirs, on its surface.
  • a wellplate apparatus may optionally have other elements of structure such as a top or cover (e.g., plastic or foil), a bottom in a form such as a plate or reservoir, clamping means to secure the wellplate and prevent loss of its contained compounds.
  • the polycyclic heterocycle library of compounds formed using the solid support mediated method aspects of the invention can be used to screen compounds for biological or other activity. Myriad biological assays are known in the art and can be used to screen the polycyclic heterocycle library of compounds.
  • the libraries of diverse polycyclic heterocycle according to the solid support mediated method of the present invention may be screened for biological activity.
  • a biological substance usually a protein such as a receptor, enzyme, membrane binding protein or antibody, and the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined.
  • this will comprise determining whether the biological substance is bound to one or more of the members of the library.
  • binding may be determined by attaching a label to the biological substance.
  • Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels.
  • the degree of binding affinity may be determined by quantitating the amount or intensity of the bound label.
  • various biologically active compounds may be selected by identifying that compounds bind the particular biological substance most effectively.
  • Illustrative additional assays include but are not limited to in vitro assays such as enzymatic inhibition, receptor - ligand binding, protein - protein interaction, andprotein - DNA interaction; cell based, functional assays such as transcriptional regulation, signal transduction / second messenger, and viral infectivity; add, incubate & read assays such as scintillation proximity assays (SPA), fluorescence polarization assay, fluorescence correlation spectroscopy, colorimetric biosensors, cellular reporter assays using reporter genes such as luciferase, green fluorescent protein, ⁇ -lactamase, and the like; and electrical cell impedance sensor assays. All of the above assays are known in the art to be predictive of success for an associated disease state.
  • in vitro assays such as enzymatic inhibition, receptor - ligand binding, protein - protein interaction, andprotein - DNA interaction
  • cell based, functional assays such as transcriptional regulation, signal transduction / second
  • Table A lists representative moieties that are substituted for R on the designated position of the starting compound. Because the moieties are representative of a general class of organic substituents, it is meant that other organic substituents are chemically equivalent to those given in Table A and are within the scope of the invention and appended claims.
  • the CF 3 radical replaces R
  • the placement of the same group in position 4, 5 or 6 is an equivalent analogue.
  • the CF 3 placed at each of positions 3, 4, 5, 6 or any combination thereof is within the scope of this invention and appended claims.
  • the CF 3 group is, optionally, meant to represent an electron withdrawing group and can therefor be replaced by other electron withdrawing groups such as polyhalo-alkyl and be within the scope of this invention.
  • the solid support bound oxime is reacted with the fluorobenzonitrile of Example 1 under suitable conditions to form the solid support bound intermediate.
  • the solid support bound intermediate is optionally isolated and finally subjected to the cyclization and displacement procedure.
  • the nitrogen and oxygen of the solid support bound oxime are involved in the cyclization and displacement reaction and become part of the polycyclic heterocycle.
  • the solid support mediated method includes analogues involving substituents at each available position of the aromatic scaffold, such as a tri or tetra substituted phenyl.
  • analogues involving substituents at each available position of the aromatic scaffold, such as a tri or tetra substituted phenyl.
  • R is methoxy
  • a d to C 12 alkoxy is considered an equivalent analogue.
  • the substituent group is Br
  • another halo substituent such as -F or -Cl is an equivalent analogue.
  • the solid support mediated method according to the present invention further allows for the optional chemical derivatization of none, any, or all of R 1 , R 2 , R 3 , R 4 substituents of the solid support bound intermediate as provided above.
  • the solid support bound intermediate is preferably stable to a broad range of reaction conditions.
  • Table B lists substituted aromatic compounds (I), which are reacted with the solid support bound member, thereby forming the solid support bound intermediate (IV), are chemically derivatized with final cyclization and displacement of the corresponding polycyclic heterocycle.
  • the resin bound intermediates of the present invention were surprisingly stable to conditions suitable for optional derivatizations such as, BOC removal/acylation, TBS removal and Mitsunobu coupling, Suzuki coupling, Sonogashira coupling, Hoerner- Emmons olefination, and ester hydrolysis/amidation reaction conditions. These and other reactions discussed were performed according to the following detailed chemical procedure.
  • the loaded resin was removed from the oven and allowed to cool for 1 h and rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of 5% TFA/CH 2 C1 2> 2 x 5 mL of isopropanol and 4 x 5 mL of MeOH. This was dried in a 35° C vacuum oven for 12 h. Next, 4 mL of TFA and ImL of aqueous 5 N HCI were then added to the resin followed by turning for 2 h in a 55° C oven. The TFA/ ⁇ 2 O was collected and the resin was rinsed with 2 x 5 mL of CH 2 C1 2 .
  • the oven was at the most preferred temperature of 55° C.
  • the loaded resin was removed from the oven and allowed to cool for 1 h and rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of 5% TFA/CH 2 C1 2 , 2 x 5 mL of isopropanol 4 x 5 mL of MeOH. This was dried in a 35° C vacuum oven for 12 h. Next, 4 mL of TFA and ImL of aqueous 5 N HCI were then added to the resin followed by turning for 8 h in a 55° C oven. The TFA/H 2 O was collected and the resin was rinsed with 2 x 5 mL of CH 2 C1 2 .
  • This reaction vessel was placed in a 55 °C oven fitted with a rotating device for 12 h. This was removed from the oven and allowed to cool for 1 h and rinsed with 2 x 5 mL of CH 2 C1 2> 2 x 5 mL of isopropanol, and 4 x 5 mL of isopropanol, and 4 x 5 mL of MeOH. This was dried in a 35 °C vacuum oven for 12 h. BOC-deprotection was achieved using 25% TFA/CH 2 C1 2 (7 mL) followed by shaking for 2 h.
  • Resin was again rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of isopropanol, and 4 x 5 mL of MeOH.
  • the resin was suspended in DMF followed by addition of acetic anhydride and diisopropylethylamine. This was allowed to shake for 3h and was rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of isopropanol, and 4 x 5 mL of MeOH. 4 mL of TFA and 1 mL of aqueous 5 N HCI were then added to the resin followed by turning for 2 h in a 55° C oven.
  • the resin was rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL H 2 O, 4 x 5 mL of MeOH. This was dried in a 35° C vacuum oven for 12 h.
  • the resin was suspended in 8 mL of 1 : 1 THF/CH 2 C1 2 followed by addition of triphenylphosphine (700mg, 5 equiv.) and 4- chlorophenol (690 mg, 10 equiv.). This was shaken by hand followed by the slow addition of diisopropyl azodicarboxylate (0.53mL 5 equiv).
  • Method A 4: 1 TFA/5N HCI, 55 °C.
  • Method B 99: 1 TFA/H 2 0, 55 °C.
  • Method C 25% THF/CH 2 C1 2 , room temp.
  • Method D TFA, 55 °C.
  • Method E AcOH ⁇ THF/H 2 0, 55 °C.
  • Method F TsOH/THF/H 2 0, 55 °C. b Isolated yield after chromatography and based on loading. Crude purity based on HPLC analysis.
  • Acid 6 was then coupled to 4-chlorobenzylamine to give the on-resin amide. Both the resin weight increase and chlorine analysis of the intermediate suggested that the coupling reaction went essentially to completion within 12 h. The resin was then treated with the standard cyclorelease conditions to give the desired amide 7 in a 3 step yield of 81% (93% crude purity).
  • the resin was then rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 x 5 mL of H 2 O, and 4 x 5 mL of MeOH.
  • the resin was then su: pended in THF (7 mL) followed by the addition of LiOH (39 mg, 1.61 mmol) dissolved in MeOH/H 2 O (1:1, 2 mL) and rotated at room temp, for 12 h.
  • the resin was then rinsed with 2 5 ml, of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 5 mL of H 2 0, 2 x 5 mL of MeOH, and 2 x 5 mL of DMF.
  • the resin was then suspended in DMF (7 mL) and to this was added p- chlorobenzylamine (261 ⁇ L, 2.14 mmol), 1 -hydroxybenzotriazole hydrate (HOBt) (289 mg, 2.14 mmol), benzotriazole-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (947 mg, 2.14 mmol), and diisopropylethylamine (DIPEA) (467 ⁇ L, 2.68 mmol). The reaction was allowed to proceed for 12 h at room temp.
  • p- chlorobenzylamine (261 ⁇ L, 2.14 mmol
  • HOBt 1 -hydroxybenzotriazole hydrate
  • BOP benzotriazole-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate
  • DIPEA diisopropylethylamine
  • the resin was then rinsed 2 5 mL of CH 2 C1 2 , 2 5 mL of MeOH, 2 x 5 mL of H 2 O, and 4 x 5 mL of MeOH.
  • TFA (4 mL) and 5 N HCl aq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HCl aq was collected and the resin was rinsed with 2 5 mL of CH 2 C1 2 .
  • Phenolic Mitsunobu reaction On-resin carbon-oxygen bond formation via the Mitsunobu reaction has been identified as an important tool in combinatorial chemistry (Kaldor, S.W.; Siegel, M.G. Comb. Chem. Mol. Diversity Drug Discovery 1998, 307-335. .dito s: Gordon, Eric M.; Kerwin, James F., Jr. Publisher: Wiley-Liss, New York, N. Y).
  • Resin 8 was prepared by reacting the potassium anion of the Kaiser resin 1 with 2-fluoro-4-(t- butyl-dimethylsilyloxymethyl)-benzonitrile.
  • the TBS-protecting group was removed using TBAF in THF.
  • the on-resin alcohol was then treated with p-chlorophenol, triphenybhosphine and diisopropylazodicarboxylate (DIAD) in THF.
  • DIAD diisopropylazodicarboxylate
  • the best results were observed for reactions times of 3 h. Longer reaction times generally led to decreased purity in the crude cyclization product. Cyclitive removal using the standard conditions then gave aryl ether 9 in a 77%> yield (3 steps) and 83% crude purity.
  • the resin was then suspended in THF (6 mL) followed by the addition of TBAF (562 ⁇ L, 1 M in THF, 0.562 mmol) and rotated at room temperature for 12 h.
  • the resin was then rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 x 5 mL of H 2 O, 2 x 5 mL of MeOH, and 2 x 5 mL of CH 2 C1 2 .
  • the resin was then suspended in CH 2 C1 2 (7 mL) and to this was added -chlorophenol (690 mg, 5.35 mmol), triphenylphosphine (700 mg, 2.68 mmol), and diisopropyl-azodicarboxylate (DIAD) (530 ⁇ L, 2.68 mmol). The reaction was allowed to proceed for 1 h at room temp. The resin was then rinsed 2 5 mL of CH 2 C1 2 , 2 5 mL of MeOH, 2 x 5 mL of H 2 O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h.
  • Resin 10 was then reacted with a variety of nucleophiles including an alkoxide (Plenkiewicz, H.; Dmowski, W. J Fluorine Chem. 1998, 89, 213.), amine (Morales, G.A.; Corbett, J.W.; DeGrado, W.F. J. Org. Chem. 1998, 63, 1172. Vojkovsky, T.; Weichsel, A.; Patek, M. J. Org. Chem. 1998, 63, 3162. Dankwardt, S.M.; Newman, S.R.; Krstenanski, J.L. Tetrahedron Lett.
  • nucleophiles including an alkoxide (Plenkiewicz, H.; Dmowski, W. J Fluorine Chem. 1998, 89, 213.), amine (Morales, G.A.; Corbett, J.W.; DeGrado, W.F. J. Org. Chem
  • this reaction could lead either to the desired aryloxime intermediate 12 through displacement of the 2-fluoro group, or to an undesired isomer 13 through displacement of the 4-fluoro group.
  • Treatment of the unsymmetrical nitrile with the potassium salt of resin 1 gave a 85% loading yield based on weight. Although the ratio of 12 to 13 was not determined, the presumed mixture was then hydrolyzed under the standard cyclization conditions to yield a single product 14a (as determined by HPLC and NMR) in 74% isolated yield. The high selectivity observed for the formation of the desired product could arise from either of two pathways.
  • the resin loading reaction may be quite selective for the 2- position, giving primarily 12, and then subsequently 14a upon cyclorelease.
  • Scheme 6 illustrates a comparison of the relative rates of hydrolysis for analogous 2- vs 4-substituted aryloximes. Since the isolation of pure 12 and pure 13 was not practical, model resins 3a and 15 were prepared from 2-fluorobenzonitrile and 4- fluorobenzonitrile respectively. As described previously, when resin 3a was treated with the standard cyclitive removal conditions, complete conversion to 3-aminobenzisoxazole was observed after 2h.
  • the resin was then rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 * 5 mL of H 2 O, and 4 x 5 mL of MeOH.
  • the resin was then rinsed 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 x 5 mL of H 2 O, and 4 x 5 mL of MeOH.
  • the resin was dried in a 35 °C vacuum oven for 3 h.
  • TFA (4 mL) and 5 N HCl aq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven.
  • the TFA/HCl aq was collected and the resin was rinsed with 2 5 mL of CH 2 C1 .
  • the reaction vessel was rotated at 55 °C in a Robbins oven for 12 h followed by cooling for 1 h.
  • the resin was then rinsed with 2 x 5 mL of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 x 5 mL of H 2 O, and 4 x 5 mL of MeOH.
  • An IR analysis of the resin shows a nitrile stretching peak at 2230.3 cm "1 .
  • reaction parameters were carefully varied including cosolvent (DMF, DMSO, and THF) and the stoichiometry of the catalyst, boronic acid and sodium carbonate. Although the crude purities were similar for the various solvents which were evaluated, in general THF (Backes, B.J.; Ellman, J.A.; J. Am. Chem. Soc, 1994, 116, 11171) was found to give the highest rate of product formation, and therefore the highest isolated yields. Optimal results were obtained with 1.3 equivalents of 2N Na 2 CO 3 , at 55 °C, over 36h. The reaction was rather sensitive to deviation from these conditions, particularly with respect to the amount of added 2N Na 2 CO 3 .
  • the resin was then rinsed with 2 5 mL of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 x 5 mL of H 2 O, and 4 x 5 mL of MeOH.
  • the resin was then suspended in THF and to this was added phenylboronic acid (261 mg, 2.14 mmol), Na 2 CO 3 (348 ⁇ L, 2 M in H 2 O, 2.14 mmol), and Pd(PPh 3 ) 4 .
  • the vessel was then rotated for 36 h in a 55 °C oven followed by rinsing with 2 5 mL of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 x 5 mL of H 2 O, and 4 5 mL of MeOH.
  • the resin was dried in a 35 °C vacuum oven for 3 h.
  • TFA (4 mL) and 5 N HCl aq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven.
  • the TFA/HCl aq was collected and the resin was linsed with 2 x 5 mL of CH 2 C1 2 .
  • the resin was dried in a 35 °C vacuum oven for 3 h. TFA (4 mL) and 5 N HCl aq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HCl aq was collected and the resin was rinsed with 2 5 mL of CH 2 C1 2 . These washings were combined and concentrated in vacuo to give the crude product 17 (93% purity by reverse phase HPLC) which was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes.
  • the reaction vessel was rotated at 55 °C in a Robbins oven for 12 h to give resin 3b followed by cooling for 1 h.
  • the resin was then rinsed with 2 5 mL of CH 2 C1 2 , 2 x 5 mL of MeOH, 2 x 5 mL of H 2 O, and 4 x 5 mL of MeOH.
  • the resin was then suspended in THF and to this was added 4-formylphenylboronic acid (482 mg, 3.21 mmol), Na 2 CO 3 (348 ⁇ L, 2 M in H 2 O, 0.696 mmol), and Pd(PPh 3 ) 4 (31 mg, 0.027 mmol).
  • the vessel was then rotated for 36 h in a 55 °C oven followed by rinsing with 2 5 mL of CH 2 C1 2 , 2 x 5 mL of MeCN, 2 x 5 mL of H 2 O, and 4 x 5 mL of MeCN (care was taken to not rinse the resin with methanol in order to avoid methyl acetal formation).

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Abstract

This invention is directed to aminobenzisoxazole compounds, methods for producing such compounds and libraries thereof. This library is useful for screening the biological activity of the aminobenzisoxazole compounds.

Description

AMINOBENZISOXAZOLE COMPOUNDS AND LIBRARIES THEREOF
BACKGROUND OF THE INVENTION
This invention relates to novel aminobenzisoxazole compounds, libraries thereof, and methods of screening such a library for useful therapeutic compounds. Certain aminobenzisoxazole compounds of the present invention have demonstrated activity as thrombin inhibitors. Other therapeutic activities are expected, depending on the substituents attached to the aminobenzisoxazole system.
The traditional methods of drug discovery did eventually yield leads for new drugs. However, the time required for characterization of the compounds considerably limited the discovery of new pharmaceutically active compounds. In the present competitive environment of the pharmaceutical industry, the need has arisen for efficient, fast and less expensive methods for new drug discovery. Combinatorial chemistry offers a means of generating large numbers (102 - 106) of related compounds generally referred to as "libraries". See J. Hogan, Jr., Nature Biotechnology, 15, 328 (1997), "Combinatorial Chemistry and New Drugs," Scientific American, page 69, April 1997, M. Pirrung, Chem. Rev., 97, 473 (1997), "A Hail of Silver Bullets," Forbes, page 76, January 26, 1998. In simple terms, combinatorial chemistry is the parallel, simultaneous preparation of multiple derivatives of a central, fixed moiety often referred to as the "scaffold," "core" or "backbone". Theoretically, the number of compounds which may be produced to establish a library is limited only by the number of reagents available to form the derivatives.
Combinatorial chemistry may be used to generate libraries which are mixtures of individual compounds and complete identification of the individual compounds are postponed until after positive screening. However, "parallel array synthesis" whereby individual reaction products are simultaneously synthesized but each reaction is carried out in a separate vessel is generally preferred. For example, a compound library may be prepared, stored and assayed in standard microtiter plates such as the plates often used in the biological sciences. In combinatorial chemistry, the scaffold is typically attached to a solid support, e.g., a polymer, which is insoluble in the reaction solvent. When the entire reaction sequence has been completed, the library compounds are detached from the support. However, in some cases combinatorial libraries can be prepared in solution phase. It is also possible to tether the reactants to a support with the scaffold in solution. In all cases, the net effect is to produce a library of many derivatives of the scaffold each in its own small reaction vessel.
Conceptually, preparation of a combinatorial library by parallel synthesis appears to be logical and straightforward. However, in practice, efficient preparation of such libraries has demanded development of new techniques and new equipment. Often synthetic sequences which are routine in traditional organic chemistry are difficult and unpredictable, if not impossible, when applied to combinatorial chemistry. In addition to the new technique and equipment developed in recent years, the application of robotics enables an organic chemist to build libraries of several thousand compounds in relatively short periods.
The need for large combinatorial libraries has been made acute by the advent of high throughput screening (HTS) technology (see W. Janzen, Laboratory Robotics and Automation, 8, 261, (1996)). With this technology, thousands of compounds can be screened each day, thus creating a demand for large libraries to be introduced through the batteries of screens. Further, HTS uses only microgram quantities of compound for each screen, so a library can be used for several screens before it is depleted. Therefore, the marriage of combinatorial chemistry and HTS has created a powerful tool for discovering new drug leads (see "Combinatorial Chemistry", Chemical and Engineering News, page 43, February 24, 1997).
BRIEF SUMMARY OF THE INVENTION
The present invention provides combinatorial libraries of aminobenzisoxazole compounds of the formula (I):
R-A3-A2-A -NH-(CH2)C-R' wherein R1 is a substituted or unsubstituted aminobenzisoxazole group of the formula
(II):
which can be attached to one group of the formula R-A3-A2-A'-NH-(CH2)C- from any of positions 4, 5, 6, or 7, and where the remaining positions can each independently be substituted with hydrogen, (C[ to C4)alkyl, or a halogen;
A1 , A2 and A3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A1, A2 and A3 is not a bond;
R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is 0, 1, 2, 3, or 4.
Also, the present invention provides a library of 4-amidino-3-hydroxylphenyl compounds of the formula (lb):
R-A3-A -A] -NH-(CH2)C-Rlb wherein Rlb is a substituted or unsubstituted group of the formula (lib):
which can be attached to a group of the formula R-A3-A2-Ai-NH-(CH2)C- from any of positions 4, 5, 6, or 7, and where the remaining positions can each independently be substituted with hydrogen, (Ci to C )alkyl, or a halogen;
A^ A2 and A3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A1, A2 and A3 is not a bond; R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is O, 1, 2, 3 or 4.
The present invention also provides libraries of aminobenzisoxazole compounds of the formula (III):
X-CO-Y-CO-NH-(CH2)c- R1 wherein R1 is a substituted or unsubstituted aminobenzisoxazole group of the formula (II):
which is attached to a group of the formula X-CO-Y-CO-NH-(CH2)c- at position 6 and where the remaining positions can each independently be substituted with hydrogen, (Ci to C )alkyl, or a halogen;
X-CO- is D-prolinyl, D-homoprolinyl, R2-(CH2) -NH-CH2-C(O)-,
)-
* denotes a chiral center that is (D) or (DL);
# denotes a chiral center that is (L); R2 is -COOR!4, -SO2(Cι-C4 alkyl), -SO3H, -P(O)(ORl4)2 or tetrazol-5-yl; R3 is hydrogen or (Cι-C4)alkyl; R4 is carboxy or methylsulfonyl; R5 is NHR6, NHCOR6 or NHCOOR6;
R6 is (Cι-Cιo)alkyl, (C3-C8)cycloalkyl or a (C3-C8)cycloalkyl-(Cι-C6)alkyl group containing 4-10 carbons;
R7 is (C3-C8)cycloalkyl, (Cι-Cg)alkyl,
R8 is -OH, (Cι-C4)alkoxy, or -NH-R12; R9 is hydrogen or (C 1 -C4)alkyl;
R10 is hydrogen, (Cι-C4)alkyl, (Cι-C4)alkoxy, hydroxy, halo or (C1-C4) alkylsulfonylamino;
R11 is (Cι-C )alkyl, (Cι-C )fluoroalkyl bearing one to five fluoros, -(CH2)d-R2, or unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen; R12 is hydrogen, (Cι-C4)alkyl, RπSO2-, RnOC(O)-, RnC(O)-, Rl3C(O)- or -
(CH2)d-R2; R13 is -COOR14 or tetrazol-5-yl;
14 each R is independently hydrogen or (Cj-C4)alkyl;
-Y-CO- is
15
R is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b-R17;
R16 is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b- R17;
L is a bond, -O-, -S-, or -NH-;
R17 is (Cι-C4)alkyl, (C3-C8)cycloalkyl, -COOH, -CONH2, or Ar, where Ar is unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
18
R is -CH2-, -O-, -S-, or -NH-;
19 18
R is a bond or, when taken with R and the three adjoining carbon atoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom of which may be -O-, -S-, or -NH-; each a, independently, is 0, 1 or 2; each b, independently, is 0, 1, 2 or 3; each c, independently, is 0, 1, 2, 3 or 4; and each d, independently, is 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
In addition to compounds of formulae (I), (lb), and (III), the present invention provides a pharmaceutical composition comprising a compound of formula (I), (lb), or (III), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier, diluent or excipient. The aminobenzisoxazole compounds of formula (III) are useful whenever inhibition of thrombin is useful, for example, for the prophylaxis and treatment of thromboembolic diseases such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process.
The process for making libraries of compounds of the formulae (I), (lb), and (III) of the invention may be carried out in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means. The libraries of compounds (I), (lb) and (III) according to the present invention may be screened for biological activity. Generally, the library to be screened is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibody; and the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined. Typically this will comprise determining whether the biological substance is bound to one or more of the members of the library. Such binding may be determined by attaching a label to the biological substance. Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels. DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
The following definitions are used herein: "Alcohols" include groups of the formula -OH. "Aldehydes" include groups of the formula -C(=O)H.
"Alkyl" (or alkyl- or alk-) refers to a substituted or unsubstituted, straight, branched or cyclic hydrocarbon chain containing of from 1 to 20 carbon atoms. Preferred alkyl groups are lower alkyl groups, i.e., alkyl groups containing from 1 to 6 carbon atoms. Preferred cycloalkyls have from 3 to 10, preferably 3-6, carbon atoms in their ring structure. Suitable examples of unsubstituted alkyl groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso-butyl, tert-butyl, sec-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like.
"Alkylene" refers to an unbranched or branched carbon chain containing 1-20, preferably 1-10, carbon atoms. Branched carbon chains include both linear and cyclic structures.
"Alkenyl" refers to a substituted or unsubstituted, straight, branched or cyclic, unsaturated hydrocarbon chain that contains at least one double bond and 2 to 20, preferably 2 to 6, carbon atoms. Exemplary unsubstituted alkenyl groups include ethenyl (or vinyl)( -CH=CH2), 1-propenyl, 2-propenyl (or allyl)(-CH2-CH=CH2), 1,3- butadienyl (-CH=CHCH=CH2), 1 -butenyl (-CH=CHCH2CH3), hexenyl, pentenyl, 1, 3, 5-hexatrienyl, and the like. Preferred cycloalkenyl groups contain having five to eight carbon atoms and at least one double bond. Examples of cycloalkenyl groups include cyclohexadienyl, cyclohexenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cycloheptadienyl, cyclooctatrienyl and the like. "Alkoxy" refers to a substituted or unsubstituted, -O-alkyl group. Exemplary unsubstituted alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, t-butoxy, and the like.
"Alkynyl" refers to a substituted or unsubstituted, straight, branched or cyclic unsaturated hydrocarbon chain containing at least one triple bond and 2 to 20, preferably 2 to 6, carbon atoms. "Amine" refers to a unsubstituted or substituted amino (-NH2) group. The amine can be primary (-NH2), secondary (-NHR) or tertiary (-NR2), depending on the number of substituents (R). Examples of substituted amino groups include methylamino, dimethylamino, ethylamino, diethylamino, 2-propylamino, 1- propylamino, di(n-propyl)amino, di(iso-propyl)amino, methyl-n-propylamino, t- butylamino, and the like.
"Amino acid" refers to a compound that contains an amino group and a carboxylic acid group. Typically, the phrase refers to α-amino acids, although other amino acids (such as β- and (-amino acids) can be used. The amino acids can either be "D," "L," or a racemic mixture. Naturally encoded amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn. N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), ornithine (Orn, O), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Tφ, W), tyrosine (Tyr, Y), and valine (Val, V). Unnatural amino acids include synthetic amino acid residues. Synthetic amino acids can include α-amino acids containing aryl or hetaryl groups attached as side chains. Examples of synthetic amino acids include norvaline, sarcosin, n-leucine, 1-naphthylalanine, 2- indolinecarboxylic acid and the like. Synthetic amino acids also include α,α- disubstituted amino acids such aminoisobutyric acid (AIB) and those described by Scott et al, Tetrahedron Lett. 1997, 38(21^:3695; incoφorated herein by reference.
"Amino protected amino acid" refers to an amino acid which includes an amino-protecting group bonded to the nitrogen atom to prevent reaction.
"Amino acid side chain" refers to the group attached to the α-carbon of an α- amino acid. Suitable amino acid side chains include the α-side chain of the naturally encoded amino acids. Alternatively, any side chain of a synthetic amino acid can be used, including lower alkyl, alkyloxy, alkylamino, alkylthio, and the like.
"Aprotic solvent" refers to polar solvents of moderately high dielectric constant which do not contain an acidic hydrogen. Examples of common aprotic solvents are dimethyl sulfoxide (DMSO), dimethylformamide (DMF), sulfolane, tetrahydrofuran (THF), diethyl ether (Et2O), methyl-t-butyl ether, or 1,2-dimethoxyethane.
"Aryl" refers to any monovalent aromatic carbocyclic group of 6 to 10 carbon atoms. Preferred aryl groups include phenyl and naphthyl. "Assay kit" as used in accordance with the present invention refers to an assemblage of at least two cooperative elements, namely (1) a well plate apparatus and (2) biological assay materials.
"Biological assay materials" are materials necessary to conduct a biological evaluation of the efficacy of any library compound in a screen relevant to a selected disease state.
"Diverse library" means a library where the substituents on the combinatorial library scaffold are highly variable in constituent atoms, molecular weight, and structure and the library, considered in its entirety, is not a collection of closely related homologues or analogues (compare to "directed library"). "Esters" include groups of the formula -C(=O)OR, where R is a substituent, preferably an alkyl group. Exemplary ester groups include methyl ester, ethyl ester, and the like.
"Halogen" (or halo-) refers to fluorine, chlorine, iodine or bromine. The preferred halogen is fluorine or chlorine. "Heterocyclic" (Het or heterocyclic) refers to a stable, saturated, partially unsaturated, or aromatic group containing 5 to 10, preferably 5 or 6, ring atoms. The ring can be substituted 1 or more times with a substituent. The ring can be mono-, bi- or polycyclic. The heterocyclic group consists of carbon atoms and from 1 to 3 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Examples of heterocyclic groups include acridine, benzathiazoline, benzimidazole, benzofuran, benzothiapene, benzthiazole, benzothiophenyl, carbazole, cinnoline, furan, imidazole, lH-indazole, indole, isoindole, isoquinoline, isothiazole, moφholine, oxazole (i.e. 1,2,3-oxadiazole), phenazine, phenothiazine, phenoxazine, phthalazine, piperazine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinazoline, quinoline, quinoxaline, thiazole, 1,3,4-thiadiazole, thiophene, 1,3,5-triazines, triazole (i.e. 1,2,3-triazole), and the like. It will be appreciated that many of the above heterocycles may exist in tautomeric forms. All such forms are included within the scope of this invention.
"Imine" includes groups of the formula -C=N-H or -C=N-R, where R is a substituent, preferably alkyl.
"Inert atmosphere" refers to reaction conditions in which the mixture is covered with a layer of inert gas such as nitrogen or argon.
"Library" or "Combinatorial library" means a large number of chemical derivatives used in screening for biological activity or other activity. In general, a library will have greater than 20 members, preferably the library will have at least 50 members, more preferably the library will have at least 96 members and most preferably the library will have at least 1000 members.
"Library compound" is an individual reaction product (usually a single compound) in a combinatorial library. "Lead compound" means a compound in a selected combinatorial library for which the assay kit has revealed significant activity relevant to a selected disease state.
"Nitriles" (or cyano) include groups of the formula -C≡N.
"Organic solvent" includes solvents containing carbon, such as halogenated hydrocarbons, ether, toluene, xylene, benzene, and tetrahydrofuran. "Peptide" refers to a chain of amino acids connect from carboxy terminus to amino terminus via peptide bonds. Typically, the chain can be a mixture of various amino acids. Chains of α-amino acids are preferred, although other amino acids (such as β- and (-amino acids) can be used. The amino acids within the chain can either be "D," "L," or a racemic mixture. The chain can be of any length, including 2 amino acids (dipeptide), 3 amino acids (tripeptide), or more. The terms "oligopeptide" and "polypeptide" should be understood to be included within this term. The average length can vary between 2 to 100 amino acids, preferably 2-20, more preferably 2-10, most preferably 2-5.
"Pharmaceutically acceptable salt" and "salts thereof means organic or inorganic salts of the pharmaceutically important molecule. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically important organic molecule may have more than one charged atom in its structure. Situations where multiple charged atoms are part of the molecule may have multiple counterions. Hence, the molecule of a pharmaceutically acceptable salt may contain one or more than one charged atoms and may also contain, one or more than one counterion. The desired charge distribution is determined according to methods of drug administration. Examples of pharmaceutically acceptable salts are well known in the art but, without limiting the scope of the present invention, exemplary presentations can be found in the Physician's Desk Reference, The Merck Index, The Pharmacopoeia and Goodman & Gilman's The Pharmacological Basis of Therapeutics.
"Protecting group" means a group used to protect a heteroatom such as oxygen, nitrogen, sulfur or phosphorus from chemical reaction. For example, a O-protecting group is used to protect an oxygen heteroatom, such as in a hydroxy group, from reaction. Examples of O-protecting groups include Boc, t-butyl ether, benzyl ethers, and the like. Examples of amino-protecting (N-protecting) groups include acetyl (Ac), 1-adamantanesulphonyl (AdSO2), 1-adamantaneacetyl (AdAc), benzoyl (Bz), t- butoxycarbonyl (Boc), carbobenzoxy (Cbz), 2-carboxybenzoyl (2-Cbz), dansyl (DNS), isovaleryl (Iva), Fmoc, methoxysuccinyl (MeOSuc), nitropiperonyl, pyrenylethoxycarbonyl, nitroveratryl (NV), nitrobenzyl, succinyl (Sue), tosyl (Ts), and such amino protecting groups which are functionally equivalent thereto. Protecting groups are well known in the art, see for example Protective Groups in Organic Synthesis, Peter G. M. Wuts (Editor), Theodora W. Greene, 3rd ed. (April 1999), Vch Pub.; Protective Groups in Organic Synthesis, Theodora W. Greene, Peter G. Wuts (Contributor), 2nd ed., (May 1991) John Wiley & Sons. Preferred protecting groups include, but are not limited to, the "Boc" protecting group, trialkyl silyl groups such as TBS (tert-butyldimethylsilyl, Si(CH3)2C(CH3)3), MEM (2-methoxyethoxymethyl), MOM (methoxymethyl), SEM (2-(trimethylsilyl)ethoxymethyl), and THP (tetrahydropyrany 1) .
"Protic solvent" refers to a solvent containing hydrogen that is attached to oxygen, and hence is appreciably acidic. Common protic solvents include such solvents as water, methanol (MeOH), ethanol (EtOH), 2-propanol (PrOH), and 1-butanol (n- BuOH).
"Protic acid" refers to an acid having an acidic hydrogen. Preferred protic acids include acetic acid, hydrochloric acid (HC1), formic acid, perchloric acid, sulfuric acid (H2SO4), and phosphoric acid (H2PO4) in an aqueous medium. The most preferred protic acids are hydrochloric acid, sulfuric acid, and formic acid.
"Scaffold" as used in accordance with the present invention refers to the invariable region of the compounds that are members of the combinatorial library.
"Solid support" broadly refers to any structure which is capable of supporting the chemical compound and is substantially inert to the chemical reactions conducted on the surface. Exemplary solid supports include, but are not limited to, metals, resins, polymers, gels, glass beads, silica gels, ceramic supports and other solid and semi-solid compositions.
"Substantially pure" is intended to mean at least about 90 mole percent, more preferably at least about 95 mole percent, and most preferably at least about 98 mole percent of the desired enantiomer or stereoisomer is present compared to other possible configurations.
"Substituted" means that the moiety contains at least one, preferably 1-3 substituent(s). α- Amino acids can be mono-substituted at the backbone nitrogen atom, substituted at the α-carbon (to form a α,α-disubstituted α-carbon) or substituted 1-3 times at one or more side chain atoms. Suitable substituents include hydrogen (H) and hydroxyl (-OH), amino (-NR2), oxy (-O-), carbonyl
(-CO-), thiol, alkyl, alkenyl, alkynyl, alkoxy, halo, nitrile, nitro, aryl and heterocyclic groups. These substituents can optionally be further substituted with 1-3 substituents. Examples of substituted substituents include carboxamide, alkylmercapto, alkylsulphonyl, alkylamino, dialkylamino, carboxylate, alkoxycarbonyl, alkylaryl, aralkyl, alkylheterocyclic, (C l -C4)fluoroalkyl groups (such as trifluoromethyl or 2,2,2- trifluoroethyl) and the like.
"Thiols" include compounds of the formula -SH or -SR where R is a substituent, preferably alkyl. Exemplary thiols include methanethiol, ethanethiol, propanethiol, and the like.
All other acronyms and abbreviations have the corresponding meaning as published in journals relative to the art of chemistry.
2. Library of Aminobenzisoxazole compounds (I) The present invention provides combinatorial libraries of aminobenzisoxazole compounds of the formula (I):
R-A3-A2-A'-NH-(CH2)C-R1 wherein R1 is a substituted or unsubstituted aminobenzisoxazole group of the formula
(II):
which can be attached to one group of the formula R-A3-A2-A1-NH-(CH2)C- from any of positions 4, 5, 6, or 7, and where the remaining positions can each independently be substituted with hydrogen, (Cj to C4)alkyl, or a halogen;
A1 , A2 and A3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A1, A2 and A3 is not a bond;
R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is O, 1, 2, 3, or 4. Compounds of formula (I) may be made by using methods including those described in Appendix A. Briefly, the compounds may be synthesized (for example, as shown for a compound of formula (III) in which the residue is attached to the 6- position of the group of the formula (II) and in which X-CO- is R7-(CH2)a-CH(NHR12)- CO-) by:
(a) reacting a compound of the formula (IV):
where each of R1, R11, and Rm is hydrogen or a substituent; Hal is a halogen; NP is a protected amino group; and c is 0, 1, 2, 3 or 4; with a compound of the formula (V):
where ** is a solid support; and where RIV is alkyl, arylalkyl or aryl; to form a corresponding intermediate of the formula (VI)
where R1 to RIV and NP are as defined above;
(b) deprotecting and coupling the resulting solid support-bound starting material (VI) with an activated amino-protected amino acid of the formula (VII):
where P' is a protecting group; and each a, independently, is 0, 1 or 2; to form a N- protected first intermediate;
(c) deprotecting the N-protected first intermediate;
(d) coupling the first intermediate with an activated compound of the formula (VIII):
to form a N-protected second intermediate;
(e) deprotecting the N-protected second intermediate;
(f) optionally repeating steps (d) and (e);
(g) optionally derivatizing the free amino terminus of the second intermediate; and (h) cyclizing the second intermediate, whereby a compound of the formula (I) is displaced from the solid support.
The selection of appropriate protecting groups and reagents to selectively remove protecting groups, activated groups, as well as suitable coupling reagents is well known in the art. For example, see M. Bodanszky, Peptide Chemistry, A Practical Textbook, Springer- Verlag (1988); J. Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co. (1984).
The temperature for the cyclization and displacement procedure may be between about 0° and 85° C but is preferably between about 30° and 70° and is most preferably at about 55 ° C .
The solvents suitable for the cyclization and displacement procedure include protic and aprotic solvent mixtures, aqueous and anhydrous solvent mixtures. A preferred solvent is TFA, a more preferred solvent mixture is TFA:H2O a most preferred solvent mixture is TFA: 5 N HCl/H2O. Although a solvent mixture of TFA: 5 N HCl/H2O is most preferred, the ratio of this TFA:5 N HCl/Η2O mixture may vary between about 1 :1 to about 99:1 TFA: 5 N HCl/H2O, but preferably is between about 80:1 to 1:1 TFA:5 N HCl H2O, and is most preferably at about 4:1 TFA:5 N HCl/H2O.
The time for the cyclization and displacement reaction may vary, but generally is between about 1 minute and 4 days but preferably between about 1 hour and 20 hours.
Those aminobenzisoxazole compounds of the invention having a basic group may be isolated in the form of an acid addition salt. A salt of the compound of formula (I) formed with an acid such as one of those mentioned above is useful as a pharmaceutically acceptable salt for administration of the antithrombotic agent and for preparation of a formulation of the agent. Other acid addition salts may be prepared and used in the isolation and purification of the compound.
As noted above, the optically active isomers and diastereomers of the compounds of formula (I) are also considered part of this invention. Such optically active isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. This resolution can be carried out by derivatization with a chiral reagent followed by chromatography or by repeated crystallization. Removal of the chiral auxiliary by standard methods affords substantially optically pure isomers of the compounds of the present invention or their precursors. Further details regarding resolutions can be obtained in Jacques, et al., Enantiomers, Racemates, and Resolutions, John Wiley & Sons, 1981.
3. Library of 4-amidino-3-hydroxylphenyI compounds (lb)
Also, the present invention provides a library of 4-amidino-3-hydroxylphenyl compounds of the formula (lb): R-A3-A2-A'-NH-(CH2)C-Rlb wherein Rlb is a substituted or unsubstituted group of the formula (lib):
which can be attached to a group of the formula R-A3-A2-A1-NH-(CH2)C- from any of positions 4, 5, 6, or 7, and where the remaining positions can each independently be substituted with hydrogen, (Cj to C4)alkyl, or a halogen; A2 and A3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A1, A2 and A3 is not a bond;
R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is O, 1, 2, 3 or 4.
4-Amidino-3-hydroxylphenyl compounds of the formula (lb) can be formed by ring opening catalytic hydrogenation of aminobenzisoxazole compounds of the formula (I) as exemplified in the following examples.
Library of Aminobenzisoxazole compounds (III) The present invention also provides libraries of aminobenzisoxazole compounds of the formula (III):
X-CO-Y-CO-NH-(CH2)c- R1 wherein R1 is a substituted or unsubstituted aminobenzisoxazole group of the formula
(II):
which is attached to a group of the formula X-CO-Y-CO-NH-(CH2)c- at position 6, and where the remaining positions can each independently be substituted with hydrogen, (Cj to C4)alkyl, or a halogen;
X-CO- is D-prolinyl, D-homoprolinyl, R2-(CH2) -NH-CH2-C(O)-,
* denotes a chiral center that is (D) or (DL);
# denotes a chiral center that is (L);
R2 is -COOR14, -SO2(Cι-C4 alkyl), -SO3H, -P(O)(OR14)2 or tetrazol-5-yl; R3 is hydrogen or (Cι-C4)alkyl;
R4 is carboxy or methylsulfonyl;
R5 is NHR6, NHCOR6 or NHCOOR6; R is (Cι-Cιo)alkyl, (C3-C8)cycloalkyl or a (C3-C8)cycloalkyl-(Cι-C6)alkyl group containing 4-10 carbons;
R7 is (C3-C8)cycloalkyl, (Cι-C8)alkyl,
R8 is -OH, (C i -C4)alkoxy, or -NH-R12;
R9 is hydrogen or (Cι-C4)alkyl;
R10 is hydrogen, (Cι-C4)alkyl, (Cι-G4)alkoxy, hydroxy, halo or (C1-C4) alkylsulfonylamino;
R11 is (Cι-C4)alkyl, (Cι-C4)fluoroalkyl bearing one to five fluoros, -(CH2)d-R2, or unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
R12 is hydrogen, (Cι-C4)alkyl, RnSO2-, RnOC(O)-, RnC(O)-, Ri C(O)- or - (CH2)d-R2;
R13 is -COOR14 or tetrazol-5-yl;
14 each R is independently hydrogen or (C 1 -C4)alkyl; -Y-CO- is
15
R" is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b-R17;
R16 is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b- R1?;
L is a bond, -O-, -S-, or -NH-;
R17 is (Cι-C4)alkyl, (C3-C8)cycloalkyl, -COOH, -CONH2, or Ar, where Ar is unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
18
R is -CH2-, -O-, -S-, or -NH-;
R is a bond or, when taken with R and the three adjoining carbon atoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom of which may be -O-, -S-, or -NH-; each a, independently, is 0, 1 or 2; each b, independently, is 0, 1, 2 or 3; each c, independently, is 0, 1, 2, 3 or 4; and each d, independently, is 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
This library is particularly useful for screening for compounds with antithrombin activity.
In formula (III), the carbonyl functionality of group X-(CO)- is attached to the amine functionality of the -Y- group.
The , where R10 and R12 are both hydrogen, is referred to at times herein as phenylglycyl and abbreviated Phg. Compounds wherein
R12 is, e.g., methyl, are referred to as the Nα-methyl-phenylglycyl group and abbreviated MePhg. Substituted compounds wherein R10 is other than hydrogen are referred to by the type and position of the substituent group, e.g., 3'-chlorophenylglycyl or Phg(3-Cl).
The group where R10 and R12 are both hydrogen, is referred to at times herein as phenylalanyl and abbreviated Phe. Compounds wherein
R12 is, e.g., methyl, are referred to as the Nα-methyl-phenylalanyl group and abbreviated MePhe. Substituted compounds wherein R10 is other than hydrogen are referred to by the type and position of the substituent group, e.g., 3'-chlorophenylalanyl or Phe(3-Cl).
is hydrogen, are referred to at times herein as 1- and 3-tetrahydro-isoquinolinecarbonyl, respectively, and are respectively abbreviated 1-Tiq and 3-Tiq. The groups and ? when R3 is hydrogen, are referred to at times herein as 1- and 3-perhydro-isoquinolinecarbonyl, respectively, and are respectively abbreviated 1-Piq and 3-Piq. As indicated by the crooked lines, various ring fusion isomers of these substituents exist ~ this invention contemplates any individual isomer and combinations thereof.
The group , wherein a is 0, 1 , or 2 is referred to as azetidine-2-carbonyl, prolinyl, or homoprolinyl, and is abbreviated Azt, Pro or hPro, respectively.
The group represents a saturated bicyclic system of the 4,5; 5,5; 6,5; 7,5; or 8,5 type. The stereochemistry at 3a is cis to the carbonyl; the other bridgehead bond may be either cis or trans except that the 4,5 and
18 19
5,5 systems must be cis at the bridgehead. The definitions of R and R provide that the variable ring, which includes the three carbon atoms shown, is a saturated carbocyclic system of 4-8 atoms. All of the ring atoms may be carbon, or one of the ring atoms may be a hetero atom selected from -O-, -S-, and -NH-. This definition includes the moiety derived from octahydroindole-2-carboxylic acid, as represented by The various cis and trans forms of this moiety are contemplated by this invention. The preferred isomer derived from [2S- (2α,3aβ,7aβ)]-octahydroindole-2-carboxylic acid is abbreviated "Ohi" and is represented by
The asterisks in Y denote a chiral center that corresponds to (L) in the natural amino acids. The asterisk in X denotes a chiral center that is (D) or (DL); the # in X denotes a chiral center that is (L). Preferred compounds of the formula (III) (1-24) are shown in the examples.
Compounds of formula (III) can be made as described above for compounds (I).
U.S. 5,863,929 describes, in-part, certain 4-amidino-3-hydroxylphenyl compounds which are among the compounds of the formula (Illb):
X-CO-Y-CO-NH-(CH2)c- Rlb wherein Rlb is a substituted or unsubstituted 4-amidino-3-hydroxylphenyl group of the formula (lib):
which is attached to a group of the formula X-CO-Y-CO-NH-(CH2)c- at position 6, and where the remaining positions can each independently be substituted with hydrogen, (Ci to C4)alkyl, or a halogen;
X-CO- is D-prolinyl, D-homoprolinyl, R2-(CH2) -NH-CH2-C(O)-,
* denotes a chiral center that is (D) or (DL);
# denotes a chiral center that is (L);
R2 is -COOR14, -SO2(Cι-C4 alkyl), -SO3H, -P(O)(OR1 )2 or tetrazol-5-yl; R3 is hydrogen or (Cι-C4)alkyl;
R4 is carboxy or methylsulfonyl; R5 is NHR6, NHCOR6 or NHCOOR6;
R6 is (Cι-Cιo)alkyl, (C3-C8)cycloalkyl or a (C3-C8)cycloalkyl-(Cι-C6)alkyl group containing 4-10 carbons; R7 is (C3-C8)cycloalkyl, (C j -C8)alkyl,
R8 is -OH, (Cι-C4)alkoxy, or -NH-R12;
R9 is hydrogen or (Cι-C4)alkyl;
R10 is hydrogen, (Cι-C4)alkyl, (Cι-C4)alkoxy, hydroxy, halo or (C1-C4) alkylsulfonylamino;
R11 is (Cι-C4)alkyl, (Cι-C4)fluoroalkyl bearing one to five fluoros, -(CH2)d-R2, or unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
R12 is hydrogen, (Cι-C4)alkyl, RuSO2-, RnOC(O)-, RuC(O)-, R13C(O)- or - (CH2)d-R2;
R13 is -COOR14 or tetrazol-5-yl;
14 each R is independently hydrogen or (C 1 -C4)alkyl; -Y-CO- is
15
R is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b-R17;
R16 is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b- R17;
L is a bond, -O-, -S-, or -NH-;
R17 is (Cι-C4)alkyl, (C3-C8)cycloalkyl, -COOH, -CONH2, or Ar, where Ar is unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
18
R is -CH2-, -O-, -S-, or -NH-;
19 18
R is a bond or, when taken with R and the three adjoining carbon atoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom of which may be -O-, -S-, or -NH-; each a, independently, is 0, 1 or 2; each b, independently, is 0, 1, 2 or 3; each c, independently, is 0, 1, 2, 3 or 4; and each d, independently, is 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
4-Amidino-3-hydroxylphenyl compounds of the formula (Illb) can be formed by ring opening catalytic hydrogenation of aminobenzisoxazole compounds of the formula (III). U.S. 5,863,929 discloses that certain compounds of the formula (Illb) inhibit thrombin.
It will be appreciated that certain compounds of formula (I), (lb), (III) or (Illb) may exist in, and be isolated in, isomeric forms, including tautomeric forms or cis- or trans-isomers, as well as optically active racemic or diastereomeric forms. The present invention encompasses compounds of formula (I), (lb), (III) or (Illb) in any of the tautomeric forms or as a mixture thereof. It is to be understood that the present invention encompasses compounds of formula (I), (lb), (III) or (Illb) as a mixture of diastereomers, as well as in the form of an individual diastereomer, and that the present invention encompasses compounds of formula (I), (lb), (III) or (Illb) as a mixture of enantiomers, as well as in the form of an individual enantiomer, any of which mixtures or form possesses inhibitory properties against thrombin, it being well known in the art how to prepare or isolate particular forms and how to determine inhibitory properties against thrombin by standard tests including those described below. In addition, compounds of formula (I), (lb), (III) or (Illb) may exhibit polymoφhism or may form a solvate with water or an organic solvent. The present invention also encompasses any such polymoφhic form, any solvate or any mixture thereof.
5. Pharmaceutical Compositions
In addition to compounds of formulae (I), (lb), and (III), the present invention provides a pharmaceutical composition comprising a compound of formula (I), (lb), (III), or a novel compound of formula (Illb), or a pharmaceutically acceptable salt or solvate thereof, in association with a pharmaceutically acceptable carrier, diluent or excipient. This invention also provides pharmaceutical compositions for use in therapeutic methods. Pharmaceutical compositions of the invention comprise an effective amount of a compound of formula (I), (lb), (III) or a novel compound of formula (Illb) in association with a pharmaceutically acceptable carrier, excipient or diluent. For oral administration the compound is formulated in gelatin capsules or tablets which may contain excipients such as binders, lubricants, disintegration agents and the like. For parenteral administration the compound is formulated in a pharmaceutically acceptable diluent e.g. physiological saline (0.9 percent), 5 percent dextrose, Ringer's solution and the like. The compound can be formulated in unit dosage formulations comprising a dose between about 0.1 mg and about 1000 mg. Preferably the compound is in the form of a pharmaceutically acceptable salt such as for example the sulfate salt, acetate salt or a phosphate salt. An example of a unit dosage formulation comprises 5 mg of a compound of the present invention as a pharmaceutically acceptable salt in a 10 ml sterile glass ampoule. Another example of a unit dosage formulation comprises about 10 mg of a compound of the present invention as a pharmaceutically acceptable salt in 20 ml of isotonic saline contained in a sterile ampoule.
The compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. The compounds of the present invention are preferably formulated prior to administration.
The active ingredient in such formulations comprises from 0.1 percent to 99.9 percent by weight of the formulation. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The present pharmaceutical compositions are prepared by known procedures using well known and readily available ingredients. The compositions of this invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art. In making the compositions of the present invention, the active ingredient will usually be admixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
6. Methods for inhibiting thrombin An aminobenzisoxazole compound of formula (III) or a novel compound of formula (Illb) is useful whenever inhibition of thrombin is useful, for example, for the prophylaxis and treatment of thromboembolic diseases such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process.
The present invention further provides a method of inhibiting thrombin comprising administering to a mammal in need of treatment, a thrombin inhibiting dose of a compound of formula (III) or a novel compound of formula (Illb). The present invention also provides a method of inhibiting thrombosis in a mammal comprising administering to a mammal in need of treatment, an antithrombotic dose of a compound of formula (III) or a novel compound of formula (Illb).
The compounds of the invention are believed to selectively inhibit thrombin over other proteinases and nonenzyme proteins involved in blood coagulation without appreciable interference with the body's natural clot lysing ability (the compounds have a low inhibitory effect on fibrinolysis). Also, they generally exhibit increased selectivity for thrombin compound to the prior amidinophenyl compounds. Further, such selectivity is believed to permit use with thrombolytic agents without substantial interference with thrombolysis and fibrinolysis. The invention in one of its aspects provides a method of inhibiting thrombin in mammals comprising administering to a mammal in need of treatment an effective (thrombin inhibiting) dose of a compound of formula (III) or a novel compound of formula (Illb). In another of its aspects, the invention provides a method of treating a thromboembolic disorder comprising administering to a mammal in need of treatment an effective (thromboembolic disorder therapeutic and/or prophylactic amount) dose of a compound of formula (III) or a novel compound of formula (Illb).
The invention in another of its aspects provides a method of inhibiting coagulation in a mammal comprising administering to a mammal in need of treatment an effective (coagulation inhibiting) dose of a compound of formula (III) or a novel compound of formula (Illb).
The thrombin inhibition, coagulation inhibition and thromboembolic disorder treatment contemplated by the present method includes both medical therapeutic and/or prophylactic treatment as appropriate.
In a further embodiment the invention relates to treatment, in a human or other mammal, of conditions where inhibition of thrombin is required. The compounds of the invention are expected to be useful in mammals, including man, in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility are in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility, in treatment and/or prophylaxis, include venous thrombosis and pulmonary embolism, arterial thrombosis, such as in myocardial ischemia, myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis. Further, the compounds have expected utility in the treatment or prophylaxis of atherosclerotic disorders (diseases) such as coronary arterial disease, cerebral arterial disease and peripheral arterial disease. Further, the compounds are expected to be useful together with thrombolytics in myocardial infarction. Further, the compounds have expected utility in prophylaxis for reocclusion after thrombolysis, percutaneous transluminal angioplasty (PTCA) and coronary bypass operations. Further, the compounds have expected utility in prevention of rethrombosis after microsurgery. Further, the compounds are expected to be useful in anticoagulant treatment in connection with artificial organs and cardiac valves. Further, the compounds have expected utility in anticoagulant treatment in hemodialysis and disseminated intravascular coagulation. A further expected utility is in rinsing of catheters and mechanical devices used in patients in vivo, and as an anticoagulant for preservation of blood, plasma and other blood products in vitro. Still further, the compounds have expected utility in other diseases where blood coagulation could be a fundamental contributing process or a source of secondary pathology, such as cancer, including metastasis, inflammatory diseases, including arthritis, and diabetes. The anticoagulant compound is administered orally, parenterally e.g. by intravenous infusion (iv), intramuscular injection (im) or subcutaneously (sc).
The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the rate of administration, the route of administration, and the condition being treated.
A typical daily dose for each of the above utilities is between about 0.01 mg/kg and about 1000 mg/kg. The dose regimen may vary e.g. for prophylactic use a single daily dose may be administered or multiple doses such as 3 or 5 times daily may be appropriate. In critical care situations a compound of the invention is administered by iv infusion at a rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferably between about 0.1 mg/kg/h and about 5 mg/kg/h.
The method of this invention also is practiced in conjunction with a clot lysing agent e.g. tissue plasminogen activator (t-PA), modified t-PA, streptokinase or urokinase. In cases when clot formation has occurred and an artery or vein is blocked, either partially or totally, a clot lysing agent is usually employed. A compound of the invention can be administered prior to or along with the lysing agent or subsequent to its use, and preferably further is administered along with aspirin to prevent the reoccurrence of clot formation. The method of this invention is also practiced in conjunction with a platelet glycoprotein receptor (Ilb/IIIa) antagonist, that inhibits platelet aggregation. A compound of the invention can be administered prior to or along with the Ilb/IIIa antagonist or subsequent to its use to prevent the occurrence or reoccurrence of clot formation.
The method of this invention is also practiced in conjunction with aspirin. A compound of the invention can be administered prior to or along with aspirin or subsequent to its use to prevent the occurrence or reoccurrence of clot formation. As stated above, preferably a compound of the present invention is administered in conjunction with a clot lysing agent and aspirin.
7. Combinatorial Synthesis and Process for preparing libraries of compounds of the formulae (I), (lb), (III), or (Illb)
The process for making libraries of compounds of the formulae (I), (lb), (III), and (Illb) of the invention may be carried out in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means.
For small scale synthesis of multiple products, the process of the invention is preferably carried out in containers adapted for parallel array synthesis. With parallel array synthesis, individual reaction products are prepared in each of multiple reaction zones. The reaction zones are physically separated from one another in a reaction vessel.
A preferred parallel synthesis embodiment of the present invention is a diverse compound library in the form of a plurality of wellplates, each wellplate having wells containing a separate reaction product (library compound). In such cases, the library compounds are conveniently identified by their wellplate number and "x" column and "y" row coordinates. The process of making the library of phenyl ether compounds may be conveniently carried out in a conventional wellplate apparatus. It is particularly advantageous to carry out the method of the invention in a standard wellplate apparatus such as a plastic 96 well microtiter plate, or FLEXCHEM ™ 96 well Synthesis Assembly available from Robbins Scientific. Typically, the wellplate apparatus is in the form of a rigid or semi-rigid plate.
Preferably, the plate has a common surface containing openings of a plurality of reservoirs arranged in rows and columns. A standard form of wellplate apparatus is a rectangular plastic plate having 8 rows and 12 columns (total 96) of liquid retaining depressions, or reservoirs, on its surface. A wellplate apparatus may optionally have other elements or structure such as a top or cover (e.g., plastic or foil), a bottom in a form such as a plate or reservoir, clamping means to secure the wellplate and prevent loss of its contained compounds.
The amount of solid bound compound introduced into each reaction zone will depend on the desired amount of each library compound that is needed for conducting biological assays, archival storage and other related needs. Typically, the desired amount of individual reaction product is from 1 microgram to 50 milligrams.
The amount of solid bound compound in each reaction zone is represented by the symbol "(n)", where (n) represents the equivalents of compound.
Typically, from about 8 to about 800 diverse coupling agents are employed serially to synthesize a library of compound. Combinatorial techniques are preferably very robust to work well for highly diverse groups of reactants. In the diverse compound library making process described herein the reactant is used in excess. The method of the invention contemplates solution phase reactions where a stoichiometric excess of the coupling reactant is used. The amount of coupling reactant used to ensure an excess is defined as at least 1.1 (n) and preferably a larger excess in the range of from 1.25(n) to 5(n), where the variable (n) is as previously defined. The 1.1 multiplier is used to ensure at least a 10% stoichiometric excess of coupling agent is present to drive the reaction to completion.
The reaction zone is maintained at a temperature and for a time sufficient to permit reaction of solid bound compound with the coupling reactant, that is, to complete consumption of the solid bound compound and form an amount of compound necessary to conduct biological assays to determine the efficacy of the prepared library compounds.
The time, temperature, and pressure of the combinatorial reaction zones used for the creation of library compounds are not critical aspects of the invention. Reaction times for a single step of the reaction are generally from about 0.1 seconds to about 24 hours, with reaction times of 1 second to 60 minutes being most often used. The temperature of the reaction may be any temperature between the freezing point and the boiling point of the liquid reaction medium, but is generally between about -10°C and about 60°C, with 10°C to 40°C being preferred and ambient temperatures (about 20°C-30°C) being most preferred. The reactions may be conducted at subatmospheric pressure or superatmospheric pressure (viz., about 60 Kg./m2 and about 2100 Kg./m2 absolute), but ambient atmospheric pressure (about 10330 Kg./m , absolute) is most often used.
The completion of the coupling and deprotecting steps may be determined by a number of conventional techniques, including, but not limited to, chromatography (preferably, thin layer chromatography).
Cyclization and removal of the compound from the solid support is described above.
The purification of the library compound dissolved in the solvent phase of the reaction may be done by any conventional chemical or physical method. Preferred are physical methods which are applicable to all members of a diverse library. Such methods include, for example: (i) ion exchange chromatography (ii) filtration, (iii) centrifugation, (iv) decantation, and (v) washing, and combination thereof. Filtration and ion exchange chromatography are particularly preferred forms of purification.
The last purification step of the process may optionally be supplemented by a solvent removal step in which the solute library compound is removed from its solvent by conventional processes known in the art; such as solvent evaporation, distillation, salting out, solvent extraction, and etc.
8. Screening of Libraries
The libraries of compounds (I), (lb) and (III) according to the present invention may be screened for biological activity. Generally, the library to be screened is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibodies wherein the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined. Typically this will comprise determining whether the biological substance is bound to one or more of the members of the library. Such binding may be determined by attaching a label to the biological substance. Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels. The degree of binding affinity may be determined by quantitating the amount or intensity of the bound label. Thus, various biologically active compounds may be selected by identifying which compounds bind the particular biological substance most effectively.
Illustrative additional assays include, but are not limited to, in vitro assays such as enzymatic inhibition, receptor - ligand binding, protein - protein interaction, and protein - DNA interaction; cell based, functional assays such as transcriptional regulation, signal transduction / second messenger, and viral infectivity; add, incubate & read assays such as scintillation proximity assays (SPA), fluorescence polarization assay, fluorescence correlation spectroscopy, colorimetric biosensors, cellular reporter assays using reporter genes such as luciferase, green fluorescent protein, β-lactamase, and the like; and electrical cell impedance sensor assays.
All of the above assays are known in the art to be predictive of success for an associated disease state.
9. Abbreviations used herein
Azt = azetidine-2-carboxylic acid
Anal. = elemental analysis
APTT assay = Activated partial thromboplastin time.
BOC = t-butyloxycarbonyl Bn = benzyl
BOP-C1 = bis(2-oxo-3-oxazolidinyl)phosphinic chloride t-Bu = t-butyl n-BuLi = butyllithium
Cbz = benzyloxycarbonyl Cha = β=cyclohexylalanine
18-Crown-6 = 1,4,7,10,13, 16-hexaoxacyclooctadecane
DIBAL = diisobutylaluminum hydride
DIPEA =diisopropylethyl amine
DMF = dimethylformamide DMSO = dimethylsulfoxide Et = ethyl
EtOAc = ethyl acetate
Et O = diethyl ether
EtOH = ethanol FAB-MS = fast atom bombardment mass spectrum
FD-MS = field desoφtion mass spectrum
Fmoc = 9-fluorenylmethoxycarbonyl.
HC1 = hydrochloric acid; HClaq = aqueous HC1.
HPLC = High Performance Liquid Chromatography HRMS = high resolution mass spectrum
HOBT = 1-hydroxybenzotriazole hydrate hPro = homo-proline,
HTS technology = high throughput screening technology i-PrOH = isopropanol IR = Infrared Spectrum
Me = methyl
MeOH = methanol
NMR = Nuclear Magnetic Resonance
NV = nitroveratryl Ohi = [2S-(2α,3aβ,7aβ)]-octahydro-indol-2-carboxylic acid
(lR,4aR,8aR)-l-Piq = (lR,4aR,8aR)-l-perhydro-isoquinolinecarboxylate
Phe = phenylalanine,
Pro = proline,
PT assay = prothrombin time assay. RPHPLC = Reversed Phase High Performance Liquid
Chromatography
Sar = sarcosine (N-methylglycine)
Siθ2 = silica gel
TBS = t-butyldimethylsilyl. TEA = triethylamine TFA = trifluoroacetic acid THF = tetrahydrofuran TLC = thin layer chromatography TMS = trimethyl silyl. Ts = tosyl (p-toluenesulfonyl)
The following Examples are provided to further describe the invention and are not to be construed as limitations thereof.
EXAMPLES
The following compounds can be synthesized in accordance with the present invention.
10
12
13
16
18
17
20
19
22
21
24
23
Compounds 1-8 and 10-24, as well as the three pages of similar compounds that follow, can be made using analogous methods. The compound number (in bold), molecular weight, and crude purity (in parenthesis) are shown below for each of the compounds of the present invention:
The preparation of various aminobenzisoxazole peptide thrombin inhibitors in accordance with the present invention is exemplified below for compounds 5 and 9. Synthesis of compound 5
l) 10% TFA/CH2Cl220 min TFA, H20 99:1
2) 10% Et3N/CH2Cl2, rinse 55C, 2h
3) p yridine/CH2Cl2 o o To /J-nitrobenzophenone oxime polystyrene (Kaiser) resin (200 mg, 1.07 mmol/g, 0.214 mmol) in a tared 8 mL Kontes Microfilter Funnel was added THF (3 mL) and potassium t-butoxide (225 μL, 1 M in THF, 0.225 mmol). After shaking for several minutes, the resin turned a deep purple color. To this suspension was added 4-(t- butoxycarbonylaminomethyl)-2-fluorobenzonitrile (107 mg, 0.428 mmol). The reaction vessel was rotated at 55 °C in a Robbins oven for 12 h, and allowed to cool for 1 h. The resin was then rinsed with 2 x 2 mL of CH2C12, 2 x 2 mL of MeOH, 2 2 mL of H2O, and 4 x 2 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h. The resin was then suspended in 25% TFA/CH C12 (3 mL) and rotated for 2 h. The resin was again rinsed with 2 2 mL of CH2C12 and 4 x 2 mL of MeOH. The resin was then suspended in DMF (2 mL) followed by addition of (L)-BOC-proline (185 mg, 0.86 mmol) dissolved in 0.5 mL of DMF, HOBt (116 mg, 0.86 mmol) dissolved in 0.5 mL of DMF, DIPEA (187 μL, 1.07 mmol), and BOP (380 mg, 0.86 mmol) dissolved in 1.0 mL of DMF. After rotating the reaction vessel for another 12 h, the resin was rinsed with 2 2 mL of MeOH, 2 x 2 mL of H2O, 2 2 L of MeOH, 2 x 2 mL of CH2C12 and 2 x 2 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h. The resin was then suspended in 25% TFA/CH2C12 (3 mL) and rotated for 2 h. The resin was again rinsed with 2 x 2 mL of CH2- Cl2 and 4 2 mL of MeOH. The resin was then suspended in DMF (2 mL) followed by addition of (D)-BOC-cyclohexylalanine (233 mg, 0.86 mmol) dissolved in 0.5 mL of DMF, HOBt (1 16 mg, 0.86 mmol) dissolved in 0.5 mL of DMF, DIPEA (187 μL, 1.07 mmol), and BOP (380 mg, 0.86 mmol) dissolved in 1.0 mL of DMF. After rotating the reaction vessel for another 12 h, the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H2O, 2 2 mL of MeOH, 2 2 mL of CH2C12 and 2 x 2 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h. The resin was then suspended in 10% TFA/CH2C12 (3 L) and rotated for 20 min. The resin was rinsed with 2 x 2 mL of CH2C12,2 x 2 mL of 10% Et3N/CH2Cl2, 4 x 2 mL of CH2C12. The resin was then suspended in CH2C12 (2 mL) followed by addition of ethanesulfonyl chloride (403 μL, 4.28 mmol) and pyridine (346 μL, 4.28 mmol). After rotating the reaction vessel for another 8 h, the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H2O, 2 2 mL of MeOH, 2 x 2 mL of CH2C12 and 2 x 2 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h. The resin was then suspended in TFA (2 mL) and 5 N HClaq (0.5 mL) and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 2 mL of CH2- Cl2. These washings were combined and concentrated in vacuo to give the crude product 5 (54% purity by reverse phase HPLC, major impurity non-sulfonylated product (compound 1)) in >100% crude yield based on a loading yield of 50% (assuming bis-TFA salt). HPLC retention time (eluent gradient 5%A to 85%A over 45 min) = 23.9 min. MS (ESI) m/z 506 (M+H)+.
Synthesis of compound 9
To /j-nitrobenzophenone oxime polystyrene (Kaiser) resin (200 mg, 1.07 mmol/g, 0.214 mmol) in a tared 8 mL Kontes Microfilter Funnel was added THF
(3 mL) and potassium t-butoxide (225 μL, 1 M in THF, 0.225 mmol). After shaking by hand for several minutes, the resin turned a deep purple color. To this suspension was added 4-(t-butoxycarbonylaminomethyl)-2-fluorobenzonitrile (107 mg, 0.428 mmol). The reaction vessel was rotated at 55°C in a Robbins oven for 12 h, and allowed to cool for 1 h. The resin was then rinsed with 2 x 2 mL of CH2C12, 2 x 2 mL of MeOH, 2 x 2 mL of H2O, and 4 x 2 mL of MeOH. The resin was dried in a 35°C vacuum oven for 12 h. The resin was then suspended in 25%) of TFA/CH2C12 (3 mL) and rotated for 2 h. The resin was again rinsed with 2 x 2 mL of CH2C12 and 4 x 2 mL of MeOH. The resin was then suspended in DMF (2 mL) followed by addition of (L)-BOC-proline (compound A)(185 mg, 0.86 mmol) dissolved in 0.5 mL of DMF, HOBt (116 mg, 0.86 mmol) dissolved in 0.5 mL of DMF, DIPEA (187 μL, 1.07 mmol), and BOP (380 mg, 0.86 mmol) dissolved in 1.0 mL of DMF. After rotating the reaction vessel for another 12 h, the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H2O, 2 x 2 mL of MeOH, 2 x 2 mL of CH2C12 and 2 x 2 mL of MeOH. The resin was dried in a 35°C vacuum oven for 12 h. The resin was then suspended in 25% TFA/CH2C12 (3 mL) and rotated for 2 h. The resin was again rinsed with 2 x 2 mL of CH2C12 and 4 x 2 mL of MeOH. The resin was then suspended in DMF (2 mL) followed by addition of (D)- BOC-cyclohexylalanine (compound B)(233 mg, 0.86 mmol) dissolved in 0.5 mL of DMF, HOBt (116 mg, 0.86 mmol) dissolved in 0.5 mL of DMF, DIPEA (187 μL, 1.07 mmol), and BOP (380 mg, 0.86 mmol) dissolved in 1:0 mL of DMF. After rotating the reaction vessel for another 12 h, the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H2O, 2 x 2 mL of MeOH, 2 x 2 mL of CH2C12 and 2 x 2 mL of MeOH. The resin was dried in a 35°C vacuum oven for 12 h. The resin was then suspended in 25% TFA/CH2/C12 (3 mL) and rotated for 2 h. The resin was again rinsed with 2 x 2 mL of CH2C12 and 4 x 2 mL of MeOH. The resin was then suspended in DMF (2 mL) followed by addition of t-butylbromoacetate (compound C)(35 μL, 0.214 mmol) and DIPEA (112 μL, 0.642 mmol). After rotating the reaction vessel for another 12 h, the resin was rinsed with 2 x 2 mL of MeOH, 2 x 2 mL of H2O, 2 x 2 mL of MeOH, 2 x 2 mL of CH2C12 and 2 x 2 mL of MeOH. The resin was dried in a 35°C vacuum oven for 12 h.
The resin was then suspended in TFA (2 mL) and 5 N HClaq (0.5 mL) and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 x 2 mL of CH2C12. These washings were combined and concentrated in vacuo to give the crude product 9 (79% purity by reverse phase HPLC, major impurity bisalkylation product) in >100% crude yield based on a loading yield of 50%) (assuming bis-TFA salt). HPLC retention time (eluent gradient 5% A to 85%A over 45 min) = 18.6 min. MS (ESI) m/z 472 (M+H)+.
Experimental for the Preparation of 2-Hydroxybenzamidines
The above compounds can be converted into 2-hydroxybenzamidines as exemplified below:
Formation of dipeptide 9b. The 3 -aminobenzisoxazole 9 (1.49 g, 2.8 mmol) was added to 250 mL round bottom flask followed by the addition of 5%> Pd(C) (310 mg, 0.145 mmol Pd). The flask was slowly evacuated and released under a nitrogen atmosphere. To this was added EtOH (50 mL) and 0.1 N HClaq (50 mL). The flask was again evacuated and released under an atmosphere of hydrogen (balloon). The reaction was allowed to stir vigorously for 30 min and removed from the hydrogen atmosphere. The reaction mixture was then filtered through a plug of celite (6 cm dia x 4 cm height) rinsing with methanol (3 x 50 mL). The filtrate was concentrated to give 1.43 g (97%) of 9b as a tan solid. (85% purity by reverse phase HPLC). HPLC retention time (eluent gradient 2% to 50% Me3CN over 40 min) = 19.3 min. MS (ESI) m/z 474 (M+H)+. Anal Calc'd for C24H36ClN5O5: C, 56.74; H, 6.75; N, 13.79; Cl, 6.98. Found: C, 56.68; H, 6.80; N, 13.52; Cl, 6.66.
Preparative RPHPLC was conducted under the following parameters: Solvent A: 0.05% aqueous hydrochloric acid (1.5 mL concentrated hydrochloric acid in 3 L water); Solvent B: acetonitrile; Gradient: as defined in each Example; Column: Vydac Ci8 - 5 cm X 25 cm; Flow rate: 10 mL/minute.
Unless otherwise stated, pH adjustments and work up are with aqueous acid or base solutions. *H-NMR indicates a satisfactory NMR spectrum was obtained for the compound described. IR indicates a satisfactory infra red spectrum was obtained for the compound described.
Compositions The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. "Active ingredient," of course, means an aminobenzisoxazole compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.
Formulation 1 : Hard gelatin capsules are prepared using the following ingredients:
Quantity (mg/capsule
Active ingredient 250
Starch, dried 200
Magnesium stearate 10
Total 460 mg
Formulation 2: A tablet is prepared using the ingredients below:
Quantity
(ms/tablef)
Active ingredient 250
Cellulose, microcrystalline 400
Silicon dioxide, fumed 10 Stearic acid
Total 665 mg
The components are blended and compressed to form tablets each weighing 665 mg.
Formulation 3: An aerosol solution is prepared containing the following components:
Weight Active ingredient 0.25
Ethanol 25.75
Propellant 22 (Chlorodifluoromethane) 70.00
Total 100.00
The active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -30 °C and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container.
Formulation 4: Tablets, each containing 60 mg of active ingredient, are made as follows:
Active ingredient 60 mg
Starch 45 mg
Microcrystalline cellulose 35 mg
Polyvinylpyrrolidone (as 10% solution in water) 4 mg
Sodium carboxy methyl starch 4.5 mg
Magnesium stearate 0.5 mg Talc 1 mg
Total 150 mg The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The aqueous solution containing polyvinylpyrrolidone is mixed with the resultant powder, and the mixture then is passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50 °C and passed through a No. 18 mesh U.S. Sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
Formulation 5: Capsules, each containing 80 mg of active ingredient, are made as follows:
Active ingredient 80 mg
Starch 59 mg
Microcrystalline cellulose 59 mg
Magnesium stearate 2 mg
Total 200 mg
The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities.
Formulation 6: Suppositories, each containing 225 mg of active ingredient, are made as follows:
Active ingredient 225 mg
Saturated fatty acid glycerides 2,000 mg
Total 2,225 mg The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
Formulation 7: Suspensions, each containing 50 mg of active ingredient per 5 ml dose, are made as follows:
Active ingredient 50 mg
Sodium carboxymethyl cellulose 50 mg
Syrup 1.25 ml
Benzoic acid solution 0.10 ml
Flavor q.v.
Color q.v.
Purified water to total 5 ml
The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor and color are diluted with a portion of the water and added, with stirring. Sufficient water is then added to produce the required volume.
Formulation 8: An intravenous formulation may be prepared as follows: Active ingredient 100 mg
Isotonic saline 1,000 ml
The solution of the above ingredients generally is administered intravenously to a subject at a rate of 1 ml per minute.
Methods for determining thrombin inhibition The ability of an aminobenzisoxazole compound of the present invention to be an effective and orally active thrombin inhibitor is evaluated in one or more of the following assays.
The compounds provided by the invention selectively inhibit the action of thrombin in mammals. The inhibition of thrombin is demonstrated by in vitro inhibition of the amidase activity of thrombin as measured in an assay in which thrombin hydrolyzes the chromogenic substrate, N-benzoyl-L-phenylalanyl-L-valyl-L- arginyl-p-nitroanilide, N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide.
The assay is carried out by mixing 50 μl buffer (0.03M Tris, 0.15M NaCl, pH 7.4) with 25 μl of human thrombin solution (purified human thrombin, Enzyme Research Laboratories, South Bend, Indiana, at 8 NIH units/ml) and 25 μl of test compound in a solvent (50%) aqueous methanol (v:v)). Then 150 μl of an aqueous solution of the chromogenic substate (at 0.25 mg/ml) are added and the rates of hydrolysis of the substrate are measured by monitoring the reactions at 405 nm for the release of p-nitroaniline. Standard curves are constructed by plotting free thrombin concentration against hydrolysis rate. The hydrolysis rates observed with test compounds are then converted to "free thrombin" values in the respective assays by use of the standard curves. The bound thrombin (bound to test compound) is calculated by subtracting the amount of free thrombin observed in each assay from the known initial amount of thrombin used in the assay. The amount of free inhibitor in each assay is calculated by subtracting the number of moles of bound thrombin from the number of moles of added inhibitor (test compound).
The Kass value is the hypothetical equilibrium constant for the reaction between thrombin and the test compound (I).
Thrombin + I ► Thrombin - I
Kass = [Thrombin-I ] [ (Thrombin) x (I) ] Kass is calculated for a range of concentrations of test compounds and the mean value reported in units of liter per mole. In general, a thrombin inhibiting compound of formula (I) of the instant invention exhibits a Kass of 0.1 X 10^ L/mole or much greater. By substantially following the procedures described above for human thrombin, and using other human blood coagulation system serine proteases and using fibrinolytic system serine proteases, with the appropriate chromogenic substrates, identified below, the selectivity of the compounds of the present invention with respect to the coagulation factor serine proteases and to the fibronolytic serine proteases are evaluated as well as their substantial lack of interference with human plasma clot fibrinolysis.
Human factors X, Xa, IXa, XIa, and Xlla are purchased from Enzyme Research Laboratories, South Bend, Indiana; human urokinase from Leo Pharmaceuticals, Denmark; and recombinant activated Protein C (aPC) is prepared at Eli Lilly and Co. substantially according to U.S. Patent 4,981 ,952. Chromogenic substrates:
N-Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (for factor Xa); N-Cbz-D-Arg-Gly-Arg-p- nitroanilide (for factor IXa assay as the factor Xa substrate); Pyroglutamyl-Pro-Arg-p- nitroanilide (for Factor XIa and for aPC); H-D-Pro-Phe-Arg-p-nitroanilide (for factor Xlla); and Pyroglutamyl-Gly-Arg-p-nitroanilide (for urokinase); are purchased from Kabi Vitrum, Stockholm, Sweden, or from Midwest Biotech, Fishers, Indiana. Bovine trypsin is purchased from Worthington Biochemicals, Freehold, New Jersey, and human plasma kallikrein from Kabi Vitrum, Stockholm, Sweden. Chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide for plasma kallikrein is purchased from Kabi Vitrum, Stockholm, Sweden. N-Benzoyl-Phe-Val-Arg-p-nitroanilide, the substrate for human thrombin and for trypsin, is synthesized according to procedures described above for the compounds of the present invention, using known methods of peptide coupling from commercially available reactants, or purchased from Midwest Biotech, Fishers, Indiana.
Human plasmin is purchased from Boehringer Mannheim, Indianapolis, Indiana; nt-PA is purchased as single chain activity reference from American Diagnostica, Greenwich, Connecticut; modified-t-PA6 (mt-PA6) is prepared at Eli Lilly and Company by procedure known in the art (See, Burck, et al., J. Biol. Chem., 265, 5120-5177 (1990). Plasmin chromogenic substrate H-D-Val-Leu-Lys-p- nitroanilide and tissue plasminogen activator (t-PA) substrate H-D-Ile-Pro-Arg-p- nitroanilide are purchased from Kabi Vitrum, Stockholm, Sweden.
In the chromogenic substrates described above the three-letter symbols He, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys are used to indicate the corresponding amino acid group isoleucine, glutamic acid, glycine, proline, arginine, phenylalanine, valine, leucine and lysine, respectively. Thrombin inhibitors preferably should spare fibrinolysis induced by urokinase, tissue plasminogen activator (t-PA) and steptokinase. This would be important to the therapeutic use of such agents as an adjunct to streptokinase, t-PA or urokinase thrombolytic therapy and to the use of such agents as an endogenous fibrinolysis- sparing (with respect to t-PA and urokinase) antithrombotic agents. In addition to the lack of interference with the amidase activity of the fibrinolytic proteases, such fibrinolytic system sparing can be studied by the use of human plasma clots and their lysis by the respective fibrinolytic plasminogen activators.
Dog plasma is obtained from conscious mixed-breed hounds (either sex Hazelton-LRE, Kalamazoo, Michigan, U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen is prepared from fresh dog plasma and human fibrinogen is prepared from in-date ACD human blood at the fraction 1-2 according to previous procedures and specifications. Smith, Biochem. J„ 185, 1-11 (1980); and Smith, et al., Biochemistry. 11, 2958-2967, (1972). Human fibrinogen (98 percent pure/plasmin free) is from American Diagnostica, Greenwich, Connecticut. Radiolabeling of fibrinogen 1-2 preparations is performed as previously reported. Smith, et al., Biochemistry. 1_1, 2958-2967, (1972). Urokinase is purchased form Leo Pharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase is purchased from Hoechst-Roussel Pharmaceuticals, Somerville, New Jersey.
Methods - Effects on Lysis of Human Plasma Clots by t-PA Human plasma clots are formed in micro test tubes by adding 50 ul thrombin (73 NIH unit/ml) to 100 ul human plasma which contains 0.0229 uCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlaying the clots with 50 ul of urokinase or streptokinase (50, 100, or 1000 unit/ml) and incubating for 20 hours at room temperature. After incubation the tubes are centrifuged in a Beckman Microfuge. 25 ul of supernate is added into 1.0 ml volume of 0.03 M tris/0.15 M NaCl buffer for gamma counting. Counting controls 100 percent lysis are obtained by omitting thrombin (and substituting buffer). The thrombin inhibitors are evaluated for possible interference with fibrinolysis by including the compounds in the overlay solutions at 1, 5, and 10 ug/ml concentrations. Rough approximations of IC50 values are estimated by linear extrapolations from data points to a value which would represent 50 percent of lysis for that particular concentration of fibrinolytic agent.
Anticoagulant Activity Materials
Dog plasma and rat plasma are obtained from conscious mixed-breed hounds (either sex, hazelton-LRE, Kalamazoo, Michigan, U.S.A.) or from anesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, Indiana, U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen is prepared from in-date ACD human blood as the fraction 1-2 according to previous procedures and specifications. Smith, Biochem. J.. 185. 1-11 (1980); and Smith, et al., Biochemistry. 11, 2958-2967 (1972). Human fibrinogen is also purchased as 98 percent pure/plasmin free from American Diagnostica, Greenwich, Connecticut. Coagulation reagents ACTIN, Thromboplastin, and Human plasma are from Baxter Healthcare Corp., Dade Division, Miami, Florida. Bovine thrombin from Parke-Davis (Detroit, Michigan) is used for coagulation assays in plasma.
Anticoagulation Determinations
Coagulation assay procedures are as previously described. Smith, et al., Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulation instrument (American LABor, Inc.) is used for all coagulation assay measurements. The thrombin time (TT) is measured by adding 0.05 ml saline and 0.05 ml thrombin (10 NTH units/ml) to 0.05 ml test plasma. The activated partial thromboplastin time (APTT) is measured by incubation of 0.05 ml test plasma with 0.05 ml Actin reagent for 120 seconds followed by 0.05 ml CaCl2 (0.02 M). The prothrombin time (PT) is measured by adding 0.05 ml saline and 0.05 ml Thromboplastin-C reagent to 0.05 ml test plasma. The compounds of formula I are added to human or animal plasma over a wide range of concentrations to determine prolongation effects on the TT, APTT and PT assays. Linear extrapolations are performed to estimate the concentrations required to double the clotting time for each assay.
Animals
Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc., Indianapolis, IN) are anesthetized with xylazine (20 mg/kg, s.c.) and ketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37 °C). The jugular vein(s) is cannulated to allow for infusions.
Arterio- Venous shunt model
The left jugular vein and right carotid artery are cannulated with 20 cm lengths of polyethylene PE 60 tubing. A 6 cm center section of larger tubing (PE 190) with a cotton thread (5 cm) in the lumen, is friction fitted between the longer sections to complete the arterio- venous shunt circuit. Blood is circulated through the shunt for 15 min before the thread is carefully removed and weighed. The weight of a wet thread is subtracted from the total weight of the thread and thrombus (see J.R. Smith, Br J Pharmacol. 77:29, 1982).
FeC model of arterial injury
The carotid arteries are isolated via a midline ventral cervical incision. A thermocouple is placed under each artery and vessel temperature is recorded continuously on a strip chart recorder. A cuff of tubing (0.058 ID x 0.077 OD x 4 mm, Baxter Med. Grade Silicone), cut longitudinally, is placed around each carotid directly above the thermocouple. FeCl3 hexahydrate is dissolved in water and the concentration (20 percent) is expressed in terms of the actual weight of FeCl3 only. To injure the artery and induce thrombosis, 2.85 μl is pipetted into the cuff to bathe the artery above the thermocouple probe. Arterial occlusion is indicated by a rapid drop in temperature. The time to occlusion is reported in minutes and represents the elapsed time between application of FeCl3 and the rapid drop in vessel temperature (see K.D. Kurz, Thromb. Res., 60:269,1990).
Spontaneous thrombolysis model
In vitro data suggests that peptide thrombin inhibitors inhibit thrombin and at higher concentration may inhibit, other serine proteases, such as plasmin and tissue plasminogen activator. To assess if the compounds inhibit fibrinolysis in vivo, the rate of spontaneous thrombolysis is determined by implanting a labeled whole blood clot into the pulmonary circulation. Rat blood (1 ml) is mixed rapidly with bovine thrombin (4 IU, Parke Davis) and 125I human Fibrogen (5 μCi, ICN), immediately drawn into silastic tubing and incubated at 37 °C for 1 hour. The aged thrombus is expelled from the tubing, cut into 1 cm segments, washed 3X in normal saline and each segment is counted in a gamma counter. A segment with known counts is aspirated into a catheter that is subsequently implanted into the jugular vein. The catheter tip is advanced to the vicinity of the right atrium and the clot is expelled to float into the pulmonary circulation. One hour after implant, the heart and lungs are harvested and counted separately. Thrombolysis is expressed as a percentage where:
% Thrombolysis = (injected cpm - lung cpm) x 100 injected cpm
The fibrinolytic dissolution of the implanted clot occurs time-dependently (see J.P. Clozel, Cardiovas. Pharmacol. 12:520, 1988). Coagulation parameters
Plasma thrombin time (TT) and activated partial thromboplastin time (APTT) are measured with a fibrometer. Blood is sampled from a jugular catheter and collected in syringe containing sodium citrate (3.8 percent, 1 part to 9 parts blood). To measure TT, rat plasma (0.1 ml) is mixed with saline (0.1 ml) and bovine thrombin (0.1 ml, 30 U/ml in TRIS buffer; Parke Davis) at 37 °C. For APTT, plasma (0.1 ml) and APTT solution (0.1 ml, Organon Teknika) are incubated for 5 minutes (37 °C) and CaCl2 (0.1 ml, 0.025M) is added to start coagulation. Assays are done in duplicate and averaged.
Index of Bioavailability
A measure of bioactivity, plasma thrombin time (TT), serves as a substitute for the assay of parent compound on the assumption that increments in TT resulted from thrombin inhibition by parent only. The time course of the effect of the thrombin inhibitor upon TT is determined after i.v bolus administration to anesthetized rats and after oral treatment of fasted conscious rats. Due to limitations of blood volume and the number of points required to determine the time course from time of treatment to the time when the response returns to pretreatment values, two populations of rats are used. Each sample population represents alternating sequential time points. The average TT over the time course is used to calculate area under the curve (AUC). The index of bioavailability is calculated by the formula shown below and is expressed as percent relative activity.
The area under the curve (AUC) of the plasma TT time course is determined and adjusted for the dose. This index of bioavailability is termed "% Relative Activity" and is calculated as
%Relative Activity = AUC P° χ Dose iv χ100
AUC iv Dose po
Compounds
Compound solutions are prepared fresh daily in normal saline and are injected as a bolus or are infused starting 15 minutes before and continuing throughout the experimental perturbation which is 15 minutes in the arteriovenous shunt model and 60 minutes in the FeCb model of arterial injury and in the spontaneous thrombolysis model. Bolus injection volume is 1 ml/kg for i.v., and 5 ml/kg for p.o. and infusion volume is 3 ml/hr.
Statistics
Results are expressed as means +/- SEM. One-way analysis of variance is used to detect statistically significant differences and then Dunnett's test is applied to determine which means are different. Significance level for rejection of the null hypothesis of equal means is PO.05.
Animals
Male dogs (Beagles; 18 months - 2 years; 12-13 kg, Marshall Farms, North Rose, New York 14516) are fasted overnight and fed Purina certified Prescription Diet (Purina Mills, St. Louis, Missouri) 240 minutes after dosing. Water is available ad libitum. The room temperature is maintained between 66-74°F; 45-50 percent relative humidity; and lighted from 0600-1800 hours.
Pharmacokinetic model Test compound is formulated immediately prior to dosing by dissolving in sterile 0.9 percent saline to a 5 mg/ml preparation. Dogs are given a single 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5 ml) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1,2,3,4 and 6 hours after dosing. Samples are collected in citrated Vacutainer tubes and kept on ice prior to reduction to plasma by centrifligation. Plasma samples are analyzed by HPLC-MS. Plasma concentration of test compound is recorded and used to calculate the pharmacokinetic parameters: elimination rate constant, Ke; total clearance, Clt; volume of distribution, VD; time of maximum plasma test compound concentration, Tmax; maximum concentration of test compound of Tmax, Cmax; plasma half-life, tθ.5; and area under the curve, A.U.C.; fraction of test compound absorbed, F. Canine Model of Coronary Artery Thrombosis
Surgical preparation and instrumentation of the dogs are as described in Jackson, et al., Circulation, 82, 930-940 (1990). Mixed-breed hounds (aged 6-7 months, either sex, Hazelton-LRE, Kalamazoo, MI, U.S.A.) are anesthetized with sodium pentobarbital (30 mg/kg intravenously, i.v.), intubated, and ventilated with room air. Tidal volume and respiratory rates are adjusted to maintain blood PO2, PCO2, and pH within normal limits. Subdermal needle electrodes are inserted for the recording of a lead II ECG. The left jugular vein and common carotid artery are isolated through a left mediolateral neck incision. Arterial blood pressure (ABP) is measured continuously with a precalibrated Millar transducer (model (MPC-500, Millar Instruments, Houston, TX, U.S.A.) inserted into the carotid artery. The jugular vein is cannulated for blood sampling during the experiment. In addition, the femoral veins of both hindlegs are cannulated for administration of test compound.
A left thoracotomy is performed at the fifth intercostal space, and the heart is suspended in a pericardial cradle. A 1- to 2-cm segment of the left circumflex coronary artery (LCX) is isolated proximal to the first major diagonal ventricular branch. A 26- gauge needle-tipped wire anodal electrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm long is inserted into the LCX and placed in contact with the intimal surface of the artery (confirmed at the end of the experiment). The stimulating circuit is completed by placing the cathode in a subcutaneous (s.c.) site. An adjustable plastic occluder is placed around the LCX, over the region of the electrode. A precalibrated electromagnetic flow probe (Carolina Medical Electronics, King, NC, U.S.A.) is placed around the LCX proximal to the anode for measurement of coronary blood flow (CBF). The occluder is adjusted to produce a 40-50 percent inhibition of the hyperemic blood flow response observed after 10-s mechanical occlusion of the LCX. All hemodynamic and ECG measurements are recorded and analyzed with a data acquisition system (model M3000, Modular Instruments, Malvern, PA. U.S.A.). Thrombus Formation and Compound Administration Regimens
Electrolytic injury of the intima of the LCX is produced by applying 100-μA direct current (DC) to the anode. The current is maintained for 60 min and then discontinued whether the vessel has occluded or not. Thrombus formation proceeds spontaneously until the LCX is totally occluded (determined as zero CBF and an increase in the S-T segment). Compound administration is started after the occluding thrombus is allowed to age for 1 hour. A 2-hour infusion of the compounds of the present invention at doses of 0.5 and 1 mg/kg/hour is begun simultaneously with an infusion of thrombolytic agent (e.g. tissue plasminogen activator, streptokinase, APSAC). Reperfusion is followed for 3 hour after administration of test compound. Reocclusion of coronary arteries after successful thrombolysis is defined as zero CBF which persisted for at least 30 minutes.
Hematology and template bleeding time determinations Whole blood cell counts, hemoglobin, and hematocrit values are determined on a 40-μl sample of citrated (3.8 percent) blood (1 part citrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900, Sequoia-Turner. Mount View, CA, U.S.A.). Gingival template bleeding times are determined with a Simplate II bleeding time device (Organon Teknika Durham, N.C., U.S.A.). The device is used to make 2 horizontal incisions in the gingiva of either the upper or lower left jaw of the dog.
Each incision is 3 mm wide x 2 mm deep. The incisions are made, and a stopwatch is used to determine how long bleeding occurs. A cotton swab is used to soak up the blood as it oozes from the incision. Template bleeding time is the time from incision to stoppage of bleeding. Bleeding times are taken just before administration of test compound (0 min), 60 min into infusion, at conclusion of administration of the test compound (120 min), and at the end of the experiment.
All data are analyzed by one-way analysis of variance (ANOVA) followed by Student-Neuman-Kuels post hoc t test to determine the level of significance. Repeated- measures ANOVA are used to determine significant differences between time points during the experiments. Values are determined to be statistically different at least at the level of p<0.05. All values are mean ± SEM. All studies are conducted in accordance with the guiding principles of the American Physiological Society. Further details regarding the procedures are described in Jackson, et al., J. Cardiovasc. Pharmacol.. 2J, 587-599 (1993).
It should be understood that a wide range of changes and modifications can be made to the embodiments described above. It is therefore intended that the foregoing description illustrates rather than limits this invention, and that it is the appended claims, including all equivalents, which define this invention.
Appendix A:
Definition of terms
As used herein and in the appended claims the term "solid support" is intended to have a relatively broad meaning including, but not limited to, a resin, a polymer, a gel, glass beads, silica gel, a ceramic solid support or other solid composition. As used herein and in the appended claims the term "solid support bound oxime" means a solid support that at least has one oxime moiety chemically attached thereto. For example, one embodiment of this compound may be represented by the formula (IX):
IX In formula (IX), g represents a solid support, such as defined above.
As used herein and in the appended claims the term "solid support bound member" means a solid support that has at least one functional moiety chemically attached thereto. For example, this may be represented by the formula (III):
III
As used herein and in the appended claims the term "oxime resin" means a solid support where the functional moiety is an oxime, and the solid support is a resin.
As used herein and in the appended claims the term "Kaiser resin" means an oxime functionalized polystyrene resin, an example of that is defined by E.T.Kaiser in a 1980 publication (Degrado, W.F.;Kaiser. E.T.; J.Org. Chem.,1980, 45, 1295). A preferred resin is an oxime functionalized polystyrene, such as an oxime-polystyrene resin derived from p- nitrobenzophenone polystyrene resin according to the following formula.
As used herein and in the appended claims, "halo" means a member selected from the group consisting of fluoro, chloro, bromo and iodo.
"Alkyl" is the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl group, and that groups may include one or more double or triple bonds. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone, and more preferably 20 or fewer and most preferred 10 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3-6 carbons in the ring structure. Particularly preferred alkyl substituents include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso-butyl, tert- butyl, sec-butyl, cyclobutyl, pentyl, hexyl, cyclohexyl, etc. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. The aliphatic cyclic groups can be single or polycyclic containing between about 3 to 12 carbons per ring, but preferably between 3 and 9 carbons per ring.
"Haloalkyl" and "alkylhalo" both refer to mono- or poly- halogen radical substituted alkyl radicals, with the alkyl radicals having the analogous length and possible substitution as described above. Typically, these terms refer to groups of the formula Xn-(CX'X")m-, where n and m are each independently an integer > 1, and X, X' and X" are each independently hydrogen or halogen (so long as at least one of X, X' and X" are halogen).
"Hydroxyalkyl" and "alkylhydroxide" and "alkyl alcohol" all refer to a mono or poly hydroxide radical substituted alkyl radical, with the alkyl radicals having the analogous length and possible substitution as described above. "Alkyloxyalkyl ether" and "alkyloxyaryl ether" both refer to ether functional radicals of either the dialkyl radical or the alkyl, aryl radical configuration, with the alkyl radicals and the aryl radicals having the analogous length and possible substitution to the alkyl and aryl radicals defined herein. "Alkenyl" and "alkynyl" refer to unsaturated aliphatic substituents analogous in length and possible substitution to the alkyl radicals described above, but that contain at least one double or triple bond, respectively.
"Amino" means an amino radical substituted with up to 2 alkyl radicals as defined above or with 1 alkyl radical and a hydrogen radical, or with two or more hydrogen radicals or with the substitution required to complete the nitrogen's valence requirements.
"Aryl" as used herein includes 5-10 membered aromatic monocyclic or fused polycyclic moieties that may include from zero to four heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthyline, benzathiazoline, benzothiapene, benzofurans, indole, quinoline, etc. The aryl group can be substituted at one or more positions with halo, alkyl, alkoxy, alkoxy carbonyl, haloalkyl, cyano, amino sulfonyl, aryl, sulfonyl, aminocarbonyl, carboxy, acylamino, alkyl sulfonyl, amino and substituted or unsubstituted substituents.
As used herein and in the appended claims, "heteroaryl" is a mono-, bi- or tricyclic, - N-, -O- or -S- heteroaryl substituent, such as benzofuran, benzothiophene, furan, imidazole, indole, isothiazole, oxazole, piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinoline, thiazole and thiophene.
As used herein and in the appended claims a "library" means a large number of chemical derivatives used in screening for biological activity or other activity. In general a library will have greater than 20 members, preferably the library will have at least 50 members, more preferably the library will have at least 96 members and most preferably the library will have at least 1000 members.
As used herein and in the appended claims "chemically derivatized" means the chemical manipulation such as addition to, oxidation of, substitution for, reduction of, or cyclization of the selected R group or R groups of the intermediate. Chemical derivatization also means the manipulation of two or more groups of the intermediate such that additional aryl or alkyl rings are formed and that rings may be fused or unfused to the intermediate ring, and that new ring may be substituted with further chemically derivatizable substituents.
As used herein and in the appended claims "pharmaceutically acceptable salt" and "salts thereof means organic or inorganic salts of the pharmaceutically important molecule. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically important organic molecule may have more than one charged atom in its structure. Situations where multiple charged atoms are part of the molecule may have multiple counterions. Hence, the molecule of a pharmaceutically acceptable salt may contain one or more than one charged atoms and may also contain, one or more than one counterion. The desired charge distribution is determined according to methods of drug administration. Examples of pharmaceutically acceptable salts are well known in the art but, without limiting the scope of the present invention, exemplary presentations can be found in the Physicia 's Desk Reference, The Merck Index, The Pharmacopoeia and Goodman & Gilman's 77ze Pharmacological Basis of Therapeutics.
As used herein and in the appended claims, "TFA" means trifluoro acetic acid, "HC1" means hydrochloric acid, "THF" means tetrahydrofuran, "DMF" means dimethylformamide, "DIPEA" means diisopropylethyl amine, "TMS" means a trimethyl silyl radical and "TBS" means a t-butyldimethyl silyl radical.
As used herein and in the appended claims "leaving group" means halo, oxo, thioxo radicals and activated alcohols such as a p-toluene sulfonyl activated alcohols and other groups that are susceptible to displacement and replacement by a nucleophile under selected conditions of temperature, solvent and time. As used herein and in the appended claims "scaffold" means a common chemical structure found within a library of organic compounds. Similarly, within a combinatorial chemical library the scaffold forms the basis for a diverse series of chemical derivatization, additions and subtractions. Importantly, regardless of the extent of the chemical derivatization performed on the scaffold, the product is within the scope of the combinatorial library. All other acronyms and abbreviations have the corresponding meaning as published in journals relative to the art of organic chemistry.
A general method for making polycyclic heterocyles according to the first aspect of the invention involves the following method.
(a) reacting a compound of the formula (I):
where each of R1, R2, R3 and R4 is a stable moiety independently selected from the group consisting of halo, haloalkyl, cyano, nitro, Ra-Q-, Ra-Q-alkyl, Ra-Q-alkenyl, Ra-Q- alkynyl, Ra-Q-arylalkyl and Ra-Q-aryl;
Ra is hydrogen, alkyl, aryl or arylalkyl;
Q is a single bond, -O-, -NRb-, -CO-, -NRb-CO-, -CO-NRb-, -CO-O-, -O-CO-, S(O)i, -S(O)j-NRb-, -NRb-S(O)j; i = 0, 1 or 2; j = l or 2;
Rb is hydrogen, alkyl, aryl or arylalkyl, where Ra and Rb may together with the nitrogen to that they are attached form a ring, R5 is selected from the group consisting of halo, nitro, and haloalkyl.
It is understood that each of R1, R2, R3 and R4 may be substituted one to three times with a substituent selected from the group consisting of alkyl, alkenyl, alkynyl, halo, hydroxy, alkoxy, alkylthio, sulfonyl, aryl, heteroaryl and where the substituents of the moieties substituted can themselves be substituted with one to three further substituents, if desired. Particularly one of R1, R2, R3 or R4 is an haloalkyl, more particularly either R1, R3 or R4 is a CF3 radical and most particularly R3 is a CF3 radical. Particularly one of R1, R2, R3 or R4 is an hydroxyalkyl, more particularly one of R1, R2, R3 or R4 is methoxy and most particularly R3 is a methoxy radical. Particularly one of R1, R2, R3 or R4 is halo, cyano or nitro. When halo, one of R1, R2,
R3 or R4 is bromo, and most particularly R3 is bromo. When one of R1, R2, R3 or R4 is a cyano radical, R3 is most particularly a cyano radical. When one of R1, R2, R3 or R4 is a nitro radical, R3 is most particularly a nitro radical.
Particularly, R5 is selected from fluoro, chloro or nitro. If a 5,6 polycycle is desired, then R5 is a halo. Also, R5 is between to Cj2 haloalkyl, particularly R5 is between Cj to C6 haloalkyl and more particularly R5 is a halomethyl and most particularly R5 is bromomethyl.
R6 of formula (I) is selected from the group consisting of cyano and a radical of formula (II) where L is selected from the group consisting of -O-, -S- and -NH-.
II
When R6 is the radical of formula (II), L is preferably -O- or -S- and most preferably -O-. R7 of formula (II) is selected from the group consisting of alkyl, aryl, arylalkyl, alkyloxyalkyl, aryloxyalkyl, alkylamino, dialkylamino, arylamino, alkoxy carbonyl, amino, alkoxy, hydroxy and heteroaryl. Where R6 is a radical of formula (II), then R7 is preferably aryl and most preferably R7 is phenyl.
In one embodiment R5 is halomethyl and R6 is cyano. In another embodiment, R5 is halo, R6 is formula (II), L is oxygen and R7 is a phenyl radical. In another embodiment R5 is halo and R6 is cyano. The compound (I) is reacted with the solid support bound member (III) under suitable conditions for a desired period of time and at a desired temperature such that the compound
(I) is reacted with the compound (III).
III Preferably in compound (III), g is a solid support selected from the group consisting of a resin, a polymer, a gel, glass beads, silica gel, a ceramic solid support or other solid composition. More preferably the solid support is a resin and most preferably the solid support is a polystyrene resin.
Likewise in compound (III), h is selected from the group consisting of alkyl and aryl. Preferably h is aryl, more preferably h is a substituted aryl and most preferably h is p- nitrophenyl. Also in compound (III) above, R is selected from the group consisting of -NH-, -O- and -S-, most preferably R is -O-.
Suitable conditions for this reaction include having a suitable solvent mixture, a suitable temperature and reacting for a suitable period of time.
A suitable solvent mixture is preferably of basic pH. Preferably the base is an aikoxide, more preferably the base is an alkoxide of fewer than 5 carbons and most preferably the base is K+OBu\ A suitable solvent is either protic or aprotic, preferably the solvent is aprotic and even more preferably the solvent is aprotic and anhydrous and most preferably the solvent is THF.
The reaction temperature is preferably between about 0° C and 85° C but more preferably between about 30° C and about 70° C and most preferably at about 55° C.
The reaction time is preferably between about 1 minute and 2 days, more preferably between about 1 hour and 1 day and most preferably between about 8 hours and 14 hours. Under the above discussed preferred conditions the compounds (I) and (III) react forming an intermediate (IV) where n > 0, and g, h, R1, R2, R3, R4, R6 and R13 are as defined above.
IV This intermediate (IV) is optionally chemically derivatized prior to the cyclization and displacement procedure.to form a corresponding intermediate (IV):
IV where the R1 , R2 , R3 and R4 substituents are each independently the same as substituents R1, R2, R3 and R4 respectively if not derivatized or are each independently the chemically derivatized substituents respectively. i 0 Examples of chemical derivatization reactions include, but are not limited to, the following general derivatizations procedures.
Chemical reaction conditions suitable for BOC (t-butyloxycarbonyl) removal/acylation, TMS (trimethylsilyl) or other silicon based alcohol-protecting group removal (FVaqueous acid), Mitsunobu reaction (O. Mitsunobu, Synthesis 1981, 1-28), Suzuki
* 5 coupling (N. Miyaura, A. Suzuki, Chem. Commun. 1979, 866), Horner-Emmons type olefinations (W.S. Wadsworth, Jr., W.D. Emmons, J. Chem. Soc. 83, 1733 (1961)), reductive aminations (Klyeuv and Khidekel, Russ. Chem. Rev. 49, 14-27 (1980)), Sonogashira coupling (S.I. Kahn, M.W. Grinstaff, Tetrahedron Lett. 39, 8031 (1998)), and ester hydrolysis/amidation reaction conditions. Specific examples of the above described chemical derivatization of the intermediate are in the included examples. The BOC protected nitrogen of the intermediate compound of Example ix is deprotected and reacted with acetic anhydride. The TBS (t-butyldimethyl silyl) protected alcohol of the intermediate compound of Example x is deprotected with tetrabutylamonium fluoride and reacted with a chlorophenol under Mitsunobu conditions to give the p-chlorophenyl ether derivatized intermediate. The methylene ester radical at R3 of the intermediate of Example xv is subjected to ester hydrolysis/amidation providing the p-chlorophenyl methylaminocarbonyl methylenamide substituent.
Although optional, examples of preferred derivatizations are as discussed above, and as described in Scheme B.
Scheme B After the optional derivatization of the intermediate, the cyclization and displacement reaction whereby the product (V) is formed.
V In product (V), the R1 , R2 , R3 and R4 substituents are each independently the same as substituents R1, R2, R3 and R4 respectively if not derivatized or are each independently the chemically derivatized substituents respectively. Hence, each of R1', R2 , R3' and R may be correspondingly the same as R1, R2, R3 and R4 or may represent the result of the optional chemical derivatization of the corresponding substituent, prior to cyclization and displacement. Also, chemical derivatization includes other ring formation or ring closure reactions such as where any two of R1 , R , R and R4 together form an aryl or an alkyl ring of between about 5 and 14 atoms. R is amino, hydroxy or the same as R above.
CONDITIONS SUITABLE FOR THE CYCLIZATION AND DISPLACEMENT PROCEDURE
The temperature for the cyclization and displacement procedure may be between about 0° and 85° C but is preferably between about 30° and 70° and is most preferably at about 55° C.
The solvents suitable for the cyclization and displacement procedure include protic and aprotic solvent mixtures, aqueous and anhydrous solvent mixtures. A preferred solvent is TFA, a more preferred solvent mixture is TFA:H2O a most preferred solvent mixture is TFA:5 N HCl/H2O. Although a solvent mixture of TFA:5 N HCl/H2O is most preferred, the ratio of this TFA:5 N HCl/H2O mixture may vary between about 1 :1 to about 99:1 TFA:5 N HCl H2O, but preferably is between about 80:1 to 1 :1 TFA:5 N HCl/H2O, and is most preferably at about 4:1 TFA:5 N HCl H2O.
The time for the cyclization and displacement reaction may vary, but generally is between about 1 minute and 4 days but preferably between about 1 hour and 20 hours.
A second aspect of the invention is directed to a solid support bound intermediate (IV) above. Optionally, the intermediate (IV) can be derivatized before the cyclization and displacement procedure to thereby provide further options for the groups R1, R2, R3 and R4 in the final product. The preferred, yet optional, derivatizations of the intermediate compound (IV), and preferred conditions whereby optional derivatizations occur are as described above. A third aspect of the invention is directed to a library of polycyclic heterocycle compounds where the library contains a plurality of diverse compounds (V):
V In compound (V), where each of R1, R2, R3 and R4 is a stable moiety independently selected from the group consisting of halo, haloalkyl, cyano, nitro, Ra-Q-, Ra-Q-alkyl, Ra-Q-alkenyl, Ra-Q- alkynyl, Ra-Q-arylalkyl and Ra-O-aryl;
Ra is hydrogen, alkyl, aryl or arylalkyl; Q is a single bond, -O-, -NRb-, -CO-, -NRb-CO-, -CO-NRb-, -CO-O-, -O-CO-, S(O)„
-S(O)j-NRb-, -NRb-S(O)j; i = 0, 1 or 2; j = 1 or 2;
Rb is hydrogen, alkyl, aryl or arylalkyl, where Ra and Rb may together with the nitrogen to that they are attached form a ring, and n > 0; and
R7 is selected from the group consisting of alkyl, aryl, arylalkyl, alkyloxy alkyl, aryloxyalkyl, alkylamino, dialkylamino, arylamino, alkoxy carbonyl, amino, alkoxy, hydroxy and heteroaryl; and
R13 is selected from the group consisting of -NH-, -O- and -S-. Preferred substituents for R1 , R2 , R3 , R4 , R7 , and values for n are as described above, in the included examples and the appended claims.
A fourth aspect according to the present invention preferably produces a library of compounds where the compounds comprise a diverse chemical library according to the general methods discussed above. All of the compounds in such a library have a common scaffold, e.g., compound (V). When preparing a combinatorial library according to the present invention, diversity is introduced via the R1 , R2 , R3 , R4 , R7 , and R13 substituents as discussed more fully above. These substituents are selected to allow the creation of a chemically diverse library that, as one goal, maximizes the exploration of molecular spatial properties. Such maximization increases the likelihood of creating compounds that will be biologically active against selected targets.
A fifth aspect of the invention is directed to an assay kit for the identification of biologically active compounds, the kit comprising assay materials and a well plate apparatus where each well in the apparatus contains a compound of the library described above.
PARALLEL SYNTHESIS
The fourth and fifth aspects of the solid support mediated method of the invention may be carried out by way of parallel synthesis in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means. For small-scale synthesis of multiple products, the solid support mediated method of the invention is preferably carried out in containers adaptable to parallel array syntheses. With parallel array synthesis, individual reaction products are prepared in each of multiple reaction zones. The reaction zones are physically separated from one another in a reaction vessel. Compounds can be added to the reaction vessel by multiple delivery apparatus, automated or robotic apparatus, any of that may be either manually or computer controlled. A preferred parallel synthesis embodiment of the present invention is a diverse polycyclic heterocycle compound library in the form of a plurality of wellplates, each wellplate having wells containing a separate reaction product (library compound). In such cases, their wellplate number and "x" column and "y" wellplate row coordinates conveniently identify the library compounds. The process of making the library of polycyclic heterocycle compounds may be conveniently carried out in a conventional wellplate apparatus. It is particularly advantageous to carry out the method of the invention in a standard wellplate apparatus such as a plastic 96 well microtiter plate.
Typically, the wellplate apparatus is in the form of a rigid or semi-rigid plate, the plate having a common surface containing openings of a plurality of reservoirs arranged in rows and columns. A standard form of wellplate apparatus is a rectangular plastic plate having 8 rows and 12 columns (total 96) of liquid retaining depressions, or reservoirs, on its surface. A wellplate apparatus may optionally have other elements of structure such as a top or cover (e.g., plastic or foil), a bottom in a form such as a plate or reservoir, clamping means to secure the wellplate and prevent loss of its contained compounds. The polycyclic heterocycle library of compounds formed using the solid support mediated method aspects of the invention can be used to screen compounds for biological or other activity. Myriad biological assays are known in the art and can be used to screen the polycyclic heterocycle library of compounds.
SCREENING OF POLYCYCLIC HETEROCYCLE DERIVATIVE LIBRARIES The libraries of diverse polycyclic heterocycle according to the solid support mediated method of the present invention (e.g., compounds (II)) may be screened for biological activity. Generally the library to be screened is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibody, and the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined. Typically this will comprise determining whether the biological substance is bound to one or more of the members of the library. Such binding may be determined by attaching a label to the biological substance. Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels. The degree of binding affinity may be determined by quantitating the amount or intensity of the bound label. Thus, various biologically active compounds may be selected by identifying that compounds bind the particular biological substance most effectively.
Illustrative additional assays include but are not limited to in vitro assays such as enzymatic inhibition, receptor - ligand binding, protein - protein interaction, andprotein - DNA interaction; cell based, functional assays such as transcriptional regulation, signal transduction / second messenger, and viral infectivity; add, incubate & read assays such as scintillation proximity assays (SPA), fluorescence polarization assay, fluorescence correlation spectroscopy, colorimetric biosensors, cellular reporter assays using reporter genes such as luciferase, green fluorescent protein, β-lactamase, and the like; and electrical cell impedance sensor assays. All of the above assays are known in the art to be predictive of success for an associated disease state.
EXAMPLES
The following examples are provided as illustration only, and are not intended to limit this invention in any way.
General. Reagents obtained from commercial sources were used without further purification. Kaiser oxime resin 1 was purchased from Novabiochem with a loading capacity of 1.07 mmol/g. All NMR spectra (400 MHz) were recorded on a Varian Gemini- 400 spectrometer. Mass spectra were obtained with either ESI or FAB as the ionization method. All purifications were carried out by radial chromatography (Chromatotron® model 8924, Harrison Research) using 1 mm silica gel plates (Analtech). Crude purities were estimated from integrated peak areas of HPLC chromatographs with the UV detector monitoring at λ = 215 ran. Analytical HPLC setup: C]8 Vydac® column with solvent gradient A = acetonitrile (0.1% TFA) and B = water (0.1% TFA) at 1 ml/min flow-rate. Unless otherwise noted, all HPLC retention times are given for an eluent gradient of 10% A to 60% A over 40 min. The nomenclature of 3 -aminobenzisoxazole compounds is based on the heterocycle numbering system (Shutske, G.M.; Kapples, K.J. J. Heterocyclic Chem. 1989, 26, 1293. For other examples of solution phase intramolecular nucleophilic additions tj nitrile by nitrogen see: Kwok, R.; Pranc, P. J Org. Chem. 1967, 32, 738. Blicke, F.F.; Zarnbito, A.J.; Stenseth, R.E.; J. Org. Chem. 1961, 26, 1826.) shown below:
Table A lists representative moieties that are substituted for R on the designated position of the starting compound. Because the moieties are representative of a general class of organic substituents, it is meant that other organic substituents are chemically equivalent to those given in Table A and are within the scope of the invention and appended claims. For example, where the CF3 radical replaces R , the placement of the same group in position 4, 5 or 6 is an equivalent analogue. Hence, the CF3 placed at each of positions 3, 4, 5, 6 or any combination thereof is within the scope of this invention and appended claims. Furthermore the CF3 group is, optionally, meant to represent an electron withdrawing group and can therefor be replaced by other electron withdrawing groups such as polyhalo-alkyl and be within the scope of this invention.
Although not wanting to be bound by a theoretical explanation of chemical mechanistic analysis, the following proposed mechanism is useful for explaining an embodiment of the invention.
In the proposed mechanism, the solid support bound oxime is reacted with the fluorobenzonitrile of Example 1 under suitable conditions to form the solid support bound intermediate. The solid support bound intermediate is optionally isolated and finally subjected to the cyclization and displacement procedure. As illustrated in Scheme III, the nitrogen and oxygen of the solid support bound oxime are involved in the cyclization and displacement reaction and become part of the polycyclic heterocycle. Table A
Example R % yield of the resin % isolated yield of the
Number bound intermediate corresponding R substituted benzisoxazoles i H 64 76 ii R4=CF3 83 62 iii R3=MeO 80 85 iv R3=CF3 90 86
V R3=Br 90 78 vi R3=CN 69 68 vii R'=CF3 69 75 viii R2=NO2 95 70
Although not limited to these exemplary reactions, it will become apparent to one of skill in the art through these examples and appended claims the broad range of chemical derivations and transformations that can, optionally, be performed on the solid support bound intermediate. The optional chemical derivatization of the solid support bound intermediate according to a broad range of chemistry with neither destruction of the solid support nor diminishment of the derivatized polycyclic heterocycle yield is a surprising result. Indeed, it is within the scope of this invention to allow the optional chemical derivatization of the solid support bound intermediate according to methods known in the art.
Although Table A demonstrates some examples, it is to be understood that the solid support mediated method according to one aspect of this invention includes analogues involving substituents at each available position of the aromatic scaffold, such as a tri or tetra substituted phenyl. Moreover, where R is methoxy, then a d to C12 alkoxy is considered an equivalent analogue. And where the substituent group is Br, then another halo substituent such as -F or -Cl is an equivalent analogue.
The solid support mediated method according to the present invention further allows for the optional chemical derivatization of none, any, or all of R1, R2, R3, R4 substituents of the solid support bound intermediate as provided above. Indeed the solid support bound intermediate is preferably stable to a broad range of reaction conditions.
Table B lists substituted aromatic compounds (I), which are reacted with the solid support bound member, thereby forming the solid support bound intermediate (IV), are chemically derivatized with final cyclization and displacement of the corresponding polycyclic heterocycle.
I The compounds of Table B are synthesized according to the solid support mediated method of the present invention. This list is intended to demonstrate the diversity of compounds that are synthesized according to this invention and is not intended to limit the scope in any way.
Table B
The resin bound intermediates of the present invention were surprisingly stable to conditions suitable for optional derivatizations such as, BOC removal/acylation, TBS removal and Mitsunobu coupling, Suzuki coupling, Sonogashira coupling, Hoerner- Emmons olefination, and ester hydrolysis/amidation reaction conditions. These and other reactions discussed were performed according to the following detailed chemical procedure.
General Synthetic Procedures
The following general synthetic procedures where used to synthesize the above mentioned products. Although the following procedures are specific to the formation of an intermediate and product with a designated "R" group, the desired intermediate and product can be obtained through replacement of the desired "R" group for the one designated in the procedure.
General Example 1
Formation of 3-aminobenzisoxazole : to 500 mg of high loading (1.07 mmol/g, 0.54 mmol)/?-nitrobenzophenone oxime polystyrene resin (obtained from Novabiochem) in a 25 mL capacity Kontes Microfilter Funnel is added 7 mL of THF and 640 μL of potassium t-butoxide (1 M in THF, 0.642 mmol). This was shaken by hand for several minutes whereupon the resin turned a purple color. To this was added 2-fluorobenzonitrile (1.07 mmol, 214 mg) neat. The reaction vessel is placed in an oven fitted with a rotating device for 12 h. The oven was at the most preferred temperature of 55° C. The loaded resin was removed from the oven and allowed to cool for 1 h and rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of 5% TFA/CH2C12> 2 x 5 mL of isopropanol and 4 x 5 mL of MeOH. This was dried in a 35° C vacuum oven for 12 h. Next, 4 mL of TFA and ImL of aqueous 5 N HCI were then added to the resin followed by turning for 2 h in a 55° C oven. The TFA/Η2O was collected and the resin was rinsed with 2 x 5 mL of CH2C12. These were combined and concentrated in vacuo to give the crude product (>98%> purity by reverse phase HPLC) that was radial chromatographed on a 2 mm plate using 25% EtOAc/Hexanes. Concentration ofthe product containing fractions gave pure (35 mg, 2 step yield = 49%, 74% yield based on loading). General Example 2
Formation of 4-amino-l H-2,3-benzoxazine : to 500 mg of high loading (1.07 mmol/g, 0.54 mmol) t nitrobenzophenone oxime polystyrene resin (obtained from Novabiochem) in a 25 mL capacity Kontes Microfilter Funnel was added 7 mL of THF and 640 μL of potassium t-butoxide (1 M in THF, 0.642 mmol). This was shaken by hand for several minutes whereupon the resin turned a purple color. To this was added 2-(l- bromomethyl)benzonitrile (1.07 mmol) neat. The reaction vessel was placed in an oven fitted with a rotating device for 12 h. The oven was at the most preferred temperature of 55° C. The loaded resin was removed from the oven and allowed to cool for 1 h and rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of 5% TFA/CH2C12, 2 x 5 mL of isopropanol 4 x 5 mL of MeOH. This was dried in a 35° C vacuum oven for 12 h. Next, 4 mL of TFA and ImL of aqueous 5 N HCI were then added to the resin followed by turning for 8 h in a 55° C oven. The TFA/H2O was collected and the resin was rinsed with 2 x 5 mL of CH2C12. These were combined and concentrated in vacuo to give the crude product (>98% purity by reverse phase HPLC) that was radial chromatographed on a 2 mm plate using 25% EtOAc/Hexanes. Concentration of the product containing fractions gave pure product.
General Example 3 Formation of 3-amino-6- (N-acetylaminomethyl) benzisoxazole: to 500mg of high loading (1.07 mmol/g, 0.54 mmol) jo-nitrobenzophenone oxime polystyrene resin (obtained from Novabiochem) in a 25 mL capacity Kontes Microfilter Funnel was added 7 mL of THF and 640 μL of potassium t-butoxide (1 M in THF, 0.642 mmol). This was shaken by hand for several minutes whereupon the resin turned a purple color. To this was added 4-(N-BOC-aminomethyl)-2-fluorobenzonitrile (1.07 mmol, 214 mg) neat. This reaction vessel was placed in a 55 °C oven fitted with a rotating device for 12 h. This was removed from the oven and allowed to cool for 1 h and rinsed with 2 x 5 mL of CH2C12> 2 x 5 mL of isopropanol, and 4 x 5 mL of isopropanol, and 4 x 5 mL of MeOH. This was dried in a 35 °C vacuum oven for 12 h. BOC-deprotection was achieved using 25% TFA/CH2C12 (7 mL) followed by shaking for 2 h. Resin was again rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of isopropanol, and 4 x 5 mL of MeOH. The resin was suspended in DMF followed by addition of acetic anhydride and diisopropylethylamine. This was allowed to shake for 3h and was rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of isopropanol, and 4 x 5 mL of MeOH. 4 mL of TFA and 1 mL of aqueous 5 N HCI were then added to the resin followed by turning for 2 h in a 55° C oven. The TFA/H2O was collected and the resin was rinsed with 2 x 5 mL of CH2C12. These were combined and concentrated in vacuo to give the crude benziosoxazole product (98%> purity by reverse phase HPLC) that was radial chromatographed on a 2 mm plate using 25% EtOAc/Hexanes. Concentration of the product containing fractions gave pure benzisoxazole product (35 mg, 4 step yield = 49%).
General Example 4
Formation of 3-amino-6- (p-chlorophenoxymethyl) benzisoxazole: to 500mg of high loading (1.07 mmol/g, 0.54 mmol) /?-nitrobenzophenone oxime polystyrene resin (obtained from Novabiochem) in a 25 mL capacity Kontes Microfilter Funnel was added 7 mL of THF and 640 μL of potassium t-butoxide (1 M in THF, 0.642 mmol). This was shaken by hand for several minutes whereupon the resin turned a purple color. To this was added 4-(tert-butyldimethylsilyloxymethyl)-2-fluorobenzonitrile (1.07 mmol, 214 mg) neat. This reaction vessel was placed in a 55 °C oven fitted with a rotating device for 12 h. This was removed from the oven and allowed to cool for 1 h and rinsed with 2 x 5 mL of CH2C12> 2 x 5 mL of isopropanol, and 4 x 5 mL of MeOH. This was dried in a 35 °C vacuum oven for 12 h to give a Δwt = 82 mg (63%> loading yield).TBS-deprotection was achieved using 1 M TBAF in THF (1.07 mL, 2 equiv.) followed by shaking for 18 h. The resin was rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL H2O, 4 x 5 mL of MeOH. This was dried in a 35° C vacuum oven for 12 h. The resin was suspended in 8 mL of 1 : 1 THF/CH2C12 followed by addition of triphenylphosphine (700mg, 5 equiv.) and 4- chlorophenol (690 mg, 10 equiv.). This was shaken by hand followed by the slow addition of diisopropyl azodicarboxylate (0.53mL 5 equiv). This was allowed to shake for 3h and was rinsed with 2 x 5 mL of MeOH, 2 x 5 mL H2O, and 4 x 5 mL of MeOH. 4 mL of TFA and 1 mL of aqueous 5 N HCI were then added to the resin followed by turning for 2 h in a 55 °C oven. The TFA/H2O was collected and the resin was rinsed with 2 x 5 mL of
CH2C12. These were combined and concentrated in vacuo (68% purity by reverse phase HPLC) that was radial chromato graphed on a 2 mm plate using 25%> EtOAc/Hexanes. Concentration of the product containing fractions gave pure benzisoxazole (49 mg, 4 step yield = 33%).
General Example 5
Loading Reaction. Nucleophilic aromatic substitution reactions have been extensively studied in solution. (Nudelman, N; Mancini, P.M.E.; Martinez, R.D.; Vortero, L.R. J. Chem. Soc. Perkin Trans. 2 1987, 951. Miller, j. "Aromatic Nucleophilic Substitution"; Elsevier: Amsterdam, 1968. Bernasconi, C.F. Ace. Chem. Res. 1978, 11, 147) However, the extent to that this precedent could be applied to predict the outcome of these heterogenous reactions was not clear given the importance of resin swelling properties, site accessibility, etc. (Yan, B; Fell J. B.; Gnanasambandam, K. j. Org. Chem. 1996, 61, 7467.) Therefore, we studied the effect of the solvent, leaving group, and counterion on the solid phase loading reaction (Table 1). In comparing THF, MeCN, DMF, and DMSO, THF gave the best results, providing a 68%> loading yield with 2-fluorobenzonitrile (2a) and 72% with 2-nitrobenzonitrile (2b). (Palermo, M.G. Tetrahedron Lett. 1996, 37, 2885.) A 20 to 30% decrease in the loading yield was observed for both 2a and 2b when DMF or DMSO was used as the solvent. Loading yields also decreased dramatically with the use of chloro- (2c), bromo- (2d), and iodo- (2e) benzonitriles.
The effect of the counterion on the loading reaction was evaluated with 2- fluorobenzonitrile as the substrate in THF (Table 1, entry 1). The potassium salt of resin 1 gave the highest loading yields with potassium tert-butoxide (KOBu1) giving slightly better results than potassium hexamethyldisilazide (KHMDS). Treatment of 1 with sodium hexamethyldisilazide (NaHMDS), gave a loading yield of 42% and, interestingly, no loading was observed with lithium hexamethyldisilazide (LiHMDS).
As described above, a variety of 2-fiuorobenzonitriles can be loaded on the Kaiser resin (Lepore, S.D.; Wiley, M.R. J. Org. Chem. 1999, 64, 4547.). Although the presence of an electron withdrawing group facilitates loading at room temperature, substrates bearing either electron withdrawing groups or electron donating groups can be loaded in high yield in about 2 h at 55 °C. We also found that the loading reaction of 2-fluorobenzonitriles is not particularly sensitive to steric hindrance around the site of nucleophilic substitution
Table 1. Effect of leaving group, solvent, and counterion on loading reactions.
% loading yield of 3a entry X base THF MeCN DMF DMSO
F KOBu1 68 13 41 47
KHMDS 55
NaHMDS 42
LiHMDS <5
NO2 KOBu1 72 28 41 54
Cl KOBu1 <5 <5 <5 15
Br KOBu1 <5
KOBu1 <5
determined by resin weight difference (average of 3 experiments)
General Example 6
Studies on the acid stability of the aryl oxime linker. As previously reported, the use of 4: 1 TF A/aqueous 5 N HCI at 55 °C for 2 h (Table 2, Method A) led to an efficient cyclorelease of a variety of substituted aminobenzisoxazoles. By contrast, we saw that the use of 99:1 TFA/H2O at 55 °C (Table 2, Method B) with intermediates bearing either inductively neutral or electron withdrawing substituents required significantly longer reaction times to reach completion. For example, hydrolysis of the resin bearing a bromo substituent para to the nitrile (Table 2, entry 2) using Method B required 4 days to give the corresponding aminobenzisoxazole 4b in 65% yield. On the other hand, treatment of the /j rα-methoxybenzonitrile derivative 3c (Table 2, entry 3), under the same conditions, gave the methoxy-substituted product 4c in 87 %> isolated yield after only 2 h.
While the studies summarized above were important for identifying useful cyclorelease conditions, further experiments were performed in order to determine the acid stability profile of the linker under a variety of conditions commonly used for the removal of acid-sensitive protecting groups. The methoxy-substituted resin 3c was selected for this study, since it is the most acid labile and therefore represents the worst case scenario. Upon treatment of the methoxy-substituted resin 3c with 25% anhydrous TFA/CH2C12 at room temp for 2 h (Table 2, entry 3, Method C), conditions precedented for on-resin Boc removal, <5% of the cyclization product 4c was removed from the resin. Even under more forcing conditions, with 100 % anydrous TFA at 55 °C (Table 2, entry 3, Method D) only 9% of the 3 -aminobenzisoxazole product (>96%> purity) was removed from the resin after 2 h. The aryl oxime linker was also stable to a number of milder aqueous acidic conditions that have been used for the removal of THP, silyl, and acetal protecting groups in solution. Thus, treatment of the resin 3c with AcOH/THF/H2O (3/1/1) at 55 °C for 12h (Corey, E.J.; Venkateswarlu, A. J. Am. Chem. Soc, 1972, 94, 6190.) (Table 2, entry 3, Method E), or with TsOH/THF/H2O at 55 °C for 12h (Thomas, E.J.; Williams, A.C. J. Chem. Soc. Chem. Comm., 1987, 992.) (Table 2, entry 3, Method F) gave no detectable release of material from the resin.
Table 2. Acid stability profile of the aryl oxime linker.
entry X n pri odiuπrc-tt m ^etho^d" („ yipeuldr b ity)
"Method A = 4: 1 TFA/5N HCI, 55 °C. Method B = 99: 1 TFA/H20, 55 °C. Method C = 25% THF/CH2C12, room temp. Method D = TFA, 55 °C. Method E = AcOHΛTHF/H20, 55 °C. Method F = TsOH/THF/H20, 55 °C. bIsolated yield after chromatography and based on loading. Crude purity based on HPLC analysis.
General Example 7
Amide bond formation. Due to the importance of solid phase peptide synthesis, amide bond forming reactions have been the most widely performed and most highly developed solid phase reactions. (Kaldor, S.W.; Siegel, M.G. Comb. Chem. Mol. Diversity Drug Discovery 1998, 307-335. Editors: Gordon, Eric M.; Kerwin, James F., Jr. Publisher: Wiley-Liss, New York, N. Y.) We have previously shown above that amides can be formed in the presence of an aryl oxime linker by the reaction of an acid chloride or an acid anhydride with an aryl oxime-linked benzylamine. In the present study, we set out to demonstrate that an aryl oxime-linked acid could be coupled to an amine under standard peptide coupling conditions. By proceeding through the intermediacy of methyl ester 5, we also sought to demonstrate the compatibility of the aryl oxime linker with the aqueous basic conditions required for saponification (Scheme 2). Thus, the potassium anion of the Kaiser resin 1 as coupled with methyl 3-fluoro-4-cyanobenzoate to give resin 5 in a 69% loading yield. Treatment of resin 5 with LiOH, in THF/MeOH/H2O (3/1/1) at room temperature gave the corresponding acid (6) and no removal of the substrate from the resin was observed, demonstrating the stability of the aryl oxime linker under these conditions. Acid 6 was then coupled to 4-chlorobenzylamine to give the on-resin amide. Both the resin weight increase and chlorine analysis of the intermediate suggested that the coupling reaction went essentially to completion within 12 h. The resin was then treated with the standard cyclorelease conditions to give the desired amide 7 in a 3 step yield of 81% (93% crude purity).
Scheme 2. On-resin hydrolysis and amide bond formation.
isolated yield = 81% crude purity = 93%
Synthesis of -[(p-chlorophenyl)methylaminocarbonyl]-3-amino-l,2- benzisoxazole (7): to -niιr benzophenone oxime polystyrene (Kaiser) resin (500 mg, 1.07 mmol/g, 0.54 mmol) in a tared 25 mL Kontes Microfilter Funnel was added THF (7 mL) a:ιd potassium t-butoxide (640 μL, 1 M in THF, 0.642 mmol). After shaking by hand for several minutes, the resin turned a deep purple color. To this suspension was added 2- fluoro-<-A,nethoχ- 'caιbony'.)benzonitrile (1.07 mmol, 192 mg). The reaction vessel was rotated at 55 °C in a Robbins oven for 12 h to give resin 5 followed by cooling for 1 h.
The resin was then rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h to give a Δwt = 50.0 mg (58%o loading yield). To effect ester hydrolysis, the resin was then su: pended in THF (7 mL) followed by the addition of LiOH (39 mg, 1.61 mmol) dissolved in MeOH/H2O (1:1, 2 mL) and rotated at room temp, for 12 h. The resin was then rinsed with 2 5 ml, of CH2C12, 2 x 5 mL of MeOH, 2 5 mL of H20, 2 x 5 mL of MeOH, and 2 x 5 mL of DMF. The resin was then suspended in DMF (7 mL) and to this was added p- chlorobenzylamine (261 μL, 2.14 mmol), 1 -hydroxybenzotriazole hydrate (HOBt) (289 mg, 2.14 mmol), benzotriazole-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (947 mg, 2.14 mmol), and diisopropylethylamine (DIPEA) (467 μL, 2.68 mmol). The reaction was allowed to proceed for 12 h at room temp. The resin was then rinsed 2 5 mL of CH2C12, 2 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h to give a Δwt = 84 mg. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 5 mL of CH2C12. These washings were combined and concentrated in vacuo to 5 give the crude product 7 (93%) purity by reverse phase HPLC) that was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes. Concentration of the product containing fractions gave pure 6-[(p-chlorophenyl)-methylaminocarbonyl]-3- amino-l,2-benzisoxazole (7) (76 mg, 3 step yield = 81% based on the ester loading yield): HPLC retention time (eluent gradient 10%A to 60%A over 45 min) = 24.9 min. Η NMR 0 (400 MHz, DMSO-d6) δ 9.19 (t, J= 5.6 Hz, IH), 7.84 - 7.91 (m, 2H), 7.72 (dd, J= 8.0, 1.2 Hz, IH), 7.37 - 7.31 (m, 4H), 6.50 (bs, 2H), 4.45 (d, J= 6.0 Hz, 2H). MS (ESI) m/z 300 [35C1, (M+H)+], 302 [37C1, (M+H)+]. HRMS Calcd for Cl7Hι5N2O3 295.1083, found 295.1079.
5 General Example 8
Phenolic Mitsunobu reaction. On-resin carbon-oxygen bond formation via the Mitsunobu reaction has been identified as an important tool in combinatorial chemistry (Kaldor, S.W.; Siegel, M.G. Comb. Chem. Mol. Diversity Drug Discovery 1998, 307-335. .dito s: Gordon, Eric M.; Kerwin, James F., Jr. Publisher: Wiley-Liss, New York, N. Y). ( Application of this reaction to an aryl oxime-linked substrate is shown in Scheme 3. Resin 8 was prepared by reacting the potassium anion of the Kaiser resin 1 with 2-fluoro-4-(t- butyl-dimethylsilyloxymethyl)-benzonitrile. The TBS-protecting group was removed using TBAF in THF. The on-resin alcohol was then treated with p-chlorophenol, triphenybhosphine and diisopropylazodicarboxylate (DIAD) in THF. (Krchnak, V.; 5 Flegelova, Z.; Weichsel, A.S.; Lebl, M. Tetrahedron Lett. 1995, 38, 3345.) The best results were observed for reactions times of 3 h. Longer reaction times generally led to decreased purity in the crude cyclization product. Cyclitive removal using the standard conditions then gave aryl ether 9 in a 77%> yield (3 steps) and 83% crude purity.
0 Scheme 3. On-resin Mitsunobu reaction.
Synthesis of 6-[(p-chlorophenyl)oxymethyI]-3-amino-l,2-benzisoxazoIe (9): to t> nitrobenzophenone oxime polystyrene (Kaiser) resin (500 mg, 1.07 mmol/g, 0.54 mmol) in a tared 25 mL Kontes Microfilter Funnel was added THF (7 mL) and potassium t-butoxide (640 μL, 1 M in THF, 0.642 mmol). After shaking by hand for several minutes, the resin turned a deep purple color. To this suspension was added 2-fluoro-4-(tert- butyldimethylsilyloxymethly)-benzonitrile (1.07 mmol, 285 mg). The reaction vessel was rotated at 55 °C in a Robbins oven for 12 h to give resin 8 followed by cooling for 1 h. The resin was then rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 L of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h to give a Δwt = 71.1 mg (54%o loading yield). To effect TBS removal, the resin was then suspended in THF (6 mL) followed by the addition of TBAF (562 μL, 1 M in THF, 0.562 mmol) and rotated at room temperature for 12 h. The resin was then rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, 2 x 5 mL of MeOH, and 2 x 5 mL of CH2C12. The resin was then suspended in CH2C12 (7 mL) and to this was added -chlorophenol (690 mg, 5.35 mmol), triphenylphosphine (700 mg, 2.68 mmol), and diisopropyl-azodicarboxylate (DIAD) (530 μL, 2.68 mmol). The reaction was allowed to proceed for 1 h at room temp. The resin was then rinsed 2 5 mL of CH2C12, 2 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 x 5 mL of CH2C12. These washings were combined and concentrated in vacuo to give the crude product 7 (83% purity by reverse phase HPLC) that was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes. Concentration of the product containing fractions gave pure 6-[(p-chlorophenyl)oxymethyl]-3-amino-l,2-benzisoxazole (9) (61 mg, 3 step yield = 77% based on loading yield): HPLC retention time (eluent gradient 10%A to 60%A over 45 min) = 37.5 min. 1H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J = 8.0 Hz, IH), 7.84 - 7.91 (m, 2H), 7.72 (dd, J= 8.0, 1.2 Hz, IH), 7.44 (s, IH), 7.30 (d, J= 8.0 Hz, IH), 7.25 - 7.21 (m, 2H), 6.99 - 6.95 (m, 2H), 5.17 (bs, 2H), 1.22 (d, J= 6.4 Hz, 2H). MS (ESI) m/z 274 [35C1, (M+H)+], 276 [37C1, (M+H)+]. Anal Calc'd for Cι4H,ιClN2O2: C, 61.21; H, 4.04; N, 10.2; Cl, 12.91. Found: C, 61.08; H, 4.32; N, 10.19; Cl, 12.47.
General Example 9
On-resin nucleophilic aromatic substitution. Recently, examples of on-resin nucleophilic aromatic displacement reactions have appeared in the literature. (Wijkmans, J.C.H.M.; Culshaw, A.J.; Baxter, A.D. Mol. Diversity 1998, 3, 117.) In order to evaluate the compatability of the aryl oxime linker with this reaction, a resin-attached fluorobenzonitrile (10) was prepared (Scheme 4). In order to avoid any regioselectivity issues in the loading reaction, the symmetrical 2,6-difluorobenzonitrile was chosen for this initial investigation. Resin 10 was then reacted with a variety of nucleophiles including an alkoxide (Plenkiewicz, H.; Dmowski, W. J Fluorine Chem. 1998, 89, 213.), amine (Morales, G.A.; Corbett, J.W.; DeGrado, W.F. J. Org. Chem. 1998, 63, 1172. Vojkovsky, T.; Weichsel, A.; Patek, M. J. Org. Chem. 1998, 63, 3162. Dankwardt, S.M.; Newman, S.R.; Krstenanski, J.L. Tetrahedron Lett. 1995, 36, 4923.), and phenol (Kiselyov, A.S.; Eisenberg, S.; Luo, Y Tetrahedron Lett. 1999, 40, 2465.. Burgess, K.; Lim, D.; Bois-Choussy, M.; Zhu, J. Tetrahedron Lett. 1997, 38, 3345.)) (Table 3). The potassium alkoxide of 4-chlorophenethanol (Table 3, entry 1) was prepared by treatment with 2 equivalents potassium t-butoxide. This salt was then added to a THF suspension of resin 10 to give the corresponding product 11a after cyclorelease in a two step yield of 50%.
Scheme 4. Regioselective synthesis of 6-fluoro-3-aminobenzisoxazole.
, crude purity > 96% isolated yield = 74%
Table 3. On-resin nucleophilic aromatic substitution.
isolated yield after chromatography and based on loading.
Crude purity based on HPLC analysis. bMajor impurity was the unreacted fluorine product
(4-fluoro-3-aminobenzisoxazole)
Addition of pyrrolidine to 1C proved more challenging (Table 3, entry 2). In our initial attempt (THF, 6 h, 55 °C) the desired product lib was produced in low isolated yield as a result of incomplete displacement. The major impurity in this reaction was 4-fluoro-3- aminobenzisoxazole, the cyclization product of the unreacted starting material (10). In an attempt to increase the rate of the SNAΓ reaction in THF, variations in the reaction time, number of equivalents of nucleophile, and the temperature were evaluated. Significant improvements were obtained with the use of alternate solvents. Although only a slight improvement was observed with acetonitrile (studies on the fluorine kinetic isotope effect in S AΓ reactions with aiyl fluorides have shown that the use of acetonitrile as the reaction solvent leads to a different rate limiting step in the overall mechanism when compared with THF. Persson, J.; Axelsson, S.; Matsson, O. J. Am. Chem. Soc. 1996, 118, 20.), both DMF and DMSO produced a significant increase in the reaction rate. After 6 h at 55 °C in DMSO, followed by the cyclorelease reaction, compound lib was obtained in 94 % crude purity and subsequently isolated in 80 % yield. Similar to the pyrrolidine SNAΓ reaction discussed above, the nucleophilic addition of phenol to resin 10 (Table 3, entry 3) proceeded at a faster rate in DMSO and DMF compared with THF. However, even in these solvents, the addition reaction required 36 h to bring about high conversion of the phenol adduct. Aryl ether lie was then obtained in 48%) yield (2 steps) and 93% crude purity after cyclitive removal. In order to explore the question of regioselectivity, the loading reaction was next attempted with 2,4-difluorobenzonitrile. As illustrated in Scheme 4, this reaction could lead either to the desired aryloxime intermediate 12 through displacement of the 2-fluoro group, or to an undesired isomer 13 through displacement of the 4-fluoro group. Treatment of the unsymmetrical nitrile with the potassium salt of resin 1 gave a 85% loading yield based on weight. Although the ratio of 12 to 13 was not determined, the presumed mixture was then hydrolyzed under the standard cyclization conditions to yield a single product 14a (as determined by HPLC and NMR) in 74% isolated yield. The high selectivity observed for the formation of the desired product could arise from either of two pathways. On one hand, the resin loading reaction may be quite selective for the 2- position, giving primarily 12, and then subsequently 14a upon cyclorelease. Alternatively, mixtures of the two isomeric aryloxime adducts could be formed with the hydrolysis of intermediate 12 occurring much more rapidly than 13. In order gain insight into the relative contributions of these two pathways, several experiments were performed. Scheme 5 depicts a solution phase model study for the loading reaction of 2,4- difluorobenzonitrile. In this experiment, the potassium salt of acetone oxime was treated with 2,4-difluorobenzonitrile and showed only a slight preference for the addition to the 2- position (1.4: 1 ). (For related regioselectivity studies on solution phase SNAr reactions, see (a) Wells, K.M.; Shi, Y.J.; Lynch, J.E.; Humphrey, G.R.; Volante, R.P.; Reider, P.J.; Tetrahedron Lett., 1996, 37, 6439. (b) Sasajima, K.; Ona, K.; Katsube, J. ; Yamamoto, H.; Chem. Pharm. Bull, 1978, 26, 2502.) Obviously such poor selectivity, if produced in the solid phase loading reaction, could not account for the >96% purity of cyclorelease product 14a.
Scheme 5. Solution phase model of the loading reaction of 2,4-difluorobenzonitrile.
1.4 : 1
Scheme 6 illustrates a comparison of the relative rates of hydrolysis for analogous 2- vs 4-substituted aryloximes. Since the isolation of pure 12 and pure 13 was not practical, model resins 3a and 15 were prepared from 2-fluorobenzonitrile and 4- fluorobenzonitrile respectively. As described previously, when resin 3a was treated with the standard cyclitive removal conditions, complete conversion to 3-aminobenzisoxazole was observed after 2h.
Scheme 6. Comparison of the relative rates of oxime hydrolysis for 2- versus 4- substituted aryl oximes.
no reaction
IR analysis of the recovered resin after hydrolysis showed complete disappearance of the nitrile peak, which had clearly been present in the starting material. Alternatively, upon exposure of the isomeric resin 15 to the same hydrolysis conditions, no organic material was released from the solid-phase. In this experiment, IR analysis of the recovered resin confirmed that the nitrile remained intact, apparently unaffected by exposure to the aqueous acid.
These observations led us to reexamine the reaction shown in Scheme 4. This time, resin 12 and 13 were recovered after the cyclorelease reaction was complete, and characterize by IR spectroscopy. The analysis revealed that a nitrile peak was still present on the resin, although the intensity of the nitrile was diminished relative to that observed prior to hydrolysis. These data support the hypothesis that the difference in the hydrolysis rates of intermediate 12 vs 13 serves as the primary source of selectivity in this reaction.
Having successfully achieved the selective functionalization of 2,4- difluorobenzonitrile a variety of additional unsymmetrical polyfluorobenzonitriles were carried through the same sequence. The results are presented in Table 4. In all cases tested, HPLC analyses of the crude reaction mixtures confirmed that the desired product is selectively released from the solid phase.
Table 4. Regioselective synthesis of fluoro-3 -amino-benzisoxazoles. eatiy ekctoφhife ^ product ^jj^
CN
N
66 27 (78) F F 14e
"Isol^dyMl sltercliTOmto^sp y' aiid' ise oii loi iig. Crude puriy based an. HPL C anafysis
Synthesis of resin 10: to / nitrobenzophenone oxime polystyrene (Kaiser) resin (500 mg, 1.07 mmol/g, 0.54 mmol) in a tared 25 mL Kontes Microfilter Funnel was added THF (7 mL) and potassium t-butoxide (640 μL, 1 M in THF, 0.642 mmol). After shaking by hand for several minutes, the resin turned a deep purple color. To this suspension was added 2,6-difluorobenzonitrile (150 mg, 1.07 mmol). The reaction vessel was rotated at 55 °C in a Robbins oven for 8 h to give resin 10 followed by cooling for 1 h. The resin was then rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 * 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h to give a Δwt = 40.1 mg (63% loading yield).
Synthesis of 4-[2-(p-chlorophenyl)ethoxy]-3-amino-l,2-benzisoxazole (11a): In a separate vial, 2-(p-chlorophenyl)ethanol (128 μL, 1.07 mmol) was dissolved in 1 mL of THF followed by the addition of potassium t-butoxide (1.07 mL, 1 M in THF, 1.07 mmol). This alkoxide solution was then added to a THF suspension (6 mL) of resin 10 (540.1 mg, assume 0.535 mmol). The reaction vessel was rotated for 12 h in a 55 °C oven. The resin was then rinsed 2 x 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 3 h. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 5 mL of CH2C1 . These washings were combined and concentrated in vacuo to give the crude product 11a (>96% rurity by reverse phase HPLC) which was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes. Concentration of the product containing fractions gave pure 6-[(p-chlorophenyl)oxymethyl]-3-amino-l,2-benzisoxazole (11a) (66 rrnv, 2 step yield = 68% based on loading yield): HPLC retention time (eluent gradient 10%A to 80%A over 45 min) = 31.8 min. 1H NMR (400 MHz, DMSO-d6) δ 7.39 - 7.31 (m, 5H), 6.93 (d, J= 8.4 Hz, 2H), 6.69 (d, J= 8.4, IH), 5.72 (bs, 2H), 4.30 (t, J= 6.4 Hz, 2H), 3.12 (t, J= 6.4 Hz, 2H). MS (ESI) m/z 289 [35C1, (M+H)+], 291 [37C1, (M+H)+]. Anal Calc'd for C15Hι3ClN2O2: C, 62.41; H, 4.54; N, 9.70; Cl, 12.28. Found: C, 62.08; H, 4.61; N, 9.53; Cl, 12.41.
Synthesis of 4-pyrrolidino-3-amino-l,2-benzisoxazole (lib): to a DMSO suspension (7 mL) of resin 10 (540.1 mg, assume 0.535 mmol) was added pyrrolidine (134 μL, 1.61 mmol). The reaction vessel was rotated for 8 h in a 55 °C oven. The resin was then rinsed 2 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 3 h. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 x 5 mL of CH2C12. These washings were combined and concentrated in vacuo to give the crude product lib (94%> purity by reverse phase HPLC) which was purified by radial chromatography on a 2 mm plate eluting with 50%) EtOAc/Hexanes. Concentration of the product containing fractions gave pure 4- pyrrolidino-3-amino-l,2-benzisoxazole (lib) (54 mg, 2 step yield = 80%) based on loading yield): HPLC retention time (eluent gradient 5%A to 40%A over 45 min) = 27.9 min. 1H NMR (400 MHz, DMSO-d6) δ 7.27 (t, J= 8.0 Hz, IH), 6.84 (d, J= 8.0 Hz, IH), 6.55 (d, J = 7.6, IH), 5.62 (bs, 2H), 3.27- 3.20 (m, 4H), 1.92 - 1.83 (m, 4H). MS (ESI) m/z 204 (M+H)+. Anal Calc'd for CnH13N3O: C, 65.01; H, 6.45; N, 20.67. Found: C, 64.46 H, 6.22; N, 20.11.
Synthesis of 4-phenoxy-3-amino-l,2-benzisoxazole (lie): to a DMF suspension (7 mL) of resin 10 (540.1 mg, assume 0.535 mmol) was added phenol (503 mg, 5.35 mmol) and K2CO3 (738 mg, 5.35 mmol). The reaction vessel was rotated for 36 h in a 55 °C oven. The resin was then rinsed 2 x 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 3 h. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 x 5 mL of CH2C12. These washings were combined and concentrated in vacuo to give the crude product lie (93% purity by reverse phase HPLC) which was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes. Concentration of the product containing fractions gave pure 4-phenoxy-3 -amino- 1 ,2-benzisoxazole (lie) (38 mg, 2 step yield = 48%) based on loading yield): HPLC retention time (eluent gradient 10%A to 90%A over 45 min) = 23.7 min. Η NMR (400 MHz, DMSO-d6) δ 7.44 (t, J= 7.6 Hz, 2H), 7.36 (t, J= 9.6 Hz, IH), 7.26 - 7.15 (m, 3H), 7.10 (d, J= 8.4 Hz, IH), 6.36 (d, J= 7.6 Hz, IH), 6.02 (bs, 2H). MS (ESI) m/z 227 (M+H)+. Anal Calc'd for Cι3H10N2O2: C, 69.02; H, 4.46; N, 12.38. Found: C, 69.21 H, 4.06; N, 12.75.
General procedure for the synthesis of fluoro-3-amino-l,2-benzisoxazoles 14a - f: to 7-nitrobenzophenone oxime polystyrene (Kaiser) resin (500 mg, 1.07 mmol/g, 0.54 mmol) in a tared 25 mL Kontes Microfilter Funnel was added THF (7 mL) and potassium t-butoxide (640 μL, 1 M in THF, 0.642 mmol). After shaking by hand for several minutes, the resin turned a deep purple color. To this suspension was added 2,4- difluorobenzonitrile (150 mg, 1.07 mmol). The reaction vessel was rotated at 55 °C in a Robbins oven for 12 h followed by cooling for 1 h. The resin was then rinsed with 2 x 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h to give a Δwt = 52.4 mg (90%> loading yield). An IR analysis of the resin shows a nitrile stretching peak at 2230.3 cm"1. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 x 5 mL of CH2C12. An IR analysis of the resin after the cyclorelease reaction shows a diminished nitrile stretching peak. The CH2C1 washings were combined and concentrated in vacuo to give the crude product 14a (>96% purity by reverse phase HPLC) that was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes. Concentration of the product containing fractions gave pure 6-fluoro-3-aminobenzisoxazole (14a): (40mg, 2 step yield = 74% based on loading yield): HPLC retention time (eluent gradient 10% A to 60%A over 45 min) = 13.3 min. 1H NMR (400 MHz, DMSO-d6) δ 7.81 (dd, J= 8.8, 5.2 Hz, IH), 7.36 (dd, J= 9.6, 2.0 Hz, IH), 7.11 (ddd, J= 11.2, 8.8, 2.4 IH), 6.44 (bs, 2H).
MS (FD) m/z 152 M+. Anal Calc'd for C7H5FN2O: C, 55.27; H, 3.31; N, 18.41. Found: C, 55.21 H, 3.27; N, 18.20.
7-fluoro-3-aminobenzisoxazole (14b): (30mg, 2 step yield = 56%> based on loading yield): >96% crude purity. HPLC retention time (eluent gradient 5%A to 40%A over 45 min) = 20.1 min. Η NMR (400 MHz, DMSO-d6) δ 7.63 (dd, J= 8.4, 1.2 Hz, IH), 7.40 (ddd, J= 11.6, 8.4, 0.8 Hz, IH), 7.21 (td, J= 8.0, 4.4 IH), 6.58 (bs, 2H). MS (FD) m/z 152 M+. Anal Calc'd for C7H5FN2O: C, 55.27; H, 3.31; N, 18.41. Found: C, 55.19 H, 3.45; N, 18.36.
6,7-difluoro-3-aminobenzisoxazole (14e): (23mg, 2 step yield = 27% based on loading yield): 78% crude purity. HPLC retention time (eluent gradient 5%A to 40%A over 45 min) = 27.6 min. 1H NMR (400 MHz, DMSO-d6) δ 7.65 - 7.61 (m, IH), 7.36 - 7.30 (m, IH), 6.62 (bs, 2H). MS (FD) m/z 171 M+. HRMS Calcd for C7H4F2N2O 171.0370, found 171.0368. 4,6-difluoro-3-aminobenzisoxazole (14f): (22mg, 2 step yield = 31% based on loading yield): >96%> crude purity. HPLC retention time (eluent gradient 5%A to 40%A over 45 min) = 21.1 min. 1H NMR (400 MHz, DMSO-d6) δ 7.31 (dd, J= 8.8, 1.2 Hz, IH), 7.10 (td, J= 10.0, 2.0 Hz, IH), 6.34 (bs, 2H). MS (FD) m/z 171 M+. Anal Calc'd for C7H4F2N2O: C, 49.42; H, 2.37; N, 16.47. Found: C, 49.36 H, 2.38; N, 16.39.
General Example 10
Carbon-carbon bond forming reactions. Compatibility with versatile methods for forming carbon-carbon bonds on-resin is an important measure of the suitability of any new linker. (Hanessian, S.; Xie, F.; Tetrahedron Lett., 1998, 39, 737.) Our initial efforts focused on the application of catalytic palladium coupling chemistry such as the Suzuki and Sonogashira reactions. Thus, the arylbromide resin 3b was prepared (Lepore, S.D.; Wiley, M.R. J. Org. Chem. 1999, 64, 4547.) and reacted with phenylboronic acid under a variety of palladium catalyzed coupling conditions, then hydro lyzed to give the biaryl product 16a (Table 5). Initially, two sets of conditions that have previously been reported for on-resin Suzuki reactions were evaluated. Use of Pd(PPh3)4 with triethylamine/DMF for 12 h (Han, Y.; Walker S.D.; Young, R.N.; Tetrahedron Lett., 1996, 37, 2703. Ruhland, B.; Bombrun, A.; Gallop, M.A.; J. Org. Chem., 1997, 62, 7820.) produced 16a, but led to the formation of numerous biproducts. Alternatively, the use of Pd(PPh3)4, with Na2CO3 in 1 : 1 DME/H2O at reflux for 12 h (Frenette, R. ; Friesen, R. W. ; Tetrahedron Lett. , 1994, 35, 9177.), produced 16a in 25 % isolated yield, contaminated only by the cyclization product of the unreacted arylbromide. Due to the superior purity observed with aqueous carbonate, optimization efforts focused on these conditions. Numerous reaction parameters were carefully varied including cosolvent (DMF, DMSO, and THF) and the stoichiometry of the catalyst, boronic acid and sodium carbonate. Although the crude purities were similar for the various solvents which were evaluated, in general THF (Backes, B.J.; Ellman, J.A.; J. Am. Chem. Soc, 1994, 116, 11171) was found to give the highest rate of product formation, and therefore the highest isolated yields. Optimal results were obtained with 1.3 equivalents of 2N Na2CO3, at 55 °C, over 36h. The reaction was rather sensitive to deviation from these conditions, particularly with respect to the amount of added 2N Na2CO3. In control experiments, resin 3b was treated with varying amounts of 2N Na CO3 in THF at 55 °C over 36h, and no removal of organic material from the resin was observed. As Table 5 shows, the application of the optimized Suzuki conditions to several other substrates (Table 5, entries 2 - 4) provided similar yields and purities. Table 5. On-resin Suzuki reaction.
boronic , . entry acid product method3 yieldb (% purity)
"Conditions A - D all use 4.0 eq of boronic acid and 5% Pd(PPh3)4, and 36 h.
A = 2.5 eq 2 M Na2C03, DME, reflux; B = 1.0 eq 2 M Na2C03, THF, 55 °C;
C = 1.3 eq 2 M Na2C03, THF, 55 °C; D = 1.5 eq 2 M Na2C03) THF, 55 °C. isolated yield after chromatography and based on loading. Crude purity based on HPLC analysis. Major impurity in all cases is 6-bromo-3-aminobenzisoxazole.
General procedure for the Suzuki coupling reactions with resin 3b: top- nitrobenzophenone oxime polystyrene (Kaiser) resin (500 mg, 1.07 mmol/g, 0.54 mmol) in a tared 25 mL Kontes Microfilter Funnel was added THF (7 mL) and potassium t-butoxide (640 μL, 1 M in THF, 0.642 mmol). After shaking by hand for several minutes, the resin turned a deep purple color. To this suspension was added 4-bromo-2-fluorobenzonitrile (214 mg, 1.07 mmol). The reaction vessel was rotated at 55 °C in a Robbins oven for 12 h to give resin 3b followed by cooling for 1 h. The resin was then rinsed with 2 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h to give a Δwt = 64 mg (66%> loading yield). The resin was then suspended in THF and to this was added phenylboronic acid (261 mg, 2.14 mmol), Na2CO3 (348 μL, 2 M in H2O, 2.14 mmol), and Pd(PPh3)4. The vessel was then rotated for 36 h in a 55 °C oven followed by rinsing with 2 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 3 h. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was linsed with 2 x 5 mL of CH2C12. These washings were combined and concentrated in vacuo to give the crude product 16a (91% purity by reverse phase HPLC) which was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes. Concentration of the product containing fractions gave pure 6-phenyl-3-amino-l,2- benzisjj.azole (16a): (46 mg, 2 step yield = 58%) based on loading yield): HPLC retention time (eluent gradient 10%A to 60%A over 45 min) = 13.3 min. Η NMR (400 MHz, DMSO-d6) δ 7.86 (d, J= 8.4 Hz, IH), 7.74 - 7.70 (m, 2H), 7.68 (s, IH), 7.53 (d, J= 8.0 Hz, 2H), 7.48 - 7.43 (m, 2H), 7.40 - 7.35 (m, IH), 6.42 (bs, 2H). MS (ESI) m/z 211 (M+FI)+. Anal Calc'd for C13H10N2O: C, 74.27; H, 4.79; N, 13.32. Found: C, 72.59 H, 4.86; N, 13.36.
6-(3,5-dichlorophenyl)-3-amino-l,2-benzisoxazole (16b): 2 step yield = 51% (based on loading yield). 95% crude purity. HPLC retention time (eluent gradient 20%A to 80%A over 45 min) = 30.4 min. 1H NMR (400 MHz, DMSO-d6) δ 7.87 (d, J= 8.8 Hz, IH), 7.82 (s, IH), 7.81 (d, J= 1.6 Hz, 2H), 7.62 - 7.58 (m, 2H), 6.46 (bs, 2H). MS (FD) m/z 278 [35C1+35C1, M+], 280 [35C1+37C1, (M+H)+]. Anal Calc'd for C]3H8Cl2N2O: C, 55.94; H, 2.89; N, 10.04. Found: C, 55.93 H, 2.60; N, 9.85. 6-(3,5-ditrifluoromethylphenyl)-3-amino-l,2-benzisoxazole (16c): 2 step yield = 45%) (based on loading yield). 81% crude purity. HPLC retention time (eluent gradient 20%A to 80%A over 45 min) = 31.0 min. Η NMR (400 MHz, DMSO-d6) δ 8.40 (s, 2H), 8.11 (s, IH), 7.98 (s, 2H), 7.93 (d, J= 8.4 Hz, IH), 7.76 - 7.71 (m, 2H), 6.49 (bs, 2H). MS (ESI) m/z 347 (M+H)+. Anal Calc'd for C15H8F6N2O: C, 52.04; H, 2.33; N, 8.09. Found: C, 52.00 H, 2.06; N, 7.99.
6-(< -formylphenyl)-3-amino-l,2-benzisoxazole (16d): 2 step yield = 43% (based on loading yield). 92%> crude purity. HPLC retention time (eluent gradient 5%A to 40%A over 45 min) = 37.6 min. 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, IH), 7.98 (s, 4H), 7.90 (d, J= 8.4 Hz, IH), 7.82 (s, IH), 7.63 (dd, J= 8.4, 1.6 Hz, IH), 6.46 (bs, 2H). MS (ESI) m/z 239 (M+H)+. HRMS Calcd for Cι4H10N2O2 (M+H)+ 239.0821, found 239.1816.
General Example 11 Application of the Sonogashira reaction to resin 3b proved less difficult than the Suzuki coupling. We were able to couple phenethylacetylene to 3b to give alkyne 17 in a 58% 2-step yield using Pd(PPh3)4/CuI in THF (Scheme 8) (Kahn, S.I.; Grinstaff, M.W.; Tetrahedron left. ; 1998, 39, 8031.) Again, as in the Suzuki chemistry, significantly better results were obtained with THF as the reaction solvent. The Pd2(dba) /CuI Et3N conditions described by Moore (Nelson, J.C; Young, J.K.; Moore, J.S.; J. Org. Chem., 1996, 61, 8160.) failed to give reasonable yields and purities.
Scheme 8. On-resin Sonogashira coupling.
Sonogashira coupling with resin 3b: resin 3b was prepared in 66%> loading yield from 0.535 mmol of Kaiser oxime 1 as detailed above. The resin was then suspended in THF and to this was added 4-phenylbutyne (280 mg, 2.14 mmol), Et3N (500 μL), Cul (5.1 mg, 0.027 mmol), and Pd(PPh3)4 (31 mg, 0.027 mmol). The vessel was then rotated for 36 h in a 55 °C oven followed by rinsing with 2 x 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 3 h. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 5 mL of CH2C12. These washings were combined and concentrated in vacuo to give the crude product 17 (93% purity by reverse phase HPLC) which was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes. Concentration of the product containing fractions gave pure 6-(4-phenylbutynyI)-3-amino-l,2-benzisoxazole (17): (58 mg, 2 step yield = 58%) based on loading yield): HPLC retention time (eluent gradient 20%A to 85%A over 45 min) = 26.2 min. 1H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J= 8.0 Hz, IH), 7.38 (s, IH), 7.29 - 7.27 (m, 4H), 7.21 - 7.15 (m, 2H), 6.43 (bs, 2H), 2.84 (t, J= 7.6 Hz, 2H), 2.70 (t, J= 7.2 Hz, 2H). MS (ESI) m/z 263 (M+H)+. HRMS Calcd for C13Hi0N2O 263.1184, found 263.1172.
General Example 12
The acquisition of aldehyde resin 18 (Scheme 9) from the Suzuki reaction with 3b (Table 5, entry 4) provided an opportunity to evaluate the compatibility of the aryloxime linker with the Horner-Emmons olefination. Thus, treatment of 18 with the anion of trimethylphosphonoacetate in THF (preformed with n-BuLi at 0 °C) gave olefin 19 in 52% isolated yield (3-steps, based on loading of resin 1) and 92% purity. (Chin, J.; Fell, B.; Shapiro, M.J.; Tomesch, J.; Wareing, J.R.; Bray, A.M.; J. Org. Chem., 1997, 62, 538. Rotella, D.P.; J. Am. Chem. Soc, 1996, 118, 12246.) The major impurity in this reaction is the cinnamic acid derivative which likely results from competing ester hydrolysis in the cyclitive removal step.
Scheme 9. On-resin Horner-Emmons olefination
Horner-Emmons olefination to give 19: to /7-nitrobenzophenone oxime polystyrene (Kaiser) resin (500 mg, 1.07 mmol/g, 0.54 mmol) in a tared 25 mL Kontes Microfilter Funnel was added THF (7 mL) and potassium t-butoxide (640 μL, 1 M in THF, 0.642 mmol). After shaking by hand for several minutes, the resin turned a deep purple color. To this suspension was added 4-bromo-2-fluorobenzonitrile (214 mg, 1.07 mmol). The reaction vessel was rotated at 55 °C in a Robbins oven for 12 h to give resin 3b followed by cooling for 1 h. The resin was then rinsed with 2 5 mL of CH2C12, 2 x 5 mL of MeOH, 2 x 5 mL of H2O, and 4 x 5 mL of MeOH. The resin was dried in a 35 °C vacuum oven for 12 h to give a Δwt = 64 mg (66% loading yield). The resin was then suspended in THF and to this was added 4-formylphenylboronic acid (482 mg, 3.21 mmol), Na2CO3 (348 μL, 2 M in H2O, 0.696 mmol), and Pd(PPh3)4 (31 mg, 0.027 mmol). The vessel was then rotated for 36 h in a 55 °C oven followed by rinsing with 2 5 mL of CH2C12, 2 x 5 mL of MeCN, 2 x 5 mL of H2O, and 4 x 5 mL of MeCN (care was taken to not rinse the resin with methanol in order to avoid methyl acetal formation). The resin was dried in a 35 °C vacuum oven for 3 h to give aldehyde resin 18. In a separate vial, trimethylphosponoacetate (172 μL, 1.07 mmol) was dissolved in THF (4 mL) and cooled to 0 °C. To this vial was added BuLi (602 μL, 0.96 mmol, 1.6 M in hexanes) dropwise. This THF solution was then added dropwise by syringe to a suspension of resin 18 in THF (4 mL) and the vessel was rotated for 6 h at room temp. This was followed by rinsing with 2 x 5 mL of CH2C12, 2 5 mL of MeOH, 2 x 5 mL of H2O, and 4 5 mL of MeOH. The resin was then dried in a 35 °C vacuum oven for 3 h. TFA (4 mL) and 5 N HClaq (1 mL) were then added to the resin and the vessel was rotated for 2 h in a 55 °C oven. The TFA/HClaq was collected and the resin was rinsed with 2 5 mL of CH2C1 . These washings were combined and concentrated in vacuo to give the crude product 19 (92% purity by reverse phase HPLC) which was purified by radial chromatography on a 2 mm plate eluting with 50% EtOAc/Hexanes. Concentration of the product containing fractions gave pure 6-[4-(methoxycarbonylethylenyI)phenyI]-3-amino-l,2-benzisoxazole (19): (47 mg, 3 step yield = 52% based on loading yield of resin 1): HPLC retention time (eluent gradient 20%A to 85%A over 45 min) = 24.3 min. Η NMR (400 MHz, DMSO-d6) δ 7.86 (d, J= 8.0 Hz, IH), 7.80 (s, 4H), 7.76 (s, IH), 7.69 (d, J= 16.4 Hz, IH), 7.59 (d, J= 8.0 Hz, IH), 6.69 (d, J= 16.4 Hz, IH), 6.43 (bs, 2H), 3.71 (s, 3H). HRMS Calcd for Cι7Hι5N2O3 295.1082, found 295.1088.
It should be understood that a wide range of changes and modifications can be made to the embodiments described above. It is therefore intended that the foregoing description illustrates rather than limits this invention, and that it is the appended claims, including all equivalents, which define this invention.

Claims

WHAT IS CLAIMED IS:
1. A library of structurally related aminobenzisoxazole compounds, wherein each library compound is of formula (I):
R-A3- A2-A' -NH-(CH2)C-R' wherein R1 is a substituted or unsubstituted aminobenzisoxazole group of the formula
(II):
which can be attached to one group of the formula R-A3-A2-A'-NH-(CH2)C- from any of positions 4, 5, 6, or 7, and where the remaining positions can each independently be substituted with hydrogen, (C! to C4)alkyl, or a halogen; A2 and A3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A1, A2 and A3 is not a bond;
R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is O, 1, 2, 3, or 4.
2. The library of claim 1, comprising from 8 to 100,000 aminobenzisoxazole compounds of the formula (I).
3. A reaction vessel on which a plurality of compounds are physically separated from each other, wherein the compounds are aminobenzisoxazole compounds of the formula (I), as defined in claim 1.
4. The reaction vessel of claim 3, which is a wellplate apparatus.
5. The reaction vessel of claim 4, which is a 96 well microtiter plate.
6. An assay kit for identification of pharmaceutical lead compounds, said kit comprising biological assay materials and a wellplate apparatus, wherein each well in said apparatus contains a unique library compound of Claim 1.
7. The assay kit of Claim 6, containing biological materials for performing assay tests selected from the group of in vitro assays, cell based functional assays and add, incubate and read assays.
8. An apparatus suitable as a replacement element in an automated assay machine as a source of individual members of a library of structurally related compounds, said apparatus comprising a 2-dimensional array of defined reservoirs, each reservoir containing a unique compound of said library, wherein said structurally related compounds are of the formula (I):
R- A3- A2- A1 -NH-(CH2)C-R' wherein R1 is a substituted or unsubstituted aminobenzisoxazole group of the formula (II):
which can be attached to one group of the formula R-A3-A2-A'-NH-(CH2)C- from any of positions 4, 5, 6, or 7, and where the remaining positions can each independently be substituted with hydrogen, (C\ to C4)alkyl, or a halogen; A2 and A3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A1, A2 and A3 is not a bond;
R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is 0, 1, 2, 3, or 4.
9. A library of structurally related 4-amidino-3 -hydroxy Iphenyl compounds, wherein each library compound is of formula (lb):
R-A3-A2-A'-NH-(CH2)C-Rlb wherein Rlb is a substituted or unsubstituted group of the formula (lib):
which can be attached to a group of the formula R-A -A -A -NH-(CH2)C- from any of positions 4, 5, 6, or 7, and where the remaining positions can each independently be substituted with hydrogen, ( to C4)alkyl, or a halogen; A1 A2 and A3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A1, A2 and A3 is not a bond;
R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is 0, 1, 2, 3 or 4.
10. The library of claim 9, comprising from 8 to 100,000 4-amidino-3- hydroxy Iphenyl compounds of the formula (lb).
11. A reaction vessel on v/nich a plurality of compounds are physically separated from each other, wherein the compounds are 4-amidino-3 -hydroxy Iphenyl compounds of the formula (lb), as defined in claim 9.
12. The reaction vessel of claim 11, which is a wellplate apparatus.
13. The reaction vessel of claim 12, which is a 96 well microtiter plate.
14. An assay kit for identification of pharmaceutical lead compounds, said kit comprising biological assay materials and a wellplate apparatus, wherein each well in said apparatus contains a unique library compound of Claim 9.
15. The assay kit of Claim 14, containing biological materials for performing assay tests selected from the group of in vitro assays, cell based functional assays and add, incubate and read assays.
16. An apparatus suitable as a replacement element in an automated assay machine as a source of individual members of a library of structurally related compounds, said apparatus comprising a 2-dimensional array of defined reservoirs, each reservoir containing a unique compound of said library, wherein said structurally related compounds are of the formula (lb):
R-A3-A2-A1-NH-(CH2)C-Rlb wherein Rlb is a substituted or uns bstitutsd group of the formula (lib):
which can be attached to a group of the formula R-A -A -A -NH-(CH2)C- from any of positions 4, 5, 6, or 7, and where the remaining positions can each independently be substituted with hydrogen, (Ci to C4)alkyl, or a halogen; A2 and A3 are each, independently, a bond or a substituted or unsubstituted amino acid, provided at least one of A1, A2 and A3 is not a bond; R is hydrogen, an amino protecting group, a substituted or unsubstituted amino acid, a substituted or unsubstituted amino protected amino acid, a substituted or unsubstituted peptide, or a substituted or unsubstituted amino protected peptide; and c is 0, 1, 2, 3 or 4.
17. A pharmaceutically acceptable salt of an aminobenzisoxazole compound of the formula (III):
X-CO-Y-CO-NH-(CH2)c- R1 wherein R1 is a substituted or unsubstituted aminobenzisoxazole group of the formula
(II):
which is attached to a group of the formula X-CO-Y-CO-NH-(CH2)c- at position 6 and where the remaining positions can each independently be substituted with hydrogen, (Ci to C )alkyl, or a halogen;
X-CO- is D-prolinyl, D-homoprolinyl, R2-(CH2)d-NH-CH2-C(O)-,
* denotes a chiral center that is (D) or (DL);
# denotes a chiral center that is (L);
R2 is -COOR14, -SO2(Cι-C4 alkyl), -SO3H, -P(O)(ORl4)2 or tetrazol-5-yl; R3 is hydrogen or (Cι-C4)alkyl;
R4 is carboxy or methylsulfonyl;
R5 is NHR6, NHCOR6 or NHCOOR6; R is (Cι-Cιo)alkyl, (C3-C8)cycloalkyl or a (C3-C8)cycloalkyl-(Cι-C6)alkyl group containing 4-10 carbons;
R7 is (C3-C8)cycloalkyl, (Cι-C8)alkyl,
R8 is -OH, (C i -C4)alkoxy , or -NH-R12;
R9 is hydrogen cr (Cι-C4)alkyl;
R10 is hydrogen, (Cι-C4)alkyl, (Cι-C4)alkoxy, hydroxy, halo or (C1-C4) alkylsulfonylamino;
R11 is (Cι-C )alkyl, (Cι-C4)fluoroalkyl bearing one to five fluoros, -(CH2)d-R2, or unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
Rι: :s hydrogen, (Cι-C4)alkyl, RnSO2-, RuOC(O)-, RnC(O , R13C(O)- or - (CH2)d-R2;
R13 is -COOR14 or tetrazol-5-yl;
14 each R is independently hydrogen or (Cι-C4)alkyl; -Y-CO- is
is
R is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b-R17;
R16 is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b- R17; L is a bond, -O-, -S-, or -NH-;
R17 is (D-C4)alkyl, (C3-C8)cycloalkyl, -COOH, -CONH2, or Ar, where Ar is unsubstituted or subs tuled aryf where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms whicl are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
R18 is -CH -, -O-, -S-, or -NH-;
R is a bond or, when taken with R and the three adjoining carbon atoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom of which may be -O-, -S-, or -NH-; each a, independently, is 0, 1 or 2; each b, independently, is 0, 1, 2 or 3; each c, independently, is 0, 1, 2, 3 or 4; and each d, independently, is 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
18. The pharmaceutically acceptable salt of the aminobenzisoxazole compound of the formula (III) as defined in claim 17, which salt is an acid addition salt with an acid affording a physiologically acceptable counterion or, for a compound of formula (I) in which X or Y bears an acidic moiety, a salt made with a base which affords a pharmaceutically acceptable cation selected from alkali metal salts, alkaline earth metal salts, aluminum salts and ammonium salts, and salts made from physiologically acceptable organic bases.
19. A pharmaceutical composition comprising an aminobenzisoxazole compound of the formula (III) as defined in Claim 17, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, diluent or excipient.
20. A library of structurally related aminobenzisoxazole compounds of formula (ID):
X-CO-Y-CO-NH-(CH2)c- R1 wherein R1 is a substituted or unsubstituted aminobenzisoxazole group of the formula
(II):
which is attached to a group of the formula X-CO-Y-CO-NH-(CH2)c- at position 6 and where the remaining positions can each independently be substituted with hydrogen, (Ci to C4)alkyl, or a halogen;
X-CO- is D-prolinyl, D-homoprolinyl, R2-(CH2) -NH-CH2-C(O)-, )-
* denotes a chiral center that is (D) or (DL);
# denotes a chiral center that is (L);
R2 is -COOR14, -SO2(Cι-C4 alkyl), -SO3H, -P(0)(ORl4)2 or tetrazol-5-yl; R3 is hydrogen or (Cι-C4)alkyl; R4 is carboxy or methylsulfonyl; R5 is NHR6, NHCOR6 or NHCOOR6;
R6 is (Cι-Cιo)alkyl, (C3-C8)cycloalkyl or a (C3-C8)cycloalkyl-(Cι-C6)alkyl group containing 4-10 carbons;
R7 is (C3-C8)cycloalkyl, (Cι-C8)alkyl,
R8 is -OH, (Cι-C4)alkoxy, or -NH-R12; R9 is hydrogen or (Cι-C4)alkyl; R10 is hydrogen, (Cι-C4)alkyl, (Cι-C4)alkoxy, hydroxy, halo or (C1-C4) alkylsulfonylamino;
R11 is (Cι-C4)alkyl, (Cι-C4)fluoroalkyl bearing one to five fluoros, -(CH2)d-R2, or unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
R12 is hydrogen, (Cι-C4)alkyl, RnSO2-, RπOC(O)-, RπC(O)-, Rl3C(O)- or - (CH2)d-R2;
R13 is -COOR14 or tetrazol-5-yl;
14 each R is independently hydrogen or (Cι-C4)alkyl; -Y-CO- is
R is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b-R17;
R16 is (Cι-C6)alkyl, (C3-C8)cycloalkyl, or -(CH2)c-L-(CH2)b- R17; L is a bond, -O-, -S-, or -NH-; R17 is (Cι-C4)alkyl, (C3-C8)cycloalkyl, -COOH, -CONH2, or Ar, where Ar is unsubstituted or substituted aryl, where aryl is phenyl, naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocyclic ring, having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocyclic group having one or two heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;
18
R is -CH2-, -O-, -S-, or -NH-;
R is a bond or, when taken with R and the three adjoining carbon atoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom of which may be -O-, -S-, or -NH-; each a, independently, is 0, 1 or 2; each b, independently, is 0, 1, 2 or 3; each c, independently, is 0, 1, 2, 3 or 4; and each d, independently, is 1, 2, or 3; or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable salt thereof.
21. The library of claim 20, comprising from 8 to 100,000 aminobenzisoxazole compounds of the formula (III).
22. A reaction vessel on which a plurality of compounds are physically separated from each other, wherein the compounds are aminobenzisoxazole compounds of the formul?. (Ill), as defined in claim 20.
23. The reaction vessel of claim 22, which is a wellplate apparatus.
24. The reaction vessel of claim 23, which is a 96 well microtiter plate.
25. An assay kit for identifying compounds with thrombin inhibitory activity, said kit comprising biological assay materials and a well plate apparatus, wherein each well in said apparatus contains a different aminobenzisoxazole compound of the formula (III), as defined in claim 20.
26. The assay kit of claim 25, wherein the biological materials are selected for performing at least one assay test selected from the group consisting of in vitro assays, cell based functional assays, and add, incubate, and read assays.
27. An apparatus suitable as a replaceable element in an automated assay machine as a source of individual members of a library of structurally related compounds, said apparatus comprising a 2-dimensional array of defined reservoirs, each reservoir containing a library compound from said library, wherein said structurally related compounds are aminobenzisoxazole compounds of the formula (III), as defined in claim 20.
28. A method of inhibiting thrombin comprising administering to a mammal in need of treatment, a thrombin inhibiting dose of a compound of formula (III), or a pharmaceutically acceptable salt thereof, as defined in Claim 20.
29. A method of inhibiting thrombosis in a mammal comprising administering to a mammal in need of treatment, an antithrombotic dose of a compound of formula (III), or a pharmaceutically acceptable salt thereof, as defined in Claim 20.
30. A method of inhibiting coagulation in a mammal comprising administering to a mammal in need of treatment an effective dose of a compound of formula (III), or a pharmaceutically acceptable salt thereof, as defined in Claim 20.
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