WO2000037022A2 - Combination therapy for the treatment of sepsis - Google Patents

Combination therapy for the treatment of sepsis Download PDF

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Publication number
WO2000037022A2
WO2000037022A2 PCT/US1999/030433 US9930433W WO0037022A2 WO 2000037022 A2 WO2000037022 A2 WO 2000037022A2 US 9930433 W US9930433 W US 9930433W WO 0037022 A2 WO0037022 A2 WO 0037022A2
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Prior art keywords
methyl
alkyl
oxyacetic acid
acid
phenyl
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PCT/US1999/030433
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French (fr)
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WO2000037022A3 (en
Inventor
Ronald Steven Maciak
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Eli Lilly And Company
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Priority to JP2000589136A priority Critical patent/JP2002542148A/en
Priority to AU19408/00A priority patent/AU1940800A/en
Priority to CA002358492A priority patent/CA2358492A1/en
Priority to EP99963109A priority patent/EP1214041A2/en
Publication of WO2000037022A2 publication Critical patent/WO2000037022A2/en
Publication of WO2000037022A3 publication Critical patent/WO2000037022A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4866—Protein C (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to the field of medicine and specifically to the treatment of sepsis.
  • Reaction to infection involves a great many bodily responses. Onset of a systemic inflammatory response to causes such as microbial invasion is called sepsis. When the body is overwhelmed and the defensive mechanisms fail (viz., sepsis, severe sepsis, septic shock) the body is a great risk.
  • the prior art has recently disclosed two relatively new pharmaceutical agents (and related methods of use) for treating sepsis. These agents are (a) Activated Protein C, and (b) sPIJ ? inhibitors.
  • Activated Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of vascular homeostasis by inactivating Factors V a and VIII a in the coagulation cascade.
  • Human Protein C is made in vivo primarily in the liver as a single polypeptide of 461 amino acids.
  • Protein C functions as an important down-regulator of blood coagulation factors that promote thrombosis.
  • the Protein C enzyme system represents a major physiological mechanism of anticoagulation.
  • the critical role of protein C in controlling hemostasis is exemplified by the increased rate of thrombosis in heterozygous deficiency, protein C resistance (e.g., due to the common Factor V Leiden mutation) and the fatal outcome of untreated homozygous protein C deficiency.
  • Human activated protein C both plasma-derived and recombinant, have been shown to be effective and safe antithrombotic agents in a variety of animal models for both venous and arterial thrombosis.
  • Activated protein C in recent clinical studies has been shown to be effective in human thrombotic diseases including the treatment of protein C deficiencies and microvascular thrombosis, such as disseminated intravascular coagulation associated with sepsis.
  • sPLA2 human non-pancreatic secretory phospholipase A2
  • sPLA2 is a rate limiting enzyme in the arachidonic acid cascade which hydrolyzes membrane phospholipids .
  • fatty acids e.g., arachidonic acid
  • Such compounds would be of value in general treatment of conditions induced and/or maintained by overproduction of sPLA2, such as sepsis.
  • This invention is a pharmaceutical composition comprising: (a) an SPLA2 inhibitor, and
  • This invention is also a method of treating or preventing sepsis by administering to a mammal in need thereof a therapeutically effective amount of (a) an
  • SPLA2 inhibitor and a therapeutically effective amount of (b) Activated Protein C; wherein (a) and (b) are both administered within a therapeutically effective interval.
  • Activated Protein C aPC - Activated human Protein C, also called, Activated Protein C.
  • Protein C Activity any property of activated human Protein C or its derivatives responsible for proteolytic, amidolytic, esterolytic, and biological (anticoagulant or pro-fibrinolytic) activities. Methods for testing for Protein C anticoagulant and amidolytic activity are well known in the art, i.e., see Grinnell et.al., 1987, Bio/Technology 5:1189-1192.
  • rhaPC Recombinant activated human protein C, produced by activating r-HPC in vitro or by direct secretion of the activated form of Protein C from prokaryotic cells, eukaryotic cells, or from transgenic animals .
  • zymogen an enzymatically inactive precursor of a proteolytic enzyme.
  • Protein C zymogen refers to secreted, inactive forms, whether one chain or two chain, of protein C.
  • sPIJJ?- secretary phospholipase A2 SPL 2 inhibitor means a compound which inhibits SPLA2 mediated release of fatty acid.
  • sepsis - Sepsis is defined as a systemic inflammatory response to infection, associated with and mediated by the activation of a number of host defense mechanisms including the cytokine network, leukocytes, and the complement and coagulation/fibrinolysis systems (Mesters et al . , Blood 88:881-886, 1996).
  • DIC Disseminated intravascular coagulation
  • terapéuticaally effective amount is an amount of (a) sPIJJ? inhibitor or an amount of (b) aPC which is effective to prevent or ameliorate sepsis.
  • terapéuticaally effective interval is a period of time beginning when one of either (a) the sPI .2 inhibitor or (b) aPC is administered to a mammal and ending at the limit of the beneficial effect in preventing or ameliorating psesis of (a) or (b) .
  • terapéuticaally effective combination means administration of both (a) SPLA2 inhibitor and (b)
  • Activated protein C either simultaneously or separately.
  • Active ingredient refers to a combination of (a) sPIA? inhibitor and (b) Activated Protein C co-present in a pharmaceutical formulation for the delivery of a treatment regimen that applies this invention.
  • injectable liquid carrier refers to a liquid medium containing either or both of (a) sPLA2 inhibitor, or (b) Activated Protein C; wherein (a) and (b) are independently dissolved, suspended, dispersed, or emulsified in the liquid medium.
  • alkyl - a straight or branched chain monovalent hydrocarbon radical such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tertiary butyl, isobutyl, sec- butyl, n-pentyl, and n-hexyl.
  • alkenyl - a straight chain or branched monovalent hydrocarbon group having the stated number range of carbon atoms, and typified by groups such as vinyl, propenyl, crotonyl, isopentenyl, and various butenyl isomers .
  • hydrocarbyl - an organic group containing only carbon and hydrogen. halo - fluoro, chloro, bro o, or iodo.
  • heterocyclic radical- radicals derived from monocyclic or polycyclic, saturated or unsaturated, substituted or unsubstituted heterocyclic nuclei having 5 to 14 ring atoms and containing from 1 to 3 hetero atoms selected from the group consisting of nitrogen, oxygen or sulfur, phenylpyridinyl, benzylpyridinyl, pyrimidinyl, phenylpyrimidinyl, pyrazinyl, 1, 3, 5-triazinyl, quinolinyl, phthalazinyl, quinazolinyl, morpholino, thiomorpholino, homopiperazinyl, tetrahydrofuranyl, tetrahydropyranyl, oxacanyl, 1, 3-dioxolanyl, 1,3- dioxanyl, 1, 4-dioxanyl, tetrahydrothiopheneyl, pentamethylenesulfadyl, 1, 3-dithianyl,
  • carbocyclic radical - a radical derived from a saturated or unsaturated, substituted or unsubstituted 5- to 14-membered organic nucleus whose ring forming atoms (other than hydrogen) are solely carbon atoms.
  • Typical carbocyclic radicals are cycloalkyl, cycloalkenyl, phenyl, naphthyl, norbornanyl, bicycloheptadienyl, tolulyl, xylenyl, indenyl, stilbenyl, terphenylyl, diphenylethylenyl, phenyl-cyclohexenyl, acenaphthylenyl, and anthracenyl, biphenyl, bibenzylyl and related bibenzylyl homologues represented by the formula (bb) ,
  • n is a number from 1 to 8.
  • substituent - radicals suitable for substitution at positions 4, 5, 6, and/or 7 on the indole nucleus (as hereinafter depicted in Formula I) and radical (s) suitable for substitution on the heterocyclic radical and carbocyclic radical as defined above.
  • Illustrative non-interfering radicals are Ci-C ⁇ alkyl, C2-C5 alkenyl, C2 ⁇ Cg alkenyl, C7-C12 aralkyl, C7-C12 alkaryl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, phenyl, tolulyl, xylenyl, biphenyl, C ⁇ -C6 alkoxy, C2 ⁇ Cg alkenyloxy, C2-C5 alkenyloxy, C2 ⁇ Ci2 alkoxyalkyl, C2 _ C12 alkoxyalkyloxy, C2-C12 alkylcarbonyl, C2-C12 alkylcarbonylamino, C2-C12 alkoxyamino, C2-C12 alkoxyaminocarbonyl, C1-C12 alkylamino, C ⁇ -C6 alkylthio, C2-C12 alkylthiocarbonyl, C1-C6
  • acidic group an organic group which when attached to an indole nucleus, through suitable linking atoms (hereinafter defined as the "acid linker"), acts as a proton donor capable of hydrogen bonding.
  • suitable linking atoms hereinafter defined as the "acid linker”
  • acid linker - a divalent linking group symbolized as, -(L a )-, which has the function of joining the 4 or 5 position of the indole nucleus to an acidic group in the general relationship:
  • acid linker length the number of atoms (excluding hydrogen) in the shortest chain of the linking group -(La)- that connects the 4 or 5 position of the indole nucleus with the acidic group.
  • the presence of a carbocyclic ring in -(L a )- counts as the number of atoms approximately equivalent to the calculated diameter of the carbocyclic ring.
  • a benzene or cyclohexane ring in the acid linker counts as 2 atoms in calculating -lithe length of - (L a ) - .
  • Illustrative acid linker groups are;
  • groups (a) , (b) , and (c) have acid linker lengths of 5, 7, and 2, respectively.
  • mammal - includes human mammalian - includes human. alkylene chain of 1 or 2 carbon atoms - the divalent radicals, -CH2-CH2- and -CH2-.
  • pharmaceutically acceptable - the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • carbazole SPLA2 inhibitors includes SPLA2 inhibitors having either a carbazole or a tetrahydrocarbazole nucleus.
  • Active ingredient refers to a combination of (a) SPLA2 inhibitor and (b) Activated Protein C existing as compounds or mixture in a pharmaceutical formulation for the delivery of a treatment regimen that applies this invention.
  • SPLA2 inhibitors are generally useful in the practice in this invention.
  • SPLA2 classes of suitable SPLA2 useful in the the method of the invention for treatment of sepsis are the following:
  • United States Patent Application Serial No. 09/105381 discloses the following process having steps (a) thru (i) : Preparing a compound of the formula (I) or a pharmaceutically acceptable salt or prodrug derivative thereof
  • R-- is selected from the group consisting of -C7-C20 alkyl
  • R-LO is selected from the group consisting of halo, C ⁇ -C ⁇ o alkyl, C ⁇ -C ⁇ o alkoxy, -S- (C ⁇ -C ⁇ o alkyl) and halo (C -C ⁇ ) alkyl, and t is an integer from 0 to 5 both inclusive;
  • R2 is selected from the group consisting of hydrogen, halo, C -C 3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (C ⁇ -C2 alkyl), -S- (C ⁇ -C2 alkyl), aryl, aryloxy and HET;
  • R ⁇ is selected from the group consisting of -CO2H, -SO3H and -P(O) (OH) 2 or salt and prodrug derivatives thereof;
  • R ⁇ , R6 a ⁇ d R 7 are each independently selected from the group consisting of hydrogen, (C -Cg) alkyl, (C ⁇ Cg) alkoxy, halo (C ⁇ -Cg) alkoxy, halo (C2 ⁇ C ) alkyl, bromo, chloro, fluoro, iodo and aryl; which process comprises the steps of: a) halogenating a compound of formula X
  • R 8 is (C ⁇ -Cg) alkyl, aryl or HET; with SO2CI2 to form a compound of formula
  • the synthesis methodology for making the 1H- indole-3-glyoxylamide SPLA2 inhibitor may be by any suitable means available to one skilled in the chemical arts. However, such methodology is not part of the present invention which is a method of use, specifically, a method of treating mammal afflicted or susceptible to sepsis.
  • the method of the invention is for treatment of a mammal, including a human, afflicted sepsis, said method comprising administering to said human a therapeutically effective amount of the compound represented by formula (la), or a pharmaceutically acceptable salt or prodrug derivative thereof;
  • R]_Q is a radical independently selected from halo, C -C ⁇ o alkyl, C ⁇ -C ⁇ o alkoxy, -S-(C ⁇ -C ⁇ o alkyl), and C ⁇ -C ⁇ o haloalkyl and t is a number from 0 to 5;
  • R2 is selected from the group; halo, cyclopropyl, methyl, ethyl, and propyl; R4 and R5 are independently selected from hydrogen, a non-interfering substituent, or the group, - (L a ) - (acidic group), wherein _ (L a )- is an acid linker; provided, the acid linker group, -(L a )-, for R4 is selected from the group consisting of;
  • the acid linker, _ (L a )- / for R5 is selected from group consisting of;
  • Rg and Rg5 are each independently selected from hydrogen, C -C g alkyl, aryl, C -C ⁇ o alkaryl, C ⁇ C aralkyl, carboxy, carbalkoxy, and halo; and provided, that at least one of R4 and R5 must be the group, - (L a ) - (acidic group) and wherein the (acidic group) on the group - (L a ) - (acidic group) of R4 or R5 is selected from -CO2H, -SO3H, or -P(O) (OH) 2; Rg and R7 are each independently selected form hydrogen and non-interfering substituents, with the non-interfering substituents being selected from the group consisting of the following: C ⁇ -Cg alkyl, C2 ⁇ Cg alkenyl, C2 ⁇ C alkynyl, C7-C12 aralkyl, C7-C12 alkaryl, C3-C8 cycloal
  • n is from 1 to 8.
  • compositions of the invention are 1H- indole-3-glyoxylamide compounds and all corresponding pharmaceutically acceptable salts, solvates and prodrug derivatives thereof which are useful in the method of the invention include the following:
  • prodrugs of the compounds of formula (I) and named compounds (A) thru (0) are prodrugs of the compounds of formula (I) and named compounds (A) thru (0) .
  • the preferred prodrugs are the aromatic and aliphatic esters, such as the methyl ester, ethyl ester, n-propyl ester, isopropyl ester, n-butyl ester, sec- butyl, tert-butyl ester, N,N-diethylglycolamido ester, and morpholino-N-ethyl ester.
  • Methods of making ester prodrugs are disclosed in U.S. Patent No. 5,654,326. Additional methods of prodrug synthesis are disclosed in U.S. Provisional Patent Application Serial No.
  • 60/063280 filed October 27, 1997 (titled, N, N-diethylglycolamido ester Prodrugs of Indole SPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference;
  • U.S. Provisional Patent Application Serial No. 60/063646 filed October 27, 1997 (titled, Morpholino-N-ethyl Ester Prodrugs of Indole SPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference;
  • U.S. Provisional Patent Application Serial No. 60/063284 filed October 27, 1997 (titled, Isopropyl Ester Prodrugs of Indole SPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference.
  • the aniline, 2, on heating with di-tert-butyl dicarbonate in THF at reflux temperature is converted to the N-tert-butylcarbonyl derivative, 3, in good yield.
  • the dilithium salt of the dianion of 3 is generated at -40 to -20 °C in THF using sec-butyl lithium and reacted with the appropriately substituted N-methoxy-N-methylalkanamide.
  • This product, 4, may be purified by crystallization from hexane, or reacted directly with trifluoroacetic acid in methylene chloride to give the 1, 3-unsubstituted indole 5.
  • the 1, 3-unsubstituted indole 5 is reacted with sodium hydride in dimethylformamide at room temperature (20-25 °C) for 0.5-1.0 hour.
  • the resulting sodium salt of 5 is treated with an equivalent of arylmethyl halide and the mixture stirred at a temperature range of 0-100 °C, usually at ambient room temperature, for a period of 4 to 36 hours to give the 1-arylmethylindole, 6.
  • This indole, 6, is 0- demethylated by stirring with boron tribro ide in methylene chloride for approximately 5 hours (see ref. Tsung-Ying Shem and Charles A Winter, Adv. Drug Res., 1977, 12, 176, the disclosure of which is incorporated herein by reference) .
  • the 4-hydroxyindole, 7, is alkylated with an alpha bromoalkanoic acid ester in dimethylformamide (DMF) using sodium hydride as a base, with reactions conditions similar to that described for the conversion of 5 to 6.
  • the a-[(indol-4- yl) oxy] alkanoic acid ester, 8, is reacted with oxalyl chloride in methylene chloride to give 9, which is not purified but reacted directly with ammonia to give the glyoxamide 10.
  • This product is hydrolyzed using IN sodium hydroxide in MeOH.
  • the final glyoxylamide, 11, is isolated either as the free carboxylic acid or as its sodium salt or in both forms.
  • the most preferred compound, [ [3- (2-Amino-l, 2- dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4- yl] oxy] acetic acid (as well as its sodium salt and methyl ester) useful in the practice of the method of the invention, may be prepared by the following procedure: Preparation of [ [3- (2-Amino-l, 2-dioxoethyl) -2-ethyl- 1- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, a compound represented by the formula:
  • reaction mixture was stirred 5 minutes, the cooling bath removed and stirred an additional 18 hours. It was then poured into a mixture of 300 mL of ether and 400 L of 0.5N HCl. The organic layer was separated, washed with water, brine, dried over MgS ⁇ 4, and concentrated at reduced pressure to give 25.5g of a crude of l-[2-(tert- butoxycarbonylamino) -6-methoxyphenyl] -2-butanone . This material was dissolved in 250 mL of methylene chloride and 50 mL of trifluoroacetic acid and stirred for a total of 17 hours. The mixture was concentrated at reduced pressure and ethyl acetate and water added to the remaining oil.
  • lH-indole-3-hydrazide SPLA2 inhibitors useful in practicing the method of the invention are described in U.S. Patent No. 5,578,634; the entire disclosure of which is incorporated herein by reference.
  • the method of the invention is for treatment of a mammal, including a human, afflicted with sepsis, said method comprising administering to said human a therapeutically effective amount of the described as lH-indole-3-acetic acid hydrazides represented by the formula (lb) , and pharmaceutically acceptable salts, and prodrugs thereof;
  • X is oxygen or sulfur
  • Rx is selected from groups (i) , (ii) and (iii) where;
  • (i) is C4-C20 alkyl, C4-C20 alkenyl, C4-C20 alkynyl, C4-C2 0 haloalkyl, C4-CX2 cycloalkyl, or
  • (ii) is aryl or aryl substituted by halo, -CN, -CHO, -OH, -SH, C ⁇ -C 10 alkylthio, C ⁇ -C 10 alkoxy, CI-CIQ alkyl, carboxyl, amino, or hydroxya ino; (iii) is
  • R74 is, independently, hydrogen or C ⁇ -C ⁇ o alkyl
  • R75 is aryl or aryl substituted by halo, -CN, -CHO, -OH, nitro, phenyl, -SH, C ⁇ -C ⁇ o alkylthio, C -C ⁇ g alkoxy, C -C ⁇ o alkyl, amino, hydroxyamino or a substituted or unsubstituted 5- to 8- membered heterocyclic ring;
  • R2 is halo, C -C3 alkyl, ethenyl, C -C2 alkylthio, C -C2 alkoxy, -CHO, -CN; each R3 is independently hydrogen, C -C3 alkyl, or halo;
  • R4 R5, Rg, and R7 are each independently hydrogen, C ⁇ -C ⁇ o alkyl, C ⁇ -C ⁇ o alkenyl, C -C ⁇ o alkynyl, C3-C8 cycloalkyl, aryl, aralkyl, or any two adjacent hydrocarbyl groups in the set R4 R5, Rg, and R7 combined with the ring carbon atoms to which they are attached to form a 5- or 6-membered substituted or unsubstituted carbocyclic ring; or C ⁇ -C ⁇ g haloalkyl, C -C ⁇ g alkoxy, C ⁇ C o haloalkoxy, C4-C8 cycloalkoxy, phenoxy, halo, hydroxy, carboxyl, -SH, -CN, -S(C ⁇ -C ⁇ o alkyl), arylthio, thioacetal, -C (0) 0 (C ⁇ -C ⁇ o alkyl), hydrazino,
  • Z is a bond, -0-, -N(C ⁇ -C 10 alkyl)-, -NH, or -S-; and Q is -C0N(Rg2 R 83) / -5-tetrazolyl, -S0 3 H,
  • Rsg is independently selected from hydrogen, a metal, or C -C ⁇ o alkyl.
  • the lH-indole-3-acetic acid ester can be readily alkylated by an alkyl halide or arylalkyl halide in a solvent such as N, N-dimethylformamide (DMF) in the presence of a base (meth a) to give the intermediate 1- alkyl-lH-indole-3-acetic acid esters, III.
  • a solvent such as N, N-dimethylformamide (DMF)
  • a base meth a
  • Bases such as potassium t-butoxide and sodium hydride were particularily useful. It is advantageous to react the indole, II, with the base to first form the salt of II and then add the alkylating agent. Most alkylations can be carried out at room temperature.
  • Useful inhibitors are represented by formula (lib) , and pharmaceutically acceptable salts and prodrug derivatives thereof,
  • X is oxygen or sulfur
  • R ⁇ is selected from groups (i) , (ii) (iii) and (iv) where;
  • (i) is C -C20 alkyl, Cg-C20 alkenyl, Cg-C2o alkynyl, Cg-C20 haloalkyl, C4-CX2 cycloalkyl, or
  • (ii) is aryl or aryl substituted by halo, nitro, -CN, -CHO, -OH, -SH, C ⁇ -C 10 alkyl, C ⁇ -C 10 alkylthio, C ⁇ -C ⁇ o alkoxyl, carboxyl, amino, or hydroxyamino; or (iii) is -(CH 2 ) n -( R 8 ⁇ ) / or -(NH)-(R 8 ⁇ ), where n is 1 to 8, and Rso is a group recited in (i), and Rsi is selected from a group recited in (i) or (ii) ;
  • Rg7 is hydrogen or C ⁇ -C o alkyl
  • Rg8 is selected from the group; phenyl, naphthyl, indenyl, and biphenyl, unsubstituted or substituted by halo, -CN, - CHO, -OH, -SH, C ⁇ -C 10 alkylthio, C ⁇ -C 10 alkoxyl, phenyl, nitro, C ⁇ -C ⁇ o alkyl, C ⁇ -C ⁇ o haloalkyl, carboxyl, amino, hydroxyamino; or a substituted or unsubstituted 5 to 8 membered heterocyclic ring;
  • R 2 s halo, C -C2 alkylthio, or C -C2 alkoxy; each R 3 is independently hydrogen, halo, or methyl; R 14 R 15' R 16' anc Rl7 are each independently hydrogen, C ⁇ -C ⁇ g alkyl, C ⁇ -C ⁇ o alkenyl, C ⁇ -C ⁇ o alkynyl, C 3 -C 8 cycloalkyl, aryl, aralkyl, or any two adjacent hydrocarbyl groups in the set R 4 15.
  • Ri6' and R 17' combine with the ring carbon atoms to which they are attached to form a 5 or 6 membered substituted or unsubstituted carbocyclic ring; or C ⁇ -C ⁇ o haloalkyl, C ⁇ C o alkoxy, C ⁇ -C ⁇ o haloalkoxy, C4-C8 cycloalkoxy, phenoxy, halo, hydroxy, carboxyl, -SH, -CN, C ⁇ -C ⁇ o alkylthio, arylthio, thioacetal, -C (0) 0 (C ⁇ -C ⁇ o alkyl), hydrazide, hydrazino, hydrazido, -NH2, -NO2, -NR82 R 83' and -C (0) NR82 R 83' where, R82 an d R83 are independently hydrogen, C ⁇ -C ⁇ o alkyl, C ⁇ -C ⁇ o hydroxyalkyl, or taken together with N, R82 and R83
  • Z is a bond, -0-, -N(C 1 -C ⁇ 0 alkyl)-, -NH-, or -S-, and
  • Q is -CON(R 8 2R83) / -5-tetrazolyl, -SO3H,
  • Rgg is independently selected from hydrogen, a metal, or C ⁇ -C ⁇ o alkyl
  • R99 is selected from hydrogen or C ⁇ -C ⁇ o alkyl.
  • the lH-indole-3-acetamide II may be alkylated by an alkyl halide or arylalkyl halide in a solvent such as N,N- dimethylformamide (DMF) in the presence of a base (method a) to give intermediate l-alkyl-lH-indole-3-acetic acid esters, III.
  • a solvent such as N,N- dimethylformamide (DMF)
  • a base (method a) to give intermediate l-alkyl-lH-indole-3-acetic acid esters, III.
  • Bases such as potassium t-butoxide and sodium hydride are useful. It is advantageous to react the indole, II, with the base to first form the salt of II and then add alkylating agent.
  • the intermediate acetic acid esters, III can be first hydrolyzed to the acetic acid derivatives, V
  • lH-indole-1-functional SPLA2 inhibitors of the hydrazide, amide, or glyoxylamide types as described in United States Patent No. 5,641,800, the entire disclosure of which is incorporated herein by reference. These inhibitors are useful ingredients in the compositons of the invention and the method of the invention for treatment of a mammal, including a human, afflicted with sepsis .
  • X is oxygen or sulfur; each R is independently hydrogen, or C -C3 alkyl; R3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or
  • (c) is the group -(L)-Rso/ where, -(L)- is a divalent linking group of 1 to 12 atoms and where Rso is a group selected from (a) or (b) ;
  • R2 is hydrogen, halo, C1-C 3 alkyl, C 3 -C4 cycloalkyl, C 3 -C4 cycloalkenyl, -0- (C ⁇ -C2 alkyl), -S- (C ⁇ C2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
  • Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group, - (L a ) - (acidic group) .
  • -(L a )- is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, - (L a ) - (acidic group) ;
  • R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
  • lH-indole-1-hydrazide compounds useful as SPLA2 inhibitors in the practice of the method and formulation of the compositions of the invention are as follows:
  • X is oxygen or sulfur; each R is independently hydrogen, or C -C3 alkyl;
  • R3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C 7 -C 2 o alkenyl, C 7 -C o alkynyl, carbocyclic radical, or heterocyclic radical, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituent; or (c) is the group -(L)-Rg ⁇ '' where, - (L) - is a divalent linking group of 1 to 12 atoms and where Rso is a group selected from (a) or (b) ;
  • R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (C -C2 alkyl), -S- (C ⁇ C2 alkyl), or a non-interfering substituent having a total of ltto 3 atoms other than hydrogen;
  • Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group,
  • R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents.
  • Phospholipase A2 Inhibitor Compounds Having an Indolizine Type Nucleus, Parmaceutical Formulations Containing Said compounds, and Therapeutic Methods of Using said Compounds are useful in the formulation of the compositions of the invention and in the practice of the method of the invention is for treatment of a mammal, including a human, afflicted with sepsis.
  • Useful lH-indole-1-functional compounds or pharmaceutically acceptable salts, solvates or prodrug derivatives are represented by the formula (Id) ;
  • R 3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C2 0 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or
  • (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or (c) is the group -(L)-Rgo; where, - (L) - is a divalent linking group of 1 to 12 atoms and where Rgo i a group selected from (a) or (b) ;
  • R 2 i hydrogen, halo, C -C 3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (C ⁇ -C2 alkyl), -S- (C ⁇ C2 alkyl) , or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
  • R 7 and R 8 are independently selected from hydrogen, a non-interfering substituent, or the group, - (L a )- (acidic group) .
  • -(L a )- is an acid linker having an acid linker length of 1 to 10; provided, that at least one of R 7 and R 8 must be the group, - (L a ) - (acidic group) ; and
  • R ⁇ 5 and R are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
  • X is selected from oxygen or sulfur; each R3 is independently hydrogen, C1-C3 alkyl, or halo;
  • R l is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or
  • (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or (c) is the group -(L)-Rgo; where, -(L)- is a divalent linking group of 1 to 12 atoms and where Rso i a group selected from (a) or (b) ;
  • R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0-(C ⁇ -C2 alkyl), -S- (C ⁇ -C 2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
  • R5 and Rg are independently selected from hydrogen, a non-interfering substituent, or the group, -(L a )- (acidic group) ; wherein -(L a )-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of R5 and Rg must be the group, - (L a ) - (acidic group);
  • R7 and Rs are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
  • the indolizine compounds may be made by one of more of the following reaction schemes:
  • Compound 12 (N. Desidiri, A. Galli, I. Sestili, and M. L. Stein, Arch. Pharm. (Weinheim) 325, 29, (1992)) is reduced by hydrogen in the presence of Pd/C to 14 which gives 15 on ammonolysis using ammonium hydroxide.
  • Compound 23 (N. Desideri F. Manna, M. L. Stein, G. Bile, W. Filippeelli, and E. Marmo, Eur. J. Med. Chem. Chi . Ther., 18, 295, (1983)) is O-alkylated using sodium hydride and benzyl chloride to give 24. N-alkylation of 24 by l-bromo-2-butanone or chloromethylcyclopropyl ketone and subsequent base catalyzed cyclization gives 25 which is acylated by aroyl halide to give 26. Hydrolysis of the ester function of 26 followed by acidification forms an acid which is thermally decarboxylated to give 27. Reduction of the ketone function of 27 by LAH yields indolizines 28.
  • R 2 Ph 33a R-pH
  • R 2 Ph b R-
  • OBn
  • R 2 Ph c R-pOMe
  • R 2 Ph c R-
  • OBn
  • R 2 cyclo-Hex d
  • OBn
  • R 2 cyclo-Hex
  • the hydroxypyridine is O-alkylated to give 44 which is heated with 2-haloketones to produce 45.
  • Treatment of 45 with base causes cyclization to 46 which on heating with acid chlorides yields acylindolizines 47 which are reduced by aluminum hydride to the corresponding alkylindolizines 48.
  • Sequential treatment of 48 with oxalyl chloride and then ammonia gives 49.
  • Cleavage of the ether functionality of 49 yields 50.
  • the oxyacetic ester derivatives 51 are formed by O-alkylation of 50 and then hydrolyzed to the oxyacetic acids 52.
  • Pyridine 43 is O-alkylated to produce 53. Heating 53 with 2-haloketones gives intermediate N-alkylated pyridinium compounds which are cyclized to 54 on treatment with base. Heating 54 with acyl chlorides gives the acylindolizines 55 which are reduced to the alkylindolizines 56 by sodium borohydride-aluminum chloride. Alternatively, 56 are produced by C-alkylation of 54 using alkyl halides. Sequential treatment of 56 with oxalyl chloride and then ammonia gives 57 which are hydrolyzed to produce 58. Compound 58b is converted to its sodium salt 59a which yields 59b-k on reaction with the appropriate alkyl halide. Scheme 6e - Part 2
  • Compound 36b is O-alkylated to give 591-p.
  • Pyridine 60 is N-alkylated by 2-haloketones to produce intermediate pyridinium compounds which are cyclized by base to give 61.
  • Reaction of 61 with acyl chlorides produces 62 which are reduced to 63 by tert butylamine-borane and aluminum chloride.
  • Sequential treatment of 63 with oxalyl chloride and then ammonia yields 64 which are O-demethylated by BBr3 to give 65.
  • the sodium salt of 65 is reacted with ethyl 4- bromobutyrate to give 66 which is hydrolyzed to the acid 67.
  • Compounds 36d and 65c are O-alkylated by omega- bromocarboxylic esters to give 68 which are hydrolyzed to the acids 69.
  • Compounds 36d and 65c produce 70 on treatment with propiolactone and base.
  • Pyridine 44b reacts with ethyl bromoacetate to produce 72 which is treated with CS2 and base and then with ethyl acrylate to form 73.
  • Reaction of 73 with base and ethyl bromoacetate yields a mixture of regioisomers 74a+b, 6- and 8-benzyloxy compounds.
  • Base treatment of 74a+b eliminates ethyl acrylate to form 75 which is separated from the isomer of 6-benzyloxy derivative and S-alkylated to give 76. Hydrolysis of 76 forms 77 which is thermally decarboxylated to yield 78.
  • Aminopicoline 84 is converted to its N-CBZ derivative 85 whose anion is alkylated by methyl bromoacetate to produce 86.
  • Reaction of 86 with methyl alpha-bromoalkyl ketones in the presence of base yields 87.
  • Sequential treatment of 87 with oxalyl chloride and then ammonia gives 88 which is converted to 89 by hydrogenolysis of the N-CBZ function. Hydrolysis of 89 yields acids 90.
  • Pyridine 24 is N-alkylated by methyl bromoacetate, cyclized with base, and o-methylated using dimethysulfate to give 94.
  • Hydrolysis of the ester function of 94 followed by thermal decarboxylation yields 2-methoxy-8- benzyloxyindolizine which is C-alkylated at position 3 and then reacted sequentially with oxalyl chloride and ammonia to produce 95.
  • Hydrogenolysis of the 8-benzyloxy group followed by O-alkylation gives 96 which is hydrolyzed to 97.
  • the method of the invention is for treatment of a mammal, including a human, afflicted with sepsis, said method comprising administering to said human a therapeutically effective amount of an indene-1-acetamide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (If);
  • X is oxygen or sulfur; each R is independently hydrogen, C1-C3 alkyl, or halo;
  • R3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or
  • (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or (c) is the group -(L)-Rgo; where, -(L)- is a divalent linking group of 1 to 12 atoms and where RQQ i- s a group selected from (a) or (b) ;
  • R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (C ⁇ -C 2 alkyl), -S- (C ⁇ -C 2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
  • Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group, - (L a ) - (acidic group) .
  • _ ( a )- is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, - (L a ) - (acidic group); and
  • R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
  • Suitable indene compounds also include the following:
  • X is oxygen or sulfur; each Rx is independently hydrogen, C -C3 alkyl, or halo;
  • R3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or
  • (c) is the group -(L)-Rg ⁇ ' where, - (L) - is a divalent linking group of 1 to 12 atoms and where Rgo is a group selected from (a) or (b) ;
  • R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0-(C ⁇ -C2 alkyl), -S- (C ⁇ -C2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
  • Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group, -(L a )- (acidic group) ; wherein ⁇ (L a )-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, - (L a ) - (acidic group); and
  • R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical
  • Suitable indene compounds for use in the method of the invention also include the following: An indene-1-glyoxylamide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula
  • X is oxygen or sulfur
  • R3 is selected from groups (a) , (b) and (c) where;
  • (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or
  • (b) is a member of (a) substituted with one or more independently selected non-interfering substituents;
  • (c) is the group -(L)-Rgo; where, - (L) - is a divalent linking group of 1 to 12 atoms and where Rgo is a group selected from (a) or (b) ;
  • R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (C ⁇ -C2 alkyl), -S- (C ⁇ -C2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
  • Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group, -(L a )- (acidic group) ; wherein -( a )-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, - (L a ) - (acidic
  • the method of making the indene compounds is as follows:
  • R 2-N
  • R b i s - ( CH 2 ) n COOH -Ill- Compound 7 is condensed with benzaldehyde and its derivatives in the presence of base to give 10.
  • Indenes 10 are converted to an active ester using benzotriazo-1- yloxytris (dimethylamino) hexafluorophosphonate and then reacted with ammonium hydroxide to form 11.
  • Demethylation of 11 with BBr3 forms 12 which is O-alkylated using sodium hydride and an omega-bromoalkanoic acid ester to produce 13.
  • Aqueous base hydrolysis of 13 yields 14.
  • Compound 12c is O-alkylated using sodium hydride and methylbromoacetate to product 15 which is reduced by hydrogen in the presence of Pd/C to give a mixture of isomers 16a and 16b.
  • Aqueous base hydrolysis of 16a and 16b gives 17a and 17b, respectively.
  • Compound lOd is treated with lithium diisopropylamine, then air is bubbled into the solution to give 18.
  • the indene 18 is converted to an active ester using benzotriazo-1- yloxytris (dimethylamino) hexafluorophosphonate and then reacted with ammonium hydroxide to form the hydroxy acetamide 19.
  • Compound 19 is oxidized to 20 using N-methylmorpholine N-oxide in the presence of tetrapropylammonium perruthenate .
  • A is phenyl or pyridyl wherein the nitrogen is at the
  • B or D is nitrogen and the other is carbon
  • Z is cyclohexenyl, phenyl, pyridyl, wherein the nitrogen is at the 1-, 2-, or 3-position, or a 6- membered heterocyclic ring having one heteroatom selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position, and nitrogen at the 1-, 2-, 3- or 4-position; is a double or single bond;
  • R20 is selected from groups (a) , (b) and (c) where; (a) is - (C5-C20) alkyl, - (C5-C20) alkenyl,
  • R21 is a non-interfering substituent
  • RI' is -NHNH 2 , -NH 2 or -CONH 2 ;
  • R2' is selected from the group consisting of -OH, and
  • R 5 ' is H, -CN, -NH 2 , -CONH 2/ -CONR 9 R 10 -NHS0 2 R 15 ;
  • R 15 is - (Ci-Cg) alkyl or -CF 3 ; phenyl or phenyl substituted with -CO2H or -CO2 (C1-C4) alkyl; and - (L a ) - (acidic group), wherein ⁇ (L a )- is an acid linker having an acid linker length of 1 to 7 and t is 1-5; R is selected from non-interfering substituent, carbocyclic radicals, carbocyclic radicals substituted with non-interfering substituents, heterocyclic radicals, and heterocyclic radicals substituted with non-interfering substituents; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt thereof; provided that; when R ⁇ ' is H, R20 j_ s benzyl and m is 1 or 2; R2 ' cannot be -0(CH2) m H; and provided that
  • compositions and method of the invention are compounds represented by the formula (He) :
  • Z is cyclohexenyl, or phenyl
  • R21 is a non-interfering substituent
  • R 1 is -NHNH 2 or -NH 2 ;
  • R is selected from the group consisting of -OH and
  • R 5 is H, -C0 2 H, -CONH 2 , -C0 2 (C]_-C alkyl); P(R R ) , where R ⁇ and R 7 are each independently -OH or -0(02-04) alkyl; -S0 3 H, -SO3 (C1-C4 alkyl), tetrazolyl, ⁇ CN ' ⁇ NH 2' -NHS0 2 R15; -CONHSO R15, where R15 is - (C, -C fi ) alkyl or -CF_, phenyl or phenyl substituted with -C0 2 H or -C0 2 (C..-C.) alkyl where m is 1-3; R 3 is H, -0(C ⁇ -C4) alkyl, halo, - (C -Cg) alkyl, phenyl, - (C1-C ) alkylphenyl; phenyl substituted with - (
  • R ⁇ is H, - (C5-C14) alkyl, - (C3-C14) cycloalkyl, pyridyl, phenyl or phenyl substituted with - (C ⁇ -Cg) alkyl, halo, -CF3, -OCF 3 , - (C1-C4) alkoxy, -CN, - (C ⁇
  • Preferred specific compounds including all salts and prodrug derivatives thereof, for the compositions and method of the invention are as follows:
  • carbazole inhibitors suitable for the compositions and method of thein invention are selected from those represented by the formula (XXX) :
  • R2 is selected from the group consisting of -OH and - 0(CH 2 ) m R 5 wherein:
  • R 5 is H, -C0 2 H, -C0 2 (C1-C4 alkyl); , where R 6 and
  • R ⁇ are each independently -OH or -0 (C1-C4 ) alkyl
  • R 15 is - (Ci-Cg) alkyl or -CF3, phenyl or phenyl substituted with -C02H or -CO2 (C ⁇ -C4) alkyl where m is 1-3;
  • R 3 is H, -0(C ⁇ -C4) alkyl, halo, - (C ⁇ -Cg) alkyl, phenyl,
  • R 4 is H, - (C5-C14) alkyl, - (C3-C14 ) cycloalkyl, pyridyl, phenyl or phenyl substituted with - (C ⁇ -Cg) alkyl, halo, -CF 3 , -OCF3 , - (C1-C4) alkoxy, -CN, - (C ⁇ C ) alkylthio, phenyl (CI-C4) alkyl, - (Ci- C4) alkylphenyl, phenyl, phenoxy or naphthyl;
  • A is phenyl or pyridyl wherein the nitrogen is at the 5-, 6-, 7- or 8-position;
  • Z is cyclohexenyl, phenyl, pyridyl wherein the nitrogen is at the 1-, 2- or 3-position or
  • compositions and method of the invention are selected from the following:
  • cabozole/tetrahydrocarbazole inhibitors for the compositions and method of treating sepsis are represented by the formulae (Xe) and (Xle) below:
  • Prodrugs are derivatives of sPIJJ? inhibitors used in the method of the invention which have chemically or metabolically cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo.
  • Derivatives of the compounds of this invention have activity in both their acid and base derivative forms, but the acid derivative form often offers advantages of solubility, tissue compatibility, or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, 7Amsterdam 1985) .
  • Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acidic compound with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a suitable amine. Simple aliphatic or aromatic esters derived from acidic groups pendent on the compounds of this invention are preferred prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy) alkyl esters or ( (alkoxycarbonyl) oxy) alkyl esters.
  • Specific preferred prodrugs are ester prodrugs inclusive of methyl ester, ethyl ester, n-propyl ester, isopropyl ester, n-butyl ester, sec-butyl, tert-butyl ester, N, N-diethylglycolamido ester, and morpholino-N-ethyl ester.
  • Methods of making ester prodrugs are disclosed in U.S. Patent No. 5,654,326. Additional methods of prodrug synthesis are disclosed in U.S. Provisional Patent Application Serial No.
  • 60/063280 filed October 27, 1997 (titled, N, N-diethylglycolamido ester Prodrugs of Indole sPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference;
  • U.S. Provisional Patent Application Serial No. 60/063646 filed October 27, 1997 (titled, Morpholino-N-ethyl Ester Prodrugs of Indole sPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference;
  • US Provisional Patent Application Serial No. 60/063284 filed October 27, 1997 (titled, Isopropyl Ester Prodrugs of Indole sPIJJ? Inhibitors) , the entire disclosure of which is incorporated herein by reference.
  • Carbazole and tetrahydrocarbazole SPL 2 inhibitor compounds useful for practicing the method of the invention may be made by the following general methods:
  • the compounds of formula le where Z is cyclohexene are prepared according to the following reaction Schemes Ig(a)and (c) .
  • R 1 is -NH 2/ R 3 (a) is H, -0 (C1-C4 ) alkyl, halo, - (C ⁇ Cg) alkyl, phenyl, - (C1-C4) alkylphenyl; phenyl substituted with - (C ⁇ -Cg) alkyl, halo, or -CF3; - CH2 ⁇ Si (C ⁇ -Cg) alkyl, furyl, thiophenyl, - (C ⁇
  • R 8 is H, -CONH 2 , -NR 9 R 10 , -CN or phenyl where R 9 and R 10 are independently hydrogen, -CF3, phenyl, - (C1-C4 ) alkyl, - (C1-C4) alkylphenyl or -phenyl (C1-C4 ) alkyl and n is 1 to 8; when R 1 is -NHNH 2/ R 3 (a) is H, -0 (C ⁇ -C ) alkyl, halo,
  • R 8 is H, -NR 9 R 10 , -CN or phenyl where R 9 and R 10 are independently hydrogen, -CF3, phenyl, - (C1-C4) alkyl, - (C1-C4) alkylphenyl or -phenyl (C1-C4 ) alkyl and n is 1 to 8; R 2 ( a ) s _0CH 3 or -OH.
  • An appropriately substituted nitrobenzene (1) can be reduced to the aniline (2) by treatment with a reducing agent, such as hydrogen in the presence of Pd/C, preferably at room temperature.
  • a reducing agent such as hydrogen in the presence of Pd/C, preferably at room temperature.
  • Compound (2) is N-alkylated at temperatures of from about 0 to 20 °C using an alkylating agent such as an appropriately substituted aldehyde and sodium cyanoborohydride to form (3) .
  • an appropriately substituted benzyl halide may be used for the first alkylation step.
  • the resulting intermediate is further N-alkylated by treatment with 2-carbethoxy- 6-bromocyclohexanone, preferably at temperatures of about 80 °C to yield (4) or by treatment with potassium hexamethyldisilazide and the bromoketoester .
  • the product (4) is cyclized to the tetrahydrocarbazole (5) by refluxing with ZnCl2 in benzene for from about 1 to 2 days, preferably at 80 °C.
  • Compound (5) is converted to the hydrazide (6) by treatment with hydrazine at temperatures of about 100 °C, or to the amide (7) by reacting with methylchloroaluminum amide in benzene.
  • Ref 2 Alternatively, (7) may be produced by treatment of (6) with Raney nickel active catalyst.
  • Compounds (6) and (7) may be dealkylated, preferably at 0 °C to room temperature, with a dealkylating agent, such as boron tribromide or sodium thioethoxide, to give compound (7) where R2 ( a ) j_ s -OH, which may then be further converted to compound (9), by realkylating with a base, such as sodium hydride, and an alkylating agent, such as Br(CH2) m R ⁇ , where R ⁇ is the carboxylate or phosphonic diester or nitrile as defined above. Conversion of R2 to the carboxylic acid may be accomplished by treatment with an aqueous base.
  • a dealkylating agent such as boron tribromide or sodium thioethoxide
  • R2 When R2 is nitrile, conversion to the tetrazole may be achieved by reacting with tri-butyl tin azide or conversion to the carboxamide may be achieved by reacting with basic hydrogen peroxide.
  • R When R is the phosphonic diester, conversion to the acid may be achieved by reacting with a dealkylating agent such as trimethylsilyl bromide. The monoester may be accomplished by reacting the diester with an aqueous base.
  • R2 and R 3 are both methoxy, selective demethylation can be achieved by treating with sodium ethanethiolate in dimethylformamide at 100 °C.
  • R 3a is as defined in Scheme 1, above.
  • the aniline (2) is N-alkylated with 2-carbethoxy-6- bromocyclohexanone in dimethyl formamide in the presence of sodium bicarbonate for 8-24 hours at 50 °C.
  • Preferred protecting groups include methyl, carbonate, and silyl groups, such as t-butyldimethylsilyl .
  • the reaction product (4') is cyclized to (5') using the ZnCl2 in benzene conditions described in Scheme 1(a), above.
  • N- alkylation of (5' ) to yield (5) is accomplished by treatment with sodium hydride and the appropriate alkyl halide in dimethylformamide at room temperature for 4-8 hours .
  • carbazole (5) is hydrolyzed to the carboxylic acid (10) by treatment with an aqueous base, preferably at room temperature to about 100 °C.
  • the intermediate is then converted to an acid chloride utilizing, for example, oxalyl chloride and dimethylformamide, and then further reacted with a lithium salt of (S) or (R) -4-alkyl-2-oxazolidine at a temperature of about -75 °C, to give (11a) and (lib), which are separable by chromatography.
  • the diastereomers are converted to the corresponding enantiomeric benzyl esters (12) by brief treatment at temperatures of about 0 °C to room temperature with lithium benzyl oxide.
  • esters (12) are then converted to (7) preferably by treatment with methylchloroaluminum amide (Ref 2, above) or, alternately, by hydrogenation using, for example, hydrogen and palladium on carbon, as described above, to make the acid and then reacting with an acyl azide, such as diphenylphosphoryl azide followed by treatment with ammonia.
  • an acyl azide such as diphenylphosphoryl azide followed by treatment with ammonia.
  • a 1, 2, 3, 4-tetrahydrocarbazole-4-carboxamide or 4- carboxhydrazide (13) is dehydrogenated by refluxing in a solvent such as carbitol in the presence of Pd/C to produce the carbazole-4-carboxamide.
  • a solvent such as carbitol
  • Pd/C a solvent such as Pd/C
  • treatment of (13) with DDQ in an appropriate solvent such as dioxane yields carbozole (14) .
  • oxidation as described above may result in de-alkylation of the nitrogen.
  • R 3 is substituted at the 8- position with methyl
  • oxidation results in dealkylation of the nitrogen which may be realkylated by treatment with sodium hydride and the appropriate alkyl halide as described in Scheme I (a) above to prepare the deired product (14) .
  • Benzoic acid derivative (16) where X is preferably chlorine, bromine or iodine and the protecting group is preferably -CH3, are reduced to the corresponding aniline (25) with a reducing agent, such as stannous chloride in the presence of acid under the general conditions of Sakamoto et al, Chem Pharm . Bull . 35 (5), 1823-1828 (1987) .
  • a reducing agent such as stannous chloride
  • the reactions are conducted at temperatures from about 0 to 100 °C. preferably at ambient temperature, and are substantially complete in about 1 to 48 hours depending on conditions.
  • the aniline (25) and dione (15) are condensed under dehydrating conditions, for example, using the general procedure of Iida, et al., (Ref 5), with or without a noninterfering solvent, such as toluene, benzene, or methylene chloride, under dehydrating conditions at a temperature about 10 to 150 °C.
  • a noninterfering solvent such as toluene, benzene, or methylene chloride
  • the water formed in the process can be removed by distillation, azetropic removal via a Dean-Stark apparatus, or the addition of a drying agent, such as molecular sieves, magnesium sulfate, calcium carbonate, sodium sulfate, and the like.
  • the process can be performed with or without a catalytic amount of an acid, such a p-toluenesulfonic acid or methanesulfonic acid.
  • an acid such as a p-toluenesulfonic acid or methanesulfonic acid.
  • suitable catalysts include hydrochloric acid, phenylsulfonic acid, calcium chloride, and acetic acid.
  • solvents examples include tetrahydrofuran, ethyl acetate, methanol, ethanol, 1, 1, 2, 2-tetrachloroethane, chlorobenzene, bromobenzene, xylenes, and carbotetrachloride .
  • the condensation of the instant process is preferably carried out neat, at a temperature about 100 to 150 °C with the resultant water removed by distillation via a stream of inert gas, such as, nitrogen or argon. The reaction is substantially complete in about 30 minutes to 24 hours.
  • Intermediate (26) may then be readily cyclized in the presence of a palladium catalyst, such as Pd(OAc)2 or Pd(PPh_ 3 )4 and the like, a phosphine, preferably a trialkyl- or triarylphosphine, such as triphenylphosphine, tri-o-tolylphosphine , or tricyclohexylphosphine, and the like, a base, such as, sodium bicarbonate, triethylamine, or diisopropylethylamine, in a noninterfering solvent, such as, acetonitrile, triethylamine, or toluene at a temperature about 25 to 200°C to form (19) .
  • a palladium catalyst such as Pd(OAc)2 or Pd(PPh_ 3 )4 and the like
  • a phosphine preferably a trialkyl- or triarylphosphine, such as triphenylpho
  • solvents examples include tetrahydrofuran, benzene, dimethylsulfoxide, or dimethylformamide .
  • Examples of other suitable palladium catalysts include Pd(PPh 3 )Cl 2 , Pd(OCOCF 3 ) 2 , [ (CH 3 CgH 4 ) 3 P] 2 PdCl 2 , [ (CH 3 CH 2 )3P]2PdCl2, [ (C 6 H 1:L ) 3 P] 2 PdCl 2 , and [(C 6 H5) 3 P] 2 PdBr 2 .
  • phosphines examples include triisopropylphosphine, triethylphosphine, tricyclopentylphosphine, 1,2- bis (diphenylphosphino) ethane, 1, 3-bis (diphenylphosphino) propane, and 1,4- bis (diphenylphosphino) butane.
  • Examples of other suitable bases include tripropyl amine, 2, 2, 6, 6-tetramethylpiperidine, 1,5- diazabicyclo[2.2.2]octane (DABCO) , 1,8- diazabicyclo [5.4.0] undec-7-ene (DBU) , 1,5- diazabicyclo [4.3.0]non-5-ene, (DBN) sodium carbonate, potassium carbonate, and potassium bicarbonate.
  • DABCO 1,8- diazabicyclo[2.2.2]octane
  • DBU 1,8- diazabicyclo [5.4.0] undec-7-ene
  • DBN 1,5- diazabicyclo [4.3.0]non-5-ene
  • the cyclization of the instant process is preferably carried out with palladium (II) acetate as catalyst in the presence of either triphenylphosphine, tri-o- tolylphosphine, 1, 3-bis (diphenylphosphino) propane, or tricyclohexylphosphine in acetonitrile as solvent and triethylamine as base at a temperature about 50 to 150 °C.
  • the reaction is substantially complete in about 1 hour to 14 days.
  • a preferred process for cyclization consists of the reaction of intermediate (26) with a palladacycle catalyst such as trans-di ( ⁇ -acetato) -bis [o- (di-o-tolylphosphino) benzyl] dipalladium (II) in a solvent such as dimethylacetamide (DMAC) at 120-140 °C in the presence of a base such as sodium acetate.
  • a palladacycle catalyst such as trans-di ( ⁇ -acetato) -bis [o- (di-o-tolylphosphino) benzyl] dipalladium (II) in a solvent such as dimethylacetamide (DMAC) at 120-140 °C in the presence of a base such as sodium acetate.
  • Intermediate (19) may be alkylated with an alkylating agent XCH2R4, where X is halo in the presence of a base to form (20) .
  • Suitable bases include potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, potassium hydroxide, sodium hydroxide, sodium hydride, potassium hydride, lithium hydride, and Triton B (N-benzyltrimethylammonium hydroxide) .
  • the reaction may or may not be carried out in the presence of a crown ether. Potassium carbonate and Triton B are preferred.
  • the amount of alkylating agent is not critical, however, the reaction is best accomplished using an excess of alkyl halide relative to the starting material .
  • a catalytic amount of an iodide such as sodium iodide or lithium iodide may or may not be added to the reaction mixture.
  • the reaction is preferably carried out in an organic solvent, such as, acetone, dimethylformamide, dimethylsulfoxide, or acetonitrile .
  • organic solvents include tetrahydrofuran, methyl ethyl ketone, and t-butyl methyl ether.
  • the reaction is conducted at temperatures from about -10 to 100 °C. preferably at ambient temperature, and is substantially complete in about 1 to 48 hours depending on conditions.
  • a phase transfer reagent such as tetrabutylammonium bromide or tetrabutylammonium chloride may be employed.
  • Intermediate (20) May by dehydrogenated by oxidation with 2, 3-dichloro-5, 6-dicyano-l, 4-benzoquinone in a noninterfering solvent to form (21) .
  • Suitable solvents include methylene chloride, chloroform, carbon tetrachloride, diethyl ether, methyl ethyl ketone, and t-butyl methyl ether. Toluene, benzene, dioxane, and tetrahydrofuran are preferred solvents.
  • the reaction is carried out at a temperature about 0 to 120 °C. Temperatures from 50 to 120 °C are preferred.
  • the reaction is substantially complete in about 1 to 48 hours depending on conditions .
  • Intermediate (21) may be a inated with ammonia in the presence of a noninterfering solvent to form a (22).
  • Ammonia may be in the form of ammonia gas or an ammonium salt, such as ammonium hydroxide, ammonium acetate, ammonium trifluoroacetate, ammonium chloride, and the like.
  • Suitable solvents include ethanol, methanol, propanol, butanol, tetrahydrofuran, dioxane, and water. A mixture of concentrated aqueous ammonium hydroxide and tetrahydrofuran or methanol is preferred for the instant process.
  • the reaction is carried out at a temperature about 20 to 100 °C . Temperatures from 50 to 60 °C are preferred.
  • the reaction is substantially complete in about 1 to 48 hours depending on conditions.
  • Alkylation of (22) is achieved by treatment with an alkylating agent of the formula XCH2R ⁇ where X is halo and R 70 is -C0 2 R 71 , -SO3R 71 , -P (0) (OR 71 ) 2 ' or _ P(O) (OR 71 )H, where R 71 is an acid protecting group or a prodrug function, in the presence of a base in a noninterfering solvent to form (23) .
  • Methyl bromoacetate and t-butyl bromoacetate are the preferred alkylating agents .
  • Suitable bases include potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, potassium hydroxide, sodium hydroxide, sodium hydride, potassium hydride, lithium hydride, and Triton B (N-benzyltrimethylammonium hydroxide) .
  • the reaction may or may not be carried out in the presence of a crown ether. Cesium carbonate and Triton B are preferred.
  • the amount of alkylating agent is not critical, however, the reaction is best accomplished using an excess of alkyl halide relative to the starting material.
  • the reaction is preferably carried out in an organic solvent, such as, acetone, dimethylformamide, dimethylsulfoxide, or acetonitrile .
  • Other suitable solvents include tetrahydrofuran, methyl ethyl ketone, and t-butyl methyl ether.
  • the reaction is conducted at temperatures from about
  • phase transfer reagent such as tetrabutylammonium bromide or tetrabutylammonium chloride may be employed.
  • Intermediate (23) may be optionally hydrolyzed with a base or acid to form desired product (24) and optionally salified.
  • Hydrolysis of (23) is achieved using a base such as sodium hydroxide, potassium hydroxide, lithium hydroxide, aqueous potassium carbonate, aqueous sodium carbonate, aqueous lithium carbonate, aqueous potassium bicarbonate, aqueous sodium bicarbonate, aqueous lithium bicarbonate, preferably sodium hydroxide and a lower alcohol solvent, such as, methanol, ethanol, isopropanol, and the like.
  • a base such as sodium hydroxide, potassium hydroxide, lithium hydroxide, aqueous potassium carbonate, aqueous sodium carbonate, aqueous lithium carbonate, aqueous potassium bicarbonate, aqueous sodium bicarbonate, aqueous lithium bicarbonate, preferably sodium hydroxide and a lower alcohol solvent, such as, methanol, ethanol, isopropanol, and the like.
  • the acid protecting group may be removed by organic and inorganic acids, such as trifluoroacetic acid and hydrochloric acid with or without a noninterferring solvent.
  • Suitable solvents include methylene chloride, tetrahydrofuran, dioxane, and acetone.
  • the t-butyl esters are preferably removed by neat trifluoroacetic acid.
  • the reaction is conducted at temperatures from about
  • the preferred alkyl halide is methyl iodide.
  • the reaction is conducted at temperatures from about 0 to 100 °C . preferably at ambient temperature, and is substantially complete in about 1 to 48 hours depending on conditions.
  • the starting material (16) may be prepared by condensation with an alcohol HOPG, where PG is an acid protecting group, in the presence of a dehydrating catalyst such as, dicyclohexylcarbodiimide (DCC) or carbonyl diimidazole.
  • a dehydrating catalyst such as, dicyclohexylcarbodiimide (DCC) or carbonyl diimidazole.
  • a palladium catalyst such as Pd(Ph3P)4
  • a base such as sodium bicarbonate
  • Compound (28) is converted to the carbazole product (29) by treatment with a trialkyl or triaryl phosphite or phosphine, such as, triethylphosphite or triphenyl phosphine, according to the general procedure of Cadogan, et al. (Ref 6) .
  • a trialkyl or triaryl phosphite or phosphine such as, triethylphosphite or triphenyl phosphine
  • Compound (29) is N-alkylated with an appropriately substituted alkyl or aryl halide XC ⁇ R ⁇ in the presence of a base, such as sodium hydride or potassium carbonate, in a noninterfering solvent, such as toluene, dimethylformamide, or dimethylsulfoxide to afford carbazole (30) .
  • a base such as sodium hydride or potassium carbonate
  • a noninterfering solvent such as toluene, dimethylformamide, or dimethylsulfoxide
  • Compound (30) is converted to the corresponding amide (22) by treatment with boron tribromide or sodium thioethoxide, followed by ammonia or an ammonium salt, such as ammonium acetate, in an inert solvent, such as water or alcohol, or with methylchloroaluminum amide in an inert solvent, such as toluene, at a temperature between 0 to 110 °C .
  • N-alkylation using for example a base such as sodium hydride and an appropriately substituted alkyl or aryl halide in dimethoxy formamide.
  • Compound (22) can be converted to product carbazole product (24) as described previously in Scheme I ⁇ Ig(b) above.
  • Conversion to the desired prodrug may be accomplished by techniques known to the skilled artisan, such as for example, by treatment with a primary or secondary halide to make an ester prodrug.
  • aprotic solvent preferably tetrahydrofuran
  • reduction of (40) is achieved using a reducing agent such as aluminum trihydride.
  • a reducing agent such as aluminum trihydride.
  • the reaction is conducted under inert atmosphere such as nitrogen, at room temperature.
  • Sulfonylation may be achieved with an appropriate acylating agent in the presence of an acid scavenger such as triethyl amine.
  • an acid scavenger such as triethyl amine.
  • intermediate (50) prepared as described in Scheme I (a) above, is first activated with an activating agent such as carbonyl diimidazole.
  • the reaction is preferably run in an aprotic polar or non-polar solvent such as tetrahydrofuran.
  • Acylation with the activated intermediate is accomplished by reacting with H2 SOR 1 ⁇ in the presence of a base, preferably diazabicycloundecene .
  • PG is an acid protecting group
  • R 22 is (C j _-C 6 ) alkoxy (C;L-C 6 ) alkyl is (Cl-Cg) alkoxy
  • Starting material (20) is O-alkylated with an alkyl halide or alkenyl halide, using a base such as NaH, in an aprotic polar solvent preferably anhydrous DMF, at ambient temperature under a nitrogen atmosphere.
  • a base such as NaH
  • an aprotic polar solvent preferably anhydrous DMF
  • the process of aromatization from a cyclohexenone functionality to a phenol functionality can be performed by treating the tetrahydrocabazole intermediate (60) with a base such as NaH in the presence of methyl benzenesulfinate in an anhydrous solvent, such as 1,4-dioxane or DMF, to form the ketosulfoxide derivative.
  • anhydrous solvent such as 1,4-dioxane or DMF
  • Conversion of the ester (61) to the amide (62) can be achieved by treating a solution of (61) in an aprotic polar solvent such as tetrahydrofuran with ammonia gas.
  • Phenolic O-alkylation of (62) with, for example, methyl bromoacetate can be carried out in anhydrous DMF at ambient temperature using CS2CO3 or K2CO3 as a base to form (63) .
  • Desired product (64) can be derived from the basic hydrolysis of ester (63) using LiOH or NaOH as a base in an H 2 0/CH 3 OH/THF solution at 50 °C for 1-2 hours .
  • R 22 is - (Ci-Cg) alkoxy (C ⁇ -Cg) alkenyl
  • hydrogenation of the double bond can be performed by treating (63) in THF using Pt ⁇ 2 as a catalysis under a hydrogen atmosphere. Desired product can then be derived as described above in Scheme I ⁇ I(g) from the basic hydrolysis of ester (63) using LiOH or NaOH as a base in an H 2 0/CH 3 OH/THF solution at 50°C for 1-2 hours.
  • PG is an acid protecting group.
  • X is halo
  • R 3 (a) is H, -0(C!-C ) alkyl, halo, - (C ⁇ Cg) alkyl, phenyl, - (C1-C4) alkylphenyl; phenyl substituted with - (Ci-Cg) alkyl, halo or -CF 3 ; -CH 2 OSi (C ⁇
  • the alcohol is alkylated to provide the formaldehyde acetal (104) which is cyclized by Lewis acid to produce the pyrano [3, 4-b] indole (105).
  • the ester is converted to the amide (106) by methylchloroaluminum amide, and then to the phenol (107) with boron tribromide.
  • the phenol is O-alkylated to give (108) which is hydrolyzed to the acid (109) .
  • PG is an acid protecting group
  • W is halo, alkyl or aryl sulfonyl
  • R 3 (a) is H, -CXC1-C4) alkyl, halo, - (C ⁇ Cg) alkyl, phenyl, - (C -C4) alkylphenyl; phenyl substituted with - (Ci-Ce) alkyl, halo or -CF 3 ; -CH 2 OSi (Ci-Cg) alkyl, furyl, thiophenyl, - (C ⁇ -Cg) hydroxyalkyl; or -
  • R 9 and R 0 are independently - (C1-C4) alkyl or
  • Conversion of the hydroxyl function of (103) to a halide or sulfate functionality is achieved by treatment with triphenylphosphine and CH3X (where X is a halogen) to make compounds of formula (111) where X is a halide; or by treatment with triethylamine and methanesulfonyl chloride to make the sulfonate.
  • Displacement with the sodium salt of thiol acetic acid gives (114) which in turn is hydrolyzed by base to the thiol (115) which is reacted with an appropriately substituted aldehyde and acid to produce the thiopyranoindoles (116) .
  • Intermediate (111) may also be reacted with sodium azide to give the azido derivative (112) which is reduced by hydrogen catalytically to give the amine which is converted to the carboline (113) with aldehyde and acid.
  • Intermediates (113), (110) and (116) may be N-alkylated, using sodium hydride and an appropriately substituted alkylhalide XCH2R 4 .
  • Lewis acids convert (126) to the thiopyrano [3, 4-b] indole (127).
  • the ester function is converted to amide using methylchloroaluminum amide, the methyl ether cleaved by boron tribromide, and the product phenol O-alkylated with bromoacetic ester to give (130) which is hydrolyzed to (131) .
  • X is halo
  • R is - (CH 2 )mR 5 .
  • Alkylation of the indole-nitrogen can then be achieved by reacting (134) with a suitable alkyl halide in the presence of potassium bis (trimethylsilyl) amide to prepare (135) .
  • the ester functionality of (135) is converted to a trimethylsilylketene acetal (136) by treatment with potassium bis (trimethylsilyl) amide and trimethylsilyl chloride.
  • Treatment of the ketene acetal (136) with bis (chloromethyl) sulfide and zinc bromide in methylene chloride affords the cyclized product (137).
  • Conversion to amide (138) can be accomplished by a einreb reaction with methylchloroaluminum amide.
  • R 3 (a) is as described in Scheme I (a) and R is as described in Scheme IV (d) .
  • N-alkylation of commercially available 4-methoxy indole (231) under basic conditions using an alkyl halide affords the N-alkyl indole (232) .
  • Acylation with a suitable acid chloride provides the glyoxalate ester product (233) which can be reduced with a variety of hydride reducing agents to give intermediate alcohols (234).
  • Conversion of the alcohol to a suitable leaving group and displacement with sulfur nucleophiles affords the thioether product (235) .
  • Conversion to the acid chloride and spontaneous cyclization affords the thioketone product (236) .
  • Cleavage of the ester can be effected under basic conditions to give the correponding acid which upon formation of the acid chloride and reaction with an appropriate amine gives the amide product (237) .
  • Cleavage of the methyl ether gives the phenol (238) which can be alkylated under basic conditions using alkyl halides to give the O-alkylated product (239) .
  • Cleavage of the ester under basic conditions gives the desired product (240) .
  • reduction of the benzylic ketone with a hydride reducing agent and subsequent deoxygenation of the resulting alcohol gives the deoxygenated product (244) .
  • Cleavage of the oxyacetic ester proceeds under basic conditions to give the desired oxyacetic acid (242) .
  • Substituted haloaniline (145) is condensed with N-benzyl- 3-piperidone to provide enamine (146). Ring closure is effected by treatment of (146) with palladium (II) acetate and the resultant product is converted to (147) by treatment with cyanogen bromide. Alkylation of (147) is accomplished by treatment with the appropriate alkyl bromide using sodium hydride as base. Hydrolysis of this N-alkylated product with basic hydrogen peroxide under standard conditions provides (148). Demethylation of (148) is carried out by treatment with boron tribromide in methylene chloride.
  • the resulting phenol (149) is converted by the standard sequence of O-alkylation with methyl bromoacetate in the presence of a base, hydrolysis with hydroxide to provide the intermediate salt which is then protonated in aqueous acid to provide desired ⁇ - carboline (150) .
  • X is halo
  • R is as defined in Scheme IV (d)
  • R3(a) j_s as defined in Scheme 1(a) .
  • Ketene acetal (136), prepared as described in Scheme I (d), is reacted with benzyl bis (methoxymethyl) amine in the presence of zinc chloride to give the tetrahydro-beta-carboline (151) .
  • amide (152) R 20 is t-butyldimethylsilyl
  • ester tetra-n-butylammonium fluoride and alkylated with, for example, ethyl bromoacetate to give ester (153).
  • Lithium hydroxide-mediated hydrolysis gives acid (154), which may be hydrogenated over an appropriate catalyst in the presence of hydrochloride acid to give the tetrahydro-beta-carboline as the hydrochloride salt (155) .
  • Compound (155) may in turn be aromatized by refluxing in carbitol with palladium on carbon to provide beta-carboline (156).
  • X is halo
  • R is as defined in Scheme IV (d); and R 3( a ) is as defined in Scheme 1(a).
  • indole (133) is successively treated with one equivalent n- butyllithium, carbon dioxide gas, one equivalent of t- butyllithium, and l-dimethylamino-2-nitroethene to give (157) .
  • Nitroalkene (157) is reduced with lithium aluminum hydride to amine (158), which is cyclized with methyl glyoxylate (Ref. 9) in refluxing ethanol to give tetrahydrocarboline (159) .
  • X is halo
  • R3 ( a ) is as defined above Scheme V(e) provides ⁇ -carboline (198) by the indicated sequence of reactions.
  • N-alkylation of 2- carboethoxyindole (190) followed by a standard two carbon homologation sequence provides 2- (3-propenoic acid) indoles (194).
  • the condensation of aldehyde (193) with malonic acid utilized a mixture of pyridine and piperidine as the base.
  • ring closure (196) was effected by treatment with bis (2,2,2- trichloroethyl) azodicarboxylate (BTCEAD) followed by zinc in acetic acid.
  • BTCEAD bis (2,2,2- trichloroethyl) azodicarboxylate
  • Reverse indoles i.e., compounds where B is carbon and D is nitrogen can be prepared as described in Scheme VIg, below.
  • Aryl hydrazines (200) are condensed with substituted prpionaldehydes to form hydrazones which are cyclized to indoles (201) by treatment with phosphorous trichloride at room temperature (Ref 1) .
  • the indoles are N-alkylated on reaction with a base such as sodium hydride and an alph-bromo ester to give indoles (202) which are cyclized to tetrahydrocarbazoles (203) by Lewis acids (e.g., aluminum chloride) or by radical initiators (e.g., tributyltin hydride) .
  • Lewis acids e.g., aluminum chloride
  • radical initiators e.g., tributyltin hydride
  • Compounds (203) can be converted to carbazoles by, for example, refluxing in a solvent such as carbitol in the presence of Pd/C.
  • X is halo
  • R is ;CH 2 ) m R ⁇
  • 4-chloroindole (210) is treated with 3 equivalents of t-butyllithium followed by carbon dioxide, 1 equivalent of n-butyllithium, l-dimethylamino-2-nitroethene, and acid to provide carboxylic acid (211), which may be esterified to give (212) .
  • Alkylation at the 1-position followed by hydrogenation provides aminoethyl indole (214).
  • Cyclization with phosgene to (215) followed by aromatization gives carboline (216) .
  • Treatment of (216) with the appropriate Weinreb reagent provides amide (217), which may be alkylated with, for example, ethyl bromoacetate and saponified with sodium hydroxide to give the carboline (218) .
  • R3(a) is as defined in Scheme 1(a), X is halo, and
  • R is (CH 2 )mR-
  • the 1,3-dione structures (228) are either commercially available or readily prepared by known techniques from commercially available starting materials.
  • a reducing agent such as SnCl2 in hydrochloric acid in an inert solvent such as ethanol
  • the amino group of (228) is protected with an appropriate protecting group, such as the, carboethoxyl, benzyl, CBZ (benzyloxycarbonyl) or BOC (tert-butoxycarbonyl) protecting group, and the like.
  • the dione (228) and aniline derivative (220) are condensed according to the general procedure of Chen, et al., (Ref 10) or Yang, et al., (Ref 11), with or without a noninterfering solvent, such as methanol, toluene, or methylene chloride, with or without an acid, such as p-toluenesulfonic acid or trifluoroacetic acid, with or without N-chlorosuccinimide and dimethyl sulfide, to afford the coupled product (221).
  • a noninterfering solvent such as methanol, toluene, or methylene chloride
  • an acid such as p-toluenesulfonic acid or trifluoroacetic acid
  • N-chlorosuccinimide and dimethyl sulfide to afford the coupled product (221).
  • Compound (221) is cyclized under basic conditions with a copper (I) salt in an inert solvent according to the general procedure of Yang, et al . , (Reft8).
  • the derivative (221) is treated with a base, such as sodium hydride, in an inert solvent, such as HMPA, at a temperature between 0 and 25 °C.
  • a copper (I) salt, such as copper (I) iodide is added and the resultant mixture stirred at a temperature between 25 and 150 °C for 1 to 48 hours to afford compound (222) .
  • Compound (221) may also be cyclized according to the general procedure of Chen, et al., (Ref 10) .
  • the derivative (221) is treated with a base, such as sodium bicarbonate, and a palladium catalyst, such as Pd(PPh_3)4, in an inert solvent, such as HMPA, at a temperature between 25 and 150 °C to afford compound (222) .
  • a base such as sodium bicarbonate
  • a palladium catalyst such as Pd(PPh_3)4
  • an inert solvent such as HMPA
  • intermediate (171) is treated with a transition metal catalyst, such as Pd (OAc) 2 (O-tol) 3P in the presence of a base such as triethylamine using a cosolvent of DMF/acetonitrile to prepare (222) .
  • a transition metal catalyst such as Pd (OAc) 2 (O-tol) 3P
  • OAc Pd
  • O-tol O-tol
  • a base such as triethylamine
  • Compound (222) is N-alkylated with an appropriately substituted benzyl halide in the presence of a base, such as sodium hydride or potassium carbonate, in a noninterfering solvent, such as dimethylformamide or dimethylsulfoxide to afford ketone (223) .
  • a base such as sodium hydride or potassium carbonate
  • a noninterfering solvent such as dimethylformamide or dimethylsulfoxide to afford ketone (223) .
  • one pot process (222) is aromatized by treatment with acetic acid and palladium on carbon in a noninterfering solvent, such as carbitol or cymene, followed by treatment with hydrogen gas and palladium on carbon to cleave the nitrogen protecting group and produce the phenolic derivative (224).
  • the ester (224) is converted to the corresponding amide (225) under standard conditions with ammonia (preferably) or an ammonium salt, such as ammonium acetate, in an inert solvent, such as water or alcohol, preferably methanol, or with MeClAlNH2 in an inert solvent, such as toluene, at a temperature between 0 to 110 °C.
  • an inert solvent such as water or alcohol, preferably methanol
  • MeClAlNH2 in an inert solvent, such as toluene
  • Alkylation of the phenolic oxygen of compound 38 with an appropriate haloester, such as methyl bromoacetate, in the presence of a base, such as cesium carbonate, potassium or sodium carbonate, in an inert solvent, such as dimethylformamide or dimethylsulfoxide affords the ester-amide (226) .
  • Other haloesters such as ethyl bromoacetate, propyl bromoa
  • compositions and method of the invention may be prepared and practiced using pyrazole sPI -2 inhibitors, which are described (together with the method of making) in US Patent Application No. 08/984261, filed December 3, 1997, the entire disclosure of which is incorporated herein by reference.
  • Suitable pyrazole compounds are represented by formula (Ih)
  • R! is phenyl, isoquinolin-3-yl, pyrazinyl, pyridin- 2-yl, pyridin-2-yl substituted at the 4- position with - (C]_-C4 ) alkyl, (Ci-C/j) alkoxyl, - CN or -(CH 2 ) n CONH 2 where n is 0-2;
  • R ⁇ is phenyl; phenyl substituted with 1 to 3 substituents selected from the group consisting of - (C1-C4) alkyl, -CN, halo, -NO2, C0 2 (C ⁇ C4) alkyl and -CF3; naphthyl; thiophene or thiophene substituted with 1 to 3 halo groups;
  • R 3 is hydrogen; phenyl; phenyl (C2-Cg) alkenyl; pyridyl; naphthyl; quinolinyl; (C1-C4) al
  • R ⁇ is cyclopentyl, cyclohexenyl, or phenyl substituted with halo or (C1-C4) alkoxy; or phenyl substituted with two substituents which, when taken together with the phenyl ring to which they are attached form a methylenedioxy ring; and m is 1 to 5; pharmaceutically acceptable salt thereof.
  • Particularly preferred are pyrazole type sPIA.2 inhibitors as follows:
  • Ri is pyridine-2-yl or pyridine-2-yl substituted at the 4-position with - (C ⁇ -C4) alkyl, (C]_- C4) alkoxy, -CN or -(CH 2 ) n CONH 2 where n is 0-2;
  • R2 is phenyl substituted with 1 to 3 substituents selected from the group consisting of - (C_- C4) alkyl, -CN, halo, -N0 2 , C0 2 (C1-C4) alkyl and -CF 3 ; and
  • R3 is phenyl; phenyl (C2-C5) alkenyl; phenyl substituted with 1 or 2 substituents selected from the group consisting of - (C1-C4 ) alkyl, -CN, -CONH2, -NO2, - CF3, halo, (C1-C4) alkoxy, CO2 (C1-C4) alky
  • pyrazole type sPIA? inhibitors useful in the method of the invention are as follows: Compounds selected from the group consisting of 3- (2- chloro-6-methylphenylsulfonylamino) -4- (2- (4- acetamido) pyridyl) -5- (3- (4-fluorophenoxy) benzylthio) - (IH) -pyrazole and 3- (2, 6-dichlorophenylsulfonylamino) -4- (2- (4-acetamido) pyridyl) -5- (3- (4- fluorophenoxy) benzylthio) - ( IH) -pyrazole .
  • the pyrazole compounds of formula Ih are prepared as described in Scheme Ih below.
  • L is a leaving group
  • an acetonitrile compound (1) is deprotonated by treatment with an excess of a strong base, such as sodium hydride, preferably under an inert gas, such as nitrogen.
  • a strong base such as sodium hydride
  • the deprotonated intermediate is treated with carbon disulfide and then alkylated twice with an appropriately substituted alkyl halide (2) of the formula R 3 (CH2) m L, where L is a leaving group, preferably bromine, and R 3 and m are as defined above, to prepare intermediate compound (3) .
  • the reaction is conducted at ambient temperatures and is substantially complete in 1 to 24 hours.
  • Cyclization to form the amino substituted pyrazole (4) is achieved by reacting intermediate (3) with hydrazine at room temperature for from about 1 to 24 hours .
  • Selective sulfonylation of the amino group of intermediate (4) can be accomplished by treatment with a sulfonyl chloride (5) of the formula R2s ⁇ 2Cl, where R ⁇ is as defined above, to prepare product (6).
  • the reaction is preferably conducted in a solvent, such as pyridine, at ambient temperature for a period of time of from 1 to 24 hours.
  • Preparation of 2, 6-dimethylphenylsulfonyl chloride can be accomplished as described in J. Org.
  • Phenyl glyoxamide sPIJV? inhibitors are described in U.S. Patent Application Serial No. 08/979446, filed November 24, 1997 (titled, Phenyl Glyoxamides as SPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference.
  • compositions and method of the invention is for treatment of a mammal, including a human, afflicted with sepsis may be practiced using phenyl glyoxamide type sPIA.2 inhibitors described as follows:
  • X is -0- or -(CH2) m -/ where m is 0 or 1; Y is -C0 2 -, -PO3-, -SO3-; R is independently -H or - (C1-C ) alkyl; Ri and R2 are each independently -H, halo or -(Ci-C 4 ) alkyl; R 3 and R 4 are each independently -H, - (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkylthio, halo, phenyl or phenyl substituted with halo; n is 1-8 ; and p is 1 when Y is -CO2- or -SO3- and 1 or 2 when Y is -PO3-; or a pharmaceutically acceptable salt thereof.
  • Phenyl glyoxylamide compounds useful in the compositons and method of the invention are prepared as follows:
  • R * is - (C1-C4 ) alkyl
  • compound (2) is internally cyclized to form compound (3).
  • the reaction is preferably conducted at temperatures from about 0 °C to room temperature and allowed to proceed for about 24 hours.
  • Aminolysis of (3) to amide (4) can be achieved by treatment with concentrated ammonium hydroxide.
  • Alkylation of the hydroxyl of compound (4) can be readily achieved by treatment with an appropriate alkylating agent, such as Br(CH2) n Y, where Y is -CO2R, - PO3R2 or SO3R and R is - (C1-C4 ) alkyl, to form intermediate (5) .
  • an appropriate alkylating agent such as Br(CH2) n Y, where Y is -CO2R, - PO3R2 or SO3R and R is - (C1-C4 ) alkyl.
  • the reaction is preferably conducted in an aprotic polar solvent, such as dimethyl formamide, in the presence of potassium carbonate and a suitable catalyst, such as potassium iodide.
  • Conversion of (5) to the carboxylic or sulfonic acid or acid salt (6) may be achieved by treatment with an appropriate base, such as aqueous sodium hydroxide, in a polar protic solvent, such as methanol.
  • a base such as aqueous sodium hydroxide
  • a polar protic solvent such as methanol.
  • n 2
  • a bromoacetal must be employed as an alkylating agent to achieve the carboxylic acid (6).
  • the alkylated moiety (5) is then converted to the acid (6) by oxidizing with sodium dichromatate in aqueous conditions.
  • conversion to the acid (6) is preferably conducted in an alkyl halide solvent, such as methylene chloride, using a dealkylating agent, such as trimethylsilyl bromide, and an excess of potassium carbonate, followed by treatment with methanol.
  • alkyl halide solvent such as methylene chloride
  • dealkylating agent such as trimethylsilyl bromide
  • R' is as defined in Scheme Ii.
  • An appropriately R ⁇ , R ⁇ substituted phenol (7) is converted to lactone (8) following the procedures described in Scheme Ii, steps (a-b) above.
  • Conversion to the intermediate (9) is accomplished by reacting (2a) with an aqueous acid, such as hydrochloric acid which affords removal of aluminum chloride from the reaction.
  • Acid (9) is converted to the corresponding acid chloride using oxalyl chloride with dimethyl formamide as a catalyst.
  • the acid chloride is recyclized to the lactone (10) on removal of the solvent, preferably under vacuum.
  • the lactone (10) is converted to the glyoxamide (11) by treatment with an excess of ammonia as described in Schemetl, step (c) , above.
  • Alkylation of (11) to prepare the ester (12), followed by conversion to the acid is accomplished according to the procedure outlined in Scheme I, steps (d) and (e) .
  • conversion of (10) to (12) can be accomplished in a one-pot procedure by treating the lactone (10) with sodium amide in an aprotic polar solvent, such as dimethylformamide, preferably at temperatures of from about 0 °C to 20 °C, followed by alkylation with an appropriate alkyl halide.
  • an aprotic polar solvent such as dimethylformamide
  • R! is hydrogen, (C -C4) alkyl, phenyl or phenyl substituted with one or two substituents selected from the group consisting of - (C ⁇ -C4) alkyl, (C -C4 ) alkoxy, phenyl (C -C4) alkyl, (C -C4 ) alkylthio, halo and phenyl;
  • R 2 is hydrogen, - (C -C4) alkyl, halo, (C -C4) alkoxy or (C ⁇ -C 4 ) alkylthio;
  • R 5 is -NH 2 or -NHNH 2 ;
  • X is R 8 (Ci-Ce) alkyl; R 8 (C 2 -C 6 ) alkenyl or phenyl substituted at the ortho position with R 8 where R 8 is
  • R 10 is -CO2R 11 , -P0 3 (R 11 ) 2 , -P0 4 (R ) or -SO3R 11 , R 11 and n is 1 to 4 as defined above, and additionally substituted with one or two substituents selected from the group consisting of hydrogen, - (C1-C4) alkyl, halo, (C1-C4) alkoxy, or two substituents which, when taken together with the phenyl ring to which they are attached, form a naphthyl group; and
  • R 9 is hydrogen or methyl or ethyl; or a pharmaceutically acceptable salt thereof.
  • Preferred pyrrole SPLA2 inhibitors useful in the method of the invention are compounds of formula Ij wherein;
  • R! is phenyl
  • R2 is methyl or ethyl
  • R 5 is -NH 2 ;
  • R ⁇ and R 7 are each hydrogen;
  • X is R 8 (C]_-Cg) alkyl or phenyl substituted at the ortho position with R 8 where
  • R 8 is -CO2R 11 ;
  • R9 is methyl or ethyl.
  • a specific suitable pyrrole SPLA2 inhibitors useful in the method of the invention is 2- [l-benzyl-2, 5-dimethyl- 4- (2-carboxyphenylmethyl) pyrrol-3-yl] glyoxamide .
  • the pyrrole compounds are prepared as follows:
  • pyrrole (2) An appropriately substituted gamma-diketone (1) is reacted with an alkylamine of the formula NHCH2R 1 to give pyrrole (2) .
  • a suitable Lewis-acid catalyst such as stannic chloride, aluminum chloride, or titanium tetrachloride (preferably stannic chloride)
  • pyrrole (2) is ring alkylated with an alkyl or arylalkyl halide compound of the formula ZCR ⁇ R 7 X where Z is a suitable halogen and R 8 of X is a protected acid or ester.
  • the reaction is preferably conducted in a halogenated hydrocarbon solvent, such as dichloromethane, at ambient temperatures and allowed to proceed for from about 1 to about 24 hours.
  • Intermediate (3) is converted to (4) by sequential treatment with oxalyl chloride followed by ammonia.
  • Selective reduction of (4) is accomplished in a two step process.
  • a hydride reduction using, for example, sodium borohydride the hydroxy intermediate (5) is prepared which can be further reduced using either catalytic or hydride reduction (preferably palladium on carbon) to prepare (6).
  • Deprotection of R 8 to the acid may be readily achieved by conventional techniques. For example, when an alkyl ester is used as a protecting group, deprotection can be accomplished by treatment with a base, such as sodium hydroxide.
  • Naphthyl glyoxamide SPLA2 inhibitors and methods of making them are described in U.S. Patent Application Serial No. 09/091079, filed December 9, 1966 (titled, “Naphthyl Glyoxamides as sPLA2 Inhibitors”) , the entire disclosure of which is incorporated herein by reference.
  • compositions and method of the invention for treatment of a mammal, including a human, afflicted with sepsis may be practiced with a naphthyl glyoxamide SPLA2 inhibitors described as follows:
  • R! and R2 are each independently hydrogen or a non- interfering substituent with the proviso that at least one of R! or R 2 must be hydrogen;
  • X is -CH2 ⁇ or -0-; and Y is (CH2) n Z where n is a number from 1-3 and Z is an acid group selected from the group consisting of CO2H, -SO 3 H or -PO(OH) 2 -
  • a specific suitable naphthyl glyoxamide SPLA2 inhibitors useful in the method of the invention has the following structural formula:
  • the naphthyl glyoxamide compounds are prepared as follows:
  • the 1, 5-dihydroxy napthalene starting material (1) is dispersed in water and then treated with 2 equivalents of potassium hydroxide.
  • the resultant solution is chilled in an ice bath and one equivalent of a strong mineral acid, such as hydrochloric acid, is added to produce the potassium saltt(2).
  • Alkylation of the radical (2) can then be accomplished by treatment with a methylating agent such as dimethyl sulfate to prepare the ether (3) .
  • Preparation of (4) is achieved by reacting the ether (3) with an appropriately substituted phenol in an Ullman-type reaction using potassium carbonate and cupric oxide .
  • De-methylation of (4) can be accomplished by treating (4) with a 40% HBr/HOAC solution at reflux in a protic polar solvent such as acetic acid, to prepare (5) . Reflux of compound (5) with oxalyl chloride and
  • Alkylation and hydrolysis of the cyclized compound (7) can be achieved by reacting (7) with an alkaliamide base, such as sodium amide, followed by treatment with an alkylating agent, such as methyl bromoacetate, using potassium iodide as a catalyst.
  • an alkaliamide base such as sodium amide
  • an alkylating agent such as methyl bromoacetate
  • the acid (9) is achieved by treating the ester (8) with an alkali base, such as aqueous sodium hydroxide, followed by treatment with a dilute aqueous mineral acid such as hydrochloric acid.
  • an organic solvent such as ethyl acetate.
  • the final product (9) can be purified using standard recrystallization procedures in a suitable organic solvent such as methylene chloride/hexane.
  • a Grignard reagent is prepared.
  • the phenyl Grignard is then reacted with 4-methoxy naphthylnitrile and the resultant compound is hydrolyzed with a dilute acid such as hydrochloric acid to form the benzoyl naphthylene compound (la) .
  • Reduction of (la) to form compound (2a) is accomplished by treatment with a reducing agent such as sodium borohydride.
  • a reducing agent such as sodium borohydride.
  • the reaction is conducted in a solvent-catalyst such as trifluoroacetic acid and initiated in an ice bath which is allowed to warm to room temperature as the reaction proceeds.
  • the desired naphthyl glyoxamide may then be prepared from (2a) according to the procedure in Scheme I starting with the chloromethylation step.
  • Phenyl acetamide SPLA2 inhibitors and methods of making them are disclosed in US Patent Application 08/976858, filed November 24 1997 (titled, "Phenyl Acetamides as SPLA2 Inhibitors”) , the entire disclosure of which is incorporated herein by reference.
  • compositions and method of the invention for treatment of a mammal, including a human, afflicted with sepsis may be practiced using a phenyl acetamide sPLA? inhibitor represented by formula (II) as follows:
  • R 1 is -H or -0(CH 2 ) n Z;
  • R 2 is -H or -OH;
  • R 3 and R ⁇ are each independently -H, halo or - (C1-C4 ) alkyl
  • R ⁇ and R ⁇ is -YR 7 and the other is -H, where Y is -0- or -CH2- and R 7 is phenyl or phenyl substituted with one or two substituents selected from the group consisting of halo, - (C -C4) alkyl, (C ⁇ -C4 ) alkoxy, phenyl or phenyl substituted with one or two halo groups;
  • Z is -CO2R. -PO3R2 or -SO3R where R is -H or - (C1-C4) alkyl; and n is 1-8; or a pharmaceutically acceptable salt, racemate or optical isomer thereof; provided that when R ⁇ is YR 7 , R ⁇ is hydrogen; and when Ri, R 2 , R 3 , R 4 and R ⁇ are hydrogen and R ⁇ is YR 7 where Y is -0-, R 7 cannot be phenyl; and when R 1 , R 2 , R 3 , R 4 and R 6 are hydrogen, R 5 is YR 7 where Y is CH2, R 7 cannot be phenyl substituted with one methoxy or two chloro groups .
  • Preferred suitable phenyl acetamide SPLA2 inhibitors useful in the composition and method of the invention are as follows:
  • R 2 , R 3 and R 4 is H, Y is oxygen or CH2, R 7 is phenyl or phenyl substituted at the meta position with one or two substituents selected from halo, - (C -C4) alkyl, (C1-C ) alkoxy, phenyl or phenyl substituted with halo and n is 4-5.
  • a specific suitable phenyl acetamide SP A2 inhibitor useful in the method of the invention is 2- (4- carboxybutoxy) -4- ( 3-phenylphenoxy) phenylacetamide .
  • phenyl acetamide inhibitors are prepared as follows:
  • X is halo
  • R 8 and R ⁇ are each independently -H, halo, - (C1-C4) alkyl, (C1-C4) alkoxy, phenyl or phenyl substituted with one or two halo groups; and PG is a carboxyl protecting group
  • An appropriately substituted carboxy-protected halophenyl compound (1), where the halogen is preferably bromine, is coupled with an appropriately substituted phenol (2) under modified Ullmann conditions, by refluxing with potassium carbonate and cupric oxide in an aprotic polar solvent, such as pyridine, under an inert gas such as argon. The reaction is substantially complete in 1-24 hours.
  • Intermediate (3) is deprotected by treatment with a base such as aqueous potassium hydroxide using a solvent, such as diethylene glycol.
  • a base such as aqueous potassium hydroxide
  • a solvent such as diethylene glycol.
  • amide (5) Conversion to the amide (5) can then be readily achieved by treatment first with oxalyl chloride in an alkyl halide solvent, such as methylene chloride, using dimethylformamide as a catalyst, at temperatures of from about 0 °C to ambient temperature, followed by treatment with an excess of ammonia gas, again in an alkyl halide solvent .
  • alkyl halide solvent such as methylene chloride
  • dimethylformamide as a catalyst
  • compounds of formula I can be prepared according to the procedure of Scheme I (b) , below.
  • the substituted phenol (2) is coupled with an appropriately substituted benzyl halide (6) as described in Scheme 1(a), step a, above, to prepare (7).
  • Halogenation of (7) is achieved using a halogenating agent, such as N-bromosuccinimide and a catalyst, such as
  • R 8 and R ⁇ are as shown in Scheme I (a) , X is halo.
  • X is a halogen .
  • diphenyl compound (11) is treated with paraformaldehyde and a halogenating agent, such as 40% hydrogen bromide in acetic acid.
  • a halogenating agent such as 40% hydrogen bromide in acetic acid.
  • Two positional isomers result with the X substituent at either the meta or para position of the phenyl ring to which it is attached.
  • Displacement of the halogen to prepare the nitrile isomers (13) can be achieved by treatment of (12) with sodium cyanide in dimethylformamide as described in Schemetl (b) , step (c) , above.
  • the isomers can then be readily separated by conventional chromatographic techniques and each isomer may be converted to its respective amide (14) by treatment with hydrogen peroxide and potassium carbonate in an aprotic polar solvent, such as dimethylsulfoxide.
  • Intermediate (16) is prepared by refluxing an appropriately substituted diphenyl compound (15) with oxalyl chloride in an alkyl halide solvent, such as chloroform.
  • an alkyl halide solvent such as chloroform.
  • the reaction is catalyzed with 4, 4-N-dimethylaminopyridine.
  • Cyclization to the lactone (17) can be achieved under Friedel-Crafts conditions using a suitable metal halide, such as aluminum chloride, as the catalyst.
  • Conversion to the glyoxamide (18) can be achieved by aminolysis of the lactone ring using concentrated ammonium hydroxide .
  • Alkylation of the hydroxy group to prepare the desired alkyl-linked ester (19) occurs by treatment of
  • (19) is achieved by treatment with a suitable reducing agent, such as sodium borohydride in methanol, preferably at temperatures of from 0°-20 °C, to prepare the intermediate (20) .
  • a suitable reducing agent such as sodium borohydride in methanol
  • the desired acid or acid salt (21) can be accomplished by treatment with a suitable base, such as sodium hydroxide.
  • composition and method of the invention for treatment of a mammal, including a human, afflicted with sepsis is practiced using a naphthyl acetamide SPLA2 inhibitor represented by formula (Im)as follows:
  • R and R 2 are each independently hydrogen or a non- interfering substituent with the proviso that at least one of R! and R 2 must be hydrogen;
  • R 3 is hydrogen, -0(CH 2 ) n Y, - ⁇ 0 n m Y where n is from 2 to 4 and Y is -CO2H, -PO3H2 or SO3H; and X is -0- or -CH2-.
  • an appropriately substituted l-bromo-4-methylnapthalene and an appropriately substituted phenol are dissolved in an aprotic polar solvent such as pyridine.
  • the mixture is treated with an excess of potassium carbonate and an excess of copper-bronze and refluxed under a nitrogen blanket to produce (1).
  • Bromination of compound (1) to produce (2) is accomplished by refluxing (1) with a brominating agent, such as N-bromosuccinamide, in a non-polar alkyl halide solvent, such as carbon tetrachloride, using 2,2- azobisisobutyronitrile as a catalyst.
  • Treatment of (2) with sodium cyanide produces (3) .
  • This reaction is best conducted in an aprotic polar solvent, such as dimethyl sulfoxide (DMSO) , while heating to a temperature of about 60 °C.
  • DMSO dimethyl sulfoxide
  • Hydrolysis of the cyano compound (3) to produce the acid (4) is accomplished in two steps.
  • a polar protic solvent such as diethylene glycol as a cosolvent
  • the cyano compound (3) is treated with an alkali metal base, such as potassium hydroxide, and the mixture is heated to about 90-95 °C.
  • the resultant product is then reacted with a strong mineral acid such as hydrochloric acid.
  • Conversion of (4) to the desired naphthyl acetamide compound (5) is accomplished by another two-step process.
  • the acid (4) is dissolved in an alkyl halide solvent such as methylene chloride.
  • the acid/alkyl halide solution is chilled in an ice bath then treated with oxalyl chloride, using dimethylformamide (DMF) as a catalyst, to produce the acid chloride.
  • the solution is allowed to warm to room temperature and then treated with ammonia gas at room temperature to produce (5) .
  • the desired product (5) can be purified using standard recrystallization procedures in a suitable organic solvent, preferably methylene chloride/hexane .
  • Compound (la) is prepared by a grignard reaction.
  • the Grignard reagent starting material is prepared by reacting an appropriately substituted phenyl bromide with magnesium and ether.
  • the reagent is then reacted with an appropriately substituted naphthyl nitrile and the resultant compound is hydrolyzed with an aqueous acid such as hydrochloric acid to form the benzoyl napthyl (la) .
  • Reduction of (la) is accomplished by treatment with a molar excess of a reducing agent such as sodium borohydride.
  • a reducing agent such as sodium borohydride.
  • the reaction is initiated in an ice bath using a solvent-catalyst such as trifluoroacetic acid and then allowed to warm to room temperature as the reduction proceeds .
  • Chloromethylation of (2a) is achieved by treatment with an excess of formaldehyde and concentrated hydrochloric acid in a polar acidic solvent such as an acetic/phosphoric acid mixture.
  • the reaction is best conducted at a temperature of about 90 °C.
  • the nitrile 4 (a) is prepared by a nucleophilic displacement of the chloride compound (3a) ith cyanide.
  • the reaction is conducted by refluxing (3a) with a slight molar excess in an aprotic polar solvent of sodium cyanide such as dimethylformamide (DMF) for about five hours, then allowing the reaction to continues while it cools to room temperature.
  • an aprotic polar solvent of sodium cyanide such as dimethylformamide (DMF)
  • the desired naphthylamide (5a) is then prepared from the nitrile (4a) in a three-step process.
  • a solution of nitrile (4a) dissolved in an aprotic polar solvent such as DMSO, potassium carbonate is added to make the nitrile solution slightly basic.
  • Hydrolysis of the nitrile is then achieved by treatment with an aqueous hydrogen peroxide solution. Crystallization of the naphthyl acetamide may be accomplished by adding water to the peroxide solution.
  • R 3 is other than hydrogen
  • a protected phenol such as a methoxy group
  • the process is conducted, as described above, to prepare compounds (1) - (3) .
  • Acid hydrolysis of the cyano group (3) and deprotection of the protected phenol can be accomplished by treating (3) with a 40% hydrogen bromide solution in acetic acid.
  • the deprotected phenol can then be reacted to prepare the appropriate substituent at the 6-position of the napthyl ring.
  • preparation of compounds where R 3 is -0(CH2) n COOH can be achieved by alkyalting the phenol with an appropriate alkyl halide followed by conversion to the acid by treatment with a base such as aqueous sodium hydroxide followed by dilute hydrochloric acid.
  • substituted phenol and phenyl bromide starting materials are either commercially available or can be readily prepared by known techniques from commercially available starting materials. All other reactants and reagents used to prepare the compounds of the present invention are commercially available. Most Preferred SPLA2 inhibitors: lH-indole-3-glyoxylamide SPLA2 inhibitors and carbazole sPLA2 inhibitors (as described, supra.) are most preferred for the compositions and method this invention.
  • Recombinant human Protein C was produced in Human Kidney 293 cells by techniques well known to the skilled artisan such as those set forth in Yan, U.S. Patent No. 4,981,952, the entire disclosure of which is herein incorporated by reference.
  • the gene encoding human Protein C is disclosed and claimed in Bang et al., U.S. Patent No. 4,775,624, the entire disclosure of which is incorporated herein by reference.
  • the plasmid used to express human Protein C in 293 cells was plasmid pLPC which is disclosed in Bang et al./ U.S. Patent No. 4,992,373, the entire disclosure of which is incorporated herein by reference.
  • the construction of plasmid pLPC is also described in European Patent Publication No.
  • the human Protein C was separated from the culture fluid by an adaptation of the techniques of Yan, U.S.
  • Patent No. 4,981,952 the entire disclosure of which is herein incorporated by reference.
  • the clarified medium was made 4 mM in EDTA before it was absorbed to an anion exchange resin (Fast-Flow Q, Pharmacia) .
  • an anion exchange resin Frazier-Flow Q, Pharmacia
  • the eluted protein was greater than 95% pure after elution as judged by SDS-polyacrylamide gel electrophoresis .
  • Bovine thrombin was coupled to Activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 4 °C. The coupling reaction was done on resin already packed into a column using approximately 5000 units thrombin/ml resin. The thrombin solution was circulated through the column for approximately 3 hours before adding MEA to a concentration of 0.6 ml/1 of circulating solution. The MEA-containing solution was circulated for an additional 10-12 hours to assure complete blockage of the unreacted amines on the resin.
  • the thrombin-coupled resin was washed with 10 column volumes of 1 M NaCl, 20 mM Tris, pH 6.5 to remove all non- specifically bound protein, and was used in activation reactions after equilibrating in activation buffer.
  • Purified rHPC was made 5 mM in EDTA (to chelate any residual calcium) and diluted to a concentration of 2 mg/ml with 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5. This material was passed through a thrombin column equilibrated at 37°C with 50 mM NaCl and either 20 mM Tris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate was adjusted to allow for approximately 20 min. of contact time between the rHPC and thrombin resin. The effluent was collected and immediately assayed for amidolytic activity.
  • the material did not have a specific activity (amidolytic) comparable to an established standard of aPC, it was recycled over the thrombin column to activate the rHPC to completion. This was followed by 1:1 dilution of the material with 20 mM buffer as above, with a pH of either 7.4 or 6.5 to keep the aPC at lower concentrations while it awaited the next processing step.
  • Removal of leached thrombin from the aPC material was accomplished by binding the aPC to an anion exchange resin (Fast Flow Q, Pharmacia) equilibrated in activation buffer (either 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5) with 150 mM NaCl. Thrombin does not interact with the anion exchange resin under these conditions, but passes through the column into the sample application effluent.
  • activation buffer either 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5
  • a 2-6 column volume wash with 20 mM equilibration buffer is done before eluting the bound aPC with a step elution using 0.4 M NaCl in either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Higher volume washes of the column facilitated more complete removal of the dodecapeptide.
  • the material eluted from this column was stored either in a frozen solution (-20 °C) or as a lyophilized powder.
  • the anticoagulant activity of activated Protein C was determined by measuring the prolongation of the clotting time in the activated partial thromboplastin time (APTT) clotting assay.
  • a standard curve was prepared in dilution buffer (1 mg/ml radioimmunoassay grade BSA, 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3) ranging in Protein C concentration from 125-1000 ng/ml, while samples were prepared at several dilutions in this concentration range.
  • dilution buffer 1 mg/ml radioimmunoassay grade BSA, 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3
  • compositions of the invention comprises as essential ingredients:
  • composition (a) an SPLA2 inhibitor, and (b) Activated Protein C.
  • the composition must be in a form which; (i) is itself in a liquid form suitable for administration by injection or, (ii) is in a form which is easily dissolved or suspended, or dispersed or emulsified into a liquid medium which is then suitable for administration by injection.
  • the pharmaceutical composition of the invention is prepared in injectable form it is a composition comprising as ingredients:
  • the essential ingredients (a) an sPLA? inhibitor and (b) Activated Protein C are present in the formulation in such proportion that a dose of the formulation provides a pharmaceutically effective amount of each ingredient to the patient being treated.
  • the weight ratio of sPLA 2 to activated protein C is from 1000:1 to 10000000:1 and preferably from 100:1 to 1000000:1.
  • An effective dosage of activated Protein C in human patients is considered to be between O.land 100 ⁇ g/kg/day. Preferably, the dosage is between 1 and 50 ⁇ g/kg/day. A most preferred dosage of activated Protein C is between 1 and 25 ⁇ g/kg/day.
  • An effective dosage of an SPLA2 inhibitor in human patients is considered to be between 0.1 and 2000 mg/kg/day. Preferably, the dosage is between 1 and 100 mg/kg/day.
  • Activated Protein C are co-present and may be mixed in any homogeneous or non-homogeneous manner or adjacently or otherwise promixately placed together in an individual dosage unit suitable for practicing the method of the invention.
  • the dosage unit of the SPLA2 inhibitor will usually be admixed with a carrier or inert ingredients, or diluted by a carrier, or enclosed within a carrier which may be in the form of a ampoule, capsule, time release dosing device, sachet, paper or other container.
  • the carrier when it serves as a diluent, it may be a solid, semi-solid, paste, or liquid material which acts as a vehicle, or can be in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , or ointment, containing, for example, up to 10% by weight of the active compound.
  • the dosage unit of the Activated Protein C will usually be admixed with a liquid carrier and/or other inert ingredients or enclosed within a carrier which may be in the form of a ampoule, bottle, time release dosing device or other container.
  • a carrier which may be in the form of a ampoule, bottle, time release dosing device or other container.
  • the carrier serves as a diluent, it may be a liquid material which acts as a vehicle, or can be in the form of solutions containing, for example, up to 10% by weight of the active compound.
  • the Activated Protein C ingredient should be in an injectable liquid form immediately prior to use, however, it may be made in a storable form which is not a liquid but is easily convertable to a liquid (e.g., paste, liquid adsorbed on a solid, etc.)
  • the carrier may be an injectable liquid medium such as is well known in the art.
  • the injectable liquid must be such that permits parenteral administration, that is, introduction of substances to a mammal being treated by intervenous, subcuataneous, intramuscular, or intramedullary injection. Intravenous injection is most preferred as a means of administration.
  • the Active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile water containing saline and/or sugars and/or suspension agents or a mixture of both.
  • a pharmaceutically acceptable carrier such as sterile water, sterile water containing saline and/or sugars and/or suspension agents or a mixture of both.
  • the compounds of the invention may be dissolved in at a concentration of 2 mg/ml in a 4% dextrose/0.5% Na citrate aqueous solution.
  • the SPLA2 inhibitor when separate from the
  • Activated Protein C may be in the form of powder, tablet or capsule.
  • a solid carrier can be one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, binders, tablet disintegrating agents and encapsulating material. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting waxes, and cocoa butter.
  • compositions are useful (as stated) for either the SPLA2 inhibitor alone, or the Active Ingredient which is a combination of (a) SPLA2 inhibitor and (b) Activated Protein .
  • the SPLA2 inhibitor typically, from 10 mg to 1000 mg of the SPLA2 inhibitor is used in a unit dose of the formulation.
  • Hard gelatin capsules are prepared using the following ingredients:

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Abstract

The present invention provides a method of prevention and treatment for sepsis for mammals. Said treatment is a combination therapy of Activated Protein C and an sPLA2 inhibitor.

Description

Combination Therapy for the Treatment of Sepsis
Field of the Invention
This invention relates to the field of medicine and specifically to the treatment of sepsis.
Background of the Invention
Reaction to infection involves a great many bodily responses. Onset of a systemic inflammatory response to causes such as microbial invasion is called sepsis. When the body is overwhelmed and the defensive mechanisms fail (viz., sepsis, severe sepsis, septic shock) the body is a great risk.
The prior art has recently disclosed two relatively new pharmaceutical agents (and related methods of use) for treating sepsis. These agents are (a) Activated Protein C, and (b) sPIJ ? inhibitors.
A. Activated Protein C and its Role in Sepsis
Activated Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of vascular homeostasis by inactivating Factors Va and VIIIa in the coagulation cascade. Human Protein C is made in vivo primarily in the liver as a single polypeptide of 461 amino acids.
In concert with other proteins, Protein C functions as an important down-regulator of blood coagulation factors that promote thrombosis. In other words, the Protein C enzyme system represents a major physiological mechanism of anticoagulation.
The critical role of protein C in controlling hemostasis is exemplified by the increased rate of thrombosis in heterozygous deficiency, protein C resistance (e.g., due to the common Factor V Leiden mutation) and the fatal outcome of untreated homozygous protein C deficiency. Human activated protein C, both plasma-derived and recombinant, have been shown to be effective and safe antithrombotic agents in a variety of animal models for both venous and arterial thrombosis. Activated protein C in recent clinical studies has been shown to be effective in human thrombotic diseases including the treatment of protein C deficiencies and microvascular thrombosis, such as disseminated intravascular coagulation associated with sepsis.
B. SPLA2 and SPL 2 Inhibitors and Their Role in Sepsis
The structure and physical properties of human non-pancreatic secretory phospholipase A2 (hereinafter called, "sPLA2") has been thoroughly described in two articles, namely, "Cloning and Recombinant Expression of Phospholipase A2 Present in Rheumatoid Arthritic Synovial Fluid" by Seilhamer, Jeffrey J.; Pruzanski,
Waldemar; Vadas Peter; Plant, Shelley; Miller, Judy A.; Kloss, Jean; and Johnson, Lorin K.; The Journal of Biological Chemistry, Vol. 264, No. 10, Issue of April 5, pp. 5335-5338, 1989; and "Structure and Properties of a Human Non-pancreatic Phospholipase A2" by Kramer, Ruth M.; Hession, Catherine; Johansen, Berit; Hayes, Gretchen; McGray, Paula; Chow, E. Pingchang; Tizard, Richard; and Pepinsky, R. Blake; The Journal of Biological Chemistry, Vol. 264, No. 10, Issue of April 5, pp. 5768-5775, 1989; the disclosures of which are incorporated herein by reference. It is believed that sPLA2 is a rate limiting enzyme in the arachidonic acid cascade which hydrolyzes membrane phospholipids . Thus, it is important to develop compounds which inhibit sPLA2 mediated release of fatty acids (e.g., arachidonic acid). Such compounds would be of value in general treatment of conditions induced and/or maintained by overproduction of sPLA2, such as sepsis.
Summary of the Invention
It is a discovery of this invention that sepsis is prevented and treated in an advantageous or superior manner by a combination therapy using (i) activated human Protein C and (ii) SPLA2 inhibitors. The combination therapy of aPC with an SPLA2 inhibitor improves the human body's response to the disease state of sepsis by mounting a timely, multi- faceted attack as both an anti-inflammatory agent and an anticoagulant. This combination therapy results in either a multi-mechanistic or synergistically effective treatment than is obtained with use of the individual aPC or SPLA2 pharmaceutical agents. The benefits are an increased efficacy of treatment or prevention, a decreased effective dosage, and/or a decreased duration of therapy.
This invention is a pharmaceutical composition comprising: (a) an SPLA2 inhibitor, and
(b) Activated Protein C.
This invention is also a method of treating or preventing sepsis by administering to a mammal in need thereof a therapeutically effective amount of (a) an
SPLA2 inhibitor and a therapeutically effective amount of (b) Activated Protein C; wherein (a) and (b) are both administered within a therapeutically effective interval.
Detailed Description
I. DEFINITIONS:
For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below. aPC - Activated human Protein C, also called, Activated Protein C.
APTT - activated partial thromboplastin time. hPC - human Protein C zymogen. rhPC - recombinant human Protein C zymogen. The terms "aPC, " "Activated human Protein C, " "Activated Protein C, " "raPC, " "recombinant Activated Protein C" are synonymous for the purpose and practice of this invention.
Protein C Activity - any property of activated human Protein C or its derivatives responsible for proteolytic, amidolytic, esterolytic, and biological (anticoagulant or pro-fibrinolytic) activities. Methods for testing for Protein C anticoagulant and amidolytic activity are well known in the art, i.e., see Grinnell et.al., 1987, Bio/Technology 5:1189-1192. rhaPC - Recombinant activated human protein C, produced by activating r-HPC in vitro or by direct secretion of the activated form of Protein C from prokaryotic cells, eukaryotic cells, or from transgenic animals . zymogen - an enzymatically inactive precursor of a proteolytic enzyme. Protein C zymogen, as used herein, refers to secreted, inactive forms, whether one chain or two chain, of protein C. sPIJJ?- secretary phospholipase A2 SPL 2 inhibitor means a compound which inhibits SPLA2 mediated release of fatty acid. sepsis - Sepsis is defined as a systemic inflammatory response to infection, associated with and mediated by the activation of a number of host defense mechanisms including the cytokine network, leukocytes, and the complement and coagulation/fibrinolysis systems (Mesters et al . , Blood 88:881-886, 1996). Disseminated intravascular coagulation (DIC) , with widespread deposition of fibrin in the microvasculature of various organs, is an early manifestation of sepsis/septic shock. DIC is an important mediator in the development of the multiple organ failure syndrome and contributes to the poor prognosis of patients with septic shock (Fourrier et al . , Chest 101:816-823, 1992). "Sepsis" includes severe sepsis, septic shock, septisemia, and related disease states.
The phrase "therapeutically effective amount" is an amount of (a) sPIJJ? inhibitor or an amount of (b) aPC which is effective to prevent or ameliorate sepsis.
The phrase "therapeutically effective interval" is a period of time beginning when one of either (a) the sPI .2 inhibitor or (b) aPC is administered to a mammal and ending at the limit of the beneficial effect in preventing or ameliorating psesis of (a) or (b) .
The phrase "therapeutically effective combination", used in the practice of this invention, means administration of both (a) SPLA2 inhibitor and (b)
Activated protein C, either simultaneously or separately.
The term, "Active ingredient" as used herein refers to a combination of (a) sPIA? inhibitor and (b) Activated Protein C co-present in a pharmaceutical formulation for the delivery of a treatment regimen that applies this invention.
The term, "injectable liquid carrier" refers to a liquid medium containing either or both of (a) sPLA2 inhibitor, or (b) Activated Protein C; wherein (a) and (b) are independently dissolved, suspended, dispersed, or emulsified in the liquid medium. Other defined chemical terms: alkyl - a straight or branched chain monovalent hydrocarbon radical such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tertiary butyl, isobutyl, sec- butyl, n-pentyl, and n-hexyl. alkenyl - a straight chain or branched monovalent hydrocarbon group having the stated number range of carbon atoms, and typified by groups such as vinyl, propenyl, crotonyl, isopentenyl, and various butenyl isomers . hydrocarbyl - an organic group containing only carbon and hydrogen. halo - fluoro, chloro, bro o, or iodo. heterocyclic radical- radicals derived from monocyclic or polycyclic, saturated or unsaturated, substituted or unsubstituted heterocyclic nuclei having 5 to 14 ring atoms and containing from 1 to 3 hetero atoms selected from the group consisting of nitrogen, oxygen or sulfur, phenylpyridinyl, benzylpyridinyl, pyrimidinyl, phenylpyrimidinyl, pyrazinyl, 1, 3, 5-triazinyl, quinolinyl, phthalazinyl, quinazolinyl, morpholino, thiomorpholino, homopiperazinyl, tetrahydrofuranyl, tetrahydropyranyl, oxacanyl, 1, 3-dioxolanyl, 1,3- dioxanyl, 1, 4-dioxanyl, tetrahydrothiopheneyl, pentamethylenesulfadyl, 1, 3-dithianyl, 1, 4-dithianyl, 1, 4-thioxanyl, azetidinyl, hexamethyleneiminium, heptamethyleneiminium, piperazinyl and quinoxalinyl . carbocyclic radical - a radical derived from a saturated or unsaturated, substituted or unsubstituted 5- to 14-membered organic nucleus whose ring forming atoms (other than hydrogen) are solely carbon atoms. Typical carbocyclic radicals are cycloalkyl, cycloalkenyl, phenyl, naphthyl, norbornanyl, bicycloheptadienyl, tolulyl, xylenyl, indenyl, stilbenyl, terphenylyl, diphenylethylenyl, phenyl-cyclohexenyl, acenaphthylenyl, and anthracenyl, biphenyl, bibenzylyl and related bibenzylyl homologues represented by the formula (bb) ,
Figure imgf000009_0001
where n is a number from 1 to 8. non-interfering substituent - radicals suitable for substitution at positions 4, 5, 6, and/or 7 on the indole nucleus (as hereinafter depicted in Formula I) and radical (s) suitable for substitution on the heterocyclic radical and carbocyclic radical as defined above. Illustrative non-interfering radicals are Ci-Cβ alkyl, C2-C5 alkenyl, C2~Cg alkenyl, C7-C12 aralkyl, C7-C12 alkaryl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, phenyl, tolulyl, xylenyl, biphenyl, Cχ-C6 alkoxy, C2~Cg alkenyloxy, C2-C5 alkenyloxy, C2~Ci2 alkoxyalkyl, C2_ C12 alkoxyalkyloxy, C2-C12 alkylcarbonyl, C2-C12 alkylcarbonylamino, C2-C12 alkoxyamino, C2-C12 alkoxyaminocarbonyl, C1-C12 alkylamino, Cχ-C6 alkylthio, C2-C12 alkylthiocarbonyl, C1-C6 alkylsulfinyl, Cχ-C6 alkylsulfonyl, C2-C6 haloalkoxy, C1-C6 haloalkylsulfonyl, C2~C6 haloalkyl, Cχ-C6 hydroxyalkyl, -C(0)0(Cι-C6 alkyl), - (CH2) n~0- (C1-C6 alkyl), benzyloxy, phenoxy, phenylthio, - (CONHSO2R) , - CHO, amino, amidino, bromo, carbamyl, carboxyl, carbalkoxy, - (CH2) n~C02H, chloro, cyano, cyanoguanidinyl, fluoro, guanidino, hydrazide, hydrazino, hydrazido, hydroxy, hydroxyamino, iodo, nitro, phosphono, -SO3H, thioacetal, thiocarbonyl, and Cχ-C6 carbonyl; where n is from 1 to 8. acidic group - an organic group which when attached to an indole nucleus, through suitable linking atoms (hereinafter defined as the "acid linker"), acts as a proton donor capable of hydrogen bonding. Illustrative of an acidic group are the following: -5-tetrazolyl,
-SO3H, 0
-p t. OH
OR89 0
-0- -P t OH
OR89
0
Figure imgf000011_0001
0
•o- OH
OH
Figure imgf000011_0002
0
R99
•0 P 0 (CH2)ii N R99
OR89 R99
Figure imgf000012_0001
0
c- OH
Figure imgf000012_0002
where n is 1 to 8, R89 is a metal or Cχ-Cιo alkyl, and R99 is hydrogen or Cχ-Cιo alkyl. acid linker - a divalent linking group symbolized as, -(La)-, which has the function of joining the 4 or 5 position of the indole nucleus to an acidic group in the general relationship:
Indole Nucleus "(La)" Acidic Group
acid linker length - the number of atoms (excluding hydrogen) in the shortest chain of the linking group -(La)- that connects the 4 or 5 position of the indole nucleus with the acidic group. The presence of a carbocyclic ring in -(La)- counts as the number of atoms approximately equivalent to the calculated diameter of the carbocyclic ring. Thus, a benzene or cyclohexane ring in the acid linker counts as 2 atoms in calculating -lithe length of - (La) - . Illustrative acid linker groups are;
Figure imgf000013_0001
Figure imgf000013_0002
wherein, groups (a) , (b) , and (c) have acid linker lengths of 5, 7, and 2, respectively. amine - primary, secondary and tertiary amines. mammal - includes human mammalian - includes human. alkylene chain of 1 or 2 carbon atoms - the divalent radicals, -CH2-CH2- and -CH2-. pharmaceutically acceptable - the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The term, "carbazole SPLA2 inhibitors" includes SPLA2 inhibitors having either a carbazole or a tetrahydrocarbazole nucleus.
The term, "Active ingredient" as used herein refers to a combination of (a) SPLA2 inhibitor and (b) Activated Protein C existing as compounds or mixture in a pharmaceutical formulation for the delivery of a treatment regimen that applies this invention.
II. sPLA2 INHIBITORS USEFUL IN THE METHOD OF THE INVENTION:
All types of SPLA2 inhibitors are generally useful in the practice in this invention.
Exemplary of classes of suitable SPLA2 useful in the the method of the invention for treatment of sepsis are the following:
1H-indole-3-glyoxylamides lH-indole-3-hydrazides
1H-indole-3-acetamides
1H-indole-1-glyoxylamides
1H-indole-1-hydrazides
1H-indole-1-acetamides indolizine-1-acetamides indolizine-1-acetic acid hydrazides indolizine-1-glyoxylamides indene-1-acetamides indene-1-acetic acid hydrazides indene-1-glyoxylamides carbazoles & tetrahydrocarbazoles pyrazoles phenyl glyoxamides pyrroles naphthyl glyoxamides phenyl acetamides naphthyl acetamides
Each of the above SPLA2 inhibitor types is discussed in the following sections (a) through (m) wherein details of their molecular configuration are given along with methods for their preparation. a) The lH-indole-3-glyoxylamide SPLA2 inhibitors and method of making them are described in U.S. Patent 5,654,326, the entire disclosure of which is incorporated herein by reference. Another method of making lH-indole-3-glyoxylamide SPLA2 inhibitors is described in United States Patent Application Serial No. 09/105381, filed June 26, 1998 and titled, "Process for Preparing 4-substituted l-H-Indole-3-glyoxyamides" the entire disclosure of which is incorporated herein by reference.
United States Patent Application Serial No. 09/105381 discloses the following process having steps (a) thru (i) : Preparing a compound of the formula (I) or a pharmaceutically acceptable salt or prodrug derivative thereof
Figure imgf000015_0001
wherein:
R-- is selected from the group consisting of -C7-C20 alkyl,
Figure imgf000016_0001
Figure imgf000016_0002
Figure imgf000016_0003
where
R-LO is selected from the group consisting of halo, Cχ-Cχo alkyl, Cχ-Cχo alkoxy, -S- (Cχ-Cχo alkyl) and halo (C -Cχø) alkyl, and t is an integer from 0 to 5 both inclusive; R2 is selected from the group consisting of hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (Cχ-C2 alkyl), -S- (Cχ-C2 alkyl), aryl, aryloxy and HET;
R^ is selected from the group consisting of -CO2H, -SO3H and -P(O) (OH) 2 or salt and prodrug derivatives thereof; and
R^, R6 aπd R7 are each independently selected from the group consisting of hydrogen, (C -Cg) alkyl, (C ~ Cg) alkoxy, halo (Cχ-Cg) alkoxy, halo (C2~C ) alkyl, bromo, chloro, fluoro, iodo and aryl; which process comprises the steps of: a) halogenating a compound of formula X
Figure imgf000017_0001
X where R8 is (Cχ-Cg) alkyl, aryl or HET; with SO2CI2 to form a compound of formula
IX
Figure imgf000017_0002
b) hydrolyzing and decarboxylating a compound of formula IX
Figure imgf000017_0003
to form a compound of formula VIII
Figure imgf000017_0004
I c) alkylating a compound of formula VII
Figure imgf000018_0001
with a compound of formula VIII
Figure imgf000018_0002
II
to form a compound of formula VI
Figure imgf000018_0003
d) aminating and dehydrating a compound of formula VI
Figure imgf000018_0004
with an amine of the formula R^NH2 in the presence of a solvent that forms and azeotrope with water to form a compound of formula V;
e) oxidizing a compound of formula V
Figure imgf000019_0001
by refluxing in a polar hydrocarbon solvent having a boiling point of at least 150 °C and a dielectric constant of at least 10 in the presence of a catalyst to form a compound of formula IV
Figure imgf000019_0002
alkylating a compound of the formula IV
Figure imgf000019_0003
with an alkylating agent of the formula XCH2R4a where X is a leaving group and R4a is -Cθ2R4b, -S03R b, -P(O) (OR4b)2, or -P (0) (OR4b) H, where R4b is an acid protecting group to form a compound of formula III
Figure imgf000020_0001
g) reacting a compound of formula III
Figure imgf000020_0002
with oxalyl chloride and ammonia to form a compound of formula II
Figure imgf000020_0003
h) optionally hydrolyzing a compound of formula II
Figure imgf000021_0001
to form a compound of formula I; and i) optionally salifying a compound of formula
I.
The synthesis methodology for making the 1H- indole-3-glyoxylamide SPLA2 inhibitor may be by any suitable means available to one skilled in the chemical arts. However, such methodology is not part of the present invention which is a method of use, specifically, a method of treating mammal afflicted or susceptible to sepsis.
The method of the invention is for treatment of a mammal, including a human, afflicted sepsis, said method comprising administering to said human a therapeutically effective amount of the compound represented by formula (la), or a pharmaceutically acceptable salt or prodrug derivative thereof;
Figure imgf000021_0002
wherein ; both X are oxygen; R is selected from the group consisting of
Figure imgf000022_0001
and
Figure imgf000022_0002
where R]_Q is a radical independently selected from halo, C -Cχo alkyl, Cχ-Cχo alkoxy, -S-(Cχ-Cχo alkyl), and Cχ-Cχo haloalkyl and t is a number from 0 to 5;
R2 is selected from the group; halo, cyclopropyl, methyl, ethyl, and propyl; R4 and R5 are independently selected from hydrogen, a non-interfering substituent, or the group, - (La) - (acidic group), wherein _(La)- is an acid linker; provided, the acid linker group, -(La)-, for R4 is selected from the group consisting of;
Figure imgf000023_0001
and provided, the acid linker, _(La)-/ for R5 is selected from group consisting of;
Figure imgf000024_0001
Figure imgf000024_0002
Figure imgf000024_0003
Figure imgf000025_0001
Figure imgf000025_0002
Figure imgf000025_0003
wherein Rg and Rg5 are each independently selected from hydrogen, C -C g alkyl, aryl, C -Cχo alkaryl, Cχ~ C aralkyl, carboxy, carbalkoxy, and halo; and provided, that at least one of R4 and R5 must be the group, - (La) - (acidic group) and wherein the (acidic group) on the group - (La) - (acidic group) of R4 or R5 is selected from -CO2H, -SO3H, or -P(O) (OH) 2; Rg and R7 are each independently selected form hydrogen and non-interfering substituents, with the non-interfering substituents being selected from the group consisting of the following: Cχ-Cg alkyl, C2~Cg alkenyl, C2~C alkynyl, C7-C12 aralkyl, C7-C12 alkaryl, C3-C8 cycloalkyl, C3-C3 cycloalkenyl, phenyl, tolulyl, xylenyl, biphenyl, Cχ-Cg alkoxy, C2~C alkenyloxy, C2- Cg alkynyloxy, C2-C12 alkoxyalkyl, C2~C 2 alkoxyalkyloxy, C2-C12 alkylcarbonyl, C2_C 2 alkylcarbonylamino, C2_C 2 alkoxyamino, C2-C12 alkoxyaminocarbonyl, C2-C12 alkylamino, Cχ-Cg alkylthio, C2~Cχ2 alkylthiocarbonyl, Cχ-Cg alkylsulfinyl, C -Cg alkylsulfonyl, C2~C haloalkoxy, Cχ-Cg haloalkylsulfonyl, C2~Cg haloalkyl, Cχ-Cg hydroxyalkyl, -C(0)0(Cχ-Cg alkyl), - (CH2) n-0- (Cχ-Cg alkyl), benzyloxy, phenoxy, phenylthio, - (CONHSO2R) . - CHO, amino, amidino, bromo, carba yl, carboxyl, carbalkoxy, - (CH2) n-Cθ2H, chloro, cyano, cyanoguanidinyl, fluoro, guanidino, hydrazide, hydrazino, hydrazido, hydroxy, hydroxyamino, iodo, nitro, phosphono, -SO3H, thioacetal, thiocarbonyl, and Cχ-Cg carbonyl; where n is from 1 to 8.
Preferred for practicing the method of the invention and preparing compositions of the invention are 1H- indole-3-glyoxylamide compounds and all corresponding pharmaceutically acceptable salts, solvates and prodrug derivatives thereof which are useful in the method of the invention include the following:
(A) [ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid,
(B) dl-2- [ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl-l-
(phenylmethyl) -lH-indol-4-yl] oxy]propanoic acid, (C) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1, 1 ' -biphenyl] -2- ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid,
(D) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1, 1 ' -biphenyl] -3- ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid, (E) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1,1 '-biphenyl] -4- ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid, (F) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [ (2, 6- dichlorophenyl) methyl] -2-methyl-lH-indol-4- yl] oxy] acetic acid (G) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [4 (- fluorophenyl ) methyl ] -2-methyl-lH-indol-4- yl] oxy] acetic acid, (H) [ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl-l- [ (1- naphthalenyl) methyl] -lH-indol-4-yl] oxy] acetic acid, (I) [ [3- (2-Amino-l, 2-dioxoethyl) -2-ethyl-l-
(phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, ( J) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [ (3- chlorophenyl) methyl] -2-ethyl- lH-indo1-4- yl] oxy] acetic acid, (K) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1, 1 ' -biphenyl] -2- ylmethyl) -2-ethyl-lH-indol-4-yl] oxy] acetic acid, (L) [ [3- (2-amino-l, 2-dioxoethyl) -1- ( [ 1, 1 ' -biphenyl] -2- ylmethyl) -2-propyl-lH-indol-4-yl] oxy] acetic acid, (M) [ [3- (2-Amino-l, 2-dioxoethyl) -2-cyclopropyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid,
(N) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [ 1, 1 ' -biphenyl] -2- ylmethyl) -2-cyclopropyl-lH-indol-4-yl] oxy] acetic acid, (O) 4- [ [3- (2-Amino-l, 2-dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-5-yl] oxy] butanoic acid, (P) mixtures of (A) through (P) in any combination. Particularly useful as SPLA2 inhibitors are prodrugs of the compounds of formula (I) and named compounds (A) thru (0) . The preferred prodrugs are the aromatic and aliphatic esters, such as the methyl ester, ethyl ester, n-propyl ester, isopropyl ester, n-butyl ester, sec- butyl, tert-butyl ester, N,N-diethylglycolamido ester, and morpholino-N-ethyl ester. Methods of making ester prodrugs are disclosed in U.S. Patent No. 5,654,326. Additional methods of prodrug synthesis are disclosed in U.S. Provisional Patent Application Serial No. 60/063280 filed October 27, 1997 (titled, N, N-diethylglycolamido ester Prodrugs of Indole SPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference; U.S. Provisional Patent Application Serial No. 60/063646 filed October 27, 1997 (titled, Morpholino-N-ethyl Ester Prodrugs of Indole SPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference; and U.S. Provisional Patent Application Serial No. 60/063284 filed October 27, 1997 (titled, Isopropyl Ester Prodrugs of Indole SPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference. Most preferred in the practice of the method of the invention are the acid, sodium salt, methyl ester, and morpholino-N-ethyl ester forms of [ [3- (2-Amino-l, 2- dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4- yl] oxy] acetic acid as represented by the following formulae:
Figure imgf000029_0001
Figure imgf000030_0001
Another highly preferred compound is the indole-3- glyoxylamide morpholino ethyl ester of represented by the formula:
Figure imgf000031_0001
the preparation of which is further described in United States provisional patent application SN 60/063,646 filed October 27, 1997.
Synthesis methods for lH-indole-3-glyoxylamide SPLA2 inhibitors are additionally depicted in the following reaction scheme:
lH-indole-3-glyoxylamide Reaction Scheme
Figure imgf000032_0001
Figure imgf000032_0002
Figure imgf000032_0004
Figure imgf000032_0003
Figure imgf000033_0001
Explanation of Reaction Scheme: To obtain the glyoxylamides substituted in the 4-position with an acidic function through an oxygen atom, the reactions outlined in scheme 1 are used (for conversions 1 through 5, see ref. Robin D. Clark, Joseph M. Muchowski, Lawrence E. Fisher, Lee A. Flippin, David B. Repke, Michel Souchet, Synthesis, 1991, 871-878, the disclosures of which are incorporated herein by reference) . The ortho-nitrotoluene, 1, is readily reduced to the 2-methylaniline, 2, using Pd/C as catalyst. The reduction can be carried out in ethanol or tetrahydrofuran (THF) or a combination of both, using a low pressure of hydrogen. The aniline, 2, on heating with di-tert-butyl dicarbonate in THF at reflux temperature is converted to the N-tert-butylcarbonyl derivative, 3, in good yield. The dilithium salt of the dianion of 3 is generated at -40 to -20 °C in THF using sec-butyl lithium and reacted with the appropriately substituted N-methoxy-N-methylalkanamide. This product, 4, may be purified by crystallization from hexane, or reacted directly with trifluoroacetic acid in methylene chloride to give the 1, 3-unsubstituted indole 5. The 1, 3-unsubstituted indole 5 is reacted with sodium hydride in dimethylformamide at room temperature (20-25 °C) for 0.5-1.0 hour. The resulting sodium salt of 5 is treated with an equivalent of arylmethyl halide and the mixture stirred at a temperature range of 0-100 °C, usually at ambient room temperature, for a period of 4 to 36 hours to give the 1-arylmethylindole, 6. This indole, 6, is 0- demethylated by stirring with boron tribro ide in methylene chloride for approximately 5 hours (see ref. Tsung-Ying Shem and Charles A Winter, Adv. Drug Res., 1977, 12, 176, the disclosure of which is incorporated herein by reference) . The 4-hydroxyindole, 7, is alkylated with an alpha bromoalkanoic acid ester in dimethylformamide (DMF) using sodium hydride as a base, with reactions conditions similar to that described for the conversion of 5 to 6. The a-[(indol-4- yl) oxy] alkanoic acid ester, 8, is reacted with oxalyl chloride in methylene chloride to give 9, which is not purified but reacted directly with ammonia to give the glyoxamide 10. This product is hydrolyzed using IN sodium hydroxide in MeOH. The final glyoxylamide, 11, is isolated either as the free carboxylic acid or as its sodium salt or in both forms.
The most preferred compound, [ [3- (2-Amino-l, 2- dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4- yl] oxy] acetic acid (as well as its sodium salt and methyl ester) useful in the practice of the method of the invention, may be prepared by the following procedure: Preparation of [ [3- (2-Amino-l, 2-dioxoethyl) -2-ethyl- 1- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, a compound represented by the formula:
Figure imgf000035_0001
Part A. Preparation of 2-Ethyl-4-methoxy-lH-indole . A solution of 140 mL (0.18 mol) of 1.3M sec-butyl lithium in cyclohexane was added slowly to N-tert- butoxycarbonyl-3-methoxy-2-methylaniline (21.3g, 0.09 mol) in 250 mL of THF keeping the temperature below -40°C with a dry ice-ethanol bath. The bath was removed and the temperature allowed to rise to 0°C and then the bath replaced. After the temperature had cooled to -60°C, 18.5g (0.18 mol) of N-methoxy-N-methylpropanamide in an equal volume of THF was added dropwise. The reaction mixture was stirred 5 minutes, the cooling bath removed and stirred an additional 18 hours. It was then poured into a mixture of 300 mL of ether and 400 L of 0.5N HCl. The organic layer was separated, washed with water, brine, dried over MgSθ4, and concentrated at reduced pressure to give 25.5g of a crude of l-[2-(tert- butoxycarbonylamino) -6-methoxyphenyl] -2-butanone . This material was dissolved in 250 mL of methylene chloride and 50 mL of trifluoroacetic acid and stirred for a total of 17 hours. The mixture was concentrated at reduced pressure and ethyl acetate and water added to the remaining oil. The ethyl acetate was separated, washed with brine, dried (MgSU4) and concentrated. The residue was chromatographed three times on silica eluting with 20% EtOAc/hexane to give 13.9g of 2-ethyl-4-methoxy-lH- indole . Analyses for C11H13N0:
Calculated: C, 75.40; H, 7.48; N, 7.99;
Found: C, 74.41; H, 7.64; N, 7.97.
Part B. Preparation of 2-Ethyl-4-methoxy-l- (phenylmethyl) -lH-indole.
2-Ethyl-4-methoxy-lH-indole (4.2g, 24 mmol) was dissolved in 30 mL of DMF and 960mg (24 mmol) of 60% NaH/minerial oil was added. After 1.5 hours, 2.9 mL(24 mmol) of benzyl bromide was added. After 4 hours, the mixure was diluted with water and extracted twice with ethyl acetate. The combined ethyl acetate was washed with brine, dried (MgS04) and concentrated at reduced pressure. The residue was chromatographed on silica gel and eluted with 20% EtOAc/hexane to give 3.1g (49% yield) of 2-ethyl-4-methoxy-l- (phenylmethyl) -lH-indole.
Part C. Preparation of 2-Ethyl-4-hydroxy-l- (phenyl ethyl) -lH-indole.
3.1g (11.7 mmol) of 2-ethyl-4-methoxy-l- (phenylmethyl) -lH-indole was O-demethylated by treating it with 48.6 mL of IM BBr in methylene chloride with stirring at room temperature for 5 hours, followed by concentration at reduced pressure. The residue was dissolved in ethyl acetate, washed with brine and dried (MgSθ ) . After concentrating at reduced pressure, the residue was chromatographed on silica gel eluting with 20% EtOAc/hexane to give 1.58g (54% yield) of 2-ethyl-4- hydroxy-1- (phenylmethyl) -lH-indole, mp, 86-90°C. Analyses for C-^H-^ O: Calculated: C, 81.24; H, 6.82; N, 5.57; Found: C, 81.08; H, 6.92; N, 5.41.
Part D. Preparation of [ [2-Ethyl-l- (phenylmethyl) - lH-indol-4-yl] oxy] acetic acid methyl ester. 2-ethyl-4-hydroxy-l- (phenylmethyl) -lH-indole (1.56g, 6.2 mmol) was added to a mixture of 248mg (6.2 mmol) of 60% NaH/mineral oil in 20mL DMF and stirred for 0.67 hour .
Then 0.6 mL ( 6.2 mmol) of methyl bromoacetate was added and stirring was continued for 17 hours. The mixture was diluted with water and extracted with ethyl acetate. The ethyl acetate solution was washed with brine, dried (MgSθ4), and concentrated at reduced pressure. The residue was chromatographed on silica gel eluting with 20% EtOAc/hexane, to give 1.37g (69% yield) of [ [2-ethyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid methyl ester, 89-92°C.
Analyses for C2QH21N03:
Calculated: C, 74.28; H, 6.55; N, 4.33; Found: C, 74.03; H, 6.49; N, 4.60.
Part E. Preparation of [ [3- (2-Amino-l, 2- dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4- yl] oxy] acetic acid methyl ester. Oxalyl chloride (0.4 mL, 4.2 mmol) was added to
1.36g (4.2 mmol) of [ [2-ethyl-l- (phenylmethyl) -lH-indol- 4-yl] oxy] acetic acid methyl ester in 10 mL of methylene chloride and the mixture stirred for 1.5 hours. The mixture was concentrated at reduced pressure and residue taken up in 10 mL of methylene chloride. Anhydrous ammonia was bubbled in for 0.25 hours, the mixture stirred for 1.5 hours and evaporated at reduced pressure. The residue was stirred with 20 mL of ethyl acetate and the mixture filtered. The filtrate was concentrated to give 1.37g of a mixture of [ [3- (2-amino-l, 2-dioxoethyl) - 2-ethyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid methyl ester and ammonium chloride. This mixture melted at 172-187°C.
Part F. Preparation of [ [3- (2-Amino-l, 2-dioxoethyl) -2- ethyl-1- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid. A mixture of 788 mg (2 mmol) of [3- (2-amino-l, 2- dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] - acetic acid methyl ester, 10 mL of In NaOH and 30 mL of MeOH is heated to maintain reflux for 0.5 hour, stirred at room temperature for 0.5 hour and concentrated at reduced pressure. The residue is taken up in ethyl acetate and water, the aqueous layer separated and made acidic to pH 2-3 with IN HCl. The precipitate is filtered and washed with ethyl acetate to give 559 mg (74% yield) of [ [3- (2-amino-l, 2-dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, mp, 230- 234 °C. Analyses for C21H20N2O5: Calculated: C, 65.96; H, 5.80; N, 7.33; Found: C, 66.95; H, 5.55; N, 6.99.
b) lH-indole-3-hydrazide SPLA2 inhibitors useful in practicing the method of the invention are described in U.S. Patent No. 5,578,634; the entire disclosure of which is incorporated herein by reference. The method of the invention is for treatment of a mammal, including a human, afflicted with sepsis, said method comprising administering to said human a therapeutically effective amount of the described as lH-indole-3-acetic acid hydrazides represented by the formula (lb) , and pharmaceutically acceptable salts, and prodrugs thereof;
Figure imgf000039_0001
wherein;
X is oxygen or sulfur;
Rx is selected from groups (i) , (ii) and (iii) where;
(i) is C4-C20 alkyl, C4-C20 alkenyl, C4-C20 alkynyl, C4-C20 haloalkyl, C4-CX2 cycloalkyl, or
(ii) is aryl or aryl substituted by halo, -CN, -CHO, -OH, -SH, Cχ-C10 alkylthio, Cχ-C10 alkoxy, CI-CIQ alkyl, carboxyl, amino, or hydroxya ino; (iii) is
Figure imgf000039_0002
where y is from 1 to 8, R74 is, independently, hydrogen or Cχ-Cχo alkyl, and R75 is aryl or aryl substituted by halo, -CN, -CHO, -OH, nitro, phenyl, -SH, Cχ-Cχo alkylthio, C -Cχg alkoxy, C -Cχo alkyl, amino, hydroxyamino or a substituted or unsubstituted 5- to 8- membered heterocyclic ring;
R2 is halo, C -C3 alkyl, ethenyl, C -C2 alkylthio, C -C2 alkoxy, -CHO, -CN; each R3 is independently hydrogen, C -C3 alkyl, or halo;
R4 R5, Rg, and R7 are each independently hydrogen, Cχ-Cχo alkyl, Cχ-Cχo alkenyl, C -Cχo alkynyl, C3-C8 cycloalkyl, aryl, aralkyl, or any two adjacent hydrocarbyl groups in the set R4 R5, Rg, and R7 combined with the ring carbon atoms to which they are attached to form a 5- or 6-membered substituted or unsubstituted carbocyclic ring; or Cχ-Cχg haloalkyl, C -Cχg alkoxy, C ~ C o haloalkoxy, C4-C8 cycloalkoxy, phenoxy, halo, hydroxy, carboxyl, -SH, -CN, -S(Cχ-Cχo alkyl), arylthio, thioacetal, -C (0) 0 (Cχ-Cχo alkyl), hydrazino, hydrazido, - NH2 - O2, -NRg2R83' an ~c (°) NR82R83' where, Rs2 and RQ3 are independently hydrogen, Cχ-Cχg alkyl, Cχ-Cχo hydroxyalkyl, or taken together with N, R82 and R83 form a 5- to 8-membered heterocyclic ring; or a group having the formula;
Figure imgf000040_0001
where, each R7 is independently selected from hydrogen, Cχ-Cχg alkyl, hydroxy, or both R7 taken together are =0; p is 1 to 8,
Z is a bond, -0-, -N(Cχ-C10 alkyl)-, -NH, or -S-; and Q is -C0N(Rg2R83) / -5-tetrazolyl, -S03H,
0
-p. OR86
OR86 0
■o- OR86
OR86
Figure imgf000041_0001
O
R 86
■0- •0- !CH2)fT -N- ■R 86
OR8. R: 86
Figure imgf000041_0002
where Rsg is independently selected from hydrogen, a metal, or C -Cχo alkyl.
The synthesis of the lH-indole-3-acetic acid hydrazides of structure (I) can be accomplished by known methods such as outlined in the following reaction schemes :
Scheme 1
Figure imgf000042_0001
Figure imgf000042_0002
The lH-indole-3-acetic acid ester can be readily alkylated by an alkyl halide or arylalkyl halide in a solvent such as N, N-dimethylformamide (DMF) in the presence of a base (meth a) to give the intermediate 1- alkyl-lH-indole-3-acetic acid esters, III. Bases such as potassium t-butoxide and sodium hydride were particularily useful. It is advantageous to react the indole, II, with the base to first form the salt of II and then add the alkylating agent. Most alkylations can be carried out at room temperature. Treatment of the l-alkyl-lH-indole-3-acetic acid esters, III, with hydrazine or hydrazine hydrate in ethanol (meth b) gives the desired l-alkyl-lH-indole-3-acetic acid hydrazides, I. This condensation to form I is usually carried out at the reflux temperature of the solvent for a period of 1 to 24 hours.
c) lH-indole-3-acetamide SPLA2 inhibitors and methods of making these inhibitors are set out in U.S. Patent No. 5,684,034, the entire disclosure of which is incorporated herein by reference. These inhibitors are useful ingredients in the compositons of the invention and the method of the invention for treatment of a mammal, including a human, afflicted with sepsis.
Useful inhibitors are represented by formula (lib) , and pharmaceutically acceptable salts and prodrug derivatives thereof,
Figure imgf000043_0001
wherein ;
X is oxygen or sulfur;
Rχι is selected from groups (i) , (ii) (iii) and (iv) where;
(i) is C -C20 alkyl, Cg-C20 alkenyl, Cg-C2o alkynyl, Cg-C20 haloalkyl, C4-CX2 cycloalkyl, or
(ii) is aryl or aryl substituted by halo, nitro, -CN, -CHO, -OH, -SH, Cχ-C10 alkyl, Cχ-C10 alkylthio, Cχ-Cχo alkoxyl, carboxyl, amino, or hydroxyamino; or (iii) is -(CH2)n-(R8θ) / or -(NH)-(R8χ), where n is 1 to 8, and Rso is a group recited in (i), and Rsi is selected from a group recited in (i) or (ii) ;
(iv) is
R87
C R88
R87
where Rg7 is hydrogen or Cχ-C o alkyl, and Rg8 is selected from the group; phenyl, naphthyl, indenyl, and biphenyl, unsubstituted or substituted by halo, -CN, - CHO, -OH, -SH, Cχ-C10 alkylthio, Cχ-C10 alkoxyl, phenyl, nitro, Cχ-Cχo alkyl, Cχ-Cχo haloalkyl, carboxyl, amino, hydroxyamino; or a substituted or unsubstituted 5 to 8 membered heterocyclic ring;
R 2 s halo, C -C2 alkylthio, or C -C2 alkoxy; each R 3 is independently hydrogen, halo, or methyl; R14 R15' R16' anc Rl7 are each independently hydrogen, Cχ-Cχg alkyl, Cχ-Cχo alkenyl, Cχ-Cχo alkynyl, C3-C8 cycloalkyl, aryl, aralkyl, or any two adjacent hydrocarbyl groups in the set R 4 15. Ri6' and R17' combine with the ring carbon atoms to which they are attached to form a 5 or 6 membered substituted or unsubstituted carbocyclic ring; or Cχ-Cχo haloalkyl, Cχ~ C o alkoxy, Cχ-Cχo haloalkoxy, C4-C8 cycloalkoxy, phenoxy, halo, hydroxy, carboxyl, -SH, -CN, Cχ-Cχo alkylthio, arylthio, thioacetal, -C (0) 0 (Cχ-Cχo alkyl), hydrazide, hydrazino, hydrazido, -NH2, -NO2, -NR82R83' and -C (0) NR82R83' where, R82 and R83 are independently hydrogen, Cχ-Cχo alkyl, Cχ-Cχo hydroxyalkyl, or taken together with N, R82 and R83 form a 5- to 8-membered heterocyclic ring; or a group having the formula;
Figure imgf000045_0001
where,
R84 and R85 are each independently selected from hydrogen, Cχ-Cχo alkyl, hydroxy, or Rβ4 and R85 taken together are =0; p is 1 to 5,
Z is a bond, -0-, -N(C1-Cχ0 alkyl)-, -NH-, or -S-, and
Q is -CON(R82R83) / -5-tetrazolyl, -SO3H,
0
OR86
OR86 0
■0- OR86
OR86
0 R99
-0- (CH; •N R99
OR86 R99 0
R99
Figure imgf000046_0001
0
■C OR; 86
Figure imgf000046_0002
where n is 1 to S, Rgg is independently selected from hydrogen, a metal, or Cχ-Cχo alkyl, and R99 is selected from hydrogen or Cχ-Cχo alkyl.
The synthesis of the lH-indole-3-acetamides of structure (lib) useful in the method of the invention can be accomplished by known methods. A procedure useful for the syntheses of these compounds is shown in the following reaction scheme:
Figure imgf000047_0001
V
The lH-indole-3-acetamide II may be alkylated by an alkyl halide or arylalkyl halide in a solvent such as N,N- dimethylformamide (DMF) in the presence of a base (method a) to give intermediate l-alkyl-lH-indole-3-acetic acid esters, III. Bases such as potassium t-butoxide and sodium hydride are useful. It is advantageous to react the indole, II, with the base to first form the salt of II and then add alkylating agent. Treatment of the 1-alkyl- lH-indole-3-acetic acid esters, III, with hydrazine or hydrazine hydrate in ethanol (method b) gives the desired l-alkyl-lH-indole-3-acetic acid hydrazides, IV. This condensation to form IV may be carried out at the reflux temperature of the solvent for a period of 1 to 24 hours. The acetic acid hydrazides, IV, are hydrogenated to give the acetamides, I, by heating with Raney nickel in ethanol
(method c) . The intermediate acetic acid esters, III, can be first hydrolyzed to the acetic acid derivatives, V
(method d) , which on treatment with an alkyl chloroformate followed by anhydrous ammonia, also give amides, I (method e) .
d) lH-indole-1-functional SPLA2 inhibitors of the hydrazide, amide, or glyoxylamide types as described in United States Patent No. 5,641,800, the entire disclosure of which is incorporated herein by reference. These inhibitors are useful ingredients in the compositons of the invention and the method of the invention for treatment of a mammal, including a human, afflicted with sepsis . A lH-indole-1-acetamide or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (Ic);
Figure imgf000048_0001
wherein for Formula (Ic);
X is oxygen or sulfur; each R is independently hydrogen, or C -C3 alkyl; R3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or
(c) is the group -(L)-Rso/ where, -(L)- is a divalent linking group of 1 to 12 atoms and where Rso is a group selected from (a) or (b) ;
R2 is hydrogen, halo, C1-C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (Cχ-C2 alkyl), -S- (Cχ~ C2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen; Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group, - (La) - (acidic group) . wherein -(La)-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, - (La) - (acidic group) ;
R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
lH-indole-1-hydrazide compounds useful as SPLA2 inhibitors in the practice of the method and formulation of the compositions of the invention are as follows:
A lH-indole-1-hydrazide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (He) ;
Figure imgf000050_0001
wherein for formula (lie) ;
X is oxygen or sulfur; each R is independently hydrogen, or C -C3 alkyl;
R3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C2o alkenyl, C7-C o alkynyl, carbocyclic radical, or heterocyclic radical, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituent; or (c) is the group -(L)-Rgθ'' where, - (L) - is a divalent linking group of 1 to 12 atoms and where Rso is a group selected from (a) or (b) ;
R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (C -C2 alkyl), -S- (Cχ~ C2 alkyl), or a non-interfering substituent having a total of ltto 3 atoms other than hydrogen;
Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group,
- (La) - (acidic group); wherein -(La)-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, -(La)-
(acidic group) ; R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents.
e) Indolizine SPLA2 inhibitors and their method of preparation are described in US Patent Application Serial No. 08/765566, filed July 20, 1995 (titled, "Synovial
Phospholipase A2 Inhibitor Compounds Having an Indolizine Type Nucleus, Parmaceutical Formulations Containing Said compounds, and Therapeutic Methods of Using said Compounds"), the entire disclosure of which is incorporated herein by reference; and also in European Patent Publication No. 0772596, published May 14, 1997. These inhibitors are useful in the formulation of the compositions of the invention and in the practice of the method of the invention is for treatment of a mammal, including a human, afflicted with sepsis.
Useful lH-indole-1-functional compounds or pharmaceutically acceptable salts, solvates or prodrug derivatives are represented by the formula (Id) ;
Figure imgf000051_0001
wherein; X is oxygen or sulfur; each R is independently hydrogen, C -C3 alkyl, or halo;
R 3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or
(b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or (c) is the group -(L)-Rgo; where, - (L) - is a divalent linking group of 1 to 12 atoms and where Rgo i a group selected from (a) or (b) ;
R 2 i hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (Cχ-C2 alkyl), -S- (Cχ~ C2 alkyl) , or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
R 7 and R 8 are independently selected from hydrogen, a non-interfering substituent, or the group, - (La)- (acidic group) . wherein -(La)-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of R 7 and R 8 must be the group, - (La) - (acidic group) ; and
Rχ5 and R are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
Particularly preferred lH-indole-1-functional compounds useful as SPLA2 inhibitors in the practice of the method of the invention are as follows: An indolizine-1-acetic acid hydrazide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof where said compound is represented by the formula (lid) ;
Figure imgf000053_0001
Particularly preferred lH-indole-1-functional compounds useful as SPLA2 inhibitors in the practice of the method of the invention are as follows:
An indolizine-1-glyoxylamide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (Hid) ;
Figure imgf000053_0002
Another preferred lH-indole-1-functional compounds useful as SPLA2 inhibitors in the practice of the method of the invention are as follows:
An indolizine-3-acetamide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (IVd), as set out below:
Figure imgf000054_0001
wherein;
X is selected from oxygen or sulfur; each R3 is independently hydrogen, C1-C3 alkyl, or halo;
Rl is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or
(b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or (c) is the group -(L)-Rgo; where, -(L)- is a divalent linking group of 1 to 12 atoms and where Rso i a group selected from (a) or (b) ;
R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0-(Cχ-C2 alkyl), -S- (Cχ-C2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
R5 and Rg are independently selected from hydrogen, a non-interfering substituent, or the group, -(La)- (acidic group) ; wherein -(La)-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of R5 and Rg must be the group, - (La) - (acidic group); R7 and Rs are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
Particularly preferred lH-indole-1-functional compounds useful as SPLA2 inhibitors in the practice of the method of the invention are as follows: An indolizine-3-hydrazide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (Vd) , as set out below:
Figure imgf000055_0001
Particularly preferred lH-indole-1-functional compounds useful as SPLA2 inhibitors in the practice of the method of the invention are as follows: An indolizine-3-glyoxylamide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (VId) , as set out below:
Figure imgf000056_0001
Particularly preferred lH-indole-1-functional compounds useful as SPL 2 inhibitors in the practice of the method of the invention are as follows: An indolizine-1-acetamide functional compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is selected from the group represented by the following formulae:
Figure imgf000056_0002
Figure imgf000057_0001
Figure imgf000057_0002
Figure imgf000058_0001
Figure imgf000058_0002
Figure imgf000059_0001
Figure imgf000059_0002
and mixtures of the above compounds.
Other particularly preferred lH-indole-1-functional compounds useful as SPLA2 inhibitors in the practice of the method of the invention are as follows: An indolizine-1-glyoxylamide functional compound and a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is selected from the group represented by the following formulae:
Figure imgf000060_0001
Figure imgf000060_0002
Figure imgf000061_0001
Figure imgf000061_0002
Figure imgf000062_0001
Figure imgf000062_0002
Figure imgf000063_0001
Figure imgf000063_0002
Figure imgf000064_0001
Figure imgf000064_0002
Figure imgf000065_0001
Figure imgf000065_0002
Figure imgf000065_0003
Figure imgf000066_0001
Figure imgf000066_0002
Figure imgf000066_0003
Figure imgf000067_0001
Figure imgf000067_0002
Figure imgf000068_0001
Figure imgf000068_0002
Figure imgf000069_0001
Figure imgf000069_0002
Figure imgf000070_0001
Figure imgf000070_0002
Figure imgf000070_0003
Figure imgf000071_0001
Figure imgf000071_0002
Figure imgf000072_0001
Figure imgf000072_0002
Figure imgf000072_0003
Figure imgf000073_0001
Figure imgf000073_0002
Figure imgf000074_0001
Figure imgf000074_0002
Figure imgf000075_0001
Figure imgf000075_0002
Figure imgf000076_0001
Figure imgf000076_0002
Figure imgf000077_0001
Figure imgf000077_0002
Figure imgf000078_0001
Figure imgf000078_0002
Figure imgf000079_0001
Figure imgf000079_0002
Figure imgf000080_0001
Figure imgf000080_0002
Figure imgf000081_0001
Figure imgf000081_0002
Figure imgf000082_0001
Figure imgf000082_0002
Figure imgf000083_0001
Figure imgf000083_0002
Figure imgf000084_0001
Figure imgf000084_0002
Figure imgf000085_0001
COCONH2
and mixtures of the above compounds.
The indolizine compounds may be made by one of more of the following reaction schemes:
The following abbreviations are used:
Bn benzyl
THF tetrahydrofuran
LAH lithium aluminum hydride
LDA lithium diiopropyl amine
DBU 1, 8-diazabicyclo 5.4.0] undec-7-une
Scheme le - Part 1
Figure imgf000086_0001
6: R=Li, 7:R=H
Figure imgf000086_0002
The anion of 2-methyl-5-methoxypyridine is formed in THF using lithium diisopropyl amide and reacted with benzonitrile to produce 2. Alkylation of the nitrogen of 2tby l-bromo-2-butanone followed by base catalyzed cyclization forms 3 which is reduced by LAH to 4. Sequential treatment of 4 with oxalyl chloride and ammonia gives 8. Alternatively, 4 is acylated with ethyl oxalyl chloride to give 5 which is converted to 6 with lithium hydroxide and then to 8 by sequential treatment with ethyl chloroformate and ammonium hydroxide. Demethylation of 8 by BBr3 yields 9 which is O-alkylated using base and ethyl 4-bromobutyrate to form 10, Hydrolysis of 10 by aqueous base produces 11.
Scheme 2e - Part 1
Figure imgf000087_0001
Figure imgf000087_0002
17 Ri R? R3 18 R1 R2 R3 R4 a OEt Et Bn a OEt Et Bn o-Ph-Ph b NH2 Et Bn b NH2 Et Bn o-Ph-Ph c NH2 Et CH2COEt c NH2 Et Bn m-CI-Ph d NH2 cyclo-Pr Bn d NH2 Et CH2COEt m-CI-Ph e NH2 cyclo-Pr Bn o-Ph-Ph f NH2 Et Bn Ph g NH2 Et Bn 1 -Naphthyl
Compound 12 (N. Desidiri, A. Galli, I. Sestili, and M. L. Stein, Arch. Pharm. (Weinheim) 325, 29, (1992)) is reduced by hydrogen in the presence of Pd/C to 14 which gives 15 on ammonolysis using ammonium hydroxide.
O-alkylation of 15 using benzyl chloride and base affords 16. Alkylation of the nitrogen atom of 13 or 16 by 1- bromo-2-ketones followed by base catalyzed cyclization yields indolizines 17 which are acylated by aroyl halides to form 18.
Scheme 2e - Part 2
Figure imgf000088_0001
19a-g 20v-z
Figure imgf000088_0002
19 R R2 R3 R4 a CH2OH Et Bn o-Ph-Ph b CONH2 Et Bn o-Ph-Ph c CONH2 Et CH2CH(OH)Et m-CI-Ph d CONH2 Et Bn m-CI-Ph e CONH2 cyclo-Pr Bn o-Ph-Ph f CONH2 Et Bn Ph g CONH2 Et Bn 1 -Naphthyl
20-22 R2 R3 R4 v: Et Et Ph w: Et Me 1-Naphthy I x: Et Bn o-Ph-Ph y: Et Bn m-CI-Ph z: cyclo-Pr Me o-Ph-Ph
Reduction of 18 by tert-butylamme-borohydride and aluminum chloride yields 19 which is reduced by hydrogen in the presence of Pd/C to give 20. O-alkylation of 20 by benzyl bromoacetate and base forms 21 which is converted to the acid 22 by debenzylation using hydrogen in the presence of Pd/C. Scheme 3e - Part 1 χ- , Ri
OH BnCI BnO 0
Jϋai±, NaHCO-,
I COOEt I COOEt N ___N
23 24
Figure imgf000089_0001
25a- R-fEt b R- cyclo-Pr 26a-f
Figure imgf000089_0002
27a-f 28a-f
26-28 R1 R2 a Et Ph b Et o-Ph-Ph c Et m-CI-Ph d Et m-CF3-Ph e Et 1-Naphthyl f cyclo-Pr o-Ph-Ph
Compound 23 (N. Desideri F. Manna, M. L. Stein, G. Bile, W. Filippeelli, and E. Marmo, Eur. J. Med. Chem. Chi . Ther., 18, 295, (1983)) is O-alkylated using sodium hydride and benzyl chloride to give 24. N-alkylation of 24 by l-bromo-2-butanone or chloromethylcyclopropyl ketone and subsequent base catalyzed cyclization gives 25 which is acylated by aroyl halide to give 26. Hydrolysis of the ester function of 26 followed by acidification forms an acid which is thermally decarboxylated to give 27. Reduction of the ketone function of 27 by LAH yields indolizines 28.
Figure imgf000090_0001
29a R2=Ph 24a R-,=OBn 31a R- H, R2=Ph b R^cyclo-Hex b R-]=OMe b R^OBn, R2=Ph
30a Rι=H c- R-|=OMe, R2=Ph d R-|=OBn, R2=cyclo-Hex
Figure imgf000090_0002
32a R^H, R2=Ph 33a R-pH, R2=Ph b R-|=OBn, R2=Ph b R-,=OBn, R2=Ph c R-pOMe, R2=Ph c R-|=OBn, R2=cyclo-Hex d R-|=OBn, R2=cyclo-Hex
Heating a mixture of 3-bromo-4-phenyl-butan-2-one or 3-bromo-4-cyclohexyl-butan-2-one and ethyl pyridine-2- acetate, or a substituted derivative, in the presence of base yields indolizine 31. Treatment of 31 with aqueous base in DMSO at elevated temperature followed by acidification gives 32 which is thermally decarboxylated to 33. Scheme 4e - Part 1
Figure imgf000091_0001
39d,i,k, I Rs=Me
Figure imgf000091_0002
f Me Me Et o-Ph-Ph g H H Me Ph h H H Et m-CI-Ph i H H Et m-CF3-Ph j H H Et 1 -Naphthyl k H H cyclo- Pr o-Ph-Ph
I H H Me cyclo-Hex
Sequential treatment of 28 or 33 with oxalyl chloride and ammonium hydroxide forms 35 which is debenzylated by hydrogen in the presence of Pd/C to give 36. Indolizines 36 are O-alkylated using sodium hydride and bromoacetic acid esters to form 37, 38, or 39 which are converted to indolizines 40 by hydrolysis with aqueous base followed by acidification. Scheme 4e - Part 2
Figure imgf000092_0001
41 42
The O-alkylation of 36h produces nitrile 41 which is converted to 42 on reaction with trialkyltin azide.
Scheme 5e
Figure imgf000093_0001
RCOCI
Figure imgf000093_0002
a-m, o-w
45, 46 R., R2 47-52 Ri 2 3 a Bn Et a-o Bn Et a-o (see below b Me Et P Bn Me 1-adamantyl c Bn Me q Bn Me o-biphenyl d Me cyclo-Pro r Bn cycloPro phenyl e Bn cyclo-Pro s Me Et p-n-Q,Hg-Ph t Bn Me cyclo-Hex u Me cycloPro cyclo-Hex
V Bn cycloPro cyclopentyl w Bn Me cyclolpentyl
Figure imgf000094_0001
48a-t, v, w 49a-t, v, w
Figure imgf000094_0002
50a-t, v, w 51a-t, v, w 52a-t, v, w
or BBr3
Figure imgf000094_0003
48a-t 49a-t
Figure imgf000094_0004
50a-t 51a-t 52a-t
47-52 R,
Figure imgf000095_0001
Figure imgf000095_0002
Figure imgf000095_0003
The hydroxypyridine is O-alkylated to give 44 which is heated with 2-haloketones to produce 45. Treatment of 45 with base causes cyclization to 46 which on heating with acid chlorides yields acylindolizines 47 which are reduced by aluminum hydride to the corresponding alkylindolizines 48. Sequential treatment of 48 with oxalyl chloride and then ammonia gives 49. Cleavage of the ether functionality of 49 yields 50. The oxyacetic ester derivatives 51 are formed by O-alkylation of 50 and then hydrolyzed to the oxyacetic acids 52. Scheme 6e - Part 1
Figure imgf000096_0001
R2CH2
54,55 Ri 2 a Me o-biphenyl b Et o-biphenyl c iPro o-biphenyl d cyclo-Pro cyclohexyl e tBu o-biphenyl f cyclopenty o-biphenyll
Figure imgf000096_0002
-58 a Me o-biphenyl f cyclopentyl o-biphenyl b Et o-biphenyl g Et m-biphenyl c iPr o-biphenyl h Et cmnamyl d cycloPro o-biphenyl i Et phenethyl e tBu o-biphenyl j cyclopropyl 1 -naphthyl k cyclopropyl cyclohexyl
Figure imgf000097_0001
59a-k
59a-k
a -ONa b -OCH(Me)OCOOMe c -OCH(Me)OCOOiPr d -OCH2OCOtBu
e -OCH (Me) OCOO-(~ f-OCH (Me) OCOO-Q g -OCH (Me) OCO-Q
h:-OCH (Me ) OCO-Q i -0 (CH2) J -(ChjfcO- (dimer) k COEt
Figure imgf000097_0002
Pyridine 43 is O-alkylated to produce 53. Heating 53 with 2-haloketones gives intermediate N-alkylated pyridinium compounds which are cyclized to 54 on treatment with base. Heating 54 with acyl chlorides gives the acylindolizines 55 which are reduced to the alkylindolizines 56 by sodium borohydride-aluminum chloride. Alternatively, 56 are produced by C-alkylation of 54 using alkyl halides. Sequential treatment of 56 with oxalyl chloride and then ammonia gives 57 which are hydrolyzed to produce 58. Compound 58b is converted to its sodium salt 59a which yields 59b-k on reaction with the appropriate alkyl halide. Scheme 6e - Part 2
Figure imgf000098_0001
36d 591-p
591-p
Figure imgf000098_0002
p 0
Compound 36b is O-alkylated to give 591-p.
Scheme 7e
Figure imgf000098_0003
tBuNH2BH3
Aicia
Figure imgf000098_0004
Figure imgf000098_0005
62a"d 63a-d
Figure imgf000099_0001
64a-d 65a-d
Figure imgf000099_0002
66a-d 67a-d
62-67 R1 R2
a Et Ph b cyclo Pro o-Ph-Ph c Et o-Ph-Ph d Et cyclohexyl
Pyridine 60 is N-alkylated by 2-haloketones to produce intermediate pyridinium compounds which are cyclized by base to give 61. Reaction of 61 with acyl chlorides produces 62 which are reduced to 63 by tert butylamine-borane and aluminum chloride. Sequential treatment of 63 with oxalyl chloride and then ammonia yields 64 which are O-demethylated by BBr3 to give 65. The sodium salt of 65 is reacted with ethyl 4- bromobutyrate to give 66 which is hydrolyzed to the acid 67. Scheme 8e
Figure imgf000100_0001
Compounds 36d and 65c are O-alkylated by omega- bromocarboxylic esters to give 68 which are hydrolyzed to the acids 69. Compounds 36d and 65c produce 70 on treatment with propiolactone and base.
Scheme 9e
Figure imgf000100_0002
66c 71a: R=Et b: R=H
Compounds 66 are reduced to 71 by tert-butylamine- borane and aluminum chloride. Scheme lOe
OBn OBn
Br COOEt 1)CS, s>_.N .COOEt
2) "^ COOEt
Br
44b
72
Figure imgf000101_0001
b: 6-substituted
Figure imgf000101_0002
75a,b 76 77 a: 8-substituted b: 6-substituted
Figure imgf000101_0003
BBr3 BrCH2COOMe
Figure imgf000102_0002
Figure imgf000102_0001
Figure imgf000102_0003
82 83
Pyridine 44b reacts with ethyl bromoacetate to produce 72 which is treated with CS2 and base and then with ethyl acrylate to form 73. Reaction of 73 with base and ethyl bromoacetate yields a mixture of regioisomers 74a+b, 6- and 8-benzyloxy compounds. Base treatment of 74a+b eliminates ethyl acrylate to form 75 which is separated from the isomer of 6-benzyloxy derivative and S-alkylated to give 76. Hydrolysis of 76 forms 77 which is thermally decarboxylated to yield 78. Compound 78 is C-alkylated to form 79 which on sequential treatment with oxalyl chloride and then ammonia forms 80. Ether cleavage of 80 gives 81 whose sodium salt is alkylated by methyl bromoacetate to form 82 which are hydrolyzed to acids 83. Scheme l ie - Part 1
Figure imgf000103_0001
84 85 86
Figure imgf000103_0002
89a-b 90a-b
Aminopicoline 84 is converted to its N-CBZ derivative 85 whose anion is alkylated by methyl bromoacetate to produce 86. Reaction of 86 with methyl alpha-bromoalkyl ketones in the presence of base yields 87. Sequential treatment of 87 with oxalyl chloride and then ammonia gives 88 which is converted to 89 by hydrogenolysis of the N-CBZ function. Hydrolysis of 89 yields acids 90. Scheme lie - Part 2
Figure imgf000104_0001
Figure imgf000104_0002
Compounds 88 are reduced by tert-butylamine-borane and aluminum chloride to 91 which are hydrolyzed to acids 92 .
Scheme 12e
2co3 >-
Figure imgf000104_0003
Figure imgf000105_0001
Figure imgf000105_0002
Pyridine 24 is N-alkylated by methyl bromoacetate, cyclized with base, and o-methylated using dimethysulfate to give 94. Hydrolysis of the ester function of 94 followed by thermal decarboxylation yields 2-methoxy-8- benzyloxyindolizine which is C-alkylated at position 3 and then reacted sequentially with oxalyl chloride and ammonia to produce 95. Hydrogenolysis of the 8-benzyloxy group followed by O-alkylation gives 96 which is hydrolyzed to 97.
f) Indene SPLA2 inhibitors as described in US Patent Application 08/776618 filed July 20 1995, (titled, Synovial Phospholipase A2 Inhibitor Compounds having an Indene Type Nucleus, Pharmaceutical Formulations Containing said Compounds, and Therapeutic Methods of Using Said Compounds") , the entire disclosure of which is incorporated herein by reference. These inhibitors are useful in making the compositions of the invention and practicing the method of the invention for the treatment of sepsis.
The method of the invention is for treatment of a mammal, including a human, afflicted with sepsis, said method comprising administering to said human a therapeutically effective amount of an indene-1-acetamide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (If);
Figure imgf000106_0001
wherein;
X is oxygen or sulfur; each R is independently hydrogen, C1-C3 alkyl, or halo;
R3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or
(b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or (c) is the group -(L)-Rgo; where, -(L)- is a divalent linking group of 1 to 12 atoms and where RQQ i-s a group selected from (a) or (b) ;
R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (Cχ-C2 alkyl), -S- (Cχ-C2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen;
Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group, - (La) - (acidic group) . wherein _( a)-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, - (La) - (acidic group); and
R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
Suitable indene compounds also include the following:
An indene-1-acetic acid hydrazide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula (Ilf);
Figure imgf000108_0001
wherein:
X is oxygen or sulfur; each Rx is independently hydrogen, C -C3 alkyl, or halo;
R3 is selected from groups (a) , (b) and (c) where; (a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or
(c) is the group -(L)-Rgθ' where, - (L) - is a divalent linking group of 1 to 12 atoms and where Rgo is a group selected from (a) or (b) ;
R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0-(Cχ-C2 alkyl), -S- (Cχ-C2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen; Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group, -(La)- (acidic group) ; wherein ~(La)-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, - (La) - (acidic group); and R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents .
Suitable indene compounds for use in the method of the invention also include the following: An indene-1-glyoxylamide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula
(iiif) ;
Figure imgf000109_0001
X is oxygen or sulfur;
R3 is selected from groups (a) , (b) and (c) where;
(a) is C7-C20 alkyl, C7-C20 alkenyl, C7-C20 alkynyl, carbocyclic radical, or heterocyclic radical, or
(b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or
(c) is the group -(L)-Rgo; where, - (L) - is a divalent linking group of 1 to 12 atoms and where Rgo is a group selected from (a) or (b) ; R2 is hydrogen, halo, C -C3 alkyl, C3-C4 cycloalkyl, C3-C4 cycloalkenyl, -0- (Cχ-C2 alkyl), -S- (Cχ-C2 alkyl), or a non-interfering substituent having a total of 1 to 3 atoms other than hydrogen; Rg and R7 are independently selected from hydrogen, a non-interfering substituent, or the group, -(La)- (acidic group) ; wherein -( a)-, is an acid linker having an acid linker length of 1 to 10; provided, that at least one of Rg and R7 must be the group, - (La) - (acidic group); R4 and R5 are each independently selected from hydrogen, non-interfering substituent, carbocyclic radical, carbocyclic radical substituted with non- interfering substituents, heterocyclic radical, and heterocyclic radical substituted with non-interfering substituents.
The method of making the indene compounds is as follows:
Scheme-lf
Figure imgf000110_0001
4 5 6a:6-OMe, Ra=Me
6b:7-OMe, R^Me 6c:7-OMe, Ra=Et
Figure imgf000111_0001
7a 8b-c
8b:7-OMe, R^Me 8c:7-OMe, R?=Et
OEt
Figure imgf000111_0002
8b,c 9b,c 7b,c
catΗ2S0
CHCb
A mixture of an anisaldehyde 1, propionic anhydride, and sodium propionate is heated to produce 2 which is reduced by hydrogen in the presence of Pd/C to give 3. Acid cyclization of 3 yields 6. Alternatively, the aromatic position para to the methoxy group of 3 is blocked by bromination to give 4 which is cyclized to 5 by acid and then debrominated using hydrogen and Pd/C to give 6. Reaction of 6 with the anion of triethyl phosphonoacetate produces 7 and/or 8. Radical bromination of 8 gives 9, which on reduction with hydrogen in the presence of Ptθ2 yields 7. Alternatively, treatment of 8 with acid gives 7. Scheme-2 f
Figure imgf000112_0001
7a-c 10a-j 11a-j
Figure imgf000112_0002
12a-j 13a-j
Figure imgf000112_0003
14a-j
Figure imgf000112_0004
a : R=Ph Ra=Me δ-R^ n=3 b : R=Ph Ra=Me l- ^ n=1 c : R=Ph Ra=Et 7-RtO n=1 d : R=o-Ph-Ph Ra=Et 7-Rte n=1 e : R=o-Bn-Ph Ra=Et 7-R*t> n=1 f : R=m-CI-Ph Ra=Et 7-Rlt> π=1 g : R=o,m-di-CI-Ph Ra=Et 7-RO n=1 h : R=m-CF3-Ph Ra=Et γ-RtO n=1 i : R=1 -Naphthyl Ra=Et 7-Rto n=1 j . R=2-Naphthyl Ra=Et 7-R*t> n=1
where Rb i s - ( CH2 ) nCOOH -Ill- Compound 7 is condensed with benzaldehyde and its derivatives in the presence of base to give 10. Indenes 10 are converted to an active ester using benzotriazo-1- yloxytris (dimethylamino) hexafluorophosphonate and then reacted with ammonium hydroxide to form 11. Demethylation of 11 with BBr3 forms 12 which is O-alkylated using sodium hydride and an omega-bromoalkanoic acid ester to produce 13. Aqueous base hydrolysis of 13 yields 14.
Scheme-3 f
Figure imgf000113_0001
12c 15
Figure imgf000113_0002
Compound 12c is O-alkylated using sodium hydride and methylbromoacetate to product 15 which is reduced by hydrogen in the presence of Pd/C to give a mixture of isomers 16a and 16b. Aqueous base hydrolysis of 16a and 16b gives 17a and 17b, respectively.
Figure imgf000114_0001
10d 18
Figure imgf000114_0002
19 20
Compound lOd is treated with lithium diisopropylamine, then air is bubbled into the solution to give 18. The indene 18 is converted to an active ester using benzotriazo-1- yloxytris (dimethylamino) hexafluorophosphonate and then reacted with ammonium hydroxide to form the hydroxy acetamide 19. Compound 19 is oxidized to 20 using N-methylmorpholine N-oxide in the presence of tetrapropylammonium perruthenate .
g) Carbazole and tetrahydrocarbazole sPIJJ? inhibitors and methods of making these compounds are set out in United States Patent Application SN 09/063066 filed April 21, 1998 (titled, "Substituted Carbazoles and 1, 2, 3, 4-Tetrahydrocarbazoles") , the entire disclosure of which is incorporated herein by reference. These inhibitors are useful in making the compositons of the invention and practicing the method of the invention for treating a mammal affliced with sepsis.
Useful carbazole or tetrahydrocarbazole inhibitors are represented by the following formulae: A compound of the formula (Ie)
Figure imgf000115_0001
wherein;
A is phenyl or pyridyl wherein the nitrogen is at the
5-, 6-, 7- or 8-position; one of B or D is nitrogen and the other is carbon; Z is cyclohexenyl, phenyl, pyridyl, wherein the nitrogen is at the 1-, 2-, or 3-position, or a 6- membered heterocyclic ring having one heteroatom selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position, and nitrogen at the 1-, 2-, 3- or 4-position; is a double or single bond;
R20 is selected from groups (a) , (b) and (c) where; (a) is - (C5-C20) alkyl, - (C5-C20) alkenyl,
- (C5-C20) alkynyl, carbocyclic radicals, or heterocyclic radicals, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or (c) is the group -(L)-R^O; where, -(L)- is a divalent linking group of 1 to 12 atoms selected from carbon, hydrogen, oxygen, nitrogen, and sulfur; wherein the combination of atoms in - (L) - are selected from the group consisting of (i) carbon and hydrogen only,
(ii) one sulfur only, (iii) one oxygen only,
(iv) one or two nitrogen and hydrogen only,
(v) carbon, hydrogen, and one sulfur only, and (vi) and carbon, hydrogen, and oxygen only; and where R^O is a group selected from
(a) or (b) ;
R21 is a non-interfering substituent;
RI' is -NHNH2, -NH2 or -CONH2; R2' is selected from the group consisting of -OH, and
-0(CH2).R5* where
R5' is H, -CN, -NH2, -CONH2/ -CONR9R10 -NHS02R15;
-CONHS02R15, where R15 is - (Ci-Cg) alkyl or -CF3; phenyl or phenyl substituted with -CO2H or -CO2 (C1-C4) alkyl; and - (La) - (acidic group), wherein ~(La)- is an acid linker having an acid linker length of 1 to 7 and t is 1-5; R is selected from non-interfering substituent, carbocyclic radicals, carbocyclic radicals substituted with non-interfering substituents, heterocyclic radicals, and heterocyclic radicals substituted with non-interfering substituents; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt thereof; provided that; when R^ ' is H, R20 j_s benzyl and m is 1 or 2; R2 ' cannot be -0(CH2)mH; and provided that when D is nitrogen, the heteroatom of Z is selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position and nitrogen at the 1-, 2-, 3- or 4-position.
Preferred in the compositions and method of the invention are compounds represented by the formula (He) :
Figure imgf000117_0001
wherein;
Z is cyclohexenyl, or phenyl;
R21 is a non-interfering substituent;
R1 is -NHNH2 or -NH2 ;
R is selected from the group consisting of -OH and
-0(CH2)m R5 where
O _ II
R5 is H, -C02H, -CONH2, -C02 (C]_-C alkyl); P(R R ), where R^ and R7 are each independently -OH or -0(02-04) alkyl; -S03H, -SO3 (C1-C4 alkyl), tetrazolyl, ~CN' ~NH2' -NHS02R15; -CONHSO R15, where R15 is - (C, -Cfi) alkyl or -CF_, phenyl or phenyl substituted with -C02H or -C02 (C..-C.) alkyl where m is 1-3; R3 is H, -0(Cχ-C4) alkyl, halo, - (C -Cg) alkyl, phenyl, - (C1-C ) alkylphenyl; phenyl substituted with - (Cχ_Cg) alkyl, halo, or -CF ; -CH2OSi (Cχ-Cg) alkyl, furyl, thiophenyl, - (C^-Cg) hydroxyalkyl; or - (CH2)nR8 where R8 is H, -CONH2, -NR9R10, -CN or phenyl where R9 and R^-O are independently - (C]_- C4) alkyl or -phenyl (C1-C4) alkyl and n is 1 to 8;
R^ is H, - (C5-C14) alkyl, - (C3-C14) cycloalkyl, pyridyl, phenyl or phenyl substituted with - (Cχ-Cg) alkyl, halo, -CF3, -OCF3, - (C1-C4) alkoxy, -CN, - (Cι~
C4) alkylthio, phenyl (CI-C4) alkyl, - (Cι~
C4) alkylphenyl, phenyl, phenoxy or naphthyl; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt, thereof.
Preferred specific compounds including all salts and prodrug derivatives thereof, for the compositions and method of the invention are as follows:
9-benzyl-5, 7-dimethoxy-l, 2, 3, 4-tetrahydrocarbazole-4- carboxylic acid hydrazide; 9-benzyl-5, 7-dimethoxy-l, 2, 3, 4-tetrahydrocarbazole-4- carboxamide; [9-benzyl-4-carbamoyl-7-methoxy-l, 2,3,4- tetrahydrocarbazol-5-yl] oxyacetic acid sodium salt; [ 9-benzyl-4-carbamoyl-7-methoxycarbazol-5-yl ] oxyacetic acid; methyl [9-benzyl-4-carbamoyl-7-methoxycarbazol-5- yl] oxyacetic acid; 9-benzyl-7-methoxy-5-cyanomethyloxy-l, 2,3,4- tetrahydrocarbazole-4-carboxamide; -benzyl-7-methoxy-5- (lH-tetrazol-5-yl-methyl) oxy) -
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; { 9- [ (phenyl) ethyl] -5-carbamoyl-2-methyl-carbazol-4- yl}oxyacetic acid; {9-[ (3-fluorophenyl) methyl ] -5-carbamoyl-2-methyl- carbazol-4-yl}oxyacetic acid; { 9- [ (3-methylphenyl) ethyl] -5-carbamoyl-2-methyl- carbazol-4-yl }oxyacetic acid; { 9- [ (phenyl) methyl] -5-carbamoyl-2- (4- trifluoromethylphenyl) -carbazol-4-yl }oxyacetic acid; 9-benzyl-5- (2-methanesulfonamido) ethyloxy-7-methoxy-
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; 9-benzyl-4- (2-methanesulfonamido) ethyloxy-2- methoxycarbazole-5-carboxamide; 9-benzyl-4- (2-trifluoromethanesulfonamido) ethyloxy-2- methoxycarbazole-5-carboxamide; 9-benzyl-5-methanesulfonamidoylmethyloxy-7-methoxy-
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; 9-benzyl-4-methanesulfonamidoylmethyloxy-carbazole-5- carboxamide;
[5-carbamoyl-2-pentyl-9- (phenylmethyl) carbazol-4- yl] oxyacetic acid; [5-carbamoyl-2- (1-methylethyl) -9- (phenylmethyl) carbazol- 4-yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2- [ (tri (-1- methylethyl) silyl) oxymethyl] carbazol-4-yl] oxyacetic acid; [5-carbamoyl-2-phenyl-9- (phenylmethyl) carbazol-4- yl] oxyacetic acid [5-carbamoy1-2- (4-chlorophenyl) -9- (phenylmethyl) carbazol-4-yl] oxyacetic acid;
[5-carbamoyl-2- (2-furyl) -9- (phenylmethyl) carbazol-4- yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2- [ (tri (-1- methylethyl) silyl) oxymethyl] carbazol-4-yl] oxyacetic acid, lithium salt; { 9- [ (phenyl) methyl] -5-carbamoylcarbazol-4-yl }oxyacetic acid; { 9- [ (3-fluorophenyl) methyl] -5-carbamoylcarbazol-4- yl }oxyacetic acid; { 9- [ (3-phenoxyphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-Fluorophenyl) methyl] -5-carbamoylcarbazol-4- yl } oxyacetic acid; {9- [ (2-trifluoromethylphenyl) methyl] -5-carbamoylcarbazol-
4-yl } oxyacetic acid; { 9- [ (2-benzylphenyl) methyl] -5-carbamoylcarbazol-4- yl } oxyacetic acid; { 9- [ (3-trifluoromethylphenyl) methyl] -5-carbamoylcarbazol-
4-yl} oxyacetic acid; { 9- [ ( 1-naphthyl ) methyl ] -5-carbamoylcarbazol-4- yl } oxyacetic acid; { 9- [ (2-cyanophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ ( 3-cyanophenyl ) methyl ] -5-carbamoylcarbazol-4- yl } oxyacetic acid; { 9- [ (2-methylphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (3-methylphenyl) methyl] -5-carbamoylcarbazol-4- yl} oxyacetic acid; { 9- [ (3, 5-dimethylphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- f (3-iodophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-Chlorophenyl) ethyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2, 3-difluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2, 6-difluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2, 6-dichlorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ ( 3-trifluoromethoxyphenyl ) methyl ] -5- carbamoylcarbazol-4-yl Joxyacetic acid;
{ 9- [ (2-biphenyl) methyl] -5-carbamoylcarbazol-4- yl Joxyacetic acid; { 9- [ (2-Biphenyl ) methyl ] -5-carbamoylcarbazol-4- yl}oxyacetic acid; the { 9- [ (2-Biphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; [9-Benzyl-4-carbamoyl-l, 2, 3, 4-tetrahydrocarbaole-5- yl] oxyacetic acid; { 9- [ (2-Pyridyl) methyl] -5-carbamoylcarbazol-4-yl }oxyacetic acid;
{ 9- [ (3-Pyridyl) methyl] -5-carbamoylcarbazol-4-yl Joxyacetic acid; [9-benzyl-4-carbamoyl-8-methyl-l, 2,3,4- tetrahydrocarbazol-5-yl ] oxyacetic acid; [9-benzyl-5-carbamoyl-l-methylcarbazol-4-yl] oxyacetic acid; [9-benzyl-4-carbamoyl-8-fluoro-l, 2, 3, 4- tetrahydrocarbazol-5-yl] oxyacetic acid; [9-benzyl-5-carbamoyl-l-fluorocarbazol-4-yl] oxyacetic acid;
[9-benzyl-4-carbamoyl-8-chloro-l, 2,3,4- tetrahydrocarbazol-5-yl ] oxyacetic acid; [9-benzyl-5-carbamoyl-l-chlorocarbazol-4-yl] oxyacetic acid; [ 9- [ (Cyclohexyl ) methyl ] -5-carbamoylcarbazol-4- yl] oxyacetic acid; [9- [ (Cyclopentyl) ethyl] -5-carbamoylcarbazol-4- yl] oxyacetic acid; 5-carbamoyl-9- (phenylmethyl) -2- [ [ (propen-3- yl) oxy]methyl] carbazol-4-yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2- [ (propyloxy) methyl] carbazol-4-yl] oxyacetic acid; 9-benzyl-7-methoxy-5- ( (carboxamidomethyl) oxy) -1,2,3,4- tetrahydrocarbazole-4-carboxamide; 9-benzyl-7-methoxy-5-cyanomethyloxy-carbazole-4- carboxamide; 9-benzyl-7-methoxy-5- ( (lH-tetrazol-5-yl-methyl) oxy) - carbazole-4-carboxamide; 9-benzyl-7-methoxy-5- ( (carboxamidomethyl) oxy) -carbazole-
4-carboxamide; and [9-Benzyl-4-carbamoyl-l, 2, 3, 4-tetrahydrocarbaole-5- yl] oxyacetic acid or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt, thereof.
Other desirable carbazole inhibitors suitable for the compositions and method of thein invention are selected from those represented by the formula (XXX) :
Figure imgf000123_0001
(XXX)
wherein:
Figure imgf000123_0002
R2 is selected from the group consisting of -OH and - 0(CH2)mR5 where
0
—P (R^R^ ) R5 is H, -C02H, -C02(C1-C4 alkyl); , where R6 and
R^ are each independently -OH or -0 (C1-C4 ) alkyl;
-SO3H, -S03(C1-C alkyl), tetrazolyl, -CN, -NH2' -NHSO2R15; -CONHS02R15. where R15 is - (Ci-Cg) alkyl or -CF3, phenyl or phenyl substituted with -C02H or -CO2 (Cχ-C4) alkyl where m is 1-3;
R3 is H, -0(Cχ-C4) alkyl, halo, - (Cχ-Cg) alkyl, phenyl,
- (C1-C4) alkylphenyl; phenyl substituted with - (Cx-Cg) alkyl, halo, or -CF3; -CH2OSi (Cχ-Cg) alkyl, furyl, thiophenyl, - (C]_-Cg) hydroxyalkyl; or -
(CH2)nR8 where R8 is H, -CONH2, -NR9R10, -CN or phenyl where R9 and R^^ are independently - (Cχ~
C4) alkyl or -phenyl (C1-C4 ) alkyl and n is 1 to 8; R4 is H, - (C5-C14) alkyl, - (C3-C14 ) cycloalkyl, pyridyl, phenyl or phenyl substituted with - (Cχ-Cg) alkyl, halo, -CF3, -OCF3 , - (C1-C4) alkoxy, -CN, - (Cι~ C ) alkylthio, phenyl (CI-C4) alkyl, - (Ci- C4) alkylphenyl, phenyl, phenoxy or naphthyl; A is phenyl or pyridyl wherein the nitrogen is at the 5-, 6-, 7- or 8-position; Z is cyclohexenyl, phenyl, pyridyl wherein the nitrogen is at the 1-, 2- or 3-position or a 6-membered heterocyclic ring having one heteroatom selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position and nitrogen at the 1-, 2-, 3- or 4-position, or wherein one carbon on the heterocyclic ring is optionally substituted with =0; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt thereof; provided that one of A or Z is a heterocyclic ring.
Further desirable specific cabazole and tetrahydrocarbazole inhibitors suitable for the compositions and method of the invention are selected from the following:
(R, S) - (9-benzyl-4-carbamoyl-l-oxo-3-thia-l, 2, 3, 4- tetrahydrocarbazol-5-yl) oxyacetic acid; (R, S)-(9- benzyl-4-carbamoyl-l-oxo-3-thia-l, 2,3,4- tetrahydrocarbazol-5-yl) oxyacetic acid; [N-benzyl-1- carbamoyl-l-aza-1, 2, 3, 4-tetrahydrocarbazol-8- yl] oxyacetic acid; 4-methoxy-6-methoxycarbonyl-10- phenylmethyl-6, 7, 8, 9-tetrahydropyrido [1, 2-a] indole; (4- carboxamido-9-phenylmethyl-4, 5-dihydrothiopyrano [3, 4- b] indol-5-yl) oxyacetic acid; 3, 4-dihydro-4- carboxamidol-5-methoxy-9-phenylmethylpyrano [3, 4- b] indole; 2- [(2, 9 bis-benzyl-4-carbamoyl-l, 2, 3, 4- tetrahydro-beta-carbolin-5-yl) oxy] acetic acid or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt thereof.
The most preferred cabozole/tetrahydrocarbazole inhibitors for the compositions and method of treating sepsis are represented by the formulae (Xe) and (Xle) below:
Figure imgf000125_0001
and
Figure imgf000126_0001
For all of the above compounds of the carbazole or tetrahydrocarbazole type it is advantageous to use them in their (i)acid form, or (ii) pharmaceutically acceptable (e.g., Na, K) form, or (iii) and prodrugs derivatives (e.g., methyl ester, ethyl ester, n-butyl ester, morpholino ethyl ester) .
Prodrugs are derivatives of sPIJJ? inhibitors used in the method of the invention which have chemically or metabolically cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo. Derivatives of the compounds of this invention have activity in both their acid and base derivative forms, but the acid derivative form often offers advantages of solubility, tissue compatibility, or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, 7Amsterdam 1985) . Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acidic compound with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a suitable amine. Simple aliphatic or aromatic esters derived from acidic groups pendent on the compounds of this invention are preferred prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy) alkyl esters or ( (alkoxycarbonyl) oxy) alkyl esters. Specific preferred prodrugs are ester prodrugs inclusive of methyl ester, ethyl ester, n-propyl ester, isopropyl ester, n-butyl ester, sec-butyl, tert-butyl ester, N, N-diethylglycolamido ester, and morpholino-N-ethyl ester. Methods of making ester prodrugs are disclosed in U.S. Patent No. 5,654,326. Additional methods of prodrug synthesis are disclosed in U.S. Provisional Patent Application Serial No. 60/063280 filed October 27, 1997 (titled, N, N-diethylglycolamido ester Prodrugs of Indole sPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference; U.S. Provisional Patent Application Serial No. 60/063646 filed October 27, 1997 (titled, Morpholino-N-ethyl Ester Prodrugs of Indole sPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference; and US Provisional Patent Application Serial No. 60/063284 filed October 27, 1997 (titled, Isopropyl Ester Prodrugs of Indole sPIJJ? Inhibitors) , the entire disclosure of which is incorporated herein by reference.
Carbazole and tetrahydrocarbazole SPL 2 inhibitor compounds useful for practicing the method of the invention may be made by the following general methods: The compounds of formula le where Z is cyclohexene are prepared according to the following reaction Schemes Ig(a)and (c) .
Scheme Ig ( a)
Figure imgf000128_0001
wherein;
R1 is -NH2/ R3(a) is H, -0 (C1-C4 ) alkyl, halo, - (Cχ~ Cg) alkyl, phenyl, - (C1-C4) alkylphenyl; phenyl substituted with - (Cχ-Cg) alkyl, halo, or -CF3; - CH2θSi (Cχ-Cg) alkyl, furyl, thiophenyl, - (Cχ~
Cg) hydroxyalkyl, - (Cι_Cg) alkoxy (Cχ-Cg) alkyl, -(Cι~ Cg) alkoxy (Cχ_Cg) alkenyl; or -(CH2)nR8 where R8 is H, -CONH2, -NR9R10, -CN or phenyl where R9 and R10 are independently hydrogen, -CF3, phenyl, - (C1-C4 ) alkyl, - (C1-C4) alkylphenyl or -phenyl (C1-C4 ) alkyl and n is 1 to 8; when R1 is -NHNH2/ R3(a) is H, -0 (Cχ-C ) alkyl, halo,
- (Cχ-Cg) alkyl, phenyl, - (C1-C4) alkylphenyl; phenyl substituted with - (Cχ-Cg) alkyl, halo or -CF3; -CH20Si (Ci-Ce) alkyl, furyl, thiophenyl,
- (Cχ-Cg) hydroxyalkyl,- (Cχ-Cg) alkoxy (Cχ-Cg) alkyl, - (Cχ-Cg) alkoxy (Cχ-Cg) alkenyl; or -(CH2)nR8 where R8 is H, -NR9R10, -CN or phenyl where R9 and R10 are independently hydrogen, -CF3, phenyl, - (C1-C4) alkyl, - (C1-C4) alkylphenyl or -phenyl (C1-C4 ) alkyl and n is 1 to 8; R2 (a) s _0CH3 or -OH.
An appropriately substituted nitrobenzene (1) can be reduced to the aniline (2) by treatment with a reducing agent, such as hydrogen in the presence of Pd/C, preferably at room temperature.
Compound (2) is N-alkylated at temperatures of from about 0 to 20 °C using an alkylating agent such as an appropriately substituted aldehyde and sodium cyanoborohydride to form (3) . Alternately, an appropriately substituted benzyl halide may be used for the first alkylation step. The resulting intermediate is further N-alkylated by treatment with 2-carbethoxy- 6-bromocyclohexanone, preferably at temperatures of about 80 °C to yield (4) or by treatment with potassium hexamethyldisilazide and the bromoketoester . The product (4) is cyclized to the tetrahydrocarbazole (5) by refluxing with ZnCl2 in benzene for from about 1 to 2 days, preferably at 80 °C. (Ref 1) . Compound (5) is converted to the hydrazide (6) by treatment with hydrazine at temperatures of about 100 °C, or to the amide (7) by reacting with methylchloroaluminum amide in benzene. (Ref 2) Alternatively, (7) may be produced by treatment of (6) with Raney nickel active catalyst.
It will be readily appreciated that when R3(a) is:
0 -(CH2)nCO(C1-C4 alkyl), conversion to the amide will also be achieved in this procedure .
Compounds (6) and (7) may be dealkylated, preferably at 0 °C to room temperature, with a dealkylating agent, such as boron tribromide or sodium thioethoxide, to give compound (7) where R2 (a) j_s -OH, which may then be further converted to compound (9), by realkylating with a base, such as sodium hydride, and an alkylating agent, such as Br(CH2)mR^, where R^ is the carboxylate or phosphonic diester or nitrile as defined above. Conversion of R2 to the carboxylic acid may be accomplished by treatment with an aqueous base. When R2 is nitrile, conversion to the tetrazole may be achieved by reacting with tri-butyl tin azide or conversion to the carboxamide may be achieved by reacting with basic hydrogen peroxide. When R is the phosphonic diester, conversion to the acid may be achieved by reacting with a dealkylating agent such as trimethylsilyl bromide. The monoester may be accomplished by reacting the diester with an aqueous base.
When R2 and R3 are both methoxy, selective demethylation can be achieved by treating with sodium ethanethiolate in dimethylformamide at 100 °C.
Ref 1 Julia, M.; Lenzi, J. Preparation d' acides tetrahydro-1, 2, 3, -carbazole-l ou -4. Bull . Soc . Chim . France, 1962, 2262-2263.
Ref 2 Levin, J.I.; Turos, E . ; Weinreb, S.M. An alternative procedure for the aluminum-mediated conversion of esters to amides. Syn . Co m. , 1982, 12, 989-993.
An alternative synthesis of intermediate (5) is shown in Scheme 1(b), as follows.
Scheme Ig(b)
Figure imgf000132_0001
Figure imgf000132_0002
where PG is a protecting group; R3a is as defined in Scheme 1, above.
The aniline (2) is N-alkylated with 2-carbethoxy-6- bromocyclohexanone in dimethyl formamide in the presence of sodium bicarbonate for 8-24 hours at 50 °C. Preferred protecting groups include methyl, carbonate, and silyl groups, such as t-butyldimethylsilyl . The reaction product (4') is cyclized to (5') using the ZnCl2 in benzene conditions described in Scheme 1(a), above. N- alkylation of (5' ) to yield (5) is accomplished by treatment with sodium hydride and the appropriate alkyl halide in dimethylformamide at room temperature for 4-8 hours .
Scheme Ilg
Figure imgf000133_0001
p3(a) 2.s as defined in Scheme Ig.
As discussed in Scheme I above, carbazole (5) is hydrolyzed to the carboxylic acid (10) by treatment with an aqueous base, preferably at room temperature to about 100 °C. The intermediate is then converted to an acid chloride utilizing, for example, oxalyl chloride and dimethylformamide, and then further reacted with a lithium salt of (S) or (R) -4-alkyl-2-oxazolidine at a temperature of about -75 °C, to give (11a) and (lib), which are separable by chromatography. The diastereomers are converted to the corresponding enantiomeric benzyl esters (12) by brief treatment at temperatures of about 0 °C to room temperature with lithium benzyl oxide. (Ref 3) The esters (12) are then converted to (7) preferably by treatment with methylchloroaluminum amide (Ref 2, above) or, alternately, by hydrogenation using, for example, hydrogen and palladium on carbon, as described above, to make the acid and then reacting with an acyl azide, such as diphenylphosphoryl azide followed by treatment with ammonia. Using the procedure described above in Scheme I, compound (9a) or (9b) may be accomplished.
Ref 3 Evans, D.A.; Ennis, M.D.; Mathre, D.J. Asymmetric alkylation reactions of chiral imide enolates. A practical approach to the enantioselective synthesis of alp a-substituted carboxylic acid derivatives. J. Am . Chem . Soc . , 1982, 1 04, 1737-1738.
Compounds of formula le where Z is phenyl can be prepared as follows in Schemes III (a) -(f), below.
Scheme III (a)
Figure imgf000134_0001
13) (14) A 1, 2, 3, 4-tetrahydrocarbazole-4-carboxamide or 4- carboxhydrazide (13) is dehydrogenated by refluxing in a solvent such as carbitol in the presence of Pd/C to produce the carbazole-4-carboxamide. Alternately, treatment of (13) with DDQ in an appropriate solvent such as dioxane yields carbozole (14) .
Depending on the substituent pattern oxidation as described above may result in de-alkylation of the nitrogen. For example when R3 is substituted at the 8- position with methyl, oxidation results in dealkylation of the nitrogen which may be realkylated by treatment with sodium hydride and the appropriate alkyl halide as described in Scheme I (a) above to prepare the deired product (14) .
Figure imgf000136_0001
(15) (16) (25)
,
Figure imgf000136_0002
(26) (19)
Figure imgf000136_0003
(20) (21)
Figure imgf000136_0004
(22) (23)
defined in Scheme I (a) above cid protecting group
Figure imgf000136_0005
(24) Benzoic acid derivative (16) where X is preferably chlorine, bromine or iodine and the protecting group is preferably -CH3, are reduced to the corresponding aniline (25) with a reducing agent, such as stannous chloride in the presence of acid under the general conditions of Sakamoto et al, Chem Pharm . Bull . 35 (5), 1823-1828 (1987) .
Alternatively, reduction with sodium dithionite in the presence of a base, such as sodium carbonate in a noninterferring solvent, such as water, ethanol, and/or tetrahydrofuran affords starting material (16).
Alternatively, reduction by hydrogenation over a sulfided platinum catalyst supported on carbon with hydrogen at 1 to 60 atmospheres in a noninterfering solvent, preferably ethyl acetate, to form a starting material (16) .
The reactions are conducted at temperatures from about 0 to 100 °C. preferably at ambient temperature, and are substantially complete in about 1 to 48 hours depending on conditions.
The aniline (25) and dione (15) are condensed under dehydrating conditions, for example, using the general procedure of Iida, et al., (Ref 5), with or without a noninterfering solvent, such as toluene, benzene, or methylene chloride, under dehydrating conditions at a temperature about 10 to 150 °C. The water formed in the process can be removed by distillation, azetropic removal via a Dean-Stark apparatus, or the addition of a drying agent, such as molecular sieves, magnesium sulfate, calcium carbonate, sodium sulfate, and the like. The process can be performed with or without a catalytic amount of an acid, such a p-toluenesulfonic acid or methanesulfonic acid. Other examples of suitable catalysts include hydrochloric acid, phenylsulfonic acid, calcium chloride, and acetic acid.
Examples of other suitable solvents include tetrahydrofuran, ethyl acetate, methanol, ethanol, 1, 1, 2, 2-tetrachloroethane, chlorobenzene, bromobenzene, xylenes, and carbotetrachloride . The condensation of the instant process is preferably carried out neat, at a temperature about 100 to 150 °C with the resultant water removed by distillation via a stream of inert gas, such as, nitrogen or argon. The reaction is substantially complete in about 30 minutes to 24 hours.
Intermediate (26) may then be readily cyclized in the presence of a palladium catalyst, such as Pd(OAc)2 or Pd(PPh_3)4 and the like, a phosphine, preferably a trialkyl- or triarylphosphine, such as triphenylphosphine, tri-o-tolylphosphine , or tricyclohexylphosphine, and the like, a base, such as, sodium bicarbonate, triethylamine, or diisopropylethylamine, in a noninterfering solvent, such as, acetonitrile, triethylamine, or toluene at a temperature about 25 to 200°C to form (19) .
Examples of other suitable solvents include tetrahydrofuran, benzene, dimethylsulfoxide, or dimethylformamide .
Examples of other suitable palladium catalysts include Pd(PPh3)Cl2, Pd(OCOCF3)2, [ (CH3CgH4) 3P] 2PdCl2, [ (CH3CH2)3P]2PdCl2, [ (C6H1:L) 3P] 2PdCl2, and [(C6H5)3P]2PdBr2. Examples of other suitable phosphines include triisopropylphosphine, triethylphosphine, tricyclopentylphosphine, 1,2- bis (diphenylphosphino) ethane, 1, 3-bis (diphenylphosphino) propane, and 1,4- bis (diphenylphosphino) butane.
Examples of other suitable bases include tripropyl amine, 2, 2, 6, 6-tetramethylpiperidine, 1,5- diazabicyclo[2.2.2]octane (DABCO) , 1,8- diazabicyclo [5.4.0] undec-7-ene (DBU) , 1,5- diazabicyclo [4.3.0]non-5-ene, (DBN) sodium carbonate, potassium carbonate, and potassium bicarbonate.
The cyclization of the instant process is preferably carried out with palladium (II) acetate as catalyst in the presence of either triphenylphosphine, tri-o- tolylphosphine, 1, 3-bis (diphenylphosphino) propane, or tricyclohexylphosphine in acetonitrile as solvent and triethylamine as base at a temperature about 50 to 150 °C. The reaction is substantially complete in about 1 hour to 14 days.
Alternatively, a preferred process for cyclization consists of the reaction of intermediate (26) with a palladacycle catalyst such as trans-di (μ-acetato) -bis [o- (di-o-tolylphosphino) benzyl] dipalladium (II) in a solvent such as dimethylacetamide (DMAC) at 120-140 °C in the presence of a base such as sodium acetate.
Intermediate (19) may be alkylated with an alkylating agent XCH2R4, where X is halo in the presence of a base to form (20) . Suitable bases include potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, potassium hydroxide, sodium hydroxide, sodium hydride, potassium hydride, lithium hydride, and Triton B (N-benzyltrimethylammonium hydroxide) .
The reaction may or may not be carried out in the presence of a crown ether. Potassium carbonate and Triton B are preferred. The amount of alkylating agent is not critical, however, the reaction is best accomplished using an excess of alkyl halide relative to the starting material .
A catalytic amount of an iodide, such as sodium iodide or lithium iodide may or may not be added to the reaction mixture. The reaction is preferably carried out in an organic solvent, such as, acetone, dimethylformamide, dimethylsulfoxide, or acetonitrile . Other suitable solvents include tetrahydrofuran, methyl ethyl ketone, and t-butyl methyl ether.
The reaction is conducted at temperatures from about -10 to 100 °C. preferably at ambient temperature, and is substantially complete in about 1 to 48 hours depending on conditions. Optionally, a phase transfer reagent such as tetrabutylammonium bromide or tetrabutylammonium chloride may be employed.
Intermediate (20) May by dehydrogenated by oxidation with 2, 3-dichloro-5, 6-dicyano-l, 4-benzoquinone in a noninterfering solvent to form (21) . Suitable solvents include methylene chloride, chloroform, carbon tetrachloride, diethyl ether, methyl ethyl ketone, and t-butyl methyl ether. Toluene, benzene, dioxane, and tetrahydrofuran are preferred solvents. The reaction is carried out at a temperature about 0 to 120 °C. Temperatures from 50 to 120 °C are preferred. The reaction is substantially complete in about 1 to 48 hours depending on conditions . Intermediate (21) may be a inated with ammonia in the presence of a noninterfering solvent to form a (22). Ammonia may be in the form of ammonia gas or an ammonium salt, such as ammonium hydroxide, ammonium acetate, ammonium trifluoroacetate, ammonium chloride, and the like. Suitable solvents include ethanol, methanol, propanol, butanol, tetrahydrofuran, dioxane, and water. A mixture of concentrated aqueous ammonium hydroxide and tetrahydrofuran or methanol is preferred for the instant process. The reaction is carried out at a temperature about 20 to 100 °C . Temperatures from 50 to 60 °C are preferred. The reaction is substantially complete in about 1 to 48 hours depending on conditions.
Alkylation of (22) is achieved by treatment with an alkylating agent of the formula XCH2R^ where X is halo and R70 is -C02R71, -SO3R71, -P (0) (OR71) 2 ' or _ P(O) (OR71)H, where R71 is an acid protecting group or a prodrug function, in the presence of a base in a noninterfering solvent to form (23) . Methyl bromoacetate and t-butyl bromoacetate are the preferred alkylating agents .
Suitable bases include potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, potassium hydroxide, sodium hydroxide, sodium hydride, potassium hydride, lithium hydride, and Triton B (N-benzyltrimethylammonium hydroxide) . The reaction may or may not be carried out in the presence of a crown ether. Cesium carbonate and Triton B are preferred. The amount of alkylating agent is not critical, however, the reaction is best accomplished using an excess of alkyl halide relative to the starting material. The reaction is preferably carried out in an organic solvent, such as, acetone, dimethylformamide, dimethylsulfoxide, or acetonitrile . Other suitable solvents include tetrahydrofuran, methyl ethyl ketone, and t-butyl methyl ether.
The reaction is conducted at temperatures from about
-10 to 100 °C. preferably at ambient temperature, and is substantially complete in about 1 to 48 hours depending on conditions. Optionally, a phase transfer reagent such as tetrabutylammonium bromide or tetrabutylammonium chloride may be employed.
Intermediate (23) may be optionally hydrolyzed with a base or acid to form desired product (24) and optionally salified. Hydrolysis of (23) is achieved using a base such as sodium hydroxide, potassium hydroxide, lithium hydroxide, aqueous potassium carbonate, aqueous sodium carbonate, aqueous lithium carbonate, aqueous potassium bicarbonate, aqueous sodium bicarbonate, aqueous lithium bicarbonate, preferably sodium hydroxide and a lower alcohol solvent, such as, methanol, ethanol, isopropanol, and the like. Other suitable solvents include acetone, tetrahydrofuran, and dioxane.
Alternatively, the acid protecting group may be removed by organic and inorganic acids, such as trifluoroacetic acid and hydrochloric acid with or without a noninterferring solvent. Suitable solvents include methylene chloride, tetrahydrofuran, dioxane, and acetone. The t-butyl esters are preferably removed by neat trifluoroacetic acid.
The reaction is conducted at temperatures from about
-10 to 100 °C. preferably at ambient temperature, and is substantially complete in about 1 to 48 hours depending on conditions.
The starting material (16) is prepared by esterifying compound (15) with a alkyl halide = XPG; where X is halo and PG is an acid protecting group, in the presence of a base, preferably potassium carbonate or sodium cabonate, in a noninterferring solvent, preferably dimethylformamide or dimethylsulfoxide . The preferred alkyl halide is methyl iodide. The reaction is conducted at temperatures from about 0 to 100 °C . preferably at ambient temperature, and is substantially complete in about 1 to 48 hours depending on conditions.
Alternatively the starting material (16) may be prepared by condensation with an alcohol HOPG, where PG is an acid protecting group, in the presence of a dehydrating catalyst such as, dicyclohexylcarbodiimide (DCC) or carbonyl diimidazole.
In addition, U.S. Patent No. 4,885,338 and Jpn. Kokai Tokkyo Koho 05286912, Nov 1993 Hesei teach a method for preparing 2-fluoro-5-methoxyaniline derivatives.
Scheme IIIg(c]
Figure imgf000144_0001
(22) (23)
Figure imgf000144_0002
(24 )
R is as defined in Scheme IΙIg(b),
^3(a) j_s as defined in Scheme Ig(a), above; and X is halo.
Benzoic acid derivatives (16) (X= Cl, Br, or I) and boronic acid derivative (27) (either commercially available or readily prepared by known techniques from commercially available starting materials) are condensed under the general procedure of Miyaura, et al., (Ref 8a) or Trecourt, et al., (Ref 8b) in the presence of a palladium catalyst, such as Pd(Ph3P)4, a base, such as sodium bicarbonate, in an inert solvent, such as THF, toluene or ethanol, to afford compound (28).
Compound (28) is converted to the carbazole product (29) by treatment with a trialkyl or triaryl phosphite or phosphine, such as, triethylphosphite or triphenyl phosphine, according to the general procedure of Cadogan, et al. (Ref 6) .
Compound (29) is N-alkylated with an appropriately substituted alkyl or aryl halide XC^R^ in the presence of a base, such as sodium hydride or potassium carbonate, in a noninterfering solvent, such as toluene, dimethylformamide, or dimethylsulfoxide to afford carbazole (30) .
Compound (30) is converted to the corresponding amide (22) by treatment with boron tribromide or sodium thioethoxide, followed by ammonia or an ammonium salt, such as ammonium acetate, in an inert solvent, such as water or alcohol, or with methylchloroaluminum amide in an inert solvent, such as toluene, at a temperature between 0 to 110 °C .
When R3(a) is substituted at the 8-position with chloro, de-alkylation of (30) with boron tribromide results in de-benzylation of the nitrogen as described above. Alkylation may be readily accomplished in a two step process. First, an O-alkylation by treatment with a haloalkyl acetate such as methyl bromo acetate using sodium hydride in tetrahydrofuran, followed by
N-alkylation using for example a base such as sodium hydride and an appropriately substituted alkyl or aryl halide in dimethoxy formamide. Compound (22) can be converted to product carbazole product (24) as described previously in Scheme IΙIg(b) above. Conversion to the desired prodrug may be accomplished by techniques known to the skilled artisan, such as for example, by treatment with a primary or secondary halide to make an ester prodrug.
Scheme I Ι Ig (d)
Figure imgf000146_0001
( 29 )
Alternatively, reduction of the nitro group of compound (28) with a reducing agent, such as hydrogen in the presence of palladium on carbon, in a noninterfering solvent, such as ethanol, at 1 to 60 atmospheres, at a temperature of 0 to 60°C affords the corresponding aniline (32) . Compound (32) is converted to the carbazole (29) according to the general procedure described by Trecourt, et al. (Ref 8b). The aniline is treated with sulfuric acid and sodium nitrite, followed by sodium azide to form an intermediate azide which is cyclized to carbazole (29) by heating in an inert sovent, such as toluene. Compound (29) is converted to carbazole product (24) as described previously in Schemes IΙIg(b) and IΙIg(c).
References :
8) a. N. Miyaura, et al., Synth. Commun. 11, 513 (1981) b. F. Trecourt, et al., Tetrahedron, 51, 11743 6) 6) J. Cadogan et al . , J. Chem. Soc, 4831 (1965)
Scheme IΙIg(e)
Figure imgf000147_0001
1 40 ) ( 41 )
Figure imgf000147_0002
In an aprotic solvent, preferably tetrahydrofuran, reduction of (40) is achieved using a reducing agent such as aluminum trihydride. Preferably, the reaction is conducted under inert atmosphere such as nitrogen, at room temperature.
Sulfonylation may be achieved with an appropriate acylating agent in the presence of an acid scavenger such as triethyl amine. Scheme IΙIg(f) activating agent
Figure imgf000148_0001
(50)
Figure imgf000148_0002
(5i;
In a two-step, one-pot process, intermediate (50) , prepared as described in Scheme I (a) above, is first activated with an activating agent such as carbonyl diimidazole. The reaction is preferably run in an aprotic polar or non-polar solvent such as tetrahydrofuran. Acylation with the activated intermediate is accomplished by reacting with H2 SOR1^ in the presence of a base, preferably diazabicycloundecene .
Scheme I Ι Ig ( g)
Figure imgf000149_0001
Figure imgf000149_0002
Figure imgf000149_0003
( 63 ) ( 64 )
PG is an acid protecting group;
R22 is (Cj_-C6) alkoxy (C;L-C6) alkyl is (Cl-Cg) alkoxy
(Cχ-C6) alkenyl
Starting material (20) is O-alkylated with an alkyl halide or alkenyl halide, using a base such as NaH, in an aprotic polar solvent preferably anhydrous DMF, at ambient temperature under a nitrogen atmosphere. The process of aromatization from a cyclohexenone functionality to a phenol functionality can be performed by treating the tetrahydrocabazole intermediate (60) with a base such as NaH in the presence of methyl benzenesulfinate in an anhydrous solvent, such as 1,4-dioxane or DMF, to form the ketosulfoxide derivative. Upon heating at about 100 °C for 1-2 hours, the ketosulfoxide derivative (60) is converted to the phenol derivative (61). Conversion of the ester (61) to the amide (62) can be achieved by treating a solution of (61) in an aprotic polar solvent such as tetrahydrofuran with ammonia gas. Phenolic O-alkylation of (62) with, for example, methyl bromoacetate can be carried out in anhydrous DMF at ambient temperature using CS2CO3 or K2CO3 as a base to form (63) . Desired product (64) can be derived from the basic hydrolysis of ester (63) using LiOH or NaOH as a base in an H20/CH3OH/THF solution at 50 °C for 1-2 hours .
When R22 is - (Ci-Cg) alkoxy (C^-Cg) alkenyl, hydrogenation of the double bond can be performed by treating (63) in THF using Ptθ2 as a catalysis under a hydrogen atmosphere. Desired product can then be derived as described above in Scheme IΙI(g) from the basic hydrolysis of ester (63) using LiOH or NaOH as a base in an H20/CH3OH/THF solution at 50°C for 1-2 hours.
Compounds of formula le where the A ring is phenyl and the heteroatom in Z is sulfur, oxygen or nitrogen can be prepared as described in Schemes IV(a)-(f), below. Scheme IVg(a)
Figure imgf000152_0001
MeClAlNH2
Figure imgf000152_0002
Figure imgf000152_0003
Figure imgf000152_0004
(107; 108)
1. NaOH
2. HCl
Figure imgf000152_0005
(109) PG is an acid protecting group.
X is halo.
R3(a) is H, -0(C!-C ) alkyl, halo, - (C^Cg) alkyl, phenyl, - (C1-C4) alkylphenyl; phenyl substituted with - (Ci-Cg) alkyl, halo or -CF3; -CH2OSi (Cι~
Cg) alkyl, furyl, thiophenyl, - (C^-Cg) hydroxyalkyl; or -(CH2)n R8 where R8 is H, -NR9R10, -CN or phenyl where R9 and R^O are independently - (C1-C4) alkyl or -phenyl (C1-C4) alkyl and n is 1 to 8; An indole-3-acetic ester (101), Ref 10, is alkylated by treatment with alkalai metal amide and benzyloxymethyl chloride to give (102) which is converted to the alcohol (103) by catalytic hydrogenation. The alcohol is alkylated to provide the formaldehyde acetal (104) which is cyclized by Lewis acid to produce the pyrano [3, 4-b] indole (105). The ester is converted to the amide (106) by methylchloroaluminum amide, and then to the phenol (107) with boron tribromide. The phenol is O-alkylated to give (108) which is hydrolyzed to the acid (109) .
10) Dillard, R. et al . , J, Med Chem. Vol 39, No. 26, 5119-5136.
Scheme IVg (b)
Figure imgf000154_0001
( 112 ;
Figure imgf000154_0002
PG is an acid protecting group
W is halo, alkyl or aryl sulfonyl
R3(a) is H, -CXC1-C4) alkyl, halo, - (C^Cg) alkyl, phenyl, - (C -C4) alkylphenyl; phenyl substituted with - (Ci-Ce) alkyl, halo or -CF3; -CH2OSi (Ci-Cg) alkyl, furyl, thiophenyl, - (C^-Cg) hydroxyalkyl; or -
(CH2)nR8 where R8 is H, -NR9R10, -CN or phenyl where
R9 and R 0 are independently - (C1-C4) alkyl or
-phenyl (Cχ-C4) alkyl and n is 1 to 8; Reaction of this alcohol (103) with aldehyde and acid produces the pyranoindole (110) .
Conversion of the hydroxyl function of (103) to a halide or sulfate functionality is achieved by treatment with triphenylphosphine and CH3X (where X is a halogen) to make compounds of formula (111) where X is a halide; or by treatment with triethylamine and methanesulfonyl chloride to make the sulfonate. Displacement with the sodium salt of thiol acetic acid gives (114) which in turn is hydrolyzed by base to the thiol (115) which is reacted with an appropriately substituted aldehyde and acid to produce the thiopyranoindoles (116) .
Intermediate (111) may also be reacted with sodium azide to give the azido derivative (112) which is reduced by hydrogen catalytically to give the amine which is converted to the carboline (113) with aldehyde and acid.
Intermediates (113), (110) and (116) may be N-alkylated, using sodium hydride and an appropriately substituted alkylhalide XCH2R4.
Scheme Ivg(c)
Figure imgf000156_0001
ecting
above
Figure imgf000157_0001
(131) 4-Methoxyindole (117) is converted to the indole acetic acid derivative (118) by alkylation with an epoxy propionate. Treatment of (118) with a brominating reagent affords the mixture of bromo isomers (119) and (120) which give the spiro compound (121) upon basic treatment. Heating (121) with benzyl bromide provides a mixture of the isomeric bromo compounds (122) and (123) which react with potassium thioacetate to give a mixture of isomers from which (124) may be separated. Solvolysis of the thioester produces the thiol (125) which is alkylated to give (126) . Lewis acids convert (126) to the thiopyrano [3, 4-b] indole (127). The ester function is converted to amide using methylchloroaluminum amide, the methyl ether cleaved by boron tribromide, and the product phenol O-alkylated with bromoacetic ester to give (130) which is hydrolyzed to (131) .
Scheme INg ( d)
Figure imgf000159_0001
(134) ( 135)
Figure imgf000159_0002
X is halo ,
R3(a) j_s as defined in Scheme 1(a) above; and
R is - (CH2)mR5.
Protection of the oxygen by treatment of (132) with tert-butyldimethylsilyl chloride and imidazole in an aprotic polar solvent such as tetrahydrofuran or methylene chloride accomplishes (133) . Alkylation at the 3-position of the indole (133) is achieved by treatment with n-butyllithum then zinc chloride at temperatures starting at about 10 °C and warming to room temperature, followed by reaction with an appropriate haloalkyl ester such as methyl or ethyl bromoacetate. The reaction is preferably conducted at room temperature in an appropriate aprotic polar solvent such as tetrahydrofuran.
Alkylation of the indole-nitrogen can then be achieved by reacting (134) with a suitable alkyl halide in the presence of potassium bis (trimethylsilyl) amide to prepare (135) .
The ester functionality of (135) is converted to a trimethylsilylketene acetal (136) by treatment with potassium bis (trimethylsilyl) amide and trimethylsilyl chloride. Treatment of the ketene acetal (136) with bis (chloromethyl) sulfide and zinc bromide in methylene chloride affords the cyclized product (137). Conversion to amide (138) can be accomplished by a einreb reaction with methylchloroaluminum amide. Removal of the oxygen protecting group with a fluoride source, such as tetrabutylammonium fluoride (TBAF) , and concommitant reaction of the resulting anion with, for example, ethyl bromoacetate yields the ester (139) . Deprotection of the ester yields the desired acid (140) . Scheme IVg ( e )
Figure imgf000161_0001
.
Figure imgf000161_0002
R3 (a) is as described in Scheme I (a) and R is as described in Scheme IV (d) .
Treatment of the ketene acetal (136) with bis (chloromethyl) ether and zinc bromide in methylene chloride affords the cyclized product (141) . Conversion to amide (142) can be accomplished by a einreb reaction with methylchloroaluminum amide. Removal of the oxygen protecting group with a fluoride source, such as tetrabutylammonium fluoride, and concommitant reaction of the resulting anion with ethyl bromoacetate yields the ester (143) . Deprotection of the ester yields the desired acid (144) . Scheme IVg(f)
Figure imgf000162_0001
(231) (232)
Figure imgf000162_0002
(236)
Figure imgf000163_0001
(241) (242)
N-alkylation of commercially available 4-methoxy indole (231) under basic conditions using an alkyl halide affords the N-alkyl indole (232) . Acylation with a suitable acid chloride provides the glyoxalate ester product (233) which can be reduced with a variety of hydride reducing agents to give intermediate alcohols (234). Conversion of the alcohol to a suitable leaving group and displacement with sulfur nucleophiles affords the thioether product (235) . Conversion to the acid chloride and spontaneous cyclization affords the thioketone product (236) . Cleavage of the ester can be effected under basic conditions to give the correponding acid which upon formation of the acid chloride and reaction with an appropriate amine gives the amide product (237) . Cleavage of the methyl ether gives the phenol (238) which can be alkylated under basic conditions using alkyl halides to give the O-alkylated product (239) . Cleavage of the ester under basic conditions gives the desired product (240) . Alternatively, reduction of the benzylic ketone with a hydride reducing agent and subsequent deoxygenation of the resulting alcohol gives the deoxygenated product (244) . Cleavage of the oxyacetic ester proceeds under basic conditions to give the desired oxyacetic acid (242) .
Compounds where Z is an aromatic or heterocyclic ring containing nitrogen can be prepared as described in Schemes Vg(a)-(e), below.
Scheme Vg(a;
Figure imgf000165_0001
(145) (146)
Figure imgf000165_0002
(149) (150)
Substituted haloaniline (145) is condensed with N-benzyl- 3-piperidone to provide enamine (146). Ring closure is effected by treatment of (146) with palladium (II) acetate and the resultant product is converted to (147) by treatment with cyanogen bromide. Alkylation of (147) is accomplished by treatment with the appropriate alkyl bromide using sodium hydride as base. Hydrolysis of this N-alkylated product with basic hydrogen peroxide under standard conditions provides (148). Demethylation of (148) is carried out by treatment with boron tribromide in methylene chloride. The resulting phenol (149) is converted by the standard sequence of O-alkylation with methyl bromoacetate in the presence of a base, hydrolysis with hydroxide to provide the intermediate salt which is then protonated in aqueous acid to provide desired δ- carboline (150) .
Scheme Vg(b)
Figure imgf000167_0001
.3
Figure imgf000167_0002
X is halo, R is as defined in Scheme IV (d) , and R3(a) j_s as defined in Scheme 1(a) . Ketene acetal (136), prepared as described in Scheme I (d), is reacted with benzyl bis (methoxymethyl) amine in the presence of zinc chloride to give the tetrahydro-beta-carboline (151) . Treatment of (151) with lithium hydroxide, neutralization with hydrochloric acid and subsequent treatment with 1- (3-dimethylaminopropyl) -3- ethylcarbodiimide hydrochloride and ammonia provides the desilyated amide (152) where R^O is hydrogen, which can be alkylated with, for example, ethylbromoacetate to give ester (153) .
Alternatively, treatment of (115) with the appropriate einreb reagent provides amide (152) (R20 is t-butyldimethylsilyl) which is desilylated with tetra-n-butylammonium fluoride and alkylated with, for example, ethyl bromoacetate to give ester (153). Lithium hydroxide-mediated hydrolysis gives acid (154), which may be hydrogenated over an appropriate catalyst in the presence of hydrochloride acid to give the tetrahydro-beta-carboline as the hydrochloride salt (155) . Compound (155) may in turn be aromatized by refluxing in carbitol with palladium on carbon to provide beta-carboline (156).
Scheme Vg(c)
2)LiAlH,
Figure imgf000169_0001
NO.
(157)
Figure imgf000169_0002
Figure imgf000169_0003
(164)
X is halo, R is as defined in Scheme IV (d); and R3(a) is as defined in Scheme 1(a). In a one-pot reaction, indole (133) is successively treated with one equivalent n- butyllithium, carbon dioxide gas, one equivalent of t- butyllithium, and l-dimethylamino-2-nitroethene to give (157) . Nitroalkene (157) is reduced with lithium aluminum hydride to amine (158), which is cyclized with methyl glyoxylate (Ref. 9) in refluxing ethanol to give tetrahydrocarboline (159) . Alkylation of both nitrogens of (159) leads to intermediate (160), which is treated with the appropriate Weinreb reagent to provide amide (161) . Fluoride-assisted desilylation and alkylation with, for example, ethyl iodoacetate gives ester (162), which may be hydrogenated over a suitable catalyst and base-hydrolyzed to give acid (163). Aromatization of (163) to carboline (164) is achieved by refluxing in carbitol in the presence of palladium-on-carbon.
Reference 9: Kelley, T. R.; Schmidt, T. E.; Haggerty, J. G. A convenient preparation of methyl and ethyl glyoxylate, Synthesi s, 1972, 544-5.
Scheme Vg(d)
Figure imgf000171_0001
(170) (171)
Figure imgf000171_0002
(172) (173)
-3
Figure imgf000171_0003
(178)
Figure imgf000172_0001
MeAlClNH- MeAlClNH,
Figure imgf000172_0002
DF- 1)F-
2)RX/K2C03 2)RX/K2C03 3) -OH 3) NaOH 4)H+
Figure imgf000172_0003
(182) (185) The commercially available acid (170) is reduced with lithium aluminum hydride, oxidized with pyridinium chlorochromate, and silylated with t-butyldimethylsilyl chloride to give (171) . Treatment with sodium azide provides azide (172) , which is reacted with nitromethane and potassium hydroxide in ethanol, followed by treatment with acetic anhydride and pyridine to give nitroolefin (173) . Heating in xylene induces cyclization to produce indole (174). Alkylation with, for example, benzyl iodide and sodium hydride gives (175), which is hydrogenated in the presence of palladium-on-carbon to give amine (176) . Acylation with the acid chloride of commercially available oxalacetic acid monoethyl ester gives (177), which is thermally cyclized to lactam (178). Selective reduction of the lactam carbonyl may be accomplished by treatment with aB^Sβ to provide amine (179) .
Protection of amine (179) with di-t-butyl dicarbonate and pyridine produces (180), which is converted via the appropriate Weinreb reagent to amide
(181) . Fluoride-assisted desilylation, alkylation with, for example, ethyl iodoacetate and potassium carbonate, base hydrolysis, and acid hydrolysis produce the tetrahydro-alpha-carboline (182) . Alternatively, amine (179) may be aromatized by refluxing in carbitol or some other suitable high boiling solvent to give alpha-carboline (183), which is converted via the appropriate Weinreb reagent to amide (184) . Fluoride-assisted desilylation, alkylation with ethyl iodoacetate and potassium carbonate, and base hydrolysis as described above provides alpha-carboline (185) . Scheme Vg (e)
Figure imgf000174_0001
(190) (191)
Figure imgf000174_0002
(193)
(192)
Figure imgf000174_0003
l)BTCEAD/Et.O l)LiAlH./THF
2 ) Zn/HOAc 2 ) TMSNCO
Figure imgf000174_0004
Figure imgf000174_0005
(197)
X is halo
R3 (a) is as defined above Scheme V(e) provides δ-carboline (198) by the indicated sequence of reactions. N-alkylation of 2- carboethoxyindole (190) followed by a standard two carbon homologation sequence provides 2- (3-propenoic acid) indoles (194). In this sequence, the condensation of aldehyde (193) with malonic acid utilized a mixture of pyridine and piperidine as the base. After methyl ester formation and hydrogenation (195), ring closure (196) was effected by treatment with bis (2,2,2- trichloroethyl) azodicarboxylate (BTCEAD) followed by zinc in acetic acid. Reduction of the cyclic amide with lithium aluminum hydride followed by treatment with trimethylsilylisocyanate provided the urea (197). Conversion to the desired d-carboline (198) was accomplished under the usual conditions of demethylation and subsequent alkylation and ester hydrolysis steps.
Reverse indoles, i.e., compounds where B is carbon and D is nitrogen can be prepared as described in Scheme VIg, below.
Scheme VIg
Figure imgf000176_0001
Aryl hydrazines (200) are condensed with substituted prpionaldehydes to form hydrazones which are cyclized to indoles (201) by treatment with phosphorous trichloride at room temperature (Ref 1) . The indoles are N-alkylated on reaction with a base such as sodium hydride and an alph-bromo ester to give indoles (202) which are cyclized to tetrahydrocarbazoles (203) by Lewis acids (e.g., aluminum chloride) or by radical initiators (e.g., tributyltin hydride) . Compounds (203) can be converted to carbazoles by, for example, refluxing in a solvent such as carbitol in the presence of Pd/C.
Compounds of formula I wherein A is pyridyl can be prepared as described in Schemes VIIg(a)-(b), below. Scheme VΙIg(a)
Figure imgf000177_0001
(218)
X is halo and
R is ;CH2)mR^ Commercially available 4-chloroindole (210) is treated with 3 equivalents of t-butyllithium followed by carbon dioxide, 1 equivalent of n-butyllithium, l-dimethylamino-2-nitroethene, and acid to provide carboxylic acid (211), which may be esterified to give (212) . Alkylation at the 1-position followed by hydrogenation provides aminoethyl indole (214). Cyclization with phosgene to (215) followed by aromatization gives carboline (216) . Treatment of (216) with the appropriate Weinreb reagent provides amide (217), which may be alkylated with, for example, ethyl bromoacetate and saponified with sodium hydroxide to give the carboline (218) .
Scheme VΙIg(b)
)3P or
Figure imgf000179_0001
Figure imgf000179_0002
(226) (227)
R3(a) is as defined in Scheme 1(a), X is halo, and
R is (CH2)mR- The 1,3-dione structures (228) are either commercially available or readily prepared by known techniques from commercially available starting materials. Preparation of the aniline derivatives (220) (X=C1, Br, or I) are accomplished by reducing an appropriately substituted benzoic acid derivative to the corresponding aniline by treatment with a reducing agent such as SnCl2 in hydrochloric acid in an inert solvent such as ethanol or by hydrogenation using hydrogen gas and sulfided platinum or carbon or palladium on carbon. The amino group of (228) is protected with an appropriate protecting group, such as the, carboethoxyl, benzyl, CBZ (benzyloxycarbonyl) or BOC (tert-butoxycarbonyl) protecting group, and the like.
The dione (228) and aniline derivative (220) are condensed according to the general procedure of Chen, et al., (Ref 10) or Yang, et al., (Ref 11), with or without a noninterfering solvent, such as methanol, toluene, or methylene chloride, with or without an acid, such as p-toluenesulfonic acid or trifluoroacetic acid, with or without N-chlorosuccinimide and dimethyl sulfide, to afford the coupled product (221).
Compound (221) is cyclized under basic conditions with a copper (I) salt in an inert solvent according to the general procedure of Yang, et al . , (Reft8). The derivative (221) is treated with a base, such as sodium hydride, in an inert solvent, such as HMPA, at a temperature between 0 and 25 °C. A copper (I) salt, such as copper (I) iodide, is added and the resultant mixture stirred at a temperature between 25 and 150 °C for 1 to 48 hours to afford compound (222) . Compound (221) may also be cyclized according to the general procedure of Chen, et al., (Ref 10) . The derivative (221) is treated with a base, such as sodium bicarbonate, and a palladium catalyst, such as Pd(PPh_3)4, in an inert solvent, such as HMPA, at a temperature between 25 and 150 °C to afford compound (222) .
In a preferred method, intermediate (171) is treated with a transition metal catalyst, such as Pd (OAc) 2 (O-tol) 3P in the presence of a base such as triethylamine using a cosolvent of DMF/acetonitrile to prepare (222) .
Compound (222) is N-alkylated with an appropriately substituted benzyl halide in the presence of a base, such as sodium hydride or potassium carbonate, in a noninterfering solvent, such as dimethylformamide or dimethylsulfoxide to afford ketone (223) . In a two step, one pot process (222) is aromatized by treatment with acetic acid and palladium on carbon in a noninterfering solvent, such as carbitol or cymene, followed by treatment with hydrogen gas and palladium on carbon to cleave the nitrogen protecting group and produce the phenolic derivative (224).
The ester (224) is converted to the corresponding amide (225) under standard conditions with ammonia (preferably) or an ammonium salt, such as ammonium acetate, in an inert solvent, such as water or alcohol, preferably methanol, or with MeClAlNH2 in an inert solvent, such as toluene, at a temperature between 0 to 110 °C. Alkylation of the phenolic oxygen of compound 38 with an appropriate haloester, such as methyl bromoacetate, in the presence of a base, such as cesium carbonate, potassium or sodium carbonate, in an inert solvent, such as dimethylformamide or dimethylsulfoxide affords the ester-amide (226) . Other haloesters, such as ethyl bromoacetate, propyl bromoacetate, butyl bromoacetate, and the like can also be used to prepare the corresponding esters.
Saponification of compound (226) , with lithium hydroxide in an inert solvent, such as methanol-water, affords (227) . The intermediate and final products may isolated and purified by conventional techniques such as chromatography or recrystallization. Regioisomeric products and intermediates can be separated by standard methods, such as, recrystallization or chromatography.
References:
10) L.-C. Chen et al . , Synthesis 385 (1995)
11) S.-C. Yang et al . , Heterocycles, 32, 2399 (1991)
h) Pyrazole sPI .2 inhibitors The compositions and method of the invention may be prepared and practiced using pyrazole sPI -2 inhibitors, which are described (together with the method of making) in US Patent Application No. 08/984261, filed December 3, 1997, the entire disclosure of which is incorporated herein by reference. Suitable pyrazole compounds are represented by formula (Ih)
Figure imgf000182_0001
wherein: R! is phenyl, isoquinolin-3-yl, pyrazinyl, pyridin- 2-yl, pyridin-2-yl substituted at the 4- position with - (C]_-C4 ) alkyl, (Ci-C/j) alkoxyl, - CN or -(CH2)nCONH2 where n is 0-2; R^ is phenyl; phenyl substituted with 1 to 3 substituents selected from the group consisting of - (C1-C4) alkyl, -CN, halo, -NO2, C02 (Cι~ C4) alkyl and -CF3; naphthyl; thiophene or thiophene substituted with 1 to 3 halo groups; R3 is hydrogen; phenyl; phenyl (C2-Cg) alkenyl; pyridyl; naphthyl; quinolinyl; (C1-C4) alkylthiazolyl; phenyl substituted with 1 to 2 substituents selected from the group consisting of - (C1-C4) alkyl, -CN, -CONH2, -N02, -CF3, halo,
(C1-C4) alkoxy, CO2 (C1-C4) alkyl, phenoxy and SR4 where R4 is - (C1-C4) alkyl or halophenyl; phenyl substituted with one substituent selected from the group consisting of -0(CH2)pR5 where p is 1 to 3 and R5 is -
CN, -C02H, -CONH2, or tetrazolyl, phenyl and
-OR^ where R^ is cyclopentyl, cyclohexenyl, or phenyl substituted with halo or (C1-C4) alkoxy; or phenyl substituted with two substituents which, when taken together with the phenyl ring to which they are attached form a methylenedioxy ring; and m is 1 to 5; pharmaceutically acceptable salt thereof. Particularly preferred are pyrazole type sPIA.2 inhibitors as follows:
A pyrazole compound of formula (I), supra, wherein: Ri is pyridine-2-yl or pyridine-2-yl substituted at the 4-position with - (Cχ-C4) alkyl, (C]_- C4) alkoxy, -CN or -(CH2)nCONH2 where n is 0-2; R2 is phenyl substituted with 1 to 3 substituents selected from the group consisting of - (C_- C4) alkyl, -CN, halo, -N02, C02 (C1-C4) alkyl and -CF3; and R3 is phenyl; phenyl (C2-C5) alkenyl; phenyl substituted with 1 or 2 substituents selected from the group consisting of - (C1-C4 ) alkyl, -CN, -CONH2, -NO2, - CF3, halo, (C1-C4) alkoxy, CO2 (C1-C4) alkyl, phenoxy and SR4 where R4 is - (C1-C4 ) alkyl or halo phenyl; phenyl substituted with one substituent selected from the group consisting of -0(CH2)pR^ where p is 1 to 3 and R5 is -CN, -C02H, -C0NH2 or tetrazolyl, phenyl and -OR^ where R^ is cyclopentyl, cyclohexenyl or phenyl substituted with halo or (C1-C4 ) alkoxy; or phenyl substituted with two substituents which when taken together with the phenyl ring to which they are attached form a methylenedioxy ring.
Specific suitable pyrazole type sPIA? inhibitors useful in the method of the invention are as follows: Compounds selected from the group consisting of 3- (2- chloro-6-methylphenylsulfonylamino) -4- (2- (4- acetamido) pyridyl) -5- (3- (4-fluorophenoxy) benzylthio) - (IH) -pyrazole and 3- (2, 6-dichlorophenylsulfonylamino) -4- (2- (4-acetamido) pyridyl) -5- (3- (4- fluorophenoxy) benzylthio) - ( IH) -pyrazole . The pyrazole compounds of formula Ih are prepared as described in Scheme Ih below.
Scheme Ih
Figure imgf000185_0001
L is a leaving group.
In an aprotic polar solvent, such as tetrahydrofuran, an acetonitrile compound (1) is deprotonated by treatment with an excess of a strong base, such as sodium hydride, preferably under an inert gas, such as nitrogen. The deprotonated intermediate is treated with carbon disulfide and then alkylated twice with an appropriately substituted alkyl halide (2) of the formula R3(CH2)mL, where L is a leaving group, preferably bromine, and R3 and m are as defined above, to prepare intermediate compound (3) . The reaction is conducted at ambient temperatures and is substantially complete in 1 to 24 hours.
Cyclization to form the amino substituted pyrazole (4) is achieved by reacting intermediate (3) with hydrazine at room temperature for from about 1 to 24 hours .
Selective sulfonylation of the amino group of intermediate (4) can be accomplished by treatment with a sulfonyl chloride (5) of the formula R2sθ2Cl, where R^ is as defined above, to prepare product (6). The reaction is preferably conducted in a solvent, such as pyridine, at ambient temperature for a period of time of from 1 to 24 hours. Preparation of 2, 6-dimethylphenylsulfonyl chloride can be accomplished as described in J. Org.
Chem . 25, 1996 (1960) . All other sulfonyl chlorides are commercially available.
i) Phenyl glyoxamide sPIJV? inhibitors (and the method of making them) are described in U.S. Patent Application Serial No. 08/979446, filed November 24, 1997 (titled, Phenyl Glyoxamides as SPLA2 Inhibitors) , the entire disclosure of which is incorporated herein by reference.
The compositions and method of the invention is for treatment of a mammal, including a human, afflicted with sepsis may be practiced using phenyl glyoxamide type sPIA.2 inhibitors described as follows:
A compound of the formula (Ii;
Figure imgf000187_0001
wherein: X is -0- or -(CH2)m-/ where m is 0 or 1; Y is -C02-, -PO3-, -SO3-; R is independently -H or - (C1-C ) alkyl; Ri and R2 are each independently -H, halo or -(Ci-C4) alkyl; R3 and R4 are each independently -H, - (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkylthio, halo, phenyl or phenyl substituted with halo; n is 1-8 ; and p is 1 when Y is -CO2- or -SO3- and 1 or 2 when Y is -PO3-; or a pharmaceutically acceptable salt thereof.
A specific suitable phenyl glyoxamide type sPIJJ? inhibitors is 2- (4-carboxybut-l-yl-oxy) -4- (3- phenylphenoxy) phenylglyoxa ide . Phenyl glyoxylamide compounds useful in the compositons and method of the invention are prepared as follows:
Compounds where R1, R^, R3 and R4 are H, and X, Y and n and p are as defined above can be prepared according to the following Scheme Ii.
Scheme Ii
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000189_0002
R * is - (C1-C4 ) alkyl
Reflux of (1) with oxalyl chloride in an alkyl halide solvent, such as chloroform, using 4-N,N' dimethylamino pyridine as a catalyst achieves intermediate (2) .
Under Friedel-Crafts conditions, using a suitable Lewis-acid catalyst such as aluminum chloride, compound (2) is internally cyclized to form compound (3). The reaction is preferably conducted at temperatures from about 0 °C to room temperature and allowed to proceed for about 24 hours. Aminolysis of (3) to amide (4) can be achieved by treatment with concentrated ammonium hydroxide.
Alkylation of the hydroxyl of compound (4) can be readily achieved by treatment with an appropriate alkylating agent, such as Br(CH2)nY, where Y is -CO2R, - PO3R2 or SO3R and R is - (C1-C4 ) alkyl, to form intermediate (5) . The reaction is preferably conducted in an aprotic polar solvent, such as dimethyl formamide, in the presence of potassium carbonate and a suitable catalyst, such as potassium iodide.
Conversion of (5) to the carboxylic or sulfonic acid or acid salt (6) may be achieved by treatment with an appropriate base, such as aqueous sodium hydroxide, in a polar protic solvent, such as methanol. When n is 2, a bromoacetal must be employed as an alkylating agent to achieve the carboxylic acid (6). The alkylated moiety (5) is then converted to the acid (6) by oxidizing with sodium dichromatate in aqueous conditions. When Y is -PO3--, conversion to the acid (6), is preferably conducted in an alkyl halide solvent, such as methylene chloride, using a dealkylating agent, such as trimethylsilyl bromide, and an excess of potassium carbonate, followed by treatment with methanol.
When Ri, R^, R3 or R4 are other than hydrogen, the preparation proceeds as described in Scheme Hi on the following page.
Figure imgf000191_0001
(9)
Figure imgf000191_0002
(12) (13) R' is as defined in Scheme Ii. An appropriately R^, R^ substituted phenol (7) is converted to lactone (8) following the procedures described in Scheme Ii, steps (a-b) above. Conversion to the intermediate (9) is accomplished by reacting (2a) with an aqueous acid, such as hydrochloric acid which affords removal of aluminum chloride from the reaction. Acid (9) is converted to the corresponding acid chloride using oxalyl chloride with dimethyl formamide as a catalyst. The acid chloride is recyclized to the lactone (10) on removal of the solvent, preferably under vacuum. The lactone (10) is converted to the glyoxamide (11) by treatment with an excess of ammonia as described in Schemetl, step (c) , above. Alkylation of (11) to prepare the ester (12), followed by conversion to the acid is accomplished according to the procedure outlined in Scheme I, steps (d) and (e) .
Alternately, conversion of (10) to (12) can be accomplished in a one-pot procedure by treating the lactone (10) with sodium amide in an aprotic polar solvent, such as dimethylformamide, preferably at temperatures of from about 0 °C to 20 °C, followed by alkylation with an appropriate alkyl halide.
j) Pyrrole SPLA2 inhibitors and methods of making them are disclosed in U.S. Patent Applicaton Serial No. 08/985518 filed December 5, 1997 (titled, "Pyrroles as SPLA2 Inhibitors") , the entire disclosure of which is incorporated herein by reference. The compositions and method of the invention for treatment of a mammal, including a human, afflicted with sepsis may be practiced with a pyrrole sPLA? described as follows :
A compound of the formula (Ij)
Figure imgf000193_0001
R! is hydrogen, (C -C4) alkyl, phenyl or phenyl substituted with one or two substituents selected from the group consisting of - (Cχ-C4) alkyl, (C -C4 ) alkoxy, phenyl (C -C4) alkyl, (C -C4 ) alkylthio, halo and phenyl; R2 is hydrogen, - (C -C4) alkyl, halo, (C -C4) alkoxy or (Cχ-C4) alkylthio;
R3 and R4 are each hydrogen or when taken together are =0;
R5 is -NH2 or -NHNH2;
R6 and R7 are each hydrogen or when one of R^ and R7 is hydrogen, the other is - (C1-C4) alkyl, -(CH2)nR10 where R10 is -C02R1:L, -Ε'θ2 (R1 1 ) 2 , -P04(R1:L)2 or -SO3R11 where R1! is independently hydrogen or - (C1-C4) alkyl and n is 0 to 4; or R6 and R7, taken together, are =0 or =S;
X is R8 (Ci-Ce) alkyl; R8 (C2-C6) alkenyl or phenyl substituted at the ortho position with R8 where R8 is
(CH2)nR10 where R10 is -CO2R11, -P03(R11)2, -P04(R ) or -SO3R11, R11 and n is 1 to 4 as defined above, and additionally substituted with one or two substituents selected from the group consisting of hydrogen, - (C1-C4) alkyl, halo, (C1-C4) alkoxy, or two substituents which, when taken together with the phenyl ring to which they are attached, form a naphthyl group; and
R9 is hydrogen or methyl or ethyl; or a pharmaceutically acceptable salt thereof.
Preferred pyrrole SPLA2 inhibitors useful in the method of the invention are compounds of formula Ij wherein;
R! is phenyl;
R2 is methyl or ethyl;
R5 is -NH2;
R^ and R7 are each hydrogen; X is R8 (C]_-Cg) alkyl or phenyl substituted at the ortho position with R8 where
R8 is -CO2R11; and
R9 is methyl or ethyl.
A specific suitable pyrrole SPLA2 inhibitors useful in the method of the invention is 2- [l-benzyl-2, 5-dimethyl- 4- (2-carboxyphenylmethyl) pyrrol-3-yl] glyoxamide .
The pyrrole compounds are prepared as follows:
Compounds of formula I where R^ is -NH2 can be prepared as shown in Scheme Ij, below. Scheme I j
Figure imgf000195_0001
( c )
Figure imgf000195_0002
( 6 ) ( 5 !
An appropriately substituted gamma-diketone (1) is reacted with an alkylamine of the formula NHCH2R1 to give pyrrole (2) . Under Friedel-Crafts conditions, using a suitable Lewis-acid catalyst such as stannic chloride, aluminum chloride, or titanium tetrachloride (preferably stannic chloride) pyrrole (2) is ring alkylated with an alkyl or arylalkyl halide compound of the formula ZCR^R7X where Z is a suitable halogen and R8 of X is a protected acid or ester. The reaction is preferably conducted in a halogenated hydrocarbon solvent, such as dichloromethane, at ambient temperatures and allowed to proceed for from about 1 to about 24 hours.
Intermediate (3) is converted to (4) by sequential treatment with oxalyl chloride followed by ammonia. Selective reduction of (4) is accomplished in a two step process. In a hydride reduction using, for example, sodium borohydride, the hydroxy intermediate (5) is prepared which can be further reduced using either catalytic or hydride reduction (preferably palladium on carbon) to prepare (6). Deprotection of R8 to the acid may be readily achieved by conventional techniques. For example, when an alkyl ester is used as a protecting group, deprotection can be accomplished by treatment with a base, such as sodium hydroxide.
k) Naphthyl glyoxamide SPLA2 inhibitors and methods of making them are described in U.S. Patent Application Serial No. 09/091079, filed December 9, 1966 (titled, "Naphthyl Glyoxamides as sPLA2 Inhibitors") , the entire disclosure of which is incorporated herein by reference.
The compositions and method of the invention for treatment of a mammal, including a human, afflicted with sepsis may be practiced with a naphthyl glyoxamide SPLA2 inhibitors described as follows:
A naphthyl glyoxamide compound or a pharmaceutically acceptable salt, solvate or prodrug derivative thereof; wherein said compound is represented by the formula Ik
Figure imgf000196_0001
wherein: R! and R2 are each independently hydrogen or a non- interfering substituent with the proviso that at least one of R! or R2 must be hydrogen;
X is -CH2~ or -0-; and Y is (CH2)nZ where n is a number from 1-3 and Z is an acid group selected from the group consisting of CO2H, -SO3H or -PO(OH)2-
A specific suitable naphthyl glyoxamide SPLA2 inhibitors useful in the method of the invention has the following structural formula:
Figure imgf000197_0001
The naphthyl glyoxamide compounds are prepared as follows:
Compounds of formula I where X is oxygen can be prepared by the following reaction Scheme Ik. Scheme Ik
Figure imgf000198_0001
Figure imgf000198_0002
Figure imgf000199_0001
In the above depicted reaction scheme, the 1, 5-dihydroxy napthalene starting material (1) is dispersed in water and then treated with 2 equivalents of potassium hydroxide. The resultant solution is chilled in an ice bath and one equivalent of a strong mineral acid, such as hydrochloric acid, is added to produce the potassium saltt(2). Alkylation of the radical (2) can then be accomplished by treatment with a methylating agent such as dimethyl sulfate to prepare the ether (3) .
Preparation of (4) is achieved by reacting the ether (3) with an appropriately substituted phenol in an Ullman-type reaction using potassium carbonate and cupric oxide .
De-methylation of (4) can be accomplished by treating (4) with a 40% HBr/HOAC solution at reflux in a protic polar solvent such as acetic acid, to prepare (5) . Reflux of compound (5) with oxalyl chloride and
4-demethylamino pyridine, in an alkylhalide solvent such as methylene chloride, prepares the oxalyl chloride (6).
Internal cyclization of (6) can be achieved under Friedel-Crafts condition using aluminum chloride or other similar metal halide as the catalyst. The reaction can be conveniently conducted in an alkyl halide solvent, such as 1, 2-dichloro ethane.
Alkylation and hydrolysis of the cyclized compound (7) can be achieved by reacting (7) with an alkaliamide base, such as sodium amide, followed by treatment with an alkylating agent, such as methyl bromoacetate, using potassium iodide as a catalyst.
Finally, the acid (9) is achieved by treating the ester (8) with an alkali base, such as aqueous sodium hydroxide, followed by treatment with a dilute aqueous mineral acid such as hydrochloric acid The acid compound (9) is then extracted with an organic solvent such as ethyl acetate.
The final product (9) can be purified using standard recrystallization procedures in a suitable organic solvent such as methylene chloride/hexane.
Compounds of formula I where X is methylene can be prepared as shown in the following Scheme Ilk
Scheme Ilk
Figure imgf000201_0001
(2a)
Using an appropriately substituted phenyl bromide, a Grignard reagent is prepared. The phenyl Grignard is then reacted with 4-methoxy naphthylnitrile and the resultant compound is hydrolyzed with a dilute acid such as hydrochloric acid to form the benzoyl naphthylene compound (la) .
Reduction of (la) to form compound (2a) is accomplished by treatment with a reducing agent such as sodium borohydride. The reaction is conducted in a solvent-catalyst such as trifluoroacetic acid and initiated in an ice bath which is allowed to warm to room temperature as the reaction proceeds.
The desired naphthyl glyoxamide may then be prepared from (2a) according to the procedure in Scheme I starting with the chloromethylation step.
It will be readily appreciated by a person skilled in the art that the substituted benzyl bromide, substituted phenol and substituted naphthylnitrile compounds of Schemes I and II are either commercially available or can be readily prepared by known techniques from commercially available starting materials.
1) Phenyl acetamide SPLA2 inhibitors and methods of making them are disclosed in US Patent Application 08/976858, filed November 24 1997 (titled, "Phenyl Acetamides as SPLA2 Inhibitors") , the entire disclosure of which is incorporated herein by reference.
The compositions and method of the invention for treatment of a mammal, including a human, afflicted with sepsis may be practiced using a phenyl acetamide sPLA? inhibitor represented by formula (II) as follows:
Figure imgf000203_0001
wherein:
R1 is -H or -0(CH2)nZ; R2 is -H or -OH;
R3 and R^ are each independently -H, halo or - (C1-C4) alkyl;
One of R^ and R^ is -YR7 and the other is -H, where Y is -0- or -CH2- and R7 is phenyl or phenyl substituted with one or two substituents selected from the group consisting of halo, - (C -C4) alkyl, (Cχ-C4 ) alkoxy, phenyl or phenyl substituted with one or two halo groups;
Z is -CO2R. -PO3R2 or -SO3R where R is -H or - (C1-C4) alkyl; and n is 1-8; or a pharmaceutically acceptable salt, racemate or optical isomer thereof; provided that when R^ is YR7, R^ is hydrogen; and when Ri, R2, R3, R4 and R^ are hydrogen and R^ is YR7 where Y is -0-, R7 cannot be phenyl; and when R1, R2, R3, R4 and R6 are hydrogen, R5 is YR7 where Y is CH2, R7 cannot be phenyl substituted with one methoxy or two chloro groups . Preferred suitable phenyl acetamide SPLA2 inhibitors useful in the composition and method of the invention are as follows:
Compounds of formula I wherein R2, R3 and R4 is H, Y is oxygen or CH2, R7 is phenyl or phenyl substituted at the meta position with one or two substituents selected from halo, - (C -C4) alkyl, (C1-C ) alkoxy, phenyl or phenyl substituted with halo and n is 4-5.
A specific suitable phenyl acetamide SP A2 inhibitor useful in the method of the invention is 2- (4- carboxybutoxy) -4- ( 3-phenylphenoxy) phenylacetamide .
The phenyl acetamide inhibitors are prepared as follows:
Compounds of formula I where R^ and R2 are H, R^ or R^ are YR7 where R7 is phenyl or substituted phenyl and Y is oxygen can be prepared as illustrated in Scheme II (a) , below.
Sche e 11(a)
Figure imgf000205_0001
X is halo;
R8 and R^ are each independently -H, halo, - (C1-C4) alkyl, (C1-C4) alkoxy, phenyl or phenyl substituted with one or two halo groups; and PG is a carboxyl protecting group An appropriately substituted carboxy-protected halophenyl compound (1), where the halogen is preferably bromine, is coupled with an appropriately substituted phenol (2) under modified Ullmann conditions, by refluxing with potassium carbonate and cupric oxide in an aprotic polar solvent, such as pyridine, under an inert gas such as argon. The reaction is substantially complete in 1-24 hours.
Intermediate (3) is deprotected by treatment with a base such as aqueous potassium hydroxide using a solvent, such as diethylene glycol. The reaction, preferably conducted at about 100°-150 °C, is substantially complete in 1-24 hours.
Conversion to the amide (5) can then be readily achieved by treatment first with oxalyl chloride in an alkyl halide solvent, such as methylene chloride, using dimethylformamide as a catalyst, at temperatures of from about 0 °C to ambient temperature, followed by treatment with an excess of ammonia gas, again in an alkyl halide solvent . Alternately, compounds of formula I can be prepared according to the procedure of Scheme I (b) , below. The substituted phenol (2) is coupled with an appropriately substituted benzyl halide (6) as described in Scheme 1(a), step a, above, to prepare (7). Halogenation of (7) is achieved using a halogenating agent, such as N-bromosuccinimide and a catalyst, such as
2, 2 ' azobisisobutyronitrile, in an alkyl halide solvent, such as chloroform, to prepare (8) .
Treatment of (8) with sodium cyanide in an aprotic polar solvent, such as dimethyl formamide produces the nitrile (9) which can then be readily converted to the amide (10) by treatment with an aqueous acid, such as hydrochloric acid. Scheme II (b)
Figure imgf000207_0001
R8 and R^ are as shown in Scheme I (a) , X is halo.
In another procedure, compounds of formula I where R1, R2, R3 and R4 are hydrogen, Y is -0- or -CH2~ and R7 is phenyl can be prepared as portrayed in Scheme III.
Scheme I I I
Figure imgf000208_0001
Figure imgf000208_0002
X is a halogen .
An appropriate diphenyl compound (11) is treated with paraformaldehyde and a halogenating agent, such as 40% hydrogen bromide in acetic acid. Two positional isomers result with the X substituent at either the meta or para position of the phenyl ring to which it is attached.
Displacement of the halogen to prepare the nitrile isomers (13) can be achieved by treatment of (12) with sodium cyanide in dimethylformamide as described in Schemetl (b) , step (c) , above. The isomers can then be readily separated by conventional chromatographic techniques and each isomer may be converted to its respective amide (14) by treatment with hydrogen peroxide and potassium carbonate in an aprotic polar solvent, such as dimethylsulfoxide.
Compounds where R^ is -0(CH2)nZ can be prepared as illustrated in Scheme III1, below.
Scheme I I II
Figure imgf000209_0001
Figure imgf000210_0001
R is - (C1-C4 ) alkyl and p = 1 or 2 .
Intermediate (16) is prepared by refluxing an appropriately substituted diphenyl compound (15) with oxalyl chloride in an alkyl halide solvent, such as chloroform. Preferably the reaction is catalyzed with 4, 4-N-dimethylaminopyridine.
Cyclization to the lactone (17) can be achieved under Friedel-Crafts conditions using a suitable metal halide, such as aluminum chloride, as the catalyst. Conversion to the glyoxamide (18) can be achieved by aminolysis of the lactone ring using concentrated ammonium hydroxide . Alkylation of the hydroxy group to prepare the desired alkyl-linked ester (19) occurs by treatment of
(18) with an appropriate alkylating agent, such as
(X) (CH2)nB where B is C02PG, -PO3PG or -SO3PG, X is halo and PG is an acid protecting group, preferably methyl. Partial reduction of the carbonyl in the glyoxamide
(19) is achieved by treatment with a suitable reducing agent, such as sodium borohydride in methanol, preferably at temperatures of from 0°-20 °C, to prepare the intermediate (20) . The desired acid or acid salt (21) can be accomplished by treatment with a suitable base, such as sodium hydroxide.
Further reduction of intermediate (20) can be achieved by treatment with triethylsilane in a strong acid, such as trifluroacetic acid, under an inert gas, such as argon, to prepare (22) followed, again, by conversion to the acid or salt (23) with a strong base. m) Naphthyl acetamide sPIA? inhibitors and the method of making them are described in U.S. Patent Application Serial No. 09/091077, filed December 9, 1996 (titled, "Benzyl naphthalene SPLA2 Inhibitors") , the entire disclosure of which is incorporated herein by reference.
The composition and method of the invention for treatment of a mammal, including a human, afflicted with sepsis is practiced using a naphthyl acetamide SPLA2 inhibitor represented by formula (Im)as follows:
Figure imgf000212_0001
wherein:
R and R2 are each independently hydrogen or a non- interfering substituent with the proviso that at least one of R! and R2 must be hydrogen;
R3 is hydrogen, -0(CH2)nY, -~0
Figure imgf000212_0002
nm Y where n is from 2 to 4 and Y is -CO2H, -PO3H2 or SO3H; and X is -0- or -CH2-.
Compounds where X is oxygen can be prepared by the following Scheme Im. Scheme Im
Figure imgf000213_0001
Figure imgf000214_0001
In the first step of the above reaction scheme, an appropriately substituted l-bromo-4-methylnapthalene and an appropriately substituted phenol are dissolved in an aprotic polar solvent such as pyridine. The mixture is treated with an excess of potassium carbonate and an excess of copper-bronze and refluxed under a nitrogen blanket to produce (1). Bromination of compound (1) to produce (2) is accomplished by refluxing (1) with a brominating agent, such as N-bromosuccinamide, in a non-polar alkyl halide solvent, such as carbon tetrachloride, using 2,2- azobisisobutyronitrile as a catalyst. Treatment of (2) with sodium cyanide produces (3) . This reaction is best conducted in an aprotic polar solvent, such as dimethyl sulfoxide (DMSO) , while heating to a temperature of about 60 °C.
Hydrolysis of the cyano compound (3) to produce the acid (4) is accomplished in two steps. Using a polar protic solvent, such as diethylene glycol as a cosolvent, the cyano compound (3) is treated with an alkali metal base, such as potassium hydroxide, and the mixture is heated to about 90-95 °C. The resultant product is then reacted with a strong mineral acid such as hydrochloric acid.
Conversion of (4) to the desired naphthyl acetamide compound (5) is accomplished by another two-step process. First, the acid (4) is dissolved in an alkyl halide solvent such as methylene chloride. The acid/alkyl halide solution is chilled in an ice bath then treated with oxalyl chloride, using dimethylformamide (DMF) as a catalyst, to produce the acid chloride. The solution is allowed to warm to room temperature and then treated with ammonia gas at room temperature to produce (5) .
The desired product (5) can be purified using standard recrystallization procedures in a suitable organic solvent, preferably methylene chloride/hexane .
Compounds where X is methylene can be prepared by the following Scheme Ilm
Scheme I lm
Figure imgf000215_0001
3) aqueous acid
Figure imgf000215_0002
Figure imgf000216_0001
(1) KCN
(2) DMF
Figure imgf000216_0002
Compound (la) is prepared by a grignard reaction. The Grignard reagent starting material is prepared by reacting an appropriately substituted phenyl bromide with magnesium and ether. The reagent is then reacted with an appropriately substituted naphthyl nitrile and the resultant compound is hydrolyzed with an aqueous acid such as hydrochloric acid to form the benzoyl napthyl (la) .
Reduction of (la) is accomplished by treatment with a molar excess of a reducing agent such as sodium borohydride. The reaction is initiated in an ice bath using a solvent-catalyst such as trifluoroacetic acid and then allowed to warm to room temperature as the reduction proceeds .
Chloromethylation of (2a) is achieved by treatment with an excess of formaldehyde and concentrated hydrochloric acid in a polar acidic solvent such as an acetic/phosphoric acid mixture. The reaction is best conducted at a temperature of about 90 °C.
The nitrile 4 (a) is prepared by a nucleophilic displacement of the chloride compound (3a) ith cyanide.
The reaction is conducted by refluxing (3a) with a slight molar excess in an aprotic polar solvent of sodium cyanide such as dimethylformamide (DMF) for about five hours, then allowing the reaction to continues while it cools to room temperature.
The desired naphthylamide (5a) is then prepared from the nitrile (4a) in a three-step process. To a solution of nitrile (4a) , dissolved in an aprotic polar solvent such as DMSO, potassium carbonate is added to make the nitrile solution slightly basic. Hydrolysis of the nitrile is then achieved by treatment with an aqueous hydrogen peroxide solution. Crystallization of the naphthyl acetamide may be accomplished by adding water to the peroxide solution. Compounds where R3 is other than hydrogen can be readily prepared by using a l-bromo-4-methyl-napthalene with a protected phenol, such as a methoxy group, on the 6-position of the napthalene ring as a starting material. The process is conducted, as described above, to prepare compounds (1) - (3) . Acid hydrolysis of the cyano group (3) and deprotection of the protected phenol can be accomplished by treating (3) with a 40% hydrogen bromide solution in acetic acid. The deprotected phenol can then be reacted to prepare the appropriate substituent at the 6-position of the napthyl ring. For example, preparation of compounds where R3 is -0(CH2)nCOOH can be achieved by alkyalting the phenol with an appropriate alkyl halide followed by conversion to the acid by treatment with a base such as aqueous sodium hydroxide followed by dilute hydrochloric acid.
It will be readily appreciated by one skilled in the art that the substituted phenol and phenyl bromide starting materials are either commercially available or can be readily prepared by known techniques from commercially available starting materials. All other reactants and reagents used to prepare the compounds of the present invention are commercially available. Most Preferred SPLA2 inhibitors: lH-indole-3-glyoxylamide SPLA2 inhibitors and carbazole sPLA2 inhibitors (as described, supra.) are most preferred for the compositions and method this invention.
III. Preparation of the Activated Protein C Ingredient
a. Preparation of Human Protein C
Recombinant human Protein C (r-hPC) was produced in Human Kidney 293 cells by techniques well known to the skilled artisan such as those set forth in Yan, U.S. Patent No. 4,981,952, the entire disclosure of which is herein incorporated by reference. The gene encoding human Protein C is disclosed and claimed in Bang et al., U.S. Patent No. 4,775,624, the entire disclosure of which is incorporated herein by reference. The plasmid used to express human Protein C in 293 cells was plasmid pLPC which is disclosed in Bang et al./ U.S. Patent No. 4,992,373, the entire disclosure of which is incorporated herein by reference. The construction of plasmid pLPC is also described in European Patent Publication No. 0 445 939, the teachings of which are also incorporated herein by reference and in Grinnell et al., 1987, Bio/Technology 5:1189-1192. Briefly, the plasmid was transfected into 293 cells, then stable transformants were identified, subcultured and grown in serum-free media. After fermentation, cell-free medium was obtained by microfiltration.
The human Protein C was separated from the culture fluid by an adaptation of the techniques of Yan, U.S.
Patent No. 4,981,952, the entire disclosure of which is herein incorporated by reference. The clarified medium was made 4 mM in EDTA before it was absorbed to an anion exchange resin (Fast-Flow Q, Pharmacia) . After washing with 4 column volumes of 20 mM Tris, 200 mM NaCl, pH 7.4 and 2 column volumes of 20 mM Tris, 150 mM NaCl, pH 7.4, the bound recombinant human Protein C zymogen was eluted with 20 mM Tris, 150 mM NaCl, 10 mM CaCl2/ pH 7.4. The eluted protein was greater than 95% pure after elution as judged by SDS-polyacrylamide gel electrophoresis .
Further purification of the protein was accomplished by making the protein 3 M in NaCl followed by adsorption to a hydrophobic interaction resin (Toyopearl Phenyl 650M, TosoHaas) equilibrated in 20 mM Tris, 3 M NaCl, 10 mM CaCl2/ pH 7.4. After washing with 2 column volumes of equilibration buffer without CaCl2/ the recombinant human Protein C was eluted with 20 mM Tris, pH 7.4. The eluted protein was prepared for activation by removal of residual calcium. The recombinant human Protein C was passed over a metal affinity column (Chelex-100, Bio-Rad) to remove calcium and again bound to an anion exchanger (Fast Flow Q, Pharmacia) . Both of these columns were arranged in series and equilibrated in 20 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.4. Following loading of the protein, the Chelex-100 column was washed with one column volume of the same buffer before disconnecting it from the series. The anion exchange column was washed with 3 column volumes of equilibration buffer before eluting the protein with 0.4 M NaCl, 20 mM Tris-acetate, pH 6.5. Protein concentrations of recombinant human protein C and recombinant activated Protein C solutions were measured by UV 280 nm extinction E°-1%=1.85 or 1.95, respectively.
b. Activation of recombinant human Protein C
Bovine thrombin was coupled to Activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 4 °C. The coupling reaction was done on resin already packed into a column using approximately 5000 units thrombin/ml resin. The thrombin solution was circulated through the column for approximately 3 hours before adding MEA to a concentration of 0.6 ml/1 of circulating solution. The MEA-containing solution was circulated for an additional 10-12 hours to assure complete blockage of the unreacted amines on the resin. Following blocking, the thrombin-coupled resin was washed with 10 column volumes of 1 M NaCl, 20 mM Tris, pH 6.5 to remove all non- specifically bound protein, and was used in activation reactions after equilibrating in activation buffer.
Purified rHPC was made 5 mM in EDTA (to chelate any residual calcium) and diluted to a concentration of 2 mg/ml with 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5. This material was passed through a thrombin column equilibrated at 37°C with 50 mM NaCl and either 20 mM Tris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate was adjusted to allow for approximately 20 min. of contact time between the rHPC and thrombin resin. The effluent was collected and immediately assayed for amidolytic activity. If the material did not have a specific activity (amidolytic) comparable to an established standard of aPC, it was recycled over the thrombin column to activate the rHPC to completion. This was followed by 1:1 dilution of the material with 20 mM buffer as above, with a pH of either 7.4 or 6.5 to keep the aPC at lower concentrations while it awaited the next processing step.
Removal of leached thrombin from the aPC material was accomplished by binding the aPC to an anion exchange resin (Fast Flow Q, Pharmacia) equilibrated in activation buffer (either 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5) with 150 mM NaCl. Thrombin does not interact with the anion exchange resin under these conditions, but passes through the column into the sample application effluent. Once the aPC is loaded onto the column, a 2-6 column volume wash with 20 mM equilibration buffer is done before eluting the bound aPC with a step elution using 0.4 M NaCl in either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Higher volume washes of the column facilitated more complete removal of the dodecapeptide. The material eluted from this column was stored either in a frozen solution (-20 °C) or as a lyophilized powder. The anticoagulant activity of activated Protein C was determined by measuring the prolongation of the clotting time in the activated partial thromboplastin time (APTT) clotting assay. A standard curve was prepared in dilution buffer (1 mg/ml radioimmunoassay grade BSA, 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3) ranging in Protein C concentration from 125-1000 ng/ml, while samples were prepared at several dilutions in this concentration range. To each sample cuvette, 50 μl of cold horse plasma and 50 μl of reconstituted activated partial thromboplastin time reagent (APTT Reagent, Sigma) were added and incubated at 37 °C for 5 min. After incubation, 50 μl of the appropriate samples or standards were added to each cuvette. Dilution buffer was used in place of sample or standard to determine basal clotting time. The timer of the fibrometer (CoA Screener Hemostasis Analyzer, American Laboratory) was started immediately after the addition of
50 μl 37 °C 30 mM CaCl2 to each sample or standard. Activated Protein C concentration in samples are calculated from the linear regression equation of the standard curve. Clotting times reported here are the average of a minimum of three replicates, including standard curve samples.
IV. Pharmaceutical Compositions of the Invention The pharmaceutical composition of the invention comprises as essential ingredients:
(a) an SPLA2 inhibitor, and (b) Activated Protein C. When these two ingredients are combined as a pharmaceutical composition the composition must be in a form which; (i) is itself in a liquid form suitable for administration by injection or, (ii) is in a form which is easily dissolved or suspended, or dispersed or emulsified into a liquid medium which is then suitable for administration by injection. When the pharmaceutical composition of the invention is prepared in injectable form it is a composition comprising as ingredients:
(a) an SPLA2 inhibitor,
(b) Activated Protein C, and
(c) an injectable liquid carrier.
a. Ratio and Amount of Ingredients in the Composition of the Invention
The essential ingredients (a) an sPLA? inhibitor and (b) Activated Protein C are present in the formulation in such proportion that a dose of the formulation provides a pharmaceutically effective amount of each ingredient to the patient being treated. Typically, the weight ratio of sPLA2 to activated protein C is from 1000:1 to 10000000:1 and preferably from 100:1 to 1000000:1.
An effective dosage of activated Protein C in human patients is considered to be between O.land 100 μg/kg/day. Preferably, the dosage is between 1 and 50 μg/kg/day. A most preferred dosage of activated Protein C is between 1 and 25 μg/kg/day. An effective dosage of an SPLA2 inhibitor in human patients is considered to be between 0.1 and 2000 mg/kg/day. Preferably, the dosage is between 1 and 100 mg/kg/day. In making compositions of the invention the essential ingredients;
(a) an sPLA2 inhibitor, and
(b) Activated Protein C are co-present and may be mixed in any homogeneous or non-homogeneous manner or adjacently or otherwise promixately placed together in an individual dosage unit suitable for practicing the method of the invention. The dosage unit of the SPLA2 inhibitor will usually be admixed with a carrier or inert ingredients, or diluted by a carrier, or enclosed within a carrier which may be in the form of a ampoule, capsule, time release dosing device, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semi-solid, paste, or liquid material which acts as a vehicle, or can be in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , or ointment, containing, for example, up to 10% by weight of the active compound.
The dosage unit of the Activated Protein C will usually be admixed with a liquid carrier and/or other inert ingredients or enclosed within a carrier which may be in the form of a ampoule, bottle, time release dosing device or other container. When the carrier serves as a diluent, it may be a liquid material which acts as a vehicle, or can be in the form of solutions containing, for example, up to 10% by weight of the active compound. The Activated Protein C ingredient should be in an injectable liquid form immediately prior to use, however, it may be made in a storable form which is not a liquid but is easily convertable to a liquid (e.g., paste, liquid adsorbed on a solid, etc.)
For the pharmaceutical formulations containing both (a) SPLA2 inhibitor and (b) Activated Protein C the carrier may be an injectable liquid medium such as is well known in the art. The injectable liquid must be such that permits parenteral administration, that is, introduction of substances to a mammal being treated by intervenous, subcuataneous, intramuscular, or intramedullary injection. Intravenous injection is most preferred as a means of administration.
The Active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile water containing saline and/or sugars and/or suspension agents or a mixture of both. For example, for intravenous injection the compounds of the invention may be dissolved in at a concentration of 2 mg/ml in a 4% dextrose/0.5% Na citrate aqueous solution. The SPLA2 inhibitor (when separate from the
Activated Protein C) may be in the form of powder, tablet or capsule. A solid carrier can be one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, binders, tablet disintegrating agents and encapsulating material. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting waxes, and cocoa butter.
The following pharmaceutical formulations are useful (as stated) for either the SPLA2 inhibitor alone, or the Active Ingredient which is a combination of (a) SPLA2 inhibitor and (b) Activated Protein .
Typically, from 10 mg to 1000 mg of the SPLA2 inhibitor is used in a unit dose of the formulation.
Formulation 1 Hard gelatin capsules are prepared using the following ingredients:
Quantity (mg/capsule) sPLA inhibitor 250
Starch, dried 200
Magnesium stearate 1_0
Total 460 mg
Formulation 2
A tablet is prepared using the ingredients below:
Quantity (mg/tablet) sPLA2 inhibitor 250
Cellulose, macrocrystalline 400
Silicon dioxide, fumed 10
Stearic acid 5
Total 665 mg
The components are blended and compressed to form tablets each weighing 665 mg
Formulation 3 An aerosol solution is prepared containing the following components: Wei<jht sPLA2 inhibitor 0 .25
Ethanol 25 .75
Propellant 22 (Chlorodifluoromethane) 74 .00
Total 100.00
The SPLA2 inhibitor is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -30 °C and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container.
Formulation 4 Tablets, each containing 60 mg of sPIA? inhibitor, are made as follows:
sPLA2 inhibitor 60 mg
Starch 45 mg
Microcrystalline cellulose 35 mg
Polyvinylpyrrolidone (as 10% solution in water! 4 mg
Sodium carboxymethyl starch 4 .5 mg
Magnesium stearate 0. .5 mg
Talc 1 mg
Total 150 mg
The SPLA2 inhibitor , starch and cellulose are passed through a No . 45 mesh U.S. sieve and mixed thoroughly. The aqueous solution containing polyvinylpyrrolidone is mixed with the resultant powder, and the mixture then is passed through a No. 14 mesh U.S sieve. The granules so produced are dried at 50 °C and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
Formulation 5
Capsules, each containing 80 mg of SPLA2 inhibitor, are made as follows:
SPLA2 inhibitor 80 mg
Starch 59 mg
Microcrystalline cellulose 59 mg
Magnesium stearate 2 mg
Total 200 mg
The SPLA2 inhibitor, cellulose, starch, and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities.
Formulation 6
Suppositories, each containing 225 mg of SPLA2 inhibitor, are made as follows:
SPLA2 inhibitor 225 mg
Saturated fatty acid glycerides 2,000 mg
Total 2,225 mg The SPLA2 inhibitor is passed through a No . 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
Formulation 7
Suspensions, each containing 50 mg of Active Ingredient per 5 ml dose, are made as follows:
Active Ingredient 50 mg
Sodium carboxymethyl cellulose 50 mg
Syrup 1.25 ml
Benzoic acid solution 0.10 ml
Flavor q.v.
Color q.v.
Purified water to total 5 ml
The active ingredient is mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste, The benzoic acid solution, flavor and color are diluted with a portion of the water and added, with stirring. Sufficient water is then added to produce the required volume .
Formulation 8 An intravenous formulation may be prepared as follows :
Active Ingredient 100 mg
Isotonic saline 1,000 ml The solution of the above Active Ingredient generally is administered intravenously to a subject at a rate of 1 ml per minute.
Typically, from 10 mg to 1000 mg of the Active Ingredient is used in a unit dose of the formulation.
A unit dosage formulation suitable for administration by continuous infusion is prepared by mixing at pH 6.0, Activated Protein C, an SPLA2 inhibitor compound, a salt
(NaCl) , a bulking agent (sucrose) , and a buffer (citrate) . The active ingredient, salt, and bulking agent are mixed in a weight to weight ratio of about 1 part Active ingredient, between about 7 and 8 parts salt, and between about 5 to 7 parts bulking agent.
After mixing, 4 ml of the solution is transferred to vials and lyophilized. The vials comprising the active ingredients is sealed. and stored until use.
V. Treating Sepsis by The Method of the Invention
This invention is a method of treating or preventing sepsis by administering to a mammal in need thereof a therapeutically effective amount of (a) an SPLA2 inhibitor and a therapeutically effective amount of (b) Activated Protein C; wherein (a) and (b) are both administered within a therapeutically effective interval. The administration of (a) or (b) to a septic patient may be either continuous or intermittent.
A. Method of the Invention using simultaneous delivery of Activated Protein C and SPLA2 inhibitor
The Activated Protein C and an SPLA2 inhibitor can be delivered simultaneously. One convenient method of simultaneous delivery is to use the compositions of the invention described in section IV, supra, wherein the Active ingredient has the essential ingredients co-present in a unit dosage form. Solution or suspensions of mixed essential ingredients may, if desired, be delivered from the same IV liquid holding bag.
Another method of simultaneous delivery of the Activated Protein C and an SPLA2 inhibitor is to deliver them to the patient separately but simultaneously. Thus, for example, the sPLA? inhibitor may be given as an oral formulation at the same time the Activated Protein C is given parenterally.
Dosage of an sP A? inhibitor can begin simultaneously with the activated Protein C administration. The length of the SPLA2 inhibitor administration can extend past the activated Protein C administration.
B. Method of the Invention using non-simultaneous delivery of Activated Protein C and sPLA? inhibitor.
Each of the essential ingredients, viz., a therapeutically effective amount of (a) an SPLA2 inhibitor and a therapeutically effective amount of (b) Activated Protein C have a therapeutically effective interval, namely the interval of time in which each agent provides benefit for the patient being treated with sepsis. The method of the invention may be practiced by separately dosing the sepsis patient in any order with a therapeutically effective amount of (a) an sPLA? inhibitor and a therapeutically effective amount of (b) Activated Protein C provided that each agent is given within the period of time that that the other agent is therapeutically effective against sepsis.
Typically, intravenous forms of SPLA2 inhibitor (e.g. , ( (3- (2-amino-l, 2-dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4-yl) oxy) acetic acid, sodium salt) are therapeutically effective immediately upon administration and up to 5 days later, and preferably in the time interval from 5 minutes after administration to 72 hours after administration. Typically, oral forms of sPLA2 inhibitor (e.g., ( (3- (2-amino-l, 2-dioxoethyl) -2- ethyl-1- (phenylmethyl) -lH-indol-4-yl) oxy) acetic acid, methyl ester) are therapeutically effective from about 10 minutes to 5 days, and preferably from one-half hour to 72 hours after administration.
Dosage delivery of the SPLA2 inhibitor can begin up to 48 hours prior to the activated Protein C infusion with the preferred time being up to 24 hours and the most preferred being up to 12 hours. Alternatively, dosage of an SPLA2 inhibitor can begin up to 48 hours after the initiation of the activated Protein C infusion with the preferred time being up to 24 hours after and the most preferred being up to 12 hours after.
The SPLA2 inhibitor can be administered by a variety of routes including oral, aerosol, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal, injectable solution. The activated Protein C compound can be administered as an injectable solution. The Activated Protein C and SPLA2 inhibitors are preferably administered parenterally to a septic patient to insure their delivery into the bloodstream in an effective form as fast as possible.
VI. Duration of Treatment for septic patients using the Method of the Invention
The amount and relative ratio of Activated protein C and SPLA2 inhibitor to be used in the practice of the method of invention is set out in the previous section, (V) supra. It may be appreciated that it may be necessary to make routine variations to the dosage of either agent depending on the age and condition of the patient . The decision to begin the therapy will be based upon the appearance of the clinical manifestations of sepsis or laboratory tests which show initiation of the sepsis cascade (inclusive of renal complications or coagulation abnormalities or multiple organ failure) . Typical clinical manifestations are fever, chills, tachycardia, tachypnea, altered mental state, hypothermia, hyperthermia, accelerated or repressed breathing or heart rates, increased or decreased white blood cell count, and hypotension. These and other symptoms are well known in the art as set out in standard references such as, Harrison's Principles of Internal Medicine (ISBN 0-07- 032370-4) 1994, pages 511-515.
The decision to determine the length of therapy may be supported by standard clinical laboratory results from commercially available assays or instrumentation supporting the eradication of the symptoms defining sepsis. The method of the invention may be practiced by continuously or intermittently administering a therapeutically effective dose of the essential Activated Protein C and SPLA2 inhibitor ingredients for as long as deemed efficacious for the treatment of the septic episode. The administration can be conducted for up to a total of about 60 days with a preferred course of therapy lasting for up to 10 days.
The decision to end therapy by the method of the invention may be supported by standard clinical laboratory results from commercially available assays or instrumentation or the disappearance of clinical symptoms characteristic of sepsis. The decision to terminate may also be based upon the measurement of the patient's baseline protein C levels returning to a value within the range of normal.
The therapy may be restarted upon the return of sepsis. The combination therapy of activated Protein C and an SPLA2 inhibitor is also a safe and effective treatment in the prevention and treatment of pediatric forms of sepsis.
While the present invention has been illustrated above by certain specific embodiments, it is not intended that these specific examples should limit the scope of the invention as described in the appended claims.

Claims

We claim:
1. A pharmaceutical composition comprising:
(a) an sPLA? inhibitor, and
(b) Activated Protein C.
2. A pharmaceutical composition according to Claim 1 wherein the SPLA2 inhibitor is a lH-indole-3-glyoxylamide of the invention represented by the formula (la), or a pharmaceutically acceptable salt or aliphatic ester prodrug derivative thereof;
Figure imgf000236_0001
wherein ; both X are oxygen;
R]_ is selected from the group consisting of
Figure imgf000236_0002
and
Figure imgf000236_0003
where R10 is a radical independently selected from halo, Cχ-C o alkyl, Cχ-Cχo alkoxy, -S-(Cχ-Cχo alkyl), and C]_- C o haloalkyl and t is a number from 0 to 5; R2 is selected from the group; halo, cyclopropyl, methyl, ethyl, and propyl;
R4 and R5 are independently selected from hydrogen, a non-interfering substituent, or the group, ~(La)- (acidic group) ; wherein ~(La)- is an acid linker; provided, the acid linker group, -(La)-, for R4 is selected from the group consisting of;
Figure imgf000238_0001
and provided, the acid linker, (La)-, for R5 is selected from group consisting of;
Figure imgf000239_0001
Figure imgf000239_0002
Figure imgf000239_0003
Figure imgf000240_0001
Figure imgf000240_0002
Figure imgf000240_0003
wherein Rg4 and Rg5 are each independently selected from hydrogen, Cχ-Cχo alkyl, aryl, C -C o alkaryl, C -Cχg aralkyl, carboxy, carbalkoxy, and halo; and provided, that at least one of R4 and R5 must be the group, - (La) - (acidic group) and wherein the (acidic group) on the group - (La) - (acidic group) of R4 or R5 is selected from -C02H, -SO3H, or -P (0) (OH) 2; Rg and R7 are each independently selected form hydrogen and non-interfering substituents, with the non- interfering substituents being selected from the group consisting of the following: C -Cg alkyl, C2-C5 alkenyl, C2-C5 alkynyl, C7-C12 aralkyl, C7-C 2 alkaryl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, phenyl, tolulyl, xylenyl, biphenyl, C -Cg alkoxy, C2~Cg alkenyloxy, C2-Cg alkynyloxy, C2~C]_2 alkoxyalkyl, C2~C 2 alkoxyalkyloxy, c-2_c12 alkylcarbonyl, C2_C 2 alkylcarbonylamino, C2~Cχ2 alkoxyamino, C2~Cj_2 alkoxyaminocarbonyl, C2-C12 alkylamino, C -Cg alkylthio, C2~C 2 alkylthiocarbonyl, Cχ-Cg alkylsulfinyl, C -Cg alkylsulfonyl, C2-C5 haloalkoxy, Cχ-Cβ haloalkylsulfonyl, C2_Cβ haloalkyl, C ~ Cζ hydroxyalkyl, -C(0)0(C1-C6 alkyl), - (CH2) n-0- (Cχ-C6 alkyl), benzyloxy, phenoxy, phenylthio, - (CONHSO2R) / - CHO, amino, amidino, bromo, carbamyl, carboxyl, carbalkoxy, - (CH2) n-Cθ2H, chloro, cyano, cyanoguanidinyl, fluoro, guanidino, hydrazide, hydrazino, hydrazido, hydroxy, hydroxyamino, iodo, nitro, phosphono, -SO3H, thioacetal, thiocarbonyl, and C -Cg carbonyl; where n is from 1 to 8.
3. A pharmaceutical composition according to Claim 1 wherein the SPLA2 inhibitor is a lH-indole-3-glyoxylamide compound or a pharmaceutically acceptable salt, solvate, or a prodrug derivative thereof selected from the group consisting of compounds (A) through (P) :
(A) [ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl-l-
(phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, (B) dl-2-[ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl-
1- (phenylmethyl) -lH-indol-4-yl] oxy] propanoic acid, (C) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1,1'- biphenyl] -2-ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid,
(D) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1, 1'- biphenyl] -3-ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid,
(E) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ([1,1'- biphenyl] -4-ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid, (F) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [ (2, 6- dichlorophenyl) ethyl] -2-methyl-lH-indol-4-yl] oxy] acetic acid
(G) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [4 (- fluorophenyl) methyl] -2-methyl-lH-indol-4-yl] oxy] acetic acid,
(H) [ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl-l- [ (1- naphthalenyl) methyl] -lH-indol-4-yl] oxy] acetic acid,
(I) [ [3- (2-Amino-l, 2-dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, (J) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [ (3- chlorophenyl) methyl] -2-ethyl-lH-indol-4-yl] oxy] acetic acid,
(K) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ([1,1'- biphenyl] -2-ylmethyl) -2-ethyl-lH-indol-4-yl] oxy] acetic acid,
(L) [ [3- (2-amino-l, 2-dioxoethyl) -1- ( [1,1'- biphenyl] -2-ylmethyl) -2-propyl-lH-indol-4-yl] oxy] acetic acid,
(M) [ [3- (2-Amino-l, 2-dioxoethyl) -2-cyclopropyl- 1- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, (N) [ [3- (2-Amino-l, 2-dioxoethyl) -l-( [1,1'- biphenyl] -2-ylmethyl) -2-cyclopropyl-lH-indol-4- yl] oxy] acetic acid,
(0) 4- [ [3- (2-Amino-l, 2-dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-5-yl] oxy]butanoic acid, and
(P) mixtures of (A) through (0) .
4. A pharmaceutical composition according to Claim 1 wherein the sPLA? inhibitor is a lH-indole-3-glyoxylamide compound or a pharmaceutically acceptable salt, solvate, or a prodrug derivative thereof selected from the group consisting of compounds (A) through (D) :
Figure imgf000243_0001
Figure imgf000244_0001
Figure imgf000245_0001
5. A pharmaceutical composition according to Claim 1 wherein the SPLA2 inhibitor is a carbazole compound of the formula (le)
Figure imgf000245_0002
wherein;
A is phenyl or pyridyl wherein the nitrogen is at the
5-, 6-, 7- or 8-position; one of B or D is nitrogen and the other is carbon;
Z is cyclohexenyl, phenyl, pyridyl, wherein the nitrogen is at the 1-, 2-, or 3-position, or a 6-membered heterocyclic ring having one heteroatom selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position, and nitrogen at the 1-, 2-, 3- or 4-position; is a double or single bond; R2^ is selected from groups (a) , (b) and (c) where;
(a) is - (C5-C20) alkyl, - (C5-C20) alkenyl,
- (C5-C20) lkynyl, carbocyclic radicals, or heterocyclic radicals, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or
(c) is the group -(L)-R80; where, -(L)- is a divalent linking group of 1 to 12 atoms selected from carbon, hydrogen, oxygen, nitrogen, and sulfur; wherein the combination of atoms in -(L)- are selected from the group consisting of (i) carbon and hydrogen only,
(ii) one sulfur only, (iii) one oxygen only, (iv) one or two nitrogen and hydrogen only, (v) carbon, hydrogen, and one sulfur only, and (vi) and carbon, hydrogen, and oxygen only; and where R^O is a group selected from (a) or (b) ;
R21 is a non-interfering substituent; RI' is -NHNH2, -NH2 or -CONH2;
R2' is selected from the group consisting of -OH, and
-0(CH ) R5' where
R5' is H, -CN, -NH2, -CONH2, -CONR9R10 -NHSO2R15;
-CONHS02R15, where R15 is - (C^Cg) alkyl or -CF3; phenyl or phenyl substituted with -CO2H or -CO2 (Cχ_C4) alkyl; and - (La) - (acidic group), wherein -(La)- is an acid linker having an acid linker length of 1 to 7 and t is 1-5; R ' is selected from non-interfering substituent, carbocyclic radicals, carbocyclic radicals substituted with non-interfering substituents, heterocyclic radicals, and heterocyclic radicals substituted with non-interfering substituents; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt thereof; provided that; when R3 ' is H, R20 is benzyl and m is 1 or 2; R ' cannot be -0(CH2)mH; and provided that when D is nitrogen, the heteroatom of Z is selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position and nitrogen at the 1-, 2-, 3- or 4-position.
6. A pharmaceutical composition according to Claim 1 wherein the SPL 2 inhibitor is a carbazole compound represented by the formula (lie) :
Figure imgf000247_0001
wherein;
Z is cyclohexenyl, or phenyl;
R21 is a non-interfering substituent;
RI is -NHNH or -NH ;
R2 is selected from the group consisting of -OH and
-0(CH ) mR5 where
R5 is H, -C02H, -CONH , -C02 (C1~C4 alkyl);
0 -P ( R R ) Where Rg ancj_ p> 7 are each independently -OH or -0(C1-C4) alkyl; -S03H, -S03 (C1-C4 alkyl), tetrazolyl, -CN, -NH„, -NHS02R15; -CONHS02R15, where R15 is - (C, -Cfi) alkyl or -CF_, phenyl or phenyl substituted with -CO„H or -CO? (C, -C alkyl where m is 1-3;
R3 is H, -0(Cχ-C4) alkyl, halo, - (Cχ-Cg) alkyl, phenyl, - (C -C4) alkylphenyl; phenyl substituted with - (Cχ-C6) alkyl, halo, or -CF3; -CH2OSi (Cχ-C6) alkyl, furyl, thiophenyl, - (C]_-Cg) hydroxyalkyl; or - (CH2)nR8 where R8 is H, -CONH , -NR9R10, -CN or phenyl where R9 and R^1-1 are independently - (Cχ~ C4) alkyl or -phenyl (C -C4 ) alkyl and n is 1 to 8; R4 is H, - (C5-C14) alkyl, - (C3-C1 ) cycloalkyl, pyridyl, phenyl or phenyl substituted with - (Cχ-Cg) alkyl, halo, -CF3, -OCF3, - (Cχ-C ) alkoxy, -CN, - (Ci.-
C4) alkylthio, phenyl (CI-C4) alkyl, - (Cι~
C4) alkylphenyl, phenyl, phenoxy or naphthyl; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt, thereof.
7. A pharmaceutical composition according to Claim 1 wherein the SPLA2 inhibitor is a carbazole compound selected from those represented by the formula (XXX) :
Figure imgf000249_0001
(XXX)
wherein:
R R11 iiss --NNHNH2, or -NH2 ;
R2 is selected from the group consisting of -OH and
-0(CH2) mR5 where
O
R5 is H, -C02H, -C02(C1-C. alkyl); p (R6R7) , where R6 and R7 are each independently -OH or -0 (C_. -C. ) alkyl; -S03H, -S03(C1-C4 alkyl), tetrazolyl, -CN, -NH2,
-NHSO Z„R15; -C0NHS0 ZR15, where R15 is - (C l. -Crb ) alkyl or -CF.-., phenyl or phenyl substituted with -CO~H or -CO„ (C1 -C.) alkyl where m is 1-3;
R3 is H, -0(C]_-C ) alkyl, halo, - (Cx-Cg) alkyl, phenyl, - (C1-C4) alkylphenyl; phenyl substituted with - (Cχ-C6) alkyl, halo, or -CF3; -CH2OSi (Cχ-C6) alkyl, furyl, thiophenyl, - (C -Cg) hydroxyalkyl; or - (CH2)nR8 where R8 is H, -CONH2, -NR9R10, -CN or phenyl where R9 and R^- ® are independently - (C ~ C4) alkyl or -phenyl (C1-C4) alkyl and n is 1 to 8;
R4 is H, - (C5-C14) alkyl, - (C3-C1 ) cycloalkyl, pyridyl, phenyl or phenyl substituted with - (C -Cs) alkyl, halo, -CF3, -OCF3 , - (C1-C4) alkoxy, -CN, - (Cχ~ C ) alkylthio, phenyl (CI-C4) alkyl, - (C]_- C4) alkylphenyl, phenyl, phenoxy or naphthyl; A is phenyl or pyridyl wherein the nitrogen is at the 5-, 6-, 7- or 8-position; Z is cyclohexenyl, phenyl, pyridyl wherein the nitrogen is at the 1-, 2- or 3-position or a 6-membered heterocyclic ring having one heteroatom selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position and nitrogen at the 1-, 2-, 3- or 4-position, or wherein one carbon on the heterocyclic ring is optionally substituted with =0; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt thereof; provided that one of A or Z is a heterocyclic ring.
8. A pharmaceutical composition according to Claim 1 wherein the SPLA2 inhibitor is a carbazole compound selected from the following:
9-benzyl-5, 7-dimethoxy-l, 2, 3, 4-tetrahydrocarbazole-4- carboxylic acid hydrazide; 9-benzyl-5, 7-dimethoxy-l, 2, 3, 4-tetrahydrocarbazole-4- carboxamide; [ 9-benzyl-4-carbamoyl-7-methoxy-l, 2,3,4- tetrahydrocarbazol-5-yl] oxyacetic acid sodium salt; [ 9-benzyl-4-carbamoyl-7-methoxycarbazol-5-yl ] oxyacetic acid; methyl [ 9-benzyl-4-carbamoyl-7-methoxycarbazol-5- yl] oxyacetic acid;
9-benzyl-7-methoxy-5-cyanomethyloxy-l, 2, 3, 4- tetrahydrocarbazole-4-carboxamide; 9-benzyl-7-methoxy-5- (lH-tetrazol-5-yl-methyl) oxy) -
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; { 9- [ (phenyl) methyl] -5-carbamoyl-2-methyl-carbazol-4- yl}oxyacetic acid; {9-[ (3-fluorophenyl) methyl] -5-carbamoyl-2-methyl- carbazol-4-yl}oxyacetic acid; { 9- [ ( 3-methylphenyl) methyl] -5-carbamoyl-2-methyl- carbazol-4-yl }oxyacetic acid; { 9- [ (phenyl) methyl] -5-carbamoyl-2- (4- trifluoromethylphenyl) -carbazol-4-yl } oxyacetic acid; 9-benzyl-5- (2-methanesulfonamido) ethyloxy-7-methoxy-
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; 9-benzyl-4- (2-methanesulfonamido) ethyloxy-2- methoxycarbazole-5-carboxamide; 9-benzyl-4- (2-trifluoromethanesulfonamido) ethyloxy-2- methoxycarbazole-5-carboxamide; 9-benzyl-5-methanesulfonamidoylmethyloxy-7-methoxy-
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; 9-benzyl-4-methanesulfonamidoylmethyloxy-carbazole-5- carboxamide;
[5-carbamoyl-2-pentyl-9- (phenylmethyl) carbazol-4- yl] oxyacetic acid; [5-carbamoyl-2- (1-methylethyl) -9- (phenylmethyl) carbazol-
4-yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2- [ (tri (-1- methylethyl) silyl) oxymethyl] carbazol-4-yl] oxyacetic acid; [5-carbamoyl-2-phenyl-9- (phenylmethyl) carbazol-4- yl] oxyacetic acid [5-carbamoyl-2- (4-chlorophenyl) -9- (phenylmethyl) carbazol-4-yl] oxyacetic acid;
[5-carbamoyl-2- (2-furyl) -9- (phenylmethyl) carbazol-4- yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2- [ (tri (-1- methylethyl) silyl) oxymethyl] carbazol-4-yl] oxyacetic acid, lithium salt; { 9- [ (phenyl) methyl] -5-carbamoylcarbazol-4-yl } oxyacetic acid; { 9- [ (3-fluorophenyl) methyl] -5-carbamoylcarbazol-4- yl }oxyacetic acid; { 9- [ (3-phenoxyphenyl) methyl] -5-carbamoylcarbazol-4- yl } oxyacetic acid; { 9- [ (2-Fluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-trifluoromethylphenyl) ethyl] -5-carbamoylcarbazol-
4-yl Joxyacetic acid; { 9- [ (2-benzylphenyl) methyl] -5-carbamoylcarbazol-4- yl} oxyacetic acid; { 9- [ (3-trifluoromethylphenyl ) methyl ] -5-carbamoylcarbazol-
4-yl}oxyacetic acid; { 9- [ ( 1-naphthyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-cyanophenyl) ethyl] -5-carbamoylcarbazol-4- yl} oxyacetic acid; { 9- [ (3-cyanophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-methylphenyl) methyl] -5-carbamoylcarbazol-4- yl Joxyacetic acid; { 9- [ (3-methylphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (3, 5-dimethylphenyl) methyl] -5-carbamoylcarbazol-4- yl joxyacetic acid; { 9- [ (3-iodophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-Chlorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2, 3-difluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; {9-[ (2, 6-difluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ ( 2 , 6-dichlorophenyl ) methyl ] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (3-trifluoromethoxyphenyl) methyl] -5- carbamoylcarbazol-4-yl }oxyacetic acid;
{ 9- [ (2-biphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-Biphenyl) methyl] -5-carbamoylcarbazol-4- yl } oxyacetic acid; the { 9- [ (2-Biphenyl) methyl] -5-carbamoylcarbazol-4- yl } oxyacetic acid; [9-Benzyl-4-carbamoyl-l, 2, 3, 4-tetrahydrocarbaole-5- yl] oxyacetic acid; { 9- [ (2-Pyridyl) methyl] -5-carbamoylcarbazol-4-yl } oxyacetic acid;
{ 9- [ (3-Pyridyl) methyl] -5-carbamoylcarbazol-4-yl } oxyacetic acid; [9-benzyl-4-carbamoyl-8-methyl-l, 2,3,4- tetrahydrocarbazol-5-yl ] oxyacetic acid; [9-benzyl-5-carbamoyl-l-methylcarbazol-4-yl] oxyacetic acid; [9-benzyl-4-carbamoyl-8-fluoro-1, 2, 3, 4- tetrahydrocarbazol-5-yl ] oxyacetic acid; [9-benzyl-5-carbamoyl-l-fluorocarbazol-4-yl] oxyacetic acid;
[9-benzyl-4-carbamoyl-8-chloro-l, 2,3,4- tetrahydrocarbazol-5-yl] oxyacetic acid; [9-benzyl-5-carbamoyl-l-chlorocarbazol-4-yl] oxyacetic acid; [ 9- [ (Cyclohexyl) methyl] -5-carbamoylcarbazol-4- yl] oxyacetic acid; [9- [ (Cyclopentyl) methyl] -5-carbamoylcarbazol-4- yl] oxyacetic acid; -carbamoyl-9- (phenylmethyl) -2- [ [ (propen-3- yl) oxy] methyl] carbazol-4-yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2- [ (propyloxy) methyl] carbazol-4-yl] oxyacetic acid; -benzyl-7-methoxy-5- ( (carboxamidomethyl) oxy) -1,2,3,4- tetrahydrocarbazole-4-carboxamide; -benzyl-7-methoxy-5-cyanomethyloxy-carbazole-4- carboxamide; -benzyl-7-methoxy-5- ( (lH-tetrazol-5-yl-methyl) oxy) - carbazole-4-carboxamide; -benzyl-7-methoxy-5- ( (carboxamidomethyl) oxy) -carbazole-
4-carboxamide; and [ 9-Benzyl-4-carbamoyl-l, 2,3, 4-tetrahydrocarbaole-5- yl] oxyacetic acid
(_R, S) - (9-benzyl-4-carbamoyl-l-oxo-3-thia-l, 2, 3, 4- tetrahydrocarbazol-5-yl) oxyacetic acid; ( R, S) - (9-benzyl-4-carbamoyl-l-oxo-3-thia-l, 2, 3, 4- tetrahydrocarbazol-5-yl) oxyacetic acid; [N-benzyl-l-carbamoyl-l-aza-1, 2,3, 4-tetrahydrocarbazol-8- yl] oxyacetic acid; 4-methoxy-6-methoxycarbonyl-10- phenylmethyl-6, 7,8, 9-tetrahydropyrido [1, 2-a] indole; (4-carboxamido-9-phenylmethyl-4, 5-dihydrothiopyrano [3,4- b] indol-5-yl) oxyacetic acid; , 4-dihydro-4-carboxamidol-5-methoxy-9- phenylmethylpyrano [3, 4-b] indole; 2- [(2, 9 bis-benzyl- 4-carbamoyl-l, 2, 3, 4-tetrahydro-beta-carbolin-5- yl) oxy] acetic acid; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug, or salt thereof.
9. A pharmaceutical composition according to Claim 1, wherein the SPLA2 inhibitor is a carbazole compound represented by the formulae (Xe) or (Xle) below:
Figure imgf000255_0001
and
Figure imgf000256_0001
or a pharmaceutically acceptable salt or ester prodrug derivative thereof.
10. The pharmaceutical composition of Claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 comprising; (a) SPLA2 inhibitor and (b) Activated Protein C in therapeutically effective amounts.
11. The pharmaceutical composition of Claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 wherein the weight ratio (a) : (b) of (a) SPLA2 inhibitor and (b) Activated Protein C is 1000:1 to 10000000:1.
12. The pharmaceutical composition of Claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 wherein the weight ratio (a) : (b) of (a) SPLA2 inhibitor and (b) Activated Protein C is 100:1 to 1000000:1.
13. The pharmaceutical composition of Claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 wherein the weight of (a) SPLA2 inhibitor is in the range of from 0.1 mg to 2000 mg and (b) Activated Protein C is in the range of
1.0 μg to 100 μg each.
14. The pharmaceutical composition of Claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 wherein the weight of (a) SPLA2 inhibitor is in the range of from 50 mg to
1000 mg and (b) Activated Protein C is in the range of
1 μg to 25 μg.
15. The pharmaceutical composition of Claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 comprising a suitable carrier, diluent or excipient therefor.
16. A method for the treatment or prevention of sepsis comprising administering within a therapeutically effective interval to a mammal in need thereof, therapeutically effective amounts of;
(a) an SPLA2 inhibitor, and
(b) Activated Protein C.
17. A method for treatment of a human afflicted with or susceptible to sepsis, said method comprising administering to said human a therapeutically effective combination of Activated Protein C and a lH-indole-3- glyoxylamide sPLA? inhibitor of the invention represented by the formula (la), or a pharmaceutically acceptable salt or aliphatic ester prodrug derivative thereof;
Figure imgf000258_0001
wherein ; both X are oxygen; R]_ is selected from the group consisting of
Figure imgf000258_0002
and
Figure imgf000258_0003
where R10 is a radical independently selected from halo, Cχ-Cιo alkyl, Cχ-Cχo alkoxy, -S- (Cχ-Cχo alkyl), and Cχ~
C o haloalkyl and t is a number from 0 to 5;
R2 is selected from the group; halo, cyclopropyl, methyl, ethyl, and propyl;
R4 and R5 are independently selected from hydrogen, a non-interfering substituent, or the group, -(La)-
(acidic group); wherein -(La)- is an acid linker; provided, the acid linker group, -(La)-, for R4 is selected from the group consisting of;
Figure imgf000259_0001
and provided, the acid linker, -(La)-, for R5 is selected from group consisting of;
Figure imgf000260_0001
Figure imgf000260_0002
Figure imgf000261_0001
Figure imgf000261_0002
Figure imgf000261_0003
wherein Rg4 and Rg5 are each independently selected from hydrogen, Cχ-Cι_o alkyl, aryl, C -C o alkaryl, Cχ-C o aralkyl, carboxy, carbalkoxy, and halo; and provided, that at least one of R4 and R5 must be the group, - (La) - (acidic group) and wherein the (acidic group) on the group - (La) - (acidic group) of R4 or R5 is selected from -C02H, -SO3H, or -P(O) (OH) 2; Rg and R7 are each independently selected form hydrogen and non-interfering substituents, with the non- interfering substituents being selected from the group consisting of the following: C -Cg alkyl, C2-C5 alkenyl, C2-C alkynyl, C7-C12 aralkyl, C7-C12 alkaryl, C3-C8 cycloalkyl, C3-C cycloalkenyl, phenyl, tolulyl, xylenyl, biphenyl, Cχ-Cg alkoxy, C2-C5 alkenyloxy, C2-C5 alkynyloxy, C2~C 2 alkoxyalkyl, C2_C 2 alkoxyalkyloxy, C2_C 2 alkylcarbonyl, C2_C 2 alkylcarbonylamino, C2~C 2 alkoxyamino, C2_C 2 alkoxyaminocarbonyl, C2~C 2 alkylamino, Cχ-Cg alkylthio, C2~Cχ2 alkylthiocarbonyl, C -Cg alkylsulfinyl, C]_-Cg alkylsulfonyl, C2-C5 haloalkoxy, Cχ-Cg haloalkylsulfonyl, C2-C5 haloalkyl, C_- C6 hydroxyalkyl, -C(0)0(Cχ-C6 alkyl), - (CH2 ) n-0- (Cχ-C6 alkyl), benzyloxy, phenoxy, phenylthio, - (CONHSO2R) - CHO, amino, amidino, bromo, carbamyl, carboxyl, carbalkoxy, - (CH2) n-Cθ2H, chloro, cyano, cyanoguanidinyl, fluoro, guanidino, hydrazide, hydrazino, hydrazido, hydroxy, hydroxyamino, iodo, nitro, phosphono, -SO3H, thioacetal, thiocarbonyl, and Cχ-Cg carbonyl; where n is from 1 to 8.
18. A method for treatment of a human afflicted with or susceptible to sepsis, said method comprising administering to said human in need of such treatment, a therapeutically effective combination of Activated Protein C and a lH-indole-3-glyoxylamide SPLA2 inhibitor or a pharmaceutically acceptable salt, solvate, or a prodrug derivative thereof, selected from the group consisting of compounds (A) through (P) :
(A) [ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, (B) dl-2- [ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl- 1- (phenylmethyl) -lH-indol-4-yl] oxy] propanoic acid,
(C) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1,1'- biphenyl] -2-ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid,
(D) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1,1'- biphenyl] -3-ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid,
(E) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ( [1,1'- biphenyl] -4-ylmethyl) -2-methyl-lH-indol-4-yl] oxy] acetic acid,
(F) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [ (2, 6- dichlorophenyl) methyl] -2-methyl-lH-indol-4-yl] oxy] acetic acid (G) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [4 (- fluorophenyl) methyl] -2-methyl-lH-indol-4-yl] oxy] acetic acid,
(H) [ [3- (2-Amino-l, 2-dioxoethyl) -2-methyl-l- [ (1- naphthalenyl) methyl] -lH-indol-4-yl] oxy] acetic acid, (I) [ [3- (2-Amino-l, 2-dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid,
( J) [ [3- (2-Amino-l, 2-dioxoethyl) -1- [ (3- chlorophenyl) methyl] -2-ethyl-lH-indol-4-yl] oxy] acetic acid, (K) [ [3- (2-Amino-l, 2-dioxoethyl) -l-( [1,1'- biphenyl] -2-ylmethyl) -2-ethyl-lH-indol-4-yl] oxy] acetic acid,
(L) [ [3- (2-amino-l, 2-dioxoethyl) -1- ([1,1'- biphenyl] -2-ylmethyl) -2-propyl-lH-indol-4-yl] oxy] acetic acid,
(M) [ [3- (2-Amino-l, 2-dioxoethyl) -2-cyclopropyl- 1- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, (N) [ [3- (2-Amino-l, 2-dioxoethyl) -1- ([1,1'- biphenyl] -2-ylmethyl) -2-cyclopropyl-lH-indol-4- yl] oxy] acetic acid,
(0) 4- [ [3- (2-Amino-l, 2-dioxoethyl) -2-ethyl-l- (phenylmethyl) -lH-indol-5-yl] oxy]butanoic acid, and
(P) mixtures of (A) through (0) .
19. A method for treatment of a human currently afflicted with sepsis or susceptible to sepsis, said method comprising administering to said human in need of such treatment a therapeutically effective combination of Activated Protein C and a lH-indole-3-glyoxylamide sPLA2 inhibitor represented by any of the formulae:
Figure imgf000264_0001
Figure imgf000265_0001
Figure imgf000266_0001
20. A method for treatment of a mammal to alleviate or prevent the pathological effects of sepsis, said method comprising administering to said mammal a therapeutically effective combination of Activated Protein C and the SPLA2 inhibitor compound [ [3- (2-amino-l, 2-dioxoethyl) -2- ethyl-1- (phenylmethyl) -lH-indol-4-yl] oxy] acetic acid, methyl ester.
21. A method for treatment of a human afflicted with sepsis or susceptible to sepsis, said method comprising administering to said human in need of such treatment, a therapeutically effective combination of Activated Protein C and a compound of the formula (le)
Figure imgf000266_0002
wherein; A is phenyl or pyridyl wherein the nitrogen is at the 5-, 6-, 7- or 8-position; one of B or D is nitrogen and the other is carbon;
Z is cyclohexenyl, phenyl, pyridyl, wherein the nitrogen is at the 1-, 2-, or 3-position, or a 6- membered heterocyclic ring having one heteroatom selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position, and nitrogen at the 1-, 2-, 3- or 4-position; is a double or single bond;
R2^ is selected from groups (a) , (b) and (c) where; (a) is - (C5-C20) alkyl, - (C5-C20) alkenyl,
- (C5-C20) alkynyl, carbocyclic radicals, or heterocyclic radicals, or (b) is a member of (a) substituted with one or more independently selected non-interfering substituents; or (c) is the group -(L)-R8^; where, - (L) - is a divalent linking group of 1 to 12 atoms selected from carbon, hydrogen, oxygen, nitrogen, and sulfur; wherein the combination of atoms in -(L)- are selected from the group consisting of (i) carbon and hydrogen only, (ii) one sulfur only, (iii) one oxygen only, (iv) one or two nitrogen and hydrogen only,
(v) carbon, hydrogen, and one sulfur only, and (vi) and carbon, hydrogen, and oxygen only; and where R8^ is a group selected from (a) or (b) ; R21 is a non-interfering substituent; RI' is -NHNH2/ -NH2 or -CONH2; R2' is selected from the group consisting of -OH, and -0(CH2) ,R5' where
R5' is H, -CN, -NH2, -CONH2, -CONR9R10 -NHS02R15;
-CONHS02R15, where R15 is - (Ci-Cg) alkyl or -CF3; phenyl or phenyl substituted with -CO2H or -CO2 (CX-.C4) alkyl; and - (La) - (acidic group), wherein - (La) - is an acid linker having an acid linker length of 1 to 7 and t is 1-5;
R3 ' is selected from non-interfering substituent, carbocyclic radicals, carbocyclic radicals substituted with non-interfering substituents, heterocyclic radicals, and heterocyclic radicals substituted with non-interfering substituents; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt thereof; provided that; when R3 ' is H, R20 is benzyl and m is 1 or 2; R2 ' cannot be -0(CH2)mH; and provided that when D is nitrogen, the heteroatom of Z is selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position and nitrogen at the 1-, 2-, 3- or 4-position.
22. A method for treatment of a human afflicted with or susceptible to affliction with sepsis, said method comprising administering to said human in need of such treatment, a therapeutically effective combination of Activated Protein C and a compound represented by the formula (lie) :
Figure imgf000269_0001
e) wherein;
Z is cyclohexenyl, or phenyl; R21 is a non-interfering substituent;
RI is -NHNH or NH,
*2'
R2 is selected from the group consisting of -OH and
-0(CH2) mR5 where
R5 is H, -C02H, -CONH2, -C02 (C1~C4 alkyl);
Figure imgf000269_0002
, where R6 and R7 are each independently -OH or -0(C1-C4 ) alkyl; -So3H, -S03 (C1-C4 alkyl), tetrazolyl, -CN, -NH , -NHS02R15; -CONHSO R15, where R15 is - (C, -C,.) alkyl or -CF,,, phenyl or phenyl substituted with -CO„H or -C0 (C-. -C alkyl where m is 1-3;
R3 is H, -0(C!-C4) alkyl, halo, - (C;L-C6) alkyl, phenyl, - (C -C4) alkylphenyl; phenyl substituted with - (Cχ-C6) alkyl, halo, or -CF3; -CH2OSi (Cχ-C6) alkyl, furyl, thiophenyl, - (C -Cg) hydroxyalkyl; or - (CH2)nR8 where R8 is H, -CONH2, -NR9R10, -CN or phenyl where R9 and R^^ are independently - (O - C4) alkyl or -phenyl (C -C4) alkyl and n is 1 to 8;
R4 is H, - (C5-C 4) alkyl, - (C3-C14 ) cycloalkyl, pyridyl, phenyl or phenyl substituted with - (C1-C ) alkyl, halo, -CF3, -OCF3, - (C1-C4) alkoxy, -CN, - (Cχ~ C ) alkylthio, phenyl (CI-C4) alkyl, - (C]_-
C4) alkylphenyl, phenyl, phenoxy or naphthyl; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt, thereof.
23. A method for treatment of a human afflicted with or susceptible to sepsis, wherein the method comprises administering to said human in need of such treatment, a therapeutically effective combination of Activated Protein C and a carbazole compound selected from those represented by the formula (XXX) :
Figure imgf000270_0001
(XXX)
wherein:
R1 is -NHNH2, or -NH ;
R2 is selected from the group consisting of -OH and
-0(CH2) mR5 where
O
R5 is H, -C02H, -C02(C1-C4 alkyl); p (R6R ) , where R6 and R7 are each independently -OH or -0 (C, -C-) alkyl; -SO3H, -S03(C1-C4 alkyl), tetrazolyl, -CN, -NH2, -NHS02R15; -CONHS02R15, where R15 is - (C^Cg) alkyl or -CF.,, phenyl or phenyl substituted with -CO„H or -CO„ (C -C.) alkyl where m is 1-3;
R3 is H, -0(Cχ-C4) alkyl, halo, - (C -Cg) alkyl, phenyl, - (C1-C4) alkylphenyl; phenyl substituted with - (Ci-Cg) alkyl, halo, or -CF3; -CH OSi (Cχ-Cg) alkyl, furyl, thiophenyl, - (C^-Cg) hydroxyalkyl; or - (CH2)nR8 where R8 is H, -CONH2, -NR9R10, -CN or phenyl where R9 and RIO are independently - (C ~ C4) alkyl or -phenyl (C1-C4) alkyl and n is 1 to 8; R4 is H, - (C5-C14) alkyl, - (C3-C1 ) cycloalkyl, pyridyl, phenyl or phenyl substituted with - (Cχ-Cg) alkyl, halo, -CF3, -OCF3 , - (C1-C4) alkoxy, -CN, - (Cχ~ C4) alkylthio, phenyl (CI-C4) alkyl, - (Cχ~ C4) alkylphenyl, phenyl, phenoxy or naphthyl; A is phenyl or pyridyl wherein the nitrogen is at the 5-, 6-, 7- or 8-position; Z is cyclohexenyl, phenyl, pyridyl wherein the nitrogen is at the 1-, 2- or 3-position or a 6-membered heterocyclic ring having one heteroatom selected from the group consisting of sulfur or oxygen at the 1-, 2- or 3-position and nitrogen at the 1-, 2-, 3- or 4-position, or wherein one carbon on the heterocyclic ring is optionally substituted with =0; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug derivative or salt thereof; provided that one of A or Z is a heterocyclic ring.
24. A method for treatment of a human afflicted with or susceptible to sepsis, comprising administering to said human in need of such treatment, a therapeutically effective combination of Activated Protein C and a compound selected from the following:
9-benzyl-5, 7-dimethoxy-l, 2, 3, 4-tetrahydrocarbazole-4- carboxylic acid hydrazide; 9-benzyl-5, 7-dimethoxy-l, 2,3, 4-tetrahydrocarbazole-4- carboxamide; [9-benzyl-4-carbamoyl-7-methoxy-l, 2, 3, 4- tetrahydrocarbazol-5-yl] oxyacetic acid sodium salt; [9-benzyl-4-carbamoyl-7-methoxycarbazol-5-yl] oxyacetic acid; methyl [ 9-benzyl-4-carbamoyl-7-methoxycarbazol-5- yl] oxyacetic acid; 9-benzyl-7-methoxy-5-cyanomethyloxy-l, 2, 3, 4- tetrahydrocarbazole-4-carboxamide; 9-benzyl-7-methoxy-5- (lH-tetrazol-5-yl-methyl) oxy) -
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; { 9- [ (phenyl) methyl] -5-carbamoyl-2-methyl-carbazol-4- yl}oxyacetic acid; { 9- [ (3-fluorophenyl) methyl] -5-carbamoyl-2-methyl- carbazol-4-yl }oxyacetic acid;
{ 9- [ (3-methylphenyl) methyl] -5-carbamoyl-2-methyl- carbazol-4-yl}oxyacetic acid; {9- [ (phenyl) methyl] -5-carbamoyl-2- (4- trifluoromethylphenyl) -carbazol-4-yl }oxyacetic acid; 9-benzyl-5- (2-methanesulfonamido) ethyloxy-7-methoxy-
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; 9-benzyl-4- (2-methanesulfonamido) ethyloxy-2- methoxycarbazole-5-carboxamide; 9-benzyl-4- (2-trifluoromethanesulfonamido) ethyloxy-2- methoxycarbazole-5-carboxamide;
9-benzyl-5-methanesulfonamidoylmethyloxy-7-methoxy-
1,2,3, 4-tetrahydrocarbazole-4-carboxamide; -benzyl-4-methanesulfonamidoylmethyloxy-carbazole-5- carboxamide; [5-carbamoyl-2-pentyl-9- (phenylmethyl) carbazol-4- yl] oxyacetic acid; [5-carbamoyl-2- (1-methylethyl) -9- (phenylmethyl) carbazol- 4-yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2- [ (tri (-1- methylethyl) silyl) oxymethyl] carbazol-4-yl] oxyacetic acid; [5-carbamoyl-2-phenyl-9- (phenylmethyl) carbazol-4- yl] oxyacetic acid [5-carbamoy1-2- (4-chlorophenyl) -9- (phenylmethyl) carbazol-4-yl] oxyacetic acid; [5-carbamoyl-2- (2-furyl) -9- (phenylmethyl) carbazol-4- yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2- [ (tri (-1- methylethyl) silyl) oxymethyl] carbazol-4-yl] oxyacetic acid, lithium salt; { 9- [ (phenyl) methyl] -5-carbamoylcarbazol-4-yl }oxyacetic acid; { 9- [ (3-fluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (3-phenoxyphenyl) ethyl] -5-carbamoylcarbazol-4- yljoxyacetic acid; { 9- [ (2-Fluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid;
{ 9- [ (2-trifluoromethylphenyl) methyl] -5-carbamoylcarbazol-
4-yl}oxyacetic acid; { 9- [ (2-benzylphenyl)methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; {9-[ (3-trifluoromethylphenyl) methyl] -5-carbamoylcarbazol- 4-yl}oxyacetic acid; { 9- [ ( 1-naphthyl ) methyl ] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-cyanophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (3-cyanophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-methylphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (3-methylphenyl) methyl] -5-carbamoylcarbazol-4- yljoxyacetic acid; { 9- [ (3, 5-dimethylphenyl) ethyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (3-iodophenyl) methyl] -5-carbamoylcarbazol-4- yl} oxyacetic acid; { 9- [ (2-Chlorophenyl) ethyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2, 3-difluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2, 6-difluorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2, 6-dichlorophenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (3-trifluoromethoxyphenyl) methyl] -5- carbamoylcarbazol-4-yl } oxyacetic acid; { 9- [ (2-biphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; { 9- [ (2-Biphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; the { 9- [ (2-Biphenyl) methyl] -5-carbamoylcarbazol-4- yl}oxyacetic acid; [9-Benzyl-4-carbamoyl-l, 2, 3, 4-tetrahydrocarbaole-5- yl] oxyacetic acid; {9- [ (2-Pyridyl) methyl] -5-carbamoylcarbazol-4-yl } oxyacetic acid; { 9- [ (3-Pyridyl) methyl] -5-carbamoylcarbazol-4-yl}oxyacetic acid; [9-benzyl-4-carbamoyl-8-methyl-l, 2, 3, 4- tetrahydrocarbazol-5-yl] oxyacetic acid; [9-benzyl-5-carbamoyl-l-methylcarbazol-4-yl] oxyacetic acid; [9-benzyl-4-carbamoyl-8-fluoro-1, 2, 3, 4- tetrahydrocarbazol-5-yl] oxyacetic acid;
[9-benzyl-5-carbamoyl-l-fluorocarbazol-4-yl] oxyacetic acid; [9-benzyl-4-carbamoyl-8-chloro-l, 2, 3, 4- tetrahydrocarbazol-5-yl ] oxyacetic acid; [9-benzyl-5-carbamoyl-l-chlorocarbazol-4-yl] oxyacetic acid; [ 9- [ (Cyclohexyl) methyl] -5-carbamoylcarbazol-4- yl] oxyacetic acid; [9- [ (Cyclopentyl) methyl] -5-carbamoylcarbazol-4- yl] oxyacetic acid;
5-carbamoyl-9- (phenylmethyl) -2- [ [ (propen-3- yl) oxy] methyl] carbazol-4-yl] oxyacetic acid; [5-carbamoyl-9- (phenylmethyl) -2-
[ (propyloxy) methyl] carbazol-4-yl] oxyacetic acid; 9-benzyl-7-methoxy-5- ( (carboxamidomethyl) oxy) -1,2,3,4- tetrahydrocarbazole-4-carboxamide; 9-benzy1-7-methoxy-5-cyanomethyloxy-carbazole-4- carboxamide; 9-benzyl-7-methoxy-5- ( (lH-tetrazol-5-yl-methyl) oxy) - carbazole-4-carboxamide;
9-benzyl-7-methoxy-5- ( (carboxamidomethyl) oxy) -carbazole-
4-carboxamide; and [9-Benzyl-4-carbamoyl-l, 2, 3, 4-tetrahydrocarbaole-5- yl] oxyacetic acid (R, S) - (9-benzyl-4-carbamoyl-l-oxo-3-thia-l, 2,3,4- tetrahydrocarbazol-5-yl) oxyacetic acid; ( R, S) - (9-benzyl-4-carbamoyl-l-oxo-3-thia-l,2, 3, 4- tetrahydrocarbazol-5-yl) oxyacetic acid; [N-benzyl-l-carbamoyl-l-aza-1, 2, 3, 4-tetrahydrocarbazol-8- yl] oxyacetic acid; 4-methoxy-6-methoxycarbonyl-10- phenylmethyl-6, 7, 8, 9-tetrahydropyrido [1, 2-a] indole; (4-carboxamido-9-phenylmethyl-4, 5-dihydrothiopyrano [3, 4- b] indol-5-yl) oxyacetic acid; 3, 4-dihydro-4-carboxamido1-5-methoxy-9- phenylmethylpyrano [3, 4-b] indole; 2- [(2, 9 bis-benzyl-
4-carbamoyl-l, 2, 3, 4-tetrahydro-beta-carbolin-5- yl) oxy] acetic acid; or a pharmaceutically acceptable racemate, solvate, tautomer, optical isomer, prodrug, or salt thereof.
25. A pharmaceutical composition according to Claim 1, wherein the SPLA2 inhibitor is a carbazole compound represented by the formulae (Xe) or (Xle) below:
Figure imgf000277_0001
and
Figure imgf000278_0001
or a pharmaceutically acceptable salt or ester prodrug derivative thereof.
26. The method according to Claims 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 wherein the Activated Protein C is delivered parenterally.
27. The method according to claims 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 wherein the sPLA2 inhibitor is delivered parenterally, orally, intra- nassally, as a supposotory, as a patch.
28. The method according to claims 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 wherein the SPLA2 inhibitor is delivered parenterally.
29. The method according to Claim 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 , wherein an sPLA2 inhibitor is administered prior to the Activated Protein C.
30. The method of Claim 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 wherein the Activated Protein C is administered prior to the sPLA? inhibitor.
31. The method of Claims 16 or 17 or 18 or 19 or 20 or
21 or 22 or 23 or 24 or 25 wherein the SPLA2 inhibitor is administered simultaneously with the Activated protein C.
32. A method according to Claim 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 wherein the dose of Activated protein C is between lμg and 100 μg/kg/day.
33. A method according to Claim 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 wherein the dose of Activated protein C is between lμg and 25 μg/kg/day
34. A method of according to Claim 1 wherein dose of an SPLA2 inhibitor is between 10 mg and 2000 mg/kg/day.
35. Use of an SPLA2 inhibitor in combination with
Activated protein C for the manufacture of a medicament for treating sepsis in a mammal, including a human, currently afflicted with or susceptible to sepsis.
PCT/US1999/030433 1998-12-21 1999-12-20 Combination therapy for the treatment of sepsis WO2000037022A2 (en)

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CA002358492A CA2358492A1 (en) 1998-12-21 1999-12-20 Combination therapy for the treatment of sepsis
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000069818A1 (en) * 1999-05-12 2000-11-23 Eli Lilly And Company MORPHOLINO-N-ETHYL ESTER DERIVATIVE OF AN INDOLE sPLA2 INHIBITOR
EP1073440A1 (en) * 1998-05-01 2001-02-07 Eli Lilly And Company sPLA 2 INHIBITOR ESTERS
WO2002005796A2 (en) * 2000-07-14 2002-01-24 Eli Lilly And Company Use of a spla2 inhibitor for the treatment of sepsis
WO2012127885A1 (en) 2011-03-18 2012-09-27 小野薬品工業株式会社 Tetrahydrocarboline derivative
US8728512B2 (en) 2001-06-13 2014-05-20 Christopher John Jackson Treatment and composition for wound healing
WO2016116527A1 (en) * 2015-01-20 2016-07-28 Cynora Gmbh Organic molecules, in particular for use in optoelectronic components

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5093117A (en) * 1989-01-24 1992-03-03 Baxter International Inc. Compositions and method for the treatment or prophylaxis of sepsis or septic shock
US5571786A (en) * 1990-08-16 1996-11-05 Immuno Aktiengesellschaft Use of protein C or the activation peptide of protein C for preparing a pharmaceutical preparation
US5654326A (en) * 1994-04-01 1997-08-05 Eli Lilly And Company 1H-indole-3-glyoxylamide SPLA2 inhibitors
EP0952149A2 (en) * 1998-04-17 1999-10-27 Eli Lilly And Company Substituted carbazoles, process for their preparation and their use as sPLA2 inhibitiors

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5093117A (en) * 1989-01-24 1992-03-03 Baxter International Inc. Compositions and method for the treatment or prophylaxis of sepsis or septic shock
US5571786A (en) * 1990-08-16 1996-11-05 Immuno Aktiengesellschaft Use of protein C or the activation peptide of protein C for preparing a pharmaceutical preparation
US5654326A (en) * 1994-04-01 1997-08-05 Eli Lilly And Company 1H-indole-3-glyoxylamide SPLA2 inhibitors
US5733923A (en) * 1994-04-01 1998-03-31 Eli Lilly And Company 1H-indole-3-glyoxylamide sPLA2 inhibitors
US5919943A (en) * 1994-04-01 1999-07-06 Eli Lilly And Company Process for preparing 1H-indole-3-glyoxylamides
US5919810A (en) * 1994-04-01 1999-07-06 Eli Lilly And Company 1H-indole-3-glyoxylamide sPLA2 inhibitors
EP0952149A2 (en) * 1998-04-17 1999-10-27 Eli Lilly And Company Substituted carbazoles, process for their preparation and their use as sPLA2 inhibitiors

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1073440A1 (en) * 1998-05-01 2001-02-07 Eli Lilly And Company sPLA 2 INHIBITOR ESTERS
EP1073440A4 (en) * 1998-05-01 2002-07-17 Lilly Co Eli sPLA 2 INHIBITOR ESTER
WO2000069818A1 (en) * 1999-05-12 2000-11-23 Eli Lilly And Company MORPHOLINO-N-ETHYL ESTER DERIVATIVE OF AN INDOLE sPLA2 INHIBITOR
WO2002005796A2 (en) * 2000-07-14 2002-01-24 Eli Lilly And Company Use of a spla2 inhibitor for the treatment of sepsis
WO2002005796A3 (en) * 2000-07-14 2002-09-06 Lilly Co Eli Use of a spla2 inhibitor for the treatment of sepsis
US8728512B2 (en) 2001-06-13 2014-05-20 Christopher John Jackson Treatment and composition for wound healing
WO2012127885A1 (en) 2011-03-18 2012-09-27 小野薬品工業株式会社 Tetrahydrocarboline derivative
WO2016116527A1 (en) * 2015-01-20 2016-07-28 Cynora Gmbh Organic molecules, in particular for use in optoelectronic components

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