Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
  • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
  • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
  • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
  • C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
  • C07D217/26—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
  • C07D233/56—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
  • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
  • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
  • C07D471/14—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/11—Compounds covalently bound to a solid support
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

• the present invention relates generally to the synthesis of heterocyclic compounds based on the isoquinoline ring. More specifically, the invention provides novel isoquinoline derivatives as well as novel libraries comprised of such compounds.
• the process of discovering new therapeutically active compounds for a given indication involves the screening of all compounds from available compound collections. From the compounds tested one . or more structure (s) is selected as a promising lead. A large number of related analogs are then synthesized in order to develop a structure-activity relationship and select one or more optimal compounds. With traditional one-at-a-time synthesis and biological testing of analogs, this optimization process is long and labor intensive. Adding significant numbers of new structures to the compound collections used in the initial screening step of the discovery and optimization process cannot be accomplished with traditional one-at-a-time synthesis methods, except over a time frame of months or even years. Faster methods are needed that allow for the preparation of up to thousands of related compounds in a matter of days or a few weeks.
• the present invention overcomes the known limitations to classical organic synthesis of isoquinolines as well as the shortcomings of combinatorial chemistry with heterocycles.
• the present invention combines the techniques of solid-phase synthesis of heterocycles and the general techniques of synthesis of combinatorial libraries to prepare new isoquinoline compounds.
• the present invention relates to novel isoquinoline compounds, as well as libraries containing such compounds, of the following formula:
• Figure 1 shows the assembly of building blocks for preparing the isoquinoline derivative compounds of the present invention. It should be noted that, in Figure 1, R ⁇ corresponds with R 11 of the subject invention; R 2 corresponds with R 2 of the subject invention; and R 3 corresponds with X of the subject invention.
• the present invention provides novel derivatives and libraries of novel derivatives of isoquinoline compounds of the following formula:
• R 1 is the structure
• R 10 is attached to each of the four carbon ring atoms and is, independently, a hydrogen atom, C x to C 6 alkyl, C ⁇ to C 6 substituted alkyl, C 7 to C 12 phenylalkyl or C 7 to C 12 substituted phenylalkyl; and
• R 11 is a hydrogen atom, C 1 to C 6 alkyl, C x to C 6 substituted alkyl, C 2 to C 7 alkenyl, C 2 to C 7 substituted alkenyl, C 2 to C 7 alkynyl, C 2 to C 7 substituted alkynyl, C 3 to C 7 cycloalkyl, C 3 to C 7 substituted cycloalkyl, C 5 to C 7 cycloalkenyl, C 5 to C 7 substituted cycloalkenyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl, C-, to C 12 phenylalkyl, C 7 to C 12 substituted phenylalkyl, heterocyclic ring or substituted heterocyclic ring;
• R 2 is a hydrogen atom, C x to C 6 alkyl, Cj_ to C 6 substituted alkyl, C 2 to C 7 alkenyl, C 2 to C 7 substituted alkenyl, C 2 to C 7 alkynyl, C 2 to C 7 substituted alkynyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl, C 3 to C 7 cycloalkyl, C 3 to C 7 substituted cycloalkyl, C 7 to C 12 phenylalkyl, C 5 to C 7 cylcoalkenyl, C 5 to C 7 substituted cycloalkenyl, C 7 to C 12 substituted phenylalkyl or a heterocyclic ring;
• R 3 R 4 , R 5 and R 6 are, independently, a hydrogen atom, halo, hydroxy, protected hydroxy, cyano, nitro, C ⁇ to C 6 alkyl, C 2 to C 7 alkenyl, C 2 to C 7 alkynyl, C x to C 6 substituted alkyl, C 2 to C 7 substituted alkenyl, C 2 to C 7 substituted alkynyl, C x to C 7 alkoxy, C : to C 7 acyloxy, C 1 to C 7 acyl, C 3 to C 7 cycloalkyl, C 3 to C 7 substituted cycloalkyl, C 5 to C 7 cycloalkenyl, C 5 to C 7 substituted cycloalkenyl, a heterocyclic ring, C 7 to C 12 phenylalkyl, C 7 to C 12 substituted phenylalkyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl,
• R 7 and R 9 are, independently, absent or present and, if present, are C 1 to C 6 alkylene or C x to C 6 substituted alkylene;
• R 8 is absent or present and, if present, is C : to C 6 alkylene, C : to C 6 substituted alkylene, C 2 to C 7 alkenylene, C 2 to C 7 substituted alkenylene, C 2 to C 7 alkynylene, C 2 to C 7 substituted alkynylene, C 3 to C 7 cycloalkylene, C 3 to C 7 substituted cycloalkylene, C 5 to C 7 cycloalkenylene, C 5 to C 7 substituted cycloalkenylene, phenylene, substituted phenylene, naphthylene, substituted naphthylene, heterocyclene or substituted heterocyclene;
• X is hydroxy, protected carboxy, amino, protected amino, (monosubstituted) amino, (disubstituted) amino, an amino acid, aniline, substituted aniline or an amino- substituted heterocyclic ring;
• Y is C0 2 H, CH 2 OH, SH, CH 2 NHR 12 , C(0)NHR 12 , or CH 2 NHR 12 , wherein R 12 is a hydrogen atom, C ⁇ to C 6 alkyl, C x to C substituted alkyl or a functionalized resin; and
• Z 0 or H 2 .
• R 1 is the structure
• R 10 is attached to each of the four carbon ring atoms and is, independently, a hydrogen atom or C x to C 6 alkyl;
• R 11 is a hydrogen atom, C, to C 6 alkyl, C x to C 6 substituted alkyl, C 2 to C 7 alkenyl, C 2 to C 7 substituted alkenyl, C 3 to C 7 cycloalkyl, C 3 to C 7 substituted cycloalkyl, C 5 to C 7 cycloalkenyl, C 5 to C 7 substituted cycloalkenyl, phenyl, substituted phenyl, C 7 to C 12 phenylalkyl, C 7 to C 12 substituted phenylalkyl, heterocyclic ring or substituted heterocyclic ring;
• R 2 is a hydrogen atom, C x to C 6 alkyl, ⁇ to C 6 substituted alkyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl, C 7 to C 12 phenylalkyl, C 7 to C 12 substituted phenylalkyl or a heterocyclic ring;
• R 3 R 4 , R 5 and R 6 are, independently, a hydrogen atom, halo, hydroxy, protected hydroxy, cyano, nitro, C t to C 6 alkyl, C to C 6 substituted alkyl, C 1 to C 7 alkoxy, C ⁇ to C 7 acyl, a heterocyclic ring, C 7 to C 12 phenylalkyl, C 7 to C 12 substituted phenylalkyl, phenyl, substituted phenyl, cyclic C 2 to C 7 alkylene, substituted cyclic C 2 to C 7 alkylene, cyclic C 2 to C 7 heteroalkylene, substituted cyclic C 2 to C 7 heteroalkylene, carboxy, protected carboxy, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino, carboxamide or protected carboxamide;
• X is hydroxy, amino, protected amino
• Y is C0 2 H , CH 2 NHR 12 or C (0 ) NHR 12 , wherein R 12 is a hydrogen atom or C : to C 6 al kyl ;
• the isoquinoline derivatives are wherein:
• R 1 is the structure
• R is attached to each of the four carbon ring atoms and is a hydrogen atom
• R 11 is (2-piperidinyl) ethyl, 3- (imidazoyl) propyl, 2, 3-dimethoxyphenylmethyl, 2, 4-dichlorophenethyl, 2- (2-pyridinyl) ethyl, 2- (4-methoxyphenyl) ethyl,
• R 2 is 1-naphthyl, 2-chloro-5-nitrophenyl, 2-chloro-6-fluorophenyl, 2-cyanophenyl, 2-imidazolyl, 2-naphthyl, 2-pyridinyl, 2-quinolinyl, 3,4, 5-trimethoxyphenyl, 3,4- (methylenedioxy) phenyl, 3, 5-bis (trifluoromethyl) phenyl, 3-methylphenyl, 3-nitrophenyl, 3-phenoxyphenyl, 4- (3-dimethylaminopropoxy) phenyl, 4- (methylthio) phenyl, 4- (trifluoromethyl) phenyl, 4-biphenylyl, 4-bromophenyl, 4-dimethylaminophenyl, 4-ethylphenyl, 4-hydroxyphenyl, 4-propoxyphenyl, 5-nitrovanillin, 6-methyl-2-pyridinyl, phenyl, 2-methoxyphenyl or 4-methylphen
• R 3 R 4 , R 5 and R 6 are each a hydrogen atom
• R 7 and R 9 are each absent
• R 8 is absent or present and, if present, is 1, 2-ethylenyl, 1, 4-cyclohexylenyl or 1, 4-phenylenyl;
• X is (r) - (-) -1-cyclohexylethylamino, 1,2,3, 4-tetrahydroisoquinolyl,
• Y is C(0)NHR 12 , wherein R 12 is a hydrogen atom
• the isoquinoline derivatives are wherein:
• R 2 is 1, 4-benzodioxan-6-yl, l-methylindol-3-yl, 2,3- difluorophenyl, 2-bromophenyl, 2-chloro-5-nitrophenyl, 2- furyl, 2-imidazolyl, 2-naphthyl, 2-pyridinyl, 2- thiophenyl, 3, 4-dichlorophenyl, 3,5- bis (trifluoromethyl) phenyl, 3, 5-dihydroxyphenyl, 3,5- dimethoxyphenyl, 3, 5-dimethyl-4-hydroxyphenyl, 3- (4- methoxyphenoxy) phenyl, 3-furyl, 3-hydroxyphenyl, 3- methyl-4-methoxyphenyl, 3-methylphenyl, 3-nitrophenyl, 3- pyridinyl, 3-thiophenyl, 4- (3-dimethylaminoprop-l- oxy) phenyl, 4- (dimethylamino) phenyl, 4- (methylthi
• R 3 , R 4 , R 5 , R 6 are each a hydrogen atom
• X is aminocyclopropyl, aminoisopropyl, 3-aminopropyl, aminoethanolyl, (aminomethyl) cyclopropyl, pyrrolidilyl, aminodiethyl, amino-2-methoxyethyl, aminocyclopentyl, piperidinyl, 1- (pyrrolidin-3-ol) , aminoamyl, amino- (2- (N,N-dimethyl) ) ethyl, azetidinyl, aminofurfuryl, aminodiallyl, 2-aminothiazolyl, 1-aminopiperidinyl, 1-methylpiperazinyl, 4-aminomorpholinyl, a inodiethanol, 2- (aminomethyl) pyridinyl, histaminyl, 1- (2-aminoethyl) pyrrolidinyl, (+) -3-hydroxy piperidine, (s) -l-amino-2- (methoxymethyl) pyrrol
• Y is C(0)NH 2 .
• R 2 is 1, 4-benzodioxan-6-yl, l-methylindole-3-yl, 2,3- difluorophenyl, 2-bromophenyl, 2-chloro-5-nitrophenyl, 2- furyl, 2-imidazolyl, 2-naphthyl, 2-pyridinyl, 2- thiophenyl, 3, 4-dichlorophenyl, 3,5- bis (trifluoromethyl) phenyl, 3, 5-dihydroxyphenyl, 3,5- dimethoxyphenyl, 3, 5-dimethyl-4-hydroxyphenyl, 3- (4- methoxyphenoxy) phenyl, 3-furyl, 3-hydroxyphenyl, 3- methyl-4-methoxyphenyl, 3-methylphenyl, 3-nitrophenyl, 3- pyridinyl, 3-thiophenyl, 4- (3- di ethylaminopropoxy) phenyl, 4- (dimethylamino) phenyl, 4- (methylthio)
• R 3 , R 4 , R 5 , R 6 are each a hydrogen atom
• X is anilinyl, 2-fluoroanilinyl, 3-fluoroanilinyl, 4- fluoroanilinyl, 2-chloroanilinyl, 3-chloroanilinyl, 4- chloroanilinyl, 2-bromoanilinyl, 3-bromoanilinyl, 4- bromoanilinyl, 2-methoxyanilinyl, 3-methoxyanilinyl, 4- methoxyanilinyl, 2-hydroxyanilinyl, 3-hydroxyanilinyl, 4- hydroxyanilinyl, 2-carboethoxyanilinyl, 3- carboethoxyanilinyl, 4-carboethoxyanilinyl, 2- trifluoromethylanilinyl, 3-trifluoromethylanilinyl, 4- trifluoromethylanilinyl, 2-dimethylaminoanilinyl, 3- dimethyla inoanilinyl, 4-d
• R 2 is phenyl, 2-bromophenyl, 2-cyanophenyl, 2- fluorophenyl, 2-hydroxyphenyl, 2-methoxyphenyl, 3- bromophenyl, 3-carboxyphenyl, 3-cyanophenyl, 3- fluorophenyl, 3-hydroxyphenyl, 3-methoxyphenyl, 3- methylphenyl, 3-nitrophenyl, 3- (trifluoromethyl) phenyl, 4-acetamidophenyl, 4-bromophenyl, 4-carboxyphenyl, 4- cyanophenyl, 4- (3-dimethylaminopropoxy) phenyl, 4- fluorophenyl, 4- (dimethylamino) phenyl, 4-hydroxyphenyl, 4-isopropylphenyl, 4-methoxyphenyl, 4-methylphenyl, 4- (methylcarboxylate) phenyl, 4-methylsulphonylphenyl, 4- (methylthio) phenyl,
• R 3 , R 4 , R 5 , R 6 are each a hydrogen atom
• X is pyridoxamino, 4- (dimethylamino) benzylamino , 2- chloro-4-fluoroanilino, 3-pyridylmethylamino, 4-
• Y is C(0)NH 2 .
• the stereochemistry of chiral centers associated with the attached groups can independently be in the R or S configuration, or a mixture of the two.
• C 1 to C 6 alkyl denotes such radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, amyl, tert- amyl, hexyl and the like.
• a preferred "C 1 to C 6 alkyl” group is methyl.
• C 2 to C 7 alkenyl denotes such radicals as vinyl, allyl, 2-butenyl, 3-butenyl, 2- pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, as well as dienes and trienes of straight and branched chains.
• C 2 to C 7 alkynyl denotes such radicals as ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, as well as di- and tri-ynes of straight and branched chains.
• C 1 to C 6 substituted alkyl denotes that the above C 1 to C 6 alkyl groups and C 2 to C 7 alkenyl and alkynyl groups are substituted by one or more, and preferably one or two, halogen, hydroxy, protected hydroxy, oxo, protected oxo, cyclohexyl, naphthyl, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino, guanidino, heterocyclic ring, substituted heterocyclic ring, imidazolyl, indolyl, pyrrolidinyl, C : to C 7 alkoxy, Cj to C 7 acyl, C x to C 7 acyloxy, nitro, C- to C 7 alkyl ester, carboxy, protected carboxy,
• alkyl, alkenyl and alkynyl groups may be substituted once or more and, preferably, once or twice, with the same or with different substituents.
• Examples of the above substituted alkyl groups include the 2-oxo-prop-l-yl, 3-oxo-but-l-yl, cyanomethyl, nitromethyl, chloromethyl, hydroxymethyl, tetrahydropyranyloxymethyl, trityloxymethyl, propionyloxymethyl, amino, methylamino, aminomethyl, dimethylamino, carboxymethyl, allyloxycarbonylmethyl, allyloxycarbonylaminomethyl, methoxymethyl, ethoxymethyl, t-butoxymethyl, acetoxymethyl, chloromethyl, bromomethyl, iodomethyl, trifluoromethyl, 6-hydroxyhexyl, 2,4- dichloro (n-butyl) , 2-aminopropyl, chloroethyl, bro oethyl, fluoroethyl, iodoethyl, chloropropyl, bromopropyl, fluoropropyl, iod
• Examples of the above substituted alkenyl groups include styrenyl, 3-chloro-propen-l-yl, 3-chloro- buten-1-yl, 3-methoxy-propen-2-yl, 3-phenyl-buten-2-yl, l-cyano-buten-3-yl and the like.
• the geometrical isomerism is not critical, and all geometrical isomers for a given substituted alkenyl can be used.
• Examples of the above substituted alkynyl groups include phenylacetylen-1-yl, l-phenyl-2-propyn-l- yl and the like.
• oxo denotes a carbon atom bonded to two additional carbon atoms substituted with an oxygen atom doubly bonded to the carbon atom, thereby forming a ketone moiety.
• protected oxo denotes a carbon atom bonded to two additional carbon atoms substituted with two alkoxy groups or twice bonded to a substituted diol moiety, thereby forming an acyclic or cyclic ketal moiety.
• C ⁇ to C 7 alkoxy denotes groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t- butoxy and like groups.
• a preferred alkoxy is methoxy.
• C 1 to C 7 acyloxy denotes groups such as formyloxy, acetoxy, propionyloxy, butyryloxy, pentanoyloxy, hexanoyloxy, heptanoyloxy and the like.
• C 1 to C 7 acyl encompasses groups such as formyl, acetyl, propionyl, butyryl, pentanoyl, pivaloyl, hexanoyl, heptanoyl, benzoyl and the like.
• Preferred acyl groups are acetyl and benzoyl.
• C 3 to C 7 cycloalkyl includes the cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings.
• C 3 to C 7 substituted cycloalkyl indicates the above cycloalkyl rings are substituted by one or two halogen, hydroxy, protected hydroxy, C 2 to C 6 alkyl, C x to C 7 alkoxy, oxo, protected oxo, (monosubstituted) amino, (disubstituted) amino, trifluoromethyl, carboxy, protected carboxy, phenyl, substituted phenyl, amino, or protected amino groups.
• C 5 to C 7 cycloalkenyl indicates a 1,2, or 3-cyclopentenyl ring, a 1,2,3 or 4-cyclohexenyl ring or a 1,2,3,4 or 5-cycloheptenyl ring
• substituted C 5 to C 7 cycloalkenyl denotes the above C 5 to C 7 cycloalkenyl ring is substituted by a C-, to C 6 alkyl radical, halogen, hydroxy, protected hydroxy, C x to C 7 alkoxy, trifluoromethyl, carboxy, protected carboxy, oxo, protected oxo, (monosubstituted) amino, protected (monosubstituted) amino (disubstituted) amino, phenyl, substituted phenyl, amino, or protected amino.
• heterocyclic ring or “heterocyclic” denotes an optionally substituted five-membered or six- membered ring that has 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen and, in particular, nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms.
• the five-membered or six-membered ring may be saturated, fully saturated or partially unsaturated, with a fully saturated ring being preferred.
• An "amino-substituted heterocyclic ring” means any one of the above-described heterocyclic rings is substituted with at least one amino group.
• Preferred heterocyclic rings include morpholino, piperidinyl, piperazinyl, tetrahydrofurano, pyrrolo, and tetrahydrothiophen-yl .
• substituted heterocyclic ring means the above-described heterocyclic ring is substituted with, for example, one or more, and preferably one or two, substituents which can be the same or different.
• the substituents can be halogen, hydroxy, protected hydroxy, cyano, nitro, C- to C 5 alkyl, Cj_ to C 7 alkoxy, C ⁇ to C 7 substituted alkoxy, C x to C 7 acyl, C ⁇ to C 7 acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino carboxamide, protected carboxamide or trifluoromethyl groups.
• Aryl group which can be used with present invention include phenyl, substituted phenyl, as defined above, heteroaryl, and substituted heteroaryl.
• heteroaryl means a heterocyclic aromatic derivative which is a five-membered or six-membered ring system having from 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen, in particular nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms.
• heteroaryls examples include pyridinyl, pyrimidinyl, and pyrazinyl, pyridazinyl, pyrrolo, furano, oxazolo, isoxazolo, thiazolo and the like.
• substituted heteroaryl means the above-described heteroaryl is substituted with, for example, one or more, and preferably one or two, substituents which are the same or different which substituents can be halogen, hydroxy, protected hydroxy, cyano, nitro, C : to C 6 alkyl, C 1 to C 7 alkoxy, C x to C 7 acyl, C x to C 7 acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino carboxamide, protected carboxamide, N-(C X to C 6 alkyl) carboxamide, protected N- (C 1 to C 6 alkyl) carboxamide, N, N-di ( C 1 to C 6 alkyl) , trifluoromethyl, N- ( ⁇ C 1 to C 6 alkyl)
• C 7 to C 12 phenylalkyl denotes a C x to C 6 alkyl group substituted at any position by a phenyl ring.
• Examples of such a group include benzyl, 2- phenylethyl, 3-phenyl (n-propyl ) , 4-phenylhexyl, 3- phenyl (n-amyl) , 3-phenyl (sec-butyl) and the like.
• Preferred C 7 to C 12 phenylalkyl groups are the benzyl and the phenylethyl groups.
• C 7 to C 12 substituted phenylalkyl denotes a C 7 to C 12 phenylalkyl group substituted on the C ⁇ to C 6 alkyl portion with one or more, and preferably one or two, groups chosen from halogen, hydroxy, protected hydroxy, oxo, protected oxo, amino, protected amino, monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino, guanidino, heterocyclic ring, substituted heterocyclic ring, C 1 to C 7 alkoxy, Cj to C 7 acyl, Cj_ to C 7 acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carboxamide, protected carboxamide, N- (C 1 to C 6 alkyl) carboxamide, protected N- ⁇ to C 6 alkyl) carboxamide, N, N- (C l to C 6 dialkyl) carboxamide, cyano, N- ⁇
• C 7 to C 12 substituted phenylalkyl examples include groups such as 2-phenyl-l- chloroethyl, 2- (4-methoxyphenyl) ethyl, 4- (2, 6-dihydroxy phenyl) -n-hexyl, 2- (5-cyano-3-methoxyphenyl) -n-pentyl, 3- (2, 6-dimethylphenyl) -n-propyl, 4-chloro-3-aminobenzyl, 6- ( -methoxyphenyl) -3-carboxy (n-hexyl) , 5- (4- aminomethylphenyl) -3- (aminomethyl) -n-pentyl, 5-phenyl-3- oxo-n-pent-1-yl and the like.
• substituted phenyl specifies a phenyl group substituted with one or more, and preferably one or two, moieties chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, C x to C 6 alkyl, C- to C 7 alkoxy, C 1 to C 7 acyl, C, to C 7 acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino, carboxamide, protected carboxamide, N-(Cj_ to C 6 alkyl) carboxamide, protected N- (C x to C 6 alkyl) carboxamide, N, N-di (Cj to C 6 alkyl) carboxamide, trifluoromethyl, N-((C ⁇ to C 6 alkyl) sulfony
• substituted phenyl examples include a mono- or di (halo) phenyl group such as 2, 3 or 4-chlorophenyl, 2, 6-dichlorophenyl, 2, 5-dichlorophenyl, 3, 4-dichlorophenyl, 2, 3 or 4-bromophenyl, 3,4- dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4- fluorophenyl and the like; a mono or di (hydroxy) phenyl group such as 2, 3 or 4-hydroxyphenyl, 2,4- dihydroxyphenyl, the protected-hydroxy derivatives thereof and the like; a nitrophenyl group such as 2, 3 or 4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl; a mono- or di (alkyl) phenyl group such as 2, 3 or 4-methylphenyl, 2, 4-dimethylphenyl, 2, 3 or 4- (iso-propy
• substituted phenyl represents disubstituted phenyl groups wherein the substituents are different, for example, 3-methyl-4-hydroxyphenyl, 3-chloro-4- hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2- hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy 4- chlorophenyl and the like.
• substituted aniline specifies an aniline group substituted with one or more, and preferably one or two, moieties chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, C x to C 6 alkyl, C 1 to C 7 alkoxy, C to C 7 acyl, C a to C 7 acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino,
• substituted anilines include, for example, 2-fluoroanilinyl, 3-fluoroanilinyl, 4- fluoroanilinyl, 2-chloroanilinyl, 3-chloroanilinyl, 4- chloroanilinyl, 2-bromoanilinyl, 3-bromoanilinyl, 4- bromoanilinyl, 2-methoxyanilinyl, 3-methoxyanilinyl, 4- methoxyanilinyl, 2-hydroxyanilinyl, 3-hydroxyanilinyl, 4- hydroxyanilinyl, 2-carboethoxyanilinyl, 3- carboethoxyanilinyl, 4-carboethoxyanilinyl, 2- trifluoromethylanilinyl, 3-trifluoromethylanilinyl, 4- trifluoromethylanilinyl, 2-dimethylaminoanilinyl, 3- dimethylaminoanilinyl, 4-
• substituted naphthyl specifies a naphthyl group substituted with one or more, and preferably one or two, moieties either on the same ring or on different rings chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, C, to C 6 alkyl, C-, to C 7 alkoxy, C 1 to C 7 acyl, C ⁇ to C-, acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino,
• substituted naphthyl examples include a mono or di (halo) naphthyl group such as 1, 2, 3, 4, 5, 6, 7 or 8-chloronaphthyl, 2, 6-dichloronaphthyl, 2, 5-dichloronaphthyl, 3, 4-dichloronaphthyl, 1, 2, 3, 4, 5, 6, 7 or 8-bromonaphthyl, 3, 4-dibromonaphthyl, 3-chloro- 4-fluoronaphthyl, 1, 2, 3, 4, 5, 6, 7 or 8-fluoronaphthyl and the like; a mono or di (hydroxy) naphthyl group such as 1, 2, 3, 4, 5, 6, 7 or 8-hydroxynaphthyl, 2, 4- dihydroxynaphthyl, the protected-hydroxy derivatives thereof and the like; a nitronaphthyl group such as 3- or 4-nitronaphthyl; a cyanonap
• substituted naphthyl represents disubstituted naphthyl groups wherein the substituents are different, for example, 3-methyl-4-hydroxynaphth-l-yl, 3-chloro-4- hydroxynaphth-2-yl, 2-methoxy-4-bromonaphth-l-yl, 4- ethyl-2-hydroxynaphth-l-yl, 3-hydroxy-4-nitronaphth-2-yl, 2-hydroxy-4-chloronaphth-l-yl, 2-methoxy-7-bromonaphth-l- yl, 4-ethyl-5-hydroxynaphth-2-yl, 3-hydroxy-8- nitronaphth-2-yl, 2-hydroxy-5-chloronaphth-l-yl and the like.
• halo and halogen refer to the fluoro, chloro, bromo or iodo groups. There can be one or more halogen, which are the same or different. Preferred halogens are chloro and fluoro.
• (monosubstituted) amino refers to an amino group with one substituent chosen from the group consisting of phenyl, substituted phenyl, C x to C 5 alkyl, Ci to C 6 substituted alkyl, x to C 7 acyl, C 2 to C 7 alkenyl, C 2 to C 7 substituted alkenyl, C 2 to C 7 alkynyl, C 2 to C 7 substitued alkynyl, C 7 to C 12 phenylalkyl, C 7 to C 12 substituted phenylalkyl and heterocyclic ring.
• the (monosubstituted) amino can additionally have an amino- protecting group as encompassed by the term "protected (monosubstituted) amino.
• (disubstituted) amino refers to amino groups with two substituents chosen from phenyl, substituted phenyl, C 2 to C 6 alkyl, C x to C 6 substituted alkyl, C x to C 7 acyl, C 2 to C 7 alkenyl, C 2 to C 7 alkynyl, C 7 to C 12 phenylalkyl or C 7 to C 12 substituted phenylalkyl.
• the two substituents can be the same or different.
• amino-protecting group refers to substituents of the amino group commonly employed to block or protect the amino functionality while reacting other functional groups of the molecule.
• protected (monosubstituted) amino means there is an amino-protecting group on the monosubstituted amino nitrogen atom.
• protected carboxamide means there is an amino-protecting group on the carboxamide nitrogen.
• amino-protecting groups include the formyl ("For") group, the trityl group, the phthalimido group, the trichloroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetyl groups, urethane-type blocking groups, such as t-butoxycarbonyl ("Boc”), 2- (4-biphenylyl) propyl-2-oxycarbonyl ("Bpoc”), 2-phenylpropyl-2-oxycarbonyl (“Poc”), 2- (4- xenyl) isopropoxycarbonyl, 1, 1-diphenylethyl-l- oxycarbonyl, 1, 1-diphenylpropyl-l-oxycarbonyl, 2- (3, 5- dimethoxyphenyl) propyl-2-oxycarbonyl ("Ddz”), 2- (p- toluyl) propyl-2-oxycarbonyl, cyclopentanyloxycarbonyl, 1-methylcyclo
• amino-protecting group employed is not critical so long as the derivatized amino group is stable to the conditions of the subsequent reaction (s) and can be removed at the appropriate point without disrupting the remainder of the compounds.
• Preferred amino- protecting groups are Boc, Cbz and Fmoc.
• Further examples of amino-protecting groups embraced by the above term are well known in organic synthesis and the peptide art and are described by, for example, T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis," 2nd ed., John Wiley and Sons, New York, NY, 1991, Chapter 7, M.
• carboxy-protecting group refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound.
• carboxylic acid protecting groups include t-butyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4- dimethoxybenzyl, 2, 4-dimethoxybenzyl, 2,4,6- trimethoxybenzyl, 2, 4, 6-trimethylbenzyl, pentamethylbenzyl, 3, 4-methylenedioxybenzyl, benzhydryl, 4, 4 ' -dimethoxytrityl, 4 , 4 ' , 4 "-trimethoxytrityl, 2- phenylpropyl, trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2, 2 , 2-trichloroethyl, ⁇ - (trimethylsilyl ) ethyl, ⁇
• carboxy-protecting group employed is not critical so long as the derivatized carboxylic acid is stable to the conditions of subsequent reaction (s) and can be removed at the appropriate point without disrupting the remainder of the molecule. Further examples of these groups are found in E. Haslam, "Protective Groups in Organic Chemistry,” J.G.W. McOmie, Ed., Plenum Press, New York, NY, 1973, Chapter 5, and T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis," 2nd ed., John Wiley and Sons, New
• protected hydroxy denotes a hydroxy group bonded to a "hydroxy-protecting group.”
• hydroxy-protecting group refers to a readily cleavable group bonded to a hydroxy group, such as the tetrahydropyranyl, 2-methoxypropyl, 1-ethoxyethyl, methoxymethyl, 2-methoxyethoxymethyl, methylthiomethyl, t-butyl, t-amyl, trityl, 4-methoxytrityl, 4,4'- dimethoxytrityl, 4 , 4 ', 4 "-trimethoxytrityl, benzyl, allyl, trimethylsilyl, (t-butyl) dimethylsilyl, 2,2,2- trichloroethoxycarbonyl groups and the like.
• protected hydroxymethyl is similarly defined.
• hydroxy-protecting group is not critical so long as the derivatized hydroxyl group is stable to the conditions of subsequent reaction (s) and can be removed at the appropriate point without disrupting the remainder of the molecule.
• Further examples of hydroxy-protecting groups are described by C.B. Reese and E. Haslam, "Protective Groups in Organic Chemistry,” J.G.W. McOmie, Ed., Plenum Press, New York, NY, 1973, Chapters 3 and 4, respectively, and T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis," 2nd ed., John Wiley and Sons, New York, NY, 1991, Chapters 2 and 3.
• C x to C 4 alkylthio refers to sulfide groups such as methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, t-butylthio and like groups.
• C x to C 4 alkylsulfoxide indicates sulfoxide groups such as methylsulfoxide, ethylsulfoxide, n-propylsulfoxide, isopropylsulfoxide, n-butylsulfoxide, sec-butylsulfoxide and the like.
• C x to C 4 alkylsulfonyl encompasses groups such as methylsulfonyl, ethylsulfonyl, n- propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl, t- butylsulfonyl and the like.
• substituted phenylthio By “substituted phenylthio, " “substituted phenyl sulfoxide,” and “substituted phenylsulfonyl” is meant that the phenyl can be substituted as described above in relation to “substituted phenyl.”
• substituted cyclic C 2 to C 7 alkylene, " “cyclic C 2 to C 7 heteroalkylene, “ and “substituted cyclic C 2 to C 7 heteroalkylene, " define such a cyclic group bonded to the phenyl radical resulting in a bicyclic ring system.
• the cyclic group may be saturated or contain one or two double bonds.
• the cyclic group may have one or two methylene or methine groups replaced by one or two oxygen, nitrogen or sulfur atoms which are the the cyclic C 2 to C 7 heteroalkylene.
• the cyclic alkylene or heteroalkylene group may be substituted once or twice by the same or different substituents selected from the group consisting of the following moieties: hydroxy, protected hydroxy, carboxy, protected carboxy, oxo, protected oxo, C x to C 4 acyloxy, formyl, C x to C 7 acyl , C x to C 6 alkyl, carbamoyl, C, to C 7 alkoxy, C ⁇ to C 4 alkylthio, C-, to C 4 alkylsulfoxide, C, to C 4 alkylsulfonyl, halo, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino, hydroxymethyl or a protected hydroxymethyl.
• the cyclic alkylene or heteroalkylene group fused onto the benzene radical preferably contains three to six members.
• saturated cyclic groups are when the resultant bicyclic ring system is 2,3- dihydro-indanyl and a tetralin ring.
• unsaturated examples occur when the resultant bicyclic ring system is a naphthyl ring or indolyl.
• fused cyclic groups which each contain one nitrogen atom and one or more double bond, preferably one or two double bonds, are when the phenyl is fused to a pyridino, pyrano, pyrrolo, pyridinyl, dihydropyrrolo, or dihydropyridinyl ring.
• fused cyclic groups which each contain one oxygen atom and one or two double bonds are when the phenyl ring is fused to a furo, pyrano, dihydrofurano, or dihydropyrano ring.
• fused cyclic groups which each have one sulfur atom and contain one or two double bonds are when the phenyl is fused to a thieno, thiopyrano, dihydrothieno or dihydrothiopyrano ring.
• cyclic groups which contain two heteroatoms selected from sulfur and nitrogen and one or two double bonds are when the phenyl ring is fused to a thiazolo, isothiazolo, dihydrothiazolo or dihydroisothiazolo ring.
• Examples of cyclic groups which contain two heteroatoms selected from oxygen and nitrogen and one or two double bonds are when the benzene ring is fused to an oxazolo, isoxazolo, dihydrooxazolo or dihydroisoxazolo ring.
• Examples of cyclic groups which contain two nitrogen heteroatoms and one or two double bonds occur when the benzene ring is fused to a pyrazolo, imidazolo, dihydropyrazolo or dihydroimidazolo ring or pyrazinyl .
• any substituent term ending in “ene” means that such substituent connects two separate additional groups.
• C 3 to C 7 cycloalkylene means a cycloalkyl, as defined above, where the cycloalkyl radical connects two separate additional groups.
• salt encompasses those salts that form with the carboxylate anions and amine nitrogens and include salts formed with the organic and inorganic anions and cations discussed below. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and organic or inorganic acids.
• Such acids include hydrochloric, sulfuric, phosphoric, acetic, succinic, citric lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D- glutamic, d-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.
• organic or inorganic cation refers to counterions for the carboxylate anion of a carboxylate salt.
• the counter-ions are chosen from the alkali and alkaline earth metals, (such as lithium, sodium, potassium, barium, aluminum and calcium) ; ammonium and mono-, di- and tri-alkyl amines such as trimethylamine, cyclohexylamine; and the organic cations, such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis (2-hydroxyethyl) ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations.
• the compounds of the above formula can also exist as solvates and hydrates. Thus, these compounds may crystallize with, for example, waters of hydration, or one, a number of, or any fraction thereof of molecules of the mother liquor solvent.
• the solvates and hydrates of such compounds are included within the scope of this invention.
• One or more isoquinoline derivatives, even when in a combinatorial library, can be in the biologically active ester form, such as the non-toxic, metabolically- labile ester-form. Such ester forms induce increased blood levels and prolong the efficacy of the corresponding non-esterified forms of the compounds.
• Ester groups which can be used include the lower alkoxymethyl groups, for example, methoxymethyl, ethoxymethyl, isopropoxymethyl and the like; the ⁇ - (C x to C 7 ) alkoxyethyl groups, for example methoxyethyl, ethoxyethyl, propoxyethyl, isopropoxyethyl and the like; the 2-oxo-l, 3-diooxlen-4-ylmethyl groups, such as 5- methyl-2-oxo-l, 3-dioxolen-4-ylmethyl, 5-phenyl-2-oxo-l, 3- dioxolen-4-ylmethyl and the like; the C ⁇ to C 4 alkylthiomethyl groups, for example methylthiomethyl, ethylthiomethyl, iso-propylthiomethyl and the like; the acyloxymethyl groups, for example pivaloyloxymethyl, pivaloyloxyethyl, ⁇ -
• amino acid includes any one of the twenty naturally-occurring amino acids or the D-form of any one of the naturally-occurring amino acids.
• amino acid also includes other non-naturally occurring amino acids besides the D-amino acids, which are functional equivalents of the naturally- occurring amino acids.
• non-naturally-occurring amino acids include, for example, norleucine ("Nle”), norvaline (“Nva”), ⁇ -Alanine, L- or D-naphthalanine, ornithine ("Orn”), homoarginine (homoArg) and others well known in the peptide art, such as those described in M.
• amino acids are indicated herein by either their full name or by the commonly known three letter code. Further, in the naming of amino acids, "D-" designates an amino acid having the "D" configuration, as opposed to the naturally occurring L-amino acids. Where no specific configuration is indicated, one skilled in the art would understand the amino acid to be an L-amino acid.
• the amino acids can, however, also be in racemic mixtures of the D- and L-configuration.
• any one of the twenty naturally-occurring amino acids means any one of the following: Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
• the language "the D-form of a naturally-occurring amino acid” means the D-isomer of any one of these naturally-occurring amino acids, with the exception of Gly, which does not occur as D or L isomers.
• the term "functionalized resin” means any resin, crosslinked or otherwise, where functional groups have been introduced into the resin, as is common in the art. Such resins include, for example, those functionalized with amino, alkylhalo, formyl or hydroxy groups. Such resins which can serve as solid supports are well known in the art and include, for example, 4-methylbenzhydrylamine-copoly (styrene-1% divinylbenzene) (MBHA) , 4-hydroxymethylphenoxymethyl-copoly (styrene-1% divinylbenzene) , 4-oxymethyl-phenyl-acetamido- copoly (stryene-1% divinylbenzene) (Wang) , 4- (oxymethyl) - phenylacetamido methyl (Pam), and TentagelTM, from Rapp Polymere Gmbh, trialkoxy-diphenyl-methyl ester- copoly (styrene-1% divinylbenzene) (RINK) all of which
• a "combinatorial library” is an intentionally created collection of differing molecules which can be prepared by the means provided below or otherwise and screened for biological activity in a variety of formats (e.g., libraries of soluble molecules, libraries of compounds attached to resin beads, silica chips or other solid supports).
• a "combinatorial library,” as defined above, involves successive rounds of chemical syntheses based on a common starting structure.
• the combinatorial libraries can be screened in any variety of assays, such as those detailed below as well as others useful for assessing their biological activity.
• the combinatorial libraries will generally have at least one active compound and are generally prepared such that the compounds are in equimolar quantities.
• a combinatorial library of the invention can contain two or more of the above-described compounds.
• the invention further provides a combinatorial library containing five or more of the above-described compounds.
• a combinatorial library can contain ten or more of the above-described compounds.
• a combinatorial library can contain fifty or more of the above-described compounds.
• a combinatorial library of the invention can contain 100,000 or more, or even 1,000,000 or more, of the above-described compounds.
• the isoquinoline compounds of the present invention were prepared as follows.
• a solid support resin-bound amine was reacted with bromoacetic acid to produce resin bound -CH 2 NHC (0) CH,Br (i.e., -"Y"CH,Br) .
• the R 1 group for example, R n -NH 2 , was then coupled, displacing the bromine and thus yielding resin bound -CH 2 NHC(0)CH 2 NH-R 11 .
• aldehydes used in the above reaction scheme are 1, -benzodioxan-6-carboxaldehyde, 1- 15 methylindole-3-carboxaldehyde, 2, 3-difluorobenzaldehyde, 2-bromobenzaldehyde, 2-chloro-5-nitrobenzaldehyde, 2- furaldehyde, 2-imidazolecarboxaldehyde, 2-naphthaldehyde, 2-pyridinecarboxaldehyde, 2-thiophenecarboxaldehyde, 3,4- dichlorobenzaldehyde, 3, 5-bis (trifluoromethyl) benzaldehyde, 3, 5-dihydroxybenzaldehyde, 3,5- dimethoxybenzaldehyde, 3, 5, -dimethyl-4- hydroxybenzaldehyde,
• reaction is allowed to proceed for 1 to 24 hrs at 20°C to 80°C, preferably at 25°C for 3 to 5 hrs to yield various carboxamide derivatives. Finally, the compounds were cleaved from the resin as described above and tested for biological activity.
• the present invention also provides libraries and individual compounds which are the corresponding amines of the above-described isoquinoline amide derivatives.
• the mixture can be further chemically transformed to extend the range and chemical diversity of the compounds.
• libraries and compounds will have the following structure:
• the nonsupport-bound library mixtures were screened in solution in radio-receptor inhibition assays described in detail below. Deconvolution of highly active mixtures were carried out by iterative, and in one instance additionally, positional scanning methods. These techniques, the iterative approach or the positional scanning approach, can be utilized for finding other active compounds within the libraries of the present invention using any one of the below-described assays or others well known in the art.
• the identity of the third variable position in the sub-library having the highest activity is determined. If more variables exist, this process is repeated for all variables, yielding the compound with each variable contributing to the highest desired activity in the screening process. Promising compounds from this process can then be synthesized on larger scale in traditional single-compound synthetic methods for further biological investigation.
• the optimum substituent at that position is determined, pointing to the optimum or at least a series of compounds having a maximum of the desired biological activity.
• the number of sublibraries for compounds with a single position defined will be the number of different substituents desired at that position, and the number of all the compounds in each sublibrary will be the product of the number of substituents at each of the other variables.
• the new isoquinoline compounds of the present invention can be used for a variety of purposes and indications and as medicaments for any such purposes and indications.
• isoquinolines are generally known to have antimicrobial activity.
• the isoquinolines of the present invention can be used to treat infections.
• the ability of the compounds to inhibit bacterial growth can be determined by methods well known in the art.
• An exemplary in vi tro antimicrobial activity assay is described in Blondelle and Houghten, Biochemistry 30:4671-4678 (1991), which is incorporated herein by reference.
• Staphylococcus aureus ATCC 29213 (Rockville, MD) is grown overnight at 37°C in Mueller- Hinton broth, then re-inoculated and incubated at 37°C to reach the exponential phase of bacterial growth (i.e., a final bacterial suspension containing IO 5 to 5 x IO 5 colony-forming units/ml) .
• the concentration of cells is established by plating 100 ⁇ l of the culture solution using serial dilutions (e.g., IO "2 , IO "3 and IO "4 ) onto solid agar plates.
• Compounds of the present invention were shown to have antimicrobial activity by the in vi tro antimicrobial activity assay described in Example 54 below and, therefore, are useful as antimicrobial agents.
• Isoquinolines are also known to be antiarrhythmic and cardioprotective agents as described, for example, in published European Patent Application 0 590 455 to Lai et al., which is incorporated herein by reference. Therein is also described assays for assessing the antiarrhythmic and cardioprotective properties of isoquinolines, such as the reperfusion induced arrhythmias assay in isolated rat heart .
• Additional assays can be, and have been, used to test the biological activity of the instant isoquinolines.
• Such assays include a competitive enzyme- linked immunoabsorbent assay and, as described in Examples 44 and 47, radio-receptor assays.
• the radio-receptor assay can be selective for any one of the ⁇ , K, or ⁇ opiate receptors and is, therefore, an indication of isoquinolines' analgesic properties as described, for example, in Dooley et al., Proc. Natl. Acad. Sci., 90:10811-10815 (1993).
• such compounds can, and have been as described in Example 47, tested in a ⁇ receptor assay.
• Ligands for the ⁇ receptor can be useful as antipsychotic agents, as described in Abou-Gharbia et al., Annual Reports in Medicinal Chemistry, 28:1-10 (1993).
• ELISA Competitive Enzyme-Linked Immunosorbent Assay
• Radio-Receptor Assay Particulate membranes can be prepared using a modification of the method described in Pasternak et al . , Mol. Pharmacol. 11:340-351 (1975), which is incorporated herein by reference.
• Rat brains frozen in liquid nitrogen can be obtained from Rockland (Gilbertsville, PA) .
• the brains are thawed, the cerebella removed and the remaining tissue weighed.
• Each brain is individually homogenized in 40 ml Tris-HCl buffer (50 M, pH 7.4, 4°C) and centrifuged (Sorvall 0 RC5C SA-600: Du Pont, Wilmington, DE) (16,000 rpm) for 10 mins .
• the pellets are resuspended in fresh Tris-HCl buffer and incubated at 37°C for 40 mins . Following incubation, the suspensions are centrifuged as before, the resulting pellets resuspended in 100 volumes of Tris buffer and the suspensions combined. Membrane suspensions are prepared and used in the same day.
• Protein content of the crude homogenates generally range from 0.15-0.2 mg/ml as determined using the method described in M.M. Bradford, M.M., Anal. Biochem. 72:248- 254 (1976), which is incorporated herein by reference.
• reaction is terminated by filtration through GF-B filters on a Tomtec harvester (Orange, CT) .
• the filters are subsequently washed with 6 ml of Tris-HCl buffer, 4°C.
• Bound radioactivity is counted on a Pharmacia Biotech Betaplate Liquid Scintillation Counter (Piscataway, NJ) and expressed in cpm.
• standard curves in which 3 H-DAMGO is incubated in the presence of a range of concentrations of unlabeled DAMGO (0.13-3900 nM) are generally included in each plate of each assay (a 96-well format) .
• the isoquinoline compounds of the present invention are generally in a pharmaceutical composition so as to be administered to a subject at dosage levels of from 0.7 to 7000 mg per day, and preferably 1 to 500 mg per day, for a normal human adult of approximately 70 kg of body weight, this translates into a dosage of from 0.01 to 100 mg/kg of body weight per day.
• the specific dosages employed, however, can be varied depending upon the requirements of the patient, the severity of the condition being treated, and the activity of the compound being employed. The determination of optimum dosages for a particular situation is within the skill of the art.
• inert, pharmaceutically acceptable carriers are used.
• the pharmaceutical carrier can be either solid or liquid.
• Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories .
• a solid carrier can be one or more substances which can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.
• the carrier is generally a finely divided solid which is in a mixture with the finely divided active component.
• the active compound is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
• a low-melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient-sized molds and allowed to cool and solidify.
• Powders and tablets preferably contain between about 5% to about 70% by weight of the active ingredient.
• Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter and the like.
• compositions can include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component (with or without other carriers) is surrounded by a carrier, which is thus in association with it.
• a carrier which is thus in association with it.
• cachets are also included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.
• Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, or suspensions, and emulsions suitable for oral administration.
• Sterile water solutions of the active component or sterile solutions of the active component in solvents comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.
• Sterile solutions can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.
• Aqueous solutions for oral administration can be prepared by dissolving the active compound in water and adding suitable flavorants, coloring agents, stabilizers, and thickening agents as desired.
• Aqueous suspensions for oral use can be made by dispersing the finely divided active component in water together with a viscous material such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art.
• the pharmaceutical composition is in unit dosage form.
• the composition is divided into unit doses containing appropriate quantities of the active isoquinoline.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, packeted tablets, capsules, and powders in vials or ampules.
• the unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.
• This Example provides a representative solid-phase combinatorial synthesis of a library which would contain approximately 525 derivatives of dihydroisoquinolines (DHQs).
• DHQs dihydroisoquinolines
• preparation of a library containing the DHQs involves the following steps. Briefly, first, thirty five diverse amino carboxylic acid, varying at R 1 , and including various amino-protected amino acids, are coupled to MBHA resin employing the tea-bag method of Houghten, et . al, as described, for example in U.S. Patent No. 4,631,211 to Houghten and Houghten et al., Proc. Natl. Acad. Sci., 82:5131-5135 (1985), both of which are incorporated herein by reference.
• each tea-bag After coupling and thorough washing the 35 tea-bags, each containing one resin-bound amino carboxylic acid, are opened and the resin beads combined and thoroughly mixed as a suspension in dichloromethane (DCM) .
• DCM dichloromethane
• the resins are isolated by filtration and dried under vacuum, then divided into 15 equal portions and resealed in 15 labeled tea-bags, each tea-bag now having a mixture of the 35 amino carboxylic acids. This is followed by condensing 15 aldehydes, each differing by their R 2 substituent, using triethylorthoformate as dehydrating agent with the tea-bag contained mixtures of resin-bound amino carboxylic acids.
• tea-bag each containing the 35 resin-bound carboxylic acids, is used for each aldehyde in a separate reaction. After washing with an anhydrous solvent the tea-bags are collectively reacted with homophthalic anhydride and triethylamine in anhydrous dimethylformamide (DMF) to arrive at a library of 525 derivatives of DHQ. Finally, the compounds are individually cleaved from the MBHA resin using a hydrogen fluoride (HF) procedure. The individual mixtures varying at R 1 and constant at R 2 , each a mixture containing 35 individual compounds can then be tested for biological activity using any one of a variety of screening assays, such as those described above or others well known in the art.
• HF hydrogen fluoride
• the individual amino carboxylic acids which can be used to prepare a library of 525 DHQs include the following: Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, D-Ala, D-Asp, D-Cys, D-Glu, D-Ile, D-Leu, D-Lys, D-Met, D-Phe, D-Ser, D-Thr, D-Tyr, and D-Val, ⁇ -alanine, and 4-aminobutyric acid. All are amino-protected with Fmoc or Boc and carry appropriate side chain protecting group as required.
• aldehydes which can be employed are as follows: benzaldehyde, 4-methoxybenzaldehyde, 4-nitrobenzaldehyde, -chlorobenzaldehyde, 2-methoxybenzaldehyde, 2-nitrobenzaldehyde, 2-chlorobenzaldehyde, 4-phenylbenzaldehyde, furfuraldehyde, 2-propionaldehyde, 2-methyl-2-buten-l-al, cyclohexane carboxaldehyde, butanal, cinnamaldehyde, acetaldehyde .
• Each resin packet is individually coupled overnight ( ⁇ 16 hrs except for Gly, 1 hr) by adding 10X amino acid in DCM (0.2 M) or amino carboxylic acid in DMF followed by 10X diisopropylcarbodiimide/DCM (0.2 M) for a final reagent concentration of 0.1 M DMF (5%) used to solubilize the Arg and Ser derivatives. Hydroxybenzotriazole (HOBt) (10X) is added to the amino carboxylic acids couplings. Following coupling completion, resin packets are washed with DCM (IX), isopropanol (IPA) (2X) , and DCM (2X) .
• DIX isopropanol
• IPA isopropanol
• Each packet is next shaken twice in 20% (v/v) piperidine/DMF (30 ml, 5 min, then 15 min) then washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• a solution of the respective aldehyde (0.203 ml, 2 mmoles) and anhydrous trimethylorthoformate (0.438 ml, 4 mmoles) is prepared in DMF (7.5 ml) ana added to the packet. After shaking for 3 hrs the packet is washed with dry ( ⁇ 0.03% water) DMF (5 x 30 ml) .
• a solution of homophthalic anhydride (324 mg, 2 mmoles) and triethylamine (0.021 ml, 0.15 mmoles) is prepared in DMF (5 ml) and added to each packet. After heating at 80°C for 16 hrs the packets are then washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• the isoquinolines are cleaved off of the resin by treatment with HF (liquid (1)) at -15°C for 2 hrs followed by warming to room temperature while removing HF (gaseous (g) ) with a nitrogen stream.
• This Example provides the solid-phase synthesis of trans-N- (2-acetamidoyl) -3-phenyl-4-carboxy-3, 4 -dihydro-1 (2H) -isoquinolone by condensing, on a TentaGelTM resin, glycine, benzaldehyde and homophthalic anhydride.
• the isoquinoline was cleaved from the resin by trifluoroacetic acid (TFA) .
• TentaGelTM S-NH 2 resin (Rapp Polymere Gmbh, Federal Republic of Germany; 385 mg, 0.100 milliequivalents) was placed in a porous polypropylene packet.
• the packet was placed in a 60 ml bottle and washed with 5% (v/v) DIEA/DCM (3 x 30 ml) followed by DCM (5 x 30 ml).
• the packet was next shaken twice in 20% (v/v) piperidine/DMF (30 ml, 5 min, then 15 min) , then washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• a solution of N- (9-fluorenylmethoxycarbonyl) glycine (149 mg, 0.5 mmoles), HOBt (68 mg, 0.5 mmoles), and DIC (0.094 ml, 0.6 mmoles) was prepared in DMF (5 ml) and added to the resin packet. After shaking for 2 hrs the packet was washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• the packet was next shaken twice in 20% (v/v) piperidine/DMF (30 ml, 5 min, then 15 min) then washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• a solution of benzaldehyde (0.203 ml, 2 mmoles) and anhydrous trimethylorthoformate (0.438 ml, 4 mmoles) was prepared in DMF (7.5 ml) and added to the packet. After shaking for 3 hrs the packet was washed with dry ( ⁇ 0.03% water) DMF (5 x 30 ml) .
• a solution of homophthalic anhydride (324 mg, 2 mmoles) and triethylamine (0.021 ml, 0.15 mmoles) was prepared in DMF (5 ml) and added to the packet. After heating at 80°C for 16 hrs the packet was washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• the isoquinolone was cleaved off of the resin by addition of a solution of 75/20/5 (v/v/v) TFA/DCM/water (10 ml) . After shaking for 135 min the acid solution was decanted into a round bottom flask. The packet was then washed with TFA (1 x 10 ml) and this wash was also added to the round bottom flask. The solvent was removed under reduced pressure providing a clear oil. The crude oil was dissolved in DCM (20 ml and extracted with 1 N hydrochloric acid (HCl; 1 x 10 ml) . The organic layer was next extracted with aqueous saturated sodium bicarbonate (NaHC0 3 ) (2 x 10 ml) .
• This Example provides the solid-phase synthesis of trans-N- (2-acetamidoyl) -3-phenyl-4-carboxy-3, 4 -dihydro-1 (2H) -isoquinolone by condensing, on a polystyrene benzhydrylamine resin, glycine, benzaldehyde and homophthalic anhydride. The final product was cleaved from the resin using an HF procedure.
• Polystyrene benzhydrylamine (BHA) resin (189 mg, 0.100 milliequivalents) was placed in a porous polypropylene packet.
• the packet was placed in a 60 ml bottle and washed with 5% (v/v) DIEA/DCM (3 x 30 ml) followed by DCM (5 x 30 ml) .
• the packet was next shaken twice in 20% (v/v) piperidine/DMF (30 ml, 5 min, then 15 min) , then washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• a solution of benzaldehyde (0.203 ml, 2 mmoles) and anhydrous trimethylorthoformate (0.438 ml, 4 mmoles) was prepared in DMF (7.5 ml) and added to the packet. After shaking for 3 hrs the packet was washed with dry ( ⁇ 0.03% water) DMF (5 x 30 ml) .
• a solution of homophthalic anhydride (324 mg, 2 mmoles) and triethylamine (0.021 ml, 0.15 mmoles) was prepared in DMF (5 ml) and added to the packet. After heating at 80°C for 16 hrs the packet was washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• the isoquinoline was cleaved off of the resin by treatment with HF (1) at -15°C for 2 hrs followed by warming to room temperature while removing HF (g) with a nitrogen stream.
• HF HF
• the packet and HF tube were washed with TFA (2 x 8 ml) and the two washes were transferred to a round bottom flask and concentrated to a clear oil under reduced pressure.
• the crude oil was dissolved in DCM (20 ml and extracted with 1 N HCl (1 x 10 ml) .
• the organic layer was next extracted with aqueous saturated NaHC0 3 (2 x 10 ml) .
• the NaHC0 3 layers were combined and the pH of the solution was brought to 1-2 by addition of 2 N HCl, followed by extraction with DCM (2 x 10 ml) .
• the final organic layers were combined and the solvent was removed under reduced pressure, providing a quantitative yield of a clear oil which upon lyophilization provided a white crystalline solid.
• Spectral data was identical to the sample prepared in Example 2.
• MBHA resin (0.05 mmoles) was sealed in a polypropylene packet.
• the packet was shaken in 55% (v/v) TFA/DCM (30 ml, 30 min) then washed with DCM (1 x 30 ml), isopropyl alcohol (2 x 30 ml), 5% (v/v) DIEA/DCM (3 x 30 ml, 2 min each), DCM (2 x 30 ml), and anhydrous DMF (2 x 30 ml).
• a solution of benzaldehyde (10 mmoles) and anhydrous trimethylorthoformate (20 mmoles) was prepared in DMF (20 ml) and added to the packet. After shaking for 3.75 hrs the packet was washed with dry ( ⁇ 0.03% water) DMF (5 x 30 ml) .
• a solution of homophthalic anhydride (7.5 mmoles) and triethylamine (225 mmoles) was prepared in chloroform (15 ml) and added to the packet.
• This Example provides the solid-phase synthesis of trans-N- (3-propionamidoyl) -3-phenyl-4-carboxy- 3, 4-dihydro-l (2H) -isoquinolone prepared by condensing, on a RINK linker derivatized TentaGelTM resin, aminopropionic acid, benzaldehyde and homophthalic anhydride. The final product was removed from the resin by TFA cleavage.
• N- ( 9-Fluorenylmethoxycarbonyl) -3-aminopropionic acid was attached to RINK linker derivatized TentaGelTM resin as described in Example 2.
• the packet was next shaken twice in 20% (v/v) piperidine/DMF (30 ml, 5 min, then 15 min) then washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• a solution of benzaldehyde (0.203 ml, 2 mmoles) and anhydrous trimethylorthoformate (0.438 ml, 4 mmoles) was prepared in DMF (7.5 ml) and added to the packet. After shaking for 3 hrs the packet was washed with dry ( ⁇ 0.03% water) DMF (5 x 30 ml) followed by chloroform (3 x 30 ml) .
• a solution of homophthalic anhydride (324 mg, 2 mmoles) and triethylamine (0.021 ml, 0.15 mmoles) was prepared in chloroform (5 ml) and added to the packet.
• the isoquinolone was cleaved off of the resin by addition of a solution of 75/20/5 (v/v/v) TFA/DCM/water (10 ml) . After shaking for 135 min the acid solution was decanted into a round bottom flask. The packet was then washed with TFA (1 x 10 ml) and this wash was also added to the round bottom flask. The solvent was removed under reduced pressure providing a clear oil .
• Examples 6 to 28 were done using the procedures of Example 5. In place of the starting materials 3-aminopropionic acid and benzaldehyde of Example 5, Examples 6 to 28 provide all possible combinations of four different amino carboxylic acids and six unique benzaldehydes.
• the amino carboxylic acids and benzaldehydes used, along with the corresponding Example number is shown in TABLE I. As a reference, the compound prepared in Example 5 is shown in Table I at upper left.
• This Example provides the solid-phase synthesis of trans-N- (2-acetamidoyl) -3-methyl-4-carboxy- 3, 4-dihydro-l (2H) -isoquinolone prepared by condensing, on an MBHA resin, glycine, acetaldehyde and homophthalic anhydride. The final product was removed from the resin by HF cleavage.
• N- (t-Butyloxycarbonyl) glycine attached to MBHA resin (0.05 mmoles) was sealed in a polypropylene packet.
• the packet was shaken in 55% (v/v) TFA/DCM (30 ml, 30 min) then washed with DCM (1 x 30 ml), isopropyl alcohol (2 x 30 ml), 5% (v/v) DIEA/DCM (3 x 30 ml, 2 min each), DCM (2 x 30 ml), and anhydrous DMF (2 x 30 ml).
• a solution of acetaldehyde (5 mmoles) and anhydrous trimethylorthoformate (10 mmoles) was prepared in DMF (10 ml) and added to the packet. After shaking • for 3.75 hrs the packet was washed with dry ( ⁇ 0.03% water) DMF (5 x 30 ml) .
• a solution of homophthalic anhydride (5 mmoles) and triethylamine (0.075 mmoles) was prepared in chloroform (10 ml) and added to the packet.
• the isoquinolone was cleaved off of the resin using HF as in Example 3. The residue was dissolved in deuterated DMSO to obtain NMR and mass spectra after which the solvent was removed under reduced pressure providing a 2/1 mixture of the desired product and the amide of homophthalic acid and glycinamide as a clear oil (10 mg, 77%).
• MALDI-TOF MS 283 (MW + Na+) .
• HATU 3-tetramethyluronium hexafluorophosphate
• anhydrous DMF (0.93 mmoles, 3.1 ml, 300 mM solution) was added to the packet and shaken for 20 min.
• the HATU solution was decanted off of the tea-bag and anhydrous DMF (4.7 ml) and anhydrous 1-propanol (0.374 ml, 5 mmoles) were added.
• the propanol solution was removed and the bag washed with anhydrous DMF (2 x 10 ml) .
• the HATU treatment was repeated followed by decanting and addition of a second 1-propanol solution. This reaction was shaken at room temperature for 66 hrs.
• the bag was then washed with DMF (4 x 10 ml), DCM (3 x 10 ml), and allowed to dry.
• Standard HF cleavage as in Example 4 provided a clear oil shown by NMR and mass spectra to contain a half mixture of the desired ester and the free acid (18 mg, 85%) MALDI-TOF MS: 448 (MW + Na+) .
• This product was prepared as in Example 32 but with the substitution of (+/-) -3-aminoquinuclidine, bis HCl salt for 1-propanol; DIEA (200 mole % versus aminoquinuclidine) was also added to neutralize the HCl salt. Yield: 23 mg, 98%.
• TentaGelTM S-NH2 resin (385 mg, 0.100 milliequivalents) derivatized with the protected RINK linker. Following the procedure provided in Example 2, one bag each was then coupled with one each of five different amino acids, N- ( 9-fluorenylmethoxycarbonyl)
• the resin packets were dried at room temperature and cut open. The resin inside was pooled from all five bags and the resin was shaken in DCM (20 ml) for 75 min. The resin was filtered off and again dried before being divided into five equal portions and resealed in porous polystyrene packets. One packet was then reacted with first benzaldehyde and then homophthalic anhydride as in Example 5. The resin was cleaved and worked up as in Example 5, providing a clear oil, 11 mg, 61% yield based on average molecular weight).
• MALDI-TOF MS of the crude products after cleavage showed all five expected isoquinolines for each individual resin packet.
• Analysis of the final extract by proton NMR and MALDI-TOF MS indicated that only four isoquinolines were now present with the 2 , 6- (s) -diaminohexanoic acid based isoquinolone having been lost in the extraction procedure.
• EXAMPLE 38 Solid-phase synthesis of a combinatorial library pool containing isoquinolines derived from five amino acids, 4-methoxybenzaldehyde and homophthalic anhydride
• Example 37 with the exception of substituting 3,5-dimethoxybenzaldehyde for benzaldehyde used in Example 37.
• Cleavage from the resin was performed on all 100 ⁇ moles of resin and yield and mass spectra were immediately obtained on the residue with no aqueous extraction being performed. Yield: 35 mg, 84%.
• MALDI-TOF MS of the crude products after cleavage showed all five expected isoquinolines for each individual resin packet .
• Eleven porous polypropylene tea-bags were prepared each containing polystyrene MBHA/resin (974 mg, 0.750 milliequivalents) .
• One tea-bag was placed in a 60 ml bottle and washed with 5% (v/v) DIEA/DCM (3 x 30 ml) followed by DCM, 5 x 30 ml.
• a solution of N- (t-butyloxycarbonyl) glycine (657 mg, 3.75 mmoles), HOBt (507 mg, 3.75 mmoles), and DIC (0.705 ml, 4.5 mmoles) was prepared in DMF (37.5 ml) and added to the resin packet.
• the tea-bags with attached (s) -2-N- (t-butyloxycarbonyl) - 3-N- (9-fluorenylmethoxycarbonyl) -diaminopropionic acid, and (s) -2-N- (t-butyloxycarbonyl) -6-N- ( 9-fluorenylmethoxy carbonyl) -diaminohexanoic acid were washed with DCM (2 x 50 ml), shaken twice in 20% (v/v) piperidine/DMF (30 ml, 5 min then 15 min), then washed with DMF (4 x 50 ml) and DCM (4 x 50 ml) .
• the remaining nine tea-bags were placed in one bottle and washed with DCM (150 ml, 15 min) and then treated with 55% (v/v) TFA/DCM (150 ml, 30 min) .
• the bags were then washed with DCM (150 ml), isopropyl alcohol (2 x 150 ml), DCM (2 x 150 ml), 5% (v/v) DIEA/DCM (3 x 150 ml, 2 min each) and DCM (3 x 150 ml) .
• the eleven tea-bags were cut open and the contents pooled in a bottle containing DCM (70 ml) . The bottle was shaken for 90 min to thoroughly mix the resin.
• the DCM/resin slurry was then poured into a large (12 x 18 cm) tea-bag to separate the resin from the DCM and the resin was dried at 50°C.
• the resulting 11.042 g of resin (8.25 mmoles total of mixed amino acids) was divided into 39 tea-bags containing 38 x 0.241 g resin (each 180 micromoles total of mixed amino acids) and 1 x 1.204 g (900 micromoles total of mixed amino acids) .
• glycine control tea-bags 38 additional tea-bags each containing 23 mg (18 micromoles) of glycine (containing a free amino group) attached to MBHA resin (coupled and deprotected as in Example 1, subheading 1, hereinafter referred to as the "glycine control tea-bags").
• Placed in a 20 ml bottle were one of the small (0.241 g of mixed amino acid resin) tea-bags and one of the control (18 micromoles of glycine) tea-bags.
• the two tea-bags were treated with a solution of benzaldehyde (0.508 ml, 5 mmoles) and anhydrous trimethylorthoformate (1.094 ml, 10 mmoles) in anhydrous DMF (9 ml). After shaking for 3 hrs the packet was washed with anhydrous DMF (3 x 8 ml) .
• a solution of homophthalic anhydride (801 mg, 5 mmoles) and triethylamine (0.044 ml, 0.3 mmoles) was prepared in chloroform (10 ml) and added to the tea-bag. After shaking at room temperature for 15.5 hrs the packet was washed with DMF (6 x 30 ml) and DCM (4 x 30 ml) and dried at room temperature.
• the 38 tea-bags containing mixed amino acid resin (now containing mixed isoquinolines after the anhydride condensation reaction) were cut open and the contents pooled in a bottle containing DCM (70 ml) . The bottle was shaken for 75 min to thoroughly mix the resin. The DCM/resin slurry was then poured into a large (12 x 18 cm) tea-bag to separate the resin from the DCM and the resin was dried at room temperature. The resulting 10.144 g of resin (6.84 mmoles total of mixed isoquinolines) was divided into 52 tea-bags each containing 0.178 g resin (120 micromoles total of mixed isoquinolines) .
• a solution of HATU in anhydrous DMF 2.4 mmoles, 8 ml, 300 mM solution
• the HATU solution was decanted off of the tea-bags and anhydrous DMF (6.9 ml) and cyclopropyl amine (0.52 ml, 7.5 mmoles) were added. After shaking for 1 hr the cyclopropyl amine solution was removed and the bags were washed with anhydrous DMF (2 x 8 ml) . The HATU treatment was repeated followed by decanting and addition of a second cyclopropyl amine solution. This reaction was shaken at room temperature for 24 hrs. The bags were then washed with DMF (3 x 8 ml) , water (8 ml, 60 min), DMF (3 x 8 ml), DCM (3 x 8 ml), and allowed to dry.
• Also prepared as a control for the aldehyde reaction were 38 single compounds from the building blocks: glycine, one of 38 aldehydes and isopropyl amine. As described above, an additional control for the amine reaction was performed resulting in 51 pools of 11 isoquinolines each prepared from the following building blocks: a mixture of the eleven amino acids, 3,5-dimethoxybenzaldehyde and, separately, each of the 51 amines.
• Each tea-bag prepared was cleaved separately via standard HF procedures with the addition of 0.2 ml anisole to each HF cleavage reaction as a scavenger and dissolved in an appropriate solvent and for testing in a variety of assays. The control tea-bags were cleaved in the same manner and characterized by NMR or mass spectra.
• This example describes the identification of individual compounds contained within the synthetic combinatorial library of Example 43 which are selective inhibitors of the ⁇ and ⁇ -opioid ligands, [ 3 H] -DAMGO and [ 3 H] -U69, 593, respectively and the ⁇ receptor ligand, radiolabeled pentazocine. Compounds were identified using the iterative approach and radioreceptor assays as described above.
• results of the screen provide evidence that there is selectivity of certain compounds for one opioid receptor over another. More importantly, the assays identify certain classes of compounds which are particularly active. For instance, those compounds made from cyclopentylamme for X (pool # 265) are particularly good inhibitors of the [ 3 H]-U69,593 ligand at the ⁇ -opioid receptor. Compounds made from 1-adamantanemethylamine at the X position (pool # 291) were identified as significant inhibitors of pentazocine ligand at the ⁇ receptor.
• tea-bags each containing a mixture of 11 amino acids on resin (7 mg, 5 micromoles) were prepared as in Example 43.
• Each tea-bag was reacted with a single aldehyde from the list: 1, -benzodioxan-6-carboxaldehyde, 1-methylindole-3-carboxaldehyde, 2, 3-difluorobenzaldehyde, 2-bromobenzaldehyde, 2-chloro-5-nitrobenzaldehyde, 2-furaldehyde, 2-imidazolecarboxaldehyde, 2-naphthaldehyde, 2-pyridinecarboxaldehyde, 2-thiophenecarboxaldehyde, 3, 4-dichlorobenzaldehyde, 3, 5-bis (trifluoromethyl) benzaldehyde, 3, 5-dihydroxybenzaldehyde, 3, 5-dimethoxybenzaldehyde,
• 3-thiophenecarboxaldehyde 4- (3-dimethylaminopropoxy) benzaldehyde, 4- (dimethylamino) benzaldehyde, 4- (methylthio) benzaldehyde, 4- (trifluoromethyl) benzaldehyde, 4-biphenylcarboxaldehyde,
• the dried tea-bags were all placed in a 60 ml bottle and washed with anhydrous DMF (2 x 30 ml) .
• Anhydrous DMF (30 ml) was then added to the tea-bags followed by HATU (3.5 g, 9.2 mmoles) and the tea-bags were shaken for 20 min.
• the HATU solution was next decanted and the tea-bags were washed with anhydrous DMF (1 x 20 ml).
• Anhydrous DMF (18.7 ml) was added to the tea-bags followed by 1-adamantanemethylamine (2.0 g, 12 mmoles) .
• the tea-bags were washed with anhydrous DMF (2 x 20 ml) and the HATU and 1- adamantanemethylamine treatments were repeated. After shaking for 16 hrs. the tea-bags were washed with DMF (4 x 20 ml), water (1 x 20 ml for 15 min., DMF (4 x 20 ml), and DCM (4 x 20 ml) . After drying the tea-bags were cleaved as in Example 43, extracted into 1:1 water/acetonitrile, examined by mass spectrometry, and tested in the ⁇ receptor assay as described above.
• Table III provides the results of that assay and evidences that pool # M367, derived from 5-hydroxymethylfuraldehyde, are the most active compounds. It was discovered in the course of identifying the individual compounds that the R 2 group resulting from 5- (hyrdoxymethyl) furanaldehhyde, 5- (hydroxymethyl) furan-2-yl, reacted with the anisole scavenger during HF cleavage to yield the Freidel-Crafts alkylation product, 5- (4 ' -methoxybenzyl) furan-2-yl .
• Example 43 Ten tea-bags each containing one amino acid on resin (75 micromoles) were prepared as in Example 43. As identified in Example 43, one amino acid was in a racemic mixture, therefor accounting for 11 different compounds in 10 bags. All of the tea-bags were placed in a 125 ml bottle and washed with anhydrous DMF (1 x 60 ml) . Added to the tea-bags were anhydrous DMF (27 ml) , 5- hydroxymethylfurfural (1.893 g, 15 mmoles), and anhydrous TMOF (3.282 ml, 30 mmoles).
• the tea-bags were washed with anhydrous DMF (3 x 50 ml) and anhydrous chloroform (1 x 50 ml) .
• Next added to the tea-bags were anhydrous chloroform (30 ml), homophthalic anhydride (2.432 g, 15 mmoles), and triethylamine (0.133 ml, 1 mmole) .
• the tea-bags were washed with DMF (5 x 50 ml) and DCM (4 x 50 ml) .
• the tea-bags were next washed with anhydrous DMF (2 x 50 ml).
• tea-bags Added to the tea-bags were anhydrous DMF (48 ml) and HATU (5.47 g, 14.4 mmoles). After shaking for 20 min., the HATU solution was decanted and the tea-bags were washed with anhydrous DMF (1 x 50 ml) . Anhydrous DMF (25 ml) was added to the tea-bags followed by 1- adamantanemethylamine (4.429 g, 25 mmoles). After shaking for 1 hr, the tea-bags were washed with anhydrous DMF (2 x 50 ml) and the HATU and 1-adamantanemethylamine treatments were repeated. After shaking for 18 hrs.
• the tea-bags were washed with DMF (4 x 50 ml), water (1 x 50 ml for 40 min., DMF (4 x 50 ml), and DCM (4 x 50 ml). After drying the tea-bags were cleaved as in Example 43, extracted into 1:1 water/acetonitrile, examined by mass spectrometry, and screened in the ⁇ receptor assay, the results of which are shown in Table IV.
• the most active compound from the library of Example 43 is one for which R 1 is 1,6-hexyl, R 2 is 5- (4 ' -methoxybenzyl) -furan-2-yl, R 3 through R 6 are, independently a hydrogen atom, X is 1- aminomethyladamantanyl, and Y is C(0)NH 2 .
• Eleven porous polypropylene tea-bags are prepared each containing polystyrene MBHA/resin (974 mg, 0.750 milliequivalents) .
• One tea-bag is placed in a 60 ml bottle and washed with 5% (v/v)
• DCM dichloromethane
• a solution of N- (t-butyloxycarbonyl) glycine (657 mg, 3.75 mmoles), HOBt (507 mg, 3.75 mmoles), and DIC (0.705 ml, 4.5 mmoles) is prepared in DMF (37.5 ml) and added to the resin packet. After shaking for 16 hrs the tea-bag is washed with DMF (3 x 30 ml) and DCM (3 x 30 ml) .
• the resulting 11.042 g of resin (8.25 mmoles total of mixed amino acids) is divided into 39 tea-bags containing 38 x 0.241 g resin (each 180 micromoles total of mixed amino acids) and 1 x 1.204 g (900 micromoles total of mixed amino acids) . Also prepared for use as a control are 38 additional tea-bags each containing 23 mg (18 micromoles) of glycine (containing a free amino group) attached to MBHA resin (coupled and deprotected as in Example 43) .
• Placed in a 20 ml bottle are one of the small (0.241 g of mixed amino acid resin) tea-bags and one of the control (18 micromoles of glycine) tea-bags.
• the two tea-bags are treated with a solution of benzaldehyde
• the large tea-bag containing 1.204 g (900 micromoles total of mixed amino acids) of resin is reacted with 3,5-dimethoxybenzaldehyde in the same manner, but on a five times larger scale of all reagents and solvents and with no control tea-bag.
• the 38 tea-bags containing mixed amino acid resin (now containing mixed isoquinolines after the above reaction) are cut open and the contents pooled in a bottle containing DCM (70 ml) . The bottle is shaken for 75 min to thoroughly mix the resin.
• the DCM/resin slurry is then poured into a large (12 x 18 cm) tea-bag to separate the resin from the DCM and the resin is dried at room temperature.
• the resulting 10.144 g of resin (6.84 mmoles total of mixed isoquinolines) is divided into 49 tea-bags each containing 0.178 g resin (120 micromoles total of mixed isoquinolines) .
• tea-bag is left as the free carboxylic acid. Also reacted with aniline in the same manner but on a 12.5 times larger scale are the 38 glycine control tea- bags as in Example 43.
• the above procedures produced 50 tea-bags each containing a mixture of 418 isoquinoline amides or acids for a total library size of 20900 compounds.
• Also prepared as a control for the aldehyde reaction are 38 single compounds from the building blocks: glycine, one of 38 aldehydes and aniline.
• An additional control for the amine reaction is performed resulting in 49 pools of 11 isoquinolines each prepared from the following building blocks: a mixture of the eleven amino acids, 3,5-dimethoxybenzaldehyde and, separately, each of the 49 amines.
• Each tea-bag prepared is cleaved separately via standard HF procedures (Example 3) , dissolved in an appropriate solvent and tested in a variety of assays.
• the control tea-bags are cleaved in the same manner and characterized by NMR or mass spectra.
• This example describes an expanded library compared to that provided in Example 43, having even more possibilities at R 1 , R 2 and X positions.
• Seventy-two porous polypropylene tea-bags were prepared each containing polystyrene MBHA/resin (1.111 g, 1.0 milliequivalents) .
• Six tea-bags were placed in a 500 ml bottle.
• a solution of N- (t-butyloxycarbonyl) -3- aminopropionic acid (beta alanine) (6.237 g, 33 mmoles), HOBt (4.458 g, 33 mmoles), and DIC (6.2 ml, 39.6 mmoles) was prepared in DMF (275 ml) and added to the resin packet.
• N,N- dimethylaminopyridine (164 mg, 1.3 mmoles) was added and the solution shaken for an additional 18 hrs.
• the tea- bags were then washed with DMF (3 x 300 ml) and DCM (3 x 300 ml) .
• the same coupling procedure was performed on the remaining 66 tea-bags in groups of six at a time, each six being reacted with a separate amino acid from the list: N- (t-butyloxycarbonyl) glycine, N- (t-butyloxycarbonyl) -5-aminopentanoic acid, N- (t-butyloxycarbonyl) -7-aminoheptanoic acid,
• DIEA/DCM (3 x 900 ml, 2 min each set) and DCM (3 x 900 ml for each set) .
• Placed in a 60 ml bottle were one of the library (0.673 g, 550 ⁇ moles of mixed amino acid resin) tea-bags and one of the control (45 micromoles of glycine) tea-bags.
• the two tea-bags were treated with a solution of benzaldehyde (2.135 ml, 21 mmoles) and anhydrous trimethylorthoformate (4.6 ml, 42 mmoles) in anhydrous DMF (38 ml) . After shaking for 4 hrs the packets were washed with anhydrous DMF (3 x 20 ml) and anhydrous chloroform (1 x 20 ml) .
• a solution of homophthalic anhydride (2.4 g, 15 mmoles) and triethylamine (0.132 ml, 0.9 mmoles) was prepared in chloroform (30 ml) and added to the tea-bag. After shaking at room temperature for 16 hrs the packets were washed with DMF (4 x 30 ml) followed by DCM (3 x 30 ml) and dried at room temperature.
• Placed in a 20 ml bottle were one of the library (154 mg, 100 ⁇ moles of mixed isoquinoline resin) tea-bags and one of the second set of control tea-bags containing 72 mg of the isoquinoline prepared from glycine, homophthalic anhydride and benzaldehyde.
• the two tea-bags were treated with a solution of HATU in anhydrous DMF (6 mmoles, 20 ml, 300 mM solution) and shaken for 20 min.
• the HATU solution was decanted off of the tea-bags and anhydrous DMF (20 ml) and aniline (1.823 ml, 20 mmoles) were added.
• Each tea-bag prepared was cleaved separately via standard HF procedures (Example 3 with the modification of an addition of 0.2 ml anisole to each HF cleavage reaction as a scavenger) , extracted into 45:45:10 water/acetonitrile/acetic acid, examined by HPLC coupled with mass spectrometry, and tested in a variety of assays.
• the control tea-bags were cleaved in the same manner and characterized by NMR or HPLC and mass spectra.
• This example describes an initial screen of libraries prepared according to Example 46 in the ⁇ - opioid receptor assay and the ⁇ receptor assay described above. The results of those screens are provided in ensuing Tables V and VI .
• the borane solution After cooling the borane solution is decanted and the bag washed with methanol (lx 25 ml), tetrahydrofuran (1 x 25 ml), and again with methanol (4 x 25 ml) . After drying the bag is returned to a 50 ml KIMAX glass tube, submerged completely in piperidine, sealed and heated at 65°C for 16 hrs.
• Example 45 Forty nine tea-bags are prepared as in Example 45 with each containing a mixture of 418 isoquinoline amides. While still attached to the resin the isoquinoline amides are reduced to the isoquinoline amines and cleaved off the resin as in Example 48, resulting in 49 pools of 418 isoquinoline amines for a total library size of 20,482.
• Example 46 One-hundred seven tea-bags were prepared as in Example 46 but on two-fold larger scale (each bag containing 200 micromoles of resin) with each containing a mixture of isoquinoline amides or acids.
• a subset of the amines used in Example 44 were used consisting of aniline, 2-fluoroaniline, 2-methoxyaniline, 2- chlorobenzylamine, 2-methoxybenzylamine, 2- trifluoromethylbenzylamine, 3-fluoroaniline, 3- methylaniline, 3-trifluoromethylaniline, 3- (methylmercapto) aniline, 3-trifluoromethylbenzylamine, 3- methylbenzylamine, 4-propylaniline, 4-pentylaniline, 4- (methylmercapto) aniline, 4-fluorobenzylamine, 4- methoxybenzylamine, 4-methylbenzylamine, 3-(l- hydroxyethyl) aniline, 4-chloroaniline, 2,3- dimethylaniline, 4-meth
• the isoquinoline amides and acids were reduced to the isoquinoline amines and alcohols via the procedure of Cuervo et al., supra , modified for solid-phase use.
• One- half of the library tea-bags and one-half of the control tea-bags were placed in a 5 L glass reactor vessel under nitrogen gas containing boric acid (66.1 g, 1.07 moles) and anhydrous trimethyl borate (107 mL, 0.955 moles).
• boric acid 66.1 g, 1.07 moles
• anhydrous trimethyl borate 107 mL, 0.955 moles.
• a IM solution of borane-tetrahydrofuran complex in tetrahydrofuran (3.2 L) was added slowly to the reaction. After sealing the reaction vessel was heated at 65°C for 96 hrs.
• Example 48 After drying the tea-bags were cleaved as in Example 48, extracted into 45:45:10 water/acetonitrile/acetic acid and examined by HPLC coupled with mass spectrometry.
• the control tea-bags were cleaved in the same manner and characterized by NMR or HPLC and mass spectra.
• the positional scan format as described above, is used to identify additional compounds which are significant inhibitors of the ⁇ receptor ligand, pentazocine.
• Subsets of compounds were prepared as described below and screened in the ⁇ receptor assay.
• the experimental procedure was as follows. Two 0.30 ⁇ mol bags each of either FMOC or BOC protected aminoacids were prepared using MBHA resin (0.90 ⁇ mol/g) as previously described (DIC, HOBt, DMF/DCM) .
• the amino acids used were: N- (t-butyloxycarbonyl) glycine, N- (t-butyloxycarbonyl) -3-aminopropionic acid, N- (t-butyloxycarbonyl) -5-aminopentanoic acid, N- (t-butyloxycarbonyl) -7-aminoheptanoic acid, (s) -2-N- (t-butyloxycarbonyl) -3-N- ( 9-fluorenylmethoxycarbonyl) -diaminopropionoic acid,
• Subset 1 consisted of 11 bags containing 55 ⁇ mol of a mixture of all 11 the resin-aminoacids .
• Subset 2 contained 11 bags of 240 ⁇ mol of the 11 resin- aminoacids mixture.
• Subset 3 had each individual resin- aminoacid subdivided into 11x11 22 ⁇ mol bags for a total of 121 bags.
• the bags from Subset 1,2 and 3 were divided into 11 groups.
• the aldehydes used were benzaldehyde, 5-nitro-2-furaldehyde, 4- nitrobenzaldehyde, 5- (hydroxymethyl) -2-furaldehyde, 4- (dimethylamino) -benzaldehyde, 3-methylbenzaldehyde, 3,5- dimethoxybenzaldehyde, 2-pyridinecarboxaldehyde, 2- naphthaldehyde, 2-furaldehyde, and 2-bromobenzaldehyde.
• Each group was composed of one bag each from Subset 1 and 2 and 11 bags of each individual resin-aminoacid from
• Subset 3 and at least one sibling bag total 14 bags, 520 ⁇ mol.
• the formation of the i ine intermediate was performed by placing a series of bags in 75 ml of a 0.5 M solution (37.5 mmol) of the benzaldehyde.
• the solution contained 8.2 ml (75 mmol) of trimethylorthoformate as a dehydrating agent.
• the resin bags were shaken in the reaction solution for 3 to 3 1/2 hrs at room temperature then the solution decanted and bags quickly washed lx with 30 ml anhydrous DMF and lx 30 ml anhydrous CHC1 3 .
• Subsets now contained the intermediate tetrahydroisoquinoline acids with a single benzaldehyde derived fragment in combination with either a mixture of all the 11 aminoacid derived fragment (Subset 1 and 2) or a single aminoacid derived fragment (Subset 3) .
• Subset 1 all the resin-bound intermediates from original Subset 1, mixed into a single portion by combining the dry solids into a large (5cm x 5cm) resin bag and mixing for 30 min in DCM. After a MeOH wash and vacuum drying the mixed resin was divided into 12 equal portions to provide 50 ⁇ mol scale bags.
• Subset 1 now represented mixtures from both the aminoacid and benzaldehyde building blocks.
• Subset 2 each of the 240 ⁇ mol bags from original Subset 2 was divided into 12 equal portions of 20 ⁇ mol to create bags which were mixed aminoacid fragments but contained sets of individual benzaldehyde fragments.
• Subset 3 for every individual resin-aminoacid set from original Subset 3 all the different benzaldehyde derived fragments were pooled into separate bags, mixed by treatment with DCM, then MeOH and dried under vacuum. These 11 new mixtures were subdivided into 12 equal portions to generate 20 ⁇ mol bags that contained a single aminoacid fragment and a mixture of the 11 benzaldehyde fragments.
• the bags were once again separated into 11 groups which contained 1 bag from Subset 1, 11 bags from Subset 2, and 11 bags from Subset 3, and a sibling bag prepared from resin-glycine-3, 5-dimethoxybenzaldehyde .
• the groups of bags were treated with 30 ml of a 0.3 M DMF stock solution of (0- (7-azabenzotriazol-l-yl) -1, 1, 3, 3- tetramethyluronium hexafluorophosphate (HATU: 79.84 g in 700 ml anhydrous DMF) by shaking for 30 min.
• the HATU solution was decanted and the bags shaken for 2 hrs in 40 ml of a 1 M DMF solution of an amine (75 mmol in 75 ml) .
• the amines used were 1- (2-aminoethyl) pyrrolidine, 1- adamantanemethylamine, 1- (2-hydroxyethyl) piperazine, piperidine, (aminomethyl) cyclohexane, 4-aminomorpholine, amylamine, aniline, cyclopentylamme, ethanolamine, and tryptamine.
• the amine solution was decanted and the bags washed 2x 30 ml DMF.
• Subset 1 The bags from Subset 1 contained mixtures from both the aminoacid and benzaldehyde building blocks but had a single amine fragment. These 50 ⁇ mol bags were used directly in the HF cleavage step.
• Subset 2 The 11 amine bags of the same resin- benzaldehyde-mixed amino acid fragments were combined into a bag in order to create a mixture of all the amines. After shaking for 30 min in DCM, then 30 min in MeOH, the resin was dried 3-4 hrs under vacuum. A 50 ⁇ mol sample bag was prepared from each of the fixed benzaldehyde mixtures for HF cleavage.
• Subset 3 The 11 amine bags containing the same resin-aminoacid-mixed benzaldehyde were mixed with the DCM/MeOH/vacuum drying procedure. Fifty ⁇ mol sample bags were created from each of the mixtures with the fixed amino acid position.
• Standard liquid HF/anisole MBHA-resin cleavage was carried out on the 33 bags and the product mixtures extracted 3x 5 ml of 50% acetonitrile/water . The solvent was removed by lypholization (2x) to yield 12 to 26 mg of solids.
• Each sample represented a 50 ⁇ mol mixture of 242 compounds with one of the variable building blocks (aminoacid, benzaldehyde, amine) as a defined compentent of the library thus producing the postional scan format.
• Bromoacetic Acid Coupling MBHA resin containing T-bags (1.2g x 1.15 mmol/g per bag) were placed into Nalgene bottles (60 bags per 2L bottle) and washed with DMF IX ( ⁇ 500mL, 20 min), washed with 50% piperidine/DMF IX (700 mL for 2L bottle, 20 min), washed with DMF 4X, washed with 0.3M HoBt/DMF IX (700 L) and finally washed with DMF 4X containing 1% bromophenol blue in the last wash (2 L per 2L bottle) .
• a bromoacetic acid/DMFsolution (104gm, 750mmol) was prepared and to this was added DIC (140mL, 750mmol) into the bromoacetic acid solution, stirred for about 30 seconds, then poured into the 2L bottle containing the
• Rl e.g., Rll-Amine
• T-bags from the reaction above were washed with DMF 3X (500mL), with DMSO 2X (500mL).
• the appropriate number of Rll-amine/DMSO solutions (0.5mol of amine, 440mL) were prepared and placed in IL Nalgene bottles with the appropriate T-bags (30 bags per 1 L bottle). The reactions were shaken overnight.
• T-bags from the reaction above were washed with DMSO IX (500mL), and with DMF 5X (500mL) containing 1% bromophenol blue in the last wash ( 0.5mL mL per 2L bottle) .
• the amino acid/DMF solution was prepared as follows: a 0.3M HOBt /860mL DMF solution and in this was dissolved the amino acid (0.259mol), to this was added DMAP (3.2gm) and then was added DIC (40.5mL) right before use.
• the T-bags were placed into 2L Nalgene bottles (60 bags per bottle) , then the amino acid solution prepared as above was added. The reactions were shaken overnight. The T-bags were washed with DMF (4X 500mL) , with DCM (4X, 500mL) and air dried in fume hood overnight.
• T-bags were placed into 2L Nalgene bottles (50 bags per 2L bottle) and washed with DCM IX, lOOOmL, 20 min) , then washed with 55% TFA/DCM for 1 h (960mL, add slowly) .
• the TFA solution was poured off and the T-bags washed with DCM (2X, lOOOmL) , with 5% DIEA/DCM (2X, lOOOmL) , with DCM (IX, lOOOmL) , with DMF (2X, lOOOmL) , with 50% piperidine/DMF IX (700 mL, 10 min) and, finally, with DMF (5X, lOOOmL) .
• T-bags For the appropriate T-bags, an 0.8M aldehyde/DMF solution was prepared in IL bottles containing 0.225mol of aldehyde. A 1.6M TMOF trimethyl ortho formate solution in DMF was prepared. To the IL bottle of aldehyde solution and T-bags (25 bags per IL bottle) was added the TMOF solution (292mL) . The reactions were shaken 3-3.5h.
• T- bags were washed with 0.2M TMOF/DMF 2X (500 mL per IL bottle each time) .
• a homophthalic anhydride/DMF solutions in 2L bottles was prepared containing 864mL DMF and 56gm of anhydride and to this was added DIEA (4.5mL), then added in T-bags immediately (60 bags per2L bottle). The reactions were shaken overnight.
• the T-bags were washed with DMF (5X, 500mL) , and with methyl t-butyl ether 4X, 500mL) .
• the T-bags were air dried bags overnight.
• the resin from each T-bag was placed into each well of a 96-well microtiter plate (-20 mg per well) . All wells were washed with DMF (2X, l.OmL). The following solutions were prepared and added to the appropriate wells. 0.3 mL of a solution of HATU/DMF was added to each well as was 0.175mL of DIEA and the plates shaken for 20 min. . The required 48 different X-amine solutions were prepared at 0.2M in DMF. The X-amine solution (0.3mL) was added to each appropriate well. The microtiter plates were capped and shaken at rt overnight. The resin in each well was washed with DMF (2X, 1. OmL per well) . This procedure was repeated to affect a double coupling of the amine to the resin bound carboxylic acid.
• the plates in batches of -15 were treated by passing N2 through chamber for 30 min, then passing HF gas for 15 min.
• the chamber was isolated under HF for 1.5 h, then the HF was removed by passing N2 through the chamber overnight.
• the plates were removed and the residual HF removed under vacuum in desiccator overnight.
• r8 building blocks names r8 radical names beta alanine 1, 2-ethylenyl
• Tea-bags are prepared as in Example 52 with each containing a mixture of isoquinoline amides. While still attached to the resin the isoquinoline amides are reduced to the isoquinoline amines and cleaved off the resin as described in Example 48, resulting in a mixture of isoquinoline amines.
• Percent inhibition for compounds of the subject invention are listed in the table below. It should be noted that, in the table below, “rl” is the building block that corresponds with R 1 of the subject formula; “r2” is the building block that corresponds with R 2 of the subject formula; and “r3” is the building block that corresponds with X of the subject formula.
• N.N-DI-N-BUTYLETHYLENEDIAMINE S tMETHYLENEDIOXYiBENZALDEHYDE 3-BUT0XYPR0PY-AMINE

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