WO1994024133A1 - Inhibiteurs de signalisation cellulaire a cycle substitue - Google Patents

Inhibiteurs de signalisation cellulaire a cycle substitue Download PDF

Info

Publication number
WO1994024133A1
WO1994024133A1 PCT/US1994/004007 US9404007W WO9424133A1 WO 1994024133 A1 WO1994024133 A1 WO 1994024133A1 US 9404007 W US9404007 W US 9404007W WO 9424133 A1 WO9424133 A1 WO 9424133A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
substituted
cyclic
unsubstituted
compound
Prior art date
Application number
PCT/US1994/004007
Other languages
English (en)
Inventor
J. Peter Klein
Anil Kumar
Alistair Leigh
John Michnick
Glenn C. Rice
Original Assignee
Cell Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cell Therapeutics, Inc. filed Critical Cell Therapeutics, Inc.
Priority to AU67028/94A priority Critical patent/AU6702894A/en
Publication of WO1994024133A1 publication Critical patent/WO1994024133A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
    • C07D211/86—Oxygen atoms
    • C07D211/88—Oxygen atoms attached in positions 2 and 6, e.g. glutarimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
    • C07D473/10—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3 with methyl radicals in positions 3 and 7, e.g. theobromine

Definitions

  • the invention provides a group of compounds that are effective agents to inhibit specific cellular signaling events often induced by inflammatory stimuli, or to be directly or indirectly antimicrobial to yeast or fungal infections. More specifically, the inventive compounds have at least one ring-substituted chain bonded to a core moiety.
  • the inventive compounds are useful antagonists to control intracellular levels of specific sn-2 unsaturated phosphatidic acids and corresponding phosphatidic acid-derived diacylglycerols, intracellular cell signaling messengers which occur in response to pro-inflammatory proliferative stimuli.
  • Pentoxifylline (l-(5-oxohexyl)-3,7-dimethylxanthine), abbreviated PTX, is a xanthine derivative which has seen widespread medical use for the increase of blood flow.
  • PTX is disclosed in U.S. Patents Nos. 3,422,107 and 3,737,433, both to Mohler et al. Metabolites of PTX were summarized in Davis et al., Applied Environment Microbiol. 48:327, 1984.
  • a metabolite of PTX is l-(5-hydroxyhexyl)-3,7-dimethylxanthine, designated Ml. Ml was also disclosed as increasing cerebral blood flow in U.S. Patents Nos.
  • PTX and its known metabolites thereof have been shown to have in vivo activity in specific biologic systems.
  • U.S. Patent No. 4,636,507 to Kreutzer et al. describes an ability of PTX and Ml, to further promote chemotaxis in polymorphonuclear leukocytes responding to a chemotaxis stimulator.
  • PTX and related tertiary alcohol substituted xanthines inhibit activity of certain cytokines to affect chemotaxis (U.S. Patents Nos. 4,965,271 and 5,096,906 to Mandell et al.).
  • TNF tumor necrosis factor
  • the invention is directed to ring-substituted therapeutic compounds and pharmaceutical compositions and uses thereof.
  • inventive ring-substituted compounds are useful in a large variety of therapeutic indications for treating or preventing disease.
  • the inventive compounds and pharmaceutical compositions thereof provide therapy for diseases advanced via intracellular signaling through specific intracellular signaling pathways, specifically the pathways discussed herein, by mediating a signaling response to an external stimuli.
  • Abnormally-induced intracellular signaling is characteristic of diseases treatable using the inventive compounds or pharmaceutical compositions thereof.
  • inventive compounds have at least one ring-containing side chain and are preferably cyclic or heterocyclic compounds.
  • inventive compounds and pharmaceutical compositions thereof have the formula:
  • R may be selected from among: hydrogen, halogen (preferably bromine, chlorine, fluorine and iodine), hydroxyl, amino, substituted or unsubstituted CQ.
  • JO alkyl, C(2-i ⁇ ) alkenyl, cyclic or heterocyclic groups and formula I.
  • the inventive compounds have at least one R of the following formula I:
  • R 2 wherein n is an integer from one to twenty; m and p are independently zero or an an integer from one to twenty.
  • R] is selected from among hydrogen, halogen, hydroxide, and substituted or unsubstituted C(- ⁇ o) alkyl, CQ. JO) alkoxy, Co-10) alkenyl, or a ring group having at least one four- to seven-membered ring;
  • R2 is selected from among hydrogen, halogen, hydroxide, substituted or unsubstituted alkoxy and C(2-i ⁇ ) alkenyl; and
  • R3 is hydrogen or a substituted or unsubstituted ring group having at least one four- to seven- membered ring.
  • At least one of R ⁇ or R3 is the ring group, a sum of either (n + m) or (n + p), corresponding to a respective R ⁇ or R3 ring group is not greater than nineteen.
  • a non-cyclic core moiety may include, but is not limited to, for example, acetamide, amide, amine, amino acid (one or two), carboxide, ester, terminal halogen or hydrogen atom, hydroxide, glutaric acid, glycine derivative, ketone, phosphate, phosphonate, sulfate, sulfonate, sulfone, sulfoxide, simple ionic functional group, thiol, thiolester or the like.
  • a cyclic core may be at least one five- to seven-member, non-heterocyclic ring or a heterocycle.
  • the core moiety may be selected from the group consisting of substituted or unsubstituted benzene; biphenyl; cyclohexane; cyclohexanedione; cyclopentanedione; napthlalene; phenol; quinone; salicylic acid and derivatives thereof; stilbene, tricyclododecane or the like.
  • substituted or unsubstituted barbituric acid benzamide; lactam; glutarimide; homophthalimide; hydrophthalimide; imidazole; imidazole amide; indo ethacin; isocarbostyril; lumazine; N-alkylheterocyclic; N-heterocyclic; pteridine; pthalimide; piperidine; pyridine; pyrimidine; pyrrole amide; quaternized N-heterocyclic; quinolizinedione; quinazolinone; quinoline; recorsinol; succinimide; theobromine thymine; triazine; uric acid; uracil; vitamins A, E or K; or xanthine.
  • R is bonded to a nitrogen of the core moiety, if present, most preferably to the nitrogen of a glutarimide, methylthymine, thymine, uracil or xanthine core.
  • R having formula I may be bonded to an Nj nitrogen of glutarimide; N-j nitrogen of xanthine (and N3 and N7 xanthine nitrogens may be independently substituted by a member selected from the group consisting of hydrogen, ' ⁇ . ⁇ alkyl, fluoro, chloro and amino); N3 nitrogen of a thymine or methylthymine; or Nj nitrogen of uracil.
  • R having formula I may be bonded to Nj and N3 xanthine nitrogens and N7 xanthine nitrogen is substituted by a member selected from the group consisting of hydrogen, methyl, fluoro, chloro and amino;
  • compositions of the inventive compounds comprise a pharmaceutical carrier or diluent and some amount of an inventive compound.
  • the nature of the composition and the pharmaceutical carrier or diluent will, of course, depend upon the intended route of administration, for example, parenterally, topically, orally or by inhalation for treatment of a patient with disease symtoms.
  • the invention includes a method for treating an individual having a variety of diseases. The disease is characterized by or can be treated by inhibiting an immune response or a cellular response to external or in situ primary stimuli. Treatment of the disease states involves mediating the cellular response through a specific phospholipid-based second messenger acting adjacent to a cell membrane inner leaflet.
  • the second messenger pathway is activated in response to various noxious or proliferative stimuli, characteristic of disease states treatable using the inventive compounds or pharmaceutical compositions thereof. Biochemistry of this second messenger pathway is described herein. More specifically, the invention includes methods for treating or preventing clinical symptoms of various disease states or reducing toxicity of other treatments by inhibiting cellular signaling through a second messenger pathway involving signaling through phosphatidic acid and through glycan phosphatidylinostinol (Gly PI).
  • Gly PI glycan phosphatidylinostinol
  • Gly PI consists of a phosphatidylinositol-1 -phosphate (PIP) bound through the carbon 6-hydroxyl to a glucosamine residue, which in turn is bound, usually to 2-5 other glycan residues (l— 4 type, linear bonds) containing an additional one to three phosphoethanolamine moieties, the last of which may be bound to an external protein such as Thy-1.
  • PIP phosphatidylinositol-1 -phosphate
  • Gly-PI Gly-PI protein binding
  • external protein binding the purpose of which may be simple binding to the cell membrane or placement of conformational constraints on the structure of externally bound membrane proteins (e.g., so that a particular portion of the molecule faces an extracellular environment);
  • signal transduction including part of the intracellular signal sent by insulin and a detectable portion of the signal transduced by Interleukin-2 (IL-2).
  • IL-2 Interleukin-2
  • Gly-PI species are synthesized: a) GlyPIl, containing 1-myristoyl 2- palmitoyl, 1-o-tetradecanyl (myristyl) 2-palmitoyl and 1-myristyl 2-myristyl phosphatidylinositol; and b) Gly Pl2, containing 1-myristoyl 2-oleoyl and l-o- myristyl 2- linoleoyl phosphatidylinositol.
  • Fraction (a) above contains a 1 : 1 mole content of C22 or C20 acyl groups attached to the inositol phosphate.
  • the Gly-PIl fraction identified by glucosamine labeling followed by mass spectrometry, exhibits a characteristic tripartite peak (glycan-inositol: 2-OH-acyl: phosphatidic acid moieties) and is uniformly inositol 2-OH acylated. Therefore, fraction (a) conveys resistance to PjG-PLC but not to PjG-PLD, suggesting that the observed fraction, when hydrolyzed, will generate 1-myristyl and 1-o-myristyl phosphatidic acid species, subsequently observed.
  • inventive compounds useful in treating diseases and reducing toxicity of other disease treatments, would affect cellular signaling through a second messenger pathway by interacting with binding and/or signaling functions of Gly PI.
  • a disease state or treatment-induced toxicity are selected from the group consisting of: tumor progression involving tumor stimulation of blood supply (angiogenesis) by production of fibroblst growth factor (FGF), vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF); tumor invasion and formation of metastases through adhesion molecule binding, expressed by vascular endothelial cells (VCAM and ICAM); tissue invasion through tumor metalloprotease production such as MMP-9; autoimmune diseases caused by dysregulation of the T cell or B cell immune systems, treatable by suppression of the T cell or B cell responses; acute allergic reactions including, but not limited to, asthma and chronic inflammatory diseases, mediated by pro-inflammatory cytokines including tumor necrosis factor (TNF) and IL- 1, and rheumatoid arthritis, osteoarthritis, multiple sclerosis or insulin dependent diabetes mellitus (IDDM), associated with enhanced localization of inflammatory cells and relase of inflammatory cytokines and metalloproteases; smooth muscle cell,
  • AIDS and AIDS related complex human immunodeficiency virus infection
  • HIV-associated dementia kidney mesangial cell proliferation in response to IL-1, MlP-l ⁇ , PDGF or FGF
  • inflammation kidney glomerular or tubular toxicity in response to cyclosporin A or amphotericin B treatment
  • organ toxicity e.g., gastrointestinal or pulmonary epithelial
  • cytotoxic therapy e.g., cytotoxic drug or radiation
  • effects of non- alkylating anti-tumor agents inflammation in response to inflammatory stimuli (e.g., TNF, IL-1 and the like) characterized by production of metalloproteases or allergies due to degranulation of mast cells and basophils in response to IgE or R ANTES; bone diseases caused by overproduction of osteoclast-activating factor (OAF) by osteoclasts; CNS diseases resulting from over-s
  • PA species some of which are generated from lyso-PA by the enzyme lyso-PA acyl transferase and some of which are generated from 2-O-acyl glycan-PI by PjG-PLD.
  • Generation of each of these PA species (the predominant forms being: 1-acyl and 1 -alkyl 2-linoleoyl PA compounds, generated by LPAAT; and 1-myristyl 2-palmitoyl and 1-o-myristyl 2-palmitoyl, generated by PjG-PLD) serves to effect both proliferative and/or inflammatory signaling in the diseases discussed and cell systems described above.
  • IL-2 signaling inhibition is potentially useful in the treatment of numerous disease states involving T-cell activation and hyperproliferation.
  • Exemplary autoimmune diseases treated by inhibiting IL-2 signaling are lupus, scleroderma, rheumatoid arthritis, multiple sclerosis, glomerula nephritis as well as potential malignancies, including but not limited to, chronic myelogenous leukemia as well as others.
  • Figure 1 is a dose response curve prepared from results in a murine thymocyte assay, determining inhibitive effects of inventive compounds nos. 2504 and 2507 (see below for chemical name and structure) on thymocyte proliferation.
  • Figure 2 is a bar graph reporting experimentally calculated IC50 values for inventive compounds nos. 1815, 1816, 2504, 2505, 2507, 2511, 2522, 2526 and 2531.
  • Figures 3, 4 and 5 are plotted graphs of compound concentrations ( ⁇ M) and inhibition (as a function of incorporated thymidine, cpm) for compounds nos. 2504, 2522 and 2526, respectively, in a mixed lymphocyte reaction (MLR) assay.
  • Figure 6 is a bar graph of experimentally calculated IC50 values for inventive compounds tested in the MLR assay of figures 3, 4 and 5.
  • Figure 7 reports results obtained in a viability assay conducted simultaneously with the MLR assay.
  • Figure 8 illustrates data collected for inventive compounds nos. 2507, 2511, 2522, 2526, and 2531, illustrating inhibition of human stromal cell proliferation in response to stimulation with platelet derive growth factor (PDGF).
  • PDGF platelet derive growth factor
  • Figure 9 reports data obtained in a proliferation assay, assessing the ability of inventive compound no. 3527 to inhibit proliferation of Balb/3T3 cells stimulated with PDGF.
  • Figure 10 reports results obtained in a Balb/3T3 viability assay used in conjunction with a proliferation assay.
  • Figure 11 reports results obtained for inventive compounds nos. 2507, 2511, 2522 and 2526 on inhibition of blast formation from human lymphocytes stimulated by IL-2 or an anti-CD3 antibody.
  • Figure 12 reports data showing that compound no. 2507 inhibited THP-1 adhesion to IL-l ⁇ -stimulated human umbilical vein endothelial cells (HUVEC).
  • Figure 13 shows that 2507 inhibits TNF secretion in an ex vivo human TNF model.
  • Figure 14 reports inhibitive results obtained for several inventive compounds in a lipo-protein saccharide (LPS)-induced TNF release assay using whole human blood.
  • LPS lipo-protein saccharide
  • the invention provides a genus of compounds which can control cellular behavior by a particular phase of a secondary messenger pathway system (Bursten et al., J. Biol. Chem. 266:20732, 1991).
  • the second messengers are lipids or phospholipids and use the following abbreviations:
  • PE phosphatidyl ethanolamine
  • LPE lysophosphoethanolamine
  • PA phosphatidic acid
  • LPA lysophosphatidic acid
  • DAG diacylglycerol
  • LPLD lysophospholipase-D
  • LPAAT lysophosphatidic acid acyl transferase
  • PAPH phosphatidic acid phosphohydrolase
  • PLA2 phospholipase A2.
  • PLD phospholipase D
  • PA A phosphoarachidonic acid
  • PC phosphatidyl choline
  • PA, cyclic pathway PAA, LPA, PA and DAG intermediates substituted with 1-saturated, 2-linoleoyl or 1,2-dioleoyl, dioleoyl/l ,2-sn-dilinoleoyl at the indicated sn-1 and sn-2 positions.
  • Classical PI Pathway PI, DAG, PA intermediates substituted with 1-stearoyl,
  • PLD-generated PA PE, PC, LPA, PA and DAG intermediates substituted with, e.g., 1,2-sn-dioleoyl-, 1 -alkyl, 2-linoleoyl-, and 1 -alkyl, 2-docosahexaenoyl-side chains.
  • Lysophosphatidic acid transferase effects the synthesis of phosphatidic acid (PA) from lysophosphatidic acid (LPA) by incorporation of an acyl group from acyl CoA. Hydrolysis of the phosphate moiety by PA phosphohydrolase (PAPH) results in the formation of DAG.
  • PA phosphatidic acid
  • PAPH PA phosphohydrolase
  • the compounds and pharmaceutical compositions of the invention include inhibitors of subspecies of LPAAT and PAPH enzymes with substrate specificity for intermediates with 1,2-diunsaturated and 1 -alkyl, 2-unsaturated subspecies.
  • One representative example of such an inhibitor is PTX.
  • PTX blocks PAPH in a specific activation pathway that does not involve PI but rather derives from a PA that is largely composed of 1,2-diunsaturated and 1 -alkyl, 2-unsaturated subspecies. This was shown, for example, by the demonstration that human mesangial cells stimulated with TNF produce DAG from PI and regenerate PI in the absence and the presence of PTX.
  • PA or DAG are derived from sources other than PI.
  • the compounds of the invention affect that subset of PAPH and LPAAT that relates to substrates with unsaturated fatty acids other than arachidonate in the sn-2 position, not the housekeeping forms of these enzymes that serve the PI pathway.
  • Each membrane phospholipid subclass (e.g., PA, PI, PE, PC and PS) reaches a stable content of characteristic fatty acyl side chains due to cyclic remodeling of the plasma membrane as well as turnover for each subclass.
  • PA is often stable, but present in relatively small quantities.
  • PA in resting cells consists mostly of saturated acyl chains, usually consisting of myristate, stearate and palmitate.
  • PC's acyl side chains consist mostly of acyl palmitate in the sn-1 position and oleate in the sn-2 position.
  • PE and PI are predominantly composed of sn-1 stearate and sn-2 arachidonate.
  • the origin of any PA species may be deduced from the chemical nature of its acyl groups in the sn-1 and sn-2 positions. For example, if PA is derived from PC through action of the enzyme PLD, the PA will contain the characteristic acyl side chains of PC substrate passed through the second messenger pathway. Further, the origin of any 1,2 sn-substrate species may be differentiated as to its origin. It is important to know whether or not each phospholipid species passes through a PA form prior to hydrolysis to DAG. The lyso-PA that is converted to PA and then to DAG may be shown.
  • the complexities of this second messenger pathway can be sorted by suitable analyses using fatty acyl side chain chemistry (e.g., by thin layer chromatography, gas-liquid chromatography, or high pressure liquid chromatography) of intermediates in cells at various time points after stimulation of the second messenger pathway.
  • suitable analyses using fatty acyl side chain chemistry (e.g., by thin layer chromatography, gas-liquid chromatography, or high pressure liquid chromatography) of intermediates in cells at various time points after stimulation of the second messenger pathway.
  • meseachymal cells such as neutrophils and rat or human mesangial cells
  • several signaling pathways may be activated in tandem, simultaneously or both.
  • F-Met-Leu-Phe stimulates formation of PA through the action of PLD, followed in time by formation of DAG through PAPH action.
  • DAG is generated from PI through the classical phosphoinositide pathway.
  • DAG is derived from both PA that is remodeled through a cycle whereby PA is sn-2 hydrolyzed by PLA2, followed by sn-2 transacylation by LPAAT and PA that is generated in a PLD-pathway from either PE or PC or both substrates by PLD.
  • the present second messenger pathway involves substrates with unsaturated fatty acids in the sn-2 position other than arachidonate and those sub-species of PAPH and LPAAT that are not involved in normal cellular housekeeping functions that are part of the classical PI pathway.
  • the PAPH and LPAAT enzymes involved in this specific second messenger pathway are extraordinarly stereo-specific for different acyl side chains and isomeric forms of substrates. Therefore, the inventive compounds may preferably be substantially enantiomerically pure.
  • PTX in vitro blocks formation of remodeled PA through the PA/DAG pathway at high PTX concentrations (greater than those that could be achieved in patients without dose- limiting side effects) by blocking formation of PA subspecies at LPAAT.
  • PTX Even in the presence of PTX, cells continue to form PA through the action of PLD, and DAG is also formed through the action of phospholipase C on PC and PI. The latter pathway are not inhibited by the inventive compounds or PTX.
  • DAG derived from remodeled and PLA- generated PA is diminished (e.g., 1,2-sn-dioleoyl DAG, 1 -alkyl, 2-linoleoyl DAG and 1 -alkyl, 2-docosahexaneolyl DAG). Therefore, the inventive compounds and PTX inhibit the formation of only a certain species of PA and DAG by selectively inhibiting a specific second messenger pathway that is only activated in cells by noxious stimuli, but is not used to signal normal cellular housekeeping functions.
  • inventive compounds are useful in treating a wide variety of clinical indications, mediated at the cellular level by a common mechanism of action.
  • in vitro and in vivo data presented herein provides predictive data that a wide variety of clinical indications, having similar effects on the specific second messenger pathway (activated by noxious stimuli and mediated through, for example, inflammatory cytokines), may be treated by the inventive compounds, which specifically inhibit the pathway.
  • the mechanism of action for the inventive compounds explains why these compounds have multifarious clinical indications.
  • Activation of the second messenger pathway is a major mediator of response to noxious stimuli and results in cellular signals that lead to, for example, acute and chronic inflammation, immune response and cancer cell growth.
  • inventive compounds may desirably inhibit other noxious stimuli not discussed, they most effectively mediate the above conditions.
  • Signals mediated by the present second messenger pathway include, for example, those cellular responses of LPS directly; T cell activation by antigen; B cell activation by antigen, cellular responses to IL-1, mediated through the IL-1 Type I receptor (but not the DL- 1 Type II receptor), and TNF (Type I receptor), growth stimulated by transformations including, but not limited to, activated oncogenes (e.g., ras, abl, her 2-neu and the like), smooth muscle cell proliferation stimulated by PDGF, b-FGF and IL-1; T cell and B cell growth stimulation by IL-2, EL-4 or IL-7 and IL-4 or EL-6, respectively; and more generally, T cell receptor signaling.
  • activated oncogenes e.g., ras, abl, her 2-neu and the like
  • smooth muscle cell proliferation stimulated by PDGF, b-FGF and IL-1
  • the inventive compounds block IL-1 signal transduction through the Type 1 receptor as shown, for example, by preventing IL-1 and IL-1 plus PDGF (platelet derived growth factor) induction of proliferation of smooth muscle, endothelial and kidney mesengial cells; (2) suppress up-regulation of adhesion molecules as shown, for example, by blocking VCAM in endothelial cells; (3) inhibit TNF, LPS and IL-1 induced metalloproteases (an inflammation model); (4) block LPS, TNF or IL-1 induced metalloprotease and secondary cytokine production (for prevention and treatment of septic shock); (5) suppress T cell and B cell activation by antigen, for example, IL-2 and IL-4; (6) inhibit mast cell activation by IgE; (7) are cytotoxic for transformed cells and tumor cell lines, yet not for normal cells; and (8) block signaling by IL-2, IL-4, IL-6 and IL-7 on T and B cells.
  • PDGF platelet derived growth factor
  • in vitro effects give rise to the following in vivo biological effects, including, but not limited to: protection and treatment of endotoxic shock and sepsis induced by gram positive or gram negative bacteria; inhibition of tumor cell growth; synergistic immunosuppression, active in autoimmune diseases and in suppressing allograft reactions; and stimulation of hair grow through reversal of an apoptotic process.
  • inventive compounds are most potent when used to prevent and treat septic shock, treat acute and chronic inflammatory disease, treat or prevent an autoimmune disease and stimulate hair growth (when applied topically).
  • the inventive compounds also are useful as an adjuvant to inhibit toxic side effects of drugs whose side effects are mediated through the present second messenger pathway.
  • Metalloproteases mediate tissue damage such as glomerular diseases of the kidney, joint destruction in arthritis, and lung destruction in emphysema, and play a role in tumor metastases.
  • Three examples of metalloproteases include a 92 kD type V gelatinase induced by TNF, IL-1 and PDGF plus bFGF, a 72 kD type IV collagenase that is usually constitutive and induced by TNF or EL- 1 , and a stromelysin/PUMP- 1 induced by TNF and IL- 1.
  • the inventive compounds can inhibit TNF or IL-1 induction of the 92 kD type V gelatinase inducable metalloprotease. Moreover, the inventive compounds can reduce PUMP-1 activity induced by 100 U/ml of IL-1. Accordingly, the inventive compounds prevent induction of certain metalloproteases induced by IL-1 or TNF and are not involved with constitutively produced proteases (e.g., 72 kD type IV collagenase) involved in normal tissue remodeling.
  • constitutively produced proteases e.g., 72 kD type IV collagenase
  • the inventive compounds inhibit signal transduction mediated through the Type I IL-1 receptor, and are therefore considered as IL-1 antagonists.
  • a recent review article entitled "The Role of Interleukin- 1 in Disease” (Dinarello et al., N. Engl. J. Med. 328, 106, Jan. 14, 1993) described the role of IL-1 as "an important rapid and direct determinant of disease...
  • IL-1 acts directly on the blood vessels to induce vasodilatation through the rapid production of platelet activating factor and nitric oxide, whereas in autoimmune disease it acts by stimulating other cells to produce cytokines or enzymes that then act on the target tissue.”
  • the article describes a group of diseases that are mediated by IL-1, including sepsis syndrome, rheumatoid arthritis, inflammatory bowel disease, acute and myelogenous leukemia, insulin-dependent diabetes mellitus, atherosclerosis and other diseases including transplant rejection, graft versus host disease (GVHD), psoriasis, asthma, osteoporosis, periodontal disease, autoimmune thyroiditis, alcoholic hepatitis, premature labor secondary to uterine infection and even sleep disorders.
  • GVHD graft versus host disease
  • the inventive compounds inhibit cellular signaling through the IL-1 Type I receptor and are IL-1 antagonists, the inventive compounds are useful for treating all of the above-mentioned diseases.
  • the mechanism of IL- 1 -induced shock appears to be the ability of IL-1 to increase the plasma concentrations of small mediator molecules such as platelet activating factor, prostaglandin and nitric oxide. These substances are potent vasodilators and induce shock in laboratory animals. Blocking the action of IL-1 prevents the synthesis and release of these mediators. In animals, a single intravenous injection of IL-1 decreases mean arterial pressure, lowers systemic vascular resistance, and induces leukopenia and thrombocytopenia.
  • IL-1 In humans, the intravenous administration of IL-1 also rapidly decreases blood pressure and doses of 300 ng or more per kilogram of body weight may cause severe hypotension.
  • the therapeutic advantage of blocking the action of IL-1 resides in preventing its deleterious biological effects without interfering with the production of molecules that have a role in homeostasis.
  • the present inventive compounds address this need, identified by Dinarello et al., by inhibiting cellular signaling only through the IL-1 Type I receptor and not through the IL-1 Type II receptor.
  • Interleukin- 1 is present in synovial lining and synovial fluid of patients with rheumatoid arthritis, and explants of synovial tissue from such patients produce IL-1 in vitro. Intraarticular injections of interleukin- 1 induce leukocyte infiltration, cartilage breakdown, and periarticular bone remodeling in animals.
  • interleukin- 1 In isolated cartilage and bone cells in vitro, interleukin- 1 triggers the expression of genes for collagenases as well as phospholipases and cyclooxygenase, and blocking its action reduces bacterial-cell-wall-induced arthritis in rats.” Therefore, the inventive compounds, as IL-1 antagonists, are useful to treat and prevent rheumatoid arthritis.
  • EL-1 can stimulate production of inflammatory eicosanoids such as prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and IL-8, an inflammatory cytokine with neutrophil- chemoattractant and neutrophil-stimulating properties.
  • PGE2 prostaglandin E2
  • LTB4 leukotriene B4
  • IL-8 an inflammatory cytokine with neutrophil- chemoattractant and neutrophil-stimulating properties.
  • Tissue concentrations of PGE2 and LTB4 correlate to severity of disease in patients with ulcerative colitis, patients with inflammatory bowel disease having high tissue concentrations of EL-1 and IL-8. Therefore, an EL-1 antagonist, such as the inventive compounds, would be effective to treat inflammatory bowel disease.
  • the inventive compounds should be effective to prevent the growth of worsening of disease for acute and chronic myelogenous leukemias.
  • IDDM Insulin-dependent diabetes mellitus
  • Islets of animals with spontaneously occurring IDDM e.g., BB rats or NOD mice
  • BB rats or NOD mice have inflammatory cells that contain IL-1. Therefore, the inventive compounds should be useful for the preventing and treating IDDM.
  • EL-1 also plays a role in atherosclerosis development. Endothelial cells are a target of IL-1. IL-1 stimulates proliferation of vascular smooth muscle cells. Foam cells, isolated from fatty arterial plaques from hypercholesterolemic rabbits, contain IL-l ⁇ and IL-l ⁇ messenger RNA. The uptake of peripheral blood monocytes results in initiation of EL-1 production by these cells. D -1 also stimulates production of PDGF. Taken together, IL-1 plays a part in the development of atherosclerotic lesions. Therefore, an IL-1 antagonist, such as the inventive compounds should be useful in preventing and treating atherosclerosis. EL-l activates (through the Type I IL-1 receptor) a lyso-PA acyltransferase
  • LPAAT phosphatidate phosphohydrolase
  • HMC human mesangial cells
  • the generation of the sn-2 unsaturated PA fraction by LPAAT serves to activate either G-proteins, or acts directly upon PLD through alteration of its lipid microenvironment.
  • Activation of LPAAT and generation of the sn-2-unsaturated PA species is an energy sensitive pathway of PLD. This provides a mechanism for a limited-receptor system to amplify a signal and generate a cellular response by rapid synthesis of small amounts of PA. Uptake of di- unsaturated PA, which is less than about 0.1% of total membrane lipid mass, is sufficient to activate PLD activity. This quantity of PA is similar to that endogeneously synthesized by LPAAT.
  • the PA-stimulated PLD acts upon PE, which should be localized to the inner leaflet of the cell membrane, enriched in PE relative to the outer leaflet. Therefore, the cellular inflammatory response to IL-1 is mediated by the pathway: IL-1R - ⁇ PA — (PLD) - ⁇ PE.
  • a localized tissue response is: lysoPA ⁇ PI ⁇ PKC ⁇ (PLD) ⁇ PC.
  • the PLD species are likely to be different isozymes.
  • the second messenger pathway whose activation is inhibited by the inventive compounds is not a Pl-derived pathway and does not involve PKC in the time courses of inhibition.
  • PKC is acutely activated by Pi-derived DAG, but chronic activation (i.e., > 30 minutes) is maintained by PC-derived PA generated by PC-directed PLD. Therefore, the pathway inhibited by the inventive compounds is PE-directed and not PC-directed. Moreover, the PE-directed PLD favors substrates with sn-2 long-chain unsaturation.
  • DAG and PA are upregulated in oncogenically transformed cells.
  • activating ras mutations result in increased generation of DAG upon stimulation with mitogens, although the sources of DAG differ between experimental systems.
  • EL-l ⁇ stimulation increased PLA2 and LPAAT activation, resulting in generation of sn-2 unsaturated PA and subsequent hydrolysis to DAG by phosphatidate phosphohydrolase.
  • the ras transformation in NIH/3T3 cells upregulates serum-stimulated generation of DAG and PA.
  • Particular species of DAG that is stimulated by serum is dioleoyl and of PA are dilinoleoyl and dioleoyl.
  • This upregulation occurs over 4-12 hours and pretreatment of cells with an inventive compound, or PTX, blocks generation of these phospholipid second messengers.
  • the inhibition occurs either through suppressing the generation of PA de novo from lysoPA, or through inhibition of one or both arms of the Lands cycle.
  • the coordinate increase of lysoPA in the setting of diminished PA/DAG production suggests inhibition of transacylation of a precursor lipid. Therefore, the ras transformation mediates an upregulation of PA through indirect stimulation of PLA2 and/or LPAAT activity.
  • the inventive compounds inhibit the conversion of the upregulated lysoPA to PA and subsequently block the phenotypic changes induced by PA/DAG in the membrane.
  • inventive compounds to inhibit generation of unsaturated phospholipids is mirrored by the ability of inventive compounds to inhibit proliferation and tumorogenicity of r ⁇ s-transformed cells in vitro and in vivo.
  • PTX inhibits r ⁇ s-transformed
  • NIH/3T3 cells more than parental cells. This inhibition is reversible and is not associated with significant cytotoxicity.
  • TNF tumor necrosis factor
  • inventive compounds or pharmaceutically acceptable salts thereof can be used in the manufacture of a medicament for the prophylactic or therapeutic treatment of any disease state in a human or other mammal, which is exacerbated or signaled through the present second messenger cellular phospholipid-based signaling pathway and by excessive or unregulated production of "first messenger" inflammatory cytokines such as TNF or IL- 1.
  • first messenger inflammatory cytokines
  • TNF first messenger signaling there are several disease states in which excessive or unregulated TNF production by monocytes/macrophages is implicated in exacerbating or causing the disease.
  • neurodegenerative diseases such as Alzheimers disease, endotoxemia or toxic shock syndrome (Tracey et al., Nature 330:662, 1987 and Hinshaw et al., Circ. Shock 30:279, 1990); cachexia (Dezube et al., Lancet 355:662, 1990), and adult respiratory distress syndrome (Miller et al., Lancet 2(8665):712, 1989).
  • inventive compounds may be used topically in the treatment of prophylaxis of topical disease states mediated or exacerbated by excessive TNF or IL-1, such as viral infections (herpes or viral conjunctivitis), psoriasis, fungal or yeast infections (ringworm, athletes foot, vaginitis, dandruff, etc.) or other dermatologic hyperproliferative disorders.
  • TNF or IL-1 such as viral infections (herpes or viral conjunctivitis), psoriasis, fungal or yeast infections (ringworm, athletes foot, vaginitis, dandruff, etc.) or other dermatologic hyperproliferative disorders.
  • High TNF levels have been implicated in acute malaria attacks (Grau et al., N. Engl. J. Med.
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • PDGF platelet derived growth factor
  • the inventive compounds also inhibit atherogenesis because increased levels of PDGF expressed by macrophages are associated with all phases of atherogenesis (Ross et al., Science 248: 1009, 1990). Further, many human tumors express elevated levels of either PDGF, FGF, receptors for FGF or PDGF, or mutated cellular oncogenes highly homologous to these growth factors or their receptors. For example, such tumor cell lines include sarcoma cell lines (Leveen et al., Int. ./. Cancer 46: 1066, 1990), metastatic melanoma cells (Yamanishi et al., Cancer Res.
  • inventive compounds are also useful to raise the seizure threshold, to stabilize synapses against neurotoxins such as strychnine, to potentiate the effect of anti- Parkinson drugs such as L-dopa, to potentiate the effects of soporific compounds, to relieve motion disorders resulting from administration of tranquilizers, and to diminish or prevent neuron overfiring associated with progressive neural death following cerebral vascular events such as stroke.
  • the compounds of the invention are useful in the treatment of norepinephrine-def ⁇ cient depression and depressions associated with the release of endogenous glucocorticoids, to prevent toxicity to the central nervous system of dexamethasone or methylprednisolone, and to treat chronic pain without addiction to the drug. Further, the compounds of the invention are useful in the treatment of children with learning and attention deficits and generally improve memory in subjects with organic deficits, including Alzheimer's patients.
  • PBMCs peripheral blood mononuclear cells
  • PBMC sucrose density gradient
  • a sucrose density gradient such as a Ficoll-Hypaque® gradient (specific gravity 1.08)
  • PBMC are obtained from a band at a plasma-Ficoll interface, separated and washed at least twice in a saline solution, such as HBSS.
  • Contaminating red cells are lysed, such as by ACK lysis for 10 min at 37 °C, and the PBMCs are washed twice in HBSS.
  • the pellet of purified PBMCs is resuspended in complete medium, such as RPMI 1640 plus 20% human inactivated serum.
  • Proliferative response of PBMC to allogeneic stimulation is determined in a two-way MLR performed in a 96- well microtiter plate. Briefly, approximately 10 5 test purified PBMC cells in 200 ⁇ l complete medium are co-cultured with approximately 10 5 autologous (control culture) or allogeneic (stimulated culture) PBMC cells, wherein the allogeneic cells are from HLA disparate individuals. Varying doses of compounds (drug) are added at the time of addition of cells to the microtiter plate. The cultures are incubated for 6 days at 37 °C in a 5% CO2 atmosphere.
  • tritiated thymidine is added (for example, 1 ⁇ Ci/well of 40 to 60 Ci/mmole) and proliferation determined by liquid scintillation counting.
  • in vitro assays that can be used to measure immunosuppressive activity of a particular compound. These assays are a predictive model for treatment or prevention of autoimmune diseases, such as diabetes, lupus, arthritis, and the like.
  • a first assay measures immunosuppressive activity of a drug at the B cell level. Spleens from adult mice contain immature B cells that express surface IgM. Cross-linking the surface IgM with an anti- mu antibody results in B cell proliferation.
  • IL-4 interleukin-4 receptors
  • IL-4R interleukin-4 receptors
  • IL-4R interleukin-4 receptors
  • IL-4 acts as a growth factor for B cells and will increase the amount of proliferation induced by anti-mu.
  • a mixture of anti-mu and murine IL-4 is added to murine splenocytes to cause their proliferation.
  • Mice spleens are obtained from adult mice and a single cell suspension is prepared in RPMI 1640 medium supplemented with 10% FCS. Cells (200,000) are plated into flat- bottomed wells and pre-incubated for 1-2 hrs with various concentrations of drug or PBS if it is a control well.
  • a mixture of anti-mu and murine is added to the wells at a final concentration of 5 ⁇ g/ml anti-mu and 12.5 ng/ml IL-4 and plates are incubated for three days. Proliferation is determined on the third day with a pulse of tritiated thymidine.
  • the IC50 concentration of a particular drug is the concentration of drug that results in a 50% inhibition of the proliferation obtained from the positive control.
  • a second immune suppression assay measures a T cell component to the immune reaction.
  • Lymph nodes contain a mixture of cells including T cells, B cells and macrophages.
  • the proliferating cells in this assay are T cells, the response is also dependent upon an antigen presenting cell such as a macrophage as well as an elaboration of various immunoregulatory cytokines.
  • Murine T cells will proliferate in vitro in response to a soluble protein antigen if they are first primed with the antigen in vivo. In vivo priming involves emulsifying the antigen (chicken ovalbumin or OVA) in complete Freunds adjuvant and injecting 50 ⁇ g of OVA into both hind footpads of adult Balb/c mice.
  • lymph nodes Fourteen days later the draining lymph nodes (popliteal) are removed and a single cell suspension is prepared in RPMI 1640 supplemented with 10% fetal calf serum. The lymph node cells (200,000) are plated into flat-bottom wells and OVA (200 ⁇ g/ml) and/or drug is added to appropriate wells and incubated for 5 days. Proliferation is determined and IC50's calculated as above.
  • a third assay measures an ability of an inventive compound to inhibit IL-2- induced proliferation of murine thymocytes.
  • Thymus glands are obtained from 4-6 week old mice and plated as a single cell suspension into flat bottomed wells in RPMI 1640 medium supplemented with 10% fetal calf serum.
  • the inventive compounds are added to appropriate wells and the cells are incubated for 1-2 hrs.
  • Concanavilin A (Con A, 0.25 ⁇ g/ml) and IL-1 (20 ng/ml) are added and the plates are incubated for 4 days. Cell proliferation is determined as above. There are also variations for this assay that follow the same basic stimulation and measure inhibition of proliferation format.
  • splenocytes can be used instead of thymocytes to measure more of a B cell response than a T cell response (e.g., thymocytes) and stimulated by an anti-mu antibody (40 ⁇ g/ml), IL-4 or PMA (2.5 nM).
  • human lymphocytes can be used from normal human volunteers and stimulated with human IL-2 (100 U/ml, Genzyme) and/or anti-CD3 antibody (2.5 ⁇ g/ml, Boehinger Mannheim).
  • Each inventive compound is investigated for cytotoxicity to determine appropriate doses for biological activity assays and to prevent cytotoxic reactions in in vitro assays when characterizing activity.
  • Cells e.g., NIH-3T3, Ras transformed 3T3 cells, malignant melanoma LD2 cells, etc.
  • drug is added about two days after plating.
  • Cell viability is determined using a fluorescent viability stain (e.g., 2',7'-bis-(2- carboroxyethyl)-5-(and -6)- carboxyfluorescein acetoxymethyl ester, BCECF excitation 488 nm and emission 525 nm) 24, 48 or 72 hours after addition of the drug.
  • a fluorescent viability stain e.g., 2',7'-bis-(2- carboroxyethyl)-5-(and -6)- carboxyfluorescein acetoxymethyl ester, BCECF excitation 488 nm and emission 525 nm
  • PDGF platelet derived growth factor
  • Human stromal cells are plated (e.g., about 2000 cells per well) in defined media (e.g., 69% McCoy's, 12.5% fetal calf serum, 12.5% horse serum, 1% antibiotics, 1% glutamine, 1% vitamin supplement, 0.8% essential amino acids, 1 % sodium pyruvate, 1% sodium bicarbonate, 0.4% non-essential amino acids and 0.36% hydrocortisone).
  • defined media e.g., 69% McCoy's, 12.5% fetal calf serum, 12.5% horse serum, 1% antibiotics, 1% glutamine, 1% vitamin supplement, 0.8% essential amino acids, 1 % sodium pyruvate, 1% sodium bicarbonate, 0.4% non-essential amino acids and 0.36% hydrocortisone.
  • the cells are treated with a stimulating agent, such as PDGF-AA, PDGF-BB or basic FGF (fibroblast growth factor) with or without IL-l ⁇ or TNF, and tritiated thymidine.
  • a stimulating agent such as PDGF-AA, PDGF-BB or basic FGF (fibroblast growth factor) with or without IL-l ⁇ or TNF, and tritiated thymidine.
  • Cell proliferation is determined by liquid scintillation counting.
  • LPAAT lysophosphatidic acid acyltransferase
  • PAPH phosphatidic acid phosphoryl hydrolase
  • the assay involves incubating of target cells with a primary stimulus (e.g., a variety of cytokines, growth factors, oncogene products, putative therapeutic agents, irradiation, viral infection, toxins, bacterial infection and the products thereof, and any stimulus which, if not counteracted, has a deleterious effect on the target cell) in the presence or absence of an inventive compound at varying dosage levels.
  • a primary stimulus e.g., a variety of cytokines, growth factors, oncogene products, putative therapeutic agents, irradiation, viral infection, toxins, bacterial infection and the products thereof, and any stimulus which, if not counteracted, has a deleterious effect on the target cell
  • Target cells include, for example, subcellular entities, such as, microsomes derived from mesenchymal and/or ectodermal cells, particularly microsomes from marrow stromal cells or human or rat mesangial cells; microsomes or synaptosomes derived from bovine brain; plasma membrane-enriched microsomes, plasma membranes derived as described in Bursten et al. (J. Biol. Chem.
  • cell lipids are extracted and assayed by thin layer chromatography according to standard procedures. Briefly, lipids are extracted using, for example, chloroform:methanol 2: 1 (v/v), and the extracts are then subjected to HPLC as described in Bursten and Harris, Biochemistry 30:6195-6203, 1991.
  • a Rainin® mu-Porasil column is used with a 3:4 hexane:propanol organic carrier and a 1-10% water gradient during the first 10 minutes of separation. Detection of the peaks in the elution pattern is by absorption in the range of ultraviolet which detects isolated double bonds. The relevant peaks of unsaturated PA and DAG are shown in the elution pattern. It is important to note that the assay method permits discrimination between various forms of PA and DAG so that those relevant to the pathway affected by the (R) or (S) compounds of the invention can be measured directly. Confirmation of the nature of the acyl substituents of these components is accomplished using fast-atom bombardment mass spectroscopy.
  • the relevant unsaturated (non-arachidonic) PA and DAG subspecies may be detected.
  • the time periods employed are 5-60 seconds after stimulation with the primary stimulus, such as a cytokine. This technique permits assessment of the levels of various lipid components as a function of time.
  • the invention provides for a class of compounds that are effective agents to inhibit specific cellular signaling events.
  • inventive compounds and inventive pharmaceutical compositions thereof have the formula:
  • R may be selected from among: hydrogen, halogen (preferably bromine, chlorine, fluorine and iodine), hydroxyl, amino, substituted or unsubstituted C(i.iQ) alkyl, C(2-10) alkenyl, cyclic or heterocyclic groups and formula I.
  • R substituents having a structure other than formula I include, but are not limited to, 2-bromopropyl, 4-chloropentyl, cyclohexyl, cyclopentyl, 3-dimethylaminobutyl, ethyl, hexyl, 2-hydroxyethyl, 5-hydroxyhexyl, 3-hydroxy-n-butyl, 3-hydroxypropyl, isobutyl, isopropyl, 2-methoxyethyl, 4-methoxy-n-butyl, methyl, n-butyl, n-propyl, phenyl, t-butyl and the like.
  • Particularly preferred R having a structure other than formula I are ethyl, methyl, or hydrogen.
  • the inventive compounds have at least one R of the following formula I:
  • Ri is selected from among hydrogen, halogen, hydroxide, and substituted or unsubstituted C(i_io) alkyl, C(i_ ⁇ o) alkoxy, C(2-10) alkenyl, or a ring group having at least one four- to seven-membered ring;
  • R2 is selected from among hydrogen, halogen, hydroxide, substituted or unsubstituted C(i _ ⁇ o) alkyl, C(i_ ⁇ o) alkoxy and Co-lO) alkenyl; and
  • R3 is hydrogen or a substituted or unsubstituted ring group having at least one four- to seven- membered ring.
  • At least one of R or R3 is the ring group, a sum of either (n + m) or (n + p), corresponding to a respective R ] or R3 ring group is not greater than nineteen.
  • n is not less than two.
  • (CH2) n , (CH2) m and/or (CH2)n may 1) be substituted by a halogen, hydroxide, substituted or unsubstituted CQ.
  • JO alkyl, C(2-i ⁇ ) alkenyl, cyclic or heterocyclic group; 2) have one or two unsaturated bonds (preferably in a cis configuration); or 3) be interrupted by at least one oxygen atom.
  • n is an integer from about three to about eighteen, more preferably, an integer from about three to about seven.
  • p is zero
  • R3 is a substituted or unsubstituted aromatic ring group.
  • R, Rj, and R2 are substituted C(I_I Q) alkyl, Co.10) alkenyl groups; R is substituted cyclic and heterocyclic groups; R ⁇ and R2 are substituted Cn . ⁇ 0) alkoxy, and Rj and R3 are a substituted ring having at least one four- to seven-membered ring, representative substituents for these groups may be amide, primary, secondary and tertiary amine, C(2-8) alkenyl, C(i_g) alkyl (including, e.g., branched and unbranched alkyl or alkenyl groups), C(i_g) alkoxyalkyl, azide, carbonate, carbonyl, carboxylic acid, cyanide, C(i_g) haloalkyl (including, e.g., mono-, di- and tri-haloalkyl substituents, such as trihalomethyl), isocyan
  • R cyclic or heterocyclic groups and R ⁇ or R3 ring groups may be, but are not limited to: anthracene, bicyclo[4.4.0]decane, bicyclo[2.2.1] heptane, bicyclo[3.2.0]heptane, bicyclo[4.1.0]heptane, bicylo[2.2.1]hexane, bicyclo[4.3.0]nonane, bicyclo[2.2.2]octane, biphenyl, cyclopentadiene, cyclopentane, cyclobutane, cyclobutene, cycloheptane, cyclohexane, cyclooctane and cyclopropane, 1 ,2-diphenylethane, fluorene, indene, phenyl, quinone, terphenyl, napthalene, phenanthrene, terphenyl, toluene, xylene, azet
  • more preferred cyclic groups include less complex ring systems, such as, for example, cyclopentane and cyclohexane, cyclopentadiene, phenyl, indene, toluene, xylene, furan, indole, thymine and xanthine.
  • a non-cyclic core moiety may include, but is not limited to, for example, acetamide, amide, amine, amino acid (one or two), carboxide, ester, terminal halogen or hydrogen atom, hydroxide, glutaric acid, glycine derivative, ketone, phosphate, phosphonate, sulfate, sulfonate, sulfone, sulfoxide, simple ionic functional group, thiol, thiolester or the like.
  • Exemplary core moiety amino acids may include one or more of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
  • the non-cyclic core moiety may preferably be an amide, carboxyl ester, carboxide, hydrogen, hydroxide or a dipeptide comprising two amino acids selected from the foregoing exemplary list.
  • a non-cyclic, halogen-core moiety may be, for example, bromine, chlorine, fluorine or iodine.
  • a cyclic core may be at least one five- to seven-member, non-heterocyclic ring or a heterocycle.
  • the at least one five- to seven-membered cyclic core may preferably have from one to three, five- to six-membered ring structures in a predominantly planar configuration.
  • An exemplary, non-heterocyclic ring core moiety may be selected from the group consisting of substituted or unsubstituted benzene; biphenyl; cyclohexane; cyclohexanedione; cyclopentanedione; napthlalene; phenol; quinone; salicylic acid and derivatives thereof; stilbene, tricyclododecane or the like.
  • substituted or unsubstituted barbituric acid benzamide; lactam; glutarimide; homophthalimide; hydrophthalimide; imidazole; imidazole amide; indomethacin; isocarbostyril; lumazine; N-alkylheterocyclic; N-heterocyclic; pteridine; pthalimide; piperidine; pyridine; pyrimidine; pyrrole amide; quaternized N-heterocyclic; quinolizinedione; quinazolinone; quinoline; recorsinol; succinimide; theobromine; thymine; triazine; uric acid; uracil; vitamins A, E or K; or xanthine.
  • substituents for the non-heterocyclic ring and heterocyclic cores may be amide, primary, secondary and tertiary amine, C(2-8) alkenyl, C(i_8) alkyl (including, e.g., branched and unbranched alkyl or alkenyl groups), C(].g) alkoxyalkyl, azide, carbonate, carbonyl, carboxylic acid, cyanide, C(i_g) haloalkyl (including, e.g., mono-, di- and tri- haloalkyl substituents, such as trihalomethyl), isocyanate, isothiocyanate, phosphate, phosphonate, primary, secondary or tertiary alcohol (including, e.g., any one of various diols, methanol, butanol, 1-cyclopentanol, ethanol, 2-ethyl-3-methyl-l-propanol, pentanol, propanol, and
  • Preferred non-heterocyclic ring cores include substituted or unsubstituted 1,3- cyclohexanedione, 1,3-cyclopentanedione; 1,3-dihydroxynaphthalene; orthophenol.
  • Preferred heterocyclic cores include substituted or unsubstituted 3,7- dimethylxanthine, glutarimide, 3-methyl-7-pivoloylxanthine, methylthymine, methyluracil, 3- methylxanthine, tetrahydrophthalimide, thymine, uracil and xanthine, most preferably halogen- substituted xanthine.
  • Exemplary preferred cores include: C( ⁇ -6) alkyl-substituted thymine; C ⁇ . 6) alkyl-substituted uracil; 1,3-dihydroxynapthalene; 3,3-dimethylglutarimide; dihydrothymine; 2,4-dioxohexahydro-1.3.5-tetrazine; hexahydrophthalimide; homophthalimide; 2- hydroxypyridine; ⁇ -ionone as vitamin A methylbarbituric acid; 2,6,6-methyl-l-cyclohexene-l- acetaldehyde as vitamin A; methyldihydroxypyrazolopyrimidine, specifically, 1,3- dimethyldihydroxypyrazolo[4,3-d]pyrimidine;l-methyl-5,6-dihydrouracil; 1,7-dimethylxanthine, 3,7-dimethylxanthine; 7-methylhypoxanthine; 1-methyllumazine; 3-
  • R is bonded to a nitrogen of the core moiety, if present, most preferably to the nitrogen of a glutarimide, methylthymine, thymine, uracil or xanthine core.
  • R having formula I may be bonded to an Nj nitrogen of glutarimide; Nj nitrogen of xanthine (and N3 and N7 xanthine nitrogens may be independently substituted by a member selected from the group consisting of hydrogen, C .5) alkyl, fluoro, chloro and amino); N3 nitrogen of methylthymine; or Nj nitrogen of uracil.
  • R having formula I may be bonded to Nj and N3 xanthine nitrogens and N7 xanthine nitrogen is substituted by a member selected from the group consisting of hydrogen, methyl, fluoro, chloro and amino.
  • Representative, preferred inventive compounds are compounds of formulas II, III and IV:
  • compositions of the inventive compounds comprise a pharmaceutical carrier or diluent and some amount of an inventive compound.
  • the compound may be present in an amount to effect a physiological response, or it may be present in a lesser amount such that the user will need to take two or more units of the composition to effect the treatment intended.
  • These compositions may be made up as a solid, liquid or in a gaseous form. Or one of these three forms may be transformed to another at the time of being administered such as when a solid is delivered by aerosol means, or when a liquid is delivered as a spray or aerosol.
  • compositions and the pharmaceutical carrier or diluent will, of course, depend upon the intended route of administration, for example, parenterally, topically, orally or by inhalation for treatment of a patient with disease symtoms.
  • pharmaceutical composition will be in the form of a cream, ointment, liniment, lotion, pastes, aerosols and drops suitable for administration to the skin, eye, ear or nose.
  • parenteral administration the pharmaceutical composition will be in the form of a steril injectable liquid such as an ampule or an aqueous or non-acqueous liquid suspension.
  • the pharmaceutical composition will be in the form of a tablet, capsule, powder, pellet, atroche, lozenge, syrup, liquid or emulsion.
  • the invention includes a method for treating an individual having a variety of diseases.
  • the disease is characterized by or can be treated by inhibiting an immune response or a cellular response to external or in situ primary stimuli.
  • Treatment of the disease states involves mediating the cellular response through a specific phospholipid-based second messenger acting adjacent to a cell membrane inner leaflet.
  • the second messenger pathway is activated in response to various noxious or proliferative stimuli, characteristic of disease states treatable using the inventive compounds or pharmaceutical compositions thereof. Biochemistry of this second messenger pathway is described herein.
  • the invention includes methods for treating or preventing clinical symptoms of various disease states or reducing toxicity of other treatments by inhibiting cellular signaling through a second messenger pathway involving signaling through phosphatidic acid and through glycan phosphatidylinostinol (Gly)
  • the invention includes a method for preparing the inventive compounds.
  • a compound containing a cyclic functional group (intended as a ring-substituent in compounds of the invention) is substituted with a carbon chain having the proper length sufficient to later comprise a side arm of the inventive compounds.
  • the cyclic compound undergoes a reaction to produce an anion, which is then subsequently reacted with a substituted carbon chain.
  • the substituted carbon chain has at least one functional group which may be substituted in a displacement reaction by the desired cyclic compound to form a cyclic intermediate, the cyclic intermediate later being attached to a core-containing compound.
  • a base may be used to obtain an anion of the cyclic compound.
  • Preferred bases include, but are not limited to, n-butyl lithium and lithium diisopropylamine.
  • Preferred substituted carbon chain compounds include, but are not limited to, carbon chain compounds having at least one halo group substituted on the carbon chain.
  • a compound containing a desired core undergoes a reaction to produce an anion.
  • the anion is then subsequently reacted with the cyclic intermediate to displace a targeted functional group on the carbon chain, producing the desired inventive compound.
  • a predetermined amount of a core-containing compound is reacted with a suitable base, a solvent and the cyclic intermediate, which comprises a carbon chain having at least one functional group which may be substituted in a displacement reaction by the desired core-containing compound.
  • Preferred bases include, but are not limited to, sodium hydride, sodium amide, sodium alkoxide, lithium hydride, potassium hydride, lithium amide, sodium amide and potassium amide.
  • An especially preferred base is sodium hydride.
  • Preferred solvents may be dimethylsulfoxide, dimethylformamide, or an alcohol.
  • Exemplary preferred alcohols include, but are not limited to, methanol, ethanol or isopropanol.
  • Cyclic intermediate compounds satisfying structural requirements of the inventive compounds may be used in the preliminary reaction according to the invention.
  • Preferred cyclic intermediates include, but are not limited to compounds having a halo-substituted carbon chain. Schematic A below, illustrates a representative synthesis for compounds of the invention.
  • inventive compounds provide a method for maintaining homeostasis in cells contacted by primary stimuli by mitigating the effects of these primary stimuli on the secondary signaling pathways invoked within seconds of a primary stimulus.
  • administration of an inventive compound in vivo or ex vivo provides a method to modify cellular behavior, the method comprising contacting cells (in vivo or ex vivo), whose behavior is to be modified, with an effective amount of an inventive compound or a pharmaceutical composition thereof.
  • the method is a method to: (1) inhibit proliferation of tumor cells, being; (2) suppress activation of T-cells by antigen or IL-2 stimulation being; (3) suppress activation of monocyte/macrophage cells by endotoxin, TNF, IL-1 or GM-CSF stimulation, being; (4) suppress antibody production of B-cells in response to an antigen, IL-4 or CD40 ligand, being; (5) inhibit the proliferation of smooth muscle cells in response to growth factors capable of stimulating said proliferation, being; (6) lower systemic vascular resistance conferred by endothelial cells, being; (7) lower systemic vascular resistance induced by endothelial cells, being; (8) lower expression of adhesion molecules induced by enhancers thereof, being; (9) suppress the activation of T-cells and macrophages by HIV, being; (10) inhibit the proliferation of kidney mesangial cells in response to stimulation by IL-1 and or MlP-l ⁇ and/or PDGF and or FGF, being; (11) enhance the resistance of kidney
  • Indications useful for administering compounds of the invention include, but are not limited to: the presence of a tumor burden, a hormone-related disorder, a neurological disorder, an autoimmune disease, inflammation, restenosis, coronary artery disease, atherosclerosis, hypertension, unwanted immune response (such as allograft reactions), viral infection, nephritis, mucositis, and various allergic responses.
  • Allergic responses include acute allergic response and thus rhinorrhea, sinus drainage, diffuse tissue edema, and generalized pruritus.
  • other chronic allergic responses include, dizziness, diarrhea, tissue hyperemia, and lacrimal swelling with localized lymphocyte infiltration.
  • Allergic reactions are also associated with leukotriene release and the distal effects thereof, including asthmatic symptoms (e.g., development of airway obstruction, a decrease in FEV1 , changes in vital capacity, and extensive mucus production).
  • Suitable subjects for the administration of compounds of the invention include patients: being administered other cytotoxic agents for the treatment of tumors, such as chemotherapeutic agents or irradiation therapy; suffering from neoplasias generally, whether or not otherwise treated including acute and chronic myelogenous leukemia, hairy cell leukemia, lymphomas, megakaryocytic leukemia, and the like; disease states caused by bacterial, fungal, protozoal, or viral infection; exhibiting unwanted smooth muscle cell proliferation in the form of, for example, restenosis, such as patients undergoing cardiac surgery; afflicted with autoimmune diseases, thus requiring deactivation of T and B cells, and having neurological disorders.
  • cytotoxic agents for the treatment of tumors such as chemotherapeutic agents or irradiation therapy
  • suffering from neoplasias generally, whether or not otherwise treated including acute and chronic myelogenous leukemia, hairy cell leukemia, lymphomas, megakaryocytic leukemia, and
  • the compounds of the invention further are able to decrease enhanced levels of a relevant PA and DAG resulting from stimulation of synaptosomes with acetylcholine and/or epinephrine. This suggests that the effects of the compounds of the invention are to both enhance the release of inhibitory neural transmitters such as dopamine, and to modulate the distal "slow current" effects of such neurotransmitters.
  • the drugs of the invention are also useful to raise the seizure threshold, to stabilize synapses against neurotoxins such as strychnine, to potentiate the effect of anti- Parkinson drugs such as L-dopa, to potentiate the effects of soporific compounds, to relieve motion disorders resulting from administration of tranquilizers, and to diminish or prevent neuron overfiring associated with progressive neural death following cerebral vascular events such as stroke.
  • the compounds of the invention are useful in the treatment of norepinephrine-deficient depression and depressions associated with the release of endogenous glucocorticoids, to prevent the toxicity to the central nervous system of dexamethasone or methylprednisolone, and to treat chronic pain without addiction to the drug.
  • the compounds of the invention are useful in the treatment of children with learning and attention deficits and generally improve memory in subjects with organic deficits, including Alzheimer's patients.
  • a particularly preferred regimen for use in treating leukemia is 4-50 mg/kg body weight. It is to be understood, however, that for any particular subject, specific dosage regimens should be adjusted to the individual's need and to the professional judgment of the person administering or supervising the administration of the inventive compounds.
  • a suitable formulation will depend on the nature of the disorder to be treated, the nature of the medicament chosen, and the judgment of the attending physician.
  • inventive compounds are formulated either for injection or oral administration, although other modes of administration such as transmucosal or transdermal routes may be employed.
  • Suitable formulations for these compounds can be found, for example, in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, PA.
  • inventive compounds and their pharmaceutically acceptable salts can be employed in a wide variety of pharmaceutical forms.
  • the preparation of a pharmaceutically acceptable salt will be determined by the chemical nature of the compound itself, and can be prepared by conventional techniques readily available. Thus, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
  • the amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1 gram, wherein the amount of inventive compound per dose will vary from about 25 mg to about 1 gram for an adult.
  • the preparation When a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.
  • a liquid carrier e.g., ethanol, polyethylene glycol, coconut oil, glycerine or water
  • inventive compound required for therapeutic effect on topical administration will, of course, vary with the compound chosen, the nature and severity of the disease and the discretion of the treatment provider.
  • Parenteral includes intravenous, intramuscular, subcutaneous, intranasal, intrarectal, intravaginal or intraperitoneal administration. Appropriate dosage forms for such administration may be prepared by conventional techniques.
  • a typical parenteral composition consists of a solution or suspension of the inventive compound or a salt thereof in a sterile or non-aqueous carrier, optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil, or sesame oil.
  • the daily dosage for treatment of sepsis or another severe inflammatory condition via parenteral administration is suitable from about 0.001 mg/kg to about 40 mg/kg, preferably from about 0.01 mg/kg to about 20 mg/kg of an inventive compound or a pharmaceutically acceptable salt thereof calculated as the free base.
  • the inventive compounds may be administered orally.
  • the daily dosage regimen for oral administration is suitably from about 0.1 mg/kg to about 1000 mg/kg per day.
  • the dosage is suitably from about 0.001 mg/kg to about 40 mg/kg of the inventive compound or a pharmaceutically acceptable salt thereof, calculated as the free base.
  • the active ingredient may be administered from 1 to 6 times a day, sufficient to exhibit activity.
  • inventive compounds may be administered by inhalation (e.g., intranasal or oral)
  • Appropriate dosage forms include an aerosol or a metered dose inhaler, as prepared by conventional techniques.
  • the daily dosage is suitably from about 0.001 mg/kg to about 40 mg/kg of the inventive compound or a pharmaceutically acceptable salt thereof, calculated as the free base.
  • Typical compounds for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant.
  • This example is a method of synthesis for inventive compound no. 1815 (see above for chemical name and structure).
  • Methanesulfonyl chloride (0.49 g, 4.2 mmol) and then triethylamine (0.62 g, 6.2 mmol) were added to a solution of 3-furan methanol (0.40 g, 4.0 mmol) in dichloromethane (10 ml) at 0 °C. After stirring the reaction mixture for 10 minutes at 0 °C, it was allowed to warm to 25 °C and stirred for an additional 2 hours. The reaction was poured into water (10 ml) and separated and washed with dichloromethane (10 ml).
  • Example 2 This example is a method of synthesis for inventive compound no. 2504 (see above for chemical name and structure). Methanesulfonyl chloride (1.92 g, 4.0 ml, 16.8 mmol) and then triethylamine (2.52 g, 25.0 mmol) were added to a solution of 3-furan methanol (1.50 g, 15.3 mmol) in dichloromethane (35 ml) at 0 °C. After stirring for 10 minutes at 0 °C, the reaction was allowed to warm to 25 °C and stirred for an additional 2 hours. The reaction was poured into water (50 ml) and extracted with two 30 ml aliquots of dichloromethane. The organic portions were combined and dried over magnesium sulfate. Evaporating the solvent left an oil, 3-methanesulfonyl methylfuran, used without further purification.
  • This example is a method of synthesis for inventive compound no. 2522. Over 30 minutes, a 2.0 M solution (30 ml) of butyllithium (60.0 mmol) in cyclohexane was added to a solution (300 ml) of furan (4.14 g, 60.9 mmol) in tetrahydrofuran at -15 °C and the reaction stirred at -15 °C for 30 minutes and 0 °C for 90 minutes.
  • This example is a synthesis of inventive compound no. 2526 (see above for chemical name and structure).
  • a 2.0 M solution (30 ml) of butyllithium (60.0 mmol) in cyclohexane was added to a solution (300 ml) of furan (4.14 g, 60.9 mmol) in tetrahydrofuran at -15 °C over 30 minutes and the reaction stirred at -15 °C for 30 minutes and at 0 °C for 90 minutes.
  • this solution was added via a canula over 50 minutes to a solution (450 ml) of 1 ,6-dibromohexane (25.0 g, 102 mmol) in tetrahydrofuran, also at -78 °C, and the reaction stirred at -78 °C for 1 hour and then allowed to warm to 25 °C over 5 hours.
  • Saturated ammonium chloride (100 ml) and water (100 ml) were added and the reaction mixture extracted with two 100 ml aliquots of diethyl ether.
  • This example is a method of synthesis for inventive compound no. 2533.
  • a mixture (20 ml) of l-chloro-3-cyclohexylpropane (0.8 ml, 5.0 mmol) and sodium theobromine (1.01 g, 5.0 mmol) in dimethylsulfoxide was stirred for 17 hours.
  • the mixture was heated at 55 °C for 6 hours, and after cooling to ambient temperature, ether (50 ml) was added.
  • the solution was washed with three 30 ml aliquots of water and dried over magnesium sulfate, and the ether was evaporated under vacuum.
  • a crude residue was purified by flash chromatography over silica using an ethyl acetate eluant, producing 437 mg of compound no. 2533 (29% yield).
  • This example is a method of synthesis for inventive compound no. 3527.
  • 1,9- Dibromononane (8.58 g, 3.0 mmol) was added to a suspension (50 ml) of potassium phthalimide (5.55 g, 3 mmol) in dimethyl sulfoxide and stirred overnight. After 12 hours of stirring at room temperature, the reaction was poured into a separatory funnel containing 200 ml of water and extracted with three 100 ml aliquots of ethyl acetate. The organic extracts were combined, washed with water (100 ml) and brine (100 ml), dried over anhydrous magnesium sulfate and concentrated under reduced pressure.
  • a crude product obtained was further purified by flash chromatography over silica gel using a 30% ethyl acetate/hexane eluant, producing 8.21 g of N- (9-Bromononyl)phthalimide (77.8 % yield).
  • Sodium hydride (720 mg, 30 mmol) was added to a suspension (50 ml) of theobromine (4.5 g, 25 mmol) and N-(9-bromononyl)phthalimide (7.04 g, 20 mmol) in dimethyl sulfoxide.
  • the resulting reaction mixture was stirred for 24 hours, after which, the reaction was poured into 300 ml of water and filtered.
  • a crude product obtained was further purified by flash chromatography over silica gel using a 20% methanol/dichloromethane, to obtain 1.3 g of compound no. 3527 (14.4% yield).
  • This example shows an inhibitive effect of inventive compounds nos. 1815, 1816, 2504, 2505, 2507, 2511, 2522, 2526 and 2531 on murine thymocyte proliferation stimulated by Concanavalin A (ConA) and interleukin-2 (IL-2).
  • ConA Concanavalin A
  • IL-2 interleukin-2
  • Drug was added at various doses two hours prior to activation with ConA and IL-2.
  • the cells were incubated for 4 days at 37 °C. On day 4, the cells were pulsed with tritiated thymidine and allowed to incubate for an additional 4 hours. Harvested cells were analyzed for incorporated tritiated thymidine, determined using a liquid scintillation counter. Dose response curves were prepared from the assay results and used to calculate an IC50 value for each compound tested.
  • figure 1 illustrates the inhibitive effects of these compounds on proliferation of thymocytes stimulated with ConA and IL-2. Background counts, without addition of representative inventive compounds were about 170 cpm.
  • Figure 1 illustrates an ability of the inventive compounds, particularly compound no. 2507, representative of ring substituted inventive compounds, to inhibit ConA/IL-2 stimulated proliferation at compound concentrations less than 100 ⁇ M.
  • IC50 values experimentally calculated from dose response curves prepared for compounds tested are plotted in a bar graph of figure 2. These concentrations plotted are within known in vivo concentrations useful in treating disease.
  • Example 9 This example illustrates an ability of inventive compounds nos. 1815, 1816, 2504,
  • PBMC peripheral blood mononuclear cells
  • MLR in vitro mixed lymphocyte reaction
  • PBMC peripheral blood mononuclear cells
  • a saline solution such as HBSS.
  • Contaminating red cells are lysed, for example, by ACK lysis for 10 minutes at 37 °C, and the PBMC were washed twice in HBSS.
  • the pellet of purified PBMC was resuspended in complete medium, such as RPMI 1640 plus 20% human inactivated serum.
  • Proliferative response of PBMC to allogeneic stimulation was determined in a two-way MLR performed in a 96- well microtiter plate. Approximately 10 ⁇ test-purified PBMC in 200 ⁇ l complete medium were co-cultured with approximately 10*5 autologous (control culture) or allogeneic (stimulated culture) PBMC. Allogeneic cells were from HLA disparate individuals. Varying doses of compounds nos. 1815, 1816, 2504, 2504, 2507, 2511, 2522 and 2526 were added simultaneously upon addition of cells to the microtiter plate.
  • tritiated thymidine was added (for example, 1 ⁇ Ci/well of 40 to 60 Ci/mmole) and proliferative inhibition was assessed by determining amount of tritiated thymidine taken up, using liquid scintillation counting.
  • Figures 3, 4 and 5 are plotted graphs of compound concentrations ( ⁇ M) and inhibition (as a function of incorporated thymidine, cpm) for compounds nos. 2504, 2522 and 2526, respectively.
  • Figure 5 illustrates a most pronounced inhibition of PBMC proliferation. At concentrations less than 100 ⁇ M, compound no. 2526 significantly inhibited incorporation of thymidine.
  • figures 4 and 5 illustrate inhibitive characteristics of inventive compounds nos. 2504 and 2522, in this MLR assay at compound concentrations less than 250 ⁇ M and 100 ⁇ M, respectively.
  • Figure 6 is a bar graph of experimentally calculated IC50 values for compounds tested in this MLR assay. Some IC50 values are in the 250 ⁇ M range, while others, such as 2507, exhibit substantial potency with an IC50 value of about 20 ⁇ M.
  • This example illustrates inhibitive effects of inventive compounds nos. 2507, 2511, 2522, 2526, and 2531 (see above for chemical names and structures) on proliferation of human stromal cells stimulated with platelet derive growth factor (PDGF).
  • PDGF platelet derive growth factor
  • stromal cells were starved in serum-free media for one day and then stimulated with 50 ng/ml PDGF-BB.
  • Inventive compounds were were added at predetermined concentrations one hour prior to PDGF stimulation.
  • Tritiated thymidine was added for one day at the time of PDGF stimulation and the cells were harvested and counted by liquid scintillation counting 24 hours later. Background counts (i.e., starved cells) were approximately 1% of control levels.
  • Figure 8 reporting results from this assay, shows that all inventive compounds tested were active in this predictive in vitro model.
  • Inventive compounds nos. 2507 and 2526 illustrate more potent activity, in comparison to other representative compounds tested.
  • This example illustrates inhibitive effects of the inventive compounds on Balb/3T3 cell proliferation in response to platelet derived growth factor (PDGF) stimulation.
  • PDGF platelet derived growth factor
  • Disregulated PDGF-proliferative response has been linked to a variety of diseases, including, e.g., restenosis, atherosclerosis, fibrosis, and tumor cell angiogenesis.
  • Balb/3T3 cells respond vigorously to PDGF stimulation, and are useful in vitro models for further study of PDGF-induced proliferation.
  • an assay useful in determining whether a compound would be useful in treating diseases characterized by this or similar disregulated proliferative responses research indicates that the inventive compounds inhibit PDGF-induced proliferation of Balb/3T3 cells.
  • Balb/3T3 cells were plated in low serum-containing medium for 24 hours prior to stimulation with various concentrations of inventive compound no. 3527.
  • PDGF was added at varying concentrations along with tritiated thymidine. The cells were allowed to incubate for one day, following addition of PDGF and thymidine. 24 hours later, the cells were harvested and counted by liquid scintillation counting.
  • Figure 9 reports data obtained in this proliferation assay. The results illustrate that compound no. 3527 inhibits PDGF-stimulated proliferation of Balb/3T3 cells at concentrations less than 30 ⁇ M, indicating that the inventive compounds are candidates for treating or preventing restenosis, atherosclerosis, fibrosis, tumor cell angiogenesis and other similar diseases.
  • FIG. 1 This example illustrates inhibitive effects of inventive compounds nos. 2507, 2511, 2522 and 2526 (see above for chemical name and structure) on inhibition of blast formation from human lymphocytes stimulated by IL-2 or an anti-CD3 antibody. Results from this assay are shown in figure 1 1. This is a human in vitro assay providing an additional basis for assessing whether tested compounds have immunosuppressive activity. As shown in figure 11, all inventive compounds tested demonstrate some immunosuppressive activity of blastogenesis stimulated by either IL-2 or anti-CD3 with IC50 values at or below 50 ⁇ M.
  • This example illustrates an ability of inventive compound no. 2507 to inhibit adherence of specific cells to human umbilical vein endothelial cells (HUVEC) stimulated with IL-l ⁇ .
  • HUVEC human umbilical vein endothelial cells
  • IL-l ⁇ human umbilical vein endothelial cells
  • This adhesion assay is useful in showing an ability of an inventive compound to inhibit adhesion of a specific cell to other cells in this signaling phenomenon, and thus in predicting therapeutic potential of the inventive compounds.
  • HUVEC Two days prior to conducting the assay procedure, HUVEC were plated at 4000 cells/well. After two days, HUVEC were stimulated overnight with IL-l ⁇ (20 ng/ml).
  • THP-1 a human acute monocytic leukemia cell line
  • BCECF ⁇ 2,7-bis-(2-carboxyethyl)- 5(and-6)carboxyflourescein,acetoxymethyl ester
  • BCECF ⁇ 2,7-bis-(2-carboxyethyl)- 5(and-6)carboxyflourescein,acetoxymethyl ester
  • BCECF ⁇ 2,7-bis-(2-carboxyethyl)- 5(and-6)carboxyflourescein,acetoxymethyl ester
  • the cells were allowed to adhere for 20 minutes at 37 °C, after which time, the plate was inverted and spun at 800 rpm. The wells were washed once with PBS and resuspended in 100 ⁇ l PBS prior to reading fluorescence on a Millipore fluorescence plate reader. Data was recorded as percent adherence of THP-1 cells to HUVEC at selected concentrations of compound no. 2507.
  • Figures 12 reports plotted results of data obtained in this assay.
  • Figure 12 shows that compound no. 2507 inhibited THP-1 adhesion to IL-l ⁇ stimulated HUVEC.
  • the representative, inventive compounds inhibit adherence at compound concentrations less than 10 ⁇ M, suggesting that the compounds tested, as representatives of all inventive compounds, possess therapeutic potential in diseases exhibiting biologic characteristics these assays are designed to model.
  • Examnle 14 shows that the compounds tested, as representatives of all inventive compounds, possess therapeutic potential in diseases exhibiting biologic characteristics these assays are designed to model.
  • This example illustrates the effect of CT2507 in an ex vivo human TNF model, a model predictive of compounds useful in treatment and prevention of septic shock and sepsis syndrome.
  • LPS was added to whole blood obtained from normal volunteers to trigger a dose-dependent synthesis and extracellular release of TNF according to Desch et al. (Lymphokine Res. 8: 141, 1989).
  • the ex vivo model examines whether a compound will block LPS-mediated release of TNF from monocytes in whole blood. Results in this assay are illustrated in figure 13. As shown at the concentrations tested, inventive compound no. 2507, representative of ring-substituted inventive compounds, blocked TNF release in a dose- dependent fashion.
  • whole blood was collected from a healthy human donor into Vacutainer tubes containing ACD citrate as anti-coagulant.
  • the compounds tested were diluted in RPMI medium and 5 ⁇ l of the dilute concentrations placed in tubes containing 225 ⁇ l of whole blood. The tubes were mixed and incubated for no more than lhour at 37 °C.
  • LPS Salmonella abortus equi (commercially available from Sigma) is diluted in RPMI and the dilute samples added to the whole blood/compound samples at 20 ⁇ l per tube (lOng/ml final concentration). The tubes are again mixed and incubated for an additional 4-6 hours at 37 °C.
  • Activity is stopped by adding 750 ⁇ l of RPMI to each tube, centrifuging and removing the cells. Supematants are collected and stored overnight at 4 °C. The supernatant samples are assayed for TNF release using immunoassay kits (available commercially from Biosource International, Camarillo, CA).

Abstract

On décrit des composés thérapeutiques dotés d'au moins une chaîne latérale substituée par un cycle et correspondant à la formule: FRACTION CENTRALE -- (R)j, où j est un nombre entier de un à trois, la fraction centrale est cyclique ou non, et R est sélectionné dans le groupe comprenant hydrogène, halogène, hydroxyle, amino, C(1-10) alkyle substitué ou non, C(2-10) alcényle, des groupes cycliques ou hétérocycliques et la formule (I), où n est un nombre entier de un à vingt, et m et p représentent indépendamment zéro ou un nombre entier de un à vingt. R1 est sélectionné dans le groupe comprenant hydrogène, halogène, hydroxide et C(1-10) alkyle, C(1-10) alcoxy, C(2-10) alcényle substitué ou non, et un groupe cyclique doté d'au moins un cycle à quatre à sept membres; R2 est sélectionné dans le groupe comprenant hydrogène, halogène, hydroxyle C(1-10) alkyle substitué ou non, C(1-10) alcoxy et C(2-10) alcényle; et R3 est sélectionné dans le groupe comprenant hydrogène ou un groupe cyclique, substitué ou non, doté d'au moins un cycle de quatre à sept membres. R1 ou R3 au moins représente le groupe cyclique et la somme n+m ou n+p, correspondant aux groupes cycliques R1 ou R3 respectivement, ne dépasse pas dix-neuf. Ces composés et leurs compositions pharmaceutiques se révèlent utiles dans les thérapies de maladies progressant par signalisation intracellulaire par des voies de signalisation intracellulaires spécifiques, en permettant la médiation d'une réponse de signalisation à un stimulus externe.
PCT/US1994/004007 1993-04-09 1994-04-11 Inhibiteurs de signalisation cellulaire a cycle substitue WO1994024133A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU67028/94A AU6702894A (en) 1993-04-09 1994-04-11 Ring-substituted cell signaling inhibitors

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US4539993A 1993-04-09 1993-04-09
US08/045,399 1993-04-09
US19232194A 1994-02-03 1994-02-03
US08/192,321 1994-02-03

Publications (1)

Publication Number Publication Date
WO1994024133A1 true WO1994024133A1 (fr) 1994-10-27

Family

ID=26722718

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/004007 WO1994024133A1 (fr) 1993-04-09 1994-04-11 Inhibiteurs de signalisation cellulaire a cycle substitue

Country Status (2)

Country Link
AU (1) AU6702894A (fr)
WO (1) WO1994024133A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6090816A (en) * 1994-12-13 2000-07-18 Euro-Celtique S.A. Aryl thioxanthines
WO2000061583A1 (fr) * 1995-06-07 2000-10-19 Cell Therapeutics, Inc. Derives de xanthine et analogues comme inhibiteurs de transmission des signaux intracellulaires et extracellulaires
US6248746B1 (en) 1998-01-07 2001-06-19 Euro-Celtique S.A. 3-(arylalkyl) xanthines
US6586429B2 (en) 2000-11-29 2003-07-01 Cell Therapeutics, Inc. Tricyclic fused xanthine compounds and their uses
US6774130B2 (en) 1998-01-16 2004-08-10 Cell Therapeutics, Inc. Therapeutic compounds for inhibiting interleukin-12 signaling and methods for using same
US6825180B2 (en) 2001-05-18 2004-11-30 Cell Therapeutics, Inc. Pyridopyrimidine compounds and their uses
US6878715B1 (en) 1994-02-18 2005-04-12 Cell Therapeutics, Inc. Therapeutic compounds for inhibiting interleukin-12 signals and method for using same
US7713982B2 (en) 2004-02-14 2010-05-11 Smithkline Beecham Corporation Xanthines with HM74A receptor activity
US8143264B2 (en) 2005-08-10 2012-03-27 Glaxosmithkline Llc Xanthine derivatives as selective HM74A agonists
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
WO2015072893A1 (fr) * 2013-11-14 2015-05-21 Общество С Ограниченной Ответственностью "Фарминтерпрайсез" Composition pharmaceutique contenant des dérivés de glutarimides et leur utilisation dans le traitement d'affections à éosinophiles
CN107614501A (zh) * 2015-05-20 2018-01-19 广东众生药业股份有限公司 羟基嘌呤类化合物及其应用
EP3950689A4 (fr) * 2019-03-27 2023-11-08 West China Hospital of Sichuan University Dérivé nucléosidique destiné à la prévention et au traitement d'une inflammation et son utilisation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995012396A1 (fr) * 1993-11-01 1995-05-11 Cell Therapeutics, Inc. Procede d'inhibition de replication virale intracellulaire

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4883801A (en) * 1984-09-07 1989-11-28 The General Hospital Corporation Xanthine derivative pest control agents

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4883801A (en) * 1984-09-07 1989-11-28 The General Hospital Corporation Xanthine derivative pest control agents

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. PHARMACOLOGY, Vol. 28, issued 1979, CROCE et al., "Inhibition of Alkaline Phosphatase by Substituted Xanthines", pages 1227-1231. *
J. HETEROCYCLIC CHEMISTRY, Vol. 28, issued Aug-Sept. 1991, GANJIAN et al., "Synthesis and Cytotoxic Activity of 2-Dialkylamino-Alkyl-1,3-Dihydropyrrolo[3,4-cÜQuinoline-1,3-Diones and 6-(2-Dimethylaminoethyl)-1H-DibenzÄc,eÜAzepine-5,7-Dione", pages 1173-1175. *
J. MED. CHEM., Vol. 35, issued 1993, MULLER et al., "Synthesis of Paraxanthine Analogs (1,7-Disubstituted Xanthines) and Other Xanthines Unsubstituted at the 3-Position: Structure-Activity Relationships at Adenosine Receptors", pages 3341-3349. *
J. MEDICINAL CHEM., Volume 15, No. 5, issued 1972, SENDA et al., "Pyrimidine Derivatives and Related Compounds 15. Synthesis and Analgetic and Antiinflammatory Activities of 1,3-Substituted 5-Amino-6-Methyluracil Derivatives", pages 471-476. *
J. ORG. CHEM., Vol. 25, issued April 1961, NAKANO et al., "Structure of Julocrotine", pages 1184-1191. *
J. PHARM. PHARMACOLOGY, Vol. 41, issued 1989, SEGURA et al., "The Influence of Structure on the Accumulation of Caffeine Induced by Methyl Xanthine Derivatives", pages 129-131. *
SYNTHESIS, No. 1, issued January 1993, MULLER et al., "General Synthesis and Properties of 1-Monosubstituted Xanthines", pages 125-128. *

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6878715B1 (en) 1994-02-18 2005-04-12 Cell Therapeutics, Inc. Therapeutic compounds for inhibiting interleukin-12 signals and method for using same
US6090816A (en) * 1994-12-13 2000-07-18 Euro-Celtique S.A. Aryl thioxanthines
WO2000061583A1 (fr) * 1995-06-07 2000-10-19 Cell Therapeutics, Inc. Derives de xanthine et analogues comme inhibiteurs de transmission des signaux intracellulaires et extracellulaires
US6248746B1 (en) 1998-01-07 2001-06-19 Euro-Celtique S.A. 3-(arylalkyl) xanthines
US6774130B2 (en) 1998-01-16 2004-08-10 Cell Therapeutics, Inc. Therapeutic compounds for inhibiting interleukin-12 signaling and methods for using same
US6586429B2 (en) 2000-11-29 2003-07-01 Cell Therapeutics, Inc. Tricyclic fused xanthine compounds and their uses
US7247630B2 (en) 2000-11-29 2007-07-24 Cell Therapeutics, Inc. Uses of tricyclic compounds
US6825180B2 (en) 2001-05-18 2004-11-30 Cell Therapeutics, Inc. Pyridopyrimidine compounds and their uses
US7713982B2 (en) 2004-02-14 2010-05-11 Smithkline Beecham Corporation Xanthines with HM74A receptor activity
US8268839B2 (en) 2004-02-14 2012-09-18 Glaxosmithkline Llc Compounds
US8394808B2 (en) 2004-02-14 2013-03-12 Glaxosmithkline Llc HM74 receptor agonists:xanthine derivatives, corresponding pharmaceutical compositions, treatment methods and processes
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
US8143264B2 (en) 2005-08-10 2012-03-27 Glaxosmithkline Llc Xanthine derivatives as selective HM74A agonists
EA032940B1 (ru) * 2013-11-14 2019-08-30 Общество С Ограниченной Ответственностью "Рсв Терапевтикс" Способ лечения эозинофильных заболеваний с помощью соединения производного глутаримидов
WO2015072893A1 (fr) * 2013-11-14 2015-05-21 Общество С Ограниченной Ответственностью "Фарминтерпрайсез" Composition pharmaceutique contenant des dérivés de glutarimides et leur utilisation dans le traitement d'affections à éosinophiles
JP2016540749A (ja) * 2013-11-14 2016-12-28 オブシェストヴォ・ス・オグラニチェンノイ・オトヴェトストヴェンノストジュ・“ファームエンタープライジーズ” グルタルイミド誘導体を含む医薬組成物、及び好酸球性疾患を処置するためのその適用
KR102312013B1 (ko) 2013-11-14 2021-10-15 오브셰스트보 에스 오그라니첸노이 오트베트스트벤노스티주 “파름엔터프라지즈” 글루타르이미드 유도체를 포함하는 약학적 조성물 및 호산구성 질병 치료에 있어서의 그 용도
US9949962B2 (en) 2013-11-14 2018-04-24 Obschestvo S Ogranichennoi Otvetstvennostiyu “Pharmenterprises” Pharmaceutical composition comprising glutarimide derivatives and use thereof in the treatment of eosinophilic diseases
AU2014349246B2 (en) * 2013-11-14 2018-10-18 “ChemImmune Therapeutics” Limited Liability Company Pharmaceutical composition containing glutarimide derivatives, and application thereof for treating eosinophilic diseases
EA030961B1 (ru) * 2013-11-14 2018-10-31 Общество С Ограниченной Ответственностью "Фарминтерпрайсез" Применение производных глутаримидов для лечения эозинофильных заболеваний
AU2014349246C1 (en) * 2013-11-14 2019-02-21 “ChemImmune Therapeutics” Limited Liability Company Pharmaceutical composition containing glutarimide derivatives, and application thereof for treating eosinophilic diseases
US10220029B2 (en) 2013-11-14 2019-03-05 Obschestvo S Ogranichennoi Otvetstvennostiyu “Pharmenterprises” Pharmaceutical composition comprising glutarimide derivatives and use thereof in the treatment of eosinophilic diseases
EP3466425A1 (fr) * 2013-11-14 2019-04-10 Obschestvo S Ogranichennoi Otvetstvennostiyu "Pharmenterprises" Composition pharmaceutique comprenant des dérivés de glutarimide et leur utilisation dans le traitement de maladies éosinophiles
EA032955B1 (ru) * 2013-11-14 2019-08-30 Общество С Ограниченной Ответственностью "Хемиммьюн Терапьютикс" Применение производного глутаримида для лечения эозинофильных заболеваний
KR20160078400A (ko) * 2013-11-14 2016-07-04 오브셰스트보 에스 오그라니첸노이 오트베트스트벤노스티주 “파름엔터프라지즈” 글루타르이미드 유도체를 포함하는 약학적 조성물 및 호산구성 질병 치료에 있어서의 그 용도
KR102312294B1 (ko) 2013-11-14 2021-10-15 오브셰스트보 에스 오그라니첸노이 오트베트스트벤노스티주 “파름엔터프라지즈” 글루타르이미드 유도체를 포함하는 약학적 조성물 및 호산구성 질병 치료에 있어서의 그 용도
EA032955B3 (ru) * 2013-11-14 2020-02-27 Общество С Ограниченной Ответственностью "Хемиммьюн Терапьютикс" Применение производного глутаримида для лечения эозинофильных заболеваний
AU2018264084B2 (en) * 2013-11-14 2020-05-14 Obschestvo S Ogranichennoi Otvetstvennostiyu "Pharmenterprises" Pharmaceutical composition comprising glutarimide derivatives, and application thereof for treating eosinophilic diseases
KR20200053639A (ko) * 2013-11-14 2020-05-18 오브셰스트보 에스 오그라니첸노이 오트베트스트벤노스티주 “파름엔터프라지즈” 글루타르이미드 유도체를 포함하는 약학적 조성물 및 호산구성 질병 치료에 있어서의 그 용도
AU2018264084C1 (en) * 2013-11-14 2020-08-27 Obschestvo S Ogranichennoi Otvetstvennostiyu "Pharmenterprises" Pharmaceutical composition comprising glutarimide derivatives, and application thereof for treating eosinophilic diseases
EA037447B1 (ru) * 2013-11-14 2021-03-30 Общество С Ограниченной Ответственностью "Атд Терапевтикс" Способ лечения эозинофильных заболеваний
CN107614501B (zh) * 2015-05-20 2020-01-14 广东众生睿创生物科技有限公司 羟基嘌呤类化合物及其应用
CN107614501A (zh) * 2015-05-20 2018-01-19 广东众生药业股份有限公司 羟基嘌呤类化合物及其应用
EP3950689A4 (fr) * 2019-03-27 2023-11-08 West China Hospital of Sichuan University Dérivé nucléosidique destiné à la prévention et au traitement d'une inflammation et son utilisation

Also Published As

Publication number Publication date
AU6702894A (en) 1994-11-08

Similar Documents

Publication Publication Date Title
US6043250A (en) Methods for using therapeutic compounds containing xanthinyl
US5670506A (en) Halogen, isothiocyanate or azide substituted xanthines
US5804584A (en) Therapeutic compounds containing a monocyclic five- to six- membered ring structure having one to two nitrogen atoms
US5777115A (en) Acetal-and ketal-substituted pyrimidine compounds
US5807861A (en) Amine substituted xanthinyl compounds
US5837703A (en) Amino-alcohol substituted cyclic compounds
US5889011A (en) Substituted amino alkyl compounds
US5770595A (en) Oxime substituted therapeutic compounds
US5801182A (en) Amine substituted compounds
CA2117377C (fr) Composes aminoalkyl substitues
EP0669825A1 (fr) Composes a teneur hydroxyle
CA2159640C (fr) Inhibiteurs de messages cellulaires specifiques
WO1994024133A1 (fr) Inhibiteurs de signalisation cellulaire a cycle substitue
WO1995020589A1 (fr) Inhibiteurs de la signalisation cellulaire
WO1994022863A9 (fr) Inhibiteurs de la signalisation cellulaire vehiculee par des messagers secondaires
US6020337A (en) Electronegative-substituted long chain xanthine compounds
US5866576A (en) Epoxide-containing compounds
US6103730A (en) Amine substituted compounds
US5521315A (en) Olefin substituted long chain compounds
AU737005B2 (en) Second messenger cell signalling inhibitors

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA