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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
• • A—HUMAN NECESSITIES
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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
  • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
  • C07H5/06—Aminosugars

Definitions

• the field of the invention is pharmaceutical chemistry.
• K-252a inhibits protein kinase C (PKC) , which plays a role in regulating cell functions.
• K-252a has various activities, e.g., inhibiting smooth muscle contraction ⁇ Jap . J. Pharmacol . 43 (suppl.): 284, 1987), inhibiting serotonin secretion (Biochem . Biophys . Res . Commun . 144: 35, 1987), inhibiting elongation of neuraxone (J " . Neurosci . 8:715, 1988), inhibiting histamine release (Allergy 43:100, 1988), inhibiting smooth muscle MLCK (J. Biol . Chem. 263:6215,
• R s or R 6 is hydrogen or lower alkyl, and the other is hydrogen, lower alkyl, acyl, aryl, heterocyclic, carbamoyl or lower alkylaminocarbonyl ; -NR 5 R 6 ;
• R 7 is lower alkyl or alkylene
• R 8 wherein j is 1-6, and R 8 is halogen; substituted aryl; unsubstituted aryl; substituted heteroaryl; unsubstituted heteroaryl ; N 3 ;
• R 9 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl ;
• -CH N-R 32 , wherein R 32 is hydroxyl, lower alkoxy, amino, guanidino, ureido, imidazolylamino, carbamoylamino, or NR 26A R 27A (wherein R 26A is the same as R 26 and R 27A is the same as R 27 ) ; or
• V represents hydrogen, methyl, ethyl, benzyl, acetyl, or trifluoroacetyl
• R 1 and R 2 are H.
• R 3 is hydrogen or a protecting group. Particularly preferred are Compounds VI, VII, VIII, X, XII, XIV, XV, XVI, XVII, XVIII, XIX, XXV, and XXVII, shown below:
• 3 ' -epimeric K252a derivatives are formulated into pharmaceutical compositions .
• the invention also provides a method for inhibiting the activity of a tyrosine kinase, for example, protein kinase C (PKC) .
• PKC protein kinase C
• the method includes contacting the tyrosine kinase with a compound of claim 1.
• the tyrosine kinase can be in vivo or in vitro.
• the invention also provides a method for inhibiting the phosphorylation of a tyrosine kinase by a second kinase.
• the method includes contacting the second kinase with a compound of claim 1.
• the tyrosine kinase can be in vivo or in vi tro.
• the invention also provides a method for enhancing the function of a cholinergic neuron. The method includes contacting the cholinergic neuron with a compound of claim 1. The cholinergic neuron can be in vivo or in vi tro .
• the invention also provides a method for enhancing the survival of a cholinergic neuron. The method includes contacting the cholinergic neuron with a compound of claim 1. The cholinergic neuron can be in vivo or in vi tro. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
• Fig. 1 is a schematic drawing of the synthesis of 3'-epimeric indolocarbazoles VI and VII from the known indolocarbazole Ilia.
• Fig. 2 is a drawing showing the synthesis of the
• Fig. 3 is a drawing showi;ng the synthesis of 3'-epimeric indolocabazoles Xlla, XIV, XV, XVI, XVII, and XVIII from the intermediate ketone XIa.
• Fig. 4 is a drawing showing the synthesis of 3'-epimeric indolocarbazoles XIX from the intermediate epoxide Xa .
• Fig. 5 is a drawing showing an alternate synthesis of the 3'-epimeric indolocarbazoles XIX from the intermediate ketone XI .
• Fig. 6 is a drawing showing the synthesis of the epimeric 3 ' -hydroxyindolocarbazole XXVII from the intermediate ketone XI, and alternatively by epimerization of the known 3 ' -hydroxyindolocarbazole XXVI.
• Fig. 7 is a drawing showing the synthesis of a ring- brominated 3 '-epimeric indolocarbazole XXV from the corresponding 3 '-epimeric indolocarbazole XIV.
• Fig. 8 is a graph summarizing data from experiments to determine the effect of Compound XIV on survival of neurons in cultures enriched for motoneurons .
• Cell viability (as percent of control) is plotted against concentration of Compound XIV in the cell culture medium.
• Epimeric K-252a derivatives of the invention display pharmacological activities, including inhibition of tyrosine kinase activity, e.g., inhibition of PKC or trk tyrosine kinase, which inhibition may be useful in treatment of diseases, including cancer.
• Compounds of the invention are useful for enhancing the function and/or survival of trophic factor responsive cells, e.g., cholinergic neurons. Effects on neurons can be demonstrated in assays including the following: (1) cultured spinal cord choline acetyl- transferase ("ChAT”) assay; and (2) cultured basal forebrain neuron (“BFN”) ChAT activity assay.
• ChAT cultured spinal cord choline acetyl- transferase
• BFN cultured basal forebrain neuron
• ChAT is an enzyme in the biosynthetic pathway leading to acetylcholine . ChAT activity associated with a cholinergic neuron indicates that the neuron is functional. Neuron survival can be assayed by measuring uptake and enzymatic conversion of a dye, e.g., calcein AM, by neurons.
• a dye e.g., calcein AM
• Various neurological disorders are characterized by neuronal cells that are injured, functionally comprised, undergoing axonal degeneration, dying, or at risk of dying.
• disorders include: Alzheimer's disease, motor neuron disorders such as amyotrophic lateral sclerosis, Parkinson's disease; cerebrovascular disorders such as stroke or ischaemia, Huntington's disease, AIDS dementia, epilepsy, multiple sclerosis, peripheral neuropathies, disorders induced by excitatory amino acids, and disorders associated with concussive or penetrating injuries of the brain or spinal cord.
• compounds of the invention can be used as therapeutic agents to enhance the function and/or survival of cells of neuronal lineage in a mammal, e.g., a human. In particular, they are useful in treatment of disorders associated with decreased ChAT activity or injury to spinal cord motoneurons.
• K-252a can be protected on the lactam amide nitrogen, e.g., as an acetate or as a silyl derivative.
• Thionocarbonate IVa (Fig. 1) can be prepared from diol Ilia using a procedure such as that described in U.S. Pat. No. 4,923,986. Treatment of IVa with trimethylphosphite gives the exocyclic alkene V. Alkene V can be converted to (S) -methanol derivative VI using hydroboration conditions, or converted to (R) -diol VII using
• the (R) -epoxide IXa (Fig. 2) can be prepared by converting (R) -diol VII to the tosyl intermediate VIII, followed by treatment with a base such as sodium hydride or sodium hydroxide.
• Halide or sulfonate derivative of, for example, compounds VI or VII can be displaced with various 0, S, N, or C nucleophiles to yield a suitable derivative.
• the preparation of 3 ' - (R) -K-252a XIV (Fig. 3) begins with ketone XI.
• Compound XIV differs from the natural K- 252a isomer only at the 3' sugar position.
• Treatment of ketone Xla with cyanide salts NaCN, KCN, tetrabutylammonium cyanide, or TMSCN gives a mixture of cyanohydrins XII and XIII.
• the mixture of cyanohydrin isomers can be separated by chromatography or directly converted to ester XIV or amide XV using HC1 in methanol.
• 3 ' -epi-K-252a XIV can be hydrolysed to the hydroxy acid XVI using a procedure such as that used for natural K-252a. See, e.g., J. A ⁇ tijbiot.39:1072, 1986.
• Acid XVI can be converted to a variety of ester or amide derivatives using similar procedures to those described for K-252a. See, e.g., U.S. Pat. Nos. 4,923,986; 5,461,146; and 5,654,427.
• Amide XV can be reduced to the corresponding methylamine derivative XVII using the procedure described for conversion of natural K- 252a, and XVII can be used to prepare a number of methyl- amide and -urea derivatives. See, e.g., U.S. Pat. Nos.
• 3 ' -epi-K-252a can be reduced to the aldehyde XVIII and condensed with various amines, hydrazines, or hydroxyl amines to form the corresponding analogs .
• the aldehyde XVIII may be treated with various metal alkyl, arylalkyl, aryl, or heteroarylalkyl reagents, e.g., Li, Mg, Zn or Cu reagents, to form the corresponding alcohol addition products.
• Aldehyde XVIII may be converted to functionalized olefins and their reduced products by treatment with phosphonium ylides (Quart J_ev. 17:406, 1963; Angew Int . 16:423, 1977), phosphonates (Horner-Wadsworth-Emmons reagents: Chem. Ber. 91:61, 1958; J. Am . Chem . Soc . 83:1733, 1961; Org . React . 25:73, 1977), silanes (J. Orgr. chem. 33:780, 1968; Synthesis 384, 1984) tellurium reagents (Tetrahedron Lett.
• alkene derived from aldehyde XVIII can be converted to an epoxide and treated, for example, with a nucleophile, as described for epoxide IX.
• Epoxide IX (Fig. 4) can be treated with a variety of nucleophiles to form tertiary alcohols of structure XIX. The nucleophile can be substituted.
• epoxides and tertiary 3' - epi-OH configurations of the alcohols is to convert ketone XI to an olefin of structure XX using a conventional olefination reaction, e.g., as described for aldehyde XVIII.
• the epoxide of structure XXI can be prepared asymmetrically using known methods. See, e.g., J. Org. Chem . 32:1363, Synthesis 89, 1986; 1967; J. Org. Chem . 60:3692, 1995; J. Am. Chem. Soc . 112:2801, 1990; J " . Am. Chem. Soc . 116:6937, 1994; J.
• the known (R) -alcohol XXVI (Fig. 6) can be converted to (S) -alcohol XXVII using conventional methods for inversion of a secondary alcohol. See, e.g., Tetrahedron Lett . 34:6145, 1996; Synthesis Letters, 1995, 336).
• XXVII can be prepared by treatment of epoxide XXIV with a hydride reagent such as lithium triethyl borohydride.
• Ketone XI (Fig. 6) can be converted to triflate XXII followed by treatment with tributyltin hydride to give alkene XXIII.
• Oxidation of (S) -methanol VI to an aldehyde or a carboxylic acid derivative can be achieved using appropriate oxidizing reagents (as described in Oxidations in Organic Chemistry, American Chemical Society Monograph 186, ACS Washington DC 1990) .
• the aldehyde or carboxylic acid derivatives can be further transformed using described procedures to prepare derivatives XVI and XVIII (Fig. 3) .
• a compound of the invention can be administered to a mammal, e.g., a human patient, as the sole active ingredient or in combination with other therapeutic agents.
• Compounds of the invention can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable excipients and carriers. Such compositions can be formulated for any route of administration, e.g., parenteral, oral, nasal, or topical.
• the composition can be administered in unit dosage form, following preparation by conventional methods. See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA) .
• the amount and concentration of the active ingredient can vary. The concentration will depend upon factors such as the total dosage of the active ingredient, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, the route of administration, the patient's age, the patient's weight, and the condition being treated.
• Compounds of the invention can be provided in an physiological buffer solution containing, e.g., 0.1 to 10% w/v compound for parenteral administration.
• Typical dose ranges are from about 1 ⁇ g/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day, and preferably about 0.1 to 20 mg/kg once to four times per day.
• the invention includes pharmaceutically acceptable salts of 3 ' -epimeric K252a derivatives.
• Pharmaceutically acceptable salts include pharmaceutically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts.
• Acid addition salts include inorganic acid addition salts such as hydrochloride, sulfate and phosphate, and organic acid addition salts such as acetate, maleate, fumarate, tartrate, citrate and lactate.
• metal salts are alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt.
• Examples of ammonium salts are ammonium salt and tetramethylammonium salt.
• organic amine addition salts are salts with morpholine and piperidine.
• amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine.
• lower alkyl means an alkyl group with 1 to 6 carbons.
• aryl (alone or in terms such as arylcarbonyl and arylaminocarbonyl) means a group having 6 to 12 carbon atoms, in a single ring, or two fused rings. Examples of aryl groups are phenyl, biphenyl and naphthyl .
• a heteroaryl group contains at least one hetero atom. Preferably, the hetero atom is 0, S, or N.
• heteroaryl groups are pyridyl, pyrimidyl, pyrrolyl, furyl, thienyl, imidazolyl, triazolyl, tetrazolyl, quinolyl , isoquinolyl, benzoimidazolyl, thiazolyl and benzothiazolyl .
• a substituted alkyl group has 1 to 3 independently-selected substituents .
• Preferred substituents for alkyl groups are hydroxy, lower alkoxy, substituted or unsubstituted arylalkoxy-lower alkoxy, substituted or unsubstituted heteroarylalkoxy-lower alkoxy, halogen, carboxyl, lower alkoxycarbonyl, nitro, amino, mono- or di- lower alkylamino, dioxolane, dioxane, dithiolane, and dithione.
• a substituted aryl, heteroaryl or arylalkyl group has 1 to 3 independently-selected substituents.
• Preferred substituents are lower alkyl, hydroxy, lower alkoxy, carboxy, lower alkoxycarbonyl, nitro, amino, mono- or di- lower alkylamino, and halogen.
• cholinergic neuron means a neuron that uses acetylcholine as a neurotransmitter .
• Examples of cholinergic neurons are basal forebrain neurons, striatal neurons, and spinal cord neurons.
• sensor neuron means a neuron responsive to an environmental stimulus such as temperature or movement . Sensory neurons are found in structures including skin, muscle and joints. A dorsal root ganglion neuron is an example of a sensory neuron.
• trophic factor-responsive cell means a cell to which a trophic factor binds. Trophic factor-responsive cells include cholinergic neurons, sensory neurons, monocytes and neoplastic cells.
• Epimeric K252a derivatives were tested for inhibition of kinase activity of baculovirus-expressed human trkA cytoplasmic domain using an ELISA-based assay as described by Angeles et al . (Anal. Biochem. 236:49-55, 1996) .
• a 96-well microtiter plate was coated with substrate solution (recombinant human phospholipase C- ⁇ /glutathione S-transferase fusion protein; Rotin et al . , EMBO J. , 11:559- 567, 1992) .
• Inhibition was measured in 100 ml assay mixtures containing 50 mM Hepes, pH 7.4, 40 ⁇ M ATP, 10 mM MnCl 2 , 0.1% BSA, 2% DMSO, and various concentrations of inhibitor. The reaction was initiated by addition of trkA kinase and allowed to proceed for 15 minutes at 37°C. An antibody to phosphotyrosine (UBI) was then added, followed by a secondary enzyme-conjugated antibody, alkaline phosphatase-labelled goat anti-mouse IgG (Bio-Rad) . The activity of the bound enzyme was measured using an amplified detection system (Gibco-BRL) . Inhibition data were analyzed using the sigmoidal dose-response (variable slope) equation in GraphPad Prism. The concentration that gave 50% inhibition of kinase activity was referred to as IC 50 . Results are summarized in Table 1.
• NGF-stimulated phosphorylation of trk by selected epimeric K-252a derivatives was measured using a procedure modified from that described in U.S. Pat. No. 5,516,771.
• NIH3T3 cells transfected with trkA were grown in 100 mm dishes. Subconfluent cells were serum- starved by replacing media with serum-free 0.05% BSA-DMEM containing compound (1-100 nM) or DMSO (added to controls) for one hour at 37°C. NGF (Harlan/Bioproducts for Science) was then added to the cells at a concentration of 10 ng/ml for 5 minutes. Cells were lysed in buffer containing detergent and protease inhibitors.
• Clarified cell lysates were normalized to protein using BCA method and immunoprecipitated with anti-trk antibody.
• Polyclonal anti-trk antibody was prepared against a peptide corresponding to the 14 amino acids at the carboxy terminus of trk (Martin-Zanca et al . , Aol . Cell . Biol 9:24- 33, 1989) .
• the immune complexes were collected on Protein A Sepharose beads (Sigma) , separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) , and transferred to a polyvinylidene difluoride (PVDF) membrane.
• PVDF polyvinylidene difluoride
• the membrane was immunoblotted with anti-phosphotyrosine antibody (UBI) , followed by incubation with horseradish peroxidase coupled goat anti-mouse IgG (Bio-Rad) . Phosphorylated proteins were visualized using ECL (Amersham) .
• the area of the trk protein band was measured and compared to NGF-stimulated control.
• Compound X showed a complete inhibition of trkA at ⁇ 50 nM, and an IC S0 of ⁇ 10 nM in cells.
• the natural isomer XXIX did not show complete inhibition at 100 nM.
• Diol VII displayed greater potency than the natural isomer III for trkA inhibition in NIH3T3 cells.
• 3 ' -Epimeric K-252a derivatives were tested for inhibition of the kinase activity of baculovirus-expressed VEGF receptor kinase domain, using the procedure described above.
• the kinase reaction mixture consisting of 50 mM Hepes, pH 7.4, 40 ⁇ M ATP, 10 mM MnCl 2 , 0.1% BSA, 2% DMSO, and various concentrations of inhibitor, was transferred to PLC- ⁇ /GST-coated plates.
• VEGFR kinase was added and the reaction was allowed to proceed for 15 min. at 37°C.
• Phosphorylated product was detected by anti-phosphotyrosine antibody (UBI) .
• UBI anti-phosphotyrosine antibody
• a secondary enzyme-conjugated antibody was used to capture the antibody-phosphorylated PLC- ⁇ /GST complex.
• the activity of the bound enzyme was measured by an amplified detection system (Gibco-BRL) .
• Inhibition data were analyzed using the sigmoidal dose-response (variable slope) equation in GraphPad Prism. Results are summarized in Table 3.
• Protein kinase C activity was measured using the Millipore Multiscreen TCA in-plate assay (Pitt et al . , J.
• ChAT was employed a biochemical marker for functional cholinergic neurons. ChAT activity has been used to study the effects of neurotrophins (e.g., NGF or NT-3) on the survival and/or function of cholinergic neurons. The ChAT assay also has been used as an indication of the regulation of ChAT levels within cholinergic neurons. 3 '-Epimeric K-252a derivatives increased ChAT activity in the dissociated rat embryonic spinal cord culture assay (Table 5) . Compound XVII increased ChAT activity 195% over control cultures (not treated with the epimeric K-252a derivative) after allowing a 2-3 hour plating period for cells to attach to control tissue culture wells.
• neurotrophins e.g., NGF or NT-3
• the ChAT assay also has been used as an indication of the regulation of ChAT levels within cholinergic neurons.
• 3 '-Epimeric K-252a derivatives increased ChAT activity in the dissociated rat embryonic spinal cord culture assay (Table
• Fetal rat spinal cord cells were dissociated, and experiments were performed, essentially as described by Smith et al . , J “ . Cell Biology 101:1608-1621, 1985), and Glicksman et al . , J. Neurochem . 61:210-221, 1993).
• Dissociated cells were prepared from spinal cords dissected from rats (embryonic day 14-15) by conventional trypsin dissociation techniques. Cells were plated at 6 x 10 s cells/cm 2 on poly-1-ornithine coated plastic tissue culture wells in serum-free N2 medium supplemented with 0.05% bovine serum albumin (BSA) (Bottenstein et al . , Proc . Natl . Acad. Sci .
• BSA bovine serum albumin
• calcein AM Molecular Probes, Eugene, OR
• fluorimetric viability assay Bozyczko-Coyne et.al., J. Neurosci . Meth . 50:205-216, 1993
• Culture medium was serially diluted in Dulbeccos phosphate buffered saline (DPBS) .
• DPBS Dulbeccos phosphate buffered saline
• reaction mixture was extracted with EtOAc (2 x 20 mL) , dried over sodium sulfate, filtered and concentrated in vacuo to give a light brown film.
• the mixture was purified by flash chromatography on silica gel using ethyl acetate to yield compound Vila (TBDMS-VII) as a yellow solid (280 mg, 76% yield).
• reaction mixture was diluted with ethyl acetate (30 mL) and washed with water (3 x 15 mL) .
• the organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo to yield the tosyl intermediate Villa as a yellow film.
• the reaction mixture was further purified by flash chromatography on silica gel using hexane: ethyl acetate (2:1) to yield a light yellow film (0.16 g, 55% yield).
• Compound XXIII displayed the following IC S0 values in the assays described above: inhibition of trkA kinase, 4 nM; inhibition of VEGF receptor kinase, 25 nM, and inhibitiion of Protein Kinase C, >1000 nM.

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