MXPA00006500A - 3'-epimeric k-252a derivatives - Google Patents

Abstract

Compounds defined by general structure (II) are disclosed. These compounds display pharmacological activities, including inhibition of tyrosine kinase activity and enhancement of the function and/or survival of trophic factor responsive cells, e.g., cholinergic neurons.

Description

DERIVATIVES OF K-2S2A 3'-EPIMERICOS This application claims the benefit of the provisional application Series No. 60 / 070,263, filed on December 31, 1997.

FIELD OF THE INVENTION The field of the invention on pharmaceutical chemistry.

BACKGROUND OF THE INVENTION K252a is an indolocarbazole whose stereochemistry is shown below (Formula I): K-252a inhibits protein C kinase (PKC), which plays an important role in the regulation of cellular functions. K-252a has several activities, that of inhibiting the contraction of smooth muscle (Jap.

J. Pharmacol. 43 (suppl.): 284, 1987), the inhibition of serotonin secretion (Biochem. Biophys., Res. Commun. 144: 35, 1987), the inhibition of elongation of neuraxone (J. Neurosci. S: 715, 1988) , inhibition of histamine release (Allergy 43: 100, 1988), inhibition of smooth muscle MLCK (J. Biol. Chem. 263: 6215, 1988), anti-inflammatory action (Acta Physiol. Hung. 80: 423, 1992 ), and promotion of cell survival (J. Neurochem, 64: 1502, 1995). K-252a also inhibits the production of I L-2 (Exper. Cell Res. 193: 175-182, 1991). The total synthesis of the natural (+) isomer of K252a and its enantiomeric isomer (-) (the three chiral carbons of the invert sugar portion) has been achieved (Wood et al., J Am. Soc. 17: 10413, 1995, and WO 97/07081).

COMPENDIUM OF THE INVENTION It has been found that certain 3'-epimeric derivatives of K-252a are biologically active. These compounds have the following general formula (Formula I I): p where: R1 and R2 independently are: Hydrogen; lower alkyl; halogen; acyl; nitro; sulfonic acid; -CH = NR4, wherein R4 is guanidino, heterocyclic or -NR5R6, wherein R5 or R6 is hydrogen or inner alkyl, and the other is hydrogen, lower alkyl, acyl, aryl, heterocyclic, carbamoyl or lower alkylaminocarbonyl; -NR5R6; -CH (SR7) 2, wherein R7 is lower alkyl or alkylene; - (CH2) jR8, wherein j is 1 -6, and R8 is halogen; substituted aryl; unsubstituted aryl; substituted heteroaryl; unsubstituted heteroaryl; N3; -CO2R9, wherein R9 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl; -C (= O) N R 10 R 1 1, wherein R 10 and R 1 1 independently are hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted aralkyl, unsubstituted aralkyl, lower alkylaminocarbonyl, or lower alkoxycarbonyl, or R 10 and R 11 combine with a nitrogen atom to form a heterocyclic group; -OR12, wherein R12 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted alkyl, unsubstituted aryl; or -C (= O) R13, wherein R13 is hydrogen, NR10C1 1, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted aralkyl, or unsubstituted aralkyl; -NR10R11; -C (= O) R 14, wherein R 14 is hydrogen, lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl or unsubstituted heteroaryl; -S (= O) rR15, wherein r is from oa to 2, and R15 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted aralkyl, unsubstituted aralkyl , thiazolinyl, (CH2) aCO2R16, wherein a is 1 or 2, and R16 is hydrogen or lower alkyl, or - (CH2) aC (= O) NR10R11); -OR17, wherein R17 is hydrogen, lower alkyl or -C (= O) R18, wherein R18 is substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, or unsubstituted aryl; -C (= O) (CH2) jR19, wherein R19 is hydrogen, halogen, NR10R11, N3, SR15, or OR20, wherein R20 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, or C (= O) R14; -CH (OH) (CH2) jR19; - (CH2) dCHR21CO2R16A, where d is 0-5, and R21 is hydrogen, CONR10R11, or CO2R16A, wherein R16A is equal to R16; - (CH2) dCHR21CONR10R11; -CH = CH (CH2) mR22, wherein m is 0-4, and R22 is hydrogen, lower alkyl, CO2R9, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, OR12, or NR10R11; -CH = C (CO2R16A) 2; -C = C (C H2) mR22; -SO2N R23R24, wherein R23 and R24 independently are hydrogen, lower alkyl, or groups that form a heterocycle with the adjacent nitrogen atoms; -OCO2R13A, wherein R13A is equal to R13; or -OC (= O) N R 10 R 1 1; R3 is hydrogen; lower alkyl; carbamoyl; Not me; tetrahydropyranyl; hydroxyl; C (= O) H; aralkyl; lower alkanoyl; or C H2CH2R25, wherein R25 is halogen, amino, di-lower alkylamino, hydroxy or lower alkylamino substituted with hydroxy; X is hydrogen; formyl; carboxyl; lower alkoxycarbonyl; lower alkylhydrazinocarbonyl; -CN; lower alkyl; -C (= O) N R 26 R 27, wherein R 26 and R 27 independently are hydrogen, unsubstituted lower alkyl or unsubstituted aryl; or R26 and R27 are combined with a nitrogen atom to form a heterocyclic group; -C H (R34) W, wherein R34 is hydrogen or lower alkyl, and W is -N = CH N (alkyl) 2; guanidino; N3; N R28R29, wherein R28 or R29 is hydrogen or lower alkyl, and the other is hydrogen, allyl, alkanoyl, aryloxycarbonyl, unsubstituted alkyl, or the residue of an a-amino wherein the hydroxy group of the carboxyl group is excluded; -CO2R9; -C (= O) N R 10 R 1 1; -S (= O) rR30, wherein R30 is substituted or unsubstituted lower alkyl, aryl, or heteroapio; or -OR31, wherein R31 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkanoyl; -CH = N-R32, wherein R32 is hydroxyl, lower alkoxy, amino, guanidino, ureido, imidazolylamino, carbamoylamino, or NR26AR27A (wherein R26A is equal to R26 and R27A is equal to R27); or -CH2Q is a sugar residue represented by wherein V represents hydrogen, methyl, ethyl, benzyl, acetyl or trifluoroacetyl; And it's hydrogen; -OH; -OC (= O) R33, wherein R33 is alkyl, aryl or amino; -OCH2O-alkyl, -O-alkyl; aralkyloxy; or X and Y combine as -X-Y- to form -CH2OCO2- or -CH2N (R16B) CO2-, (wherein R16B is equal to R16); A1 and A2 are hydrogen, or both combine to present O; or B1 and B2 are hydrogen, or both combine to represent O; or a pharmaceutically acceptable salt thereof; provided that at least one of A1, A2 or B1, B2, represents O; and also with the proviso that both X and Y simultaneously are not hydrogen. Preferably, it is -C (= O) NR ^ R 'carboxyl, lower alkoxycarbonyl, formyl, lower alkyl, -CH2OR31, -CH2N R28R29, or -CH2S (O) rR30. Preferably, R1 and R2 are H. Preferably, R3 is hydrogen or a protecting group. Particularly preferred are compounds VI, VII, VIII, X, XII, XIV, XV, XVI, XVII, XVIII, XIX, XXV, and XXVII, which are shown below: vi vp v XV xvi XD W - OH,. O, S, NH, or * ** W - H R * 1 - H or lower alkyl R 4 I - lower alkyl In some embodiments of the invention, the 3'-epimeric K252a derivatives are formulated into pharmaceutical compositions.

The invention also provides a method for inhibiting tyrosine kinase activity, for example, protein C kinase (PKC). 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 through 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 vitro. The invention also provides a method for improving the function of a cholinergic neuron. The method includes contacting the cholinergic neuron with a compound of claim 1. The cholinergic neuron may be in vivo or in vitro. The invention also provides a method for improving the survival of a cholinergic neuron. The method includes contacting the cholinergic neuron with a compound of claim 1. The cholinergic neuron may be in vivo or in vitro. Unless otherwise defined, all the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In case of conflict, the present application, including definitions, will control it. All publications patent applications, patents and other references mentioned herein are incorporated herein by reference. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other aspects and advantages of the invention will be apparent from the detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic drawing of the synthesis of indolocarbazoles VI and Vil 3'-epimeric from known indolocarbazole Illa. Figure 2 is a drawing showing the synthesis of the X 3'-epimeric indolocarbazole starting from the indigocarbazole Vi l 3'-epimeric, prepared as shown in Figure 1. Figure 3 is a drawing showing the synthesis of indolocarbazoles XI, XIV, XV, XVI, XVII, and XVII I 3'-epimeric from the intermediate ketone Xla. Figure 4 is a drawing showing the synthesis of 3'-epimeric indolocarbazoles XIX from the intermediate epoxide IXa. Figure 5 is a drawing showing an alternating synthesis of the 3'-epimeric indolocarbazoles XIX from the intermediate ketone XI. Figure 6 is a drawing showing the synthesis of the 3'-hydroxyindolocarbazoles epimeric XXVII from the intermediate ketone XI, and alternatively through the epimerization of 3'-hydroxyindolocarbazole XVI known. Figure 7 is a drawing showing the synthesis of an indolocarbazole XXV 3'-epimeric brominated ring from the corresponding XIV 3'-epimeric indolocarbazole. Figure 8 is a graph that summarizes the data from experiments to determine the effect of compound XIV on the survival of neurons in cultures rich in motor neurons. The availability of cells (as a percentage of control) was plotted against the concentration of compound XIV in the cell culture medium. The epimeric derivatives of K-252a of the invention exhibit pharmacological activities, including the inhibition of tyrosine kinase activity, for example, the inhibition of PKC or tyrosine kinase, trk, said inhibition may be useful in the treatment of diseases , including cancer. The compounds of the invention are useful for improving the function and / or survival of trophic factor sensitive cells, for example, cholinergic neurons. Effects on neurons can be demonstrated in trials including the following: (1) choline acetyltransferase assay of cultured spinal cord ("ChAT"); and (2) assay of cultured basal forebrain neqrona ChAT activity ("BFN"). ChAT is an enzyme in the biosynthetic path that leads to acetylcholine. The activity of ChAT associated with a cholinergic neuron indicates that the neuron is functional. The survival of neurons can be analyzed by measuring the consumption and enzymatic conversion of a dye, for example, calcein AM, through neurons. Several neurological disorders are characterized through neuronal cells that are damaged, functionally compromised, that undergo axonal degeneration, death, or are in danger of death. These disorders include: Alzheimer's disease, motor neuron disorders such as trophic lateral sclerosis, Parkinson's disease; cerebrovascular disorders such as shock or ischemia, Huntington's disease, dementia for SI DA, epilepsy, multiple sclerosis, peripheral neuropathies, disorders induced by exciting amino acids, and disorders associated with damage by concussion or penetration of the brain or spinal cord. Since these enhance the activities induced by the trophic factor of trophic factor sensitive cells, the compounds of the invention can be used as therapeutic agents to improve the function and / or survival of neuronal lineage cells in a mammal, for example, a human being. In particular, they are useful for the treatment of disorders associated with reduced ChAT activity or spinal cord motor neuron damage.

Chemical Synthesis The compounds of the invention can be prepared as described below (Figures 1-7). The compounds can be prepared starting with a suitably protected K-252a derivative. The K-252a derivative can be protected on the lactam amide nitrogen, for example, as an acetate or as a silyl derivative. Thionocarbonate IVa (Figure 1) can be prepared from the diol I using a process such as that described in the U.S. patent. A. No. 4,923,986. The treatment of IVa with trimethylphosphite gives the exocyclic alkene V. The alkene V can be converted to the derivative (S) -methanol VI using hydroboration conditions, or converted to (R) -diol VII using osmium tetraoxide in tetrahydrofuran (TH F) . In the compound VI, the configuration at the 3 'position of the sugar is opposite to that reported as (S) -diol l l in the patent of E. U. A. No. 4,923,986. The (R) -ephoxide IX (Figure 2) can be prepared by converting (R) -diol VI to the tosyl intermediate VI I I, followed by treatment with a base such as sodium hydride or sodium hydroxide. Treatment of (R) -ephoxide IXa with hydride reducing agents such as triethyl borohydride provides the (S) -tertiary alcohol X after deprotection of the t-butyldimethylsilyl group (TBDMS). The chiral alcohol derivatives such as the compounds VI, Vi I or X can be further converted to ether derivatives through reaction with a base and a halide or tosyl standard using conventional techniques. The alcohol derivatives can also be converted to ester derivatives through treatment with acid chlorides or anhydrides, or carbamates, through reaction with an appropriate isocyanate through known procedures. The halide or sulfonate derivative of, for example, compounds VI or VII can be displaced with several nucleophiles O, S, N, or C to produce a suitable derivative. The preparation of 3 '- (R) -K-252a XIV (Figure 3) begins with ketone IX. Compound XIV differs from the natural K-252a isomer only at the 3 'sugar position. Treatment of the Xla ketone with cyanide salts (NaCN, KCN, tetrabutylammonium cyanide, or TMSCN) provides a mixture of cyanohydrins XI I and XI I I. The mixture of isomers of cyanohydrins can be prepared through chromatography or directly converted to ester XIV or amide XV using HCL in methane. The 3'-epi-K-252a XIV can be hydrolyzed to the hydroxy acid XVI using a method, such as that used for natural K252a. See, for example, J. Antibiot.39: 1072, 1986. Acid XVI can be converted to a variety of ester or amide derivatives using procedures similar to those described for K-252a. See for example, patents of E. U. A. Nos. 4,923,986; 5,461, 146; and 5,654m427. The amide XV can be reduced to the corresponding methylamine derivative XI I using the procedure described for the conversion of natural K-252a, and XI I can be used to prepare a number of methyl-amide and urea derivatives. See, for example, patents of E. U. A. Nos. 4,923,986, 5,461, 146; and 5,654,427. 3'-epi-K252a can be reduced to aldehyde XVII I and condensed as various amines, hydrazines or hydroxylamines to form the corresponding analogs. The aldehyde XVIII can be treated with various metal alkyl, arylalkyl, aryl or heteroarylalkyl reagents, for example, Li, Mg, Zn or Cu reagents, to form the corresponding alcohol addition products. The aldehyde XVI II can be converted to functionalized olefins and their reduced products through treatment with phosphonium ylides (Quart Rev. 17: 406, 1963; Angew Int. 16: 423, 1977), phosphonates (Horner-Wadsworth-Emmons Reagents : Chem. Ver. 91:61, 1958; J. Am. Chem. Soc. 83: 1733, 1961; Org. React 25:73, 1977), silanes (J. Org. Chem. 33: 780, 1968; Synthesis 384, 1984), tellurium reagents (Tetrahedron Lett, 28: 801, 1987), or boron reagents (Tetrahedron Lett, 24: 635, 1983), followed by the reduction of alkene, for example, through catalytic hydrogenation . The alkene derived from the aldehyde XVIII can be converted to an epoxide and treated, for example, with a nucleophile, as described for epoxide IX. The epoxide IX (Figure 4) can be treated with a variety of nucleophiles to form tertiary alcohols of structure XIX. The nucleophile can be substituted. An alternative method (Figure 5) for preparing epoxides and tertiary 3'-epi-OH configurations of the alcohols is to convert the ketone XI to an olefin of structure XX using a conventional olefination reaction, for example, as described for aldehyde XVIII. The epoxide of structure XXI can be prepared asymmetrically using known methods. See, for example, 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. Org. Chem. 58: 7615, 1993). The nucleophilic epoxide opening in a manner similar to that used with epoxide IX provides the tertiary alcohol substituted with the OH group in the 3'-epi configuration. The known (R) -alcohol XXVI (Figure 6) can be converted to (S) -alcohol XXVII using conventional methods for inversion of a secondary alcohol, see, for example, Tetrahedron Left. 34: 6145; nineteen ninety six; Synthesis Letters, 1995, 336). Alternatively, compound XXII can be prepared through the treatment of epoxide XXIV with a hydride reagent such as lithium triethyl borohydride. Ketone XI (Figure 6) can be converted to triflate XI I followed by treatment with tributyltin hydride to give alkene XXI II. Known methods used to prepare K-252a derivatives with substituents at the R and R2 positions can be used to obtain the corresponding Ri and R2 substituents at 3'-epi-K-252a. See, for example, patent of E. U. A. Nos. 4, 923,986; 5,461, 146; and 5,654,427. For example, treatment of XIV (Figure 7) with an equivalent of N-bromosuccinimide (N BS) yields the XXV derivative where R is Br. Two equivalents of NBS could give the derivative where both Ri and R2 are Br. Oxidation of (S) -methanol VI to an aldehyde or a carboxylic acid derivative can be achieved using appropriate oxidation reagents (as described in Oxidations in Organic Chemistry, American Chemical Society Monograph 186, ACS Washington DC 1990). The aldehyde or carboxylic acid derivatives can further be transformed using described procedures to prepare derivatives XVI and XVIII (Figure 3).

Pharmaceutical Compositions A compound of the present invention can be administered to a mammal, for example, a human patient, as the sole active ingredient or in combination with other therapeutic agents. The compounds of the invention can be formulated into pharmaceutical compositions through admixture with pharmaceutically acceptable excipients and vehicles. Said compositions can be formulated for any route of administration, for example, parenteral, oral, or topical. The composition can be administered in unit dosage form, after preparation by conventional methods. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA). The amount and concentration of the active ingredient may vary. The concentration will depend on factors such as the total dose of the active ingredient, the chemical characteristics (eg, hydrophobic character) of the compounds employed and the route of administration, the age of the patient, the weight of the patient, and the condition being treated. trying. The compounds of the invention can be provided in a physiological pH regulating solution containing, for example, 0.1 to 10% w / v of the compound for parenteral administration. Typical dose scales are from about 1 μg / kg to about 1 g / kg of body weight per day; A preferred dose scale 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 during 4 times a day. The invention includes pharmaceutically acceptable salts of 3'-epimeric K-252a derivatives. The 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. Examples of metal are alkali metal salts such as sodium salt, lithium salt and potassium salt, alkaline earth metal salts such as magnesium salt, calcium salt, aluminum salt and zinc salt. Examples of ammonium salts are the ammonium salt and the tetramethylammonium salt. Examples of organic amine addition salts are salts with morpholine and piperidine. Examples of amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine. As used herein, "lower alkyl" means an alkyl group with 1 to 6 carbon atoms. As used herein "aryl" (alone or in such terms as arylcarbonyl and arylaminocarbonyl) means a group having from 6 to 12 carbon atoms, in an individual ring or in two fused rings. Examples of aryl groups are phenyl, biphenyl and naphthyl. A heteroaryl group contains at least one heterogeneous atom. Preferably, the heterogeneous atom is O, S, or N. Examples of heteroaryl groups are pyridyl, pyrimidyl, pyrrolyl, furyl, thienyl, imidazolyl, triazolyl, tetrazolyl, quinolyl, isoquinolyl, benzoimidazolyl, thiazolyl and benzothiazolyl. A substituted alkyl group has from 1 to 3 substituents independently selected. Preferred substituents for the 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-dialkylamino lower, dioxolane, dioxane, dithiolane and dithione. A substituted aryl, heteroaryl or arylalkyl group has from 1 to 3 independently selected substituents. Preferred substituents are lower alkyl, hydroxy, lower alkoxy, carboxy, lower alkoxycarbonyl, nitro, amino, mono or lower dialkylamino, and halogen. As used in this"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. As used herein, "sensory neuron" means a neuron responsive to an environmental stimulus such as temperature or movement. Sensory neurons are found in structures that include the skin, muscle and joints. A dorsal root ganglion neuron is an example of a sensory neuron. As used herein, "trophic factor responsive cell" means a cell to which a trophic factor binds. The cells sensitive to trophic factor include cholinergic neurons, sensory neurons, monocytes and neoplastic cells. The invention is further illustrated by the following examples. The examples are not constructed to limit the scope or content of the present invention in any way.

EXPERIMENTAL EXAMPLES Inhibition of Tyrosine Kinase Activity Epimeric K-252a derivatives were tested for the inhibition of the kinase activity of the human trkA cytoplasmic domain expressed by baculovirus 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 a substrate solution (recombinant human phospholipase C-β / glutathione S-transferase fusion protein; Rotin et al., EMBO J., 1 1: 559-567, 1992). Inhibition was measured in 100 ml assay mixtures containing 50 mM Hepes, pH 7.4, 40 μM ATP, 10 mM MnCl 2, 0.1% BSA, 2% DMSO, and various concentrations of the inhibitor. The reaction was initiated through the addition of trkA kinase and allowed to process for 15 minutes at 37 ° C. An antibody to phosphotyrosine (BI) was then added, followed by a secondary enzyme-conjugated antibody, goat anti-mouse IgG labeled with alkaline phosphatase (Bio-Rad). The activity of the bound enzyme was measured using an amplified detection system (Gibco-BRL). The inhibition data were analyzed using the sigmoidal dose-response equation (variable tilt) in GraphPad Prism. The concentration that gave 50% inhibition of kinase activity was referred to as IC50. The results are summarized in Table 1.

TABLE 1 Inhibition of TrkA Kinase Activity through 3'-epimeric K-252a Derivatives Inhibition of Trk Phosphorylation stimulated by NGF The inhibition of NGF-stimulated phosphorylation of trk through selected epimeric K252a derivatives was measured using a modified procedure from that described in the patent of US Pat. No. 5,516,771. NIH3T3 cells transfected with trkA were developed in 100 mm dishes. The confluent cells were deprived of serum by replacing the medium with a compound containing 0.05% serum free BSA-DMEM (1-100 nM) or DMSO (added to the controls for 1 hour at 37 ° C. Then NGF was added. (Harlan / Bioproducts for Science) to the cells at a concentration of 10 ng / ml for 5 minutes.The cells were used in pH regulator containing detergent and protease inhibitors.The clarified cell lysates were normalized to protein using the BCA method and they were immunoprecipitated with the anti-trk antibody.An anti-trk polyclonal antibody was prepared against a peptide corresponding to the 14 amino acids in the carboxy terminus of trk (Martin-Zanca et al., Mol. Cell. Biol 9: 24-33 , 1989) .The immune complexes were collected in Sepharose protein A beads (Sigma), separated through SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane (PVDF). ). The membrane was immunostained with the anti-phosphotyrosine antibody (UBI), followed by incubation with goat anti-mouse IgG coupled to horseradish peroxidase (Bio-Rad). The phosphorylated proteins were visualized using ECL (Amersham). The area of the trk protein band was measured and compared to the stimulated control with NGF. The measurement classification system used, based on percent reduction in the trk protein band, was as follows: 0 = no reduction; 1 = 1 -25%; 2 = 26-49%; 3 = 50-75%; 4 = 76-100%. The inhibition data of trk (Table 2) revealed that the 3'e? I-OH isomers were more potent to inhibit trk in an entire cell preparation than the corresponding natural isomers. 3'-epi-K-252a (XIV) displayed an IC50 of < 10 nM, while K-252a displayed an IC 50 of approximately 50 nM. Compound X showed complete inhibition of trkA at < 50 nM, and an IC50 of < 10 nM in cells. The natural XXIX isomer did not show complete inhibition at 100 nM. The diol VII displayed a higher potency than the natural isomer III for the inhibition of trkA in NIH3T3 cells.

TABLE 2 Effects of 3'-epimeric k-252 derivatives on the phosphorylation of trkA stimulated by NGF in NIH3T3 cells Inhibition of VEGF Receptor Kinase Activity The 3'-epimeric K-252a derivatives were tested for inhibition of the kinase activity of the VEGF receptor kinase domain expressed in baculovirus, using the procedure described above. The kinase reaction mixture, consisting of 50 nM Hepes, pH 7.4, 40 μM ATP, 10 mM MnCl 2, 0.1% BSA, 2% DMSO, and various concentrations of inhibitor, was transferred to plates coated with PLC-γ / GST. VEGFR kinase was added and the reaction was allowed to proceed for 15 minutes at 37 ° C. The phosphorylated product was detected by the anti-phosphotyrosine antibody (UBI). A secondary enzyme conjugated antibody was used to capture the phosphorylated PLC-α / GST antibody complex. The activity of the bound enzyme was measured through an amplified detection system (Qibco-BRL). The inhibition data were analyzed using the sigmoidal dose-response equation (variable tilt) in GraphPad Prism. The results are summarized in Table 3.

TABLE 3 Inhibition of VEGF Receptor Kinase Activity through 3'-epimeric K-252a Derivatives Inhibition of Protein C Kinase Activity The activity of kinase C protein was measured using the Millipore Multiscreen TCA plate assay (Pitt et al., J. Biomol. Screening, 1: 47-51, 1996). The assays were performed on 96-well multiscreen-DP plates (Millipore). Each 40 ml test mixture contained 20 mM Hepes, pH 7.4, 10 mM MgCl 2, 2.5 mM EGTA, 2.5 mM CaCl 2, 80 mg / ml phosphatidyl serine, 3.2 mg / ml dielecine, 200 mg / ml histone H- 1 (Fluka) 5 mM [? -32P] ATP, 1.5 ng of protein C kinase (UBI, mixed isozymes of a, b, g), 0.1% BSA, 2% DMSO, and derivatives of K-252a 3 '-epimeric. The reaction was allowed to proceed for 10 minutes at 37 ° C. The reaction was quenched with 50% trichloroacetic acid cooled with TCA ice). The plates were equilibrated for 30 minutes at 4 ° C, then washed with 25% TCA cooled with ice. The scintillation cocktail was added to the plates, and the radioactivity was determined using a Wallac MicroBeta 1450 PLUS scintillation counter. The IC5o values were calculated by fitting the data to the sigmoidal dose-response equation (variable tilt) in GraphPad Prism. The results are summarized in Table 4.

TABLE 4 Inhibitory effects of 3'-epimeric K-252a derivatives on protein C kinase activity Improvement of the ChAT Activity in the Spine ChAT, a biochemical marker for functional cholinergic neurons, was used. The activity of ChAT has been used to study the effects of neurotrophins (for example, NGF or NT3) on the survival and / or function of cholinergic neurons. The ChAT assay was also used as an indication of the regulation of ChAT levels within cholinergic neurons. The 3'-epimeric K-252a derivatives increased ChAT activity in the dissociated rat embryonic spinal cord culture assay (Table 5). Compound XVII increased the ChAT activity by 195% over control cultures (not treated with the epimeric K-252a derivatives) after a plaque period of 2-3 hours for the cells to bind to the culture cavities of the control tissue. In these trials, a compound was directly added to a dissociated spinal cord culture. Compounds with increased ChAT activity for at least 120% of the control activity were considered active. An increased ChAT activity was observed after a single application of a selected epimeric K-252a derivative. The results are summarized in Table 5.

TABLE 5 Improvement of ChAT activity in the spinal cord through 3'-epimeric K-252a derivatives Spinal cord cells from rat fetuses were dissociated, and the experiments were performed, essentially as described by Smith and others, J. Cell Biology 101: 1608-1621, 1985), and Glicksman et al., J. Neurochem. 61: 210-221, 1993). Dissociated cells were prepared from spinal cords cut from rats (embryonic day 14-15) through standard trypsin dissociation techniques. Cells were plated at 6 x 10 5 / cm 2 in plastic tissue culture wells coated with poly-1 -ornithine in serum free N 2 medium supplemented with 0.05% bovine serum albumin (BSA) (Bottenstein and others, Proc. Matl. Acad. Sci. USA 76: 514-517, 1979). The cultures were incubated at 37 ° C in a humid atmosphere of 5% CO2 / 95% air for 48 hours. The activity of ChAT was measured after 2 days in vitro using a modification of the Fonnum / Fonnum, J procedure. Neurochem. 24: 407-409, 1975) according to McManaman et al., (Developmental Biology 125: 31 1 -320, 1988 and Glicksman et al., (Supra).

Survival assay using rat spinal cord motor neurons Selected 3'-epimeric K-252a derivatives were analyzed for survival activity in rat spinal cord motor neuron survival. Compound XVI significantly improved the survival of spinal cord motor neurons (Figure 8). Spinal cords were excised from Sprague-Dawley rat fetuses (Charles River Laboratories, Wilmington, MA) with an embryonic age of (E) 14.5-15. Cells from only the ventral portion of the spinal cord were separated, and further enriched for motor neurons through centrifugation in a step gradient of 6.5% metrizamide, and analyzed for purity by staining with the receptor antibody. low affinity neurotrophin (IgG-192, Boehringer-Mannheim). The cells were seeded in 96-well plates previously coated with poly-1-nithin and laminin (5 μg / ml each) at a density of 6 × 10 4 cells / cm 2 in a chemically-defined serum-free N 2 medium (Bottensatein and Sato, 1979). To distinguish the binding of survival effects, 3,9-bis [(ethylthio) methyl] -K-252a (Kaneko et al., J. Med. Chem. 40: 1863-1869, 1997) was added to the cultures after an initial binding period of 1 -3 hours. Neuronal survival was determined after 4 days via calcein AM (Molecular Probes, Eugene, OR) in a fluorimetric viability assay (Bozyczko-Coyne et al., J Neurosci, Meth.50: 205-26, 1993). The culture medium was serially diluted in saline regulated in its pH with Dulbeccos phosphate (DPBS). A concentration of 6 μM of calcein supply material AM was added to each cavity. Plates were incubated for 30 minutes at 37 ° C, followed by serial dilution washes in DPBS. The fluorescent signal was read using a plate reading fluorometer (Cytofluor 2350) (excitation = 485 nm, emission = 538 nm). For each plate, the average antecedent derived from cavities that received calcein AM, but without containing cells, was subtracted from all values. The linearity of the fluorescence signal was verified for the concentration and incubation time for the cell density scale. The microscopic accounts of neurons were directly correlated with relative fluorescence values. Preparation of Compounds V Compound IVb (US Patent No. 4,923,986) was dissolved in 2 ml of trimethylphosphite and heated to reflux for 3 hours. The reaction mixture was cooled to room temperature and washed through a flash silica gel column using chloroform-methanol (20: 1) to remove the trimethylphosphite. The product was purified by flash chromatography (silica gel; ethyl acetate: hexane; 1: 1) to give compound V as a pale yellow solid (15 mg, 95% yield). MS (ESI +): m / e 406 (M + H) +, 1 H NMR (CDCl 3, 300 MHz): d 2.62 (s, 3H), 2.85 (d, 1 H), 3.37-3.45 (m, 1 H ), 4.95 (s, 1 H), 5.00 (S, 2H), 5.09 (s, 1 H), 6.29 (s, 1 H), 6.90 (d, 1 H), 7.33-7.53 (m, 5H), 7.91 (d, 1 H), 9.41 (d, 1 H).

Preparation of Compound VI To a stirred solution of compound V (161 mg, 0.397 mmol) in 8 ml of THF at 0 ° C under nitrogen, BH3 THF (1.59 ml of a 1 M solution, 1.59 mmol) was added. The reaction mixture was stirred for 30 minutes at 0 ° C and then was warmed to room temperature overnight. The mixture was again cooled to 0 ° C and 10% NaOH (0.1 ml) was added with vigorous gas evolution. Then 80 ml of hydrogen peroxide were added dropwise. After stirring at 0 ° C for 30 minutes, the mixture was diluted with 15 ml of ethyl acetate and washed with water (3 x 10 ml). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to a light green solid. The product was purified by chromatography (silica gel: hexane: ethyl acetate: 1: 1) to give compound VI as a white solid (0.12 g, 71% yield). MS (ES): m / e 424 (M + H) +, 1 H NMR (CDCl 3, 300 MHz): d 2.54 (s, 3H), 2.51 (m, 1 H), 2.99-3.01 (m, 2H) , 3.47 (m, 1 H), 3.8 (m, 1 H), 5049 (s, 2H), 6.21 (broad 2, 1 H), 6.98 (m, 1 H), 7.13-7.49 (m, 6H), 7.94-8.02 (m, 2H), 9.34 (d, 1 H).

Preparation of Compound VII To a stirred solution of Compound Va (TBDMS-V) (350 mg, 0.673 mmol) in 10 mL of THF at room temperature under nitrogen, pyridine (0.435 mL, 5.39 mmol) was added followed by osmium tetroxide. (6.73 ml, 0.673, 0.1 M in CCI). The reaction mixture was stirred at room temperature for 36 hours. During this time, the mixture changed color from yellow to orange-coffee. 30 ml of aqueous sodium bisulfite was added to the reaction mixture and the reaction was stirred for 30 minutes. The 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 via flash chromatography on silica gel using ethyl acetate to yield the Vlla compound (TBDMS-VII) as a yellow solid (280 mg, 76% yield). MS (ESI +): m / e 544 (M + H) 1 H NMR (CDCl 3, 300 MHz): d 0.56 (d, 6 H), 1079 (s, 9 H), 2.04 (dd, 1 H), 2.12 ( broad s, 1 H), 2.40 (s, 3H), 2.86 (dd, 1 H), 3.52 (broad s, 3H), 4.99 (s, 2H), 6.98 (dd, 1 H), 7.32 (t, 1 H), 7.39-7.46 (m, 4H), 7.97 (dd, 2H), 9.35 (d, 1 H). To a flask containing 2 ml of methanol at 0 ° C under nitrogen, 4 drops of acetyl chloride were added. The Vlla compound (40 mg, 0.072 mmol) in 1 ml of methanol was added dropwise to the methanolic HCl solution. The reaction mixture was stirred at 0 ° C for 1 hour, then was warmed to room temperature overnight. The solvent was removed in vacuo to give the compound Vi 1 as a tan solid (21 mg, 66% yield) MS (EST): m / e 440 (M + H) +, 1 H NMR (CDCl 3, 300 MHz ): d 2.052 (dd, 1 H), 2.43 (s, 3H), 2.90 (dd, 1 H), 3.57 (s, 1 H), 3.61 (s, 2H), 5.04 (s, 2H), 6.28 ( s, 1 H), 7.02 (dd, 1 H), 7.33- 7.54 (m, 6H), 7.95 (d, 1 H), 8.02 (d, 1 H), 9.32 (d, 1 H).

Preparation of Compound X To a stirred solution of intermediate Vlla (Figure II); 0.23 g, 0.415 mmole) in 10 ml of THF at 0 ° C under nitrogen was added triethylamine (57.9 ml, 0.415 mmole), DMAP (25.4 mg, 0.208 mmole) and p-toluenesulfonyl chloride (79.1 mg, 0.415 mmole). The reaction mixture was stirred at 0 ° C for 1 hour. Then it was slowly heated to room temperature overnight. The reaction mixture was heated for 1 hour, while verifying by thin layer chromatography (hexane: ethyl acetate, 2: 1). The reaction mixture was diluted with 30 ml of linden acetate 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 VII as a yellow film. The reaction mixture was further purified via flash chromatography on silica gel using hexane: ethyl acetate (2: 1) to produce a light yellow film (0.16 g, 55% yield). MS (APCI): m / e 708 (M + H) H NMR (CDCl 3, 300 MHz): d 0.57 (d, 6 H), 1.08 (s, 9 H), 2.01 (dd, 1 H), 2.33 (s) , 3H), 2.42 (s, 3H), 3.88 (dd, 1 H), 3.66 (dd, 2 H), 4.98 (s, 2H), 6.97 (dd, 1 H), 7.14 (d, 2H), 7.24 -7.49 (m, 7H, 7.75 (d, 1 H), 7.91 (d, 1 H), 9.35 (d, 1 H) To a stirred solution of the Villa intermediate (0.14 g, 0.198 mmol) in 5 ml of THF at 0 ° C under nitrogen, sodium hydride (15.8 mg, 0.395 mmol) was added, a vigorous evolution of gas was observed, and the reaction mixture became cloudy, 2 additional equivalents of sodium hydride and the contents were added. of the flask were stirred for a further 2 hours, then heated moderately for 4 hours.The reaction mixture was then cooled to 0 ° C and quenched with water.The reaction mixture was diluted with ethyl acetate and washed with water and brine The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo to give compound IXa as a cobalt film. Yellow lor (100.2 mg, 95% yield). MS (APCI): m / e 536 (M + H) +, 1 H NMR (CDCl 3, 300 MHz): d 0.56 (d, 6H), 1.08 s, 9H), 2.32-2.38 (m, 4H), 2.57 (d, 2H), 3.01 (dd, 1 H) , 4.99 (s, 2H), 7.01 (d, 1 H), 7.33-7.56 (m, 5H), 7.73 (d, 1 H), 7.94 (d, 1 H), 9.46 (d, 1 H). To a stirred solution of compound IXa (100 mg, 0.187 mmol) in 5 mL of THF at 0 ° C under nitrogen was added lithium-triethyl borohydride (0.37 mL of a 1 M solution in THF, 0.374 mmol) drop to drop, with evolution of gas. An additional 2 equivalents of lithium triethyl borohydride was added at 0 ° C and the reaction was stirred at 0 ° C for 30 minutes and then warmed to room temperature. The reaction mixture was cooled to 0 ° C and quenched with water, diluted with ethyl acetate and washed with water and brine. The organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo. The reaction mixture was purified via flash chromatography on silica gel using hexane: ethyl acetate (1: 1) to give Xa (R = TBDMS) as a pale yellow film. To a stirred solution of compound Xa in methanol at 0 ° C under nitrogen, a solution made of acetyl chloride (5 drops) in methanol was added. The reaction mixture was stirred at 0 ° C for 30 minutes, then warmed to room temperature overnight. The solvent was removed in vacuo leaving a yellow solid, which was purified through silica gel chromatography (hexane: ethyl acetate; 1: 1) to give compound X (30 mg, 42%). MS (ES): m / e 424 (M + H) 1 H NMR (CDCl 3, 300 MHz): d 1.39 (s, 3 H), 2.29 (dd, 1 H), 2.37 (s, 3 H), 2.92 ( dd, 1 H), 5.05 (s, 2H), 6.19 (s, 1 H), 6.95 (t, 1 H), 7.32-7.50 (m, 5H), 7.78 (d, 1 H), 7.95 (d, 1 H), 9.33 (d, 1 H).

Preparation of Compounds XIV v XV To a stirred solution of Ketone XI (Patent of US Pat. No. 4,923,986) (Figure 5; 451 mg, 1.11 mmol) in a mixture of CH2Cl2-dioxane (6 mL, 5: 1) under nitrogen, tetrabutylammonium cyanide (740 mg, 2.77 mmol) and acetic acid (95 ml, 1.66 mmol) were added at room temperature. The dark reaction mixture was stirred for 24 hours, and then concentrated in vacuo. The dark oil was dissolved in 20 ml of ethyl acetate and 2 ml of dioxane and washed with water (3 x 10 ml) and brine (1 x 10 ml). The organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo to a brown solid. Analysis by HPLC showed the presence of two cyanohydrin intermediates, Xlla and XI llb. To a flask containing 4 ml of methanol was added HCl (g) for 10 minutes. A crude cyanohydrin mixture solution from step 1 (459 mg, 1.04 mmol) in methane: dioxane (2: 1, 3 ml) was added to HCl in a methanol solution at 0 ° C. The reaction mixture was sealed and stirred at 0 ° C for 2 hours, then placed in a refrigerator for 48 hours. The flask was warmed to room temperature and carefully added in 6 N HCl. The mixture was stirred for 30 minutes and then concentrated to dryness. The residue was dissolved in 50% methanol: water and a precipitate formed while stirring overnight at room temperature. The reaction mixture was concentrated to dryness. The residue was purified via flash chromatography on silica gel using hexane: ethyl acetate (1: 1) to give compound XIV as an off-white solid. MS (ES): m / e 468 (M + H) +, 1 H NMR (CDCl 3, 300 MHz): d 2.416 (s, 3 H), 2.77 (dd, 1 H), 2.91 (s, 3 H), 2,952 ( dd, 1 H), 4.99 (s, 2H), 7.13 (dd, 1 H), 7.33 (t, 1 H), 7.44 (dd, 2H), 7.64 (t, 2H), 7.98 (d, 1 H) , 9.16 (d, 1 H). The column was eluted with ethyl acetate to obtain amide XV as a light orange product (13 mg). MS (ESI): m / e 453 (M + H) +, 1 H NMR (DMSO-d 3, 300 MHz): d 2.33 (s, 3H), 2.37 (m, 2H), 4.94 (s, 2H), 6.58 (s, 1 H), 7.19-7.64 (m, 6H), 7.81 (m, 3H), 7.96 (d, 1 H), 8.59 (s, 1 H), 9.17 (d, 1 H).

Preparation of Compound XXIII To a stirred solution of the ketone Xla (Figure 6; R = TBDMS) (95.4 mg, 0.183 mmol) in 5 mL of TH F at -78 ° C under nitrogen was added lithium diethylamide (0.12 mL, 1.5 M of solution in hexane). The reaction mixture was stirred at -78 ° C for 30 minutes. A solution of N-phenyltrifluoromethanesulfonimide (71.9 mg, 0.201 mmol) in 1.5 ml of THF was added dropwise to the reaction mixture and the mixture was stirred at -78 ° C for 30 minutes. The reaction mixture was allowed to warm to 0 ° C, stirred for 1 hour and then warmed to room temperature overnight. The reaction mixture was quenched with ammonium chloride (saturated aqueous 2 ml), diluted with ethyl acetate and washed with water. The mixture was purified via flash chromatography on silica gel using hexane: ethyl acetate (2: 1) to give compound XXIIa (R = TBDMS) as an off-white solid (66 mg, 61% yield). MS (ESI +): m / e 654 (M + H) 1H NMR (CDCl 3, 300 MHz): d 0.58 (s, 6H), 1.12 (s, 9H), 2.66 (s, 3H), 5.02 ( dd, 2H), 6.14 (s, 1H), 7.31-7.62 (m, 6H), 7.83 (d, 1H), 7.98 (d, 1H), 9.44 (d, 1H). To a stirred solution of the product from step 1 (compound XXIIa) (75 mg, 0.115 mmol) in 10 mL of THF was added lithium chloride (14.6 mg, 0.345 mmol) and tetrakis (triphenylphosphine) palladium (0) (2.6 mg, 0.0023 mmol). Tributyltin hydride (37 ml, 0.139 mmol) was added dropwise and the contents were heated to 60 ° C. The reaction mixture was heated for 4 hours, during which time the color of the reaction changed from yellow to red-black. The reaction was concentrated in vacuo and purified by flash chromatography on silica gel using hexane: ethyl acetate (2: 1). Two products were isolated: the product XXIIIa protected with TBDMS contaminated with tributyltin (60 mg) and the product XXIII deprotected (10 mg, 22%). MS (ESI): m / e 392 (M + H) +, 1 H NMR (CDCl 3 >; 300 MHz): d 2.65 (s, 3H), 4978 (s, 2H), 6.19 (d, 1H), 6.28 (d, 1H), 6.41 (s, 1H), 7.26-7.62 (m, 5H), 7.65 -7.69 (m, 1H), 7.87 (dd, 2H), 9.39 (d, 1H). Compound XXIII exhibited the following IC5o values in the assays described above: an inhibition of trkA kinase, 4 nM; inhibition of VEGF receptor kinase, 25 nM and an inhibition of protein C kinase, > 1000 nM.

Preparation of Compound XXV To a stirred solution of compound XIV (30.4 mg, 0.65 mmol) in 5 ml of THF at room temperature under nitrogen was added N-bromosuccinimide (1.6 1.6 mg, 0.065 mmol) in one portion. The reaction mixture had a light orange color initially and gradually became light purple. The reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo and the solid was purified by flash chromatography on silica gel using hexane / ethyl acetate (2: 1). This compound XXV was produced as an off-white solid (31.2 mg, 88% yield). MS (ESI): m / e 547 (M + H) 1 H NMR (CDCl 3, 300 MHz): d 2.42 (s, 3 H), 2.77-2.83 (m, 1 H), 2.86 (3 H, s), 31.66 (dd, 1 H), 4.105 (s, 1 H), 5.07 (s, 2H), 6.39 (s, 1 H), 7.01 (dd, 1 H), 7.95 (d, 1 H), 7.58 (d) , 1 H), 7.34-7.57 (m, 4H), 9.50 (s, 1 H).

Preparation of Compound XXIX XXIX Method AA a stirred solution of the epoxide XXVIII (US patent 4,923,986, compound 1-27) (90.1 mg, 0.152 mmol) in 4 ml of THF at 0 ° C under nitrogen was added lithium-triethyl borohydride (0.455 ml of 1M in THF solution, 0.455 mmol), dropwise. The reaction mixture was stirred at 0 ° C for 1 hour, then was warmed to room temperature overnight. The mixture was cooled to 0 ° C and quenched through the slow addition of methanol. Stirring was continued at 0 ° C for 15 minutes, after which the reaction mixture was warmed to room temperature. The solvent was removed in vacuo. This produced a yellow oil. The oil was purified via flash chromatography on silica gel using ethyl acetate: hexane (1: 1). This compound XXIX was produced as a white solid (75 mg, 83% yield). MS (ESI): m / e 424 (M + H) +, 1 H NMR (CDCl 3, 300 MHz): d 1.69 (s, 3H), 1.99 (s, 3H), 2.86 (dd, 1H), 3.03 (dd , 1H), 4.37 (m, 3H), 4.93 (s, 2H), 6.43 (t, 1H), 6.95 (d, 1H), 7.03 (t, 1H), 7.18 (t, 1H), 7.44 (t, 1H), 7.79 (d, 1H), 7.99 (d, 1H), 8.69 (d, 1H).

Method B To a stirred solution of Ketone XI (Figure 3, R = H) (212 mg, 0. 41 mmol) in 6 ml of THF at 0 ° C under nitrogen, methylmagnesium iodide (0.27 ml, 0.82 mmol) was added dropwise. The reaction mixture was stirred at 0 ° C for 1 hour, then was warmed to room temperature overnight. The mixture was then heated to reflux for 24 hours, then cooled to room temperature. The reaction was quenched with ammonium chloride (saturated aqueous), diluted with ethyl acetate (20 ml) and washed with water (3 x 10 ml). The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo to give a yellow residue. The product was purified via flash chromatography on silica gel using hexane: ethyl acetate (1: 1) to give compound XXIX as a tan solid (0.1 1 g, 50% yield). The 1 H NMR and the mass spectrometry data were consistent with the product obtained in Method A. Other embodiments are within the following claims.

Claims (2)

1 .- A compound of the formula: wherein: R1 and R2 independently are: Hydrogen; lower alkyl; halogen; acyl; nitro; sulfonic acid; -CH = NR4, wherein R4 is guanidino, heterocyclic or -NR5R6, wherein R5 or R6 is hydrogen or lower alkyl, and the other is hydrogen, lower alkyl, acyl, aryl, heterocyclic, carbamoyl or lower alkylaminocarbonyl; -N R5R6; -CH (SR7) 2, wherein R7 is lower alkyl or alkylene; - (CH2) jR8, wherein j is 1 -6, and R8 is halogen; substituted aryl; unsubstituted aryl; substituted heteroaryl; unsubstituted heteroaryl; N3; -CO2R9, wherein R9 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, ^ Substituted heteroaryl, or unsubstituted heteroaryl; -C (= O) N R 10 R 1 1, wherein R 10 and R 1 1 are independently hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, aralkyl substituted, aralkyl not substituted, lower alkylaminocarbonyl, or lower alkoxycarbonyl, or R 10 and R 1 1 combine with a nitrogen atom to form a heterocyclic group; -OR12, wherein R12 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted alkyl, unsubstituted aryl; or -C (= O) R13, wherein R13 is hydrogen, NR10C11, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted aralkyl, or unsubstituted aralkyl; -N R10R1 1; -C (= O) R 14, wherein R 14 is hydrogen, lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl or unsubstituted heteroaryl; -S (= O) rR15, wherein r is from oa to 2, and R15 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted aralkyl, unsubstituted aralkyl , thiazolinyl, (CH2) aCO2R16, wherein a is 1 or 2, and R16 is hydrogen or lower alkyl, or - (C H2) aC (= O) N R 10 R 1 1); -OR17, wherein R17 is hydrogen, lower alkyl or -C (= O) R18, wherein R18 is substituted lower alkyl, unsubstituted lower alkyl, substituted aryl, or unsubstituted aryl; -C (= O) (CH2) jR19, wherein R19 is hydrogen, halogen, NR10R11, N3, SR15, or OR20, wherein R20 is hydrogen, substituted lower alkyl, unsubstituted lower alkyl, or C (= O) R14; - (CH2) dCHR21CO2R16A, wherein d is 0-5, and R21 is hydrogen, CONR10R11, or CO2R16A, wherein R16A is equal to R16; - (CH2) dCHR21CONR10R11; -CH = CH (CH2) mR22, wherein m is 0-4, and R22 is hydrogen, lower alkyl, CO2R9, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, OR12, or NR10R11; -CH = C (CO2R16A) 2; -C = C (CH2) mR22; -SO2NR23R24, wherein R23 and R24 independently are hydrogen, lower alkyl, or groups that form a heterocycle with the adjacent nitrogen atoms; -OCO2R13A, wherein R13A is equal to R13; or -OC (= O) NR10R11; R3 is hydrogen; lower alkyl; carbamoyl; Not me; tetrahydropyranyl; hydroxyl; C (= O) H; aralkyl; lower alkanoyl; or CH2CH2R25, wherein R25 is halogen, amino, di-lower alkylamino, hydroxy or lower alkylamino substituted with hydroxy; X is hydrogen; formyl; carboxyl; lower alkoxycarbonyl; lower alkylhydrazinocarbonyl; -CN; lower alkyl; -C (= O) NR26R27, wherein R26 and R27 independently are hydrogen, unsubstituted lower alkyl or unsubstituted aryl; or R26 and R27 are combined with a nitrogen atom to form a heterocyclic group; -CH (R34) W, wherein R34 is hydrogen or lower alkyl, and W is -N = CHN (alkyl) 2; guanidino; N3; NR28R29, wherein R28 or R29 is hydrogen or lower alkyl, and the other is hydrogen, allyl, alkanoyl, aryloxycarbonyl, unsubstituted alkyl, or the residue of an a-amino wherein the hydroxy group of the carboxyl group is excluded; -CO2R9; -C (= O) NR10R11; -S (= O) rR30, wherein R30 is substituted or unsubstituted lower alkyl, aryl, or heteroaryl; or -OR31, wherein R31 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkanoyl; -CH = N-R32, wherein R32 is hydroxyl, lower alkoxy, amino, guanidino, ureido, imidazolylamino, carbamoylamino, or NR26AR27A (wherein R26A is equal to R26 and R27A is equal to R27); or -CH Q is a sugar residue represented by wherein V represents hydrogen, methyl, ethyl, benzyl, acetyl or trifluoroacetyl; And it's hydrogen; -OH; -OC (= O) R33, wherein R33 is alkyl, aryl or amino; -OCH2O-alkyl, -O-alkyl; aralkyloxy; or X and Y combine as -X-Y- to form -CH2OCO2- or -CH2N (R16B) CO2-, (wherein R16B is equal to R16); A1 and A2 are hydrogen, or both combine to present O; or B1 and B2 are hydrogen, or both combine to represent O; or a pharmaceutically acceptable salt thereof; provided that at least one of A1, A2 or B, B2, represents O; and also with the proviso that both X and Y simultaneously are not hydrogen. 2 - The compound according to claim 1, wherein X is -C (= O) NR26R27, carboxyl, lower alkoxycarbonyl, formyl, lower alkyl -CH2OR31, -CH2NR28R29, or -CH2S (O) rR30. 3. The compound according to claim 1, wherein R1 and R2 are H. 4. The compound according to claim 1, wherein R3 is hydrogen or a protecting group. 5. The compound according to claim 1, wherein the compound is selected from the group consisting of (compounds VI, VII, VIII, X, XII, XIV, XV, XVI, XVII, XVIII, XIX, XXV, and XXVII): vi vp vm XIX - CH ,, O, S, NH, OÍR "R41" H or lower alkyl R42 - lower alkyl XXVII XXV 6 - A pharmaceutical composition comprising a compound according to claim 1. 7. A method for inhibiting the activity of a tyrosine kinase, comprising contacting the tyrosine kinase with a compound of claim 1. 8. The method according to claim 7, wherein the tyrosine kinase is a tyrosine kinase C. The method according to claim 7, wherein the tyrosine kinase is trkA. 10. A method for inhibiting the phosphorylation of a tyrosine kinase through a second kinase, comprising contacting the second kinase with a compound of claim 1. 1 1 .- A method for improving the function of a cholinergic neuron, comprising contacting the cholinergic neuron with a compound of claim 1. 1

2. A method for improving the survival of a cholinergic neuron, comprising contacting the cholinergic neuron with a compound of claim 1.