CA2264020A1 - Inhibitors of phospholipase enzymes - Google Patents

Inhibitors of phospholipase enzymes Download PDF

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CA2264020A1
CA2264020A1 CA002264020A CA2264020A CA2264020A1 CA 2264020 A1 CA2264020 A1 CA 2264020A1 CA 002264020 A CA002264020 A CA 002264020A CA 2264020 A CA2264020 A CA 2264020A CA 2264020 A1 CA2264020 A1 CA 2264020A1
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Jean Bemis
Yibin Xiang
Neelu Kaila
John Mckew
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Genetics Institute LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/12—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07D401/06—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 carbon chain containing only aliphatic carbon atoms
    • 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
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

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  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Compounds having a chemical formula selected from the group consisting of formulae (I), (II) and (III) or a pharmaceutically acceptable salt thereof, wherein: A is independent of any other group and is selected from the group consisting of -CH2- and -CH2-CH2-; B is independent of any other group and is selected from the group consisting of -(CH2)n-, -(CH2O)n-, -(CH2S)n-, -(OCH2)n-, -(SCH2)n-, -(CH=CH)n-, -(CC)n-, -CON(R6)-, -N(R6)CO-, -O-, -S- and -N(R6)-;
R2 is independent of any other R group and is selected from the group consisting of -H, -COOH, -COR5, -CONR5R6, -(CH2)n-W-(CH2)m-Z-R5, -(CH2)n-W-R5, -Z-R5, C1-C10 alkyl, alkenyl and substituted aryl; R3 is independent of any other R group and is selected from the group consisting of -H, -COOH, -COR5, -CONR5R6, -(CH2)n-W-(CH2)m-Z-R5, -(CH2)n-W-R5, -Z-R5, C1-C10 alkyl, alkenyl and substituted aryl; which inhibit the activity of phospholipase enzymes, particularly cytosolic phospholipase A2. Pharmaceutical compositions comprising such compounds and methods of treatment using such compositions are also disclosed.

Description

?101520253035WO 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943INHIBITORS OF PHOSPHOLIPASE ENZYMESBackground of the InventionThe present invention relates to chemical inhibitors of the activity of variousphospholipase enzymes, particularly phospholipase A3 enzymes.Leukotrienes and prostaglandins are important mediators of in?ammation.Leukotrienes recruit in?ammatory cells such as neutrophils to an in?amed site, promote theextravasation of these cells and stimulate release of superoxide and proteases whichdamage the tissue. Leukotrienes also play a pathophysiological role in the hypersensitivityexperienced by asthmatics [See, e.g. B. Samuelson et al., Science, 1:] 171-76 (1987)].Prostaglandins enhance in?ammation by increasing blood ?ow and therefore in?ltration ofleukocytes to in?amed sites. Prostaglandins also potentiate the pain response induced bystimuli. Prostaglandins and leukotrienes are unstable and are not stored in cells. but areinstead synthesized [W. L. Smith, Biochem. 1., 2_5_2:3l5-324 (1989)] from arachidonic acidin response to stimuli. Likewise arachidonic acid is not free in cells but is released from thesn-2 position of membrane phospholipids by phospholipase A3 (hereinafter PLAZ). Thereaction catalyzed by PLA3 is believed to represent the rate-limiting step in the process oflipid mediated biosynthesis. When the phospholipid substrate of PLA3 is of thephosphotidyl choline class with an ether linkage in the sn-1 position, the lysophospholipidproduced is the immediate precursor of platelet activating factor (hereafter called PAF),another potent mediator of in?ammation [S.I. Wasserman, Hospital Practice, 15:49-58( 1988)]. Consequently the direct inhibition of the activity of PLA3 has been suggested as auseful mechanism for a therapeutic agent. i.e.. to interfere with the in?ammatory response.[See, e.g., J. Chang et al, Biochem. Pharmacol., }_§:2429—2436 (l987)].A family of PLA2 enzymes characterized by the presence of a secretion signalsequenced and ultimately secreted from the cell have been sequenced and structurallyde?ned. These secreted PLAZS have an approximately 14 kD molecular weight and containseven disul?de bonds which are necessary for activity. These PLA3s are found in largequantities in mammalian pancreas, bee venom, and various snake venom. [See, e.g.,references 13-15 in Chang et al, cited above; and E. A. Dennis, Druo Devel. Res., Q205- 22O (1 987).] However, the pancreatic enzyme is believed to serve a digestive function and,as such, should not be important in the production of the in?ammatory mediators whoseproduction must be tightly regulated.The primary structure of the first human non-pancreatic PLA3 has been determined.This non-pancreatic PLA3 is found in platelets, synovial ?uid, and spleen and is also a1SUBSTITUTE SHEET (RULE 25)?1015202530WO 98/08818CA 02264020 1999-02-24PCT/US97/14943secreted enzyme. This enzyme is a member of the aforementioned family. [See, J. J.5eilhamer et al, J. Biol. Chem., ._2§§:5335—5338 (1989); R. M. Kramer et al, J. Biol. Chem.,_2_6?:5768-5775 (1989); and A. Kando et al, Biochem. Biophvs. Res. Comm.. l_6_3:42—48( 1989)]. However, it is doubtful that this enzyme is important in the synthesis ofprostaglandins, leukotrienes and PAF, since the non-pancreatic PLA3 is an extracellularprotein which would be dif?cult to regulate, and the next enzymes in the biosyntheticpathways for these compounds are intracellular proteins. Moreover, there is evidence thatPLA3 is regulated by protein kinase C and G proteins [R. Burch and J. Axelrod, Proc. Natl.Acad. Sci. U.S.A., §41:6374-6378 (I989)] which are cytosolic proteins which must act onintracellular proteins. It would be impossible for the non-pancreatic PLA3 to function in thecytosol, since the high reduction potential would reduce the disul?de bonds and inactivatethe enzyme.A murine PLA3 has been identi?ed in the murine macrophage cell line, designatedRAW 264.7. A speci?c activity of 2 timols/min/mg, resistant to reducing conditions, wasreported to be associated with the approximately 60 kD molecule. However, this proteinwas not puri?ed to homogeneity. [See, C. C. Leslie et al, Biochem. Biophys. Acta.,26§:476-492 (1988)]. The references cited above are incorporated by reference herein forinformation pertaining to the function of the phospholipase enzymes, particularly PLA3.A cytosolic phospholipase A3 (hereinafter "cPLA1") has also been identi?ed andcloned. See, U.S. Patent Nos. 5,322,776 and 5,354,677, which are incorporated herein byreference as if fully set forth. The enzyme of these patents is an intracellular PLA2 enzyme,puri?ed from its natural source or otherwise produced in puri?ed form, which functionsintracellularly to produce arachidonic acid in response to inflammatory stimuli.Now that several phospholipase enzymes have been identi?ed. it would bedesirable to identify chemical inhibitors of the action of enzymes, which inhibitors could beused to treat in?ammatory conditions. I-lowever, there remains a need in the art for anidenti?cation of effective anti—inflammatory agents for therapeutic use in a variety ofdisease states.2SUBSTlTUTE SHEET (RULE 26)?l01520253035W0 98/08818CA 02264020 1999-02-24PCT/US97/ 14943Summary of the InventionThe present invention provides compounds having a chemical formula selectedfrom the group consisting of:A R2R2 ’% ‘ QW ‘*4N R] N\ \::]R3 R5Nand \>/B R4R, N\R3 R5or a pharmaceutically acceptable salt thereof, wherein:A is independent of any other group and is selected from the group consisting of—CH2- and -CH3-CH3-;B is independent of any other group and is selected from the group consisting of-(CH2)..-. -(CHg0)..-. -(CH:S ..-. -(0CH:)..-. -(SCH:)..-. -(CH=CH)..-. -(CEC)..-.-CON(R6)-, -N(R,,)CO—, -O—, —S- and -N(R6)—;R, is independent of any other R group and is selected from the group consisting of-X—R6, -H. —OH, halogen, —CN, -N03, C,—C5 alkyl, alkenyl, alkinyl, aryl and substituted aryl;R3 is independent of any other R group and is selected from the group consisting of-H, -COOH, -COR5, -CONR5R,,, —(CH3),,-W-(CH3),,,—Z-R5, -(CH3),,—W-R5, -Z-R5, C,—C,0alkyl, alkenyl and substituted aryl;R, is independent of any other R group and is selected from the group consisting of-H, -COOH, -COR5, -CONR5R6, -(CH2),,—W—(CH2),,,-Z-R5, -(CH3),,—W—R5, -Z-R5, C,—C,0alkyl, alkenyl and substituted aryl;R4 is independent of any other R group and is selected from the group consisting of-H. -OH, -OR6, -SR6, —CN, —COR6, -NHR6, -COOH, —CONR6R,, -N03, -CONHSOZRR, C,-C5 alkyl, alkenyl and substituted aryl;R5 is independent of any other R group and is selected from the group consisting of—H, -OH, -O(CH3),,R(,, —SR6, —CN, —COR,,, -NHR6, —COOH, -N03, -COOH, -CONRGR7,-CONHSOZR8, C,-C5 alkyl, alkenyl, alkinyl, aryl, substituted aryl, -CF_,, —CF3CF3 and3SUBSTITUTE SHEET (RULE 26)?l01520253035W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943R9RIOR6 isindependent of any other R group and is selected from the group consisting of-H, C,-C5 alkyl, alkenyl, alkinyl, aryl and substituted aryl;R7 is independent of any other R group and is selected from the group consisting of-H, C,-C5 alkyl, alkenyl, alkinyl, aryl and substituted aryl;R3 is independent of any other R group and is selected from the group consisting ofC ,-C3 alkyl, aryl and substituted aryl;R9 is independent of any other R group and is selected from the group consisting of-H, -01-], a halogen, -CN, —OR6, -COOH, -CONRGR7, tetrazole, -CONHSO1R3, —COR6,-(CH3),,CH(OH)R6 and -(CH3),,CHR6R5;Rm is independent of any other R group and is selected from the group consistingof -H, —OH, a halogen. -CN, -OR6, -COOH, -CONRGR7, tetrazole, —CONI-ISOZRS, -COR6,-(Cl-l2),,CH(OI-I)R6 and —(CH2),,CHR6R5;W is. independently each time used including within the same compound, selectedfrom the group consisting of -0-, -S—, -CH3-, —CH=CH—, -C E G and -N(R6)-;X is independent of any other group and is, independently each time used includingwithin the same compound, selected from the group consisting of —O~, -S- and -N(R6)-;Z is independent of any other group and is, independently each time used includingwithin the same compound, selected from the group consisting of -CH2—, -0-, -S—, -N(R6)-, -CO—, —CON(R,,)- and -N(R,,)CO—;m is, independently each time used including within the same compound, aninteger from 0 to 4; andn is independent of m and is, independently each time used including within thesame compound, an integer from 0 to 4.Preferably, the compounds of the invention have phospholipase enzyme inhibiting activity.Other preferrred embodiments include compounds having the following chemical fonnula:SUBSTITUTE SHEET (RULE 26)?101520 .,,2530W0 98/08818CA 02264020 1999-02-24PCT/US97ll4943compounds having the following chemical formula:R2R] N\ \ R3 R5compounds having the following chemical formula:@C N\>/B R4RI N\ \<:]R3 R5ln particularly preferred embodiments, A is 'CH2‘ and R2 is-(CI-12),,-W-(CH2),,,-ZR5 These preferred compounds includes those wherein n is l, m is 1,W is -S- and Z is -CO-; those wherein R5 is -NHR6; those wherein R6 is a substituted arylgroup and those wherein said aryl group is substituted with one or more substituentsindependently selected from the group consisting of a halogen, -CF3,-CFZCF3, -(CH2)pCOOH, —(CH2)pCH3, -O(CH3)pCH3, -(CH3)pOH, -(CH3)pS(C,,H6),—(Cl-I2)pCONH2 and -CHRHCOOH, wherein R” is selected froup the group consisting ofalkyl, alkenyl, alkynyl, -(CHZ)pOH, and -O(CH2)pCH,, and wherein p is an integer from 0 to4. Other preferred comounds include those wherein R, is selected from the groupconsisting of -H and -OCH2(C6H,,) and R, is -COR5, R5 is —OCHzR6 and R6 is a substitutedaryl group. In particularly preferred compounds, said aryl group is substituted with one ormore substituents selected from the group consisting of -CF,, -CF2CF, and-C(CH,)2CH2CH3.The present invention also provides for a method of inhibiting the phospholipaseenzyme activity of an enzyme, comprising administering to a mammalian subject atherapeutically effective amount of a compound of the present invention. Methods oftreating an in?ammatory condition, comprising administering to a mammalian subject atherapeutically effective amount of a compound of the present invention are also provided.Pharmaceutical compositions comprising compounds of the present invention and apharmaceutically acceptable carrier are also provided.5SUBSTITUTE SHEET (RULE 26)?101520W0 98l08818CA 02264020 1999-02-24PCT/US97/14943Pharmaceutically acceptable salts of the compounds of the compounds describedherein are also part of the present invention and may be used in practicing the compoundsand methods disclosed herein.Brief Description of the FiguresFigs. I-13 depict schemes for synthesis of compounds of the present invention.The depicted schemes are described in further detail below.Detailed Description of Preferred EmbodimentsAs used herein: “halogen” includes chlorine, ?uorine, iodine and bromine; “alkyl”,“alkenyl” and “alkinyl” include both straight chain and branched moieties; “aryl” includessingle and multiple ring moieties; and “substituted” denotes the presence of one or moresimilar ot dissimilar substituent groups of any character.Preferred compounds of the present invention are disclosed in Tables I-VI below.Methods for synthesis of the compounds listed in Tables I-VI are described below.Compound Nos. in the tables correspond to example numbers below describing synthesis ofthat particular compound.Tables I-VI also report data for the listed compounds in the "LysoPC" assay andthe Coumarine assay (see Example 88 below). In the data columns of the tables, assayresults are reported as an "IC50" value, which is the concentration of a compound whichinhibits 50% of the activity of the phospholipase enzyme in such assay. Where nonumerical IC50 value appears, "NA" denotes that inhibitory activity was not detected fromsuch compound in the corresponding assay and a blank box denotes that the compound wasnot tested in such assay as of the time of ?ling of the present application.6SUBSTITUTE SHEET (RULE 26)?Table IW0 98/08818CA 02264020 1999-02-24PCT/US97/14943Cou-marine[Cm (P M)LysoPC47R4<——Cu;oHOOC<—CH20 coonKN0.7SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98/08818 PCT/US97/149433 9, o"7 <‘ M o\o Z tr‘ N‘I =0 ~ 8 5O ; O :5‘ I \ 0:.‘; V:3‘¢"3 <r to \o8SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24PCT/US97/14943WO 98/08818V‘ °‘? ‘'7 oo" 0'1 \o N_ - 35 m' T‘. r?N O OI I : I I8 8 .3 8 8 2 CU U u U U \& — Z5 OO__ u2Z O_/ 2O o_EC st“ 95L4 L:Q 1 Uu?f. g $ $xx 00 ox 29SUBSTITUTE SHEET (RULE 26)?W0 98l08818CA 02264020 1999-02-24PCT/US97/ 14943[0I2141.9(‘OOH0NH(‘Hz \ ‘/S\J\COCH3u?U,., uf, Zr: °5 <2.:12‘.L.) 0<2 /:1:U2 ‘.3 1‘ ‘*SUBSTITUTE SHEET (RULE 26)10?CA 02264020 1999-02-24W0 98/088123 PCT/US97/14943I3506 56.5coon0VANH coon(—CH;I5I61 1SUBSTITUTE SHEET (RULE 26)? Table HW0 98/08818CA 02264020 1999-02-24SUBSTITUTE SHEET (RULE 26)..E~o826“DE25-9.BoN3825c2‘ 8O “N IZz/< /5 JM.0' I\Z —<12PCT/US97/14943?CA 02264020 1999-02-24W0 98/08818 PCT/US97/ 14943O <r ox "3"" <1-3 F3 %o C: o a‘II‘.C8§ 5“ IU 8 8I (J DooUU:5“ Z 3: 0Z L) 2 2: z/ f / /°_‘’ 2‘. 8 CT,1 3SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24WO 98/08818 PCT/U S97/ 14943In In "3 ON 7|9. 3‘ \O_ C")o’ o ‘“ -‘Io8EI§ 5 3O..-» E8‘ é0X2 2 8 Z Z< g / I ,/ /8 8 § 3‘I4SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24WO 98/08818oo M .—d v—t IN m I_; as -I 5'5 8 88 U _. U" ::5 oA I I IQ 2 81 2 8 2I‘ ; I / A\ /xo :\ oo01 N 011 5SUBSTITUTE SHEET (RULE 25)PCT/U S97/ 14943?CA 02264020 1999-02-24W0 98l08818 PCT/US97/l4943: 3 gu:‘f‘ M A O U8 =2 “o 8 5” S" /Z\u:o:5 / / /z8 5% E16SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24PCT/U S97] 14943W0 98/08818-n In 53/\°°. ‘G <3-m cu N.. : N5 0 %O M 8 OU 5 = Q0 OQ E a z ‘-A Z1/ my / IQ Q 3»17SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98/08818? <1-": "3.ox NI8O: cn:O Ui :Z OOU /2 Ci)“ /25.:In \om m18SUBSTITUTE SHEET (RULE 25)Table HIPCT/US97/14943?CA 02264020 i999—02-24WO 98/08818 PCT/US97/ 149430EE o 0N1 In5 A AOU25-Bi’?0 \D3: as 2 3;z i‘U0CB0COOHCOOHHN4/P40.373819SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98/08818 PCT/US97/14943‘:9 2 R‘ 1*“'7 N. N 00<r \o N xx’~ 5\Z__ 5 5o O I35 5 8 88 8 U UZ z EI\ ‘N I‘ *\3 S3 :; 3'20SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98/08818 PCT/US97/14943__ <1)(\I .gE 85 A~ 5325‘;8B" 0~ 3%: aZL‘.9CUE-~5'LC‘0LE‘L)O<\ 5 ad 5';Z\‘ /5°o 2 oIooDO\Z 1I‘M .<r E 3SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98/08818 PCT/U S97/ 14943O OV‘: _. E 2 I?<1: K ‘D. V’! oo’ o " O Vmf":UZ5 OQ 3 ‘<7 3 3?2 2SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24wo 98/08818 PCT/US97ll4943G‘/3/\V.V/ \CV‘)2 3SUBSTITUTE SHEET (RULE 26)?Table VCA 02264020 1999-02-24W0 98/08818 PCT/US97/ 14943- 25 .._ V‘A o ‘O xo2 U E “‘ A3-Q o3%: 3 3 25? §:2‘ <2UOOUIZ0(ZI2 xO‘E.i U}Z/Q *0°‘" CO +°C52 .7: 9: :224SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98/08818 PCT/US97/ 149431’ S.’ tnV3 4. L?o o o54555625SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98l08818 PCT/US97/l494300 <r 3 3/\ /\8 3% I\. <9.o’ o °° VIoOu:0 Z91I’ O4/5oocu _.5758596026SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24 W0 98/08818ON ooA“f z\Q .._:§ 5O i O-— N M\o \o up27SUBSTITUTE SHEET (RULE 26)PCT/US97/ 14943?CA 02264020 1999-02-24W0 98/08818 PCT/US97/14943<2-‘R \D 2 a1 o‘O on N 0 <1-‘: N S “R 2:1:O0U0 OQA/ NH (‘OOH01‘/s\/coonCOOH.AI’CONHCH34’rgco4/ S\/\NH/7O64656667686928SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24WO 981088182 Na? Cu3’ V3:o8 -.1:OOU:’:"o 8 Z:3‘ o:§* mG —-(\ (\29SUBSTITUTE SHEET (RULE 26)PCT/US97/ 14943?Table VICA 02264020 1999-02-24wo 9s/ossxs PCT/US97/149435 E <1-2 8g ‘R ‘°:3.:5’ oE» E.’ 5?‘.IOOL)$32Z0::mf".as2/‘? O\ 2 O1:?0/D‘. %2 S S30SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98/08818 PCT/US97/14943(V V5'“ \o' <r M*1 O.-— t\IO IO OU : :0UI c 2U. SI*5OU”= O Q5 0:3 3 F9 I‘: 331SUBSTITUTE SHEET (RULE 26)?CA02264020 1999-02-24W0 98/08818 PCT/US97/14943°? In InQ‘ —- —n(\! -—vM o_g_ DU* 9 “°UQ §}w:2U0O/O \ 'ORnr 5U oULL“UFx‘ 8 38 Q32SUBSTITUTE SHEET (RULE 26)?CA 02264020 1999-02-24W0 98/088183‘Q .9: $4. /\ /\O 9‘Q to?! Ng C::cl/1:0U E 02:: 0E C:/ /2O‘: : O’.-LC‘ULC‘UI:5("7 V‘ V3X 00 O033SUBSTITUTE SHEET (RULE 26)PCT/U S97] 14943?WO 98/08818CA 02264020 1999-02-24176.5coonJ1 /Q/\NH NH COOHca4-Cu-<< :>—\ IQCE868734SUBSTITUTE SHEET (RULE 26)PCT/U S97/ 14943?51015202530WO 98/08818CA02264020 1999-02-24PCT/US97/14943Compounds of the present invention were also tested for in vivo activity in a ratpaw edema test according to the procedure described in Example 89. The results arereported in Table VII.Table VIICompound No. % inhibition of rat carrageenan—inducedfootpad edema8 2910 8.914 34.215 21.816 26.317 29.319 10.520 19.525 17.526 10.332 26.733 4.246 12.547 7.850 1 1.767 17.570 21.776 8.277 13.0As used herein. "phospholipase enzyme activity" means positive activity in anassay for metabolism of phospholipids (preferably one of the assays described in Example88 below). A compound has "phospholipase enzyme inhibiting activity" when it inhibitsthe activity of a phospholipase (preferably CPLA3) in any available assay (preferably an35SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/ 14943assay described below in Example 88 or Example 89) for enzyme activity. In preferredembodiments, a compound has (1) an IC50 value of less than about 25 t1M, preferably lessthan about 6 pM, in the LysoPC assay; (2) an IC50 value of less than about 50 pM in thevesicle assay; (3) an IC50 value of less than about 1 pM in the PMN assay; (4) an IC50 valueof less than about 15 uM in the Coumarine assay; and/or (5) measurable activity (preferablyat least about 5% reduction in edema, more preferably at least about 10% reduction) in therat carrageenan-induced footpad edema test.Compounds of the present invention are useful for inhibiting phospholipaseenzyme (preferably CPLA3) activity and, therefore, are useful in "treating" (i.e., treating,preventing or ameliorating) in?ammatory or in?ammation—related conditions (e.g.,rheumatoid arthritis, psoriasis, asthma, in?ammatory bowel disease, and other diseasesmediated by prostaglandins, leukotrienes or PAP) and other conditions, such asosteoporosis, colitis, myelogenous leukemia. diabetes, wasting and atherosclerosis.The present invention encompasses both pharmaceutical compositions andtherapeutic methods of treatment or use which employ compounds of the present invention.Compounds of the present invention may be used in a pharmaceutical compositionwhen combined with a phamiaceutically acceptable carrier. Such a composition may alsocontain (in addition to a compound or compounds of the present invention and a carrier)diluents, ?llers, salts, buffers, stabilizers, solubilizers, and other materials well known inthe art. The tenn "pharmaceutically acceptable" means a non-toxic material that does notinterfere with the effectiveness of the biological activity of the active ingredient(s). Thecharacteristics of the carrier will depend on the route of administration. Thepharmaceutical composition may further contain other anti—in?ammatory agents. Suchadditional factors and/or agents may be included in the pharmaceutical composition toproduce a synergistic effect with compounds of the present invention, or to minimize sideeffects caused by the compound of the present invention.The pharmaceutical composition of the invention may be in the fonn of a liposomein which compounds of the present invention are combined, in addition to otherphannaceutically acceptable carriers, with amphipathic agents such as lipids which exist inaggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal fonnulation include, without limitation,monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids,and the like. Preparation of such liposomal fomiulations is within the level of skill in theart, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728;36SUBSTITUTE SHEET (RULE 26)?101520253035CA 02264020 1999-02-24WO 98/08818 PCT/US97/14943U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporatedherein by reference.As used herein, the term "therapeutically effective amount" means the total amountof each active component of the pharmaceutical composition or method that is sufficient toshow a meaningful patient benefit, i.e., treatment. healing, prevention or amelioration of anin?ammatory response or condition, or an increase in rate of treatment, healing, preventionor amelioration of such conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. When applied to acombination, the term refers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially or simultaneously.In practicing the method of treatment or use of the present invention, atherapeutically effective amount of a compound of the present invention is administered toa mammal having a condition to be treated. Compounds of the present invention may beadministered in accordance with the method of the invention either alone or in combinationwith other therapies such as treatments employing» other anti-inflammatory agents,cytokines, lymphokines or other hematopoietic factors. When co—administered with one ormore other anti-in?ammatory agents, cytokines, lymphokines or other hematopoieticfactors, compounds of the present invention may be administered either simultaneouslywith the other anti-in?ammatory agent(s), cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti—thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriate sequence ofadministering compounds of the present invention in combination with other anti-in?ammatory agent(s), cytokine(s), lymphokine(s), other hematopoietic factor(s),thrombolytic or anti-thrombotic factors.Administration of compounds of the present invention used in the pharmaceuticalcomposition or to practice the method of the present invention can be carried out in avariety of conventional ways, such as oral ingestion, inhalation, or cutaneous,subcutaneous, or intravenous injection.When a therapeutically effective amount of compounds of the present invention isadministered orally, compounds of the present invention will be in the form of a tablet,capsule, powder, solution or elixir. When administered in tablet fonn, the pharmaceuticalcomposition of the invention may additionally contain a solid carrier such as a gelatin or anadjuvant. The tablet, capsule, and powder contain from about 5 to 95% compound of thepresent invention, and preferably from about 25 to 90% compound of the present invention.When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal37SUBSTITUTE SHEET (RULE 26)?10I520 2530W0 98I088l8CA 02264020 1999-02-24PCT/U S97/ 14943or plant origin such as peanut oil, mineral oil, soybean oil. or sesame oil, or synthetic oilsmay be added. The liquid form of the phamiaceutical composition may further containphysiological saline solution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% by weight ofcompound of the present invention, and preferably from about I to 50% compound of thepresent invention.When a therapeutically effective amount of compounds of the present invention isadministered by intravenous, cutaneous or subcutaneous injection, compounds of thepresent invention will be in the form of a pyrogen-free, parenterally acceptable aqueoussolution. The preparation of such-parenterally acceptable protein solutions, having dueregard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferredpharmaceutical composition for intravenous, cutaneous, or subcutaneous injection shouldcontain, in addition to compounds of the present invention. an isotonic vehicle such asSodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. Thephamiaceutical composition of the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additives known to those of skill in the art.The amount of compound(s) of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature and severity of thecondition being treated, and on the nature of prior treatments which the patient hasundergone. Ultimately, the attending physician will decide the amount of compound of thepresent invention with which to treat each individual patient. Initially, the attendingphysician will administer low doses of compound of the present invention and observe thepatients response. Larger doses of compounds of the present invention may beadministered until the optimal therapeutic effect is obtained for the patient, and at that pointthe dosage is not increased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention should contain about 0.1p g to about 100 mg of compound of the present invention per kg body weight.The duration of intravenous therapy using the phamiaceutical composition of thepresent invention will vary, depending on the severity of the disease being treated and thecondition and potential idiosyncratic response of each individual patient. It is contemplatedthat the duration of each application of the compounds of the present invention will be inthe range of 12 to 24 hours of continuous intravenous administration. Ultimately the38SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943attending physician will decide on the appropriate duration of intravenous therapy using thepharmaceutical composition of the present invention.Methods of SynthesisCompounds of the present invention can be prepared according to the followingmethods. Temperatures are in degrees Celsius.METHOD AIndol-2—carboxylic acid ethyl ester I is converted to aldehyde II in two steps:reduction with lithium aluminum hydride (LAH) or other hydride in a suitable solvent suchas tetrahydrofuran (THF) at 0 °C, and then oxidation with an oxidizing reagent such asmanganese dioxide in a solvent such as THF. Deprotonation of aldehyde II with a strongbase such as potassium hexamethyldisilyl amide (KHMDS) in THF. followed by reactionwith a chlorofonnate in the presence of a base, such as triethyl amine, produces carbamateIII. III is transformed into bromide IV in two steps: (1) reduction with sodium borohydridein an alcoholic solution and (2) reaction withcarbon tetrabromide in the presence of aphosphine reagent such as bis(diphenylphosphino)propane in dichloromethane.Displacement of the bromine in IV with potassium phenoxide, prepared by reaction of aphenol with KHMDS, in a suitable solvent such as THF or DMF affords ether V. V can beconverted to either tri?uoromethyl ketone VII or to carboxylic acid IX in differentprocedures. Reaction of V with tri?uoromethyl trimethylsilane (TMSCF3) in the presenceoftetrabutylammonium ?uoride gives tri?uoromethyl alcohol, which is then oxidized withperiodinane (Dess-Martin reagent) in dichloromethane to afford ketone VI. In this stagethe carbamate can be removed with either tri?uoroacetic acid (TFA) or with a base such assodium hydroxide. The indole nitrogen is then alkylated with a suitable alkyl bromide inthe presence of a base such as sodium hydride to produce VII. Alternatively, V can bedeprotected with TFA or aqueous base, and then reacted with alkyl bromide to give VIII,which is oxidized with sodium chlorite in an aqueous THF to yield acid IX.METHOD B2—Indolyl carboxylic acid ethyl esterl is deprotonated with a strong base such as sodiumhydride (NaH) in THF, and then reacted with a suitable alkyl bromide to give X.39SUBSTITUTE SHEET (RULE 26)?1015202530W0 98I088l8CA 02264020 1999-02-24PCT/US97l 14943Hydrolysis of X with a aqueous base such as sodium hydroxide and reaction with aniline ora substituted aniline in the presence of a carbodiimide such as dimethylaminopropylethylcarbodiimide hydrochloride (EDCI) in a suitable solvent such as dichloromethaneaffords amide X1. X1 is hydrolyzed to corresponding acid XII in 21 aqueous base such assodium hydroxide.METHOD CIndole I can be brominated on the 3-position by reaction with a bromine or N-bromosuccinimide in a suitable solvent such ascarbon tetrachloride or dichloromethane toyield bromide XIII. Reaction of XIII with a suitable alkyl bromide in the presence of astrong base such as NaH in THF or DMF affords indole XIV. Palladium mediatedcoupling of XIV with a suitable alkene in the presence of phosphine and a base such astriethyl amine produces 3-substituted indole XV. XV can be converted to amide XVII intwo step reactions: (1) hydrolysis with aqueous base such as NaOH and (2) coupling withan amine in the presence of carbodiimide such as EDCI. Ester XIV can be transformed tolithium salt XVIII by hydrolysis with aqueous base and then reaction with lithiumhydroxide in a suitable solvent such as ether. Lithiation with n-butyl lithium in suitablesolvent such as THF, and then acylation with an acyl chloride in THF affords ketone XIX.Carbodiimide (EDCI) catalyzed coupling of XIX and a suitable amine gives amide XX.METHOD DIndole I can be converted to XXI in two steps: (1) reduction with LAH in a solvent such asTHF and (2) silylation with t—butyldimethylsilyl chloride (TBDMSCI) in a solvent such asdichloromethane or DMF in the presence of a base such as imidazole. Treatment of XXIwith Grignard reagent such as ethyl magnesium bromide in a solvent such as THF at -60°C, acylation of the resulting magnesium salt with a suitable acyl chloride such as acetylchloride in ether and ?nally, alkylation on the nitrogen with an alkyl halide such as ethylbromide in the presence of a strong base such as NaH in DMF affords ketone XXII. Thesilyl group on XXII is removed using tetrabutylammonium ?uoride in a solvent such THF.the resulting alcohol is then converted to bromide using carbon tetrabromide andbis(diphenylphosphino)ethane in a solvent such as dichloromethane to yield bromide40SUBSTITUTE SHEET (RULE 26)?1015202530WO 98/08818CA 02264020 1999-02-24PCT/US97/14943XXIII. Displacement of the bromine of XXIII with a thiol compound in the presence of abase such as Cs2CO_,, or with an alcohol in the presence of a strong base such as NaH inDMF affords XXIV (sulfide, or ether respectively).METHOD EAldehyde II, prepared by Method A, can be alkylated by a suitable alkyl bromide (oriodide), such as benzyl bromide or ethyl iodide in the presence of a strong base such assodium hydn'de or KHMDS in a solvent such as DMF to yield XXV. XXV can beconverted to an unsaturated acid XXVI by two steps: (1) Wittig reaction with a suitablereagent such as trimethyl phosphonoacetate in the presence of a base such as sodiumhydride in a solvent such as THF and (2) Hydrolysis by aqueous sodium hydroxide.Coupling reaction of XXVI with an amine catalyzed by a diimide such as EDCI(dimethylaminopropyl ethylcarbodiimide hydrochloride), followed by hydrolysis withaqueous base such as sodium hydroxide affords XXVII.METHOD FIndole I is reduced with LAH in a solvent such as THF. A second reduction with sodiumcyanoborohydride in a solvent such as acetic acid to yield alcohol XXVIII. Protection ofthe nitrogen of XXVIII with t-butoxycarbonyl (BOC) using di-t-butyldicarbonate((BOC)3O) in the presence of a base such as triethylamine affords carbamate XXIX. Thehydroxyl group in XXIX is mesylated using mesyl chloride and triethylamine in a solventsuch as dichloromethane, and then displaced by either a thiol or an alcohol as described inMETHOD D to produce indoline XXX. Deprotection of XXX using tri?uoroacetic acidaffords XXXI, which is either acylated (acyl chloride, triethylamine. dichloromethane) oralkylated (alkyl halide. KECOJ, DMF) to afford XXXII, or XXXIII respectively.METHOD GCarboxylic acid XXXIV is converted to aldehyde XXXV in two steps: (1) reaction withN,O-dimethylhydroxy amine in the presence of EDCI in a solvent such as41SUBSTITUTE SHEET (RULE 26)?CA 02204020 1999-02-24PCT/US97/ 14943W0 98/088181015202530dichloromethane. and (2) reduction with diisobutyl aluminum hydride (DIBAL) in aI solvent such as THF. Treatment of XXXV with trimethyl phosphonoacetate in thepresence of a strong base such as KHMDS in a solvent such as THE results in theformation of ester XXXVI. Reduction of XXXVI with tin in hydrogen chloride, followedby cyclization in a heated inert solvent such as toluene gives XXXVII. Alkylation onnitrogen of XXXVII under conditions described in METHOD F, and then hydrolysis of theester with aqueous base such as NaOH affords acid XXXVIII. XXXVIII can be convertedto an amide XXXIX by coupling with a suitable amine such as benzylamine in thepresence of EDCI.METHOD HAldehyde XXXV, prepared in METHOD G, is subjected to a Wittig reaction using methyltriphenylphosphonium iodide in the presence of a strong base such as KHMDS or NaH in asolvent such as THF to afford alkene XL. Reduction of the nitro group of XL with ironpowder in an ammonium chloride solution, followed by treatment with benzylchloroforrnate in the presence of a base such as triethyl amine produces carbamate XLI.XLI is treated with iodine in a basic solution such as aqueous NaHCO_, in THF to yieldiodide XLII. Displacement of the iodine on XLII with lithium benzoate in a solvent suchas DMF, followed by hydrolysis with NaOH affords alcohol XLIII.METHOD IIndoline XXVIII, prepared in METHOD F or METHOD H. can be either acylated byreaction with an acyl chloride in the presence of a base such as triethyl amine or alkylatedusing alkyl halide in the presence of K2CO:‘ in a solvent such as DMF to produce alcoholXLIV. Treatment of XLIV with mesyl chloride and triethyl amine in a solvent such asdichloromethane, followed by displacement with a thiol such as methyl mercaptoacetate inthe presence of a base such as Cs3CO_, in a solvent such as acetonitrile yields ester XLV.Hydrolysis of XLV with an aqueous base such as NaOH gives acid XLVI, which can becoupled with an amine catalyzed by a diimide such as EDCI in a solvent such asdichloromethane to afford amide XLVII. XLVII can be alkylated on the amide nitrogen by42SUBSTITUTE SHEET (RULE 26)?W0 98/088181015202530CA 02264020 1999-02-24PCT/US97/ 14943treatment with alkyl halide and strong base such as NaH in DMF. Hydrolysis of the2 resulting amide with aqueous base such as NaOH gives acid XLIX. XLIV can also bedirectly hydrolyzed with NaOH to a carboxylic acid XLVIII.METHOD JMETHOD J illustrates the synthesis of alpha—substituted aminophenylacetic acid esters.Ester L can be deprotonated with a strong base such as lithium diisobutylamide (LDA) in asolvent such as THF, and subsequently alkylated with an alkyl halide such as methyl iodideto give LI. Reduction of L1 to amine LIII can be accomplished using hydrogenationcatalyzed by palladium in a solvent such as ethanol. L can be oxidized to alcohol LII usingLDA and oxaziridine in a solvent such as THF. Alkylation of LII with a alkylating reagentsuch as methyl iodide in the presence of a strong base such as NaH in DMF, followed bycatalytic hydrogenation in the presence of palladium produces amine LIV.METHOD KMETHOD K illustrates the synthesis of substituted aminobenzoic acid esters. Mono-acidLV can be converted to amide LVI by the following steps: (1) reaction with oxalyl chloridein dichloromethane to form acid chloride and (2) treatment with a suitable amine such asdimethyl amine. Reduction of the nitro group to the amine is accomplished withhydrogenation catalyzed by palladium as described in METHOD J. LV can be reduced toalcohol LVIII with hydroborane-THF complex in THF. Protection of the hydroxy groupas a silyl ether using TBDMSCI in the presence of imidazole and subsequently, reductionof the nitro group (H: / Pd—C) to the amine affords LIX. LVIII can be convened to thesecondary alcohol LX in two steps: (1) oxidation with a suitable reagent such asmanganese dioxide (MnO2) in ethyl acetate and (2) addition of a desired Grignard reagentsuch as methyl magnesium bromide in THF. Oxidation of LX with MnO3 in THF andreduction of the nitro group (H: / Pd—C) produces ketone LXIII. Reduction of LVII (H3 /Pd—C) yields LXI.43SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/ 14943METHOD LAlcohol LXIV, prepared in METHOD 1, can be debenzylated by hydrogenolysis catalyzedby palladium on carbon in a solvent such as ethanol. The resulting alcohol is treated withp-methoxybenzyl chloride in the presence of KZCO3 in a solvent such as THF to affordLXV. Alcohol LXV can be transformed into ether or sul?de LXVI by the proceduresdescribed in METHOD D. Deprotection of the p-methoxybenzyl group with TFA in asolvent such as dichloromethane, and subsequent alkylation on oxygen with a suitablereagent such as 4-benzylbenzyl bromide in the presence of KQCO3 in a solvent such as THFaffords LXVII.EXPERIMENTAL SECTIONThe Examples which follow further illustrate the invention. All temperatures setforth in the Examples are in degrees Celsius. All the compounds were characterized byproton magnetic resonance spectra taken on a Varian Gemini 300 spectrometer orequivalent instruments.EXAMPLE 12-(2-( I -Phenvlmethoxvcarbonvl-5-Dhenvlmethoxv)indolvl)methoxvben2oic acidStep I: 2-(5-Phenvlmethoxvlindolvl aldehvde12.3 g (42 mmol) of ethyl 2—(5-phenylmethoxy)indolyl) carboxylate was dissolvedin 100 mL of THF, to which was added 130 mL (130 mmol) of l M solution of lithiumaluminum hydride in THF at 0 °C. The reacton was stirred at this temperature for 2 hoursand quenched by adding 65 mL of 6 N NaOH solution slowly. The product was extractedwith ethyl acetate, and the organic phase was washed with aqueous ammonium chloride.Evaporation of the solvent afforded crude alcohol, which without further purification wasdissolved in 400 mL of THF, 52 g of manganese(IV) oxide was added, and the mixturewas stirred at room temperature overnight. Removal of manganese oxide by filtration and?ash chromatographic puri?cation using 3:] hexanezethyl acetate yielded 8.15 g of the titlecompound.44suasmure SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943Step 2: Benzvl (1—(2-fomwl-5-phenvlmethoxv)indolvl)fonnateTo a solution of 6.9 g (27.5 mmol) of the aldehyde of step lin 140 mL of THF wasslowly added 61 mL (30.5 mmol) of 0.5 M solution of potassium bis(trimethylsilyl)amidein toluene at-35 °C. After stirring at this temperature for 10 min, 4.4 mL (29.5 mmol) of benzylchlorofonnate was added at -35 °C, and the mixture was then warmed from -35 °C to 0 °Cfor 3.5 hours. The reaction was quenched by pouring into aqueous ammonium chloride.Aqueous work up and ?ash chromatography using 12:] toluenezethyl acetate afforded 4.8g of the title compound.SICD 3: Benzvl (l—(2-hvdroxvmethvl-5-Dhenvlmethoxvlindolvl)formateTo a solution of 2.9 g (7.5 mmol) of the aldehyde of step 2 in 40 mL of THF and20 mL of tri?uoroethanol was added 760 mg (20 mmol) of sodium borohydride at 0 °C.The mixture was stirred at 0 °C for 30 min and then quenched by adding aqueousammonium chloride. Flash chromatography using 2:] hexane-ethyl acetate afforded 2.2 gof the title compound.Step 4: Benzvl (l-(2-bromomethvl-5—DhenvlmethoxvlindolvllformateTo a solution of 2.2 g (5.7 mmol) of the alcohol of step 3 and 2.05 g (5.0 mmol) of1,3-bis(diphenylphosphino)propane in 60 mL of dichloromethane was added a solution of2.0 g (6 mmol) of carbon tetrabromide in 4 mL of dichloromethane at l5 °C. The mixturewas stirred at room temperature for 2 hours and l g (3 mmol) of 1,3-bis(diphenylphosphino)propane was added at room temperature. After 1 hour stirring, thereaction was quenched by adding aqueous ammonium chloride. Aqueous work up and?ash chromatography using 4:] hexane:ethyl acetate afforded 1.7 g of the title compound.Step 5: Benzvl (1—(2—(2-formvlphenoxvlmethvl—5—DhenvlmethoxvlindolvllformateTo a solution of 439 mg (3.6 mmol) of methyl 2-hydroxybenzoate in 18 mL ofTHF was added 6 mL (3 mmol) of 0.5 M solution of potassium bis(trimethylsily|)amide intoluene at 0 °C. The solution was stirred at 0 °C for 10 min, to which was added a solution45SUBSTITUTE SHEET (RULE 26)?l015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/14943of 1.25 g (2.8 mmol) of the bromide, prepared in step 4, in THF at 0 °C. The reaction waswarmed to room temperature and stirred at this temperature for 2 hours. After aqueouswork up (NI-I,Cl / ethyl acetate), the organic solvent was collected, dried over sodiumsulfate and evaporated. The product was solidi?ed and washed with ethyl acetatezhexane1:1. Yield 690 mg (51%).Step 6:120 mg (0.24 mmol) of the aldehyde of step 5 was dissolved in 1 1 mL of 5:l:5THF-acetonitrile—2,2-dimethylethanol. To this solution was added a solution of 56 mg (0.5mmol) of sodium chlorite in 0.5 mL water and 1 drop of aqueoues hydrogen peroxidesolution. After 4 hours, another 56 mg (0.5 mmol) of sodium chlorite was added. Themixture was stirred at room temperature for three days. Aqueous work up and ?ashchromatography using 2.5: 1 :0.05 hexane:ethyl acetate—acteic acid afforded 1 10 mg of thetitle compound.EXAMPLE 24-(2-(l -Phenvlmethoxvcarbonvl—5—Dhenvlmethoxv)indolvDmethoxvbenzoic acidThe title compound was prepared according to the procedure described in Example1, but using 4—hydroxybenzaldehyde.EXAMPLE 334241-PhenvImethoxvcarbonvl-5—phenv|methoxv)indo|vllmethoxvbenzoic acidThe title compound was prepared according to the procedure described in Example1, but using 3-hydroxybenzaldehyde.46SUBSTITUTE SHEET (RULE 26)?1015202530WO 98/08818CA 02264020 1999-02-24PCT/US97/14943EXAMPLE 4Benzvl (1-(2—(2-(1-oxo—2,2.2-trifluoroethyl)phenoxv)methvl-5-phenvlmethoxvlindolyl)formateStep 1: Benzvl (1—(2—(2—(l—hvdroxv—2,2.2-trifluoroethvl)phenoxv)methyl-5-phenylmethoxy zindolyl )—formateA solution of 0.4 g (0.8 mmol) of the aldehyde, prepared in step 1 of Example 1,in 4 mL of THF was cooled to 0 °C. To this were added 0.24 mL (1.6 mmol) oftri?uoromethyl trimethylsilane and 5 mg of tetrabutylammonium ?uoride trihydrate. Thereaction was stirred for 2.5 hpurs at 0 °C, and additional 0.2 mL (1.3 mmol) oftrifluoromethyl trimethylsilane and 5 mg of tetrabutylammonium ?uoride trihydrate wereadded. After stireed at 0 °C for 2 hours, the reaction was worked up with aqueousammonium chloride and ethyl acetate. Silica gel chromatographic purification using 4:1hexane-ethyl acetate afforded corresponding TMS ether. Treatment of TMS ether with 1.3mL of IN Hcl solution at room temperature, aqueous woukup using brine and ethyl acetateand chromatographic puri?cation using 3:1 hexane—ethyl acetate gave 230 mg of the titledcompound.Step 2:To a solution of 150 mg (0.27 mmol) of trifluoroethanol. prepared in step 1, in 5.5ml. of dichloromethane was added 255 mg (0.6 mmol) of the Dess—Martin’s periodinate.The mixture was stirred at room temperature for 1 hour, and then partitioned betweenaqueous NaHCO, and ethyl acetate. The organic phase was washed once with aqueousNaHCO3 and purified with chromatography using 3:1 hexane—ethyl acetate to yield 150 mgof the titled compound.47SUBSTITUTE SHEET (RULE 26)?WO 98/088181015202530CA 02264020 1999-02-24PCT/U S97/ 14943EXAMPLE 53—(2—(1-Benzvl-5-benzyloxv)indolecarboxamidolbenzoic acidStep 1: Ethyl 2-(l-benzvl-5-benzvloxy)indolecarboxvlateTo a solution of l g (3.4 mmol) of ethyl 5-benzyloxyindole-2-carboxylate in 12 mlof DMF, sodium hydride (0.163g, 60% oil dispersion, 4.07 mmol) was added at roomtemperature. The reaction was stirred for 30 minutes. Benzyl bromide (0.44 mL, 3.73mmol) was added at this time and the reaction stirred for another hour. On completion ofthe reaction (monitored by TLC = 0.5 Rf in 3:1 Hexane:Ethyl acetate) it was quenchedwith water, extracted with ethyl acetate (3X). Organic layers were dried over magnesiumsulfate, concentrated and used for the next step.Step 2: 2~(1—Benzyl-5—benzv|oxv)indolecarboxvic acidThe ester (3.4 mmol), prepared in step 2, was dissolved in THF (20 mL), methanol(20 mL) and then 1N NaOH (15 mL) was added. The reaction mixture was stirred at roomtemperature over night at which time it was concenterated, diluted with water, acidi?ed topH 5 with 10% HCl and extracted with ethyl acetate (3X), the organic extracts were driedover magnesium sulfate and concentrated to give the indole acid ( l.l4 g, 94.2 %, TLC =0.5 Rf in l:l Hexane:Ethyl acetate with 1% acetic acid).Step 3: Ethvl 3—(2—(l-benzvI-5—ben2y|oxv)indolecarboxamido)benzoateThe acid (0.54 g, 1.5 mmol) of step 2, 1-(3-dimethylaminopropyI)-3-ethylcarbodiimide (EDCI) (0.32 g, l.66 mmol), 4-dimethylaminopyridine (DMAP) (0.018g, 0.15 mmol) and ethyl 3-aminobenzoate (0.27 g, 1.66 mmol) were stirred intetrahydrofuran (9 mL) at room temperature overnight. The next day the reaction wasdiluted with ethyl acetate and water, extracted with ethyl acetate (3X), dried overmagnesium sulfate and concentrated. The crude material was purified on silica gel using3:] hexanezethyl acetate to give pure amide (0.578 g, 76%, TLC = 0.4 Rf in 3:1Hexane:Ethyl acetate).48SUBSTITUTE SHEET (RULE 26)?l015202530WO 98/08818CA 02264020 1999-02-24PCTIUS97/14943Step 4:The ester (0.578 g, 1.15 mmol), prepared in step 3. was dissolved in THF (13.6mL), methanol (13.6 mL) and then IN NaOH (9.6 mL) was added. The reaction mixturewas stirred at room temperature overnight at which time it was concenterated, diluted withwater, acidified to pH 5 with 10% HCl and extracted with ethyl acetate (3X), the organicextracts were dried over magnesium sulfate and concentrated to give the titled compound(0.437 g, 80 %, TLC = 0.5 Rf in 3:1 hexanezethyl acetate with 1% acetic acid).The Examples 6, 7, 8, 9, l0 and _l_l in Table I were prepared by the procedures of Example5 using suitable amines and alkyl halides.EXAMPLE 1 23-(2-(3—(2,4-bis(l , l —dimethvpropvlmhenoxvacetvl)-5-methoxy- l -methvl)indolvl)methvlthioacetamido-4-methoxvbenzoic acidStep 1: 2-(5-MethoxvlindolvlmethanolEthyl 5-methoxy-2-indolcarboxylate (30 g, 102 mmol) is dissolved in 250 mL ofTHF and cooled to 0° C and Lithium Aluminum Hydride (LAH) (255 mL of a 1.0 Msolution in THF) is added via addition funnel over 40 minutes. The reaction was stirred afurther 2 hours at 0° C and then worked up by the addition of 4N NaOI-I (190 mL). Theresulting salts are ?ltered and washed with ethyl acetate (3X400 mL), the ?ltrates arecombined and dried over MgSO4 and concentrated to yield 24.8 g of alcohol, which wasused for the next reaction directly.Step 2: 2—(5—methoxv)indolvlmethoxv~tert-buthvldimethylsilaneThe crude indole alcohol prepared in step 1 (6.2 g, 32.6 mmol) was dissolved inDMF (10.5 mL). To this solution was added imidazole (5.5g, 81.5 mmol) and t-butyldimethylsilyl chloride (5.4g, 35.8 mmol). The mixture was stirred at room49SUBSTITUTE SHEET (RULE 26)?W0 98/0881810I520\(2530CA 02264020 1999-02-24PCT/US97/14943temperature overnight. The reaction was poured into water and extracted with ethyl acetate(3X). Organic layers were dried over magnesium sulfate and concentrated. The crudematerial was puri?ed on a silica gel column using 19:] hexane:ethyl acetate to give pureproduct (9.5g, 31 mmol, 94 % yield, TLC: 0.8 Rf in toluenezethyl acetate 2:1)Step 3: 3—(2—tert-butvdimethvlsiIvloxvmethyI-5-methoxv)indolvl (2.4-bis(l,1—dimethvnropvl)Dhenoxv)methvI ketone2.32 g (7.95 mmol) of 2.4-Bis-tert-amylphenoxyacetic acid was dissolved indichloromethane (21 mL), oxalyl chloride (1.4 mL 16.] mmol) was added, followed bydimethyl fonnamide (0.5 mL) at room temperature. After one hour the reaction isconcentrated and azeotroped with toluene and left on the high vacuum for two hours.In another reaction vessel, a solution of the silyl protected indole, prepared in step2, (2 g, 6.56 mmol) in ether (20 mL) was added dropwise to ethyl magnesium bromide (2.4mL of a 3M solution in ether, 7.2 mmol) in ether ( 10 ml), the latter maintained at -78 “C.The reaction was stirred at -60°C for 2 hr. To this reaction solution, the above prepared acidchloride in ether (4 mL) was added slowly. The reaction was maintained between -50°Cand -60°C for another 2 hrs. The reaction was then quenched with saturated sodiumbicarbonate. Extracted with ethyl acetate (3X). Organic layers were dried over magnesiumsulfate and concentrated. The crude material was purified on a silica gel column using19:] Hexane:Ethyl acetate to give pure product (2.36 g, 50%, TLC: 0.15 Rf inhexane:ethyl acetate 19: l.Step 4: 3—(2—tert-butvdimethvlsilvloxvmethvl-5-methoxy—l-methvhindolvl (2.4-bis(l,|-dimethvnronvl)Dhenoxv)methvl ketoneTo the ketone (l.97g, 3.4 mmol) of in step 3 in 12 ml of DMF, sodium hydride(0.l63g, 60% oil dispersion, 4.07 mmol) was added at room temperature. The reaction wasstirred for 30 minutes. Methyl iodide (0.23 mL, 3.73 mmol) was added at this time and thereaction stirred for another hour. On completion of the reaction (monitored by TLC) it wasquenched with water, extracted with ethyl acetate (3X). Organic layers were dried overmagnesium sulfate, concentrated and the crude product was used for the next step.50SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943Step5: 3—(2-I-Ivdroxvmethvl-5-methoxv-1-methvl)indolvl (bis-2,4-11,1 gimethvlpropv1)Dhenoxv)methvl ketoneA mixture of N-methyl indole, prepared in step 4, (2.01 g, 3.4 mmol) and tetra-butyl ammoniumfluoride (TBAF) (8.5 ml. of a 1M solution in THF, 8.5 mmol) in THF(17.9 mL) were stirred at room temperature for one hour. At this time the reaction wasdiluted with ethyl acetate and water, extracted with ethyl acetate (3X), dried overmagnesium sulfate and concentrated. The crude material was puri?ed on silica gel usinghexanezethyl acetate 2:1 to yield pure alcohol (0.82 g, 60 %, TLC: 0.3 Rf in 2:1hexane:ethyl acetate).Step 6: Methyl 3-(2-13-(2.4—bis(1 ,1-dimethvpropvl)phenoxv)acetvl-5-methoxv-1-methvlindolvl)methvlthioacetamido)-4-methoxybenzoateThe indole alcohol, prepared in step 5, (0.20 g, 0.43 mmol) was dissolved indichloromethane (0.7 mL) and treated with triethylamine (0.1 mL, 0.64 mmol) and cooledto 0° C at which time mesyl chloride (0.04 mL 0.52 mmol) was added over 5 minutes,followed by addition of two drops of DMF. The reaction was stirred for a funher 2 hour at0°C, it was then concentrated and used directly for the next reaction.The above prepared mesylate was dissolved in DMF (0.8 mL). The solution wasdegassed by bubbling nitrogen through for ten min. Cesium carbonate (0.25 g, 1.29 mmol)was added and then thiol (0.1 1 g, 0.43 mmol), prepared in Intermediate 1, was added. Themixture was stirred overnight, then poured into saturated ammonium chloride and extractedwith ethyl acetate (3X), dried, concentrated. The crude material was puri?ed on a silica gelcolumn using hexanezethyl = 2:1 acetate to give pure product (0.12 g, 40%, TLC: 0.3 Rf inhexanezethyl acetate 1 :1).Step 7:The ester, prepared in step 6, (0.12 g, 0.17 mmol) was dissolved in THF (1.0 mL),methanol (1.0 mL) and then IN NaOH (0.4 mL) was added. The reaction mixture wasstirred at room temperature overnight at which time it was concenterated, diluted withwater, acidified to pH 5 with10% HCl and extracted with ethyl acetate ( 3X), the organic51SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943extracts were dried over magnesium sulfate and concentrated to give the titled compound(85 mg, 72 %, TLC = 0.3 Rf in 1:1 Hexane:Ethyl acetate with 1% acetic acid).EXAMPLES 13 14 15 and _l_§ in Table I were prepared by the procedures of Example 12 using Ethyl 2-(5-benzyloxy)indolecarboxylate, acetyl chlorides and suitable alkyl halides.EXAMPLE 173—(2-(-5-benzvloxv-1-(2.4-bisjl,1—dimethv)propvl)phenoxvacetvl)indolinvl)methylthioacetamidobenzoic acidStep 1: 2-(5-Benzyloxv)indolinvlmethanolEthyl 5—benzyloxy-2-indolecarboxylate (30 g, 102 mmol) was dissolved in 250 mL ofTHF and cooled to 0° C, to which Lithium Aluminum Hydride (LAH) (255 mL of a 1.0 Msolution in THF) was added via addition funnel over 40 minutes. The reaction was stirreda for 2 hours at O “C and then worked up by the addition of 4N N aOH (190 mL). Theresulting salts were ?ltered and washed with ethyl acetate (3X400 mL), the ?ltrates werecombined, dried over MgSO4 and concentrated to yield 24.8 g. This crude material wasthen dissolved in glacial acetic acid (260 mL) and the resulting yellow solution was cooledto 15° C, sodium cyanoborohydride (18.5 g, 294 mmol) was added ponionwise over 10minutes, and the resulting mixture was stirred for 3 hours. The reaction was quenched bypouring slowly into 1.5 liters of nearly saturated NaHCO3, extracted with ethyl acetate(3X), dried over MgSO4 and concentrated to yield a orange solid (29.6 g).Step 2: tert-Butvl 1-(5-benzvloxv—2-hvdroxvmethv)lindolinvlformate25 g (85 mmol) of crude alcohol, prepared in step 1, and 4-dimethylamino pyridine(DMAP) (1.19 g, 9.78 mmol) were dissolved in dichloromethane (180 mL). The solutionwas cooled to 0° C and then triethylamine (13.6 mL, 98 mmol) was added to it. After 10minutes of stirring a solution of di-tert~butyl dicarbonate (21.3 mL, 98mmol) dissolved indichloromethane (20 mL) was added via syringe pump over 2 hours. After 1 hour ofstirring the reaction was quenched by the addition of 1/2 saturated NH 4C1 solution and52SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/ 14943extracted with CHZCIZ (3X), dried over MgSO4 and concentrated to yield 36.3 g of a yellowoil, which was purified by column chromatography using a hexane:ethy1 acetate gradient of9:1 to 4:1 to 1:1 to deliver the product (15.25 g, 44%).Step3: Ethyl 2-(5-benzvloxv—1—tert—butoxvcarbonvl)indolinvlmethvlthioacetateThe carbamate, prepared in step 2, (15.25 g, 43 mmol) was dissolved indichloromethane (180 mL) and treated with triethylamine (9.0 mL, 64.4 mmol). Thesolution was cooled to -10° C at which time mesyl chloride (4.3 mL. 56 mmol) was addedover 5 minutes. The reaction was stirred for a further 2 hour at -10 "C, it was thenconcentrated and used directly for the next displacement reaction.The above prepared mesylate was dissolved in DMF (85 mL. degassing thesolvent is strongly reccomended) cesium carbonate (35 g, 107.3 mmol) was added and thenethyl thioacetate (4.70 mL. 42.9 mmol) was added. The mixture was stirred for 1 day, thenpoured into 1/2 sturated ammonium chloride and extracted with ethyl acetate (3X), dried,concentrated and chromatographed (hexane:ethyl acetate gradient 10:1 to 4:1) to yield 8.55g of a yellow oily product.Step 4: 2-(5-Benzvloxv-1-ten—butoxvcarbonvl)indolinvlmethvlthioacetic acidTo a solution of the indoline ester, prepared in step 3, (5g, 1 1 mmol) in 1Mpotassium hydroxide in methanol ( 100 mL), water (10 mL) was added. The reaction wasstirred at room temperature for two hours at which time it was diluted with water, acidifiedto pH 5 with10% HCl and extracted with ethyl acetate (3X), the organic extracts were driedover magnesium sulfate and concentrated to give the indoline acid ( 4.5g, 95.5%, TLC =0.5 Rf in 2:1 hexanezethyl acetate with 1% acetic acid). The crude material was used for thenext step directly.Step 5: Ethvl 3—(2-(5-benzvloxv-l-tert-butoxvcarbonvl )indolinybmethvlthioacetamidobenzoateThe acid (3g, 7 mmol), prepared in step 4, 1—(3—dimethylaminopropyl)—3—ethylcarbodiimide (l.6g, 8.4 mmol), 4—dimethylaminopyridine (0.85g, 7 mmol) and ethyl3-aminobenzoate (1.27 g, 7.7 mmol) were stirred in tetrahydrofuran (43 mL) at room53SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943temperature overnight. On next day the reaction was diluted with ethyl acetate and water,extracted with ethyl acetate (3X), dried over magnesium sulfate and concentrated. Thecrude material was purified on silica gel using 3:] hexanezethyl acetate to give the product(3.4g, 85%, TLC = 0.3 Rf in 3:1 hexanecethyl acetate).Step 6: Ethyl 3—(2-(5-benzvloxv)indolin?)methvlthioacetamidobenzoateTo the indoline (3.4g, 5.9 mmol) of step 5, trifluoroacetic acid (24 mL) was addedand the reaction stirred for 1 hour at 0°C. The reaction was quenched by the addition ofwater and the TFA neutralized by the addition of sodium bicarbonate, the aqueous layerwas extracted with ethyl acetate (3X), dried over magnesium sulfate and concentrated. Thecmde material was purified on silica gel using 2:1 hexanezethyl acetate to yield product(2.7 g, 96 %, TLC = 0.3 Rf in 2:1 hexanecethyl acetate).Step 7: Ethvl 3—(2-(5—benzvloxv-1—(2.4-bis(l.1—dimethvl)DroDvlmhenoxvacetvl)indolinvl) methvlthioacetamidobenzoateThe 2,4-bis(1,1—dimethylpropyl)phenoxyacetic acid (0.228 g, 0.78 mmol) wasdissolved in dichloromethane (2 mL), to which oxalyl chloride (0.14 mL 1.6 mmol) wasadded followed by dimethyl formamide (0.1 mL) at room temperature. After one hour thereaction is concentrated and azeotroped with toluene and left on the high vacuum for twohours. The indoline ester (0.308 g. 0.65 mmol), prepared in step 6, and 4-dimethylaminopyridine (0.008 g, 0.066 mmol) were dissolved in dichloromethane (1.2mL) and then the above prepared acid chloride in dichloromethane (0.5mL) was addedfollowed by the addition of triethylamine (O.28mL, 1.95 mmol). The reaction was stirredat room temperature overnight, and then diluted with ethyl acetate and water, extractedwith ethyl acetate (3X), dried over magnesium sulfate and concentrated. The crudematerial was puri?ed on silica gel using 2:1 hexanezethyl acetate to yield product (0.291 g,60 %, TLC = 0.4 Rf in 2:1 hexanezethyl acetate).Step 8:The ester (0.231 g, 0.31 mmol) of step 7 was dissolved in THF (4.3 mL), methanol(4.3 mL) and than 1N NaOH (3.2 mL) was added. The reaction mixture was stirred at54SUBSTITUTE SHEET (RULE 26)?W0 98/088181015202530CA 02264020 1999-02-24PCT/US97/ 14943room temperature overnight at which time it was concenterated, diluted with water,acidi?ed to pH 5 with 10% HCl and extracted with ethyl acetate (3X), the organic extractswere dried over magnesium sulfate and concentrated to give the titled product ( 0.207 g,93.2 %, TLC = 0.3 Rf in 2:1 hexanezethyl acetate with 1.5 % acetic acid).EXAMPLE l83-(2-(~5-Benzvloxv-l -(2.4-bis( l .l-dimethvloropvllnhenoxvacetvl)indolinvl)methylthioacetamido—4-methvlbenzoic acidStep 1: Ethvl 2—(5-benzvloxvlindolinvlmethvlthioacetateThe N-tembutoxycarbonyl indoline (3.0 g, 6.6 mmol), prepared in step 3 ofExample 17, was added to a flask and cooled to 0°C. To this reaction mixturetri?uoroacetic acid was added (35 mL) and the reaction was stired for 1 hour at 0 °C andthen 1 hour at rt. The reaction was quenched by the addition of water. and the TFA wasneutralized by the addition of solid sodium bicarbonate. the aqueous layer was extractedwith ethyl acetate (4X) and dried over magnesium sulfate and concentrated to an orange oil(1.85 g, 79%) that was used directly for the next step.Step 2: Ethvl 2—(5—benzvloxv-l-(2.4-bis(l.1—dimethv)propvl)phenoxvacetvl)—indolinylmethylthioacetate2,4-Bis( l ,l—dimethy)propyl)phenoxyacetic acid (2.0g, 6.8 mmol), dichloromethane(15 mL), oxalyl chloride (l.2 mL, 13.6 mmol), dimethylfonnamide (0.1 mL) were stirredat 0° C for 45 minutes at which time the reaction is concentrated and azeotroped withtoluene (IX) and concentrated on the high vac for 2 hours before use. The indoline ester(l.85g, 5.2 mmol), prepared in step], and 4-dimethylaminopyridine (0.08 g) weredissolved in dichloromethane (15 mL) and then the above generated acid chloride indichloromethane (5 mL) was added followed by the addition of triethylamine (0.95 mL,6.8 mmol). The reaction was stirred 16 hours at rt, worked up and concentrated (4.0 g,orange oil), chromatographed using a 9:1 to 6:1 gradient of hexanezethyl acetate to yieldthe product (2.5g, 75%) that was used for the next step without further purification.55SUBSTITUTE SHEET (RULE 26)?101520 5‘_2530W0 98/08818CA 02264020 1999-02-24PCT/US97/14943Step 3: 2-(5-Benzvloxv-1—(2.4-bis(1 ,1-dimethvlpropvllphenoxvacetvl)indo1invlmethvlthioacetic acidThe ester (2.5 g, 3.9 mmol), prepared in step 2, was dissolved in THF (20 mL),methanol (6 mL) and then 1N sodium hydroxide (12 mL) was added. The resultingmixture was stirred 24 hours at which time it was concentrated, diluted with water,acidi?ed to pH 4 with concentrated HCl and extracted with ethyl acetate (4X), the organicextracts were dried over magnesium sulfate, concentrated, and puri?ed via chromatography(3:l hexane:ethyl acetate with 1% acetic acid) to yield 1.17 g ( 50%) of the product aswhite solid.Step 4: Methvl 3—(2—(5-benzvloxv~l—(2.4-bis(l,1-dimethv)propvl)phenoxvacetvl)indolinvl) methvlthioacetamid0—4—methvlbenzoateThe acid (0.20 g, 0.33 mmol), prepared in step 3, EDCI (0.08 g, 0.43 mmol),DMAP (4 mg, 0.03 mmol) and methyl 3—amino—4-hydroxy benzoate (0.06 g, 0.33 mmol)were dissolved in THF (3 mL) and re?uxed16 hours. Aqueous workup with ammoniumchloride and ethyl acetate and purification via silica gel chromatography (hexane:ethylacetate 3: 1) yielded 0.13 g (52%) of the product as a white solid.Step 5:The titled compound was prepared from ester. prepared in step 4, according to theprocedure described in step 3.EXAMPLES 17 to 36 in Table 2 were prepared according to the procedurs described ineither Example 17 or Example 18.56SUBSTITUTE SHEET (RULE 25)?W0 98/0881810I5202530CA 02264020 1999-02-24PCT/US97/14943EXAMPLE 372-(5-Ben2vloxv— l —(3.5—bis(trifluoromethvl)Dhenoxvacetvl)indolinvllmethvlthioacetic acidStep 1: 2—(5-Benzvloxv-I~(3.5-bis(trifluoromethvl)phenoxvacetvl)indolinvl)methanolA 1-L oven-dried round bottom ?ask ?tted with a magnetic stirring bar andequalizing dropping funnel was charged with 17.0 g ( 59 mmol) of 3,5-bis(tri?uoromethyl)phenoxyacetic aci, DMF (5 drops) and anhydrous CHZCIZ (300 mL).Oxalyl chloride (23 mL, 263 mmol) was added dropwise over 10 min. After stirring for2.5 h at room temperature solvent, excess oxalyl chloride were removed in vacuo to affordacid chloride as a white solid. This was used immediately in the next reaction.A l—L oven-dried round bottom ?ask ?tted with a magnetic stirring bar andequalizing dropping funnel was charged with 15.3 g (60 mmol) of 2-(5-Benzyloxy)indolinylmethanol, prepared in stepl of Example 17, DMAP (0.73 g, 6 mmol)and anhydrous CH2Cl2 (300 mL). After cooling to 0 “C, a solution of above prepared acidchloride (59 mmol) in anhydrous CH3Cl3 (100 mL) was added dropwise, followed by NEt3(9 mL, 64.7 mmol). After stining for l h at 0 “C the reaction mixture was washed withsaturated NaHCO3 solution (100 mL), 1 N HCl solution (100 mL) and H30 (100 mL),dried over N%SO_‘ and ?ltered. The solvent was removed in vacuo. Purification bycolumn chromatography in silica gel using 25-40% AcOEt in hexane afforded product as alight yellow solid. Yield 22.0 g (71%).Step 2: Ethvl 2—(5-benzvloxv-I-(3.S—bis(tri?uoromethvl)phenoxvacetvl)indolinvl)methylthioacetateA 500-mL oven-dried round bottom ?ask fitted with a magnetic stirring bar wascharged with alcohol (19.0 g, 36.15 mmol), prepared in step 1, anhydrous CHZCIZ (300mL), and NEt3 (7.5 mL, 54.23 mmol). MsCl was added dropwise over 2 min and thereaction mixture was stirred at room temperature for 10 min. The solution was diluted withCH3Cl2 (500 mL) and washed with IN HCl solution (100 mL) and saturated NaHCO3solution (100 mL). The CHZCIZ solution was dried over Na3SO4 and ?ltered. The solventwas removed and the mesylate was used in the next step without further purification.57SUBSTITUTE SHEET (RULE 26)?101520WO 98/08818CA 02264020 1999-02-24PCT/US97/14943A 500—mL oven—dried round bottom ?ask fitted with a magnetic stirring bar wascharged with ethyl thioacetate (4.2 mL, 38.5 mmol), and anhydrous THF (75 mL). Aftercooling in a dry ice/acetone bath NaN(SiMe3)2 (1 M solution in THF, 50 mL, 50 mmol)was added. After 15 min a solution of above prepared mesylate (21 g, 35 mmol) inanhydrous THF (60 mL) was added. After 15 min the reaction mixture was allowed towarm to room temperature. After stirring at room temperature for 100 min the reactionwas heated at re?ux for 4 h. The solution was allowed to cool to room temperature. It wasdiluted with CHCl3 (500 mL), washed with saturated Na1CO3 solution (200 mL) and 1NHCl solution (200 mL). The organic solution was dried over Na3SO4 and ?ltered. Thesolvent was removed in vacuo. The crude material was puri?ed by columnchromatography on silica gel using15% AcOEt in hexane to afford 13.8 g (63%) ofproduct.Step 3:A 250—mL round bottom ?ask ?tted with a magnetic stirring bar was charged withester (12.45 g, 19.8 mmol), prepared in step 2, THF (100 mL), MeOH (33 mL) and H20(33 mL). LiOH'H3O (1.08 g, 25.7 mmol) was added and the reaction mixture was stirred atroom temperature for 3 h. The solvents were removed in vacuo. The residue was takeninto 1N HCl solution (200 mL) and extracted with AcOEt (2 x 400 mL). The combinedextracts were washed with 1 N HCI solution (100 mL), dried over Na2SO4 and ?ltered. Thesolvent was removed in vacuo to afford the titled compound. Yield 11.9 g (100%).58SUBSTITUTE SHEET (RULE 26)?WO 98/088181015202530CA 02264020 1999-02-24PCT/US97/14943EXAMPLE 385-(2—(—5—Benzvloxv-l -(3,5-bisttri?uoromethvllnhenoxvacetvl)indolinvllmethvlthioacetamidol benzene-1 .3-dicarboxylic acidStep l: 5—(2-(-5—Benzvloxv-l—(3,5-bis(tri?uoromethvDphenoxvacetvl)indolinvl)methvlthioacetamido)benzene- l .3—dicarboxvlateA 100-mL oven-dried round bottom ?ask ?tted with a magnetic stirring bar wascharged with acid (1.2 g, 2 mmol), prepared in step 3 of Example 37, anhydrous THF (40mL), EDCI (0.544 g, 2.8 mmol), DMAP (0.024 g, 0.2 mmol). and 5-amino-l,3-benzenedicarboxylic acid (0.46 g, 2.2 mml). The reaction mixture was heated at re?uxuntil no change was detected by TLC. The solvent was removed in vacuo. The residuewas dissolved in CHZCI2 (200 mL), washed with 1 N HCl solution (25 mL), dried overNa2SO4 and ?ltered. The solvent was removed in vacuo. The crude material was puri?edby column chromatography on silica gel using l-2% MeOH in CHZCIZ to afford 1,2 g(77%) of product.Step 2:A 25-mL round bottom ?ask ?tted with a magnetic stirring bar was charged withester (0.6 g, 0.76 mmol), prepared in step] , TI-IF (7.5 mL), MeOl-I (2.5 mL) and H20 (2.5mL). LiOI-IHZO (0.084 g, 2 mmol) was added, and the reaction mixture was stirred atroom temperature for 6 h. The solvents were removed in vacuo. The residue was takeninto IN HCI solution (10 mL) and extracted with AcOEt (2 x 50 mL). The combinedextracts were dried over Na2SO4 and filtered and removed in vacuo. The crude materialwas purified by column chromatography on silica gel (eluant: 5% MeOH in CHCI3 + 0.5-0.7% ACOH) to yield 0.28 g (46%) of the titled compound.EXAMPLES 39 40 43 in Table 3 were prepared according to the procedurs described ineither Example 38. 59SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97l14943EXAMPLE 415—(2—(—5-Benzvloxy- l —(3,5—bis(tri?uor0methvl)Dhenoxvacetvl)indolinvl)methylthioacetamido)—3-hvdroxvmethylbenzoic acidStep 1: Methvl 5-(2—(—5—benzvloxv-I-(3.5-bis(tri?uoromethv|)phenoxvacetvl)indolinvl)methylthioacetamido)-3—tert-butvldimethvlsilvloxvmethvlbenzoateThis compound was prepared according to the procedure described in step 1 ofExample 38.Step 2: Methyl 5-(2-L-5-benzvloxwl—(3,5-bis(txifluoromethyl)phenoxvacetvl)indolinvl)methylthioacetamido1-3—hydroxymethylbenzoateA 25-mL oven-dried round bottom ?ask fitted with a magnetic stirring bar wascharged with silyl propected ester (1.32 g, 1.5 mmol), prepared in step 1, anhydrous THF(10 mL), and TBAF (1 M solution in THE, 2.5 mol equiv.). The reaction mixture wasstirred at room temperature for 3 hours. The solvent was removed in vacuo. The oilyresidue was purified by column chromatography on silica gel using 0—30% ACOEI inCH3Cl3_ to afford 0.94 g (92%) of desired product.Step 3:The titled compound was prepared according to the procedure described in step 2of Example 38.EXAMPLE 42 in table 3 was prepared according to the procedures described in Example4_l.60SUBSTITUTE SHEET (RULE 26)?101520 ‘2530WO 98/08818CA 02264020 1999-02-24PCT /U S97] 1 4943EXAMPLE 445-(2-1-5-Hydroxy-l-13,}bis(tri?uoromethvl)phenoxvacetvl)indolinvDmethvlthioacetamido) benzene—1,3—dicarboxylic acidStep 1: 2—(5-Hvdroxv-l—(3.5-bis(trifluoromethyl)phenoxvacetyl)indolinyl)methanolA 500-mL Parr Hydrogenation bottle was charged with 2-(5-Benzyloxy-l—(3,5-bis(tri?uoromethyl)phenoxyacetyl)indolinyl)methanol (10 g, 19.1 mmol), prepared in step1 of Example 37, 5% Pd on carbon (l.O g), AcOEt (150 mL) and MeOH (100 mL) andsubsequently hydrogenated at 50 psi for 18 h. The reaction mixture was ?ltered throughCelite and concentrated in vacuo to afford crude product. This was used in the next stepreaction without further puri?cation.Step 2: 2-(5-(4-Methoxvlbenzvloxv- l -(3.5-bis(tri?uoromethyl)phenoxvacetvl)indolinvl)methanolA 1-L oven-dried round bottom ?ask fitted with a magnetic stirring bar and re?uxcondenser was charged with alcohol (8.56 g, 19.7 mmol), prepared in stepl , 200 meshKZCO3 (6.53 g, 47.2 mmol), KI (3.91 g, 23.6 mmol) and ?nally the p—methoxy benzylchloride (3.2 mL, 23.6 mmol) in 450 mL of anhydrous acetonitrile. The reaction mixturewas heated at reflux for 4 h. The reaction mixture was partitioned between AcOEt (500mL) and H30 (200 mL). The aqueous layer was extracted with AcOEt (3 x 500 mL). Thecombined AcOEt extracts were washed with brine (500 mL), dried over Na3SO4 and?ltered. The solvents were removed in vacuo. Puri?cation of the residue by columnchromatography on silica gel (eluant: 40% AcOEt in hexane) afforded desired product.Yield 8.7 g (83%).Step 3: Methyl 5—(2-(-5—(4-methoxv)benzvloxv—l-(3.5—bis(tri?uoromethvl)phenoxvacetvl)indolinvl)methvlthioacetamido)benzene- l .3-dicarboxvlateA l00—mL oven-dried round bottom ?ask ?tted with a magnetic stirring bar wascharged with alcohol (3.2 g, 5.77 mmol), prepared in step 2, and anhydrous CH3Cl2 (4461SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/ 14943_mL). The reaction mixture was cooled to 0°C and added anhydrous Et2N (1.2 mL. 8.6!mmol) followed by MsCl (0.53 mL, 6.84 mmol). The reaction mixture was stirred at 0 °Cfor 5 min. The reaction mixture was partitioned between CH2Cl2 (100 mL) and H20 (50mL). The aqueous layer was extracted with CH2Cl2 (3 x 100 mL). The combined CH2Cl2extracts were washed with 1 N HCl solution (100 mL), saturated NaHC02 solution ([00mL), H20 (100 mL), brine (100 mL), dried over Na2S04 and ?ltered. The solvents wereremoved in vacuo to afford mesylate. This was used in the next step reaction withoutfurther purification.A 100-mL oven—dried round bottom ?ask ?tted with a magnetic stirringbar and re?ux condenser was charged with above prepared mesylate (3.60 g, 5.70 mmol),anhydrous Cs2C02 (5.19 g, 15.9 mmol) and anhydrous DMF (20 mL). The reactionsolution was passed through N2 for 15 min. Methyl 5-thioacetamido—1 ,3-benzenedicarboxylate, prepared in Intemiediate 2, was added in one portion and thereaction mixture was heated at 50 “C for l8 h. The reaction mixture was partitionedbetween Ac0Et (500 mL) and H20 (200 mL). The aqueous layer was extracted withAc0Et (3 x 100 mL). The combined Ac0Et extracts were washed with saturated Na2C02solution (100 mL), H20 (100 mL), brine (500 mL), dried over Na2S02 and ?ltered. Thesolvents were removed in vacuo. Purification of the residue by column chromatography onsilica gel (eluant: 5% Ac0Et in CH2Cl2 ) afforded product. Yield 2.5 g (53%).Step 4: Methyl 5-(2-(-5-Hvdroxv—l-(3,5—bis(tri?uoromethyhphenoxvacetvl)indolinvl)methv|thioacetamido)benzene-l .3—dicarboxvlateA 100-mL oven—dried round bottom ?ask ?tted with a magnetic stining bar wascharged with ester (2.60 g, 3.17 mmol), prepared in step 3, and anhydrous CH2Cl2 (30 mL).To the reaction mixture was added TFA (25 mL) in several portions over 1 min. Thereaction mixture was poured onto 500 mL saturated NaHCO_, solution and extracted withCH2Cl2 (3 x 100 mL). The combined CH2Cl2 extracts were washed with saturated Na2C0_,solution (200 mL), H20 (200 mL), brine (500 mL), dried over Na2S04 and ?ltered. Thesolvents were removed in vacuo. Purification of the residue by column chromatography onsilica gel (eluant: 12.5% - 20% Ac0Et in CH2Cl2 ) afforded the product. Yield 1.5 g(68%).62SUBSTITUTE SHEET (RULE 26)?W0 98/088181015202530CA 02264020 1999-02-24PCT/US97/ 14943V§!§.L5;A 25-mL round bottom ?ask ?tted with a magnetic stirring bar was charged withester (270 mg, 0.40 mmol), prepared in step 4, LiOH hydrate (3.3 equiv.), THF (3.6 mL),MeOH (1.2 mL) and H30 (1.2 mL). The reaction mixture was heterogeneous with whitesolid suspended in the solution. After stirring for 4 h, more solvents were added in 3 : I : 1= THF : MeOH : H20 to make a clear solution. The reaction mixture was stirred at roomtemperature for 18 h and monitored by TLC. The reaction mixture was acidi?ed with 1 NHCl solution to pH = 2 or with acetic acid to pH = 4 and then partitioned between AcOEt(20 mL) and H30 (20 mL). The aqueous layer was extracted with AcOEt (3 x 20 mL).The combined AcOEt extracts were washed with H30 (20 mL), brine (20 mL), dried overNa2SO4 and filtered. The solvents were removed in vacuo. Puri?cation of the residue bycolumn chromatography on silica gel followed by recrystallization from acetone / hexaneafforded 130 mg of the titled compound (50%).EXAMPLE 455-(2-(5-(3.5—Dibromo)benzvloxy— I —(3,5-bis(trifluoromethvl)Dhenoxvacetvl)indolinvl)methvlthioacetamido)benzene— l .3-dicarboxvlic acidSteplz Methyl 5-(2-(5-(3,5-Dibromo)benzvloxv-l-(3.5-bisttrifluoromethvlmhenoxvacetvl) indolinvI)methvlthioacetamido)benzene~ l ,3-dicarboxylateA 25-ml. oven—dried round bottom ?ask fitted with a magnetic stirring bar andre?ux condenser was charged with methyl 5-(2-(-5-Hydroxy-l-(3,5-bis(tri?uoromethyl)phenoxyacetyl) indolinyl)methylthioacetamido)benzene-l ,3-dicarboxylate (0.19 g, 0.27 mmol), prepared in step 4 of Example 4, 200 mesh KZCOJ (2.4equiv.) and 3,5—dibromobenzyl bromide (1.2 equiv.) in 7.5 ml. of anhydrous acetonitrile.The reaction mixture was heated at 70 “C for 2 h. The reaction mixture was partitionedbetween AcOEt (30 mL) and H30 (20 mL). The aqueous layer was extracted with AcOEt(3 x 30 mL). The combined AcOEt extracts were washed with brine (50 mL), dried overNa3SO4 and ?ltered. The solvents were removed in vacuo. Puri?cation of the residue by63SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943column chromatography on silica gel using 15% EtOAc in dichloromethane afforded 0.20g of the product (77%).Step 2:The titled compound was prepared from the ester, prepared in step 1, according tothe procedure described in step 5 of Example 44.EXAMPLES 46 to 5_0 in table 4 were prepared according to the procedures described inExample 451, but using corresponding alkylating reagent.EXAMPLE 51Methyl 3—(2-(5-benzv|oxv—l-(4-benzvlbenzoyl)indolinvl)methvlthi0acetamido)benzoate4-Benzylbenzoic acid (0.19g, 0.91 mmol) was dissolved in dichloromethane (2.3ml), next oxalyl chloride (0.16 mL, 1.82 mmol) was added followed bydimethylfonnamide (0.5 mL) at room temperature. After one hour the reaction wasconcentrated and azeotroped with toluene and left on high vaccum for two hours.Ethyl 3-(2-(5—benzyloxy)indolinyl)methylthioacetamidobenzoate (0.308 g, 0.65mmol), prepared in step 6 of Examle 17, and 4-dimethylaminopyridine (8 mg, 0.066mmol) were dissolved in dichloromethane (1.2 mL) and then the above prepared acidchloride in dichloromethane (0.5 mL) was added followed by the addition of triethylamine(0.28 mL, 1.95 mmol). The reaction was stirred at room temperature overnight. Thereaction was diluted with ethyl acetate and water, extracted with ethyl acetate (3X), driedover magnesium sulfate and concentrated. The cmde material was puri?ed on silica gelusing 2:l hexane:ethyl acetate to yield 0.354 g of the titled product (81.7%, TLC = 0.4 Rfin 2:] hexane:ethyl acetate).64SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943EXAMPLE 523-(2-(5-Benzyloxy—l -(4-benzylbenzovhindolinvl)methvlthioacetamido)benzoic acidThe ester (0.354 g, 0.53 mmol), prepared in Example 51, was dissolved in THF(5.6 mL), methanol (5.6 mL) and than IN NaOH (4.2 mL) was added. The reactionmixture was stirred at room temperature overnight at which time it was concentrated,diluted with water, acidi?ed to pH 5 with10% HCI and extracted with ethyl acetate (3X).The organic extracts were dried over magnesium sulfate and concentrated to give the titledproduct (0.32 g, 94.4 %, TLC = 0.3 Rf in Zslhexanezethyl acetate with 1.5 % acetic acid).EXAMPLES 53 to 58 in Table 5 were prepared according to the procedures described inExample 51 and 5_2_.EXAMPLE 593-(2—(5-Benzvloxv-1—(2-naphthoxvacetvllindolinvl)methvlthioacetamido)-4-methoxybenzoic acidStep 1: Methvl 3-(2-(5—benzvloxvindolinvl)methvIthioacetamido)-4-methoxvbenzoateThis compound was prepared according to the procedures described in step 6 ofExample 17, but with methyl 4-methoxybenzoate.Step 2: Methvl 3—(2-(5-benzvloxv—1—(2~naohthoxvacetvl)indolinvl)methvlthioacetamido)-4-methoxybenzoateThe indole ester (0.22 g, 0.45 mmol), prepared in step 1, 2—naphthoxyacetic acid(0.11 g, 0.53 mmol), EDCI (0.10 g, 0.53 mmol) and DMAP (5 mg, 0.04 mmol) wereweighed into a flask that was equipped with a condenser, flushed with nitrogen, and thentetrahydrofuran (5 mL) was added and the reaction was brought to reflux for 18 hours; thereaction was diluted with 1/2 saturated ammonium chloride and ethyl acetate, extracted 3X65SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943with ethyl acetate. dried over magnesium sulfate. concentrated to yield (0.30 g, 100%crude) a white solid that was used without puri?cation.Step 3:The ester ( 0.12 g, 0.20 mmol), prepared in step 2, was dissolved in THF/ methanoland then IN sodium hydroxide (0.8 mL) was added and the resulting mixture was stirred16 hours at RT and a further 5 hours at 45°C, workup yielded 0.12 g of a yellow solid thatwas purified via preparative TLC (1 :1 hexane:ethy1 acetate with 1% acetic acid) to yield0.12 g of the titled product (95%).EXAMPLES 60 to 63 in Table 5 were prepared according to the procedures describedeither in Example 59 or in Examples 51 and 3;.EXAMPLE 643-(2—(5-benzvloxv—1—tert—butoxvcarbonvl)indo|invl)methvlsulfonvlacetamidobenzoic acidStep 1: Ethyl 3-(2-(5-benzv1oxy—1-tert-butoxvcarbonvl)indolinyl)methvlsulfonvlacetamidobenzoateTo a solution of Ethyl 3—(2—(5-benzy1oxy—1—ten-butoxycarbonyl)indolinyl)methylthioacetamidobenzoate (0.05g, 0.09 mmol), prepared in step 5 of Example 17, indichloromethane (0.1 mL) at room temperature, m-chloroperbenzoic acid (0.06g of 60%m-CPBA, 0.21 mmol) was added and the reaction stirred overnight. Next day the reactionwas quenched with an aqueous solution of sodium bicarbonate, extracted with ethyl acetate(3X), dried over magnesium sulfate and concentrated. The crude sulfone (0.52g, 98%, TLC= 0.3 Rf in 1:1 hexanezethyl acetate) was used for the next reaction directly.66SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943Step 2:The titled compound was prepared according to the procedure described in step 3of Example 59.EXAMPLES 66 and 65 were prepared according to the procedures described in ExampleQ.EXAMPLE 67..-(2—(-5-Benzvloxv-1—(2.4-bis(1.1-dimethvlpropvl)phenoxvacetyl)indolinvllmethvlthiobenzoic acidStep 1: 5-Benzy|oxv—1-(2.4-bis(1.1-dimethvlpropyl)phenoxvacetvl)-2-hydroxymethylindolineThe diisopropylethylamine (3.5 mL, 20.5 mmol), DMAP(O.25 g, 2.05 mmol) andthe indoline alcohol (4.53 g, 17.7 mmol), prepared in step 1 of Example 17, were weighedinto a flask which was ?ushed with nitrogen and cooled to 0° C at which time 21 0° Csolution of di-tert—amylphenoxyacetyl chloride (20.5 mmol) in CH2Cl2 (50 mL) was addedvia cannula. The resulting solution was left to warm to room temperature overnight andthen quenched by the addition of 1/2 saturated ammonium chloride and CH3Cl3, thesolution was extracted with Cl-13Cl3(3X). the combined layers were dried over magnesiumsulfate and concentrated to yield (10.4 g) of a yellow foam that was purified viachromatography using a gradient (hexane:ethyl acetate 7:1 to 3:1 to 1:1) to yield 3.62 g ofthe product.Step 2: 2-(5-Benzvloxv-1—(2.4—bis(1.1-dimethv)DropvImhenoxvacetvDindolinvlmethvlmethylsulfonateTo a solution of alcohol (1.2 g, 2.26 mmol) in CHZCI3 (15 mL), prepared in step 1,is added triethylamine (0.44 mL, 3.16 mmol). The solution is brought to -50 °C and then67SUBSTITUTE SHEET (RULE 26)?W0 98/0881810I520\_2530CA 02264020 1999-02-24PCT/U S97/ 14943mesyl chloride (0.23 mL, 2.93 mmol) is added. The mixture is stirred 2 h at -50 °C.quenched with saturated ammonium chloride and allowed to come to rt. The mixture istaken up in CHCIJ (50 mL), washed with saturated sodium bicarbonate (1 X 10 mL), brine(1 X 10 mL), dried (MgSO4), ?ltered and concentrated to afford the product (1.19 g, 86%).Step 3: Methyl 2—(2—(-5-benzyloxy-1-(2,4-bis(1.1-dimethv)propvl)phenoxvacetvl)indolinyl zmethylthiobenzoateTo a solution of mesylate (0.54 g, 0.89 mmol), prepared in step 2, in degassedDMF (2 mL) is added CsCO3 (0.724 g, 2.22 mmol) and methyl thiosalicylate (0.134 mL,0.98 mmol). The mixture is stirred 4 h, taken up in ethyl acetate (20 mL), washed withbrine (3 X 3 mL), dried (MgSO4), ?ltered and concentrated. Chromatography (gradient.hexanezethyl acetate 15:1 to 4:1) afforded 0.53 (86%) of the title compound as a yellow oil.Step 4:The titled compound was prepared according to the procedure described in step 3of Example 59.EXAMPLE 68 was prepared according to the procedures described in Example 67.EXAMPLE 693-(N—(2-(-5-Benzvloxv—1-(2,4-bis(1 .1—dimethv)Dropvl)phenoxvacetvl)indo1invl)methylthioethyl laminobenzoic acidThe titled product was prepared according to the procedures described in step 3 ofExample 59, but using Intermediate 15.68SUBSTITUTE SHEET (RULE 26)?1015202530W0 98l088l8CA 02264020 1999-02-24PCT/US97/14943EXAMPLE 703-N—Methy|—(2-(-5-Benzvloxv-1—(2,4-bis(l .1-dimethv)Dropvl)phenoxvacetvl)indolinvl)methvIthioacetamido-4—methoxvbenzoic acidAn oven—dried 100 mL, 3-neck round bottom flask, equipped with a stir bar andnitrogen inlet. was charged with methyl 3—(2—(—5—Benzyloxy-l-(2,4-bis(l,1-dimethy)propyI)-phenoxyacetyl)indolinyl)methylthioacetamido-4-methoxybenzate (581mg, 0.757 mmol), prepared in the synthesis of Example 20 using the procedures describedin Example 18, and 10 mL of THF was added via syringe. To the resulting yelllow solutionwas added NaH (60% suspension in mineral oil, 39 mg, 0.975 mmol). The reaction mixturewas stirred at 25 °C for 1.5 h to afford a pale suspension. Methyl iodide (161 mg, 1.14mmol) was added, and the reaction mixture was stirred at 25 °C for 2 days. After chillingto 0 °C, water was added (10 mI_.), followed by 50 mL of half saturated ammoniumchloride, and 100 ml. of EtOAc. The layers were separated. and the aqueous phase wasextracted once with EtOAc (50 mL). The combined organic phases were dried (sodiumsulfate), filtered, and concentrated to afford 0.6 g of crude product as an orange oil. Thismaterial was dissolved in 15 mL of THF and 10 mL of methanol. and 7 mL of 1N NaOHsolution was added, under nitrogen. After being stirred for 2 h at 25 °C, the reactionmixture was concentrated to dryness on the rotary, and 100 ml. of IN HCl, and 100 mL ofEtOAc were added. The layers were separated. and the organic phase was dried(magnesium sulfate), ?ltered. and concentrated. The cmde material obtained (0.565 g) waspuri?ed by column chromatography on silica gel (eluant: chloroform to 3% MeOH inchlorofonn) to afford the titled compound (0.415 g, 70% yield).EXAMPLE 71 was prepared according to the procedures described in Example 70, butusing allyl bromide.69SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/14943EXAMPLE 723—(2-(5-benzv1oxv- 1 -(2-(4-pvridinyl))ethvl)indolinvl)methylthioacetamidobenzoic acidStep 1: Ethvl 3-(2-(5—benzvloxv-1—(2-(4-pyridinyl zethyl gindolinyl zmethylthioacetamidobenzoateTo a solution of ethyl 3—(2-(5-benzyloxy)indoliny1)methylthioacetamidobenzoate(0.30 g, 0.63 mmol), prepared in step 6 of Example 17, in dichloromethane (3.0 mL) andacetic acid (2.0 mL), 4-vinylpyridine (0.08 mL, 0.75 mmol) was added. The reaction wasstirred at room temperature overnight. The reaction was quenched with half saturatedsodium bicarbonate, extracted with ethyl acetate (3X), dried over magnesium sulfate andconcentrated. The cmde material was purified on silica gel using a gradient of 2:1hexane:ethyl acetate to 100% ethyl acetate to yield 0.023 g of product (25 %, TLC = 0.7 Rfin ethyl acetate).Step 2:The titled compound was prepared according to the procedure described in step 3of Example 59.EXAMPLE 733—(2—(5—benzy1oxv—1—(2—naphthvl)methv)indolinvl)methvlthioacetamidobenzoic acidStep 1: Ethvl 3—(2-(5-benzv1oxv-l-(2-naphthvl)methv)indo1inv1)methylthioacetamidobenzoateA mixture of 3-(2-(5-benzyloxy)indolinyl)methylthioacetamidobenzoate (0.2g,0.42 mmol), prepared in step 6 of Example 17, 2-(bromomethyl)naphthalene (0.1 g, 0.42mmol) and potassium carbonate (0.17 g, 1.26 mmol) in N,N-dimethylfonnamide (2 mL)was stirred at room temperature overnight. Next the reaction was diluted with ethyl acetateand water, extracted with ethyl acetate (3X), dried over magnesium sulfate and70SUBSTITUTE SHEET (RULE 26)?1015202530WO 98/08818CA 02264020 1999-02-24PCT/US97/ 14943concentrated. The crude material was puri?ed on silica gel using 2:] hexanezethyl acetateto yield 0.22 0 of product (85 %. TLC = 0.5 Rf in 2:1 hexanezethyl acetate).Step 2:The titled compound was prepared according to the procedure described in step 3of Example 59.EXAMPLES 74 and 7_5 in Table 6 were prepared according to the procedures described inExample Q.EXAMPLE 762-(2-(-5-Benzvloxy—l—(2—naDhthvl)methvl)indo|invllmethvlthiobenzoic acidStep l : 2-(2—(—5-Ben2vloxv— l —( l , l -dimethvl)ethoxvcarbonvl)indolinvl)methvlmethylsulfonateten-Butyl l-(5-benzyloxy-2-hydroxymethy)lindolinylformate (6.72 g, 19 mmol),prepared in step 2 of Example l7, was dissolved in CH3Cl2 (80 mL, dried over MgSO3before use). The clear yellow solution was cooled in a dry-ice bath. Et_,N (4.0 mL) wasthen added followed by methanesulfonyl chloride (2.0 mL). The reaction mixture wasstirred for 2 h at -40 0C then quenched with H30. It was washed with satuarated Nal-ICO3(300 mL) and the aqueous layer extracted twice with CH3Cl3. The combined CHZCIZ layerswere dried over MgSO4, filtered and evaporated to dryness to give the product (7.30 g,89.1 % yield), which was used for the next reaction directly.Step 2: Methyl 2—(2—(5—Benzvloxv-l-(l.l-dimethvhethoxvcarbonvl)indolinvl)methvlthiobenzoateMesylate (7.2 g, 1.8 mmol), prepared in step 1, was dissolved in DMF (50 mL).The clear light brown solution was degassed by vigorously bubbling with Ar for 30 min.71SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/ 14943Cesium carbonate (13.8 g) was added followed by methyl thiosalicylate (2.4 mL). Thesolution changed to a bright yellow and the suspension was stirred overnight. Methylthiosalicylate (0.15 mL) was added to complete the reaction and the mixture was stirredovernight. The reaction was then quenched by the addition of saturated Nal-ICOJ (400mL). The mixture was extracted with CH2Cl2 (3 x) and the combined CHZCIZ solution wasback—washed with H30 (200 mL). The organic layer was dried over MgSO4, filtered andevaporated to dryness to give the product (9.71 g, 99%).Step 3: Methyl 2-(2-(5—Benzvloxv)indolinvnmethvlthiobenzoateEthyl acetate (75 mL, dried over MgSO4 before use) was charged in a 500 mLround bottom ?ask. HCI gas was bubbled through and the EtOAc/I-ICl solution was cooledin an ice bath. Methyl ester (8.4 g), prepared in step 2, was dissolved in EtOAc (25 mL,dried over MgSO4 before use). This solution was transferred to the I-ICl/EtOAc solutionby syringe. The solution turned to red and was stirred in an ice bath. A white precipitateappeared in l h and the solution was stirred overnight to complete the reaction. The solidwas collected by filtration, washed with dry EtOAc, suspended in saturated NaHCO3 (175mL) and stirred with EtOAc (400 mL). The milky emulsion gradually dissolved and themixture changed to a clear solution. The layers were separated and the aqueous layer wasextracted (2 x) with EtOAC, while the combined EtOAC layers were dried over MgSO4,filtered and evaporated to dryness to give the product (6.06 g, 90 % yield).Step 4: Methyl 2-(2-(5—Benzvloxv-l-(4-benzvl)benzvl)indolinvl)methvlthiobenzoateIn a 50 mL round bottom ?ask, ester (1 g), prepared in step 3, was dissolved inDMF (6 mL). p-Benzylbenzyl bromide was added (l eq) followed by KZCO3 (1 eq). Thereaction mixture was stirred overnight at room temperature. To complete the reactionadditional p-benzylbenzyl bromide (0.5 eq) was added and the reaction was stirred foranother 2 hours. After its completion. the reaction was diluted with H30 and extractedwith EtOAc (2 x). The organic layers were combined and dried over MgSO4. The MgSO4was ?ltered and the solvent was evaporated to give an oily material which was driedovernight on high vacuum to give the product (1.59 g, 109 % yield).72SUBSTITUTE SHEET (RULE 26)?WO 98/08818101520 2530CA 02264020 1999-02-24PCT/US97/14943The ester (1.52 g), prepared in step 4, was dissolved in THF (10 mL) in a 50 mLround bottom ?ask. To it was added NaOH (1 eq, 2N) followed by MeOH (3 mL) and thereaction mixture was stirred overnight. Additional NaOH (0.3 eq) was added to completethe reaction and the mixture was stirred throughout the weekend. Then it was acidi?ed anddiluted with H20 and extracted with EtOAc (2 x). The organic layers were combined anddried over MgSO4. The MgSO4 was filtered and the solvent was evaporated and dried onhigh vacuum to give a cmde reddish solid. This solid was dissolved in EtOAc and hexanewas added to precipitated the product. The resulting solid was ?ltered and the impure ?ltercake was combined with the ?ltrate and evaporated to dryness. This material was treatedwith EtOAc and EtOH. The resulting solid was filtered then suspended in EtOH, withstirring and heating at a low temperature. Then it was allowed to cool to roomtemperature. The suspension was filtered and washed with EtOH to give the titled product(280 mg, 19 % yield).EXAMPLES 77. 78 and 79 in Table 6 were prepared according to the proceduresdescribed in Example 76.EXAMPLE 804-(1—(5-Benzvloxv-2—(bis-2,4—tri?uoromethvl)benzvloxvmethvl)indolinvl)methvlbenzoicaidStep 1: Methyl 1-(5-Benzvloxy/-2-(hvdroxvmethvl)indolinvl)methvlbenzoate2-(5—Benzyloxy)indolinylmethanol (3.21 g, 12.6 mmol), prepared in DMF (20mL). methyl 4-(bromomethyl)benzoate (2.88 g, 14.5 mmol) and potassium carbonate (1.77g, heated to 125 0C before use) were mixed and stirred at room temperature for 2 h. Thereaction was diluted with 100 mL of H30 and extracted three times with EtOAc. Thecombined EtOAc layers were evaporated to dryness to give the cmde product (5.66 g).The crude material was purified on a silica gel column using hexane:ethyl acetate 3:1 to73SUBSTITUTE SHEET (RULE 26)?10152025W0 98/08818CA 02264020 1999-02-24PCT/US97/149432:1. The appropriate fractions were combined. evaporated to dryness and further dried onhigh vacuum to the product (3.00 g, 64%).Step 2: Methyl 4-(l—(5-Benzvloxv-2-(bis-2,4-trifluoromethvI)benzvloxvmethv|)indolinvl)methylbenzoateEster (700 mg), prepared in step l, and bis-(2,4-trifluoromethyl)benzyl bromide(0.35 mL) were dissolved in DMF (5 mL). The resulting clear yellow solution was cooledin an ice bath and then NaH (85 mg) was added in small portions over a period of 5minutes. The suspension was stirred at 0 0C for 4 h. To complete the reaction, another0.35 mL of 2,4—bis(trifluoromethyl)—benzyl bromide was added and the stirring wascontinued for another 3 h 40 min. The reaction was then diluted with H20 and extractedthree times with EtOAc. The combined EtOAc layers were evaporated to give a crudeproduct which was then putifed on a silica gel columnusing hexanezethyl acetate 8: l. Theappropriate fractions were combined and evaporated to dryness to give the product (0.417g, 38.2 % yield).Step 3:The titled compound was prepared according to the prodedure described in step 5of Example 76.EXAMPLES 81 and 82 in Table 6 were prepared according to the procedures described inExample 80.74SUBSTITUTE SHEET (RULE 26)?W0 98/088181015202530CA 02264020 1999-02-24PCT/U S97/ 14943EXAMPLE 835—(2—(1—(2.4—Bis(tri?uoromethvl)benzvl)indolinvl)carboxamido-1,3—benzenedicarboxvlicacidStep 1: 2-(1—(2.4—Bis(trifluoromethvl)benzvl)indolinvl)carboxvlic acid2-Indolinylcarboxylic acid (0.43 g, 2.6 mmol) was dissolved in DMF (5 mL),placed under N3, and cooled to 0° C, the sodium hydride (0.26 g of a 60 % dispersion, 6.5mmol) was added and stirring was continued for 1 hour at this temperature. 2,4-Bis(trifluoromethyl)benzyl bromide (1.22 mL, 6.5 mmol) was next added and the reactionwas wamied to room temperature overnight. The reaction was then diluted with 1/2saturated ammonium chloride/ethyl acetate. the aqueous layer was extracted with ethylacetate (3X). the organic layers were dried over magnesium sulfate and concentrated. Thecrude product was purified via chromatography (hexane:ethyl acetate 9:1) to yield 0.96 gof the ester. The resulting ester (0.87 g, 0.1.41 mmol) was dissolved in THF/ methanol andthen 1N sodium hydroxide (4.21 mL) was added and the resulting mixture was stirred 2hours at RT, workup and purification via Chromatography (721 hexane:ethyl acetate with1% acetic acid) yielded 0.58 g of the product.Step 2:The acid (0.25 g. 0.64 mmol), prepared in step 1, EDCI (0.16 g, 0.83 mmol),DMAP (7 mg, 0.06 mmol) and dimethyl 5—aminoisophthalate (0.16 g, 0.77 mmol) weredissolved in THF (2 mL) and refluxed 16 hours which yielded after aqueous workup 0.33 gof a crude product. The ester (0.29 g, 0.50 mmol) was dissolved in THF/ methanol andthen IN sodium hydroxide (1.5 mL) was added and the resulting mixture was stirred 16hours at RT, workup and purification via Chromatography (l:l hexane:ethyl acetate with1% acetic acid) yielded 0.22 g of the titled compound.EXAMPLE 84N-Methvlsu|fonvl—2—( 1 —(2.4-bis(tri?uoromethyIlbenzvl)indo|invl)carboxamide75SUBSTITUTE sneer (RULE 25)?WO 98/08818l015202530CA 02264020 1999-02-24PCT/US97/I 4943The acid (0.13g, 0.32 mmol), prepared in step 1 of Example 83. EDCI (0.07 g,0.39 mmol), DMAP (4 mg, 0.03 mmol) and methylsulfonanilide (0.04 g, 0.39 mmol) weredissolved in THF (5 mL) and re?uxed 16 hours which yielded after workup (0.16 g),purification via Chromatography (98:2 dichloromethane:methanol) yielded 0.04 g of thetitled compound (29%).EXAMPLE 85N-Phenvlsulfonvl—2-(I -(bis—2.4-tri?uoromethvl)benzvl)indolinvl)carboxamideThe titled compound was prepared according to the prodedure described inExample 84. but using phenylsulfonylamide.EXAMPLE 865-(2-(5—Methoxvbenzvloxy— I —(2,4-bis(trifluoromethvl)benzvl)indolinvl)methvlaminocarboxamido-I .3—benzenedicarboxvlicacidStep l: 2—Trimethvlsilvlethvl 1—(5-benzvloxv—2-hvdroxvmethvl)indolinvlfonnateAn oven—dried 1 L round bottom ?ask, equipped with a stir bar was charged with2-(5-benzyloxy)indolinylmethanol (33.2 g, 130 mmol), prepared in step] of Example 17,2-(trimethylsilyl)ethyl p—nitrophenyl carbonate 36.8 g, 130 mmol), NEt3 (38 ml, 273mmol), and 300 mL of anhydrous DMF. The reaction mixture was stirred at 60 °C for 28hours and at room temperature overnight. The resulting solution was concentrated todryness in vacuo, and l L of CHCl_,V and 200 mL of saturated NaHCO3 solution wereadded. The layers were separated, and the organic phase was dried (Na3SO4), filtered, andconcentrated. The crude material obtained (55.7 g) was puri?ed by columnchromatography on silica gel (eluant: 0-5 % MeOH in dichloromethane) to afford product(33.5 g, 60% yield).76SUBSTITUTE SHEET (RULE 26)?WO 981088181015202530CA 02264020 1999-02-24PCT/US97/14943Step 2: 2—Trimethvlsilvlethvl 1-(5-hvdroxv-2—hydroxvmethvl)indolinvlfomiateAn oven-dried 500 mL Parr pressure ?ask was charged with the alcohol (30 g, 75mmol), prepared in step 1, Pd/C (10 %, 2.2 g), 100 mL of MeOH, and 300 mL of EtOAc.After being shaken overnight in a Parr apparatus under H: atmosphere (50 psi), the reactionmixture was filtered through Florisil. The filtrate was concentrated to dryness on therotary. The crude material obtained (24 g) was purified by column chromatography onsilica gel (eluant: 0-3 % MeOH in dichloromethane) to afford product (209 g, 90% yield).Step 3: 2-Trimethvlsilvlethvl 1-(5-(4-methoxv)benzvloxv-2-hvdroxvmethvhindolinvlformateAn oven-dried 1 L round bottom ?ask. equipped with a stir bar was charged withthe diol (27.1 g, 87.7 mmol), prepared in step 2, 4-methoxybenzyl chloride (Aldrich, 15mL, 110 mmol), KZCO, (200 mesh, 30.4 g, 220 mmol), KI (Aldrich, 18.3 g, 110 mmol),and 800 mL of anhydrous acetonitrile. The reaction mixture was heated at re?ux for 4 h.The solution was allowed to cool to room temperature and water (800 mL) and CHCl, (1.5L) were added. The layers were separated, and the aqueous phase was extracted withCHCI3 (800 mL). The combined extracts were washed with water (200 mL), dried(Na2SO4), ?ltered, and concentrated. The crude material obtained (45 g) was purified bycolumn chromatography on silica gel (eluant: 20-25 % EtOAc in hexane), andrecrystallization from EtOAc/Hexane to afford product (22.2 g, 59% yield).Step 4: 2—Trimethvlsilvlethvl 1—(5—.(4-methoxv)benzvloxv-2-bromomethyl gindolinylfonnateTo a solution of 3.0 g (6.4 mmol) of the alcohol, prepared in step 3, in 30 mL ofdichloromethane was added 2.53 g (7.6 mmol) of carbon tetrabromide and 3.15 g (7.6mmol) of I,3—bis(dipheny|phosphino)propane. The reaction was stirred at roomtemperature for 18 h. The reaction was quenched with saturated aqueous NH4Cl, and theproduct was extracted with dichloromethane. The combined organic extracts were washedwith brine and dried over MgSO4. The crude product was puri?ed by ?ashchromatography using hexanezethyl acetate 3:2 to afford 1.51 g of the product.77SUBSTITUTE SHEET (RULE 25)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943(Step 5: 2—Trimethvlsilvlethvl 1-(5—(4—methoxv)benzvloxv-2-azidomethyl gindolinylfonnateTo a solution of 1.4 g (2.6 mmol) of the bromide, prepared in step 4, in 15 mL ofdimethylfonnamide was added 0.51 g (7.9 mmol) of sodium azide. The reaction washeated to 75 °C, and was stirred for 18 h. The reaction was quenched with water, and theproduct was extracted with ethyl acetate. The combined organic layers were washed withwater, brine and dried over MgSO4. The crude product was puri?ed by ?ashchromatography using hexanezethyl acetate 4:] to afford 1.08 g of the product.Step 6: 2-Trimethvlsilvlethvl 1-(5-(4-methoxv)benzvloxy-2-aminomethyl zindolinylformateTo a solution of 0.88 g (1.9 mmol) of the azide, prepared in step 5, in 20 mL ofethanol was added 90 mg (10%/wt) of Pd/CaCO3. The mixture was placed underatmospheric hydrogen. and was stirred for 18 h. The reaction was then ?ltered through apad of celite and the organic phase was concentrated. The crude product was purified by?ash chromatography using 10% MeOI-I/CH3Cl3 to afford 0.717 g of the product.Step 7: Methyl 5-(2—(5~Methoxvbenzvloxy-1-(2-trimethvlsilvloxv)ethoxvcarbony)lindolinvl) methvlaminocarboxamido-1.3-benzenedicarboxylateTo a solution of 0.164 g (0.6 mmol) of triphosgene in 5 mL of dichloromethanewas added a solution of 0.31 g (1.5 mmol) of dimethyI-5-aminoisophthalate and 0.39 g(3.0 mmol) of diisopropylethylamine in 20 mL of dichloromethane over a 30 minute periodvia a syringe pump. The reaction was stirred for 1 h at room temperature following theaddition. and then a solution of 0.64 g (1.5 mmol) of the amino, prepared in step 6, in 5 ml.of dichloromethane was added in one portion. The reaction was stirred for 2 h, and thenquenched with water. The product was extracted with ethyl acetate, and the combinedorganic layers were washed with water, saturated aqueous NaHCO,, brine and dried overMgSO4. The crude product was purified by flash chromatography using 10%MeOI-1/CHZCIZ to afford 0.78 g of the product.78SUBSTITUTE SHEET (RULE 26)?101520 "2530W0 98/08818CA 02264020 1999-02-24PC'I‘/US97/ 14943Step 8: Methvl 5-(2-(5—Methoxvbenzvloxv)indolinvl)methvlaminocarboxamido-1.3-benzenedicarboxylateTo a solution of 0.485 g (0.7 mmol) of the ester, prepared in step 7, in 20 mL ofacetonitrile was added 2.2 mL (2.2 mmol) ofa 1.0 M tetrabutylammonium ?uoridesolution in THF. The reaction was stirred at room temperature for 18 h. The reaction wasquenched with brine, and the product was extracted with ethyl acetate. The combinedorganic extracts were washed with saturated aqueous N1-14Cl, brine and dried over M gSO4.The crude product was puri?ed by ?ash chromatography using 5% MeOH/CHZCIZ toafford 0.342 g of the product.Step 9: Methvl 5—(2—(5-Methoxvbenzvloxv—1—(bis-2.4-tri?uoromethvl)benzvI)indolinvl)methvlaminocarboxamido-1.3-benzenedicarboxvlateTo a solution of 0.15 g (0.3 mmol) of the indoline diester, prepared in step 8, in 5mL of dimethylformamide was added 0.097 g (0.3 mmol) of 2,4-bis(tri?uoromethyl)benzyl bromide and 0.12 g (0.9 mmol) of potassium carbonate. Thereaction was stirred at room temperature for 18 h. The reaction was quenched with water,and the product was extracted with ethyl acetate. The combined organic extracts werewashed with water, brine and dried over MgSO4. The crude product was purified by ?ashchromatography using hexane:ethyl acetate 1:1 to afford 0.066 g of the product.Step 10:To a solution of 0.063 g (0.1 mmol) of the diester, prepared in step 9, in 5 mL oftetrahydrofuran was added 0.8 mL (0.8 mmol) of a 1.0 N NaOH solution and 0.5 mL ofmethanol. The reaction was stirred at room temperature for 18 h. The organic solventswere evaporated, and the resulting solid was suspended in water and acidified to pH 3 with10% HCl. The product was extracted with ethyl acetate, and the combined organic extractswere washed with water, brine and dried over MgSO4. The crude product was purified by?ash chromatography using 5% MeO1-I/CHZCIZ to afford 0.049 g of the titled compound.79SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943EXAMPLE 87 was prepared according to the prodedure described in Example 86, butusing 4—(3,5-bis(tri?uoromethyl)phenoxymethyl)benzyl bromide.INTERMEDIATE 1Methyl 4—methoxv~3-thioacetamidobenzoateStep 1: Bis(methvl 4-methoxv-3-dithioacetamidobenzoate)A 2-1.. oven-dried round bottom ?ask ?tted with a magnetic stirring bar wascharged with Dithioacetic acid (10.2-15.5 g, 56-85 mmol) and anhydrous CHZCIE (50 mL).Oxalyl chloride (2.1 mol equiv.) was added dropwise over 10 min. The reaction mixturewas stirred at room temperature for 4-5 h. Methyl 4-methoxy—3-amidobenzoate (2.1 molequiv.) in anhydrous CI-12Cl3 (300-500 mL) and DMAP (0.1 mol equiv.) were added atroom temperature. NB} (4.2 mol equiv.) was added dropwise over 30 min. After stirringovernight at room temperature the reaction mixture was washed with 1 N HCl solution (2 x300 mL), dried over Na2SO4 and ?ltered. The solvent was removed in vacuo. Purificationof the residue by column chromatography on silica gel using hexanezethyl acetate = 5:1afford desired product in 56% yield.Step 2:A 1-1.. round bottom ?ask fitted with a magnetic stirring bar was charged withdisul?de, prepared in step 1, (l5.7—26.3 g, 36.6-57.5 mmol) and PPh3 (1.1 mol equiv.).The reactants were suspended in dioxane/1-I30 (4/1, 375-500 mL) and concentrated I-{Clsolution (5 drops) was added. The reaction mixture was heated at 40 °C until all disulfidewas consumed. Solvents were removed in vacuo. The residue was purified immediately bycolumn chromatography on silica gel using hexane : ethyl acetate 2:1 to afford the titledproduct in 89% yield.80SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/1 4943INTERMEDIATE 2Methyl 5—thioacetamido-l ,3-benzenedicarboxvlateThe titled compound was synthesized according to the procedures described inIntennediate 1 using 5-amino—1.3-benzenedicarboxylate.UINTERMEDIATE 3Methvl 2—(3—amino-4-methoxvDhenvl)~2-methoxvacetateStep 1: Methyl 2-(3—nitro-4—methoxvnhenvI)acetateAn oven-dried 2—L, 3-neck round bottom ?ask, equipped with a mechanical stirmotor, a low-temperature thermometer and an equalizing dropping funnel, was chargedwith acetic anhydride (631 mL) and subsequently cooled to -78 °C. Fuming nitric acid(Baker, 90%. 27 mL) was added dropwise via the dropping funnel protected with a dryingtube ?lled with CaCl2. After addition was completed, the reaction temperature wasallowed to wami to 20 “C over 1 h. The reaction mixture was cooled to -78°C again andadded 4-methoxyphenylacetic acid (50 g, 0.28 mol) dropwise via the dropping funnel.After stirring at -50 °C for l h., the reaction mixture was allowed to wann to -30 °C over 20min. and then cooled to -50 “C again. The reaction mixture was quenched with H30 (500mL) at -50 “C and warmed up to room temperature and stirred for 0.5 h. The reactionmixture was partitioned between CHZCIZ (500 mL) and H30. ‘The aqueous layer wasextracted with CHIC]: (3 x 500 mL). The combined CHZCIZ extracts were concentrated invacuo to give a yellow oil. This was added slowly to a 2 M solution of NaOH (2 L) cooledat 0 °C and stirred at room temperature overnight. The reaction mixture was partitionedbetween CH3Cl3 (500 mL) and H30. The aqueous layer was extracted with CHZCIZ (3 x500 mL). The combined CH3Cl3 extracts were stirred with 2 M NaOH solution (1 L) for lh. The layers were separated and the organic layer was washed with H10 (500 mL), brine(500 mL). dried over Na3SO4 and filtered. The solvents were removed in vacuo to affordcrude product as a light yellow solid (56 g). Puri?cation by recrystallization from MeOH(600 mL) gave product. Yield 48 g (77%).81SUBSTITUTE SHEET (RULE 25)?10l5202530W0 98/08818CA 02264020 1999-02-24PCTIU S97/ 14943Step 2: Methyl 2—(3-nitro—4-methoxvphenvl)-2—hvdrox\/acetateA 25-mL oven-dried round bottom ?ask ?tted with a magnetic stirring bar wascharged with ester (2.3 g, 10 mmol). prepared in step I, and anhydrous THF (100 mL).The reaction mixture was cooled to -78 “C and a solution of NaN(SiMe3)3 (1.0 M in THF,12 mL, 12 mmol) was added dropwise over 10 min. After stirring at -78 °C for 30 min.,the deep purple solution was added dropwise a solution of racemic camphorsulfonyloxaziridine (3.4 g, 15 mmol), prepared by mixing the commercially available (IS)-(+)-(10-camphorsulfonyl)oxaziridine (1.7 g) and (IR)—(-)-( l0—camphorsulfonyl)oxaziridine(1-.7 g) in 50 mL THF. After stirring at -78 °C for 30 min., the reaction mixture wasquenched with sat. NH4Cl solution (45 mL) at -78 "C and then allowed to warm to roomtemperature. The reaction mixture was partitioned between ether (250 mL) and H30 (50mL). The aqueous layer was extracted with ether(3 x 250 mL). The combined etherextracts were washed with brine (250 mL), dried over Na3SO4 and ?ltered. The solventswere removed in vacuo. Puri?cation by column chromatography on silica gel (eluant: 50%AcOEt in hexane) afforded desired product. Yield 2.2 g (88%).Step 3: Methyl 2—(3—nitro-4-methoxvphenvl)-2-methoxvacetateA 10-mL oven-dried round bottom ?ask fitted with a magnetic stirring bar wascharged with alcohol (0.30 g, 1.24 mmol), prepared in step 2, Ag2O (0.68 g, 3.0 mmol)and toluene (3 mL). To this was added CH_,I (0.36 g, 5.75 mmol) dropwise. The reaction?ask was capped tightly and placed into a sonication chamber. The reaction mixture wassonicated for 18 h while stirring at room temperature. The reaction mixture was filteredthrough Celite and concentrated in vacuo to dryness. The residue was puri?ed by columnchromatography on silica gel (eluant: 30% AcOEt in hexane) to afford desired product.Yield 0.26 g (82%).Step 4:A 100-ml. oven-dried round bottom ?ask ?tted with a magnetic stirring bar and athree way adapter, connecting to a hydrogen balloon and a water aspirator was chargedwith nitro compound (0.7 g, 2.6 mmol), 5% Pd on Carbon (10% by weight) and MeOH (20mL). The reaction ?ask was placed under vacuum via the water aspirator and subsequently82SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/14943filled with Hz. This was repeated three times. The reaction mixture was stirred for 18 hoursunder positive I-I2 pressure until all starting material was reacted. The reaction mixture was?ltered through Celite and concentrated in vacuo to dryness. The residue was purified bycolumn chromatography on silica gel using 10% ethyl acetate in dichloromethane to affordthe titled compound (0.57 g, 97%)INTEMEDIATE 4Methyl 2—(3-amino—4-methoxvphenvl)—2-tert-butvldimethvlsilvloxvacetateA 25—mL oven-dried round bottom ?ask ?tted with a magnetic stirring bar wascharged with alcohol (0.30 g, 1.24 mmol), prepared in step 2 of Intermediate 3 andanhydrous CHZCIZ (10 mL). The reaction mixture was cooled to 0 "C and added 2,6-lutidine (dried over NaOH pellet, 0.36 mL, 3.1 1 mmol) followed by addition of'BuMe3SiOTf (0.43 mL, 1.87 mmol) dropwise. After stirring at 0 “C for 30 min., thereaction mixture was partitioned between CH3Cl2 (20 mL) and H20 (15 mL). The aqueouslayer was extracted with CHZCIZ (3 x 20 mL). The combined CH2Cl2 extracts were washedwith brine (20 mL), dried over Na3SO4 and ?ltered. The solvents were removed in vacuo.Puri?cation by column chromatography on silica gel (eluant: 30% AcOEt in hexane)afforded desired product. Yield 0.42 g (95%).Step 2:The titled compound was prepared from nitro compound of step 1 according to theprocedure described in step 4 of Intennediate 3.INTERMEDIATE 5Methyl 2-(3-amino-4-methoxvDhenvl)acetateThe titled compound was prepared from nitro compound, prepared in step 1 ofIntermediate 3, according to the procedure described in step 4 of Intermediate 3.83SUBSTITUTE SHEET (RULE 26)?10152025WO 98108818CA 02264020 1999-02-24PCTIUS97/14943INTERMEDIATE 6Methyl 2—(3—amino-4-methoxvvhenvl)—2—methvlacetateStep 1: Methyl 2-(3-nitro—4—methoxvDhenvl)-2-methvlacetateA 25-mL oven-dried round bottom ?ask ?tted with a magnetic stirring bar wascharged with redistilled diisopropylamine (0.84 mL, 6.0 mmol) and anhydrous THF (10mL) and cooled to 0 °C. A solution of n—BuLi (2.5 M in hexane, 2.4 mL, 6.0 mmol) wasadded dropwise over 5 min. After stirring at 0 °C for 15 min., the reaction temperature wasallowed to cool to -78 °C and added a solution of easter (1.13 g, 5.0 mmol), prepared instep 1 of Intemiediate 3, in 10 mL THF dropwise. After stirring at -78 °C for 45 min.,dimethylsulfate (1.60 g, 12.5 mmol) was added dropwise and the reaction mixture wasallowed to wami to room temperature and stirred overnight. The reaction mixture waspartitioned between CH2Cl2 (50 mL) and H30 (50 mL). The aqueous layer was extractedwith CH2Cl2 (3 x 50 mL). The combined CHZCIZ extracts were washed with brine (50 mL),dried over Na3SO4 and ?ltered. The solvents were removed in vacuo. Purification bycolumn chromatography on silica gel (eluant: 30% AcOEt in hexane) afforded 0.7 g ofproduct (58%).Step 2:The titled compound was prepared. from nitro compound, prepared in step 1,according tothe procedure described in step 4 of Intennediate 3.84SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/US97/ 14943INTERMEDIATE 7Methyl 2—(3—amino-4-methoxvnhenvl)-2—allvlacetateStep 1: Methyl 2-(3—nitro-4-methoxvphenvl)—2-allvlacetateThis compound was synthesized form ester, prepared in step Iof Intennediate 3,according to the procedure described in step 1 of Intermediate 6, but using allyl bromide.Step 2:A 25—mI_. oven—dried round bottom ?ask ?tted with a magnetic stirring bar wascharged with ester (0.30 g, l.l3 mmol), prepared in step I. SnCl3 21-I10 (1.28 g, 5.66mmol) and EtOH (5 mL). The reaction mixture was heated at 70 “C for 30 min. Thereaction mixture was cooled to room temperature and poured onto ice/water (20 mL) andbasi?ed with saturated Na3CO_, solution to pH = 8. AcOEt (50 mL) was added. Theresulting emulsion was filtered through Celite. The ?ltrate was partitioned between AcOEt(20 mL) and H20 (15 mL). The aqueous layer was extracted with AcOEt (3 x 50 mL).The combined AcOEt extracts were washed with brine (50 mL), dried over Na3SO4 and?ltered. The solvents were removed in vacuo. Puri?cation of the residue by columnchromatography on silica gel (eluant: 10% AcOEt in CHZCIZ) afforded the titledcompound. Yield 0.16 g (60%).INTERMEDIATE 82,4-Bis( l,1—dimethypropylyphenoxyacetic acidThe 2,4-bis(l,1-dimethy)propylphenol (12 g, 51.2 mmol) in dimethylformamide(100 mL) was cooled to -30° C, treated with solid potassium bis(trimethylsilyl)amide(l2.3g, 61.5 mmol), stirred for 30 minutes and then methyl bromoacetate (5.7 mL, 61.5mmol) was added, the reaction was stirred I hour at this temperature and ?ve hours afterremoval of the cooling bath, workup yielded (l6.6g, = l0O%) a yellow oil. The oil wasdissolved in THF/methanol and treated with 1N sodium hydroxide (155 mL) and stirred for85SUBSTITUTE SHEET (RULE 26)?1015202530WO 98/08818CA 02264020 1999-02-24PCTIUS97/1494348 hours. The reaction was concentrated, diluted with water. acidified to pH 4 withconcentrated HCI, extracted with ethyl acetate (4X), dried over magnesium sulfate andconcentrated. Crystalization from ethyl acetate and hexane yielded 12.85 g of the titledcompound. (86%).INTERMEDIATE 94-Benzylphenoxyacetic acidThe titled compound was prepared from 4-benzylphenol according to theprocedure described in of Intennediate 8.INTERMEDIATE I02—NaQhthoxyacetic acidThe titled compound was prepared from 2-naphthol according to the proceduredescribed in of Intermediate 8.INTERMEDIATE I I;5—Bis(tri?uoromethvl)Dhenoxvacetic acidThe titled compound was prepared from 3,5-bis(tri?uoromethyl)phenol accordingto the procedure described in of Intermediate 8.INTERMEDIATE 12Methvl 5—amino-3-(N.N-dimethvl)carbamoylbenzoateStep l: Methyl 5—nitro-3-(N,N—dimethvl)carbamovlbenzoate86SUBSTITUTE SHEET (RULE 26)?1015202530W0 98/08818CA 02264020 1999-02-24PCT/U S97/ 14943A 100—mL oven-dried round bottom ?ask fitted with a magnetic stirring bar wascharged with 5-nitro-3-methoxycarbonylbenzoic acid (3.15 g, 10 mmol), DMF (I drop),anhydrous CH2Cl3 (70 mL), and oxalyl chloride (3.7 mL, 42.3 mmol). The reactionmixture was stirred at room temperature for 2 h. The solvent was removed in vacuo toafford acid chloride as a white solid. This was used immediately in the next step withoutfurther purification.An oven—dried round bottom ?ask fitted with a magnetic stirring bar was chargedwith above prepared acid chloride (14 mmol), anhydrous CHIC]: (50 mL), anddimethylamine hydrochloride (70 mmol). NEt3 (2 mL, 144 mmol) was added dropwise.After stirring at room temperature for 30-60 min excess NEt3 (1 mL, 72 mmol) was addedand stirring was continued. After 30-60 min the solution was washed with saturatedNa2CO3 solution (2 x 20 mL), dried over Na3SO4 and ?ltered. The solvent was removed invacuo to afford 3.3 g of product. This was used in the next step without furtherpurification.Step 2:The titled compound was prepared from nitro compound, prepared in step 1,according to the procedure described in step 4 of Intennediate 3.INTERMEDIATE I 3Methyl 5—amino—3-acetylbenzoateStep 1: Methyl 5-nitro-3-acetvlbenzoateA 250-mL oven—dried round bottom ?ask ?tted with a magnetic stirring bar wascharged with di-tert—butyl malonate (2.16 g, 10 mmol), anhydrous toluene (50 mL), andNaH (60% suspension in mineral oil, 0.88 g, 22 mmol). The reaction mixture was heated at80 °C for l h. A solution of methyl 5—nitro-3-chloroformylbenzoate (l0 mmol), preparedin step 1 of Intennediate 12, in anhydrous toluene (20 mL) was added and heating wascontinued for 2 h. The reaction mixture was cooled to room temperature and p-toluenesulfonic acid (0.21 g, 1.2 mmol) was added. The resulting mixture was filtered and87SUBSTITUTE SHEET (RULE 26)?1015202530WO 98/08818CA 02264020 1999-02-24PCTIU S97/ 14943the oily residue was washed with toluene until a white solid was left. The ?ltrates werecombined and the solvent was removed in vacuo. The resulting oil was dissolved inanhydrous toluene (50 mL) and p—toluenesulfonic acid (0.3 g, 1.74 mmol) was added. Afterheating to re?ux for 18 h the reaction mixture was allowed to cool to room temperature,washed with saturated Na2CO3 solution (2 x 25 mL), dried over Na3SO4 and ?ltered. Thesolvent was removed in vacuo. The crude material was puri?ed by columnchromatography on silica gel (eluant: CHZCIZ) to afford product. Yield 1.06 g (50%).Step 2:The titled compound was prepared from nitro compound, prepared in step 1,according to the procedure described in step 4 of Intermediate 3.INTERMEDIATE 14Methvl 5—amino-3-(l -tert-butvldimethylsilvloxwethvlbenzoateStep 1: Methyl 5—nitro-3-(l—hvdroxv)ethvlbenzoateAn oven-dried round bottom ?ask fitted with a magnetic stirring bar was chargedwith compound methyl 5-nitro—3-acetylbenzoate (05 g), prepared in step 1 of Intermediate13, BH3_TI-IF (1 M solution in THF, 5 mol equiv.), and anhydrous THF. After stirring atroom temperature for 24 h, H30 (20 mL) was added and the solution was concentrated invacuo. The residue was taken in H30 (20 mL) and extracted with CHCI3 (3 x 100 mL).The combined CI-{C1, extracts were washed with saturated Na3CO3 solution (20 mL), driedover Na3SO4 and ?ltered. The solvent was removed in vacuo to afford product. This wasused in the next step without further puri?cation.Step 2: Methyl 5nitro-3-(1-tert-butvldimethvlsilvloxv)ethvlbenzoateAn oven-dried round bottom ?ask fitted with a magnetic stirring bar was chargedwith alcohol (0.5g, 5 mmol), prepared in step 1, tert-BuMe3SiCl (1.3 mol equiv.),imidazole (2.15 mol equiv.), and anhydrous THF. After stirring at room temperature for88SUBSTITUTE SHEET (RULE 26)?W0 98/088181015202530CA 02264020 1999-02-24PCT/U S97/ 1494328 hours the solvent was removed in vacuo. The residue was taken in H30 (50 mL) andextracted with CHCl3 (2 x 100 mL). The combined CHCl3 extracts were washed with H30(50 mL), dried over Na3SO4 and filtered. The solvent was removed in vacuo. The cmdematerial was puri?ed on silica gel using 25%-50% dicloromethane in hexane to afford theproduct (0.69 g, 91%).Step 3:The titled compound was prepared from nitro compound, prepared in step 2,according to the procedure described in step 4 of Intermediate 3.INTERMEDIATE 15Methyl 4-methoxy-3-(2-thioethyl)aminobenzoateStep 1: Bis(2—bromoethyl ldisul?deThe dithioethanol (0.79 mL, 6.48 mmol), carbon tetrabromide (4.3 g, 13.0 mmol)and 1,3 bis(diphenylphosphino)propane (5.34 g, 13.0 mmol) were weighed into a flask and?ushed with nitrogen and then taken up in CI-I2Cl2 (15 mL) and stirred for 16 hours,workup consisted of pouring into 1/2 saturated ammonium chloride and extracted withCH2Cl3 (3X) dry magnesium sulfate and concentrated to yield (9.0 g) of a cmde productthat was chromatographed (Hexane:Ethyl acetate9: l) to yield 1.49 g of product.Step 2: Bis-(methyl 4-methoxv-3—(2—dithioethvl)aminobenzoateBromide (0.39 mg, 1.387 mmol), prepared in step 1, and methyl 3—amino-4-methoxy benzoate (1.00 g, 5.51 mmol) were added into a flask, flush with nitrogen andtake up in DMF (5 mL) and then heat to 60 ° C for 24 hours at which time the reaction wasdiluted with ethyl acetate and quenched into water, extracted with ethyl acetate (3X), thecombined organic layers were washed with water (3X), dried and concentrated to yield1.27 g of a product that was puri?ed by chromatography (hexanezethyl acetate 5:1 to 3: 1)to yield 0.15 g of the desired product.89SUBSTITUTE SHEET (RULE 26)?1015202530WO 98/08818CA 02264020 1999-02-24PCT/US97/14943Step 3:The disul?de (0.15 g, 0.24 mmol), prepared in step 2, and the triphenylphoshpine(0.14 g, 0.53 mmol) were taken up in THF (3 mL). H30 (0.3 mL) and two drops of cone.HCl were added and the resulting mixture was stirred at 40 " C for 2 hours, the reactionwas diluted with water and ethyl acetate, extracted with ethyl acetate (3 X) and dried overmagnesium sulfate to yield 0.27 g of a crude product that was puri?ed by chromatography(hexanezethyl acetate 9:1 to 6:1) to yield 0.1 l g of the titled compound.Example 88Activity Assays(a) Vesicle Assay1-palmitoyl-2—["‘C] arachidonyl phosphotidylcholine (58 mCi/mmol) (?nalconcentration 6 uM) and l,2—dioleyolglycerol (?nal concentration 3 pM) were mixed anddried under a stream of nitrogen. To the lipids was added 50 mM Hepes pH 7.5 (2x ?nalconcentration of lipids) and the suspension was sonicated for 3 min. at 4°C. To thesuspension was added 50 mM Hepes pH 7.5, 300 mM NaCl, 2 mM DTT, 2 mM CaCl3 and2 mg/ml bovine semm albumin (BSA) (Sigma A7511) (l.2x ?nal concentration of lipids).A typical assay consisted of the lipid mixture (85 pl) to which was added consecutively, theinhibitor (5 pl in DMSO) and CPLA3, l0 rig for an automated system or 1 ng for a manualassay, in l0l.ll of the BSA buffer. This assay was conducted by either the manual assay orautomated assay protocol described below.(b) Soluble Substrate Assay (LysoPC)l—["C]-palmitoyl—2-hydroxyphosphotidyl-choline (57 mCi/mmol) (?nalconcentration 4.4 uM) was dried under a stream of nitrogen. The lipid was resuspended byvortexing 80 mM Hepes pH 7.5, 1 mM EDTA (1.2 x ?nal concentration). A typical assayconsisted of lipid suspension (85 pl) to which was added consecutively the inhibitor (5pl inDMSO) and CPLA3, 200 ng in 80 mM Hepes pH 7.5, 2 mM DTT and l M EDTA. Thisassay was conducted by either the manual assay or automated assay protocol describedbelow.90SUBSTITUTE SHEET (RULE 26)?101520 x2530WO 98/08818CA 02264020 1999-02-24PCT/US97/ 14943(c) Automated AssayThe lipid suspension and inhibitor were pre—incubated for 7 min. at 37°C. Enzymewas added and the incubation was continued for a further 30 mins. The reaction was thenquenched by the addition of decane: isopropanol: trifluoroacetic acid (192:8:1 w/v, 150 pl).A portion of the quench layer (50 pl) was passed through a Rainin Spheric—5 silica column(5p., 30 x 2.1 mm) eluting with heptane:methanol:TFA (97:3:0.l v/v). The level of ["‘C]-arachidonic acid was analyzed by an in—line Radiomatic Flo—One/Beta counter (Packard).(d) Manual AssayThe lipid, inhibitor and enzyme mixture were incubated at 37°C for 30 min. Thereaction was quenched by the addition of heptane:isopropanol:O.5M sulfuric acid ( lO5:20:lv/v, 200 pl). Half of the quench layer was applied to a dispoable silica gel column(Whatman SIL, 1 ml) in a vacuum manifold positioned over a scintillation vial. Free [”C]-arachidonic acid was eluted by the addition of ethyl ether (1 ml). The level of radioactivitywas measured by liquid scintillation counter.(e) PMN AssayPMNS were isolated using Ficoll—Hypaque according to the manufacturersdirections. Red blood cells contaminating the PMNS were removed by hypotonic lysis, andthe PMN pellet was washed once, and resuspended in Hanks buffered saline at aconcentration of 2 x 10'‘ cells/ml. The cells were preincubated with inhibitors for 15 min at37°C and then stimulated with 2 uM A23] 87. When monitoring LTB4 production as ameasure of CPLA3 inhibition. the reaction was quenched with an equal volume of ice coldphosphate buffered saline. Cells were removed by centrifugation, and the LTB4 present inthe cell supernatant was measured using the LTB, scintillation proximity assay provided byAmersham according to the manufacturers directions. In the assays reported in the Tablesabove, LTB4 was measured. When monitoring arachidonic acid production, the reactionwas quenched with methanol containing D8-arachidonic acid as an internal reference. Thelipids were extracted by the method of Bligh et al. ((1959) Can. J. Biochem. Physiol., 37,91 1-91 7), and the fatty acid was converted to the pentafluorobenzyl ester and analyzed byGC-MS in a manner similar to that reported by Ramesha and Taylor ((1991) Anal.Biochem. 192, 173-180).91SUBSTITUTE SHEET (RULE 26)?101520WO 98/08818CA 02264020 1999-02-24PCT /U S97! 14943(e) Coumarine (PGE, Production) AssavRBL—2H3 cells were routinely cultured as 37°C in a 5% CO3 atmosphere inminimal essential medium containing nonessential amino acids and 12% fetal calf serum.The day before the experiment, cells were seeded into spinner ?asks at 3 x 105 cells/ml and100 ng/ml DNP speci?c-IgE was added. After 20 hrs, the cells were harvested bycentrifugation and washed once in serum—free minimal essential media, and resuspended to2 x 106 cells/ml in serum free media. The cells were then preincubated with either inhibitorin DMSO (1% v/v) or DMSO (1% v/v) for 15 min at 37°C followed by stimulation withDNP-BSA (300 ng/ml). After 6 min, the cells were removed by centrifugation, and thesupernatant was assayed for PGD3 content in accordance with known methods.Example 89Rat Carrageenan-Induced Footpad Edema TestEach compound was suspended in O.3ml absolute ethanol, 0.] ml Tween-80 and2.0 ml Dulbecco’s PBS (without calcium or magnesium). To this mixture. O.lml INNaOH was added. After solution was complete, additional amounts of PBS were added toadjust the concentration to 1 mg/ml. All comounds remained in solution. Compoundswere administered i.v. in a volumne of 5 ml/kg to male Sprague Dawley rats at the sametime that edema was induced by injection of 0.05ml of 1% Type IV carrageenan into thehind footpad. Footpad volume was measured before dosing with compound and 3 hoursafter dosing with carageenan.All patent and literature references cited herein are incorporated as if fully set forhtherein.92suasrrrure sneer (RULE 25)

Claims (16)

What is claimed is:
1 . A compound having a chemical formula selected from the group consisting of:

and or a pharmaceutically acceptable salt thereof, wherein:
A is independent of any other group and is selected from the group consisting of-CH2- and -CH2-CH2-;
B is independent of any other group and is selected from the group consisting of -(CH2)n-,-(CH2O)n-,-(CH2S)n-,-(OCH2)n-,-(SCH2)n-,-(CH=CH)n-,-(C ~ C)n-,-CON(R6)-, -N(R6)CO-, -O-, -S- and -N(R6)-;
R1 is independent of any other R group and is selected from the group consisting of -X-R6, -H. -OH, halogen, -CN, -NO2 C1-C5 alkyl, alkenyl, alkinyl, aryl and substituted aryl;
R2 is independent of any other R group and is selected from the group consisting of -H, -COOH, -COR5, -CONR5R6, -(CH2)n-W-(CH2)m-Z-R5, -(CH1)n-W-R5, -Z-R5, C1-C10 alkyl, alkenyl and substituted aryl;
R3 is independent of any other R group and is selected from the group consisting of -H, -COOH, -COR5, -CONR5R6, -(CH2)n-W-(CH2),n-Z-R5, -(CH2)n-W-R5, -Z-R5, C1-C10 alkyl, alkenyl and substituted aryl;
R4 is independent of any other R group and is selected from the group consisting of -H, -OH, -OR6, -SR6, -CN2 -COR6, -NHR6, -COOH, -CONR6R7, -NO2 -CONHSO2R8, C1-C5 alkyl, alkenyl and substituted aryl;
R5 is independent of any other R group and is selected from the group consisting of -H, -OH, -O(CH2)nR6, -SR6, -CN, -COR6, -NHR6, -COOH, -NO2, -COOH, -CONR6R7, -CONHSO2R8, C1-C5 alkyl, alkenyl, alkinyh aryl, substituted aryh -CF3, -CF2CF3 and R6 is independent of any other R group and is selected from the group consisting of -H, C1-C5 alkyl, alkenyl, alkinyl, aryl and substituted aryl;
R7 is independent of any other R group and is selected from the group consisting of -H, C1-C5 alkyl, alkenyl, alkinyl, aryl and substituted aryl;
R8 is independent of any other R group and is selected from the group consisting of C1-C3 alkyl, aryl and substituted aryl;
R9 is independent of any other R group and is selected from the group consisting of -H, -OH. a halogen, -CN, -OR6, -COOH, -CONR6R7, tetrazole, -CONHSO2R8, -COR6, -(CH2)nCH(OH)R6 and -(CH2)nCHR6R5;
R10 is independent of any other R group and is selected from the group consisting of -H, -OH, a halogen, -CN, -OR6, -COOH, -CONR6R7, tetrazole, -CONHSO2R8, -COR6,-(CH2)nCH(OH)R6 and -(CH2)nCHR6R5;
W is, independently each time used including within the same compound, selected from the group consisting of -O-, -S-, -CH2-, -CH=CH-, -C ~ C- and -N(R6)-;
X is independent of any other group and is, independently each time used including within the same compound, selected from the group consisting of -O-, -S- and -N(R6)-;
Z is independent of any other group and is, independently each time used including within the same compound, selected from the group consisting of -CH,-, -O-, -S-, -N(R6)-, -CO-, -CON(R6)- and -N(R6)CO-;
m is, independently each time used including within the same compound, an integer from 0 to 4; and n is independent of m and is, independently each time used including within the same compound, an integer from 0 to 4.
2. The compound of claim 1 having phospholipase enzyme inhibiting activity.
3. The compound of claim 1 wherein said compound has the following chemical formula:

4. The compound of claim 1 wherein said compound has the following chemical formula:

5. The compound of claim 1 wherein compound has the following chemical formula:
6. The compound of claim 1 wherein A is -CH2- and R2 is -(CH2)n-W-(CH2)m-ZR5.
7. The compound of claim 6 wherein n is 1, m is 1, W is -S- and Z is -CO-.
8. The compound of claim 7 wherein R5 is -NHR6.
9. The compound of claim 8 wherein R6 is a substituted aryl group.
10. The compound of claim 9 wherein said aryl group is substituted with one or more substituents independently selected from the group consisting of a halogen, -CF3, -CF2CF3, -(CH2)pCOOH, -(CH2)pCH3, -O(CH2)pCH3, -(CH2)pOH, -(CH2)pS(C6H6), -(CH2)pCONH2 and -CHR11COOH, wherein R11 is selected froup the group consisting of alkyl, alkenyl, alkynyl, -(CH2)pOH, and -O(CH2)pCH3, and wherein p is an integer from 0 to 4.
11. The compound of claim 6 wherein R1 is selected from the group consisting of -ll and -OCH2(C6H6).
12. The compound of claim 6 wherein R3 is -COR5, R5 is -OCH2R6 and R6 is a substituted aryl group.
13. The compound of claim 12 wherein said aryl group is substituted with one or more substituents selected from the group consisting of -CF3, -CF2CF3 and -C(CH3),CH2CH3.
14. A method of inhibiting the phospholipase enzyme activity of an enzyme, comprising administering to a mammalian subject a therapeutically effective amount of a compound of claim 1.
15. A method of treating an inflammatory condition, comprising administering to a mammalian subject a therapeutically effective amount of a compound of claim 1.
16. A pharmaceutical composition comprising a compound of claim I and a pharmaceutically acceptable carrier.
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